Biblografia Endocrino BQ

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ENDOCRINOLOGY CONCEPTS FOR MEDICAL STUDENTS

H. Maurice Goodman Department of Physiology, University of Massachusetts Medical School, Worcester, Massachusetts 01655
ADV PHYSIOL EDUC 25: 213224, 2001.
WHAT IS ENDOCRINOLOGY? The explosive growth of information in endocrinology made possible by unprecedented advances in technology and by expansion of the number of investigators engaged in endocrinological research presents a difcult and growing challenge to those of us who teach endocrine physiology to medical students. The scope of research has so extended the boundaries of endocrinology and blurred the distinctions among disciplines that even dening endocrinology is problematic. Additionally, it has become increasingly difcult to decide what should be taught to rst-year medical students and in what context. Regulation of cellular functions by hormones represents only a subset of the larger eld of chemical communication that includes aspects of neurobiology, cell biology, immunology, and developmental biology. From the perspective of the target cell, what we call a hormone is quite arbitrary. Cellular and molecular processes associated with production, secretion, and actions of hormones are no different from actions of hundreds of other paracrine and autocrine factors, immunomodulators, neurotransmitters, growth factors, and so forth. The exquisite sensitivity of molecular biological tools has uncovered hormone production and hormone receptors in the most unexpected places. A host of nonendocrine tissues produce some of the same molecules secreted by the classic endocrine glands and use them to serve as local paracrine modulators or neurotransmitters. It is now apparent that hormones act on many more cells than their classically dened targets and that virtually every tissue in the body participates in some endocrine function.
Despite all of the above, the endocrine system remains a vital component of any course in medical physiology, and mastery of the principles and phenomena it encompasses is essential for later study of clinical medicine. The role of the endocrine system is to coordinate and integrate cellular activity within the whole body, regulating cellular and organ function from a distance, with factors produced locally, often in response to hormones, governing local ne tuning. From this perspective, it becomes more logical to focus on the physiological processes that are governed by the endocrine system rather than the classical morphologically based gland by gland survey, although students need to master basic facts connected with each gland and hormone. We can regard the endocrine system as having the following physiological missions
Regulation of sodium and water balance: preservation of the volume/pressure reservoir required for tissue perfusion Regulation of calcium balance: preservation of extracellular uid concentrations required for membrane integrity, intracellular signaling, hemostasis, etc., and preservation of skeletal integrity Regulation of energy balance: preserving, accessing, and interconverting metabolic fuels to meet cellular energy demands Coordination of processes for coping with a hostile environment Coordination of growth and development
1043 - 4046 / 01 $5.00 COPYRIGHT 2001 THE AMERICAN PHYSIOLOGICAL SOCIETY
VOLUME 25 : NUMBER 4 ADVANCES IN PHYSIOLOGY EDUCATION DECEMBER 2001
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Coordination of processes associated with reproduction and lactation
From this perspective, it is clear that at least some aspect of virtually every physiological system lies within the realm of endocrine control. No single hormone or endocrine gland can accomplish any of these missions alone, and virtually every hormone participates in fullling multiple missions. Consequently, students need to understand not only how hormones act but also how they interact. Some basic concepts transcend the wide range of physiological actions of hormones and may provide a foundation for understanding hormonal regulation. Many of these concepts are also the bases for diagnosis and treatment of endocrine disorders. CONCEPTS RELATED TO CONTROL OF HORMONE SECRETION Negative feedback control. Understanding negative feedback lies at the heart of understanding endocrine control systems. The essence of negative feedback control of hormone secretion is that some consequence of secretion blocks or dampens further secretion (Fig. 1). The model depicted would ensure constancy of a regulated parameter at some set point except for the transient negative deviations that initiate the cycle
FIG. 2. Negative feedback including features that allow for adaptive changes: adjustment of the set point or additional input to override the set point.
and the positive overshoots that stop it. This model is inexible and permits no opportunity for adaptation to changing environmental demands. The added elements of changing the set point or overriding the set point, shown in Fig. 2, are more likely to meet physiological requirements. Many biological examples appear more complex but are simply superimposition of the same principles. The pituitary and adrenal glands are in a negative feedback relationship (Fig. 3), with cortisol acting as an inhibitor of both pituitary secretion of ACTH and hypothalamic secretion of corticotropin-releasing hormone and arginine vasopressin. Hypothalamic input to the negative feedback system allows for episodic override and adjustment of the set point in response to environmental inputs. Input to the hypothalamus comes also from circadian elements that impose periodic adjustments of the set point. Positive drive to the system is imposed by stress, in this case hypoglycemia. Another aspect of negative feedback illustrated here is negation of the positive drive im-
FIG. 1. Simple negative feedback.
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FIG. 3. Feedback relationships governing ACTH and cortisol secretion. Note that the simple negative feedback relationship of the pituitary and adrenal cortex is extended to include the paraventricular nucleus (PVN) of the hypothalamus and corticotropin-releasing hormone (CRH) and arginine vasopressin (AVP). Adjustment of the set point throughout the day accounts for circadian rhythm, and overriding of the set point, at least transiently, is imposed by the stress of hypoglycemia. A broader negative feedback relationship between hypoglycemia and the adrenal cortex is also present as glucocorticoid hormones contribute to alleviation of the hypoglycemia.
posed by hypoglycemia when glucose production is increased as a consequence of cortisol secretion. Negative feedback systems operating in opposite directions combine to maintain blood glucose concentrations within narrow limits (Fig. 4). This illustration also incorporates feed-forward elements. Minimizing upward deviations is facilitated by the feed-forward effects of the intestinal hormone, glucagon-like peptide 1, which stimulates insulin secretion in anticipation of absorption of a dietary glucose load. A similar but perhaps more ambiguous role is played by parasympathetic stimulation of both the - and -cells during the cephalic phase of eating. Override of negative feedback to permit blood glucose to increase is provided by the sympathetic innervation of the islets and circulating catecholamines from the adrenal me-
dulla. Such a transient override ensures adequacy of high energy fuels to meet the needs of episodic muscular activity or other responses to environmentally imposed emergencies. Positive feedback. In positive feedback systems, the consequences of hormone secretion feed back to reinforce the drive for secretion rather than dampen it. Rather than maintaining matters stable and unchanging, positive feedback creates instability and leads to explosive changes (Fig. 5). Consequently, positive feedback is rare in biology. The best example is oxytocin secretion by the posterior pituitary lobe during the birthing process. Stretch exerted on the uterine cervix is a powerful stimulus for oxytocin secretion and is transmitted to the oxytocin-secreting magnocellular elements in the paraventricular and supraop-
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FIG. 4. Feedback regulation of pancreatic islets. Note that glucose has opposite effects on insulin and glucagon secretion, and override of negative feedback is achieved by parasympathetic and sympathetic input as well as by the feed-forward input of glucagon-like peptide 1.
tic hypothalamic nuclei by sensory neural inputs. With progressively greater stretch sensed by cervical nerve endings by the contractions of uterine muscle, more and more oxytocin is released, stimulating escalating contractile force and stretch on the cervix. Ultimately, the cycle is broken by the explosive evacuation of the uterus with the birth of the baby (Fig. 6). CONCEPTS RELATED TO SPECIFICITY Because of internal secretion of hormones into the blood, hormones are widely disseminated throughout the body and have access to virtually all cells. However, only certain cells respond to any particular hormone. These target cells differ from all other cells in the respect that they express receptors for that hormone (Fig. 7). In this example, only the cells that express receptors change color in response to hormones. Receptors are proteins located within a cell or on its surface and contain a hormone recognition site that
binds its hormone with high afnity and selectivity and a signal-transducing domain. Typical cells express thousands to tens of thousands of copies of receptors for a particular hormone. Binding the hormone produces some intramolecular change that activates the signal-transducing domain. Some receptors initiate signaling through their intrinsic enzymatic activity, but most do so by the change in their interactions with other proteins that may or may not have enzymatic activity. The information delivered to the target cell is present in the structure and three-dimensional conformation of the hormone and is sufcient only to activate the receptor. It appears that activation of the receptor is an all-or-none phenomenon, with gradations in response resulting from gradations in the numbers of receptors that are activated in each cell. The receptor, by virtue of the biochemical changes it triggers in transducing the signal, initiates a particular biochemical change or group of changes (Fig. 8). Once the
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FIG. 5. Positive feedback is initiated by some perturbation and culminates in some explosive event.
hormone has delivered its message by activating the receptor, except in very rare cases, it plays no role in shaping the response. Rather, the signals generated in the target cell are determined by the signal-transducing component of the receptor. In many cases, there is more than a single class of receptors for a particular hormone, and each class usually activates a different biochemical pathway. Only in rare instances can a receptor bind more than a single entity of hormone with high afnity, and when this occurs (e.g., parathyroid hormone and the parathyroid hormone-related peptide), both agents produce identical cellular responses. It is possible to express chimeric receptors that contain the hormone recognition component of one hormone with the signal-transducing component of a second hormone. The biochemical changes set in motion are invariably characteristic of the signaling domain of the receptor (Fig. 8). The nature of the nal response elicited in a target cell is not determined by the intracellular signal generated by the receptor but, rather, by the effective machinery expressed in the cell as a consequence of its
differentiated state. For example, receptors for the parathyroid hormone are present in the basal membranes of cells of both the proximal and distal portions of the nephron (Fig. 9). Binding of the hormone initiates the same signaling cascade in both cell types, but the proximal tubules respond by decreasing phosphate reabsorption from the glomerular ltrate and increasing hydroxylation of vitamin D, while the distal cells respond by increasing reabsorption of calcium. CONCEPTS RELATED TO TARGET CELL RESPONSIVENESS Responsiveness of target cells to stimulation by their hormones is not constant but may vary widely in different physiological states and is often adjusted by the actions of other hormones or local paracrine or autocrine agents as well as the primary hormone. Factors that govern the magnitude of the response to a hormone. 1) The most obvious determinant of the magnitude of the response is the concentration of hormone that is available to bind to
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FIG. 6. Positive feedback of oxytocin secretion during childbirth.
receptors. That concentration, in turn, is determined by
The number of functional receptors that are expressed The afnity of the receptor for the hormone The status of postreceptor amplication mechanisms The status and abundance of effector molecules
The rate of hormone secretion The rate of delivery by the circulation to the target cell surface, which is slower if the hormone circulates bound to plasma proteins than if it is unbound The rate at which the hormone is degraded or excreted
2) Of equal importance to the hormone concentration is the number of competent target cells that express functional receptors. 3) The sensitivity of target cells to hormonal stimulation is not constant and depends on
The relationship between the magnitude of response to a hormone and the concentration of hormone producing that response is described by a sigmoidal curve (Fig. 10). The sensitivity to a hormone is often dened as the concentration needed to produce a half-maximal response. Target organ sensitivity is not constant and is often adjusted in accordance with physiological circumstances. In the example shown in Fig. 10, we may assume that curve B is the basal sensitivity that may be
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FIG. 7. Specicity of hormone (H) action. Only cells that express receptors (R) for the hormone can respond to it (in this case by changing color).
FIG. 8. Receptors contain a hormone recognition domain and a signal-transducing domain. The nature of the signal generated is a function of the receptor and not of the hormone.
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FIG. 9. Proximal and distal renal tubular cells respond to parathyroid hormone (PTH) by increasing cAMP production, but cAMP initiates different events in each cell in accordance with the capabilities programmed during cellular differentiation.
increased (curve A) or decreased (curve C). With increased sensitivity, a lower concentration of hormone is required to produce a half-maximal response.
FIG. 10. Concentration-response curves showing 3 different levels (curves AC) of sensitivity as dened by the concentration of hormone required to produce a half-maximal response (ED50).
Sensitivity does not necessarily parallel hormone binding by the receptor and, therefore, is not necessarily a function of the afnity of the receptor for the hormone. Because it depends on many postreceptor events, the response to a hormone may be at a maximum at a hormone concentration that does not saturate all of the receptors (Fig. 11). When 100% of the receptors need to be occupied to obtain a maximum response, cells are said to express spare receptors. For example, glucose uptake by the fat cell is stimulated in a dose-dependent manner by insulin, but the response reaches a maximum when only a few percent of the available and functional insulin receptors are occupied by insulin. The afnity of the receptor for the hormone is dened as the concentration at which half of the receptors are occupied by hormone. Because the response is related to the number of receptors that are activated and can therefore produce a biochemical response, the consequence for cells that express spare receptors is that they are more sensitive to the hormone than would be predicted from their binding afnity. In the example shown in Fig. 11, A twofold excess of receptors reduced by half the concentration of hormone needed to produce a half-maximal response.
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Under a variety of circumstances, cells may increase (upregulate) or decrease (downregulate) the number of functional receptors they express. Upregulation or downregulation can be achieved by adjusting the relative rates of receptor synthesis and degradation, receptor endocytosis and sequestration, or covalent modication through phosphorylation or dephosphorylation. The consequences of upregulation or downregulation for sensitivity are shown in Fig. 12. Small changes in receptor abundance are of little consequence in the presence of a large number of spare receptors but can be quite profound for cells that express no spare receptors. It is more common for cells to adjust the number of receptors rather than for receptor afnity to regulate their sensitivity to a hormone.
FIG. 11. The presence of spare receptors lowers the concentration of hormone needed to produce a half-maximal response below that needed to produce half-saturation of receptor binding sites (i.e., the afnity of the receptor for the hormone). Kd, dissociation constant.
The apparent sensitivity to hormonal stimulation is not a function only of receptor number. Downstream events also contribute to the concentrationresponse relationship. On the cellular level, upregulation or downregulation of effector molecules such as enzymes, ion channels, and contractile proteins, etc., may increase or reduce the maximum capacity for a response even though the sensitivity, as dened earlier, is unchanged (Fig. 13). On a tissue or organ level, the measured response is the
FIG. 12. Regulation of receptor number changes the concentration of hormone needed for a half-maximal response (i.e., the sensitivity).
FIG. 13. A change in the capacity of effector elements downstream from the receptor or in the number of competent cells changes the magnitude of the response without necessarily changing the concentration of hormone needed to produce a half-maximal response.
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aggregate of the contributions of all of the responding cells, so that the magnitude of the response to a particular concentration of hormone is a function of the number of available cells as well as the competence of each cell. Thus a change in response capacity will result in the need for a greater or lesser concentration of hormone to produce a given level of response and, therefore, appears as a change in sensitivity, even though the concentration of hormone required for the half-maximal response may remain unchanged. Factors that govern the duration of the response to a hormone. Of equal concern to the magnitude of response is its duration. Factors that govern the duration of the response to a hormone include 1) the duration of hormone availability, which is determined by
FIG. 14. Responses to multiple hormonal inputs may be additive (glucagon epinephrine) or synergistic (glucagon epinephrine cortisol).
The duration of secretion
FIG. 15. Reinforcement. Different effects of cortisol in different tissues reinforce hepatic actions to increase glucose production.
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The rate of hormone clearance from the blood, usually described as its half-life
2) whether the response results from
A rapidly reversible covalent change, i.e., phosphorylation or dephosphorylation of key enzymes Or genomic events involving synthesis of proteins and the half-lives of the proteins
motes the breakdown of protein in skeletal muscle and the release of amino acids into the circulation. It also facilitates the breakdown of triglycerides in adipose tissue and the release of glycerol and fatty acids into the blood. Amino acids and glycerol (along with lactate) are the principal substrates for gluconeogenesis. Push-pull. Secretion of glucose by the liver is under both positive and negative control. In emergency situations or during exercise, there is increased demand for glucose. Just increasing the secretion of glucagon and epinephrine would increase the rate of glycogen breakdown and would, therefore, increase glucose production. Stimulation by these hormones becomes much more effective if the restraining effect of insulin is simultaneously relieved. This push-pull mechanism allows for rapid, unhindered glucose mobilization (Fig. 16). Dozens of other examples of patterns of hormone interactions at the molecular, cellular, organ, and
CONCEPTS RELATED TO INTEGRATION Hormones seldom act alone or on a single tissue in carrying out their missions of regulation and coordination. The following examples illustrate some concepts of integration. Additivity and synergy. Multiple hormones often work in concert, and in some instances, they may appear to be redundant. Figure 14 illustrates the interactions of glucagon, epinephrine, and cortisol to increase blood glucose concentrations. The data shown are redrawn from Eigler et al. (1). Both epinephrine and glucagon, when administered alone to dogs, produced an increase in blood glucose, and when given together, their effects were additive. Cortisol alone had little effect on the blood glucose concentration, but when epinephrine plus glucagon were given to cortisol-treated dogs, the rise in glucose was far greater than the sum of the increases produced by the individual hormones alone. These data illustrate the concept of synergism, wherein the response to a combination of hormones is greater than the sums of their individual actions. Reinforcement. As shown in Fig. 14, the actions of several hormones may converge to regulate the process of glucose production. Figure 15 shows an example of how the diverse actions of a single hormone exerted on several different tissues may converge to reinforce a critical action. One of the primary effects of glucocorticoids is to stimulate gluconeogenesis in the liver. Cortisol increases the enzymatic capacity of the liver for gluconeogenesis and renders the hepatocyte sensitive to gluconeogenic stimulation by glucagon and catecholamines. Efcient gluconeogenesis, however, requires a supply of substrate. Cortisol pro-
FIG. 16. Push-pull mechanism for producing a rapid increase in glucose production.
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organismal levels can be cited to underscore concepts of integration. The concept of homeostasis is a fundamental tenet of physiology, and the endocrine system is its principal defender. The everchanging challenges to maintaining the constancy of the internal environment and ensuring survival of the species demand that endocrine control be dynamic, adaptable, and precise. The concepts presented here will hopefully provide students with some insight into the workings of the endocrine system. The concepts presented here are not unique to endocrinology, however, and perhaps
will also contribute to their understanding of physiology as a whole.

Address for reprint requests and other correspondence: H. M. Goodman, Dept. of Physiology, Univ. of Massachusetts Medical School, Worcester, MA 01655 (E-mail: maurice.goodman@umassmed.edu).
REFERENCES
1. Eigler N, Sacca L, and Sherwin RS. Synergistic increments of glucagon, epinephrine, and cortisol in the dog. J Clin Invest 63: 114, 1979.
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Available online at www.sciencedirect.com
Frontiers in Neuroendocrinology
Frontiers in Neuroendocrinology 29 (2008) 211218 www.elsevier.com/locate/yfrne
Review
Mechanisms of nongenomic actions of thyroid hormone

Paul J. Davis
b c
a,b,*
, Jack L. Leonard c, Faith B. Davis
a Ordway Research Institute, Inc., 150 New Scotland Avenue, Albany, NY 12208, USA Albany Medical College and Stratton Veterans Aairs Medical Center, Albany, NY, USA Department of Physiology, University of Massachusetts Medical School, Worcester, MA, USA
Available online 5 October 2007
Abstract The nongenomic actions of thyroid hormone require a plasma membrane receptor or nuclear receptors located in cytoplasm. The plasma membrane receptor is located on integrin aVb3 at the Arg-Gly-Asp recognition site important to the binding by the integrin of extracellular matrix proteins. L-Thyroxine (T4) is bound with greater anity at this site than 3,5,3 0 -triiodo-L-thyronine (T3). Mitogen-activated protein kinase (MAPK; ERK1/2) transduces the hormone signal into complex cellular/nuclear events including angiogenesis and tumor cell proliferation. Acting at the integrin receptor and without cell entry, thyroid hormone can foster ERK1/2-dependent serine phosphorylation of nuclear thyroid hormone receptor-b1 (TRb1) and de-repress the latter. The integrin receptor also mediates actions of the hormone on intracellular protein tracking and on plasma membrane ion pumps, including the sodium/protein antiporter. Tetraiodothyroacetic (tetrac) is a T4 analog that inhibits binding of iodothyronines to the integrin receptor and is a probe for the participation of this receptor in cellular actions of the hormone. Tetrac blocks thyroid hormone eects on angiogenesis and cancer cell proliferation. Acting on a truncated form of nuclear TRa1 (TRDa1) located in cytoplasm, T4 and 3,3 0 ,5 0 -triiodothyronine (reverse T3), but not T3, cause conversion of soluble actin to brous (F) actin that is important to cell motility, e.g., in cells such as glia and neurons. Normal development of the central nervous system requires such motility. TRb1 in cytoplasm mediates action of T3 on expression of certain genes via phosphatidylinositol 3-kinase (PI 3-K) and the protein kinase B/Akt pathway. PI 3-K and, possibly, cytoplasmic TRb1 are involved in stimulation by T3 of insertion of Na,K-ATPase in the plasma membrane and of increase in activity of this pump. Because ambient thyroid hormone levels are constant in the euthyroid intact organism, these nongenomic hormone actions are likely to be contributors to basal rate-setting of transcription of certain genes and of complex cellular events such as angiogenesis and cancer cell proliferation. 2007 Elsevier Inc. All rights reserved.
Keywords: Thyroxine; 3,5,3 0 -Triiodo-L-thyronine; Integrin aVb3; Nuclear thyroid hormone receptor-b1 (TRb1); TRDa1; Actin; Protein kinase B/Akt; Mitogen-activated protein kinase (MAPK); ERK1/2
1. Introduction The mechanism of action of thyroid hormone on or in cells has been assumed to begin in the cell nucleus and to require participation of receptor proteins for thyroid hormone in the nuclear compartment. These receptors are transcriptionally active proteins that cause expression of thyroid hormoneresponsive genes. This classical or genomic mechanism (see below) has been complimented in the past decade by descrip*
Corresponding author. E-mail address: pdavis@ordwayresearch.org (P.J. Davis).
tions of thyroid hormone action that are now understood to involve novel extranuclear (nongenomic) mechanisms in a variety of cells, including those of the central nervous system (CNS). Such nongenomic mechanisms appear to be relevant to proliferation and motility of endothelial cells and certain tumor cells, including glioma cells that are models for human brain tumors. The mechanisms are also important to the state of the actin cytoskeleton and motility of normal nerve and glial cells. The current review is focused on recent insights into such nongenomic actions in a variety of cells and on interfaces of nongenomic and genomic actions of the hormone that are now known to exist.
0091-3022/$ - see front matter 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.yfrne.2007.09.003
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2. Genomic mechanisms of thyroid hormone action The classical molecular mechanism of thyroid hormone action involves uptake of L-thyroxine (T4) or 3,5,3 0 -triiodoL-thyronine (T3) by target cells, access of T3 to the cell nucleus and complexing of the hormone with nuclear thyroid hormone receptor (TR) protein [6163]. TR is found in the nucleus as a heterodimer with retinoic acid X receptor (RXR). The heterodimeric complex sheds corepressor proteins when T3 is bound and recruits coactivators that facilitate binding of the heterodimer-T3 complex to thyroid hormone response elements (TREs) of hormone-responsive genes and consequent gene transcription [63]. Activation of TR may involve phosphorylation of the receptor [12,58]. This genomic mechanism of hormone action has been demonstrated in a variety of thyroid hormone-responsive cells and leads to modulation of transcription of a hundred or more genes [24,43]. Characteristics of genomic actions of the hormone include the requirement for access of the hormone to the cell interior, translocation of the hormone to the nucleus, altered rates of gene transcription, generation of specic mRNAs, translation and changes in cell content or secretion of specic gene products. Several or more hours are usually required for genomic mechanisms to be manifest. L-Thyroxine (T4) can act via nuclear TR, but the anity of the receptor for T4 is much lower than that for T3. Thus, T3 is the natural ligand of TR [6163]. In the genomic concept of hormone action, T4 is viewed as a prohormone that yields the more metabolically active T3 via action of tissue deiodinase activities. That T4 acts as a hormone as well as a prohormone will be considered below and has been reviewed elsewhere [16]. 3. Nongenomic mechanisms of thyroid hormone action For more than two decades, actions of thyroid hormone in a variety of cells have been described that do not primarily involve nuclear TR (reviewed in [2,14]) and thus are nongenomic. The mechanisms of several of these nongenomic actions of thyroid hormone are now understood, at least in part, and depend upon cellular signal transduction systems and either novel cell surface receptors for thyroid hormone [3,14] or extranuclear TRb [33,45] or derivatives of TRa ([28]; see section on actin, below). It is important to point out that actions of thyroid hormone that begin nongenomically at a cell surface receptor may culminate in complex nuclear and cellular events. One example is phosphorylation of Ser-142 of the TRb1 isoform [12] that results in altered transcriptional activity (de-repression) of the receptor due to shedding of corepressor proteins and recruitment of coactivators [12]. Estrogen receptor-a (ERa) in the nucleus may also be specically phosphorylated under the direction of thyroid hormone that is acting exclusively at the cell surface [60]. These are examples of interfaces between nongenomic and genomic mechanisms of action of iodothyronines. Other complex
cellular events directed by thyroid hormone from the plasma membrane include cell proliferation in endothelial cells and specic tumor cells ([13,15]; see below). Acting exclusively at the cell surface, thyroid hormone can also modulate intracellular protein tracking, such as translocation from cytoplasm to the nucleus of nuclear hormone receptor superfamily membersincluding TRb1 and ERaand moieties such as signal transducer and activator of transcription-3 (STAT3) [39], STAT1a [39], and Trip230 [7], a coactivator for the nuclear receptor for thyroid hormone. Another action of the hormone that is initiated at the cell surface is modulation of the activity of the Na+/H+-antiporter or exchanger (sodium/hydrogen ion exchanger, NHE) [10,29]. This eect does not appear to involve the cell nucleus. When actions of thyroid hormone are initiated within the cell, but outside the nucleus, nongenomic mechanisms may involve a derivative of cytoplasmicTRa1 that regulates the state of actin, converting soluble into brous (F) actin, and regulates cell motility. These eects have been well-characterized in CNS cells [21,22] and are reviewed in detail below. TRb1 also exists in cytoplasm and remarkably is involved in transduction of the thyroid hormone signal through a specic signal transduction cascade into specic gene expression, e.g., the immodulatory protein, ZAKI-4 [44,45]. From the foregoing, it is apparent that nongenomic actions of thyroid hormone result in changes in cell function. These actions have usually been demonstrated in thyroprival cells or tissues in vitro that are acutely re-exposed to thyroid hormone. This paradigm has resulted in characterization of nongenomic actions of the hormone as acute or rapid onset (seconds or minutes) when compared to purely genomic mechanisms that require gene transcription and consequent translation of mRNA. This characterization is not accurate in the context of the intact organism, where ambient concentrations of thyroid hormone normally are stable. In this setting we assume that the hormone contributes to basal activity rates of the functions reported, e.g., proton eux of NHE-1 [10], rates of protein tracking [14] or background levels of phosphorylation of specic nucleoproteins [12,60]. The hormone is assumed to condition the state of actin in the cell and the rate of migration of cells toward or away from trophic factors. The term, acute actions, is also inaccurate when interfaces between nongenomic and genomic mechanisms are studied. Nongenomic mechanisms thus are best characterized as those which at initiation do not primarily depend upon intranuclear complexing of TR and thyroid hormone. Another feature of nongenomic mechanisms of thyroid hormone action is the plurality of hormone analogues that may initiate specic actions. Genomic actions, as noted above, are T3-dependent. In contrast, the nongenomic mechanism of action of the hormone on the state of actin in the cell requires T4 or reverse-T3 (rT3, 3,3 0 ,5 0 -triiodothyronine) and is insensitive to T3. On the other hand, nongenomic actions that aect the NHE are conditioned by T3
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[10,29], whereas intracellular protein tracking and the phosphorylation of nucleoproteins that is initiated at the cell surface are primarily T4-responsive eects [14]. As will be noted below, tetraiodothyroacetic acid (tetrac), a T4 derivative, is purely inhibitory of the actions of T4 and T3 that are initiated at the cell surface [3]; inside the cell, however, tetrac has low potency thyromimetic actions, e.g., suppressing TSH release by pituitary gland thyrotropes [4]. Although the nongenomic actions of thyroid hormone on mitochondria are not included in this review, it should be noted that 3,5-diiodothyronine (T2) is an eective modulator of cellular respiration [41] and perhaps more potent than T3 in this regard. 4. Receptors for nongenomic actions of thyroid hormone Studies of thyroid hormone action on cell surface events, such as calcium eux [11,49] or glucose uptake [54,55], several decades ago implied the existence of one or more plasma membrane receptors for T3 or T4. This was particularly the case when experiments were conducted in membrane vesicles from enucleate cells, such as the human mature erythrocyte (RBC) [49] or when rates of glucose transport in intact cells were increased in a time frame in vitro that precluded transcription and translation. Binding sites for the hormone were also described on RBCs that inferred the saturation of binding sites on human thyroxine-binding globulin (TBG) (T3 uptake) by a partition of labeled iodothyronine between erythrocytes and plasma [26]. The latter sites, however, were not seen to have a specic function and thus could not be characterized as receptors. Recently, a cell surface receptor for iodothyronines has been described on a structural protein of the plasma membrane of virtually all cells [3,14]. This protein is integrin aVb3, a heterodimer that interacts with a substantial number of proteins of the extracellular matrix (ECM) [1,50]. The integrin is highly plastic and is capable of transducing a number of discrete signals. These include ECM protein signals that are converted into cellular responses (outsidein conduction) and signals that originate within the cell to the external milieu (inside-out conduction). The thyroid hormone receptor domain is at or near the Arg-Gly-Asp (RGD) recognition site on the integrin [3,8]. ECM proteins such as vitronectin, bronectin and osteopontin that are bound at discrete sites on the integrin must also contain an RGD sequence to achieve the bound state [50]. The binding of T4, T3 and tetrac at this site has been modeled [8]. The Kd for T4 at this site is subnanomolar and the anity of the site for T4 is higher than for T3 [3]. T4 and T3 are agonists at this site (see below), whereas tetrac is an antagonist and inhibits T4- and T3-binding at the receptor [3]. The classication of this site on integrin aVb3 as a receptor required denition downstream of functional consequences of binding site occupancy. This denition came when T4 andless potently, T3at this site were shown to activate from the cell surface a serinethreonine kinase,
mitogen-activated protein kinase (MAPK, ERK1/2). This activation (tyrosine phosphorylation of the enzyme) results immediately in nuclear translocation of phosphoMAPK (pMAPK). In the nucleus, the activated kinase is found in a complex with TRb1 and serine phosphorylates this nuclear receptor [12]. As noted above, this phosphorylation step de-represses TR and induces a basal rate of transcription in the receptor. Full activation of transcription apparently requires the presence of T3 in the nucleus. We also pointed out above that T4, acting via the integrin receptor, can also cause MAPK to phosphorylate Ser-118 of nuclear ERa [60]. When this occurs in ERa-positive human breast cancer (MCF-7) cells in vitro in the absence of estrogen, cell proliferation is enhanced [60]. In this context, T4 acts like an estrogen. These observations re-initiated concern that thyroid hormone may have a proliferative eect on certain tumor cells in the clinical setting. This issue is discussed below in a consideration of actions of the hormone on glial tumor cells and other cancer cell lines. The mechanisms of these actions of thyroid hormone are shown in Fig. 1. Interestingly, thyroid hormone-activated MAPK appears to be a nidation factor for a complex in the nucleus of transcription factors. Immunoprecipitation of nucleoproteins with anti-pMAPK in thyroid hormone-treated cells results in the recovery of TRb1, ERa, STAT1a, p53 and retinoic acid X receptor (RXR) (H.-Y. Lin, F.B. Davis, P.J. Davis: unpublished observations). As noted above, each of these proteins is subject to serine phosphorylation by activated MAPK (ERK1/2). The function of such complexes is not known. Complexes of transcription factors and co-factors, such as corepressor proteins, have been termed enhanceosomes [6]. We speculate that pMAPK-associated nucleoproteins serve to organize the unliganded superfamily of nuclear hormone receptors (transcription factors TRb1, ERa, RXR) in anticipation of interactions with nuclear nonpeptide ligands [14]. The collaborating Refeto and Seo laboratories and Ingbar and Mariash have reported that nongenomic mechanisms of action of thyroid hormone can involve a signal transduction pathway other than MAPK (ERK1/2). For example, X Cao, Kambe, Moeller, Refeto and Seo have implicated protein kinase B (PKB)/Akt in transduction of the thyroid hormone signal that culminates in the transcription of the ZAKI-4 gene [5]. The two ZAKI-4 gene product isoforms (a and b) are calcineurin inhibitors. This group of investigators has shown the transduction pathway in this human skin broblast system to include phosphatidylinositol 3-kinase (PI 3-K)-Akt/PKB, then nuclear mammalian target of rapamycin (mTOR) and nally phosphorylation of a nuclear mTOR substrate, p70S6kinase [5]. The rst step in this interesting cascade is an interaction in cytoplasm of TRb1 and the p85a regulatory subunit of PI 3-K [5]. A number of laboratories have reported the existence of TRb1 in cytoplasm [42,64], but whether this pool of nuclear receptor was nascent or functional has not been clear. In addition, L.C. Moeller et al. from the same laboratory have shown that the same signal transduc-
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T4 or rT3
2
CYTOPLASM
v3
T4 T3
T4 TR1
1 F actin
PLC PKC
NHE
Soluble actin
MAPK
Serine phosphorylation and change in transcriptional activity
Na/H exchanger
TR1 ER
ZAKI-4 HIF1 GLUT1
Trafficking TR1 ER
STATs
NUCLEUS
Tumor cell proliferation; angiogenesis
3 TR1
PI 3-K Specific gene transcription
T3
CYTOPLASM
3 TR1
PI 3-K
NaK-ATPase
MAPK
1
v3
T4 T3
NaK-ATPase activity
and membrane insertion
T3
Fig. 1. Schematic representation of nongenomic cellular actions of thyroid hormone analogues T4, T3 and rT3. T4 can act via a plasma membrane receptor on integrin aVb3 to activate the mitogen-activated protein kinase (MAPK; ERK1/2) signal transduction cascade via phospholipase C (PLC) and protein kinase Ca (PKCa). Activated MAPK can translocate to the nucleus to phosphorylate nuclear thyroid hormone receptor TRb1 (Ser-142) or nuclear estrogen receptor ERa (Ser-118). T4activated MAPK also modulates intracellular protein tracking of ER and TR from cytoplasm to nucleus and can act locally at the plasma membrane to activate the sodium proton exchanger (NHE). Via the integrin receptor and MAPK, T4 also is pro-angiogenic and causes proliferation of certain tumor cells. T3 may also act via the integrin receptor, but the anity of the receptor for T3 is lower than for T4. Nongenomic mechanisms of action initiated at the integrin receptor are designated 1 in the gure. T4 and reverse T3 (rT3), but not T3, may interact with cytoplasmic TRa-derived polypeptide (TRDa1) to cause a change in the state of intracellular actin that supports cell migration (conversion of soluble actin to F actin). Nongenomic mechanisms that are initiated at cytoplasmic TRDa1 are designated 2 in the gure. T3, but apparently not T4, may interact with cytoplasmic TRb1 to activate the phosphatidylinositol 3-kinase (PI 3-K) signal transduction pathway. The end results of this process include change in numbers of pumps inserted in the membrane and increased activity of the sodium pump (Na,K-ATPase) in the plasma membrane and the transcription of specic genes, such as ZAKI-4, an anti-calcineurin, and hypoxia-inducible factor-1a (HIF1a). A number of genes are targets of HIF1a protein, including a glucose transporter (GLUT1). Nongenomic actions that are initiated at cytoplasmic TRb1 are designated 3 in the gure.
transporter-4 (MCT4) [45]. These nongenomic actions of thyroid hormone are depicted in Fig. 1. In interesting studies of the actions of T3 on rat lung alveolar cells, Lei, Nowbar, Mariash and Ingbar have shown that the hormone nongenomically increases (a) the insertion of the sodium pump (Na,K-ATPase) in the plasma membrane and (b) the activity of the membranebound enzyme [33]. These eects of thyroid hormone were subsequently shown by the same group to depend upon hormonal activation of PI 3-K activity via Src kinase [34]. Lei and co-workers have also described the acquisition during rat embryonic lung development of T3-sensitivity of Na,K-ATPase [35]. It is not yet clear what the rst step is, upsteam of Src kinase, in this action of T3 in lung cells. The possibilities exist that the initial step involves TRb1 in residence in the cytoplasm, as in the case of modulation of transcription of ZAKI-4, or an extranuclear TRa1 derivative, as described below in the regulation by T4 of the actin cytoskeleton and mobility in nerve and glial cells. An additional consideration is that plasma membrane receptors in addition to integrin aVb3 may exist that primarily transduce the T3 signal. As noted above, we have shown that the MAPK pathway downstream of the integrin thyroid hormone receptor transduces the hormonal signal into angiogenesis and tumor cell growth. We have recently examined the possibility in one of our tumor cell models that PI 3-K might also mediate cell proliferation. T4 activates MAPK in human glioblastoma (U87) cells. We found that PI 3-K was activated by T3, but not by T4. T3 and T4 both stimulated tumor cell proliferation, measured by proliferating cell nuclear antigen (PCNA), but a pharmacologic inhibitor of PI 3-K, LY294002 [18], did not block the proliferative eects of either hormone analogue (H.-Y. Lin, F.B. Davis, P.J. Davis: unpublished observations). Thus, PI 3-K did not mediate the action on proliferation in this human cell line of either T3 or T4 and these two analogues dier in their abilities to activate PI 3-K. 5. Complex tissue responses induced nongenomically by thyroid hormone In addition to the eects on individual nuclear proteins [12,29,39,60] that are dictated from the cell surface receptor by thyroid hormone analogues, the latter can induce from its integrin receptor certain complex cellular or tissue responses. Among these are tumor cell proliferation [15], angiogenesis [3,13] and cellular migration (S.A. Mousa, L. OConnor, P.J. Davis: unpublished observations). Tumor cell proliferation is discussed in the next section of this review. That thyroid hormone promotes new blood vessel formation has been extensively documented in models of the chick chorioallantoic membrane (CAM) [3,13,47] and tubule formation by human dermal microvascular endothelial cells (HDMEC) [46]. These actions can be mimicked by agarose-T4 that does not gain access to the cell interior. Tetrac inhibits thyroid hormone-induced angio-
tion mechanism induces the expression of several other genes [45]. The latter include hypoxia-inducible factor-1a (HIF1a). HIF1a protein targets several genes relevant to carbohydrate handling by cells and causes their expression. These include a glucose transporter (GLUT1), platelet-type phosphofructokinase (PFKAP) and monocarboxylate
215
genesis. Induction of angiogenesis by thyroid hormone is a complex process that is dependent, at least in part, on transcription of the basic broblast growth factor (bFGF) gene and release of the gene product into the medium of the CAM assay [13]. Addition of anti-bFGF to the medium of thyroid hormone-exposed CAM blocks the pro-angiogenic activity of iodothyronines (T4 and T3). Tetrac also inhibits the hormone-induced process. Because tetrac is a probe for thyroid hormone-related events that begin at the integrin aVb3, the proangiogenic activity of T4 is integrin-requiring. Anti-integrin aVb3 was also inhibitory [13]. We also know that thyroid hormone enhances mobility of HDMEC in a Boyden chamber (S.A. Mousa, L. OConnor, P.J. Davis: unpublished observations). This factor is also likely to contribute to the angiogenic process and vascular tubule formation. While it is clear that tetrac inhibits the pro-angiogenic eect of iodothyronines, it has also been shown that in the absence of thyroid hormone tetrac is anti-angiogenic [48]. This is a remarkable nding that we attribute to the proximity of the integrin thyroid hormone receptor site at which tetrac binds and the RGD recognition site on the same protein. As pointed out earlier, this recognition site is required for angiogenesis induced by a variety of vascular endothelial growth factors, including VEGF and bFGF. Thus, tetrac is a small molecule with potentially important usefulness as an anti-angiogenic agent. 6. Nongenomic actions of thyroid hormone on tumoral glial cells In 2003, Hercbergs and co-workers reported that the pharmacological induction with propylthiouracil (PTU) of mild biochemical hypothyroidism signicantly improved survival time in patients with advanced, recurrent glioblastoma multiforme (GBM) [27]. Control and treated patients also received high-dose tamoxifen that appeared to have little eect on survival. We subsequently showed that physiological concentrations of T4 and T3 were proliferation factors for several mouse glioma cell lines [15] that are models for GBM. Tetrac blocked glioma cell proliferation, as did anti-integrin aVb3. In separate studies, tetrac has been shown to inhibit C6 glioma cell migration in a Boyden chamber (S.A. Mousa, L. OConnor, P.J. Davis: unpublished observations). Because GBM is a highly vascular tumor, it is desirable to include an anti-angiogenic strategy in its management and tetrac has the desirable feature of being an anti-angiogenic small molecule. Thus, this antithyroid hormone that acts at the cell membrane receptor for thyroid hormone on glioma cells acts by at least 3 pathways to oppose glioma cell growth. Tetrac is anti-proliferative [15] and anti-angiogenic [3,46,47], as well as an inhibitor of cell migration. Because thyroid hormone tends to alkalinize tumor cells by enhancing transport activity of the NHE [10,29], endogenous hormone levels may stimulate the activity of multi-drug resistance (MDR) pumps in the plasma membrane [17]. By opposing this action of
the hormone, tetrac may support acidication of the cells, a state that decreases activity of the MDR pumps and increases retention by tumor cells of chemotherapeutic agents. Among the other cancer cell types on which iodothyronines have proliferative activity are ERa-positive human breast cancer cells [60], as mentioned above, and human papillary and follicular thyroid cancer [40], human headand-neck cancer cell lines (H.-Y. Lin, F.B. Davis, H.J. Cao et al.: unpublished observations) and lung cancer cell lines [51]. 7. Mechanisms of thyroid hormone action mediated through the actin cytoskeleton Twenty years ago, Faivre-Sarrailh and Rabie [19] found that the quantity of lamentous actin in the hypothyroid neonatal cerebellum was markedly reduced compared to normal and that acute treatment with T4 normalized this defect. This was the rst hint of the ability of thyroid hormone to regulate the dynamics of cytoskeleton remodeling, a key structural component required for cells to migrate and to interact with their environment. Coincident with Rabies work, we were examining the molecular events that participate in the nongenomic regulation of thyroid hormone metabolism in the brain [21,56]. Interestingly, the ability of astrocytes to adhere to the culture dish was found to depend on the presence of thyroid hormone and subsequent analysis of the actin cytoskeleton revealed that the prominent actin cables observed in normal astrocytes were lost when T4 was removed from the culture medium [56]. Replacement with either T4 or reverse T3 restored these actin cables to normal within 20 min, while the T3 had no eect on the actin cytoskeleton. Since astrocytes lack chromatin-bound TRs [38], but express both the truncated delta versions derived from the TRa gene, TRDa1 and TRDa2, [36], it was clear that these dynamic, structural changes in the cytoarchitecture were not mediated by direct, regulated gene expression. Subsequent work showed that the laminin receptor, a member of the integrin family of transmembrane adhesion molecules, was largely responsible for the initial binding of astrocytes to the culture dish [20,23]. In both astrocytes and neuronal growth cones, bundled actin cables anchor this transmembrane integrin receptor in place by interactions between their cytoplasmic tail and lamentous actin cytoskeleton [9,25,59]. This allows the cell to take a foothold on the extracellular matrix and for the integrin receptor to initiate intracellular signaling. Importantly, astrocytes secrete most, if not all, of laminin in brain, and subsequent work revealed that loss of the actin cables in hypothyroid astrocytes eliminated their ability to anchor this guidance molecule on their cell surface, leading to a loss of this key molecule both in vitro and in the developing cerebellum [21]. While the ability of thyroid hormone to regulate the dynamics of actin ber remodeling is a convenient biolog-
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ical event readily studied in cell culture, the biological consequences of such a molecular pathway are more far reaching. In astrocytes and neuronal growth cones, bundled actin cables anchor transmembrane integrin receptor(s), one of several classes of adhesion molecules, in place by interactions between their cytoplasmic tail and lamentous actin cytoskeleton [25,53,57]. Both neuron pathnding and guided neurite elongation rely on integrins that project from the leading edge of the neuronal growth cone and bind to guidance cues in the extracellular matrix (ECM). Thus, cell migration depends, in part, on interactions between the ECM and the extracellular domain(s) of integrins that are stabilized by anchoring to the intracellular lamentous actin cytoskeleton. One of the most obvious developmental events regulated by thyroid hormone is the programmed migration of granule cells from the external granular layer to the inner granular layer in the neonatal cerebellum [3032]. Here, just as in cultured astrocytes, hypothyroidism delays and diminishes the timing of appearance and quantity of astrocytederived laminin found in the molecular layer of the developing rat cerebellum as compared to that in the euthyroid cerebellum [20]. Thus, the inability of the hypothyroid astrocyte to anchor the laminin receptor leads to disruption of the deposition and patterning of secreted laminin and ultimately the loss of a key guidance cue for the migrating granule cell. Analysis of the ligand activation properties of this novel eector molecule responsible for the T4-dependent modulation of actin polymerization revealed that the alanine side chain played an essential role [52]. A net negative charge on the alanine side chain of T4 destroyed its ability to modulate the dynamics of microlament remodeling, whereas an uncharged or positively charged alanine side chain on T4 retained functional control. Importantly, these ligand requirements dier markedly from those determined for the TR and for other T4-binding proteins [52], indicating that the mediator of T4-dependent actin polymerization possesses a unique ligand specicity. Importantly, the failure of tetrac to initiate actin polymerization [52] diers from its ability to block glioma cell proliferation, or inhibit migration of C6 cells (see above) suggesting that the anticancer properties of this thyroid hormone analog do not utilize regulated actin polymerization to exert its biological eects. Recent studies done in astrocytes from the TR0/0 mouse that lack all TRa-derived gene products revealed a disorganized actin cytoskeleton and marginal quantities of laminin bound to the cell surface; thyroid hormone has no eect on these two processes [37]. Unlike the full length TRa gene products, both the TRDa1- and TRDa2-encoded polypeptides bind T4 and rT3 with high anity, but do not specifically bind T3. Expression of transfactor TRa1 did not repair this defect in laminin deposition, while expression of TRDa1, but not TRDa2 in TRa-null astrocytes restored T4-dependent actin polymerization and led to the T4dependent deposition of laminin on the cell surface [37].
These data imply that the delta forms derived from the TRa gene are biologically active and provide a clear molecular pathway by which thyroid hormone can regulate a key element of the developmental program of the brain. References
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M I N I R E V I E W
Minireview: The Sodium-Iodide Symporter NIS and Pendrin in Iodide Homeostasis of the Thyroid
Aigerim Bizhanova and Peter Kopp
Division of Endocrinology, Metabolism, and Molecular Medicine, Feinberg School of Medicine, Northwestern University, Chicago, Illinois 60611
Thyroid hormones are essential for normal development and metabolism. Thyroid hormone biosynthesis requires iodide uptake into the thyrocytes and efflux into the follicular lumen, where it is organified on selected tyrosyls of thyroglobulin. Uptake of iodide into the thyrocytes is mediated by an intrinsic membrane glycoprotein, the sodium-iodide symporter (NIS), which actively cotransports two sodium cations per each iodide anion. NIS-mediated transport of iodide is driven by the electrochemical sodium gradient generated by the Na/K-ATPase. NIS is expressed in the thyroid, the salivary glands, gastric mucosa, and the lactating mammary gland. TSH and iodide regulate iodide accumulation by modulating NIS activity via transcriptional and posttranscriptional mechanisms. Biallelic mutations in the NIS gene lead to a congenital iodide transport defect, an autosomal recessive condition characterized by hypothyroidism, goiter, low thyroid iodide uptake, and a low saliva/plasma iodide ratio. Pendrin is an anion transporter that is predominantly expressed in the inner ear, the thyroid, and the kidney. Biallelic mutations in the SLC26A4 gene lead to Pendred syndrome, an autosomal recessive disorder characterized by sensorineural deafness, goiter, and impaired iodide organification. In thyroid follicular cells, pendrin is expressed at the apical membrane. Functional in vitro data and the impaired iodide organification observed in patients with Pendred syndrome support a role of pendrin as an apical iodide transporter. (Endocrinology 150: 1084 1090, 2009)
he iodide-containing thyroid hormones T3 and its precursor T4 are crucial for normal development, growth, and regulation of numerous metabolic pathways. The main function of the thyroid gland is to concentrate iodide and to make it available for biosynthesis of thyroid hormones. The significance of this mechanism is evident in light of the scarcity of iodide in most of the environment and the fact that insufficient dietary supply of iodide remains a major public health issue in many parts of the world (1). The synthesis of thyroid hormones requires a normally developed thyroid gland, an adequate nutritional intake of iodide, and a series of sequential biochemical steps. Thyroid hormone synthesis takes place in the follicles, the functional units of the gland (2). Each follicle consists of a single layer of thyroid epithelial cells surrounding the follicular lumen. The follicular lumen is filled with colloid, which is predominantly composed of thyroglobulin, a large glycoprotein that serves as the scaffold for thyroid hormone synthesis (3).
The synthesis of thyroid hormones requires uptake of iodide across the basolateral membrane into the thyrocytes, transport across the cell, and efflux through the apical membrane into the follicular lumen. Uptake of iodide is mediated by the sodiumiodide symporter (NIS), which cotransports two sodium ions along with one iodide ion, with the sodium gradient serving as the driving force (Fig. 1) (1). The energy required to produce the sodium gradient is provided by the ouabain-sensitive Na/KATPase (4). The efflux of iodide across the apical membrane is mediated, at least in part, by pendrin (5). Once iodide reaches the cell-colloid interface, it is oxidized and rapidly organified by incorporation into selected tyrosyl residues of thyroglobulin. This reaction, referred to as organification, is catalyzed by thyroid peroxidase (TPO) in the presence of hydrogen peroxide and results in the formation of mono- and diiodotyrosines (MIT and DIT). The generation of hydrogen peroxide is mediated by the calcium-dependent reduced nicotinamide adenine dinucleotide phosphate (NADPH) dual oxidase type 2
Abbreviations: CICn5, Chloride channel 5; DIT, diiodotyrosine; DUOX2, dual oxidase type 2; ITD, iodide transport defect; MDCK, Madin-Darby canine kidney; MIT, monoiodotyrosine; NIS, sodium-iodide symporter; PKA, protein kinase A; SLC5A, solute carrier 5A; TPO, thyroperoxidase.
ISSN Print 0013-7227 ISSN Online 1945-7170 Printed in U.S.A. Copyright 2009 by The Endocrine Society doi: 10.1210/en.2008-1437 Received October 14, 2008. Accepted January 22, 2009. First Published Online February 5, 2009
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FIG. 1. Main steps in thyroid hormone synthesis. At the basolateral membrane of thyroid follicular cells, which form the follicles, iodide is transported into thyrocytes by the NIS. NIS is dependent on the sodium gradient created by the Na/K-ATPase. At the apical membrane, iodide efflux is, in part, mediated by pendrin (PDS/SLC26A4). At the cell-colloid interface, iodide is oxidized by TPO in the presence of H2O2. H2O2 is produced by the calcium- and reduced nicotinamide adenine dinucleotide phosphate-dependent (NADPH) enzyme DUOX2. DUOX2 requires a specific maturation factor, DUOXA2. Thyroglobulin (TG), which is secreted into the follicular lumen, serves as matrix for synthesis of T4 and T3. First, TPO catalyzes iodination of selected tyrosyl residues (organification), which results in the formation of MIT and DIT. Subsequently, two iodotyrosines are coupled to form either T4 or T3 in a reaction that is also catalyzed by TPO. Iodinated thyroglobulin is stored as colloid in the follicular lumen. Upon a demand for thyroid hormone secretion, thyroglobulin is internalized into the follicular cell by pinocytosis and digested in lysosomes, which generates T4 and T3 that are released into the bloodstream through unknown mechanisms. The unused MIT and DIT are retained in the cell and deiodinated by the iodotyrosine dehalogenase 1 (DEHAL1). The released iodide is recycled for thyroid hormone synthesis.
(DUOX2). TPO also catalyzes the coupling of two iodotyrosines to form either T3 or T4. To release thyroid hormones, thyroglobulin is engulfed by pinocytosis, digested in lysosomes, and then secreted into the bloodstream at the basolateral membrane (2). The mechanisms of hormone secretion at the basolateral membrane and the involved channel(s) have not been characterized.
NIS
NIS is an integral plasma membrane glycoprotein localized at the basolateral plasma membrane of thyrocytes (6). This protein plays an essential role in thyroid physiology by mediating uptake of iodide into the thyrocytes, a key step in thyroid hormone synthesis. The ability of the thyroid gland to accumulate radioiodine is also a cornerstone in the diagnosis and treatment of thyroid disorders (7).
The molecular characterization of NIS began in 1996 when the cDNA encoding rat NIS was isolated by expression-cloning in Xenopus laevis oocytes (6). Subsequently, the human cDNA has been cloned by a RT-PCR approach taking advantage of the homology to rat NIS (8). Rat NIS is predicted to have 618 amino acids with a relative molecular mass of 65,196 Daltons (6), and human NIS contains 643 amino acids and exhibits an 84% amino acid identity and 93% similarity to rat NIS (9). The human NIS gene is located on chromosome 19p12-13.2 and contains 14 introns and 15 exons (9). NIS (SLC5A5) belongs to the solute carrier family 5A (SLC5A). All members of this protein family depend on an electrochemical sodium gradient as the driving force for transport of anions across the plasma membrane (10). The current secondary structure model predicts that NIS contains 13 transmembrane domains with the amino terminus located extracellularly and the carboxy terminus facing the cytosol (1). The mature NIS protein is approximately 87 kDa in size and has three asparagine-linked glycosylation sites (1). Glycosylation does not seem to be required for stability, activity, or targeting of the NIS molecule to the plasma membrane (11). The expression of NIS is differentially regulated and is subjected to various posttranslational modifications in each tissue in which it is expressed (1, 12). In addition to thyroid follicular cells, NIS is expressed in several other tissues, including the salivary glands, gastric mucosa, and the lactating mammary gland, where it mediates active transport of iodide (1). In the lactating mammary gland, NIS plays an important role by concentrating iodide in the milk, thereby supplying newborns with iodide for thyroid hormone synthesis. Although NIS has a high affinity for iodide, it is able to mediate transport of other ions (13). Large anions such as thiocyanate and perchlorate can inhibit accumulation of iodide in the thyroid by competing with iodide (14, 15). Perchlorate is 10 100 times more potent than thiocyanate in inhibiting iodide accumulation in the thyroid (1). The ability of perchlorate to block iodide transport has been used in the therapy of hyperthyroidism, and it is used in the perchlorate discharge test, which serves to detect defects in iodide organification (16). In normal individuals, administration of perchlorate blocks subsequent accumulation of iodide in the thyrocytes but does not cause any discharge of previously accumulated radioiodine because of its rapid organification. In contrast, in individuals with a total or partial iodide organification defect, administration of perchlorate results in the rapid release of the unorganified fraction of the tracer from the thyrocytes (16). Until recently, it has been controversial whether perchlorate acts as a blocker or as a substrate that is transported by NIS (13, 17, 18). Two recent studies provide evidence that perchlorate is actively transported by NIS (19, 20). Perchlorate transport could be demonstrated in a polarized cell system (19) as well as by direct measurement with mass spectrometry (20). Moreover, it has been shown that perchlorate, a widely found pollutant, is transported into the milk (19). Remarkably, the stoichiometry of the NIS-mediated Na/ClO4 transport is electroneutral, which contrasts with the electrogenic transport of iodide (two Na and one I) (19). These findings indicate that NIS is able to transport different substrates with distinct stoichiometries (19).
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Regulation of NIS protein expression

TSH is the major regulator of thyroid cell proliferation, differentiation, and function, including iodide uptake (21). The effects of TSH are primarily mediated through the activation of the cAMP cascade via the GTP-binding protein Gs (22). TSH stimulates iodide accumulation by positively regulating NIS expression at the protein and mRNA level via the cAMP pathway (23). Hypophysectomized rats with low circulating levels of TSH have a decreased protein expression of NIS, whereas a single injection of TSH leads to a prompt increase in NIS expression (24). Rats maintained on an iodide-deficient diet or treated with propylthiouracil, an agent blocking iodide organification, have high concentrations of TSH, which correlates with an increase in NIS protein expression. These findings are in agreement with the results obtained in human thyroid primary cultures (25, 26) and the rat thyroid FRTL-5 cell line (23). In FRTL-5 cells, withdrawal of TSH results in a decrease in intracellular cAMP concentration and iodide uptake activity (23). Re-addition of TSH increases NIS mRNA and protein expression and subsequently restores iodide uptake activity. Recent studies have shown that TSH not only regulates NIS transcription and biosynthesis but also mediates NIS activity by posttranscriptional mechanisms (27). In the presence of TSH, NIS is active and inserted in the basolateral membrane of thyrocytes (27). Upon TSH withdrawal, NIS protein half-life decreases from 5 to 3 d, and it translocates from the plasma membrane to intracellular compartments (27). As of yet, the mechanisms regulating the subcellular distribution of NIS are only partially elucidated. It is known that NIS has several consensus sites for kinases, including protein kinase A (PKA) and protein kinase C (1). Although it has been shown that NIS is phosphorylated in vivo (27), the functional significance of this modification needs further characterization. A more recent study identified five amino acid residues in NIS that are phosphorylated in vivo, but the phosphorylation status of these amino acid residues does not affect targeting of NIS to the plasma membrane (28). NIS contains several sorting sequences that are known to play a role in targeting, retention, and endocytosis of other membrane proteins (29). For example, the PDZ motif (T/S-X-V/L) located at the carboxy terminus of NIS is one of the sequences involved in protein-protein interactions (1). The PDZ motif is recognized by PDZ-binding proteins that have been implicated in internalization of other transporters (29). NIS also has a dileucine motif, L557L558, which is known to interact with the clathrin-coated system (30). This interaction leads to incorporation of integral membrane proteins into coated vesicles that are then carried to different destinations within the cell (31). Iodide is another factor that can regulate iodide accumulation in the thyroid. In 1948, Wolff and Chaikoff (32) reported that high doses of iodide block iodide organification in the rat thyroid in vivo. This phenomenon, known as the acute Wolff-Chaikoff effect, is a reversible process, because iodide organification resumes when the iodide concentration in the serum decreases. The mechanisms underlying the Wolff-Chaikoff effect are complex and involve acute regulation of several key genes and proteins within the thyrocytes. Several studies have examined the effect of
iodide on NIS mRNA and protein expression in vivo and in vitro (3335). In vivo data suggest that high concentrations of iodide lead to reduction in both NIS mRNA and protein levels, partially by a transcriptional mechanism. In vitro results suggest that exposure to high doses of iodide results in a decrease in NIS protein levels that is, at least in part, due to an increase in NIS protein turnover (3335).
Congenital iodide transport defect (ITD)

Biallelic mutations in the NIS gene cause a congenital ITD. ITD is an autosomal recessive condition characterized by hypothyroidism, goiter, reduced or absent thyroid uptake of radioiodide, and a low saliva/plasma iodide ratio (1, 36). Currently, at least 12 ITD-causing mutations of NIS have been identified (37). Six of these mutations, namely 226delH, T354P, G395R, Q267E, G543E, and V59E have been characterized more thoroughly (38 41). The G543E substitution leads to retention of NIS in intracellular compartments as a result of improper maturation and trafficking of the protein, the other mutants are still targeted to the membrane but result in a loss of function (38 41). Structural and functional analysis of the T354P mutant protein demonstrated that a hydroxyl group at the -carbon of the residue at position 354 is crucial for proper NIS function (38). Substitutions of the glycine residue at position 395 residue with several amino acids indicated that the presence of a small and an uncharged amino acid residue at this position is required for NIS function (39). Lastly, a recent study revealed that the histidine residue at position 226 is important for the iodide transport activity of NIS (37).
Pendrin
Pendrin is a highly hydrophobic membrane protein located at the apical membrane of thyrocytes (2, 4). In addition to the thyroid, pendrin is also expressed in the kidney and in the inner ear (42, 43). In the kidney, pendrin plays an important role in acid-base metabolism as an exchanger of chloride and bicarbonate in -intercalated cells (44). In the inner ear, pendrin is important for generation of the endocochlear potential (45). Pendrin belongs to the SLC26A family, which includes several anion transporters, as well as the motor protein prestin that is expressed in outer hair cells (46, 47). Pendrin is encoded by the SLC26A4 gene, which was cloned in 1997 (48). The SLC26A4 gene is located on chromosome 7q21-31 and contains 21 exons with an open reading frame of 2343 bp (48). Pendrin is a glycoprotein composed of 780 amino acids (2). It contains three putative extracellular asparagine-glycosylation sites (4, 49). Pendrin usually appears as a single protein band with a molecular mass of 110 115 kDa when isolated from human thyroid membranes (49). Pendrin is proposed to have 12 transmembrane domains with both amino and carboxy termini located inside the cytosol (4, 50). Like other members of the SLC26A family, pendrin contains a so-called STAS (sulfate transporter and antisigma factor antagonist) domain (51). The
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exact function of this domain has not been elucidated. Recent studies, however, suggest that the STAS domain can interact with the regulatory domain of CFTR (cystic fibrosis transmembrane conductance regulator) in certain epithelial cells (5254).
Pendred syndrome
Mutations in the SLC26A4 gene lead to Pendred syndrome (2). Pendred syndrome is an autosomal recessive disorder characterized by sensorineural deafness, goiter, and a partial defect in iodide organification (55, 56). Deafness or hearing impairment is the leading clinical sign of Pendred syndrome (56). In many patients, hearing loss is prelingual; in some individuals, however, the hearing loss develops later in childhood (57). Patients with Pendred syndrome display an enlarged endolymphatic sac and duct (58 60). A subset of patients presents with a so-called Mondini defect, which is characterized by replacement of the cochlear turns by a single cavity or a rudimentary cochlea (57, 58). Variability in the hearing loss observed in patients with SLC26A4 mutations suggests that the phenotype is influenced by environmental factors and/or genetic modifiers (61). Goiter usually develops during childhood. There is, however, a substantial variation within and between families and different geographic regions (62 64). Nutritional iodide intake appears to play an important role as a modifier of the thyroidal phenotype (65, 66). Under conditions of high iodide intake, most individuals have no or only a mild enlargement of the thyroid (60, 67). If the nutritional iodide is scarce, patients with Pendred syndrome not only develop goiter but may also present with mild or overt hypothyroidism (68, 69). Mutations in the SLC26A4 gene, found in patients with Pendred syndrome, are highly heterogeneous (56). Currently, more than 150 mutations of the SLC26A4 gene have been reported (56). Most of the mutations are missense mutations, and a smaller number of mutations consists mainly of nonsense and intronic mutations (56). Loss of function of some of the mutants results from the retention of the mutated and misfolded protein in intracellular compartments, most likely the endoplasmic reticulum (70, 71).
NIS and pendrin than in cells expressing NIS alone. Electrophysiological studies with transfected COS-7 cells also indicate that pendrin mediates iodide transport and that it is more efficient at high extracellular concentrations of chloride (76). In addition, iodide efflux/chloride influx appears to be more efficient than chloride efflux/iodide influx (76). These findings are consistent with results obtained in polarized Madin-Darby canine kidney (MDCK) cells (50). MDCK cells expressing NIS and pendrin independently or simultaneously were cultured in a bicameral system, which allowed measuring iodide uptake at the basolateral membrane and iodide efflux at the apical membrane. Cells transfected with NIS alone have a significant increase in intracellular iodide uptake compared with untransfected control cells. In contrast, cells expressing NIS and pendrin show a significant increase in iodide transport into the apical chamber and consequently a significant decrease in the intracellular iodide content. These findings support the notion that pendrin may have a role in facilitating vectorial iodide transport at the apical membrane (50). The partial organification defect found in patients with Pendred syndrome suggests, however, that iodide can reach the follicular lumen independently of the presence of pendrin.
Questions concerning the role of pendrin as an apical iodide transporter

The traditional concept held that iodide simply crosses the apical membrane due to the electrochemical gradient that is present between the cytosol and the follicular lumen (77). However, autoradiography studies revealed that iodide first accumulates in the cytosol and subsequently moves to the follicular lumen (78). This transport of iodide across the apical membrane is rapidly stimulated by TSH (64, 79). Hence, it has been proposed that apical iodide efflux is mediated through a specific transporter or channel. This concept has risen based on several findings. Electrophysiological studies performed with thyroid membrane vesicles suggested the presence of two apical iodide transporters (80). Functional studies performed in heterologous cells, including polarized cells (50, 75, 76, 81), along with the iodide organification defects found in patients with Pendred syndrome (62, 74), suggested that pendrin mediates apical iodide efflux in thyrocytes. The physiological role of pendrin has, however, been questioned for several reasons (77). Patients with biallelic mutations in the SLC26A4 gene display a mild or no thyroidal phenotype under conditions of sufficient iodide intake (65). The pendrin knockout mice, studied under normal iodide intake conditions, do not develop a goiter or abnormal thyroid hormone levels (45). In addition, it is intriguing that pendrin may have distinct roles in the thyroid, the inner ear, and the kidney (77). This led to the proposal that pendrin may be a part of multiprotein complex, the composition of which may vary among different cell types, thereby potentially explaining a variability in anion selectivity of pendrin (77). Other proteins (SLC5A8 and chloride channel 5 ClCn5) have been proposed to mediate apical iodide efflux (82, 83). Functional studies performed in Xenopus oocytes and polarized MDCK cells clearly demonstrate that
Role of pendrin in the thyroid

Initial functional studies of pendrin in Xenopus oocytes have demonstrated that pendrin is able to mediate transport of chloride and iodide (5) and that it can act as a chloride/formate exchanger (72). The ability of pendrin to mediate iodide efflux (5), the localization of pendrin at the apical membrane of thyrocytes (4, 73), as well as the defect in iodide organification observed in patients with Pendred syndrome (62, 74), suggested that pendrin could function as an apical iodide transporter in thyroid cells (46). The results obtained from a number of independent studies performed in heterologous systems support the role of pendrin in mediating, at least in part, apical iodide efflux. Yoshida et al. (75) have demonstrated that iodide efflux is much higher in nonpolarized Chinese hamster ovary cells expressing
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SLC5A8, originally designated as human apical iodide transporter (hAIT) (82), does not mediate iodide uptake or efflux (84). Localization of the ClCn5 protein at the apical membrane of thyrocytes and a thyroidal phenotype of the ClCn5-deficient mice that is reminiscent of Pendred syndrome suggest that ClCn5 could be, possibly in conjunction with other chloride channels, involved in mediating apical iodide efflux or iodide/chloride exchange (83). This possibility has, as of yet, not been corroborated by further experimental data.
Regulation of pendrin expression

TSH stimulates iodide efflux across the apical membrane of thyrocytes (79, 85, 86). After exposure to TSH, iodide efflux is rapidly stimulated in FRTL-5 cells (85) and in polarized porcine thyrocytes (79, 86). In polarized porcine thyrocytes grown in a bicameral system, measurement of iodide transport in both directions demonstrates that TSH up-regulates iodide efflux at the apical membrane, whereas efflux across the basolateral membrane does not change (79). Treatment of rat thyroid PCCl3 cells with TSH for a short time results in the rapid translocation of pendrin from intracellular compartments, specifically endosomes, to the plasma membrane, thus suggesting a role of pendrin in rapid regulation of apical iodide efflux (87). Insertion of pendrin in the apical membrane correlates with the phosphorylation of pendrin, but it remains unknown whether phosphorylation is necessary or sufficient for this translocation. These events occur through the PKA pathway and can be inhibited by H89, a specific PKA inhibitor (87). Interestingly, translocation of pendrin from the cytosol to the plasma membrane through a PKC-dependent pathway has been demonstrated after exposure of cultured rat thyroid cells to insulin for 10, 20, and 40 min (88). At least in rat FRTL-5 cells, TSH does not significantly modify SLC26A4 gene expression (4). Interestingly, thyroglobulin has been shown to up-regulate SLC26A4 mRNA levels in FRTL-5 cells while suppressing expression of several thyroid-specific genes, including the TSH receptor, NIS, TPO, and TG genes (4). Treatment with iodide does not affect expression of the SLC26A4 gene (89). Exposure to thyroglobulin leads to a decreased NIS gene and protein expression and subsequently results in a reduced iodide uptake in vitro (90). In contrast, accumulation of thyroglobulin in the follicular lumen suppresses iodide uptake in vivo (90). It has been suggested that the inverse relationship between the concentration of thyroglobulin in the follicular lumen and iodide uptake in vivo may be important in the regulation of thyroid function under constant TSH levels (91) and promote iodide efflux into the follicular lumen (89).
pothyroidism, goiter, low thyroid iodide uptake, and low saliva/plasma iodide ratio. Pendrin is involved in the apical iodide efflux in thyroid cells. It can also exchange chloride and bicarbonate. Pendrin is encoded by the SLC26A4 gene. Biallelic mutations in the SLC26A4 gene cause Pendred syndrome, an autosomal recessive disorder, characterized by deafness, goiter, and impaired iodide organification. In the inner ear, pendrin is important for anion and fluid transport and for maintenance of the endocochlear potential. In the kidney, pendrin plays a role in acid-base metabolism as a chloride/bicarbonate exchanger. In addition to pendrin, other apical iodide channels or transporters may be involved in regulation of apical iodide efflux in thyrocytes.
Acknowledgments
Address all correspondence and requests for reprints to: Peter Kopp, M.D., Associate Professor, Director ad interim Center of Genetic Medicine, Division of Endocrinology, Metabolism, and Molecular Medicine, Northwestern University, Tarry 15, 303 East Chicago Avenue, Chicago, Illinois 60611. E-mail: p-kopp@northwestern.edu. Part of this work has been supported by Grant 1R01DK63024-01 from the National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health (to P.K.).
References
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NIS mediates the active transport of iodide at the basolateral membrane of thyrocytes. Biallelic mutations in NIS cause a congenital iodide transport defect, an autosomal recessive condition, characterized by hy-
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GLUCOCORTICOIDS AND MOOD
Glucocorticoid Signaling in the Cell

Expanding Clinical Implications to Complex Human Behavioral and Somatic Disorders
George P. Chrousosa,b and Tomoshige Kinob
a b
First Department of Pediatrics, Athens University Medical School, Athens, Greece
Pediatric Endocrinology Section, Program in Reproductive and Adult Endocrinology, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland, USA
Glucocorticoids contribute to the maintenance of basal and stress-related homeostasis in all higher organisms, and inuence a large proportion of the expressed human genome, and their effects spare almost no organs or tissues. Glucocorticoids regulate many functions of the central nervous system, such as arousal, cognition, mood, sleep, the activity and direction of intermediary metabolism, the maintenance of a proper cardiovascular tone, the activity and quality of the immune and inammatory reaction, including the manifestations of the sickness syndrome, and growth and reproduction. The numerous actions of glucocorticoids are mediated by a set of at least 16 glucocorticoid receptor (GR) isoforms forming homo- or hetero-dimers. The GRs consist of multifunctional domain proteins operating as ligand-dependent transcription factors that interact with many other cell signaling systems, including large and small G proteins. The presence of multiple GR monomers and homo- or hetero-dimers expressed in a cell-specic fashion at different quantities with quantitatively and qualitatively different transcriptional activities suggest that the glucocorticoid signaling system is highly stochastic. Glucocorticoids are heavily involved in human pathophysiology and inuence life expectancy. Common behavioral and/or somatic complex disorders, such as anxiety, depression, insomnia, chronic pain and fatigue syndromes, obesity, the metabolic syndrome, essential hypertension, diabetes type 2, atherosclerosis with its cardiovascular sequelae, and osteoporosis, as well as autoimmune inammatory and allergic disorders, all appear to have a glucocorticoid-regulated component. Key words: metabolic syndrome; osteoporosis; CDK5; GR phosphorylation; stress system; glucocorticoid resistance; glucocorticoid hypersensitivity
Introduction Glucocorticoids are among the most pervasive hormones in mammalian organisms.1,2 These steroid molecules reach all tissues, including the brain, readily penetrate the cell membrane, and interact with ubiquitous cytoplasmic/nuclear glucocorticoid receptors
Address for correspondence: George P. Chrousos, MD, MACP, MACE, FRCP (London), Professor and Chairman, First Department of Pediatrics, Athens University Medical School, Aghia Sophia Childrens Hospital, 115 27 Athens, Greece. Voice: +30-210-7794023; fax: +30-210-7759167. chrousge@med.uoa.gr
(GRs), through which they exert markedly diverse actions.1,2 Using DNA microarray technology, we found that about 20% of the expressed human leukocyte genome was positively or negatively affected by glucocorticoids.3 This is many-fold higher than the proportion of genes that change in the transformation of a normal cell to a tumor cell and involves a broad array of functions, affecting every aspect of resting and stress-related homeostasis, including a large number of genes expressed by the immune system.3,4 The pervasive nature of glucocorticoids, the rapid advances
Glucocorticoids and Mood: Ann. N.Y. Acad. Sci. 1179: 153166 (2009). doi: 10.1111/j.1749-6632.2009.04988.x c 2009 New York Academy of Sciences.
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in our general knowledge of the human and other mammalian genomes, and the massive amount of information that increasingly accumulates, dictate a new model of thinking and testing of hypotheses regarding the actions of these hormones and their involvement in human physiology and pathophysiology. GR Gene Polymorphisms and Complex Human Pathophysiology W ust et al . reported a convincing association between the hypothalamic-pituitaryadrenal (HPA) axis response to a standardized socio-emotional stimulus (Trier test) and polymorphisms of the GR gene.5 This study followed others that used a similar rationale and examined HPA axis indices and other endpoints, such as arterial blood pressure, body mass index and markers of the metabolic syndrome, and bone mineral density.613 These studies have had some overlap and have produced mostly concordant results, but have also shown inconsistencies. This should have been expected because these studies were performed in a limited number of subjects in different ethnic populations, and because the altered GR would have been expected to function differently in the context of different genetic backgrounds characterized by different panels of genes with differing epistatic effects upon the ability of the GR to exert its actions.14 Glucocorticoid Effects: Physiology and Pathophysiology Empirically, experimentally, and intuitively, physicians and scientists have made major advances in the general understanding of glucocorticoids and their involvement in human physiology and pathophysiology, and in using these hormones extensively and effectively in the treatment of a wide spectrum of human diseases.1,2 As the end product of the HPA axis, glucocorticoids are literally present in ev-
ery organ system of the human organism, in almost all physiologic, cellular and molecular networks, and in many crucial modules of these networks.2,3,14 Glucocorticoids, furthermore, participate in a pivotal fashion in the unfolding of vital biologic programs employing synchronously or in tandem several networks, including the behavioral and physical response to stress, the inammatory reaction and the consequent sickness syndrome, i.e., the collection of nonspecic symptoms caused by excessive inammatory cytokines during infectious or inammatory illness, as well as the process of sleep, and long-term functions, such as growth and reproduction.4 As is true with many other homeostatic systems, too much or too little HPA axis and/or glucocorticoid activity may be associated with pathologyfor instance, Cushing syndrome versus Addison disease, respectively.1517 Since the responsiveness of the target tissues to glucocorticoids is crucial for the end-effect of these hormones, similar pathology may result from hypersensitivity or resistance of these target tissues to these hormones, respectively (Table 1).16,18 However, because the brain and the pituitary are also the targets of glucocorticoids, and because the organism strives for homeostasis in time-integrated free cortisol exposure, any generalized change in the glucocorticoid signaling system would be expected to be followed by corrective, compensatory changes in the activity of the HPA axis. Indeed, in the rare GR-mediated genetic disorder Primary Glucocorticoid Resistance, the majority of the clinical manifestations are not Addisonian, but are rather due to the compensatory hyperfunction of the HPA axis leading to adrenal androgen and mineralocorticoid hypersecretion while the opposite would be expected in primary generalized glucocorticoid hypersensitivity (Fig. 1A and B).19,20 However, an absence of complete concordance between HPA axis activity and the target tissues outside those responsible for feedback regulation, be it slightly excessive or decient, could result in a state called
Chrousos & Kino: Glucocorticoids and Pathophysiology
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TABLE 1. Expected Clinical Manifestations in Target Tissue Hypersensitivity or Resistance to Glucocorticoids Target area Central nervous system Liver Fat Blood vessels Bone Inammation/immunity Glucocorticoid excess = Glucocorticoid hypersensitivity Insomnia, anxiety, depression, defective cognition +Gluconeogenesis, +lipogenesis Accumulation of visceral fat (metabolic syndrome) Hypertension Stunted growth, osteoporosis Immune suppression, anti-inammation, vulnerability to certain infection and tumors Glucocorticoid deciency = Glucocorticoid resistance Fatigue, somnolence, malaise, defective cognition Hypoglycemia, resistance to diabetes mellitus Loss of weight, resistance to weight gain Hypotension +Inammation, +autoimmunity, +allergy
Modied from Refs. 22 & 61.
allostasis, more accurately termed cacostasis, leading to target tissue pathology, as occurs in chronically stressed or depressed individuals in whom there is frequently mild chronic hypercortisolism.21,22 This has been known for several decades. The key question is whether it is possible to have discordance between the feedback regulation of the HPA axis by glucocorticoids and peripheral target tissue sensitivity to these hormones in totally normal individuals. Indeed it appears to be possible. The glucocorticoid signaling system of the suprahypothalamic, hypothalamic, and pituitary glucocorticoid-sensing network is different from the signaling systems of the reward, arousal, associative, cardiovascular, metabolic and immune systems, which are inuenced by glucocorticoids. The feedback centers of the HPA axis sense and thus determine the circulating glucocorticoid levels, while other tissues passively accept the actions of circulating glucocorticoids. Indeed, any change in one or more molecules or processes that participate in the glucocorticoid signaling system could potentially have a different impact on the HPA feedback system and other target tissues. Such discrepancies in the glucocorticoid sensing network between the HPA axis and peripheral tissues could therefore produce peripheral tissue
hypercortisolism or hypocorticosolism, depending on their combinations (Fig. 1C and Table 1).16 As an example, in a recent study by Alevizaki et al., both high HPA axis reactivity to stress and increased peripheral tissue sensitivity to glucocorticoids were associated with increased severity of coronary artery disease.23 Naturally, GR is not alone in dening the sensitivity of the feedback system and other tissues to glucocorticoids. Numerous GR isoforms with different activities, other molecules or processes with considerable input into the activity of the cellular glucocorticoid signaling system have been described (Table 2).18,24,25 In our studies of the glucocorticoid signaling system, we have identied several molecules from a variety of different signaling systems that interact in many ways with and inuence the activity of the GR and vice versa, and are depicted in Figure 2 and Table 2.18,2433 These include the subunit of the G protein trimeric complex, small G proteins, components of the tumor necrosis factor-/Fas ligand signaling systems, such as FLASH, the chaperone protein 14-3-3, the HIV accessory proteins Vpr and Tat, the adenoviral protein E1A, and molecular components of brain cyclin-dependent kinase 5 (CDK5).2631,33 In light of the major physiologic homeostatic inuence of glucocorticoids on many brain functions, as
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Figure 1. (A) Feedback-regulated compensatory changes in the activity of the HPA axis and their effects in peripheral tissues, such as the liver, fat, and blood vessels. Note that glucocorticoid sensitivity in the HPA axis and the peripheral tissues can be independently regulated, and the former determines the serum-free cortisol levels; thus, a combination of their directions inuences net peripheral action of this hormone. ACTH: adrenocorticotropic hormone; AVP: arginine vasopressin; CRH: corticotropin-releasing hormone; DOC: deoxycorticosterone; B: corticosterone. Modied from Refs. 68, 73 & 74. (B) Known GR mutations that cause familial/sporadic glucocorticoid resistance syndrome. Localization of GR mutations that cause familial/sporadic glucocorticoid resistance syndrome are shown in the human GR gene (top) and in the human GR protein (bottom). DBD: DNA-binding domain; LBD: ligand-binding domain. From Refs. 19 & 20. (C) Alteration of net glucocorticoid effects in target tissues by activity of the central HPA axis and peripheral tissue sensitivity to glucocorticoids. Net glucocorticoid action in peripheral tissues, such as the CNS, liver, fat and vasculature, is determined by two components: the central HPA axis and sensitivity of peripheral tissues to glucocorticoids. (From Ref. 68.)
157
Figure 1. Continued.
well as the major pathologic effects of these hormones on the central nervous system (CNS), we elected to summarize below our work on the interactions of CDK5 and the GR.34
Glucocorticoids and the Brain: CNS CDK5 Regulates Glucocorticoid Actions in the Brain by Phosphorylating GR The transcriptional activity of GR is regulated by direct phosphorylation of this receptor by serine/threonine kinases.35 GR has several phosphorylation sites, all of which are located in the AF-1 domain of its N-terminal domain (NTD),24,36 suggesting that AF-1 acts as an interface for phosphorylation-dependent intracellular molecular signals (Fig. 3A). For example, yeast CDK p34CDC28 phosphorylates rat GR at serines 224 and 232, which are orthologous to serines 203 and 211 of human GR, with the resultant phosphorylation enhancing rat GR transcriptional activity in the yeast.35 These residues are also phosphorylated after activation of GR with agonists
or antagonists, and the phosphorylated receptor shows reduced translocation to the nucleus and/or altered subcellular localization in mammalian cells.36,37 The p38 mitogen-activated protein kinase (MAPK) phosphorylates serine 211 of human GR and enhances its transcriptional activity.38 p38 MAPK and JNK also phosphorylate serine 226 of human GR and suppress its transcriptional activity by enhancing nuclear export of the receptor.39 Threonine 171 of rat GR is phosphorylated by p38 MAPK and glycogen synthase kinase-3. Threonine 171-phosphorylated GR demonstrates reduced transcriptional activity in yeast and human cells; however, human GR does not have a threonine residue equivalent to that of the rat GR.40 In addition to these kinases that phosphorylate GR and regulate its transcriptional activity, we recently found that CDK5, which is a member of the CDK family,41 phosphorylates GR and modulates its transcriptional activity.33 In contrast to other CDKs, which function in the control of the cell cycle, CDK5 has no activity during mitosis but is essential for brain development and neuronal morphogenesis and survival.42,43 CDK5 is expressed
158 TABLE 2. Factors Inuencing GR Functions Ligands Membrane transporters of glucocorticoids 11-hydroxysteroid dehydrogenases Agonists, antagonists (ex. RU 486) Ursodeoxycholic acid, cortivazol, thioredoxin, carnitine Phosphorylation, nitrosylation, acetylation, methylation, sumoylation Heat shock proteins, RAP46, FK506-binding proteins Glucocorticoid receptor and isoforms (16 or more, 256 or more combinations of dimers) Coactivators/corepressors, SWI/SNF, TRAP/DRIP complex, SMAD6 Viral proteins: adenoviral E1A, Human Immunodeciency virus type-1 Vpr and Tat Nuclear factor-B, activator protein-1, CREB-binding protein, p53, chicken ovalbumin upstream promoter-transcription factor-II, GATA-1, SP-1, Nuclear factor-1, CLOCK/BMAL1 PPAR 14-3-3, FLASH, Rho-type guanine nucleotide exchange factors (Brx and c-Lbc), autoimmune regulator gene (AIRE), guanine nucleotide-binding protein SRA, Gas5
Chemical compounds Chemical modications Chaperones, cochaperones Receptor isoforms Transcriptional co-regulators
Transcription factors
Other proteins
RNAs
Modied from Refs. 18, 24, 2629, 32, 6872 & 75.
ubiquitously in many tissues, but its activity is restricted primarily to the nervous system due to neuron-specic expression of its activator molecules p35 and p39.44,45 In addition to these physiologic roles of CDK5, recent evidence suggests that aberrant CDK5 activation caused by proteolytic conversion of p35 to p25 may play a role in the pathogenesis of neurodegenerative disorders, such as Alzheimers disease and amyotrophic lateral sclerosis.43,46 In these conditions, calpain-directed proteolysis of p35 deprives the membrane-associated p35 of its N-terminal myristoylated membrane tether, releasing p25 into the cytoplasm and activating CDK5.47,48
We found that the CDK5/p35 complex physically interacts with the GR ligand-binding domain (LBD) and modulates GR-induced transcriptional activity by phosphorylating serines 203, 211, and 226 of human GR.33 The C-terminal part of p35 binds the GR LBD in a ligand-dependent fashion, while both the GR LBD and NTD are necessary for CDK5/p35 to modulate GR transcriptional activity, indicating that these subdomains of GR respectively act as interactor and effector surfaces for CDK5/p35.33 We found that CDK5 positively or negatively regulates the transcriptional activity of GR on endogenous glucocorticoidresponsive genes in rat cortical neuronal cells in microarray analyses, indicating that the effect of CDK5 on GR-induced transcriptional activity is gene promoterspecic.33 Since phosphorylation of GR at serines 203, 211, and 226 alters the attraction of cofactor molecules to the promoter-bound GR,33 it is possible that phosphorylation of the receptor may respectively facilitate or inhibit the attraction of transcription cofactors on promoter-bound GR, through as yet undetermined mechanisms that appear to be gene-specic (Fig. 3B). The tumor susceptibility gene 101 (TSG101), which interacts with the co-integulator molecules p300/CBP and inhibits the transcriptional activity of GR by modulating GR-induced attraction of coactivators, preferentially interacts with a nonphosphorylated form of GR.36,40,49 Thus, TSG101 and/or similar molecules that specically bind nonphosphorylated or phosphorylated forms of GR40 may mediate phosphorylation-dependent transcriptional modulation by changing accumulation of cofactors on promoter-bound GR. Glucocorticoids play an essential role in the homeostasis of the CNS50,51 ; these hormones indeed regulate cognition, memory, mood, and sleep, and inuence the anatomic structure of the brain, causing reduction of hippocampal volume, ventricular enlargement, and reversible cortical atrophy.51 Alteration of CDK5 activity in neuronal cells can potentially inuence any of these glucocorticoid actions in
159
Figure 2. Regulation of tissue GR activity by distinct signaling pathways. Transcriptional activity of GR in glucocorticoid target tissues is regulated by numerous signaling pathways through distinct mechanisms. (From Refs. 18, 24, 2629, 32, 69, 71 & 72.) (In color in Annals online.)
the CNS. It is also particularly interesting whether the regulatory actions of CDK5 on GR transcriptional activity contribute to the development of neurodegenerative disorders, since glucocorticoids have strong activity on consolidation of memory and neuronal cell survival.51,52 Proteolytically produced cytoplasmic p25, whose excessive production has been associated with Alzheimers disease,53,54 demonstrated a much stronger effect than p35 in regulating GR transcriptional activity.33 Thus, it is possible that CDK5 may exert pathologic effects by altering GR transcriptional activity through aberrant conversion of p35 to p25.
Glucocorticoids, the Metabolic Syndrome and Other Somatic Sequelae of Stress Endogenous or exogenous Cushing syndrome is associated with the full metabolic
prole of the metabolic syndrome and with substantially increased cardiovascular morbidity and mortality.55,56 Glucocorticoids directly cause insulin resistance of peripheral target tissues in proportion to their levels and to the particular target tissues sensitivity to these hormones. Over time, glucocorticoids also cause progressive accumulation of visceral fat, leading to worsening manifestations of the metabolic syndrome. Thus, when polymorphisms of the GR gene lead to an unfavorable discordance between the activity of the HPA axis and the sensitivity of muscle, fat and/or liver to glucocorticoids, the increased glucocorticoid effect in these tissues could inuence the metabolic prole and the longevity of humans in a negative fashion, similar to that which occurs in Cushing syndrome.7,15,5760 Genetic and developmental factors, nutrition, lifestyle, and cumulative chronic or intermittent stress may lead to the development
160
Figure 3. Regulation of GR transcriptional activity through phosphorylation. (A) Human GR has three major phosphorylation sites at serines 203, 211, and 226, and two minor phosphorylation sites at serines 113 and 141. The former sites are phosphorylated by several kinases as indicated. From Refs. 24 & 3439. (B) Schematic regulation of GR-induced transcriptional activity by CDK5. CDK5 regulates GR-induced transcriptional activity by changing attraction of transcriptional cofactors to responsive promoters in the CNS, possibly through molecules that specically interact with phosphorylated or nonphosphorylated ligand-bound GR. The direction and size of the effect depends also on the presence of other gene promoter, tissue- or brain regionspecic transcriptional cofactors. (Modied from Refs. 33 & 34.)
of obesity, primarily of the visceral type with concurrent loss of lean body mass, and, hence, the metabolic syndrome with its components of insulin resistance, dyslipidemia, chronic smoldering inammation, blood hypercoagulation, arterial hypertension, and/or diabetes mellitus type 2 (Fig. 4).61 These changes lead to endothelial inammation, atherosclerosis, and cardiovascular disease, ultimately resulting in premature cardiovascular morbidity and death.61 Similarly, through decreased bone formation
and/or increased bone resorption, osteopenia or osteoporosis may ensue. Glucocorticoids contribute to the pathogenesis of obesity and the metabolic syndrome, not only through unfavorable genetic variations that increase both the activity of the HPA axis and the sensitivity of tissues to glucocorticoids, but also because of fetal programming of the HPA axis by an adverse intrauterine environment, which may lead to a postnatally hyperactive axis, and because of chronic cortisol
161
Figure 4. Endogenous/exogenous inputs to the stress system and their effects on the metabolic and cardiovascular systems and bone. ABP: arterial blood pressure; APR: acute phase reactants; AVP: arginine vasopressin; CRH: corticotropin-releasing hormone; E2: estradiol; EDS: excessive daytime sleepiness; GH: growth hormone; HDL: high-density lipoprotein; HPA axis: hypothalamic-pituitary-adrenal axis; IGF-1: insulin-like growth factor-1; IL-6: interleukin-6; LC: locus cruleus; LDL: low-density lipoprotein; LH: luteinizing hormone; NE: norepinephrine; T: testosterone; T3: triiodothyronine; TG: triglyceride; TSH: thyroid-stimulating hormone. (From Ref. 68.) (In color in Annals online.)
hypersecretion owing to concurrent real or perceived stress.15,21,62 The opposite result is possible as well. Patients may be protected from obesity, the metabolic syndrome, and premature death because of favorable genetic variations causing a decreased activity of their HPA axis and their tissue sensitivity to glucocorticoids, as well as because of normal or opposite fetal programming and decreased exposure to real or perceived stress.15,21,62 In the studies by Van Rossum et al.62a,62b Syed et al. and others,58,62a,62b,6366 favorable genetic variations in the GR gene, in which carriers of a particular polymorphism had peripheral target tissues with decreased sensitivity to glucocorticoids, resulted in increased sensitivity of the same tissues to insulin and hence a healthier metabolic prole.
Beyond Glucocorticoids and the Metabolic Syndrome Despite their obvious importance, glucocorticoids and their signaling system are only one of several physiologic and molecular networks that participate in the development of obesity and the metabolic syndrome, with a resultant adverse effect on longevity.61 Other major hormones of the stress and other systems and their receptors also participate in these phenomena (Fig. 4).4,17 The stress system includes brain nuclei, such as the paraventricular nucleus of the hypothalamus, the brainstem locus cruleus norepinephrine/autonomic nervous system nuclei, and two powerful peripheral neuroendocrine limbsthe HPA axis and the systemic sympathetic and adrenomedullary systems.4,50
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Figure 5. Central regulation of the stress system in normal (left panel) and chronically stressed and stress-hyperresponsive individuals (right panel). The stress system (PVN CRH/AVP and LC/NE) activates the amygdala and the MCLS and receives activating signals from the amygdala and suppressive signals from the MCLS and the hippocampus. Chronic stress has behavioral and somatic consequences summarized in the bottom of right panel. MCLS = mesocorticolimbic (reward) system; PVN = paraventricular nucleus; CRH = corticotropin-releasing hormone; AVP = arginine vasopressin. (Modied from Ref. 4.)
The stress system normally receives positive regulatory input from the amygdala (fear), negative tonic input from the hippocampus and negative regulation from the mesocorticolimbic dopaminergic system (MCLS, reward), while itself regulates these systems by providing positive inuences on all three (Fig. 5, left panel).4 Abnormally increased chronic activity and/or reactivity of the stress system can be primary or secondary to excessive input from the amygdala or defective input from the hippocampus and/or MCLS (Fig. 5, right panel).4 The normally positive input of the stress system to the MCLS becomes negative in response to chronic hyperactivity of the former, perhaps as a result of the characteristic tolerance of the latter.4
The main central molecular mediators of the stress system are corticotropin-releasing hormone, arginine vasopressin, and norepinephrine. The key peripheral molecular mediators are corticotropin, cortisol, arginine vasopressin, norepinephrine, epinephrine and, interestingly, interleukin-6 (IL-6).4,15,50 The genes that code for the synthesis, regulation, actions, and metabolism of these mediators and their receptors are major participants in the adaptation to stress.4,15,50 The stress system is activated in a coordinated fashion during acute, time-limited stress, inuencing central and peripheral functions that are important for adaptation and survival.4 Chronic activation of the stress system, however, is associated with many negative manifestations and
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TABLE 3. Gene Networks Subserving Functions Important for Human Survival and Species Preservation, Which May Produce Pathology in Contemporary Western Societies due to Changes in Lifestyle Response to survival threat Combat starvation Combat dehydration Combat infectious diseases Anticipate adversaries Minimize exposure to danger Prevent tissue strain and injury Modied from Refs. 61 & 68. Selective advantage Energy conservation Fluid and electrolyte conservation Potent immune reaction Arousal/fear Withdrawal from danger Retain tissue integrity and reserve Contemporary diseases Obesity Hypertension Autoimmunity/allergy Anxiety/insomnia Depression Pain and fatigue syndromes
sequelae beyond obesity/metabolic syndrome, atherosclerosis and loss of bone mineral density, which include a long list of behavioral disorders (Fig. 5, right panel, bottom).4 In addition to noninammatory stress, even very mild, asymptomatic inammation stimulates secretion of IL-6 and other inammatory cytokines, while adipose tissue is a major source of circulating tumor necrosis factor and IL-6.14,50,67 Both glucocorticoids and IL-6 synergistically stimulate the acute phase response, including C-reactive protein, brinogen, and plasminogen activator inhibitor 1, all of which increase the ability of blood to coagulate and through their pro-atherosclerosis action have a negative effect on longevity.14,50,67 Thus, chronic stress, an indolent infection, an active autoimmune process, and visceral obesity are all associated with mild hypercytokinemia and low-grade inammation, which ultimately results in blood hypercoagulability, endothelial dysfunction, atherosclerosis, and cardiovascular disease. Finally, it is evident that the metabolic syndrome, regardless of its cause, is a major risk factor for the development of diabetes type 2 and the polycystic ovary syndrome in patients with a genetic propensity to develop these very common disorders.61,68 As a species, we have survived because we have been able to adapt to potentially lethal evolutionary stressors during our life on Earth. Thus, selective pressures on our genome have allowed adaptive changes that, at this time in our evolutionary history and with our current lifestyle, have become somewhat maladaptive in a large proportion of the population
(Table 3).4,22,50,67 Thus, gene networks dedicated to adaptation and survival, with a nite number of members, are probably responsible for much of the contemporary nosology of Western societies presented in Table 3. Even though cancer is not included in this table, it is likely that modication of the immune system and the inammatory reaction by stress could increase the susceptibility of the organism to certain neoplasias. Conclusions To understand the roles of polymorphisms of multiple genes related to the HPA axis and the glucocorticoid signaling system in human physiology and pathophysiology, one will have to study large populations of normal subjects, including adequate numbers of representative racial and ethnic subpopulations, as well as populations of patients aficted by states and diseases that may result from dysfunction of this system, which are summarized in Tables 1 and 3. Once crucial genes and their polymorphisms have been dened, new, existing and constantly improving methods could be employed to screen for changes in the entire gene networks of choice, which, in the appropriate context, could predict the relative risk for developing these common disorders. Also, granted that a large subgroup of this gene network plays a major role in regulating immune function, this information could be useful in predicting vulnerability to certain infections and tumors. Finally, this knowledge might help individualize medications and doses for subjects with the
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above conditions, depending on their genetics in a rational wayan effort that is developing into the eld of pharmacogenomics.
Acknowledgments
This is a synoptic review of work supported by the University of Athens, Athens, Greece, and the Intramural Research Program of the Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD.
Conicts of Interest
The authors declare no conicts of interest. References

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Frontiers in Neuroendocrinology
Frontiers in Neuroendocrinology 29 (2008) 268272 www.elsevier.com/locate/yfrne
Review
Corticosteroid hormones in the central stress response: Quick-and-slow

E. Ronald de Kloet
a
a,*
, Henk Karst b, Marian Joe ls
Department of Medical Pharmacology, LACDR, Leiden University Medical Center, PO Box 9502, 2300 RA Leiden, The Netherlands b SILS-CNS, University of Amsterdam, The Netherlands Available online 24 October 2007
Abstract Recent evidence shows that corticosteroid hormones exert rapid non-genomic eects on neurons in the hypothalamus and the hippocampal CA1 region. The latter depend on classical mineralocorticoid receptors which are accessible from the outside of the plasma membrane and display a 10-fold lower anity for corticosterone than the nuclear version involved in neuroprotection. Consequently, this membrane receptor could play an important role while corticosteroid levels are high, i.e. during the initial phase of the stress response. We propose that during this phase corticosterone promotes hippocampal excitability and amplies the eect of other stress hormones. These permissive non-genomic eects may contribute to fast behavioral eects and encoding of stress-related information. The fast eects are complemented by slower glucocorticoid receptor-mediated eects which facilitate suppression of temporary raised excitability, recovery from the stressful experience and storage of information for future use. 2007 Elsevier Inc. All rights reserved.
Keywords: Stress; Brain; Behavior; Electrophysiology; Feedback; Glucocorticoid receptors; Mineralocorticoid receptors; Non-genomic steroid action; Genomic steroid action; CRH
1. Introduction Glucocorticoids are secreted from the adrenal in hourly pulses which are thought to synchronize and coordinate sleep related and daily events [42]. At any time a glucocorticoid response can be triggered by a stressor. In concert with other stress mediators, the stress-induced rise in glucocorticoid concentration facilitates adaptation to stress and restores homeostasis, (among other things) by enhancing emotional arousal and promoting motivational and cognitive processes [6,17,26]. A glucocorticoid response that is excessive, prolonged or inadequate impairs adaptation to stress and is considered a risk factor for stress-related diseases. The hormones have profound eects on brain development and are a signicant factor in the aging process. The key towards understanding these fundamental processes underlying homeostasis and health is in the receptors that mediate the action of the corticosteroids. These are the
Corresponding author. Fax: +31 71 5274715. E-mail address: e.kloet@lacdr.leidenuniv.nl (E.R. de Kloet).
classical glucocorticoid receptors (GR), selective for naturally occurring and synthetic glucocorticoids; and the mineralocorticoid receptors (MR), which retain corticosterone and aldosterone with a very high anity, i.e. about 10-fold higher than to the GR [6]. The MR also binds progesterone and deoxycorticosterone with relatively high anity [3] and hence this receptor is considered promiscuous in non-epithelial cells. In epithelial cells the MR is aldosterone-selective because the naturally occurring glucocorticoids are metabolized by the 11b-steroid-dehydrogenase type 1 [13]. The MR and GR are nuclear receptors that mediate genomic actions of the naturally occurring glucocorticoids corticosterone in rodents and cortisol in man [32,43]. Our contribution to this special issue of Frontiers will focus on the MR and GR. These receptors are abundantly expressed in the limbic brain where they mediate distinct and complementary actions. While most emphasis in the past decades was on their genomic action there obviously was a problem. Thus, over the years ndings were reported that showed fast eects of corticosteroids on feedback operation in the HPA axis [4]. Fast eects within minutes were also described for violent behavior propagated by a
0091-3022/$ - see front matter 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.yfrne.2007.10.002
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fast feedforward mechanism [21] which were not genomic, but obeyed the pharmacology dictated by the classical nuclear receptors. Moreover, corticosteroid eects were observed on cognitive operations in appraisal of novel situations [29,30], extinction processes [2] and more recently on retrieval processes [7,35]. Recent discoveries using an electrophysiological approach have supplied a mechanistic basis to these fast behavioral and neuroendocrine eects. We rst will review these fast cellular eects, and next examine the fast eects in the chain of events that take place from the rst encounter with a real or imagined stressor to its long-term outcome on cognitive performance. 2. Fast eects of corticosteroid hormones on neuronal function Parvocellular neurons in the PVN are critically involved in the production and release of CRH. These processes are under fast and delayed negative control of corticosterone [4]. Insight in the putative neurobiological substrate of the fast negative feedback was recently provided by Tasker and co-workers [38]. They showed that glucocorticoids rapidly and non-genomically suppress the frequency (but not amplitude) of miniature excitatory postsynaptic currents (mEPSCs), which reect the postsynaptic response to the spontaneous release of a single glutamate-containing vesicle [8]; inhibitory events were changed in magnocellular but parvocellular neurons [9,40]. It was argued that corticosterone attenuates the release probability of glutamatecontaining vesicles, a presynaptic phenomenon. Interestingly, this eect was found to depend on a postsynaptic G-protein linked pathway, a process blocked by leptin [24]. This supports the involvement of retrograde messengers. In accordance, corticosteroids stimulate endocannabinoid synthesis and release from the postsynaptic compartment which subsequently via a presynaptic CB1 receptor leads to suppression of glutamate release [8,24]. The nature of the receptors mediating these fast eects of corticosteroid hormones on PVN parvocellular neurons is still unclear. Corticosterone was only active when administered on the outside of the plasma membrane. This does not congrue with a nuclear MR or GR localization. Moreover, antagonists for the classical nuclear MR and GR did not interfere with the suppression of mEPSC frequency. Possibly a so far unknown G-protein coupled receptor is responsible for these actions. Rapid eects of corticosteroid hormones have also been seen in the hippocampus, but the characteristics dier from those seen in the hypothalamus. Thus, it was observed that rapidly after corticosterone application the frequency of mEPSCs in hippocampal CA1 neurons is enhanced [18]. No eect was observed on mEPSC amplitude or kinetic properties. As paired pulse facilitation was decreased, it was concluded that corticosterone rapidly increases the release probability of glutamate-containing vesicles. Enhanced release of glutamate from the hippocampus in vivo, shortly after administration of corticosterone, was
indeed reported [39]. Preliminary evidence indicates that the rapid eect on mEPSC frequency involves a presynaptic ERK1/2 pathway, independent of retrograde messengers [31]. The corticosterone-induced enhancement of mEPSC frequency was quickly reversed upon removal of the steroid hormone [18], which points to a non-genomic mechanism. This was conrmed in follow-up experiments where the increase in mEPSC frequency with corticosterone was also demonstrated in the presence of a protein synthesis inhibitor. Moreover, nuclear localization of the hormone-receptor complex appeared not necessary, since a corticosteroneBSA conjugate evoked eects comparable to the natural hormone itself. The eects of corticosterone were seen with 10 nM of the hormone, which is a concentration that can be reached in the hippocampus under stressful conditions. Because the hormone concentration necessary to see changes in mEPSC frequency is closer to the Kd of the GR than of the MR it was assumed that the rapid eects involve the GR. However, application of a selective GRagonist was ineective. The eect of corticosterone could not be blocked by a GR-antagonist and was still present in forebrain specic GR knockouts [18]. By contrast, the mineralocorticoid aldosterone strongly increased mEPSC frequency, an eect that was fully suppressed by the MRantagonist spironolactone. No change in mEPSC frequency was induced by corticosterone in forebrain specic MR knockouts. Collectively, these data indicate that in the hippocampus corticosterone can increase the release probability of glutamate-containing vesicles in a manner requiring the classical MR, which for some reason is inserted into the membrane and has a 10-fold lower apparent anity than the nuclear MR [18]. More recently, other rapid eects were also reported for CA1 hippocampal neurons. For instance, it has been found that corticosterone suppresses the K+-conductance IA, via a postsynaptic membrane MR coupled to a G-protein dependent pathway [31]. Eectively, this could mean that increased presynaptic release of glutamate is accompanied by an enhanced likelihood that action potentials are generated postsynaptically. This could contribute to the fact that long-term potentiation in the CA1 area is facilitated when corticosterone is present during high-frequency stimulation of the Schaer collaterals, although it should be noted that this phenomenon is not sensitive to GR- as well as MRblockers [41]. Interestingly, corticosterone was also found to rapidly facilitate the excitatory eect of b-adrenergic agonists on LTP in the dentate gyrus [34]. By contrast, if corticosterone was applied several hours before the b-agonist, so that genomic eects were allowed to develop, the steroid suppressed excitatory eects via the b-agonist on LTP [34]. 3. Corticosteroid hormones in the stress response How can these rapid cellular eects of corticosterone contribute to the central stress response?
270
With respect to the HPA regulation, it has been known already for a long time that rising levels of corticosterone can exert a fast negative control over the release of CRH and ACTH [4]. This eect may be short-lived, because as soon as corticosteroid levels plateau the negative inuence is lost, only to re-appear some time later when gene-mediated eects kick in. This transient character of fast corticosteroid eects does not seem to hold for limbic actions. Thus, in vitro studies show that the enhancement of mEPSC frequency lasts as long as corticosterone concentrations are high (Karst, unpublished observation). This could signify that during the initial stage of the central stress responsei.e. when levels of corticosterone, peptides like CRH and vasopressin, and of catecholamines are highstress-induced levels of corticosterone promote hippocampal excitability and amplify the eect of other stress hormones (Fig. 1). These permissive eects of corticosterone, which at least partly involve membrane MRs, may contribute to fast encoding of stress-related information, to appraisal processes and the selection of behavioral responses to cope with the stressor [27,29,30]. By the time that hormone levels subside, gene-mediated eects by corticosterone have developed. These slow eects are mostly mediated via nuclear GRs (reviewed in [15,17]). In the hippocampal CA1 area, activation of nuclear GRs raises the threshold for induction of LTP, so that any information reaching this area several hours after the encoding of stress-related information has started must be very sali-
ent in order to surpass the threshold for LTP induction. This may help to preserve the earlier information. Genemediated GR actions further slowly attenuate the transfer of excitatory information, suppress excitatory b-adrenergic actions and enhance inhibitory eects of serotonin (reviewed in [15,17]). This ts with the notion that at least 1 h after stress exposure the temporary raised excitability is reversed and normalized to pre-stress levels [16], thus preventing the hippocampal response from overshooting or persisting when no longer necessary. This can be regarded as the limbic correlate of the slow negative feedback in the hypothalamus. Gene patterns contributing to these delayed genomic eects of corticosteroids have been identied [5,28]. At last: what about the nuclear form of the MR? It has been demonstrated that nuclear MR-mediated actions are crucial for the stability of neuronal networks, survival of neurons in the hippocampus under adverse conditions as well as the threshold/sensitivity of the stress system and behavioral responses [20,23,37]. Recent studies using mouse lines where the MR was knocked out [1,12] or overexpressed [11,22,36] generally support this point of view. 4. Perspectives While electrophysiological studies over the past years have rmly established the existence of rapid non-genomic corticosteroid eects in brain, numerous questions are still
[corticosterone]
stressor
threshold to activate membrane MR
2 hrs
Neuroendocrine feedback in hypothalamus
Stress Reaction Rapid nongenomic corticosteroid effects: -Enhanced Glu release probability -Suppression IA -Synergy with NA -Facilitation LTP Excitability up
Recovery & Adaptation Delayed genomic corticosteroid effects: -Attenuated transfer of excitatory information -Increased 5-HT inhibition -Suppression of NA action -Impaired LTP Excitability normalized Behavioral feedback in hippocampus
Fig. 1. Shortly after exposure to a stressor (vertical arrow), corticosteroid levels rise. Rapid as well as delayed negative feedback has been described (grey bars), the latter causing normalization of corticosteroid levels some hours after stress exposure. The onset of the delayed negative feedback varies (indicated here by horizontal arrow), depending e.g. on modulatory input to the PVN. Recently, rapid non-genomic cellular eects were described in the hypothalamus, which may provide a neurobiological substrate for the fast feedback. In the hippocampus, corticosterone can also exert a number of rapid non-genomic actions which require relatively high levels of the hormone. Hence, these will take place only when a certain threshold concentration of the hormone is surpassed (striped line). We propose that overall these rapid eects in CA1 neurons promote excitability and amplify the eects of other stress hormones. These rapid eects contribute to the overall central stress reaction, comprising facets of appraisal, attention and alertness. At the same time, however, gene-mediated pathways are started [28] which some hours later (i.e. by the time that hormone levels are declining) result in attenuated neurotransmission, thus normalizing the earlier raised activity. This phase is important for recovery and adaptation.
271
unanswered. First and foremost it will be important to link the presently described cellular eects to the sets of behavioral and neuroendocrine observations: the cellular nongenomic eects are likely to underlie fast changes in memory formation and HPA activity, but causative evidence has not yet been provided. In addition to the rapid hormonal inuences on encoding of information, there is also ample evidence for non-genomic modulation by stress hormones of retrieval of information stored in limbic regions [35]. As yet, a neurobiological substrate for this phenomenon is not available at the cellular level. Clearly, a host of mechanistic questions are still open. The fast non-genomic eects in part require classical nuclear corticosteroid receptors [18]. Are these membrane receptors identical to or variants of the ones mediating genomic eects? If they are identical, then why do they not translocate to the nucleus but instead travel towards the plasma membrane? And is the proportion of receptors in (or close to) the membrane dependent on external factors, like the availability of corticosteroid hormones over a longer period of time? In part, however, the receptors mediating fast corticosteroid receptors do not display the pharmacological prole of nuclear receptors [8,41]. Therefore, the existence of a so far unknown G-protein coupled receptor to which corticosterone binds with relatively high anity cannot be excluded, as was described for estrogens [33]. The comparison between hypothalamus and hippocampus shows that rapid non-genomic eects of corticosteroids are regionally dierentiated. It will therefore be very important to also examine rapid eects in other limbic areas important for the central stress response, like amygdalar nuclei or subareas of the prefrontal cortex [10,19,25]. Extracellular recording studies in fact support rapid eects of stress on ring patterns in the prefrontal cortex [14]. Extension of the current studies to these areas will be necessary to get a full comprehension of the role of rapid nongenomic corticosteroid eects during the initial and late phases of the central stress response. Acknowledgments The support by the Netherlands Organisation for Scientic Research and the Royal Netherlands Academy of Arts and Sciences is gratefully acknowledged. References
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Diabetologia (1997) 40: 487495 Springer-Verlag 1997
Review
The pancreatic beta-cell as a fuel sensor: an electrophysiologists viewpoint*
P. Rorsman
Department of Islet Cell Physiology, Novo Nordisk A/S, Copenhagen, Denmark
Summary The pancreatic beta cell serves as the fuel
sensor of the entire body and controls, via secretion of the hypoglycaemic hormone insulin, the blood glucose concentrations within narrow limits by regulation of glucose uptake and release. During the last 30 years, a combination of biochemical and ultrastructural approaches has resulted in dramatic progress in the understanding of the processes by which glucose and other nutrients modulate the release of insulin. The beta cells have also been investigated using electrophysiological techniques and were thus found to be electrically excitable and to undergo complex changes in their membrane potential when exposed to glucose and other stimulators of secretion. The application of the patch-clamp technique to the
pancreatic islet preparations has revolutionized the understanding of how bioelectrical processes participate in the fuel-sensing of the beta cell. An important achievement was the identification of an ATP-sensitive K +-channel as the resting and glucose-sensitive membrane conductance of the beta cell. This channel also constitutes the target of the hypoglycaemic sulphonylureas: a group of compounds which have been used successfully in the treatment of insulin-dependent diabetes mellitus for several decades. [Diabetologia (1997) 40: 487495]
Keywords Ion channels, ATP, insulin, exocytosis,

pancreas.
The central role of ions in the control of cellular excitability has been recognized for more than a century and dates back to the pioneering work of Ringer [1]. For a long time, electrical excitability was a property believed to be confined to a small group of highly sophisticated cells such as nerve and muscle cells in which the role and need of electrical signalling was obvious. However, during the 1960 s and 1970 s it became obvious that a number of endocrine cells share this capacity and that they utilize changes in their membrane potential to transduce changes in their
* The 31st Minkowski lecture given in the Austria Centre, Vienna, Austria, 4 September, 1996.
environment to acceleration of hormone secretion [2, 3].
The pancreatic beta cell is electrically excitable

In 1968 Dean and Matthews [3] provided the first evidence for glucose-stimulated electrical activity in the pancreatic beta cell. The salient features of this electrical activity are summarized in Figure 1. In the absence of glucose, or at substimulatory glucose concentrations (< 78 mmol/l), the membrane potential of the beta cell is negative (60 mV). Following elevation of glucose to insulin-releasing concentrations, the beta cell depolarizes and once the cell becomes sufficiently depolarized (i. e. exceeds the threshold potential), electrical activity is generated. This electrical activity consists of slow oscillations in membrane potential between a depolarized plateau, on which action potentials are superimposed (active
Cell Physiology, Novo Nordisk A/S, Fruebjergvej 3, DK-2100 Copenhagen, Denmark Abbreviations: K-ATP channel, ATP-sensitive potassium channel; PKA, protein kinase A.
Corresponding author: Dr. P. Rorsman, Department of Islet
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P. Rorsman: The pancreatic beta-cell as a fuel sensor
Fig.1. The membrane potential (V) of a single beta cell within
an intact pancreatic islet recorded in the presence of 6.5 and 10 mM glucose as indicated by the staircase
phase), and repolarized electrically silent intervals. The beta cell responds to glucose in a graded fashion: the fraction active phase increases as the glucose concentration is raised until electrical activity becomes continuous at concentrations above 20 mmol/l. The induction of electrical activity is a key event in the sequence of events that culminates in the release of insulin and it has been possible to demonstrate that the periods of electrical activity coincide with pulsatile insulin secretion [4]. More extensive accounts of the beta cell electrical activity have been published and interested readers are referred to these for a detailed description [5, 6].
that the glucose-sensitive resting conductance of the beta cell is due to the activity of K + -channels which are inhibited by intracellular ATP (the K-ATP channel) [711]. The regulation of the K-ATP channel is extremely complex but there is now agreement that changes in the cytoplasmic ATP/ADP-ratio represents an important determinant of channel activity [11, 12]. In parallel with the work on the K-ATP channel, the voltage-dependent membrane currents participating in the generation of the beta cell action potential were characterized. In mouse beta cells, which represent the classic preparation for electrophysiological experiments, the depolarizing phase of the action potential is attributable to the activation of voltage-gated Ca2 + -channels which are sensitive to dihydropyridines such as nifedipine (L-type Ca2 + -channels) [13, 14]. The repolarization of the action potential principally results from the opening of voltage-dependent K + -channel with a time course of activation which is delayed relative that of the Ca2 + -channels (hence delayed rectifying K + -current) [13, 15, 16].
The K-ATP channel: a target of the hypoglycaemic sulphonylureas

Following the identification of the K-ATP channel as the glucose-sensitive membrane conductance of the beta cell it was proposed that this channel also represents the target of the hypoglycaemic sulphonylureas, compounds which have been used in the treatment of non-insulin-dependent diabetes for several decades. With the aid of the patch-clamp technique it was possible to show that this was indeed the case and that therapeutic concentrations of the sulphonylureas produce a concentration-dependent inhibition of the K-ATP channel [1719]. The exact nature of the interactions between the sulphonylureas and the KATP channel could not be resolved in the first patch-clamp experiments, but the observation that they remained inhibitory in isolated membrane patches enabled the conclusion that their effect is not secondary to interference with beta cell metabolism. Some of the sulphonylureas were found to be very potent inhibitors of the K-ATP channel. For example, glibenclamide was effective at nanomolar concentrations [19]. By using the sulphonylureas as ligands it was thereby possible to purify and eventually to clone the sulphonylurea receptor. Thanks to these efforts we now know that the K-ATP channel is a complex of a 145 kDa sulphonylurea receptor (SUR; [20]) and an inward rectifier K + -channel protein (KIR6.2; [21, 22]). Hopefully this novel molecular information can be exploited in current and future endeavours to develop new and more tissue-selective antidiabetic compounds. In this context it is pertinent that K-ATP channels in different tissues
ATP-sensitive K + -channels: the biophysical basis for the fuel-sensing of the beta cell
Recordings using intracellular electrodes impaled into the beta cell in intact pancreatic islets have been invaluable in determining the effects of insulin-releasing agents on the membrane potential and electrical activity of the beta cell (reviewed in [5]). However, because it was not possible to control the membrane potential of the preparation using this technique, the identity and characteristics of the ion channels underlying the electrical activity could not be determined. Such analyses had to await the application of the patch-clamp technique to pancreatic islet cells [7]. This technique, the features of which relevant to the study of pancreatic beta cells have been described at length elsewhere [6], permits the recording under voltage-clamp conditions (i. e. the membrane potential is kept constant irrespective of any activation of membrane currents) of both the minute single-channel and the whole-cell currents; the latter reflecting the summed activity of all the ion channels in the entire plasma membrane. The introduction of the patch-clamp technique revolutionized electrophysiology and in just a few years transformed it from an art, practised in only a few laboratories, into a standard technique of cell physiology. Using the various recording modes of the patchclamp technique it became possible to demonstrate
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the pancreatic beta cell. Glucose produces, via its metabolism, an increased cytoplasmic ATP/ADP-ratio which inhibits the K-ATP channels. This results in membrane depolarization and the opening of voltage-dependent Ca2 + -channels, an increase in [Ca2 + ]i and the initiation of exocytosis of the insulin-containing secretory granules. Also indicated in the model is the site of action of the hypoglycaemic sulphonylureas which inhibit the K-ATP channels by a direct effect which is not dependent on glucose metabolism
Fig.2. Schematic model for the stimulus-secretion coupling of
have distinct molecular composition. For example, the K-ATP channels in cardiac and beta cells contain distinct isoforms of SUR (denoted SUR2 and SUR1, respectively) [23]. This accounts for the different pharmacological properties of cardiac and beta cell K-ATP channels. It is not known which SUR is present in the K-ATP channels in smooth muscle cells (e. g. those in the blood vessels) but it is likely to represent yet another isoform of SUR as neither SUR1 nor SUR2 is expressed in smooth muscle preparations.
A model for glucose-stimulated insulin secretion

A simple model for the stimulus-secretion coupling of the pancreatic beta cell is shown in Figure 2. In the absence of glucose, the cytoplasmic ATP/ADP ratio is low and the K-ATP channels are active. Each beta cell is equipped with thousands of K-ATP channels and their summed activity effectively clamps the beta-cell membrane potential at the K + equilibrium potential which (with the K + -gradients existing over the beta-cell membrane) is around 70 mV. Ion channels other than the K-ATP channels are present, and perhaps active even in the absence of glucose, but they are too few to influence the membrane potential as long as the K-ATP channels remain active. When the beta cell is exposed to glucose, the associated acceleration of glucose metabolism leads to a rise in the cytoplasmic ATP/ADP-ratio and the K-ATP channels close. Once they are almost
completely inhibited ( > 90 %), the remaining KATP conductance is unable to balance the depolarizing influence of (tonically active?) background conductance(s) and the beta cell depolarizes. When this depolarization is large enough, voltage-gated Ca2 + channels become activated in a feed-forward manner (i. e. the initial depolarization up to the threshold potential causes the opening of a few voltage-gated Ca2 + -channels which in turn causes a bigger depolarization and the opening of additional Ca2 + -channels with resultant further depolarization, etc), thus accounting for the upstroke of the beta-cell action potential. The associated Ca2 + -influx causes a transient elevation of the cytoplasmic Ca2 + -concentration ([Ca2 + ]i) [24] which, via a series of poorly defined reactions, culminates in the exocytosis of the insulincontaining granules. The unique feature of the pancreatic beta cell, essential for its ability to serve as the bodys fuel sensor, is the presence of the K-ATP channels. As discussed above, the activity of these channels sets the membrane potential of the beta cell and thus determines its electrical and secretory activities. If the beta cell had not been equipped with K-ATP channels, it would have been tonically active and constantly releasing its insulin into the circulation regardless of the glucose concentration. Indeed, these are precisely the characteristics of beta-cells isolated from patients with persistent hyperinsulinaemic hypoglycaemia of infancy [25]. This rare hereditary disease is linked to mutations in SUR [26], which is part of the K-ATP channel complex (see above), and thus results in the formation of non-functional K-ATP channels. The fact that K-ATP channels play such an important role in the stimulus-secretion coupling of the beta cell does not exclude, however, that metabolic regulation of more distal processes also contributes to the overall fuel-sensing of the beta cell. For example, there is evidence suggesting that glucose metabolism may control both the functional state of the voltagegated Ca2 + -channels [27] (thus determining the amount of Ca2 + -entry and the extent of Ca2 + -induced exocytosis) as well as the insulin secretory process itself [28]. The objective of beta-cell electrical activity is to generate the intracellular signal that initiates the exocytosis of the insulin-containing granules. In the remainder of this review I shall therefore discuss the control of exocytosis in the insulin-secreting beta cell and consider various modulatory mechanisms.
Capacitance measurements of insulin secretion

Elucidation of the fundamental properties of exocytosis requires a means to record secretion in single cells with high temporal (millisecond) resolution. Unfortunately, none of the traditional biochemical
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The exocytosis of a secretory granule (left and centre) results in the incorporation of the granular membrane with the plasma membrane. The added membrane causes an increase in cell surface area which can be detected as an increase in cell capacitance (Cm), an electrical parameter of the cell which is proportionally related to the surface area (A; i. e. Cm A). The specific membrane capacitance is 10 fF/mm2. The surface area of a secretory granule can be estimated as 0.2 mm2 assuming spherical geometry and a granule diameter of 0.25 mm [30]. The fusion of a single granule accordingly results in a capacitance increase of 2 fF. When a granule is subsequently retrieved by endocytosis, a corresponding decrease in cell capacitance can be recorded (right)
Fig.3. Principle for capacitance measurements of exocytosis.
involve measurements of insulin secretion as such but changes in a physical property of the cell which hopefully reflect exocytosis. To ascertain that this is indeed the case we have combined capacitance measurements with fluorimetric and/or electrochemical detection of insulin secretion [32]. So far we have encountered no discrepancies between the capacitance measurements and secretion detected by the alternative methods making it reasonable to conclude that an increase in cell capacitance can be equated to exocytosis. The conclusion is reinforced by the observations that insulin secretion and exocytosis as reported by the capacitance measurements exhibit the same Ca2 + -dependence [32], are equally affected by cooling [33] and similarly modulated by hormones and neurotransmitters [34, 35]. Capacitance measurements finally offer the unique possibility of studying the retrieval of the secreted membranes by endocytosis. In this case, membrane retrieval results in a decreased membrane surface area and thus a reduction of the cell capacitance [36].
Exocytosis in the beta cell is rapid

Using capacitance measurements it was possible to demonstrate that the latency between the opening of the Ca2 + -channels and the onset of exocytosis is less than 50 ms. This is shorter than the time required for [Ca2 + ]i to equilibrate within the beta cell which suggests that the Ca2 + -channels, the secretory granules and the release sites are situated in the near vicinity of each other. During the first 50 ms the rate of capacitance increase approached 1 pF/s. Using the conversion factor of 2 fF/granule this can be converted to an exocytotic rate of 500 granules/s. Since a beta cell contains 13 000 granules [30], this value corresponds to a release rate of 4 % of the total granule number being released per second! Clearly, exocytosis can only continue at this high rate for a very limited period and during protracted stimulations exocytosis proceeds at much slower average rates. This decline in the exocytotic rate reflects the gradual depletion of the pool of granules which is immediately available for release (the readily releasable pool). Once this pool has been depleted other processes, such as the refilling of the readily releasable pool (see below), become rate-limiting to exocytosis. It should be made clear that the short latency (< 50 ms) observed in the capacitance measurements does not imply that the release process (including the dissolution of the Zn2 + -insulin crystal) is completed within this time span: the latency is simply a reflection of the time required for the granular membrane to fuse with the plasma membrane and thus be detected by the capacitance measurements. Even so the above considerations illustrate the high speed and capacity of the exocytotic machinery in the beta cell.
approaches for measuring insulin release have achieved this resolution. We have therefore been obliged to monitor changes in cell capacitance as an indicator of exocytosis. The membrane capacitance (C) is an electrical property of the cell which is proportionally related to the cell surface area. When the insulin-containing granules undergo exocytosis, their membranes are incorporated into the plasma membrane (Fig. 3). The resulting increase in cell surface area can be monitored as an increase in cell capacitance using electrophysiological techniques [29]. The major advantages of capacitance measurements over more traditional approaches to study secretion are: 1) the measurements can be carried out in single cells with millisecond resolution; 2) the experiments are carried out in a voltage-clamped preparation: i. e. any effects of a compound on membrane conductances and membrane potential will not influence the results; and 3) large proteins such as antibodies can be applied intracellularly by including them into the pipette solution which replaces the cytoplasm when utilizing the whole-cell recording mode of the patchclamp technique. The specific capacitance of biological membranes (including those of the plasma membrane and the secretory granules) is 10 fF/mm2. With a diameter of 250 nm [30], each beta-cell granule can be estimated to add 0.2 mm2 of membrane area which corresponds to 2 fF of capacitance. Capacitance increases with this unitary amplitude have been recorded [31] and may accordingly reflect single exocytotic events (i. e. the fusion of an individual granule with the plasma membrane). It must be kept in mind that recordings of cell capacitance do not
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Modulation of Ca2 + -induced exocytosis

Exocytosis in the beta cell is clearly Ca2 + -dependent but Ca2 + should perhaps be regarded as an initiator rather than a determinant of exocytosis. This is suggested by the observation that the amplitude of the exocytotic responses depends to a greater extent on the activity of protein kinases and phosphatases than the actual [Ca2 + ]i. For example, agents which increase cytoplasmic cyclic AMP levels, such as glucagon and glucagon-like peptide-1, potentiate glucosestimulated insulin secretion by a protein kinase A (PKA)-dependent mechanism almost 10-fold without much affecting Ca2 + -influx or [Ca2 + ] [34, unpublished data]. In the case of glucagon-like peptide-1 (GLP-1), no stimulation of exocytosis was observed in the absence of glucose suggesting that ATP derived by glucose metabolism is required for the PKA-dependent phosphorylation. Our capacitance measurements indicate that this stimulation principally results from PKA accelerating granule mobilization from the reserve pool into the readily releasable pool thus increasing the size of the latter over fivefold. When exocytosis is subsequently initiated by elevation of [Ca2 + ]i, a greater number of granules are available for release. We have previously postulated that cAMP acts by sensitizing the secretory machinery [34]. However, the fact that the relationship between [Ca2 + ]i and exocytosis remains the same in the absence and presence of cAMP is hard to reconcile with such a concept and an increased size of the readily releasable pool may produce effects on exocytosis which at first glance are difficult to distinguish from a sensitizing mechanism. The molecular mechanism by which PKA accelerates granule mobilization remains obscure. In nerve endings, PKA is known to exert a similar action as in the beta cell by phosphorylation of the protein synapsin-1 which controls the interactions between the vesicles and the cytoskeleton. Synapsin-1 is not expressed in pancreatic beta cells but a synapsin-1-like protein, which may fulfill the function of synapsin-I, has recently been characterized in insulin-secreting cells [37]. Ca2 + -induced exocytosis is also enhanced by agents which activate protein kinase C, such as ACh and the phorbol ester 4-b-phorbol-12-b-myristate13-a-acetate (PMA) [35]. In general, conditions which promote protein phosphorylation lead to enhancement of secretion. Conversely one would expect that agents which produce the activation of protein phosphatases inhibit exocytosis. Indeed, this seems to be the mechanism by which the inhibitory hormones and neurotransmitters somatostatin, galanin and adrenaline suppress glucose-stimulated insulin secretion. The action of these compounds is mediated by activation of an inhibitory (pertussis toxinsensitive) G-protein and culminates in the activation of the protein phosphatase calcineurin [38].
Hypoglycaemic sulphonylureas stimulate insulin secretion by interaction with exocytotic machinery

Perhaps the most surprising finding that has emanated from the capacitance measurements is that the sulphonylureas, in addition to closing the K-ATP channels, also stimulate insulin secretion by direct interaction with the exocytotic machinery [39] (but see [40] for conflicting data). Such an effect is not easily detected in ordinary assays of insulin secretion as the stimulation of secretion resulting from the closure of the K-ATP effectively obscures any contribution of a late mechanism. Using capacitance measurements it became possible to separate the two effects as the membrane potential was voltage-clamped and thus held constant irrespective of K-ATP channel activity. An effect of the sulphonylureas on exocytosis would also be consistent with the ultrastructural and biochemical evidence indicating that as much as 90 % of the sulphonylurea-binding in the beta cell is intracellular and localized to the secretory granules [41, 42]. The observations that the sulphonylureas interfere with exocytosis, possibly by binding to granular sulphonylurea receptors, raises several interesting questions: 1) Is the granular sulphonylurea receptor the same as the 145 kDa SUR which is part of the K-ATP channel? 2) Do the granular sulphonylurea receptors couple to ion channels in the granule membrane. Sulphonylurea-sensitive membrane currents have been demonstrated in pancreatic zymogen granules and have been proposed to control the fusion process [43]. It is attractive to speculate that the sulphonylureas modulate exocytosis in the beta cell by interference with similar conductances in the insulin-containing granules. In this context it is of interest that the 145 kDa sulphonylurea receptor has been reported to promiscuously couple to K + -channel proteins other than KIR6.2 and it is therefore possible that it may also associate with other channel proteins [44]; 3) What is the physiological role of the granular sulphonylurea receptors and do they participate in normal Ca2 + -induced exocytosis? The answers to these questions are clearly central to the understanding of how the sulphonylureas stimulate exocytosis in the beta cell. However the significance of these results may not be limited to the understanding of the control of insulin secretion. The sulphonylureas have been postulated to enhance glucose uptake in fat cells [45, 46]; an effect which seems as controversial [47] as the effect on exocytosis in the beta cell [40]. Glucose uptake in fat cells involves the insertion of the glucose transporters into the plasma membrane by exocytosis of their intracellular storage vesicles [48, 49]. Since exocytosis in various cell types appears to involve the same molecular processes it is attractive to speculate that the mechanism we have described in the pancreatic beta cell is also operational in other cells (such as
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adipocytes) and accounts for some of the reported extrapancreatic actions of the sulphonylureas.
Metabolic regulation of exocytosis

In this review I emphasize the metabolic regulation of the beta cell. I have already described how ATP, via regulation of the K-ATP channels, controls the membrane potential and thereby Ca2 + -influx and Ca2 + -induced exocytosis. However, there is evidence suggesting that ATP also controls insulin secretion in a more direct way. Experiments on permeabilized insulin-secreting cells have indicated that withdrawal of ATP from the cytoplasm results in 90 % inhibition of exocytosis [50]. Moreover, glucose exerts a strong stimulatory action (particularly at late times) in cells which are already maximally depolarized by high extracellular K +, i. e. under conditions where the sugar is unable to act via depolarization and elevation of [Ca2 + ]i [28]. Collectively these observations indicate that access to cytoplasmic ATP (or another glucose metabolite) is somehow rate-limiting to exocytosis. We have applied capacitance measurements in conjunction with photorelease of caged Ca2 + from its caged precursor Ca2 + /NP-EGTA to test this hypothesis. A representative experiment is shown in Figure 4. Here [Ca2 + ]i was elevated in two different cells which were dialysed with an ATP-containing or an ATP-free solution. Whereas elevation of [Ca2 + ]i in the presence of ATP produced a biphasic stimulation of exocytosis (seen as rapid initial increase in cell capacitance followed by a sustained second slower phase), no change in cell capacitance was observed in the absence of ATP. If anything, the response in the latter cell consisted of a transient decrease in cell capacitance which may reflect endocytosis of granules that had been inserted in the plasma membrane during the period required for the wash-in of the Ca2 + /NP-EGTA complex and the concomitant wash-out of the endogenous ATP. Ca2 + -induced exocytosis in the beta cell is clearly highly dependent on access to cytoplasmic ATP. In this respect the pancreatic beta cell differs from other neuroendocrine cells. In both chromaffin and pituitary cells, large exocytotic responses can be obtained long after complete wash-out of ATP [51]. As I shall try to explain below, this does not necessarily imply that the biochemical regulation of exocytosis in the beta cell differs from that in the other cell types in any fundamental way. As alluded to above, both ultrastructural and functional studies have suggested that the granules in endocrine cells exist in pools of different releasability [52]. A small fraction of the total granule population is immediately available for release and accordingly designated as the readily releasable pool. These granules are probably located just beneath the
an increase in cell capacitance; Cm) was elicited by photorelease of Ca2 + from its caged precursor Ca2 + /NP-EGTA which had been preloaded into the cell. Upon irradiation with ultraviolet light, the affinity of NP-EGTA for Ca2 + is dramatically reduced resulting in the release of Ca2 + . Photolysis was effected as indicated by the dotted vertical lines in the presence (right) and absence of ATP (left). Note the failure of Ca2 + to elicit exocytosis in the absence of ATP. The capacitance increase in the presence of ATP ( 1000 fF) corresponds to the release of 500 secretory granules
Fig.4. Exocytosis is ATP-dependent. Exocytosis (monitored as
membrane and are the first to undergo exocytosis when [Ca2 + ]i is elevated. The vast majority of granules are not immediately available for release and presumably located further away from the plasma membrane. These granules need to be mobilized into the readily releasable pool before they can be released and are referred to as the reserve pool. The mobilization of the granules may involve either their physical translocation within the cell, a chemical modification of the granules (such that the release probability is increased) or both. According to current biochemical models of exocytosis, hydrolysis of ATP is required in the chemical modification of the granules which precedes exocytosis (priming). When [Ca2 + ]i subsequently rises to exocytotic levels, the primed granules (and only those) can be released in a process which does not require any further consumption of ATP. However, the pool of primed/energized granules is of limited size and once these granules have been released the pool needs to be replenished in an ATP-dependent way by mobilization of granules from the reserve pool. A simple explanation to the apparent greater ATP-dependence of exocytosis in the beta cell than in the other neuroendocrine cells is therefore that the insulin-secreting cell contains fewer primed granules. In the pituitary cell, the size of this pool has been estimated both functionally and by electron microscopy. Both methods suggest that the primed pool comprises 4000 granules. This is considerably higher than the corresponding number in the pancreatic beta
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Comparison with insulin secretion

A rapid elevation of [Ca2 + ]i produces a biphasic stimulation of exocytosis consisting of an initial rapid increase in cell capacitance followed by a second slower phase (Fig. 4). This biphasic response can be interpreted as the release of different pools of granules. Whereas the first rapid phase is likely to correspond to the release of granules situated immediately beneath the membrane (the readily releasable and/or primed pool), the second slower phase reflects the mobilization of the granules from the reserve pool. The biphasic increase in cell capacitance is clearly reminiscent of the biphasic nature of glucose-stimulated insulin secretion [53] and it is attractive to speculate that it can be explained in similar terms (Fig. 6). In fact, the size of the readily releasable pool which can be released in an ATP-independent fashion compares favourably with the number of granules that can be estimated to undergo exocytosis during the first phase of glucose-stimulated insulin secretion (L. Eliasson, P. Rorsman, unpublished data). Using photorelease of ATP from a caged precursor we believe it has been possible to estimate the time required for a granule to pass from the reserve pool into the readily releasable pool and to be released. We consistently observed a delay of 10 s between the application of ATP and the onset of secretion under experimental conditions that ensure that the primed/readily releasable pool was previously depleted. The long latency argues that the process of mobilization involves physical translocation, and not just chemical modification, of the granules.
Fig.5. Comparison of the number of primed granules in the
beta cell and in a pituitary cell. The pituitary cell contains a much larger number pool of primed (energized) granules, which are located just beneath the plasma membrane and that can be rapidly released in a seemingly ATP-independent fashion when [Ca2 + ]i is elevated, than the beta cell
Fig.6. Biphasic glucose-stimulated insulin secretion can be ex-
Glucose metabolism, ATP and diabetes

In this review I have attempted to illustrate how glucose metabolism, via changes in the cytoplasmic ATP concentration, exerts its control of insulin secretion. The action of ATP is exerted at several levels. First, it controls the size of the readily releasable pool of granules by regulating the rate of granule mobilization/priming. Secondly, ATP generated by glucose metabolism determines the amplitude of Ca2 + evoked secretion via protein kinase A and C-dependent phosphorylation of exocytosis-regulating proteins. Thirdly, glucose metabolism modulates the activity of the voltage-dependent Ca2 + -channels and thus Ca2 + -entry and Ca2 + -induced secretion. Finally, by controlling the activity of the ATP-sensitive K + channel, ATP regulates the membrane potential of the beta cell and thereby the electrical and secretory activities. Because ATP exerts so many regulatory functions it is not surprising that conditions which interfere with the ability of the beta cell to generate ATP have marked effects on its secretory capacity. For example, it has been reported that increased
plained as the release of distinct pools of granules. The first phase of insulin secretion can be accounted for by the release of readily releasable (primed/docked) granules which are located immediately below the plasma membrane. The second slower phase results from the time- and ATP-dependent mobilization of granules situated further away from the plasma membrane
cell. Based on our capacitance measurements, we estimate that only 1550 granules (Fig. 5), or 0.10.3 % of the total granule population ( 13 000 [30]), exist in the primed state and are capable of being released in an ATP-independent fashion. Since the number of energized granules in the beta cell is only 1 % of that in the pituitary cell, it is not surprising that insulin secretion exhibits a higher ATP dependence. It seems possible that this represents an important functional adaptation as keeping the number of energized granules low provides the beta cell with the means of rapidly adjusting its secretory capacity to the metabolic state.
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P. Rorsman: The pancreatic beta-cell as a fuel sensor from cells and cell-free membrane patches. Pflu gers Arch 291: 85100 8. Cook DL, Hales CN (1984) Intracellular ATP directly blocks K + -channels in pancreatic B-cells. Nature 312: 271273 9. Ashcroft FM, Harrison DE, Ashcroft SJH (1984) Glucose induces closure of single potassium channels in isolated rat pancreatic b-cells. Nature 312: 446448 10. Rorsman P, Trube G (1985) Glucose-dependent K + -channels in pancreatic b-cells are regulated by intracellular ATP. Pflu gers Arch 405: 305309 11. Misler S, Falke LC, Gillis K, McDaniel ML (1986) A metabolite-regulated potassium channel in rat pancreatic Bcells. Proc Natl Acad Sci USA 83: 71197123 12. Dunne MJ, Petersen OH (1986) Intracellular ADP activates K + -channels that are inhibited by ATP in an insulin-secreting cell line. FEBS Lett 208: 5962 13. Rorsman P, Trube G (1986) Calcium and potassium currents in mouse pancreatic b-cells under voltage-clamp conditions. J Physiol 374: 531550 14. Rorsman P, Ashcroft FM, Trube G (1988) Single Ca channel currents in mouse pancreatic B-cells. Pflu gers Arch 412: 597603 15. Smith PA, Bokvist K, Arkhammar P, Berggren PO, Rorsman P (1990) Delayed rectifying and calcium activated K + -channels and their significance for action potential repolarization in mouse pancreatic b-cells. J Gen Physiol 95: 10411059 16. Bokvist K, Rorsman P, Smith PA (1990) Effects of external tetraethylammonium and quinine on delayed rectifying K + -channels in mouse pancreatic b-cells. J Physiol 423: 311325 17. Sturgess NC, Ashford MLJ, Cook DL, Hales CN (1985) The sulfonylurea receptor may be an ATP-sensitive potassium channel. Lancet II: 474475 18. Trube G, Rorsman P, Ohno-Shosaku T (1986) Opposite effects of tolbutamide and diazoxide on the ATP-dependent gers Arch K + -channel in mouse pancreatic b-cells. Pflu 407: 493499 19. Zu nkler BJ, Lenzen S, Ma nner K, Panten U, Trube G (1988) Concentration-dependent effects of tolbutamide, meglitinide, glipizide, glibenclamide and diazoxide on ATP-regulated K + -currents in pancreatic B-cells. Arch Pharmacol 337: 225230 20. Aguilar-Bryan L, Nichols CG, Herrera-Sosa H, Nguy K, Bryan J, Nelson DA (1995) Cloning of the b-cell high-affinity sulfonylurea receptor: a regulator of insulin secretion. Science 268: 423426 21. Inagaki N, Gonoi T, Clement JP IV et al. (1995) Reconstitution of IKATP: an inward rectifier subunit plus the sulfonylurea receptor. Science 270: 11661170 mma 22. Sakura H, A la C, Smith PA, Gribble FM, Ashcroft FM (1996) Cloning and the functional expression of the cDNA encoding a novel ATP-sensitive potassium channel subunit expressed in pancreatic b-cells, brain, heart and skeletal muscle. FEBS Lett 377: 338344 23. Nichols CG, Shyng SL, Nestorowicz A et al. (1996) Adenosine diphosphate as an intracellular regulator of insulin secretion. Science 272: 17851787 mma 24. Rorsman P, A la C, Berggren PO, Bokvist K, Larsson O (1992) Cytoplasmic calcium transients due to single action potentials and voltage-clamp depolarizations in mouse pancreatic B-cells. EMBO J 11: 28772884 25. Kane C, Shepherd RM, Squires PE et al. (1996) Loss of functional KATP channels in pancreatic b-cells causes persistent hyperinsulinemic hypoglycemia of infancy. Nature Med 2: 13441347
activity of ATP-consuming substrate cycles of glucose metabolism (e. g. glucose glucose 6-phosphate glucose; reactions catalysed by glucokinase and glucose 6-phosphatase, respectively) is an early sign of human non-insulin-dependent diabetes [54, 55]. Such a defect will not only interfere with the ability of glucose to depolarize the beta cell but also, as outlined above, reduce the refilling of the readily releasable pool thus possibly accounting for absence of a first phase of glucose-stimulated insulin secretion in these patients [56]. Finally, it deserves pointing out that currently available pharmacological principles for the treatment of non-insulin-dependent diabetes, such as the sulphonylureas, only rectify the inability of glucose to close the K-ATP channel but fail to correct the other ATP-dependent steps and these accordingly require alternative therapeutic approaches.
Acknowledgements. I thank the present and past members of my group (Dr. K. Bokvist, Dr. P. A. Smith, Dr. O. Larsson, Dr. mma C. A la , Dr. L. Eliasson, Dr. E. Renstro m, Dr. J. Gromada, Dr. L. Best and Dr. W.-G. Ding) for their hard work, loyalty and enthusiasm. During the last 10 years I have had a fruitful and productive collaboration with Professor F. M. Ashcroft (Oxford) and Professor P. O. Berggren (Stockholm). Finally I wish to express my gratitude to Dr. G. Trube for first introducing me to the patch-clamp technique. Supported in part by The Juvenile Diabetes Foundation, The Swedish Medical Research Council, The Nordic Insulin Foundation, The Swedish Diabetes Association and the Novo Nordisk A/S. Note added in proof. Recently Isomoto et al. (J Biol Chem 271:
24321-24324, 1996) describe an isoform of the cardiac sulphonylurea receptor (SUR2B). This isoform is present in smooth muscle cells and, when it is coexpressed with KIR6.2, forms K-ATP channels with the pharmacological properties of the smooth muscle type of the channel.
References
1. Ringer S (1883) A further contribution regarding the influence of the different constituents of blood on the contraction of the heart. J Physiol 4: 2942 2. Dean PM, Matthews EK (1968) Electrical activity in pancreatic islet cells. Nature 219: 389390 3. Taraskevich PS, Douglas WW (1977) Action potentials occur in cells of the normal anterior pituitary gland and are stimulated by the hypophysiotropic peptide thyrotropin releasing hormone. Proc Natl Acad Sci USA 74: 40644067 AR, Stamford JA, Santos 4. Barbosa RM, Silva AM, Tome RM, Rosario LM (1996) Real time electrochemical detection of 5-HT/insulin secretion from single pancreatic islets: effects of glucose and K + depolarization. Biochem Biophys Res Commun 228: 100104 5. Henquin JC, Meissner HP (1984) Significance of ionic fluxes and changes in membrane potential for stimulus-secretion coupling in pancreatic B-cells. Experientia 40: 10431052 6. Ashcroft FM, Rorsman P (1989) Electrophysiology of the pancreatic b-cell. Prog Biophys Molec Biol 54: 87143 7. Hamill OP, Marty A, Neher E, Sakmann B, Sigworth FJ (1981) Improved patch-clamp techniques for the recording
P. Rorsman: The pancreatic beta-cell as a fuel sensor 26. Thomas PM, Cote GJ, Wohlik N et al. (1995) Mutations of the sulfonylurea receptor gene in familial persistent hyperinsulinemic hypoglycemia of infancy. Science 268: 426429 27. Smith PA, Rorsman P, Ashcroft FM (1989) Modulation of dihydropyridine-sensitive Ca2 + -channels by glucose metabolism in mouse pancreatic B-cells. Nature 342: 550553 28. Detimary P, Gilon P, Nenquin M, Henquin JC (1994) Two sites of glucose control of insulin release with distinct dependence on the energy state in pancreatic B-cells. Biochem J 297: 455461 29. Neher E, Marty A (1982) Discrete changes of cell membrane capacitance observed under conditions of enhanced secretion in bovine chromaffin cells. Proc Natl Acad Sci USA 79: 67126716 30. Dean PM (1973) Ultrastructural morphometry of the pancreatic beta-cell. Diabetologia 9: 115119 mma 31. A la C, Eliasson L, Bokvist K, Larsson O, Ashcroft FM, Rorsman P (1993) Exocytosis elicited by action potentials and voltage-clamp calcium currents in individual mouse pancreatic B-cells. J Physiol 472: 665688 mma 32. Bokvist K, Eliasson L, A la C, Renstro m E, Rorsman P (1995) Co-localization of L-type Ca2 + -channels and insulin-containing granules and its significance for the initiation of exocytosis in mouse pancreatic B-cells. EMBO J 14: 50 57 33. Renstro m E, Eliasson L, Bokvist K, Rorsman P (1996) Cooling inhibits exocytosis in single mouse B-cells by suppression of granule mobilization. J Physiol 494: 4152 mma 34. A la C, Ashcroft FM, Rorsman P (1993) Ca2 + -independent potentiation of insulin release by cyclic AMP in pancreatic b-cells. Nature 363: 556558 mma 35. A la C, Eliasson L, Bokvist K et al. (1994) Activation of protein kinases and inhibition of protein phosphatases play a central role in the regulation of exocytosis in insulin-secreting mouse pancreatic B-cells. Proc Natl Acad Sci USA 91: 43434347 mma 36. Eliasson L, Proks L, A la C et al. (1996) Endocytosis of secretory granules in pancreatic b-cells evoked by transient elevation of cytosolic calcium. J Physiol 493: 755767 37. Matsumoto K, Fukunaga K, Miyazaki J, Schichiri M, Miyamoto E (1995) Ca2 + /calmodulin-dependent protein kinase II and synapsin 1-like protein in mouse insulinoma MIN-6 cells. Endocrinology 136: 37843793 38. Renstro m E, Ding WG, Bokvist K, Rorsman P (1996) Neurotransmitter-induced inhibition of exocytosis in insulin-secreting b-cells by activation of calcineurin. Neuron 17: 513 522 mma 39. Eliasson L, Renstro m E, A la C et al. (1996) PKC-dependent stimulation of exocytosis by sulfonylureas in pancreatic b cells. Science 271: 813815 40. Garciabarrado MJ, Jonas JC, Gilon P, Henquin JC (1996) Sulfonylureas do not increase insulin secretion by a mechanism other than a rise in cytoplasmic Ca2 + in pancreatic B-cells. Eur J Pharmacol 298: 279286 41. Carpentier JL, Sawano F, Ravazzola M, Malaisse WJ (1986) Internalization of 3H-glibenclamide in pancreatic islet cells. Diabetologia 29: 25926
495 42. Ozanne SE, Guest PC, Hutton JC, Hales CN (1995) Intracellular localization and molecular heterogeneity of the sulphonylurea receptor in insulin-secreting cells. Diabetologia 38: 277282 venod F, Chathadi KV, Jiang B, Hopfer U (1992) ATP43. The sensitive K + conductance in pancreatic zymogen granules: block by glyburide and activation by diazoxide. J Membr Biol 129: 253256 mma 44. A la C, Moorhouse A, Gribble F et al. (1996) Promiscuous coupling between the sulphonylurea receptor and inwardly rectifying potassium channels. Nature 379: 545548 45. Tsiani E, Ramlal T, Leiter LA, Klip A, Fantus IG (1995) Stimulation of glucose uptake and increased plasma membrane content of glucose transporters in L6 skeletal muscle cells by the sulfonylureas gliclazide and glyburide. Endocrinology 136: 25052512 46. Mu ller G, Wied S (1993) The sulfonylurea drug, glimepiride, stimulates glucose transport, glucose transporter translocation, and dephosphorylation in insulin-resistant rat adipocytes in vitro. Diabetes 42: 18521867 47. Panten U, Schwanstecher M, Schwanstecher C (1992) Pancreatic and extrapancreatic sulfonylurea receptors. Horm Metabol Res 24: 549554 48. Simpson IA, Cushman SW (1986) Hormonal regulation of mammalian glucose transport. Ann Rev Biochem 55: 10551089 49. Lienhard GE (1983) Regulation of cellular membrane transport by the exocytotic insertion and endocytotic retrieval of transporters. Trends Biochem Sci 8: 125127 50. Regazzi R, Wollheim CB, Lang J et al. (1995) Vamp-2 and cellubrevin are expressed in pancreatic b-cells and are essential for Ca2 + but not for GTPgS induced insulin secretion. EMBO J 14: 27232730 51. Parsons TD, Coorssen JR, Horstmann H, Almers W (1995) Docked granules, the exocytotic burst and the need for ATP hydrolysis in endocrine cells. Neuron 15: 10851096 52. Neher E, Zucker RS (1993) Multiple calcium dependent processes related to secretion in bovine chromaffin cells. Neuron 10: 2130 53. Grodsky GM (1994) An update on implications of phasic insulin secretion. In: Pickup J, Williams G (eds) Textbook of diabetes. Blackwell Scientific Publications, Oxford, Vol. 1, pp. 421430 54. Efendic S, Wajngot A, Vranic M (1985) Increased activity of the glucose cycle in the liver: early characteristic of type 2 diabetes. Proc Natl Acad Sci USA 82: 29652969 stenson CG, Efendic S (1994) Glucose cycling in 55. Khan A, O pancreatic islets. In: Flatt P, Lenzen S (eds) Frontiers of insulin secretion and pancreatic B-cell research. Smith-Gordon, London pp. 103111 56. Ramirez LC, Raskin P (1991) Pancreatic abnormalities in non-insulin-dependent diabetes mellitus. In: Pickup J, Williams G (eds) Textbook of diabetes. Blackwell Scientific Publications, Oxford, Vol. 1, pp. 198204
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The metabolic actions of glucagon revisited
Kirk M. Habegger, Kristy M. Heppner, Nori Geary, Timothy J. Bartness, Richard DiMarchi and Matthias H. Tschp
Abstract | The initial identification of glucagon as a counter-regulatory hormone to insulin revealed this hormone to be of largely singular physiological and pharmacological purpose. Glucagon agonism, however, has also been shown to exert effects on lipid metabolism, energy balance, body adipose tissue mass and food intake. The ability of glucagon to stimulate energy expenditure, along with its hypolipidemic and satiating effects, in particular, make this hormone an attractive pharmaceutical agent for the treatment of dyslipidemia and obesity. Studies that describe novel preclinical applications of glucagon, alone and in concert with glucagon-like peptide 1 agonism, have revealed potential benefits of glucagon agonism in the treatment of the metabolic syndrome. Collectively, these observations challenge us to thoroughly investigate the physiology and therapeutic potential of insulins long-known opponent.
Habegger, K. M. et al. Nat. Rev. Endocrinol. 6, 689697 (2010); published online 19 October 2010; doi:10.1038/nrendo.2010.187
Introduction
The isolation of insulin in 1921 by Banting, Best, Collip and Macleod transformed our understanding of the hormonal regulation of glucose metabolism and has provided a life-saving treatment for millions of patients with diabetes mellitus. Of far lesser prominence was the simultaneous description of a pancreatic contaminant observed to elicit a temporary increase in blood glucose levels.1 This contaminant was later studied by Murlin et al.2 and found to oppose insulin in the control of glucose homeostasis, both in healthy animals and in those whose pancreas had been removed. Murlin and colleagues named this new hormone glucagon, as they thought it was a glucose agonist. Later work by Sutherland, Park and Exton described the counter-regulatory actions of glucagon relative to insulin pharmacology. Specifically, the researchers determined that glucagon stimulates hepatic glycogenolysis and gluconeogenesis in hypoglycemic states to restore glucose homeostasis. 3,4 The pathophysiological role of hyperglucagonemia and unopposed glucagon action in diabetes mellitus has been emphasized.59 By contrast, the possibility that glucagon may also have benefits in the treatment of metabolic disease has received little or no attention. However, new studies that describe novel preclinical applications of glucagon, either alone or in concert with glucagon-like peptide 1 (GLP-1) agonism, have revealed the prospect of harnessing the benefits of glucagon action for the treatment of the metabolic syndrome.10,11
Glucagon Expression
Glucagon is a hormone produced in the cells of the pancreatic islets. Encoded by the proglucagon gene, the proglucagon peptide is processed by neuroendocrine convertase-2 (NEC2) to produce the 29-amino acid native glucagon peptide. In addition, the proglucagon gene contains sequences that encode GLP-1, GLP-2, oxyntomodulin and glicentin. These hormones are processed from the proglucagon peptide via NEC1-mediated cleavage in the brain and in L cells of the intestine. 12 Studies on the mechanisms of proglucagon transcription have elucidated the minimal promoter region, as well as four enhancer elements.13 Regulation of transcription has been shown to occur through homeodomain transcription factors, influenced by amino acids and cyclic AMP (cAMP) in the pancreas and intestinal cells,14 as well as Wnt signaling in the intestine.15 Insulin is also known to exert inhibitory effects on proglucagon expression in cells.16,17 Interestingly, this effect is reversed in the intestine, where insulin at pathological concentrations stimulates proglucagon expression, which results in increased GLP-1 production.18
Competing interests N. Geary declares an association with the following company: Novo Nordisk. R. DiMarchi declares an association with the following company: Marcadia Biotech. See the article online for full details of the relationships. The other authors declare no competing interests.
Secretion Glucagon secretion, similar to that of insulin, is tightly regulated and intimately tied to blood glucose levels (Figure 1). Converse to the inhibition of insulin secretion by hypoglycemia, low levels of blood glucose directly stimulate the pancreatic cells to secrete glucagon.19 The secretion of glucagon is promoted via the action of voltage-dependent sodium (Na+) and calcium (Ca2+) channels, which maintain action potentials during times of low glucose. Depolarization increases the Ca2+ influx and subsequent glucagon secretion,20 which is supported by the activity of ATP-sensitive potassium (KATP)
Department of Medicine, University of Cincinnati, Metabolic Diseases Institute, Office E-217, 2170 East Galbraith Road, Cincinnati, OH 45237, USA (K. M. Habegger, K. M. Heppner, M. H. Tschp). Institute of Food, Nutrition and Health, Swiss Federal Institute of Technology Zrich, Schorenstrae 16, 8603 Schwerzenbach, Switzerland (N. Geary). Department of Biology, Georgia State University, 33 Gilmer Street Southeast, Atlanta, GA 30303-3044, USA (T. J. Bartness). Department of Chemistry, Indiana University, 107 South Indiana Avenue, Bloomington, IN 47405-7000, USA (R. DiMarchi). Correspondence to: M. H. Tschp tschoemh@ ucmail.uc.edu
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Key points
In addition to its well-known effects on glycemia, increased glucagon signaling directly regulates triglyceride, free fatty acid, apolipoprotein and bile acid metabolism Glucagon action can be inhibited via receptor desensitization by excess dietary fat intake Energy expenditure and thermogenesis are increased by glucagon agonism Glucagon administration stimulates satiety and decreases food intake Glucagon action, in combination with incretins such as glucagon-like peptide 1, may be a crucial tool in the treatment of the metabolic syndrome
Glucose production Fatty acids Metabolites Paracrine and endocrine input Neural inputs Cell Glucagon secretion Lipolysis Fatty acid oxidation Ketogenesis Satiation Food intake Thermogenesis Energy expenditure Bile acid synthesis
Figure 1 | Physiological stimuli and outcomes of glucagon secretion.
channels.21 As glucose levels rise, secretion of glucagon is inhibited through the elevation of cytosolic ATP, blockade of KATP channels and termination of the Na+-induced and Ca2+-induced action potentials. This process inhibits Ca2+ influx and ends glucagon secretion. Although the cellular signals regulating glucagon secretion are fairly well-established, the role of glucose, whether directly, or indirectly via -cell activation, is still a matter of debate. Studies in rats suggest that mediatory, paracrine signaling from the cell is essential for the inhibition of glucagon secretion by elevated glucose levels.22,23 Investigations in mice and humans, however, suggest that glucose directly inhibits glucagon secretion at concentrations which are too low to stimulate insulin secretion.21 Furthermore, this inhibition has been demonstrated in vitro in both isolated cells and in intact pancreatic islets.24 However, a study in individuals with type 1 diabetes mellitus suggests that insulin is the primary signal that inhibits glucagon secretion in humans.25 In addition to glucose, several other physiological parameters are known regulators of glucagon secretion, including GLP-1,26 GLP-2,27 fatty acids,28 the autonomic nervous system29 and circulating amino acids.30
Signaling
Glucagon receptor The effects of glucagon are mediated by the binding of glucagon to its membrane-bound receptora seventransmembrane protein and a member of the class II guanine nucleotide-binding protein (G protein) coupled receptor superfamily.31 Glucagon receptors (encoded by the GCGR gene) are expressed abundantly in the liver and kidney and to a lesser extent in heart, adipocytes, lymphoblasts, spleen, endocrine pancreas, brain, retina,
adrenal gland and the gastrointestinal tract.32 Previous studies suggest that glucagon receptors are also expressed in islet cells.33 In the liver, glucagon receptors are located mainly in hepatocytes, but can also be found on the surface of Kupffer cells.34 Interestingly, glucagon receptors in the pancreas are predominately located on cells, which, together with the data described above, suggests a bidirectional feedback mechanism.35 Further evidence supporting this hypothesis is the fact that glucagon, at physiological concentrations, was found to stimulate insulin release via these receptors.36 Much knowledge concerning glucagon action has been gained from genetically altered animal models. Glucose homeostasis and pancreatic function has been elaborately studied in mice with a null mutation of the glucagon receptor (Gcgr/). As expected, blood glucose levels are markedly lower and glucose tolerance is improved in Gcgr/ mice compared with wild-type controls.37,38 The improvement in glucose tolerance is not owing to an increase in insulin secretion, but rather to an improvement in insulin sensitivity, as demonstrated by hyperinsulinemic euglycemic clamp studies.39 When fed a high-fat diet (HFD), Gcgr/ mice do not display dietinduced insulin resistance, which is potentially a result of enhanced insulin sensitivity in these mice. However, after prolonged fasting, Gcgr/ mice experience severe hypoglycemia,37 which illustrates the essential role of glucagon for the maintenance of blood glucose levels.38 Gcgr/ mice were observed to display an enlargement of the pancreas primarily attributed to -cell hyperplasia, which indicates glucagon receptor signaling is essential for normal endocrine-cell proliferation.37 Further indication of glucagons role in endocrinecell function was demonstrated in transgenic mice that overexpress the glucagon receptor specifically on pancreatic cells. These mice display enhanced glucagonstimulated and glucose-stimulated insulin secretion, as well as improved glucose tolerance.40 A reduction in both fasting hyperglycemia and impaired glucose tolerance is observed when these transgenic mice are exposed to a HFD,40 suggesting that enhanced glucagon signaling on cells improves the function of these cells. The studies that show improvements in glucose homeostasis in mice overexpressing the glucagon receptor on cells are in contrast to studies in mice treated with streptozotocin, which showed that ablation of glucagon signaling has protective effects. Streptozotocin treatment destroys cells, impairs insulin secretion and induces hyperglycemia in wild-type mice; however, when streptozotocin is administered to Gcgr/ mice, euglycemia is maintained even after HFD exposure. 38 This finding indicates that lack of glucagon receptor signaling results in resistance to streptozotocin-mediated -cell destruction and hyperglycemia in vivo. The mechanism behind the streptozotocin resistance in Gcgr/ mice remains unknown; however, given that other reports demonstrate that increased concentration of circulating GLP-1 results in resistance to streptozotocin-induced -cell destruction, the increased levels of circulating GLP-1 determined in Gcgr/ mice have been speculated to be an important
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factor.41,42 These data illustrate that both enhanced, as well as lack of, glucagon receptor signaling have positive effects on glucose homeostasis and pancreatic function and, therefore, further studies are needed to understand the cause of these results.
Signaling pathway and targets Binding of glucagon to its receptor elicits activation of a heterotrimeric, stimulatory G protein (Gs) in a process dependent on GTP and magnesium.43 In liver cells, the glucagon receptor and Gs proteins have been postulated to be linked in a multimeric configuration, which disengages following activation.44 The activated Gs protein undergoes a conformational change, upon which the GTP-bound subunit Gs is released from the G-protein complex that comprises two additional subunits, Gs and Gs. This activation leads to interaction and subsequent stimulation of adenylate cyclase, elevated cAMP levels and enhanced intracellular signaling via Rap guanine nucleotide exchange factor 3 (RAPGEF3; also known as EPAC1), cAMP response element-binding protein (CREB)-regulated transcription coactivator 2 (CRTC2; also known as TORC2) and protein kinase A (PKA). The activation of PKA results in the phosphorylation and nuclear localization of CREB.45,46 Once phosphorylated in the liver, CREB binds to the cAMP response element of target genes, resulting in the recruitment of coactivators, such as hepatic nuclear factor 4 (HNF-4),47 peroxisome proliferator-activated receptor coactivator 1- (PGC-1)48 and the glucocorticoid receptor.47 In addition to this well-described pathway, glucagon has also been implicated in signaling via 5'-AMP-activated protein kinase (AMPK), 49 mitogen-activated protein kinase (MAPK) and in a c-Jun N-terminal kinase (JNK)-dependent manner.50
Beyond glucose homeostasis

The role of glucagon in glucose homeostasis has been well-studied and previously reviewed elsewhere.51 However, this pancreatic peptide has additional metabolic effects of notable importance. Glucagon agonism has been shown to regulate lipid metabolism and energy expenditure, as well as reduce histamine-induced cardiac injury during reperfusion.52
Glucagon and lipid metabolism

Plasma lipid homeostasis Studies in the early 1960s investigated glucagons action independent of glucose homeostasis. These studies described a lipid-mobilizing effect for glucagon in a range of species.5357 The regulation of plasma lipids by glucagon was first highlighted in studies which suggested that glucagon acts to decrease plasma cholesterol, total esterified fatty acids and arachidonic acid levels.53,58 As epinephrine, insulin and exogenous glucose all failed to lower levels of plasma cholesterol, the investigators concluded that these effects were not an indirect result of altered carbohydrate metabolism.59 In both canines and humans, the researchers observed a total decrease in levels of plasma cholesterol, as well as plasma total lipid
concentrations, within 30 min of intravenous glucagon administration. Intriguingly, this depression was not found for whole-blood total lipid levels, suggesting a repartitioning of lipids from serum to platelet-rich fractions. Incubation of the blood at 37 C led to partial or full restoration of plasma cholesterol and total lipid levels, further supporting the hypothesis of repartitioning.58 Reports from other groups provided additional support for a role of glucagon in the regulation of plasma lipids.55,56 In the context of triglyceride metabolism, several reports indicated that triglyceride production is decreased in perfused livers treated with glucagon.6062 Although these studies suggest hepatic lipoprotein metabolism as the site of such regulation, the molecular mechanism by which glucagon achieved this result remained elusive. Building upon these observations, the role of glucagon on lipid suppression was investigated in a rat model of hyperlipidemia.63 This study demonstrated that glucagon significantly decreased levels of serum triglycerides, cholesterol, and VLDL cholesterol in hyperlipidemic rats, as well as in control eulipidemic rats. Furthermore, a decrease in synthesis of hepatic lipoprotein apoproteins was observed,63 complementing earlier studies that described depression of triglyceride synthesis in livers treated with glucagon. A caveat of this study is that it involved supraphysiological concentrations of glucagon administered over a 4-day period. Thus, the relevance of acute signaling by endogenous glucagon remained uncertain. Guettet et al.6466 continued the investigation of chronic effects of glucagon signaling in the Wistar rat. Their initial studies described a decrease in concentrations of plasma cholesterol, phospholipids and triglycerides. Interestingly, these reductions were not seen in the liver or in erythrocytes of these rats, which suggested another target tissue as the site of action. Continued investigation of cholesterol turnover revealed increased urinary secretion of cholesterol, as well as elevated transformation to bile acids. Of special importance was the reported observation that the chronic treatment of Wistar rats with twice-daily glucagon (20 g per day) for 3 weeks reduced blood glucose and insulin concentrations. This report thus indicated that chronic glucagon pharmacology was neither transient nor detrimental to glucose homeostasis.64 Further studies by Guettet et al.65,66 evaluated the effect of chronic glucagon treatment on lipoprotein composition in fed and fasted rats, as well as rats fed a cholesterol-rich diet. Cholesterol, phospholipids and total protein levels were proportionally decreased in chylomicrons, VLDL cholesterol, LDL cholesterol and HDL cholesterol, which suggested a reduction in the number of lipoprotein particles. Triglycerides were reported to be decreased only in the chylomicron and VLDL fractions. Evaluation of the lipoproteins indicated a decline in levels of apolipoprotein E (ApoE) and an increase in ApoB.65 Collectively, these findings suggested glucagon-stimulated targeting of the ApoE-rich lipoproteins to limit the accumulation that typically occurs with high-cholesterol feeding.66 A followup study showed that glucagon treatment had no effect on secretion rates of triglyceride-rich particles in either

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fed or fasted conditions. Conversely, glucagon treatment accelerated the rate of triglyceride removal (fractional catabolic rate constant) from the plasma compartment.67 Taken together, the work of Guettet et al.6466 revealed that increased glucagon signaling may directly regulate lipid catabolism. Rudling and Angelin68 extended these findings by the direct study of glucagons effects on LDL receptors (LDLRs). The investigators found that glucagon administration to rats resulted in a dose-dependent increase in binding of LDL cholesterol to the receptor, with no apparent effect on receptor mRNA expression. Concomitant with this increased binding was a decrease in levels of cholesterol, ApoB, and ApoE. Furthermore, these effects were completely antagonized by insulin administration. Rudling and Angelin hypothesized a mechanism such as post-translational receptor modification, whereby glucagon increases LDLR activity without affecting expression levels.68 The findings in rodents were complemented by studies in dairy cattle which showed that subcutaneous injections of glucagon led to variable decreases, some of sizable magnitude, in circulating plasma concentrations of VLDL-triglycerides, HDL1phospholipids and HDL2-free cholesterol.69 Improved glucose status with decreased nonesterified fatty acids and -hydoxybutyrate was also reported.70 Genetic manipulation of the glucagon receptor has led to inconclusive results regarding glucagons role in lipid metabolism. Conarello et al.38 found that Gcgr/ mice are resistant to HFD-induced liver steatosis; however, Longuet et al.71 showed that Gcgr/ mice have enhanced susceptibility to hepatic steatosis. Interestingly, these studies used genetically modified null mice generated in an identical manner and of the same background (C57B6). Furthermore, the effect was observed in mice fed a similar diet (45% fat). The length of diet differed between the two studies (8 versus 12 weeks); however, mice fed a HFD for the longer duration did not exhibit steatosis. Taken together, the contribution of glucagon receptor agonism to liver steatosis has yet to be conclusively described. Longuet et al.71 also demonstrated the importance of glucagon signaling in the adaptive response to fasting. Following exposure to a prolonged fasting period, Gcgr/ mice showed increased levels of plasma triglycerides and free fatty acids.71 During either glucagon treatment or a prolonged fast, wild-type mice had an increased expression of genes involved in fatty acid oxidation, including Decr2, carnitine O-palmitoyltransferase (Cpt) 1a, Cpt2 and Acadm. These changes in expression were correlated with an increased capacity of the liver to oxidize free fatty acids. By contrast, this increased gene expression was not observed in Gcgr/ mice, which displayed a reduced oxidation of free fatty acids in both the fed and the fasted state. These data highlight the essential physiological role of glucagon-receptor signaling in the regulation of gene expression during prolonged fasting.
Glucagon-mediated lipolysis and ketogenesis Glucagon-mediated regulation of lipid metabolism is not limited to plasma triglycerides and cholesterol. Glucagon
treatments as low as 108 mol/l have been implicated in promoting lipolysis in white adipose tissue.72 This lipolytic effect appears to be independent of sympathetic nervous system innervation,73 as denervation of white adipose tissue does not block glucagon-induced glycerol release, whereas it decreases the release of nonesterified fatty acids. The latter, however, is reported to be the result of re-esterification,73 not a blockade of glucagons lipolytic effects. Mechanistically, glucagon is known to stimulate the activity of hormone-sensitive lipase (HSL) in adipocytes, resulting in an increase of nonesterified fatty acids in the circulation.74 These fatty acids, usually bound to albumin, are transported to the heart, skeletal muscle, kidney and liver, where they are catabolyzed or, in the case of the liver, alternatively converted to ketone bodies. Ketone bodies provide up to two-thirds of the energy for the brain during times of glucose deficiency, thus sparing glucose utilization and reducing proteolysis.75 Supporting a role for glucagon in the regulation of ketogenesis, suppression of glucagon secretion via somatostatin prevented the development of ketoacidosis in patients with type 1 diabetes mellitus.76 Pegorier et al.77 elucidated the regulation of fatty acid oxidation and ketogenesis by glucagon in rabbit hepatocytes. The researchers reported increased ketone-body production in the presence of either glucagon or dibutyryl cAMP. Oxidation of exogenous oleate was increased by both glucagon and cAMP, effects which were reduced in the presence of insulin. Furthermore, exposure to glucagon or cAMP completely inhibited lipogenesis and decreased malonylCoA concentration and stimulated fatty acid oxidation. These effects were suggested to be driven by a fall in the sensitivity of CPT1 to malonyl-CoA, releasing the inhibition of this rate-limiting enzyme in the transport of fatty acids across the mitochondrial membranes.77,78 The relevance of these studies was confirmed by the observation that glucagon directly controls ketone-body production in primary human hepatocytes.79 Specifically, glucagon increased ketone-body production and fatty acid oxidation, whereas it decreased fatty acid esterification, similar to the effects of cAMP.79
Lipid-induced glucagon resistance In addition to glucagons regulation of lipid metabolism, previous studies suggest that lipids may also regulate glucagon signaling. Studies by Charbonneau et al. 80 show that rats fed a HFD display hepatic steatosis that is associated with glucagon resistance. Specifically, exercise training in rats was used to increase plasma glucagon levels and improve fatty liver. In trained rats, a negative correlation between liver adiposity and density of the glucagon receptors in the plasma membrane was observed. Additional studies described a reduction in both hepatic glucagon-receptor density and Gs protein content at the plasma membrane.81,82 The mechanism for this decrease in receptor density was explored in a study which proposed that HFD promoted glucagon receptor proteolysis.81 A reduction in glucagon receptors at the plasma membrane was notable, accompanied by a marked increase in the number of endosomal and

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lysosomal compartments. These effects were correlated with an increase of protein kinase C (PKC) on the plasma membrane, which is known to phosphorylate G-protein-related kinases. Such action, in turn, is known to inhibit receptor internalization which leads to receptor desensitization.83 The same association seems to be conserved in humans, as glucagon resistance is associated with pathological hyperlipidemia in humans.84 tissue by glucagon98 has become even more relevant, as novel findings suggest a renewed importance for brown adipose tissue in human energy metabolism.99 The mechanism by which glucagon-induced thermogenesis is regulated is probably complex and may involve the sympathetic nervous system. Supporting this hypothesis, glucagon-induced increases in oxygen consumption, blood flow and thermogenesis in brown adipose tissue were blocked by the nonselective adrenergic receptor blocker, propranolol,100,101 whereas denervation of brown adipose tissue blunted the thermogenic action of glucagon.98 Furthermore, although glucagon injection in ducklings was associated with an increase in norepinephrine, blockade of catecholamines via guanethidine sympathectomy (the interruption of the transmission of sympathetic nerve impulses by a chemical agent that blocks the secretion of epinephrine and norepinephrine at postganglionic nerve endings) decreased metabolic rate compared to nonsympathectomized birds treated with glucagon or saline.102 Glucagon might also have a direct effect on thermogenesis, as glucagon incubation of brown adipocytes of rat and mice, but not those of Syrian hamsters, markedly increased oxygen consumption.100 However, the concentration of glucagon required for such in vitro effects has been speculated to be supraphysiological.100 The role of glucagon in mediating cold-induced thermogenic responses has been further questioned by a report that denervated interscapular brown adipose tissue decreased the number of glucagon receptors as compared to the contralateral adipose tissue pad that served as an internal (within animal) control.103 Given that cold exposure is a well-known stimulator of the sympathetic nervous system in brown adipose tissue,104,105 a decrease in glucagon receptor protein or gene expression mitigates the role for glucagon in cold-triggered brown adipose tissue thermogenesis via innervation of the sympathetic nervous system. Taken together, these data suggest a pivotal role for the sympathetic nervous system in glucagon-induced thermogenesis and suggest a thermogenic basis for anti-obesity effects of glucagon.
Glucagon and bile acid metabolism Although the canonical role of bile acids is associated with the absorption of dietary lipids and cholesterol homeostasis, new studies suggest that these acids have additional signaling roles in glucose homeostasis. This emerging function was highlighted in a study by Song and Chiang,85 who investigated cholesterol 7-alphamonooxygenase (CYP7A1) as a possible target of glucagon signaling. Their study demonstrated that glucagon, as well as cAMP, represses CYP7A1 expression in human primary hepatocytes. CYP7A1 is the rate-limiting enzyme in bile acid synthesis, and its expression is tightly regulated as a means to control flux through the pathway.86 Song and Chiang 86 further showed that glucagons regulation of CYP7A1 expression is mediated via PKA signaling to HNF-4. Given that no direct measure of bile acid synthesis or secretion was available, the direct result on bile acid metabolism remains unknown. The findings of their earlier study,85 however, were complemented by work from Hylemon et al.,87 who showed similar decreases in CYP7A1 expression in response to glucagon or cAMP exposure in rat hepatocytes. These in vitro studies support initial observations that suggest a role for glucagon in the regulation of bile acid metabolism and provide a plausible mechanism for the glucagon-induced decrease in plasma cholesterol levels. Glucagon and energy expenditure In addition to regulating glucose and lipid metabolism, glucagon participates in the control of energy expenditure and thermogenesis. Early studies by Davidson et al.88,89 showed that pharmacological infusion of glucagon increased oxygen consumption in rats. This effect was mirrored by a report in human study participants, in whom infusion of a pharmacological dose of glucagon increased resting energy expenditure during acute insulin deficiency produced by the additional infusion of somatostatin.90 Moreover, a physiological dose of glucagon increased energy expenditure in humans during euinsulinemia, and hyperinsulinemia blunted glucagons thermogenic activity.91 Studies conducted in vitro showed that hyperglucagonemia may increase energy expenditure via stimulation of oxygen consumption and heat production in brown adipose tissue.92,93 Studies in rats confirmed that glucagon administration increased whole-body oxygen consumption, core body temperature, blood flow,94 as well as temperature and mass of brown adipose tissue.95,96 Furthermore, cold exposure was found to increase plasma glucagon levels,97 which implicates glucagon in nonshivering thermogenesis. The stimulation of thermogenic activity in brown adipose
Glucagon and regulation of food intake Early studies indicated that glucagon administration diminishes the sense of hunger and decreases food intake in humans106108 and rats.109 Additional studies in rats confirmed that glucagon specifically decreases meal size owing to increased satiation rather than illness or aversion. Intravenous infusions similarly produced a dose-related decrease in eating.109113 Evidence for an endogenous role of glucagon in satiation was provided by demonstrations that glucagon concentration increases physiologically during meals, in many situations,114116 and that antagonism of glucagon by preprandial administration of glucagon antibodies increases the amount of food consumed in one meal (meal size).117,118 Finally, intravenous infusion of a physiological dose of glucagon during meals was shown to reduce meal size in humans.119 A study in rats with both hepatic portal vein and vena cava infusion catheters indicated that both exogenous
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Glucagon secretion
Glugagon-specific effects Glucose production Thermogenesis
Cell Obesity GLP-1 secretion Pharmacological synergism Food intake Body weight
L Cell
GLP-1-specific effects Insulin secretion Gastric emptying
Figure 2 | Individual and synergystic effects of glucagon-like peptide 1 and glucagon in obesity and pharmacotherapy.
and endogenous glucagon act in the liver to limit meal size. 120 Furthermore, as for several other peripheral signals that control eating, vagal afferents relay the signal to the brain.121 In the case of glucagon, the hepatic branch of the abdominal vagus is critical,122 consistent with the hepatic site of action. Although the inhibitory effect of glucagon on feeding probably arises, at least in part, from a hepatic metabolic action of the hormone, novel studies in sheep suggest that glucagon acts directly in the central nervous system to inhibit food intake.123 Genetically modified animal models also demonstrate that glucagon signaling is involved in regulating food intake and body composition. Gcgr/ mice display a decreased adipose tissue mass and an increased lean mass despite similar food intake and body weight compared to their wild-type littermates.37 Furthermore, Gcgr/ mice on a HFD are resistant to diet-induced obesity,38 which can be primarily attributed to a lower food intake compared with controls.
Glucagon-based pharmacological therapy

Glucagon-based drug therapy has largely been restricted to acute emergency use to treat episodes of hypoglycemia in patients with type 1 diabetes mellitus and as an esophageal muscle relaxant to prepare patients for radiological procedures. Nevertheless, glucagons hypolipidemic, energy expenditure-stimulatory and satiating effects make it an attractive pharmaceutical agent for the treatment of dyslipidemia and obesity. Such chronic uses, however, must be considered carefully in light of glucagons ability to accelerate the development of glucose intolerance and insulin resistance. Studies in cattle suggest that glucagon may be an effective treatment for fatty liver and dyslipidemia124 without deleterious effects on glucose homestasis, as both single and multiple injections of 5 mg glucagon over 14 days consistently improve the carbohydrate status of dairy cows and decreased concentrations of plasma nonesterified fatty acids.70 Of course, these actions must be verified in nonruminant species.
Glucagon and GLP-1 coagonism Two prominent studies published in July 2009, have put forth the hypothesis that glucagon agonism may be beneficial in the pharmacological treatment of obesity and, possibly, obesity-associated glucose intolerance.10,11 Interestingly, both studies combined agonists of two proglucagon-derived peptides to elicit these effects in mouse models. Day et al.10 combined the antidiabetic properties of GLP-1 receptor (GLP-1R) agonism with the hypolipidemic properties of glucagon receptor agonism to create a dual agonist, which they conjugated to a polyethylene glycol polymer to prolong pharmacokinetic action. The researchers then compared 1-week administration of this dual agonist with an equimolar amount of a structurally comparable, but selective, GLP-1R agonist in diet-induced obese mice. Both peptides significantly decreased food intake, body weight and adipose tissue mass when compared with saline-injected controls. Significant decreases in blood glucose and insulin concentrations and a profound increase in glucose tolerance were also reported. Intriguingly, the peptide with balanced glucagon and GLP-1 coagonism exhibited significantly greater efficacy, as measured by changes in body weight, adipose tissue mass and glucose homeostasis, compared with the GLP-1R agonist monotherapy.10 Studies conducted over a 1-month period revealed similar effects on body weight, adiposity and blood glucose levels, although cumulative total food intake in this study was not impressively altered. Interestingly, energy expenditure in the 1-month study was significantly increased in the animals treated with the coagonist, with a trend for a decreased respiratory quotient. Lipid metabolism was markedly improved with decreases in levels of total cholesterol, LDL cholesterol, HDL cholesterol and trigylcerides, as well as reversal of liver steatosis and activation of HSL in white adipose tissue. Similar to the study by Day et al.,10 Pocai et al.11 tested a dual agonist of GLP-1R and the glucagon receptor in diet-induced obese mice. The investigators injected the coagonist daily for 2 weeks and compared these mice with those treated with either a GLP-1R agonist with the same degree of GLP-1R agonism as the coagonist or vehicle. Significant enhancements in measures of body weight and glucose tolerance were reported in mice treated with the coagonist. Results of animals treated only with a GLP-1R agonist were intermediate between those of mice treated with either the coagonist peptide or vehicle, for all measured parameters.11 Also, similar to the study by Day and colleagues,10 Pocai et al.11 showed decreases in liver steatosis, total cholesterol and triglyceride levels in the coagonist-treated animals, with the single-agonisttreated mice again displaying intermediate values in comparison with the other two groups. Together, these two studies exemplify a novel and potentially important new direction for therapy of obesity and metabolic disease (Figure 2). An advantage of the described approach is the use of single-agent peptides that represent two full hormone-receptor agonists that elicit additive therapeutic effects, while at the same time adverse effects can be minimized. These studies
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come with the caveat that the glucagon agonism caused by the coagonist peptide far exceeds the actions elicited by endogenous glucagon. Furthermore, these studies did not compare coagonism with glucagon monotherapy; any effect observed is, therefore, a possible, and probable, interaction between the two receptor agonists. Taken together, interpretation of glucagon physiology on the basis of these findings must be tempered. Possibly of greater importance, these studies challenge our deeply rooted understanding of the full physiology of a pancreatic hormone that partners with insulin in the management of glucose and lipid homeostasis and body weight. The fact that excessive glucagon receptor agonism leads to glucose intolerance and insulin dysregulation is wellestablished. Observations of elevated glucagon concentration in insulin-resistant individuals, as well as animal models of insulin resistance, have supported this view. The studies of Day et al.10 and Pocai et al.11 build upon a foundation of less evident metabolic actions of glucagon reported in this Review. They provide a rationale to reconsider whether glucagon administered at modest concentrations and frequency of exposure constructively contributes to proper glucose and lipid metabolism directly by acting at specific target tissues and indirectly by modulating body weight. The prospect of using glucagon in combination with other agents to address obesity-associated glucose intolerance and insulin resistance, as well as dyslipidemia, is something worthy of scholarly consideration. diabetogenic properties that render it poorly suited for the treatment of obesity and metabolic diseases. Development of stable, soluble glucagon agonists of varying pharmacokinetics and of glucagon-like coagonists in conjunction with GLP-1, which are suitable for pharmacological study, however, provide an unprecedented opportunity to explore the full physiological character and pharmacological potential of this fascinating hormone. When viewed in combination with historical observations, the first reports resulting from studies of these agonistic peptides suggest that glucagon may potently regulate glucose metabolism and lipid homeostasis, as well as energy balance, body adipose tissue mass and food intake. Clearly, excessive and unopposed glucagon action is catabolic and must be avoided if chronic use is considered. Nevertheless, collectively, novel observations challenge us to thoroughly investigate and broadly take into considera tion the physiology and therapeutic potential of this long-known counter-regulatory hormone to insulin.
Review criteria
Articles referenced in this Review were selected on the basis of relevance to the less appreciated effects of glucagon on lipid metabolism, food intake and energy expenditure. Articles were searched for in the PubMed database. Search terms used included glucagon, glucagon AND food intake, glucagon AND cholesterol, glucagon AND triglycerides, glucagon AND energy expenditure. Full-text articles published in English were chosen, and referenced papers were utilized for additional leads. Articles from all years were used; however, whenever possible, the most recent references were used.
Conclusions
The limited data available on the pharmacology of chronically administered glucagon is the result of a focus on acute glucagon action and its disadvantageous
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ENDOCRINOLOGY CONCEPTS FOR MEDICAL STUDENTS

ADV PHYSIOL EDUC 25: 213224, 2001.

1043 - 4046 / 01 $5.00 COPYRIGHT 2001 THE AMERICAN PHYSIOLOGICAL SOCIETY

VOLUME 25 : NUMBER 4 ADVANCES IN PHYSIOLOGY EDUCATION DECEMBER 2001

Coordination of processes associated with reproduction and lactation

FIG. 1. Simple negative feedback.

VOLUME 25 : NUMBER 4 ADVANCES IN PHYSIOLOGY EDUCATION DECEMBER 2001

VOLUME 25 : NUMBER 4 ADVANCES IN PHYSIOLOGY EDUCATION DECEMBER 2001

VOLUME 25 : NUMBER 4 ADVANCES IN PHYSIOLOGY EDUCATION DECEMBER 2001

VOLUME 25 : NUMBER 4 ADVANCES IN PHYSIOLOGY EDUCATION DECEMBER 2001

FIG. 6. Positive feedback of oxytocin secretion during childbirth.

receptors. That concentration, in turn, is determined by

VOLUME 25 : NUMBER 4 ADVANCES IN PHYSIOLOGY EDUCATION DECEMBER 2001

VOLUME 25 : NUMBER 4 ADVANCES IN PHYSIOLOGY EDUCATION DECEMBER 2001

VOLUME 25 : NUMBER 4 ADVANCES IN PHYSIOLOGY EDUCATION DECEMBER 2001

VOLUME 25 : NUMBER 4 ADVANCES IN PHYSIOLOGY EDUCATION DECEMBER 2001

The duration of secretion

VOLUME 25 : NUMBER 4 ADVANCES IN PHYSIOLOGY EDUCATION DECEMBER 2001

2) whether the response results from

VOLUME 25 : NUMBER 4 ADVANCES IN PHYSIOLOGY EDUCATION DECEMBER 2001

will also contribute to their understanding of physiology as a whole.

VOLUME 25 : NUMBER 4 ADVANCES IN PHYSIOLOGY EDUCATION DECEMBER 2001

Available online at www.sciencedirect.com

Mechanisms of nongenomic actions of thyroid hormone

, Jack L. Leonard c, Faith B. Davis

Available online 5 October 2007

Corresponding author. E-mail address: pdavis@ordwayresearch.org (P.J. Davis).

P.J. Davis et al. / Frontiers in Neuroendocrinology 29 (2008) 211218

P.J. Davis et al. / Frontiers in Neuroendocrinology 29 (2008) 211218

P.J. Davis et al. / Frontiers in Neuroendocrinology 29 (2008) 211218

P.J. Davis et al. / Frontiers in Neuroendocrinology 29 (2008) 211218

P.J. Davis et al. / Frontiers in Neuroendocrinology 29 (2008) 211218

Endocrinology, March 2009, 150(3):1084 1090

Endocrinology, March 2009, 150(3):1084 1090

IApical membrane PDS

Ca2+ Hydrolysis MIT DIT

Bizhanova and Kopp

Endocrinology, March 2009, 150(3):1084 1090

Regulation of NIS protein expression

Congenital iodide transport defect (ITD)

Endocrinology, March 2009, 150(3):1084 1090

Questions concerning the role of pendrin as an apical iodide transporter

Role of pendrin in the thyroid

Bizhanova and Kopp

Endocrinology, March 2009, 150(3):1084 1090

Regulation of pendrin expression

Endocrinology, March 2009, 150(3):1084 1090

46. 47. 48.

55. 56. 57.

Bizhanova and Kopp

Endocrinology, March 2009, 150(3):1084 1090

62. 63. 64. 65.

GLUCOCORTICOIDS AND MOOD

Glucocorticoid Signaling in the Cell

First Department of Pediatrics, Athens University Medical School, Athens, Greece

Annals of the New York Academy of Sciences

Chrousos & Kino: Glucocorticoids and Pathophysiology

Modied from Refs. 22 & 61.

Annals of the New York Academy of Sciences

Chrousos & Kino: Glucocorticoids and Pathophysiology

Annals of the New York Academy of Sciences

Chrousos & Kino: Glucocorticoids and Pathophysiology