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Human Biochemistry:
NOTES & OBJECTIVES
Fall 2005
last updated 1/4/2007
Christopher B. Kolar
cbkolar@wisc.edu
This study guide has been created in the course of my studies at the University of Wisconsin
School of Medicine and Public Health. It is intended as an exam review of the required learning
objectives. It references a variety of course materials, including lecture, Power Point, assigned
readings, and sometimes outside sources. While I have attempted to make it as thorough, specific,
and accurate as possible, I cannot guarantee this, so use it at your own risk. If you have any
questions or comments, or have found an error within the text, please feel free to contact me.
COLOR KEY:
red:
diseases
blue:
medications
orange:
enzymes and
compounds
pink:
microorganisms
FORMAT KEY:
margins:
1
tab stops:
0.25
font:
Times New Roman
size:
10
approximate pK
3 (C-terminal)
4
6
8
8 (N-terminal)
9 (N-terminal)
10
12
pH pK' log
[HB ]
[B ]
; pH pK' log
[B - ]
[HB]
- modifications
- phosphorylation:
attachment of a phosphoryl group to a hydroxyl group, extruding water
- N-glycosylation:
attachment of a sugar to an amine (commonly with Asn)
- O-glycosylation:
attachment of a sugar to an oxygen (commonly on Ser, Thr, modified residues)
- hydroxylation:
attachment of a hydroxyl group to the R group (commonly on Pro, Lys)
- carboxylation:
attachment of a carboxyl group to the R group (commonly on Glu)
- nomenclature
- N-terminus to C-terminus
- substitute yl for ine, except for aspartyl, asparaginyl, glutamyl, glutaminyl, cysteinyl, tryptophanyl
- peptide bond formation: carboxyl group + primary amine group peptide bond + H2O
- protein structure
- primary structure: sequence of amino acids
- secondary structure: common organizations of structure
- -helix
- 3.6 amino acids per helical turn, with each AA able to participate in up to two H-bonds
- H-bonds: between =O, H-N- within helix, connecting i to i+4
- little Pro due to incompatibility with helix angle; little Gly due to being too free to form tight conformations
- -pleated sheet
- stretched polypeptide chains running either parallel or antiparallel, depending on chain orientation
- H-bonds: between carbonyl and amide hydrogens of adjacent chains
- turn
- allows protein backbone to make abrupt turns
- abundant Pro, Gly, due to stearic considerations
- tertiary structure: three-dimensional structure
- quaternary structure: interaction of tertiary domains
- classes of proteins
- enzymes:
accelerate the attainment of equilibrium
- structural:
form biological structures
- transport:
carry biochemically important substances
- defense:
protect the body from foreign invaders
2. Enzyme Kinetics
- general features of catalysts
- enzymes do not alter the final equilibrium ratio of substrates and products
- enzymes act by lowering the activation energy of a reaction
Vmax [S]
K m [S]
- Michaelis-Menten:
1 Km
v Vmax
1
1
[S] Vmax
- inhibition
- competitive inhibition: binds only to free enzyme (typically at active site)
- Vmax:
no effect
- Km:
raises apparent Km
- saturation plot:
shifts Km to the right, graph approaches Vmax more slowly
- reciprocal plot:
affects slope: rotates graph counterclockwise on the y-intercept
- equation:
factor (1 + [I]/Ki) added to slope term
- non-competitive inhibition: binds to E or ES complex (typically at distant binding site)
- Vmax:
decreases
- Km:
no effect
- saturation plot:
flattens graph, but doesnt affect rate of achieving Vmax
- reciprocal plot:
affects slope and y-intercept: rotates graph counterclockwise on the x-intercept
- equation:
factor (1 + [I]/Ki) added to slope AND intercept terms
- uncompetitive inhibition: binds only to ES complex (typically at distant binding site); self titrating
- Vmax:
decreases
- Km:
decreases apparent Km
- saturation plot:
flattens graph, makes it achieve Vmax much more quickly
- reciprocal plot:
affects y-intercept: shifts graph leftward on a parallel line
- equation:
factor (1 + [I]/Ki) added to intercept terms
- irreversible inhibition: removes enzyme from the equation
- Vmax:
decreases stoichiometrically
- Km:
no effect
- kinetics:
- at [I] = KI, looks similar to non-competitive inhibition
- however, at [I] = 2KI, gives 100% inhibition/deactivation, compared to 66.7%
5. Thermodynamics
- general considerations
- thermodynamics indicates the favorability of a reaction given the conditions, NOT the speed or pathway
- work can only be done if a system is not at equilibrium
- equilibrium constants
- Keq=1: reaction is at equilibrium
- Keq>1: products are favored
- Keq<1: reactants are favored
- thermodynamics
- free energy:
G G' o RT ln(Q)
[products]
[reactants]
- high energy bonds: those that have a free energy of -5 kcal/mol or lower
- bond types
- pyrophosphate:
- phosphate ester:
- carboxyl phosphate:
- enol phosphate:
- guanido phosphate:
- ester:
- thioester:
- -keto acid:
- glycoside/acetal:
- amide:
- hemiacetal phosphate:
PO3-2-OPO32-R
PO3-2-OCH2
PO3-2-OOC
PO3-2-OC(=CHR)-COOH
PO3-2-NH-C(=NH)-NH-R
R-O-C(=O)-R
R-S-C(=O)-R
R-C(=O)-CH2-COOR-O-CH-R-O (O connected to CH)
NH2-C(=O)-R
PO3-2-O-CH-O
high
low
high
high
high
low
high
high
low
low
~high
- protein misfolding
- mutation: can cause reduced or non-function (e.g. CFTR transmembrane protein in cystic fibrosis)
- multiple low-energy stable states: prions
- consequences
- often, only time is necessary for folding to the desired state
- sometimes, protein misfolding can cause pathology, as in aggregation of prion plaques
- in such cases, the misfolded protein can lead to formation of holes in brain tissue, leading to CNS problems
- prion diseases
- kuru
- Creutzfeld-Jakob diseases
- scrapie (sheep and goats)
- mad cow disease (cows)
- chronic wasting disease (deer)
- aggregations impart sickle shape to cells, damaging them and affecting their ability to move in bloodstream
- this disease leads to symptoms similar to anemia, is often fatal in childhood
8. Motor Proteins
- muscle contraction
- structure
- actin (thin filament): polar array of globular proteins arranged in a filament
- myosin (thick filament): intertwined -helix coiled coil with aligned head groups
- muscle myofiber structure: Z I M H A
- Z line: attachment point of actin thin filaments
- M line: attachment point of myosin thick filaments
- I: exclusively actin
- H: exclusively myosin
- M: intermixed actin and myosin
- mechanism
- release:
ATP binds, causing myosin to release from actin
- extension:
ATP ADP + Pi, causing the head group to extend slightly
- binding:
Pi is released, causing the extended head group to bind
- power stroke:
ADP is released, returning myosin to return to conformation (now moved slightly)
- regulation
- tropomyosin sits in the binding pocket, disallowing myosin binding to actin
- upon CNS stimulation, Ca2+ stores in the cell are released
- Ca2+ binds troponin C, which leads to a conformational change that moves tropomyosin
- with sufficient ATP, muscle is able to begin power strokes
- P-loop NTPases
- molecular motors using an NTP hydrolysis reaction to drive motor function typically use P-loops
- P-loop: phosphate loop motif
- binds the triphosphate moiety of the NTP
- stabilizes the transition state for converting NTP to NDP + Pi
- frequently involves a lysine residue, metal ion, both of which stabilize the intermediate strong negative charge
- other molecular motors
- kinesins: structural homologues of actin; transport cellular organelles on intracellular myosin-like microtubules
- helicases: use NTP hydrolysis to unwind DNA
- each antibody has three hypervariable loops that are almost perfectly matched to an antibody
- note that antibodies can also be designed to bind small molecules using similar chemistry
- use in biochemistry
- polyclonal antibodies: a heterogenous collection, each of which binds a different epitope (sequence) of antigen
- monoclonal antibodies: homogenous collection that recognizes only one epitope of the antigen
- collagen: an overview
- collagen fiber: quarter-staggered array of cross-linked collagen triple helices
- structural classification
- group I:
type I, II, III, V
long polypeptide chains wound into a single helical domain
- group II: type IV, VI, VII, VIII
long chains, several helical segments with several nonhelical segments
- group III:
shorter chains in one or more helical segments
- synthesis
- (1) synthesis of pro- chain
ER/Golgi
- (2) hydroxylation of selected Pro, Lys
ER/Golgi
- (3) glycosylation of selected hydroxylysine:
ER/Golgi
- (4) self assembly of pro- chains
ER/Golgi
- (5) procollagen triple helix formation:
secretory vesicle
- (6) secretion:
plasmalemma
- (7) cleavage of procollagen into tropocollagen:
extracellular matrix
- (8) self assembly into collagen fibril:
extracellular matrix
- collagen synthesis: detail
- (1) pro- chain structure: three polypeptide chains winding around a common axis
- repeating sequence: Gly-X-Y, where X is commonly Pro, and Y is commonly HyPro
- prevalence of Gly: allows R groups to fit at the center of the axis
- prevalence of Pro, HyPro: imparts helical stability
- (2) proline hydroxylation
- prolyl residue + -ketoglutarate 4-hydroxyprolyl residue + succinate
- enzyme: prolyl 4-monooxygenase
- monooxygenase enzymes
- prolyl 3-monooxygenase, prolyl 4-monooxygenase, lysyl 5-monooxygenase
- mechanism always require a second reducing agent (e.g. -ketoglutarate)
- cofactors: Fe2+, ascorbate (vitamin C)
- vitamin C: reduces Fe2+ in hyxroxylase enzymes
- lack of Vitamin C leads to scurvy, which is characterized by collagen weaknesses
- (3) glycosylation: primarily galactose and glucosylgalactose; function unknown, may involve signaling
- (4,5) procollagen
- pro- chains each have 100-300 extra amino acids at each end
- with linkage by disulfide bonds, these stabilize the molecules during the formation of tropocollagen
- (7) tropocollagen
- formation
- amino and carboxy procollagen peptidase enzymes cleave peptide bonds to release the precursor portions
- this allows tropocollagen to self-assemble into insoluble collagen fobers
- (8) maturation of the collagen fiber
- during collagen maturation, lysyl oxidase catalyzes the formation of an aldehyde from lysine
- critical step in forming cross-links; inhibition of this step can cause lathyrism, or defects in skeletal formation
- problems in collagen synthesis
- lathyrism
- inhibition of lysyl oxidase reaction in collagen maturation
- characterized by defective skeletal formation, excretion of HyPro-rich peptides
- Ehlers-Danlos syndromes
- clinically heterogeneous category of connective tissue disorders
- type IX: low lysyl oxidase activity; causes defective skeletal formation
- type VII: decreased pro-collagen aminopeptidase activity, leading to fragile, loose, hyperextensible CT
- type VI: deficiency in lysyl-5 monoxygenase, leading to fragile, loose, hyperextensible CT
- types of Ehlers-Danlos syndromes listed here due to defects in modification, not synthesis, of collagen
- breakdown
- Clostridium histolyticum, which causes gas gangrene, contains a collagenase
- mammalian tissues undergoing reorganization often have mammalian collagenases within
46 x 2
46 x 1
46 x 2
2 x (23 x 2)
4 x (23 x 1)
- recessive: trait seen only in individuals homozygous for the causative allele
- autosomal: gene on one of the 22 autosomes
- X-linked: gene on the X chromosome
- recognizing patterns of inheritance
- autosomal dominant: found in each generation, afflicting on average half of progeny
- autosomal recessive: seemingly skips generations, appearing on average in 1/4 of progeny of unafflicted
- sex-linked: appears commonly in sons of unafflicted; females require an afflicted father and carrier mother
- genetic linkage
- Mendelian inheritance requires two individual traits to separate independently (50% separation)
- traits that are physically very close on a chromosome will often separate with one another
- the distance between traits is thus measured by separation using cM, where 1 cM = 1% separation
you should know:
- crossing over (meiotic recombination)
- occurs during division 1 of meiosis, when homologous chromosomes line up on one another
- occurs 1-2 times per homologous chromosome pair
- reasons for patterns of inheritance
- autosomal dominant: manifestation of disease requires only one copy of afflicted trait
- autosomal recessive: manifestation of disease requires two copies of the defective trait
- sex-linked: must be homozygous for mutation, but males have only one copy of the chromosome
you should understand:
- X-inactivation
- expression of genetic material requires a careful balance, one that is disrupted in the sex chromosomes
- to compensate, cells in early females inactivate one of the X chromosomes
- reactivation occurs only when gametes are being formed
- linkage and recombination
- recombination events can occur anywhere on a chromosome, so any two traits could separate independently
- traits that are very close together have a statistically smaller chance of being split by a recombination event
- traits at opposite ends of a chromosome are essentially independent
Notes: Lecture and Reading
characteristics of the human genome
- overview of the human genome
- genome: complement of genetic information stored in an organisms DNA
- length (haploid number): 3 x 109 base pairs
- number of genes: 35,000 (twice as many as in the round worm)
- humans as diploid organisms
- homologs: maternal and paternal copies of a chromosome containing similar but not identical sequences
- alleles: exact form of a gene at a given locus
- thus each human cell has two homologs of the 22 autosomes and two sex chromosomes, for a total of 46
- chromosomes
- locus: position of a gene in the genome
- chromosome: segments of a genome
- human genome: 24 chromosomes; 22 are autosomal, the other two are X and Y
- chromosomes average 1000 2000 loci each
- sister chromatids: identical copies of a chromosome that exist after replication in preparation for division
- centromere: attachment point of sister chromatids post replication
- mitochondrial chromosomes
- mitochondria contain multiple copies of a chromosome that code for ~35 mitochondrial proteins or RNA
- maternally inherited: sperm cells are devoid of cytoplasm
- certain myopathies and neuropathies result from mutations in the mitochondrial chromosome
gametogenesis, recombination, and linkage
- germ line
- germ line: the collection of cells that give rise to gametes (eggs and sperm)
- somatic cells: all other cells of the body
- only mutations in germ line cell DNA are passed onto offspring
- division
- mitosis: (46 x 2) (46 x 1), (46 x 1)
- division: sister chromatids separate
- meiosis: (46 x 2) (23 x 2), (23 x 2) (23 x 1), (23 x 1), (23 x 1), (23 x 1)
- division 1: homologous chromosomes separate
- division 2: sister chromatids separate (similar to meiotic division)
- recombination
- crossing over: meiotic recombination
- occurs once or twice per chromosome pair, resulting in patchwork chromosomes from maternal, paternal DNA
- linkage
- linkage: the tendancy of an allele at one locus to end up in the same gamete as another locus
- tightly linked: combination of alleles corresponds to combination in either of the parental homologs
- unlinked: equal proportions of all possible combinations are observed
- loci on different chromosomes are unlinked
- loci on the same chromosome are linked in proportion to their physical separation
- 1 cM: 1% chance of being separated by recombination (works to ~50 cM due to probability)
- average human chromosome is 150 cM long, so opposite ends are unlinked
general aspects of inheritance
- definitions
- homozygous: diploid individual has identical alleles at a given locus
- heterozygous: diploid individual has different alleles at a given locus
- genotype: genetic composition of an individual
- phenotype: observable characteristics of an individual resulting from the genotype
- dominant: trait seen in individuals heterozygous for the causative allele
- recessive: trait seen only in individuals homozygous for the causative allele
- autosomal: gene on one of the 22 autosomes
- X-linked: gene on the X chromosome
- limitations
- this course considers only single gene defects exhibiting simple Mendelian inheritance
- over 3000 have been identified
- half are autosomal dominant
- one third are autosomal recessive
- less than one tenth are X-linked
autosomal inheritance
- autosomal dominant
- transmitted only to offspring that have at least one afflicted parent
- both males and females suffer, and about half of the individuals from a parent are affected
- autosomal recessive
- can occur in the offspring of unafflicted parents if both are carriers (P=0.25)
- males and females afflicted with equal probability
X-linked inheritance
- inheritance
- disease is fully expressed in males due to being hemizygous
- normal male, carrier female
- son: 50% chance of being afflicted
- daughter: 50% chance of being a carrier
- afflicted male, normal female
- daughter: always at least a carrier if mother is normal
- son: always normal
- X inactivation
- to maintain genetic balance, females have one X chromosome inactivated during early fetal development
- this is random within a particular cell
- however, this relationship is maintained within subsequent mitotic divisions
- each cell can express only one X allele of an X-linked locus, though on average they are equally expressed
- components
- RNA polymerase: transcribes DNA to make RNA
- mRNA (messenger): class of RNA that is used to make protein
- ribosome: translates RNA into protein
- tRNA (transfer): class of RNA that supplies appropriate amino acids for addition to the growing chain
- additional complexities in eukaryotes
- transcription: occurs in the nucleus
- translation: occurs in the cytoplasm
- introns: extraneous internal segments of RNA that are removed prior to translation by splicing
- gene expression
- direct: level of transcription
- indirect: post-transcriptional
- DNA replication
- enzyme: DNA polymerase
- each strand is copied, producing two identical double-stranded DNA molecules
DNA and RNA as nucleotide polymers
- nucleotide structure
- 5-carbon sugar:
ribose in RNA, deoxyribose in DNA
- base:
purine or pyrimidine; attached to 1 carbon of the sugar
- phosphate:
one or more; attached to the 5 carbon of the sugar
- nucleoside structure: nucleotide without phosphate
- nucleotides in RNA
sugar
base
type
nucleoside
nucleoside
nucleotide
abbreviation
abbreviation
ribose
adenine
purine
adenosine
A
AMP
ribose
cytosine
pyrimidine
cytidine
C
CMP
ribose
guanine
purine
guanosine
G
GMP
ribose
uracil
pyrimidine
uridine
U
UMP
- nucleotides in DNA
sugar
deoxyribose
deoxyribose
deoxyribose
deoxyribose
base
type
adenine
cytosine
guanine
uracil
purine
pyrimidine
purine
pyrimidine
nucleoside
deoxyadenosine
deoxycytidine
deoxyguanosine
deoxythymidine
nucleoside
abbreviation
dA
dC
dG
dT
nucleotide
abbreviation
dAMP
dCMP
dGMP
dTMP
- DNA helicase:
ATP-based enzyme that can unwind DNA at ambient temperatures
- melting temperature Tm
- Tm: temperature at which DNA will denature into two single strands
- higher proportion of GC: higher Tm, as there is a greater number of H-bonds
- salt solutions: higher Tm, as salt shields repulsive forces between phosphate groups on opposite strands
- renaturation
- reannealing: strands were previously paired
- hybridization: strands were not previously paired
- a short fragment added in excess will find complementary regions faster than the original strand
- this is the basis for PCR, many genetic tests
DNA replication
- cell cycle
- M phase:
mitosis and creation of a daughter cell
- G1 phase:
gap phase and preparation for replication
- S phase:
DNA replication
- G2 phase:
gap phase and preparation for cell division
- replication
- helicase unwinds DNA into its leading and lagging strands
- leading strand synthesis: continuous line in 5 3 direction
- lagging strand synthesis
- primase adds an RNA primer
- polymerase III synthesizes an Okazaki fragment
- polymerase I replaces RNA with DNA
- ligase closes Okazaki fragments, giving the complete strand form
- bond formation
- DNAp promotes base pairing of appropriate dNTP to template DNA
- DNAp catalyzes attach of 3-OH on phosphate of the dNTP, forming a phosphodiester bond and releasing PPi
- pyrophosphatase hydrolyzes PPi, helping drive DNA synthesis
- proofreading
- DNA polymerase has a 35 exonuclease activity, which adds to DNA fidelity
- fidelity still results in a mistake in 1 per 108 nucleotides
- SUMMARY: enzymes of DNA replication
- DNA helicase: unwinds DNA at the replication fork (ATP-driven)
- DNA polymerase (DNAp)
- phosphodiester bond formation: catalyzes addition of nucleotides to 3-hydroxyl end of primer (53)
- 3-5 exonuclease activity: stops at mismatched bases and releases a dNMP
- RNA replacement: replaces RNA fragments with DNA
- RNA primase: adds RNA primers for use by DNAp
- pyrophosphatase: hydrolyzes pyrophosphate (PPi + H2O 2Pi), helping drive bond formation by mass action
- DNA ligase: forms phosphodiester linkage between Okazaki fragments
- RNAp binds at the promoter, and a small region of double-stranded DNA is denatured
- base-pairing guides polymerization except that an A in template DNA specifies incorporation of U in RNA
- elongation
- growing polynucleotide chain continually displaced from template, so DNA/RNA duplex is short
- as RNA is displaced, DNA duplex is reformed
- termination
- RNAp or a termination factor recognizes a specific sequence or secondary structure in the transcribed RNA
- transcription units
- transcription unit: a region of DNA containing a promoter and a termination signal, thus directing synthesis
- in eukaryotes, this is typically equivalent to a gene
- in bacteria, several genes often contained within a single transcription unit (operon)
- transcript: the RNA made from a transcription unit
- transcript sequence analogous to sequence of the non-template strand
- difference: replacement of T with U
messenger RNA and the genetic code
- overview
- codon: group of three adjacent nucleotides specifying the amino acid of a protein
- untranslated regions (UTRs): portions at 5 and 3 ends that are not translated
- range in length from <50 to several thousand nucleotides
- play a role in regulation of translation, degradation of mRNA
- primary structure of mRNA
- methylguanosine cap
- 5 UTR
- translated region
- AUG (start) codon
- protein-coding region (may contain introns that are excised prior to translation)
- stop codon
- 3 UTR
- poly-A signal (AAUAAA)
- poly-A site
- poly-A tail
- post-transcriptional modifications
- methylguanosine cap (m7G)
- 5 triphosphate remains on the first codon
- shortly after leaving RNAp II, capping enzymes add a methylated guanosine in a 5-5 triphosphate linkage
- this protects from degradation by 5 exonucleases
- this also is a binding site for proteins that facilitate:
- splicing of the pre-mRNA
- transport of the mRNA to the cytoplasm
- translation of the mRNA by ribosomes
- termination and the poly-A tail
- nascent polynucleotide is cleaved 10-30 nucleotides downstream of 5-AAUAAA-3
- poly(A) polymerase then adds 100-200 AMP residues to the 3 end, using ATP and releasing PPi
- this poly(A) tail is found only on mRNA
- thought to inhibit 3-exonucleolytic degradation, promote translation
- poly(A) site: point in the transcript where cleavage and polyadenylation occurs
- poly(A) signal: AAUAAA sequence
- mRNA splicing
- definitions
- pre-mRNA: precursor to eukaryotic mRNA, prior to excision of intervening sequences
- intron: intervening sequences that interrupt mRNA-coding sequences
- exon: mRNA-coding sequences, not necessarily protein-coding
- RNA splicing: the process of intron removal; occurs in the nucleus in the spliceosome
- process
- spliceosome composed of pre-mRNA substrate, 5 snRNAs, and >60 proteins
- spliceosome base pairs with sequences in the intron to direct splicing
- released intron is degraded and nucleotides are recycled, while mRNA is transported to the cytoplasm
- introns in a gene
- can vary from 0 to >50, though most genes have several
- example: Factor VIII gene is 186 kbp in length, and 175 kbp is contained in 25 introns
- first exon always contains at least some of the 5-UTR
- last exon always contains at least some of the 3-UTR, including the poly(A) signal
- the genetic code
- overview
- 64 trinucleotide codons to specify each of the 20 amino acids
- one start codon (AUG, or methionine), and three stop codons (UAA, UAG, UGA)
- when an amino acid is specified by multiple codons, the 3rd codon is often the only difference
- mutations
- silent mutation: does not change the sequence of the encoded protein
- missense mutation: changes the codon to specify a different amino acid
- nonsense mutation: changes a codon to a stop codon
- frameshift mutation: deletion of addition of nucleotides such that the downstream reading frame shifts
point mutation in -thalassemias
- thalassemias: an overview
- thalassemia: inherited deficiency in the production of or globin (resulting in or thalassemia)
- the globin chain present in normal amounts tends to form insoluble homotetramers that do not function well
- when either chain is nearly absent, severe anemia and death usually occur before 10 without regular transfusions
- variance in severity
- 0: alleles that are completely inactive
- +: alleles that are partially active
- thalassemia minor: 0, asymptomatic
- thalassemia major: 00, requires regular blood transfusions
- intermediate: ++, shows intermediate symptoms
- mutations and consequences
- promoter regions
- cluster in two regions, about 90 and 30 base pairs upstream of transcription start site
- CACCC at -88: regulatory protein
- ATA at -31: TATA box for binding TFIID
- point mutations generally result in + alleles, with transcription reduced ~5-fold
- splice sites
- 5 splice site of intron 1
- mutation in initial GU sequence results in 0 allele
- mutation in more weakly-conserved site at position 5 results in + allele
- 3 splice site of intron 2
- mutation in terminal AG to GG mutation results in 0 allele
- poly(A) signal: mutation of AAUAAA to AACAAA
- results in cleavage of pre-mRNA after another AAUAAA sequence 900 nt further downstream
- less -globin is made due to a loss in stability, resulting in a + allele
- other mutations
- nonsense mutation in exon 2: 0 allele
- frameshift mutations: 0 allele
- missense mutations: rarely affect levels of -globin produced, so do not result in thalassemia
- exception: Indianapolis -globin, which is highly unstable due to a single AA substitution
- generally, may result in pathological effects, such as sickle cell anemia
stable RNAs with biochemical functions
- translated vs. untranslated RNA
- mRNA makes up a small fraction of a cells RNA (10%)
- most RNA in a cell is not translated, instead serving important cellular functions
- ribosomal RNA (rRNA) (75% of total cellular RNA)
- the ribosome is 2/3 RNA and 1/3 protein
- catalysis of peptide bond formation by modern ribosomes is carried out by rRNA
- transfer RNA (tRNA) (15% of total cellular RNA by weight)
- used by ribosome to read mRNA codon, provide the corresponding amino acid
- one end: anticodon complementary to a given codon
- other end: amino acid corresponding to a codon
- undergo post-translational modification, giving them bases other than A, C, G, U (e.g. T by methylation)
- due to redundancy, more than 20 different kinds of tRNAs
- small nuclear RNA (snRNA)
- small, 100-300 nucleotide RNAs that participate in processing of pre-mRNA in the nucleus
- packaged with proteins into small nuclear ribonucleoprotein particles (snRNPs)
- some autoimmune disorders (e.g. systemic lupus erythematosis) produce antibodies that recognize snRNPs
- the disease significance of this is not known
- small nucleolar RNA (snoRNA)
- similar to snRNA, but found in nucleolus, which is dedicated to ribosome synthesis
- base pair with newly-synthesized rRNA, direct processing and modification of rRNA into mature form
- rRNA then assembles with ribosomal proteins to form the ribosomal subunits, sent to cytoplasm
- micro RNA (miRNA)
- 20-22 nucleotides long, complementary to specific mRNA
- pairing of miRNA with mRNA targets mRNA for degradation by ribonucleases
- have an important role in human gene expression
- autosomal recessive disorder caused by mutation in cystic fibrosis transmembrane regulator (CFTR)
- CFTR: regulates transport of Cl- ions across cellmembranes
- symptoms usually include respiratory and digestive problems
- lungs become clogged with mucous and are susceptible to pneumonia
- pancreatic duct becomes clogged, and digestive enzymes fail to reach the intestines
- 1/2000 U.S. newborns is afflicted
- median survival age for individuals with CF is about 25 YO
- diagnosis
- CF: causes excess salt in sweat
- CF carriers: no detectable phenotype, so a DNA test is required
- mutations
- F508: 70% of carriers; deletion of phenylalanine at position 508
- blocks proteins transit from ER to cell membrane, thus blocking its function in Cl - transport
- can be detected with ASO
- other 30% of carriers: more than 200 causative mutations, making ASO much more difficult
- linkage analysis
- situation 1
- parents: disease alleles on chromosomes with site, normal alleles on chromosomes lacking site
- children: homozygous uncut = normal, heterozygous = carrier, homozygous cut = afflicted
- situation 2
- parents: disease alleles on chromosomes with site, one parents normal allele also has site
- children: homozygous cut = afflicted OR carrier, heterozygous = carrier OR normal
- situation 3
- parents: one parent has disease allele with site and normal without, other parent has opposite
- children: homozygous cut/uncut = carrier, heterozygous = afflicted OR normal
- situation 4
- parents: both parents have restriction sites on all four chromosomes
- children: all will have restriction sites, and thus this site is not useful
- there is no obligatory relationship between RFLP and a disease
- glucagon: polypeptide hormone that signals low blood glucose, promoting PEPCK
- adrenaline: adrenal hormone that signals need for glucose, promoting PEPCK
- both cause an upregulation of cAMP, which binds CREB, and complex binds CRE
you should know:
- combinatorial control
- numerous genes may come together to repress or (more commonly) activate gene transcription
- it is the combined effect of all elements that determines the total regulation
- mechanism of Jun/Fos promoters
- -helices containing a leucine zipper and a basic region
- hydrophobic leucines at every 7th residue face the same side, come together
- (+) charged basic regions oriented to fit into the grooves of DNA, where they interact with (-) charged DNA
- bind at the AP-1 promoter element
- dimerization
- Jun/Jun: bind poorly
- Jun/Fos: bind extremely well
- Fos/Fos: do not bind at all
you should understand:
- regulation
- general transcription factor: trans-acting elements required for transcription of all protein-coding genes
- gene regulatory protein: modify basal level of transcription by TFs, in a gene-specific manner
- domains of a transcriptional activator protein
- DNA-binding: specifically interacts with and binds DNA, about 8-10 bp long
- activation: interacts with general transcription factors
- effector: alters ability to activate transcription in response to a cellular signal
Notes: Lecture and Reading
overview of regulation of gene expression
- cellular identity
- multicellular organisms must coordinate levels of gene expression
- intercellular signals: hormones, growth factors, cell to cell contact, amongst others
- controlling gene expression
- transcriptional control (most common)
- processing control
- translational control
- degradation control
- components of gene expression
- factors: proteins, RNA, or complexes thereof that act on signals or elements present in DNA, RNA, or protein
- promoter elements: DNA sequences near the gene that aid in the binding of RNAp II
- cis-acting elements: act on a local scale, with limited expression
- example: sequence elements
- inherited defects in gene expression tend to be caused by mutation of cis-acting elements
- trans-acting elements: act on a global scale, across numerous molecules
- example: transcription factors
- mutations in transcription factors are typically lethal very early on, and are often not recognized
control of gene expression: DNA sequence elements
- overview
- DNA transcription level is generally controlled by the interaction of trans-acting and cis-acting elements
- cis-acting sequence elements: collectively termed promoter
- trans-acting elements
- general transcription factors (TFs): required for transcription of protein coding genes
- gene regulatory proteins: modify basal level of transcription directed by TFs in a gene-specific manner
- activators: increase transcription
- repressors: decrease transcription
- RNAp II: initiation of transcription
- general transcription factors
- TFIID: binds sequence 5-TATAAA-3 (TATA box) at ~30 base pairs upstream of transcription start site
- TFIIB: binds adjacent to TFIID
- transcription initiation: process
- TFIID and TFIIB bind to DNA, often joined by other factors (such as TFIIA)
- RNAp II recognizes the DNA complex, binds, and begins transcription
- TFIID and TFIIB stay bound to the promoter after initiation, promoting additional recruitment of RNAp II
- process requires several activator proteins
- activators in human gene expression
- activator binding sites
- upstream elements: binding sites for activator proteins just upstream of the promoter
- enhancers: binding sites located thousands of base pairs away; orientation-independent
- activator proteins
- DNA-binding domain: recognizes a specific DNA sequence 8-10 bp long
- activation domain: interacts with general transcription factors
- effector domain: interacts with a cellular signal (e.g. hormone, phosphorylation)
- found only in certain activator proteins
- other gene regulatory proteins are always on, thus activity is determined primarily by their concentration
- combinatorial control: phosphoenolpyruvate carboxykinase (PEPCK)
- definitions
- combinatorial control: level of synthesis determined by net effect of all bound regulators
- PEPCK: key role in gluconeogenesis; produced primarily in the liver
- PEPCK structure
- TATA box:
-30
- CRE:
-100
- AP-1 promoter:
-125, -250, -275
- GRE:
-360
- HNF4 binding site:
-400
- receptors
- cyclic AMP response element (CRE)
- glucagon and adrenaline (which signal need for glycolysis) stimulate production of cAMP
- cAMP stimulates a protein kinase that activates the protein CREB
- cAMP binds CREB, complex binds CRE, promoting transcription
- AP-1 promoter: bind Jun/Fos general activators, regulated in some part by their synthesis
- glucocorticoid response element (GRE)
- DNA sequence element
- binds hormone/glucocorticoid receptor (GR) complex, which increases transcription
- hepatocyte nuclear factor 4 (HNF4)
- tissue-specific activator, present primarily in the kidney and liver
- absence of this factor restricts synthesis in other tissues, even if cortisol, glucagon, or adrenaline is high
nuclear receptors
- nuclear receptors: overview
- nuclear receptors: gene regulatory proteins that bind small, hydrophobic molecules in their effector domains
- steroid hormone receptors: have a steroid-derived hormone receptor, such as cortisol, estrogens, or androgens
- steroids are lipid soluble, and can thus diffuse through cell membrane to bind a nuclear receptor
- this allows direct action, as opposed to the indirect use of second messengers such as cAMP
- other examples of molecules using nuclear receptors
- thyroxine (thyroid hormone), vitamin D, retinoic acid (derived from vitamin A)
- ligand for these is currently unknown
- nuclear receptor structure
- structure
- variable N-terminal receptor (transcription activator)
- DNA-binding domain
- C-terminal ligand-binding domain
- ligand binding
- Hsp90: inhibitory protein that complexes nuclear receptors without bound ligand
- upon binding of ligand, Hsp90 is released, and complex can bind to DNA to regulate transcription
- components
- aminoacyl tRNA: located in A site, contains amino acid to be added
- peptidyl tRNA: located in T site, contains growing polypeptide chain (N-terminus distal to ribosome)
- codon: three letter code located on DNA
- anticodon: complementary three letter code on RNA
- peptidyl-transferase site: located between ends of the aminoacyl and peptidyl tRNAs
you should know:
- components of translation initiation
- start codon: AUG (methionine)
- tRNA: methionyl-tRNAMet(i) (Meti is specific to initiation)
- recognition of the start codon: ribosome looks for first AUG sequence downstream of the 5-mGppp cap
- EF-1 and protein elongation
- function: binds GTP, binds an aminoacyl-tRNA, and brings it to the aminoacyl site in the ribosome
- molecular clock
- peptidyl transferase can only work after EF-1 has left the site, which requires hydrolysis of GTP
- binding of aa-tRNA anticodon signals hydrolysis of GTP
- proofreading: if anticodon does not match, tRNA will dissociate before GTP hydrolysis is complete
you should understand:
- diphtheria toxin
- EF-2 is a GTP-binding protein that is required for translocation of peptidyl tRNA from A site to P site
- diphtheria toxin ADP-ribosylates (from NAD+) a specific amino acid residue in EF-2, inactivating it
- one molecule of toxin is potent enough to kill an entire cell
- tRNAs and the genetic code
- there are 20 amino acids, 20 aminoacyl-tRNA synthetases and 64 possible amino acid codons
- redundancy: multiple codons must be recognized by a single aminoacyl-tRNA synthetase
- as such, many synthetases must use structural features other than tRNA codon in order to bind
Notes: Lecture and Reading
tRNA activation: aminoacylation
- components
- definitions
- aminoacyl-tRNA synthetase: enzyme that activates tRNA by attaching amino acid
- tRNAamino acid: recognizes RNA codon, involved in transferring it to a growing polypeptide
- anticodon: sequence by which an activated tRNA recognizes and binds DNA
- there are 20 AA-tRNA synthetases, one for each amino acid
- more than 20 tRNA molecules required for all codons (some tRNA can recognize multiple anticodons)
- some synthetases must therefore recognize multiple tRNA molecules
- isoacceptors: tRNAs that have different anticodon sequences but become charged with the same amino acid
- some synthetases recognize tRNA molecules by their anticodon sequence
- synthetases that charge multiple tRNAs must recognize other structural features of the tRNA
- enzymatic process: two steps, both catalyzed by aminoacyl-tRNA synthetase
- adenylation
- amino acid + ATP aminoacyl-AMP + PPi
- this reaction activates the amino acid for use in the next step
- fidelity
- this reaction gives the synthetase another opportunity to proofread the amino acid, increasing fidelity 100X
- if aminoacyl-AMP does not fit properly, adenylate is hydrolyzed and amino acid is discarded
- aminoacylation
- aminoacyl-AMP + tRNA aminoacyl-tRNA + AMP
- 2 or 3 OH of terminal adenine in tRNA attacks the carboxyphosphate bond formed in adenylation reaction
- this attaches the amino acid to the tRNA, activating it for use in polypeptide elongation
- note that every tRNA has an adenylate residue on the 3 end
- net energy used: 2 phosphates
- PPi generated in aminoacylation step is hydrolyzed to 2Pi by pyrophosphatase
- this makes the net reaction more exothermic, driving it forward by mass action
- EF-2: GTP-regulated protein required for translocation of peptidyl-tRNA from A site to P site
- diphtheria
- diphtheria: toxin in certain strains of Corynebacterium diphtheria
- acts by catalyzing transfer of ADP-ribose from NAD+ to a specific amino acid in EF-2, inactivating the protein
- this blocks protein synthesis by halting the translocation step
- a single molecule of toxin can kill an entire cell (which contains half a million EF-2 molecules)
- erythromycin
- erythromycin: antibiotic that binds large subunit RNA in bacterial ribosome, inhibits translocation
- because this does not affect eukaryotic translocation, it is an effective antibiotic
- some bacterial strains are emerging that have a resistance to this
energy of protein synthesis
- addition of each amino acid residue to a growing polypeptide chain requires 4 high energy phosphate bonds
- aminoacyl-tRNA synthetase
amino acid activation
ATP AMP + PPi 2Pi
- EF-1
molecular clock
GTP GDP + Pi
- EF-2
translocation
GTP GDP + Pi
- cleaves the signal peptide, creating a new N-terminal end that faces the inner ER lumen
- signal sequence remains in the ER lumen until it is degraded by other enzymes
- function
- use of signal peptidases, stop sequences, and start sequences alters the orientation and position of the protein
- each odd-numbered hydrophobic segment acts as a signal peptide or start transfer sequence
- each even-numbered hydrophobic segment acts as a stop transfer sequence
- polarity of integral membrane proteins
- signal sequence, when cleaved by peptidase, places the N-terminal region within the ER lumen
- if signal sequence is further into the polypeptide and is not cleaved by peptidase, N-terminus in cytosol
- knowing this, protein orientation can be predicted
- because of how vesicles work, the ER lumen is topographically equivalent to extracellular membrane
- proper folding: molecular chaperones
- binding protein (BiP): molecular chaperone protein present in high concentration in ER lumen
- molecular chaperone: protein specialized to guide the folding and assembly of other proteins
- proteins are extruded into the ER lumen in an extended state, and are not properly folded
- this promotes non-specific aggregation of exposed hydrophobic regions
- molecular chaperones shield forming polypeptides, giving them time to fold individually
- proper folding: disulfide bonds
- protein disulfide isomerase (PDI): catalyzes cleavage of disulfide bonds
- disulfide bonds
- cytosol: reducing environment (favors removal of disulfide bonds)
- ER lumen: oxidizing environment (favors addition of disulfide bonds)
- upon entry into ER, cysteine disulfide bonds form spontaneously, often incorrectly
- PDI cleaves bonds, allowing protein to continue towards its lowest energy (most stable) state
- protein glycosylation
- N-linked glycosylation: attachment of sugars to amino group of certain asparagine residues
- most proteins entering ER are covalently modified in this way
- glycosyl transferase: catalyzes this process on luminal side of ER membrane
- only happens within the ER lumen
- secretory vesicles work such that ER side of integral membrane proteins (IMPs) will face outside cell
- because of this, only extracellular portions of IMPs are glycosylated
the Golgi apparatus and beyond
- Golgi apparatus structure
- Golgi apparatus consists of 4-8 cisternae organized in a stacked fashion
- cisternae: disk-shaped membrane bound vesicles
- movement through the Golgi
- cis face: cisterna closest to ER (also called transitional ER)
- trans face: cisterna closest to plasmalemma
- proteins move stepwise through cisterna in vesicles that bud off each face and merge with the next
- from the trans face, vesicles move to the lysosome, secretory granules, or directly to the plasmalemma
- retrograde transport: returns proteins to ER
- occurs through the use of retrograde vesicles that bud off and return to ER
- proteins to be returned to ER (e.g. BiP or PDI) are recognized by KDEL receptor
- KDEL receptor: Lys-Asp-Glu-Leu conserved sequence
- Golgi function
- localization: proteins are sent to proper places within the cell
- this often occurs by enzymes binding to specific receptors on Golgi membranes
- example: lysosome
- N-linked oligosaccharides of lysosome enzymes are recognized by phosphomannose receptors
- phosphomannose receptors bind, package enzymes into vesicles bound for lysosome
- I-cell disease: autosomal recessive disorder characterized by psychomotor and skeletal difficulties
- defect in kinase that phosphorylates mannose in N-linked oligosaccharides of lysosomal enzymes
- phosphomannose receptor fails to recognize this, and proteins are secreted instead
- lysosome is unable to complete its digestion, leading to large cellular inclusions
- glycosylation
- N-linked sugars can be trimmed, and different sugars can be added in order to modify oligosaccharides
- this may happen because the DNA to be removed is not chemically altered
DNA, RNA
ATP
cAMP, cGMP, GTP
adenosine derivatives
aminoacyl-AMP
[X B ]
[X A ]
G RTln
G ZF
G RTln
[X B ]
ZF
[X A ]
- common use:
dehydrogenation of an alkane to alkene
- origin:
riboflavin (vitamin B2) (no major associated disease)
- energetics
- reversibility
- irreversible:
G values of great magnitude
- reversible:
G values of relatively small magnitude (close to 0)
- coupled reactions
- calculate G of individual reactions, ensuring that signs are correct
- sum values to get overall G
you should know
- glycolysis
- recognize glucose and the 10 intermediates
- know the types of reactions
- (1) hexokinase:
phosphorylation (IRREVERSIBLE)
- (2) phosphoglucose isomerase:
isomerization (aldose ketose)
- (3) phosphofructokinase:
phosphorylation (IRREVERSIBLE)
- (4) aldolase:
aldol cleavage of a C-C bond
- (5) triose phosphate isomerase:
isomerization
- (6) G 3-P dehydrogenase:
dehydrogenation, phosphorylation
- (7) phosphoglycerate kinase:
ADP phosphorylation
- (8) phosphoglycerate mutase:
isomerization (mutase)
- (9) enolase:
dehydration
- (10) pyruvate kinase:
ADP phosphorylation (IRREVERSIBLE)
- draw structure of product or reactant, given the other
- irreversible reactions of glycolysis
- hexokinase:
glucose glucose 6-P
- phosphofructokinase: fructose 6-P fructose 1,6-bis-P
- pyruvate kinase:
phosphoenolpyruvate pyruvate
Notes: Lecture and Reading
introduction to metabolism
- overview
- ATP: commonly used energy storage molecule
- nutrients taken in, energy extracted to make ATP
- energy: stored in the large free energy change associated with hydrolysis of phosphoanhydride bonds
- metabolic pathways
- anabolic: biosynthetic
- reductive, require expenditure of free energy (usually as ATP)
- common electron donor: nicotinamide adenine dinucleotide phosphate (NADPH)
- catabolic: degrative
- oxidative, use organic molecules to make ATP
- oxidation: usually involved, as usable energy is derived from increasing oxidation state of carbon
- physiological importance of glucose metabolism
- glucose: only source of energy for brain, RBCs
- normal conditions: blood glucose at 65-110 mg / 100 mL blood (3.6-6.1 mM)
- eating: diet is primary glucose source
- non-eating: glucose derived from breakdown of glycogen stores, gluconeogenesis
- glucose as a fuel source
- glucose is an excellent fuel source because:
- (1) abundant in diet
- (2) large predicted standard free energy change (G= -686 kcal/mol) for complete oxidation to CO2, H2O
- subjected to glycolysis, pyruvate DH, citric acid cycle, electron transport, oxidative phosphorylation
- free energy conserved as 36-38 ATP per glucose
oxidation-reduction reactions
- catabolic strategy
- reaction:
glucose glucose 6-phosphate [ATP ADP]
- enzyme:
hexokinase
- mechanism: phosphorylation
- energetics: essentially irreversible
- regulation: inhibited by glucose 6-phosphate
- (2) phosphoglucose isomerase
- reaction:
glucose 6-phosphate fructose 6-phosphate
- enzyme:
phosphoglucose isomerase
- mechanism: isomerization (aldose ketose)
- energetics: reversible
- (3) phosphofructokinase
- reaction:
fructose 6-phosphate fructose 1,6-bisphosphate [ATP ADP]
- enzyme:
phosphofructokinase
- mechanism: phosphorylation
- energetics: irreversible, and first committed step of glycolysis
- regulation: activated by AMP, inhibited by ATP
- (4) aldolase
- reaction:
fructose 1,6-bisphosphate dihydroxyacetone phosphate + glyceraldehyde 3-phosphate
- enzyme:
aldolase
- mechanism: aldol cleavage of a C-C bond
- energetics: reversible
- (5) triose phosphate isomerase
- reaction:
dihydroxyacetone 3-phosphate glyceraldehyde 3-phosphate
- enzyme:
triose phosphate isomerase
- mechanism: isomerization
- energetics: reversible
- (6) glyceraldehyde 3-phosphate dehydrogenase
- reaction:
glyceraldehyde 3-phosphate 1,3-bisphosphoglycerate [H-OPO3H2; NAD+ NADH + H+]
- enzyme:
glyceraldehyde 3-phosphate dehydrogenase
- mechanism: oxidative phosphorylation: dehydrogenation (oxidation) of G3P accompanied by phosphorylation
- energetics: reversible under cellular conditions
- (7) phosphoglycerate kinase
- reaction:
1,3-bisphosphoglycerate 3-phosphoglycerate [ADP ATP]
- enzyme:
phosphoglycerate kinase
- mechanism: cleavage of the carboxyl phosphate to phosphorylate ADP
- energetics: reversible
- (8) phosphoglycerate mutase
- reaction:
3-phosphoglycerate 2-phosphoglycerate
- enzyme:
phosphoglycerate mutase
- mechanism: isomerization by a mutase
- energetics: reversible
- (9) enolase
- reaction:
2-phosphoglycerate phosphoenolpyruvate [H2O]
- enzyme:
enolase
- mechanism: dehydration to redistribute energy of molecule
- energetics: reversible
- (10) pyruvate kinase
- reaction:
phosphoenolpyruvate pyruvate [ADP ATP]
- enzyme:
pyruvate kinase
- mechanism: cleavage of high energy enol phosphate group to form ATP by substrate level phosphorylation
- energetics: irreversible
- regulation: inhibited by ATP
general principles
- balance of glycolysis reactions
- 2 net ATP formed
- CoA-SH:
attaches to fragment, forming a leaving group useful for later reactions
- FAD:
oxidizes and regenerates lipoic acid, becoming FADH 2 in the process
- NAD+:
oxidizes FADH2, regenerating FAD, becoming NADH + H+ in the process
- thiamine deficiency
- disease:
Beri Beri
- distribution: countries where polished rice is the staple diet (thiamine is removed in shavings)
- pathology: accumulation of pyruvate
- symptoms: irreversible brain damage
- regulation of pyruvate dehydrogenase: kinase/phosphatase system
- kinase:
p-PDH (inactive form); activated by reaction products acetyl-CoA, NADH
- phosphatase: PDH (active form)
Notes: Lecture and Reading
fates of pyruvate
- sources
- glycolysis: oxidation of glucose
- catabolism of amino acids
- fates
- complete oxidation to CO2, H2O: TCA cycle
- mitochondrion: contains enzymes for TCA cycle, electron transport, and oxidative phosphorylation
- O2: final electron acceptor during electron transport process
- incomplete oxidation
- cells must rely on glycolysis ATP
- NAD+ must be regenerated by other means
pyruvate to lactate
- conversion to lactate occurs when:
- cells lack mitochondria:
RBCs, cornea, lens, regions of retina)
- cells have few mitochondria: kidney medulla, testis, leukocytes, white muscle fiber
- cells become limited in O2:
exercising muscle
- consequence of limited oxidation potential
- glycolytic reactions become primary ATP source
- NAD+ is regenerated from NADH by donation of electrons to pyruvate, forming lactate
- enzyme: lactate dehydrogenase
- lactate dehydrogenase
- reaction: pyruvate lactate [lactate dehydrogenase: NADH + H+ NAD+]
- structure
- tetramer of two varying subunits, H and M
- possible structures: H4, H3M, H2M2, HM3, M4
- consequences
- differ in catalytic, physical, and immunological properties
- structure is tissue specific, and electrophoretic properties can be used to identify damaged organs
- subunit predominance
- H subunit: heart muscle
- M subunit: skeletal muscle, liver
oxidative fate of pyruvate
- free energy changes: aerobic vs. anaerobic
- anaerobic
- glucose 2 lactate (G = -47 kcal/mol)
- conservation: 2 ATP
- aerobic
- glucose + 6 O2 6 CO2 + 6 H2O (G = -686 kcal/mol)
- conservation: 36-38 mol ATP
- process requires:
- oxidative decarboxylation of pyruvate to acetyl CoA
- disease:
- complex
- F0: integral membrane component; contains rotating portion of enzyme
- F1: inner surface of inner mitochondrial membrane component; catalyzes ATP formation
- ATP formation
- proton reentry turns part of F0 subunit, which alters physical characteristics of F1 component
- this causes formation of ATP in 3 steps: ADP + Pi ATP release
- ATP generation: 3 for NADHmitochondria, 2 for FADH2 (succinate), 2 for NADHcytosol
- transport
- ETS proton translocation create favorable gradients for incoming H+, outgoing OH- ATP-4/ADP-3 translocase:
imports ADP-3, exports ATP-4 (electrogenic, driven by membrane potential)
- H2PO4 /OH translocase:
imports H2PO4-, exports OH- (electroneutral; driven by OH- gradient)
- ATP synthase:
uses H+ gradient to catalyze ATP formation
- thus it is the proton gradient and membrane potential that drive synthesis and transport
Notes: Lecture and Reading
the electron transport system
- electron transport system (ETS): overview
- function
- utilize reducing power of NADH, FADH2
- convert into a form of energy that can drive ATP synthesis
- method
- ETS actively transports protons out of mitochondrial matrix
- establishes an electrochemical gradient used by ATP synthase to produce ATP
- mechanism
- hydrogen atoms from NADH, FADH2 are separated into protons and electrons (2 [H] 2 H+ + 2 e-)
- electrons are sequentially transferred across carriers until reaching complex IV
- electrons are then reunited with protons to reduce molecular O2 to H2O (2 e- + 2 H+ + O2 H2O)
- composition of the electron transport chain: four membrane protein complexes
- complex I
NADH DH:
transfers electrons from NADH to CoQ, a membrane-bound shuttle
- complex II succinate DH:
transfers electrons from succinate (via FADH 2) to CoQ
- complex III ubiquinol-cyt c reductase: accepts electrons from CoQ, transfers them to cytochrome c
- complex IV cytochrome oxidase:
accepts electrons from cytochrome c, transfers to O2 to form H2O
chemistry of the electron carriers
- flavin mononucleotide (FMN)
- structure: active end similar to FAD
- function: accepts 2 [H] atoms
- ubiquinone (coenzyme Q, CoQ)
- structure: ring structure attached to hydrophobic tail composed of 10 isoprenoid units
- isoprenoid: -CH2-CH=C(CH3)-CH2- tail anchored to inner mitochondrial membrane
- function: accepts 2 [H] atoms
- cytochromes
- structure: proteins with heme as a prosthetic group
- function: accepts 1 e- iron sulfur centers
- structure: iron coordinated to Cys-S residues, inorganic sulfur in cubical structure
- distribution: NADH DH complex, ubiquinol-cyt c reductase complex, succinate DH, other flavin-linked DHs
- function: accepts 1 e- copper centers
- structure: Cu coordinated to Cys-S residues, inorganic sulfur in diamond structure
- distribution: cytochrome oxidase (complex IV)
- function: accepts 1 ethe affinity of electron carriers for electrons
- trend
- during electron transport, each carrier is alternately reduced and then oxidized
- H+ gradient:
increased
(exported protons unable to return)
- ATP synthesis:
decreased
(no substrate)
- electron transport: decreased
(coupled to ATP synthesis)
- oxygen use:
decreased
(coupled to ETS)
- NADH/NAD+
increased
(decreased ETS uses less NADH)
- ADP in matrix:
decreased
(ADP converted to ATP, unable to leave)
- feedback inhibition
- respiratory control: ADP
- when ADP is lacking (due to cellular presence as ATP), protons cannot reenter cell (slowed ATP synthase)
- when protons cannot reenter, proton gradient builds up (slowed electron transport)
- causal relationships: high ATP/ADP high NADH/NAD+ increased citrate, acetyl-CoA
- feedback inhibition
- TCA cycle
- citrate synthase:
inhibited by NADH, citrate
- isocitrate DH:
inhibited by NADH
- pyruvate DH:
inhibited by NADH, acetyl-CoA
- glycolysis
- pyruvate kinase:
inhibited by ATP
- phosphofructokinase:
inhibited by ATP, activated by AMP
- hexokinase:
inhibited by glucose 6-phosphate
Notes: Lecture and Reading
the rate of electron transport is coupled to ATP synthesis and disruption by poisons
- oxidative phosphorylation is tightly coupled to the electron transfer process
- coupling: one process requires the other
- experiment: [O2] vs. time
- setup
- isolated mitochondria depleted of ADP are incubated in buffer (which includes Pi)
- concentration of O2 in solution is measured with an O2 electrode
- observations
- in absence of ADP, decline in [O2] is negligible
- upon addition of ADP, [O2] declines heavily, but slows after all ADP is used
- further addition of ADP once again causes O2 to decline
- conclusion: electron transport (measured via [O2] vs. time) requires ADP
- experimental parameters
- P/O ratio: determine amount of ATP synthesized upon addition of ADP, read O2 consumed off graph
- respiratory control ratio:
< 1% in solution
> 99% in solution
- proteins:
degradation through proteosomes
- lipids:
peroxidation
- nucleic acids:
NER and other pathways
- removal: peroxides
- 2 GSH + HOOH GS-SG + H2O [glutathione peroxidase]
- GS-SG 2 GSH [glutathione reductase: NADPH + H+ NADP+]
- glucose 6-P 6-phosphogluconolactone [glucose 6-P DH: NADP+ NADPH + H+]
Notes: Lecture and Reading
reactive oxygen species
- molecular oxygen: two unpaired electrons, and thus fairly reactive
- cytochrome oxidase: reduction of oxygen by sequential electron addition
- reaction:
O2 + 4 H+ + 4 e- 2 H2O
- enzyme:
cytochrome oxidase
- mechanism: sequential addition of electrons
- small portion of O2 is not completely reduced
- consequently, 3 reactive oxygen species (ROS) arise
- electron chemistry of oxygen
- O2 (+ e-) O2- (+ e-) H2O2 (+ e-) OH- + OH (+ e-) H2O
- reactions reflect 1 e- transfers, but do not reflect balanced equations
- reactive oxygen species
- superoxide anion:
O-Oone unpaired electron, moderately reactive
- acts as a single electron-oxidizing agent
- hydrogen peroxide:
HOOH
no unpaired electrons, relatively stable
- oxidizes SH residues on proteins
- hydroxyl radical:
OH
one unpaired electron, most reactive ROS
- abstracts H atom or adds on to existing molecule
- short half life in solution
generation of reactive oxygen species during normal metabolism
- superoxide radicals (O2-)
- reaction: O2 + e- O2- (one electron transfer)
- electron sources
- mitochondria: normal metabolism
- inadvertent transfer of an electron to O2
- reaction: CoQH2 + O2 CoQH + O2- red blood cells: spontaneous reduction of O2 bound to Hb
- reaction: Hb(Fe2+) + O2 MetHb(Fe3+) + O2- repair: 2 MetHB(Fe3+) 2 Hb(Fe2+) [methemoglobin reductase: NADH NAD+]
- methemoglobin: result of spontaneous O2 reduction, Fe2+ oxidation
- methemoglobin reductase: reduces methemoglobin to hemoglobin
- hydrogen peroxide (H-O-O-H)
- product of some FAD-linked oxidase reactions (e.g. xanthine oxidase)
- product of catalase enzyme in peroxisomes, mitochondria
- hydroxyl radicals (OH)
- Fenton reaction: spontaneous reduction of HOOH in the presence of iron(2)
- reaction: HOOH OH + OH- [Fe2+ Fe3+]
elimination of reactive oxygen species
- antioxidants
- vitamin E: lipid-soluble vitamin that acts to trap free radicals
- prevents peroxidation of fatty acids in adipose tissue, biological membranes
- deficiencies are rare, symptoms include muscular weakness and fragile RBCs
- more important with additional dietary intake of polyunsaturated fatty acids
- vitamin A: lipid-soluble vitamin that sequesters ROS
- vitamin C: water-soluble vitamin that keeps vitamin E, vitamin A in reduced states
- antioxidant enzymes
- superoxide dismutase: reduction of superoxide
- reaction:
2 H+ + 2 O2- H2O2 + O2
- enzyme:
superoxide dismutase (SOD)
- distribution:
MnSOD mitochondria
Cu/Zn SOD: cytoplasm
- catalase: reduction of peroxide
- reaction:
H2O2 + H2O2 O2 + 2 H2O
- enzyme:
catalase
- distribution:
peroxisomes, mitochondria
- glutathione peroxidase: reduction of peroxide
- reaction:
2 GS-H + H2O2 GS-SG + 2 H2O
2 GS-H + ROOH GS-SH + HOH + ROH
- enzyme:
glutathione peroxidase
- distribution:
primary enzymatic system for control of cellular peroxide levels
- glutathione: oxidation and reduction
- glutathione (GSH): sulfhydryl component that serves as a general reducing agent
- glutathione peroxidase: generates oxidized glutathione (GS-SG) during peroxide reduction
- glutathione reductase: reduction of GS-SG, regeneration of GSH
- reaction:
GS-SG 2 GS-H [NADPH + H+ NADP+]
- enzyme:
glutathione reductase
- distribution:
near glutathione peroxidase
- NADPH produced from oxidative reactions of pentose phosphate pathway
- importance of glutathione reductase underscores need for functional PP pathway
reactive oxygen species can damage most macromolecules
- nucleic acids: OH
- DNA damage near the site of generation, due to short hydroxyl radical half life
- sugar: causes strand breakage
- base: causes base modification (alteration, oxidation) or elimination
- prevalence: 104-105 DNA base modifications per cell per day, all of which must be repaired
- proteins: O2-, HOOH, OH
- O2-:
inactivation of Fe-S centers in metabolic enzymes
- HOOH: oxidation of sulfhydryl groups
- OH:
abstraction of a H atom (esp. histidine, proline, arginine, lysine)
- lipids: OH
- hydroxyl radical causes abstraction of H atom, and extensive peroxidation occurs
- this decreases membrane fluidity by affecting packing of fatty acid chains
repair of damage
- lipid peroxidation: glutathione peroxidase
- glutathione peroxidase specificity
- highly specific for glutathione
- relatively nonspecific for peroxide substrate
- function: repair of biomolecules converted to hydroperoxides by oxidation
- prevention: detoxification of HOOH
- repair: detoxification of ROOH, giving less damaging alcohols (particularly important in lipids)
- reactions
- hydrogen peroxide: 2 GSH + HOOH GS-SG + 2 HOH
- other peroxides:
2 GSH + ROOH GS-SG + ROH + HOH
- oxidized protein: glutathione peroxidase
- reaction: 2 GS-H + enz-S-S-R GS-SG + RSH + enz-SH
- function: restore sulfhydryls in oxidized proteins
- some proteins will not be repaired, and instead will be selectively degraded by proteosomes
- damaged DNA: host excision repair
clinical relevance of oxidative stress
- red blood cells and reactive oxygen species
- tissue requirements
- brain and RBCs must have glucose, as they are not able to utilize other forms of energy
- brain:
120 g glucose / day
- RBCs: 40 g glucose / day
- total:
160 g / day
- glycogen stores
- liver:
75 g glycogen available for export to the blood (lean, 70 kg human)
- liver glycogen cannot satisfy the needs of the brain and blood for more than half a day
- producing glucose and maintaining energy
- gluconeogenesis: de novo synthesis of glucose, with carbon derived from lactate, glycerol, and amino acids
- glycerol: released from triglyceride in adipose tissue
- amino acids: released from protein breakdown, primarily from muscle
- fasting state: glucagon
- stimulates glycogenolysis, gluconeogenesis, and export of glucose to blood
- stimulates lipolysis in adipose tissue, with resultant release of fatty acids into the blood
- fatty acids are used as an energy source by many tissues in lieu of glucose
- fatty acids are also used to make ketone bodies (-hydroxylbutyrate, acetoacetate), another energy source
- glucose is thus saved for use by the brain, RBCs
- some fatty acid fuel made available to liver is used to make ATP to drive gluconeogenesis
maintenance of blood glucose during fasting
- blood glucose concentration units
- clinical preference:
mg/dL (mg/100 mL, or mg %)
- scientific preference: mM
- MWglucose:
180 g/mol
mg
g
g
mol
(1.8 ) / (180
) 0.010
10 mM
dl
L
mol
L
- example:
180
- reference:
- 180 mg/dL = 10 mM
- 90 mg/dL = 5 mM
I
exogenous
use by all tissues
glucose
II
glycogen, hepatic
gluconeogenesis
muscle and adipose
tissue: diminished
rates
glucose
III
glycogen, hepatic
gluconeogenesis
muscle and adipose
tissue: gradual
cessation
glucose
IV
hepatic and renal
gluconeogenesis
brain, RBCs, renal
medulla only
V
hepatic and renal
gluconeogenesis
brain (diminished),
RBCs, renal medulla
glucose, ketone
bodies
37. Gluconeogenesis
Study Guide
know the following:
- gluconeogenesis: the process of synthesis of glucose from substrates such as lactate, amino acids, and glycerol
- gluconeogenesis from pyruvate: four new reactions to bypass the three irreversible steps (ATP required)
- reactions
- pyruvate kinase
- pyruvate carboxylase:
pyruvate oxaloacetate [biotin: H2CO3 ; ATP ADP + Pi]
- malate dehydrogenase:
oxaloacetate malate [NADH NAD+]
- (malate shuttle):
malatemitochondria malatecytoplasm
- malate dehydrogenase:
malate oxaloacetate [NAD+ NADH]
- PEP carboxykinase:
oxaloacetate phosphoenolpyruvate [GTP GDP; CO2]
- phosphofructokinase-1
- fructose 1,6-bisphosphatase-1:
fructose 1,6-bisphosphatase fructose 6-phosphate [H2O Pi]
- hexokinase / glucokinase
- glucose 6-phosphatase:
glucose 6-phosphate glucose [H2O Pi]
- regulation
- pyruvate kinase (liver): cAMP-dependent phosphorylation of protein kinase A
- pyruvate kinase-P:
inactive
low insulin, high glucagon
slows glycolysis
- pyruvate kinase:
active
high insulin, low glucagon
hastens glycolysis
- phosphoenolpyruvate carboxykinase (PEPCK)
- high insulin:
transcriptional inhibition
- high glucagon:
transcriptional activation
- phosphofructokinase-1 / fructose bisphosphatase-1
- FBPase: active with low fructose 2,6-bisphosphate (due to active FBPase-2)
- PFK-1: active with high fructose 2,6-bisphosphate (due to active PFK-2)
- FBPase-2 / PFK-2: regulation by protein kinase A and constitutive phosphoprotein phosphatase
- high insulin: low protein kinase A, active PFK-2, inactive FBPase-2
- high glucagon: high protein kinase A, active FBPase-2, inactive PFK-2
- gluconeogenesis from glycerol: two reactions to generate dihydroxyacetone phosphate
- glycerokinase:
glycerol glycerol 3-phosphate [ATP ADP]
- glycerol 3-phosphate dehydrogenase:
glycerol 3-P dihydroxyacetone-P [NAD+ NADH + H+]
- gluconeogenesis from amino acids: converted to TCA intermediates, which are converted to pyruvate
- alanine:
alanine pyruvate
[alanine aminotransferase: -KG Glu]
- aspartate:
aspartate oxaloacetate
[aspartate aminotransferase: -KG Glu]
- glutamate:
glutamate -ketoglutarate
[glutamate DH: H2O NH3; NAD+ NADH + H+]
Notes: Lecture and Reading
- gluconeogenesis: overview
- gluconeogenesis: process of making glucose from non-sugar materials
- uses many enzymes of glycolysis, but with bypass pathways around the irreversible reactions
- phosphate bond energy used to make these bypass reactions also irreversible
- considerations
- localization
- early fasting: liver, some kidney
- prolonged fasting: primarily kidney
- fatty acid oxidation: generates NTPs used during gluconeogenesis
- amino acid catabolism: generates sources of carbon for gluconeogenesis
phosphates are required for conversion of fructose 1,6-bisphosphate to glucose
- forward pathway (glycolytic)
- glucokinase:
glucose glucose 6-P [ATP ADP]
- phosphoglucose isomerase:
glucose 6-P fructose 6-P
- phosphofructokinase-1:
fructose 6-P fructose 1,6-bisP [ATP ADP]
- reverse pathway (gluconeogenic)
- fructose 1,6-bisphosphatase-1: fructose 1,6-bisP fructose 6-P [H2O Pi]
- phosphoglucose isomerase:
frucose 6-P gluctose 6-P
- glucose 6-phosphatase:
glucose 6-P glucose [H2O Pi]
- new enzymes
- fructose 1,6-bisphosphatase-1: bypasses the second irreversible reaction of glycolysis
- glucose 6-phosphatase: bypasses the first irreversible reaction of glycolysis
- genetic deficiencies in glucose 6-phosphatase
- frequency: 1/200,000
- symptoms: hypoglycemia during fasting, glycogen accumulation in liver (hepatomegaly)
gluconeogenesis from pyruvate
- forward pathway (glycolytic)
- pyruvate kinase:
phosphoenolpyruvate pyruvate [ADP ATP]
- reverse pathway (gluconeogenic)
- pyruvate carboxylase:
pyruvate + carbonic acid oxaloacetate [biotin: ATP ADP + Pi]
- malate dehydrogenase:
oxaloacetate malate [NADH NAD+]
- (malate shuttle):
malatemitochondria malatecytoplasm
- malate dehydrogenase:
malate oxaloacetate [NAD+ NADH]
- PEP carboxykinase:
oxaloacetate phosphoenolpyruvate [GTP GDP; CO2]
- mitochondrial transport
- oxaloacetate generated by pyruvate carboxylase must be exported from the mitochondria
- however, it must be converted to malate, as oxaloacetate is unable to be transported across the membrane
- net effect
- export of pyruvate via formation of phosphoenolpyruvate (and glycolytic bypass)
- export of reducing equivalents (to supply glyceraldehyde 3-P DH reaction of gluconeogenesis)
biotin as a vitamin
- biotin
- location: bound as an amide to the -amino of a lysyl residue on the enzyme
- function: carboxylations (addition of CO2, from H2CO3, to an organic linkage)
- reaction: - CO2 forms an unstable covalent intermediate with biotin
- it is subsequently transferred to the substrate
- deficiencies
- biotin: cofactor vitamin needed in very tiny amounts
- covalently bound to enzymes, so does not diffuse out of cells
- deficiencies therefore not usually produced by dietary shortages
- avidin: protein in raw egg white that tightly binds, complexes biotin
- this can produce a biotin deficiency
- cooking: renders avidin ineffective
gluconeogenesis from glycerol
- pathway
- glycerokinase:
glycerol glycerol 3-P [ATP ADP]
- energetics: irreversible
- localization: cytoplasm
- glycerol 3-P dehydrogenase: glycerol 3-P dihydroxyacetone phosphate [NAD+ NADH + H+]
- energetics: reversible
- localization: cytoplasm
- reactions are also used in triglyceride synthesis
gluconeogenesis from amino acids
- amino acids and gluconeogenesis
- each -amino acid corresponds in structure to an -keto acid of the TCA cycle
- thus one can make TCA intermediates by replacing the amino acid -amino groups with -keto groups
- example reactions
- alanine (C3) pyruvate
- aspartate (C4) oxaloacetate
- glutamate (C5) -ketoglutarate
regulation of gluconeogenesis
- energy requirements for gluconeogenesis
- during gluconeogenesis, nutritional requirements of liver must be met before glucose can be exported
- this is done primarily by fatty acid oxidation, producing high ATP, NADH, and acetyl-CoA
- this will allosterically slow glucose oxidation and promote gluconeogenesis
- regulation: major controls
- regulation occurs at the three irreversible steps of glycolysis
- glucagon: - promotes glucose output from the liver (glycogenolysis, gluconeogenesis)
- inhibits glucose utilization in the liver (glycogenesis, glycolysis)
- insulin:
- inhibits glucose output from the liver (glycogenolysis, gluconeogenesis)
- promotes glucose utilization in the liver (glycogenesis, glycolysis)
- regulation focuses on preventing futile cycles from occuring
- futile cycle: cycle of reactions that results in hydrolysis of ATP without doing useful work
- there are three possible cycles corresponding to the three irreversible glycolytic reactions
- phosphoenolpyruvate / pyruvate cycle
- pyruvate kinase:
phosphorylated and inactivated by protein kinase A (high glucagon)
- pyruvate carboxylase:
allosterically activated by acetyl-CoA
- pyruvate dehydrogenase:
causes inhibition of acetyl-CoA (activates a kinase, producing inactive PDH-P)
- PEP carboxykinase:
transcriptionally regulated by glucagon (activated) and insulin (inhibited)
- fructose 1,6-bisP / fructose 6-P cycle
- first level: regulation by ATP
- phosphofructokinase-1:
glycolysis
allosterically activated by ATP
- fructose 1,6-bisphosphatase-1:
gluconeogenesis
allosterically inhibited by ATP
- second level: reciprocal regulation by fructose 2,6-bisphosphate
- fructose 2,6-bisphosphate: regulatory molecule not otherwise involved in metabolism
- cycle regulatory reactions
- phosphofructokinase-1:
glycolysis
allosterically activated by fructose 2,6-bisP
- fructose 1,6-bisphosphatase-1:
gluconeogenesis
allosterically inhibited by fructose 2,6-bisP
- fructose 2,6-bisphosphate: production and degradation
- phosphofructokinase-2:
fructose 2,6-bisP
activated by phosphoprotein phosphatase
inactivated by protein kinase A
- fructose 1,6-bisphosphatase-2:
fructose 6-P
activated by protein kinase A
inactivated by phosphoprotein phosphatase
- fructose 2,6-bisphosphate: regulatory reactions
- phosphoprotein phosphatase:
PFK-2-P (inactive) PFK-2 (active) [H2O Pi]
FBPase-2-P (active) FBPase-2 (inactive) [H2O Pi]
- protein kinase A:
PFK-2 (active) PFK-2-P (inactive) [ATP ADP]
FBPase-2 (inactive) FBPase-2-P (active) [ATP ADP]
- third level: hormonal regulation of phosphofructokinase-2 and fructose 2,6-bisphosphatase-2
- PFK-2 / FBPase-2
- enzymes are part of a single, bifunctional protein, with activity dependent on phosphorylation
- phosphorylation (protein kinase A) inactivates PFK-2, activates FBPase-2
- dephosphorylation (phosphoprotein phosphatase) activates PFK-2-P, inactivates FBPase-2-P
- hormonal regulation
- glucagon: elevated cAMP, active protein kinase A, which promotes phosphorylation of PFK-2 / FBPase-2
- insulin: depressed cAMP, inactive protein kinase A, and PFK-2 / FBPase-2 in dephosphorylated form
- summary: effects of glucagon
- glucagon elevates cAMP levels, leading to active protein kinase A
- protein kinase A phosphorylates complex, activating FBPase-2 and inactivating PFK-2
- FBPase-2 dephosphorylates fructose 2,6-bisphosphate, causing decreased [fructose 2,6-bisP]
- PFK-2 is inactive, does not synthesize fructose 2,6-bisphosphate, causing decreased [fructose 2,6-bisP]
- decreased [fructose 2,6-bisP] leads to:
- increased gluconeogenesis (through more active FBPase-1)
- decreased glycolysis (through less active PFK-1)
- increased gluconeogenesis increases blood [glucose]
- glucose 6-phosphate / glucose cycle
- glucokinase:
allosterically inhibited by fructose 6-phosphate
transcriptionally activated by insulin
- glucose 6-phosphatase:
transcriptionally activated by glucagon
- note: glucose 6-phosphatase is located on inside of ER, so glucose 6-P must be transported there via a carrier
TABLE: Enzyme nomenclature
classical
fructose 6-P 1-kinase
fructose 6-P 2-kinase
fructose 1,6-bisP 1-phosphatase
fructose 1,6-bisP 2-phosphatase
glucose 6-P phosphatase
phosphoenolpyruvate carboxykinase
modern
phosphofructokinase-1
phosphofructokinase-2
fructose bisphosphatase-1
fructose bisphosphatase-2
glucose 6-phosphatase
PEP carboxykinase
abbreviation
PFK-1
PFK-2
FBPase-1
FBPase-2
G6Pase
PEPCK
- step 1:
total:
129 ATP (net)
- control of -oxidation
- once inside mitochondria, only rate of FAD, NAD+ regeneration control oxidation
- thus rate of electron transport controlled by ADP availability (respiratory control)
- genetic deficiencies in -oxidation
- three types of fatty acyl-CoA dehydrogenases
- long chain:
> C12
- medium chain:
C6 C12
- short chain:
< C6
- medium chain deficiencies common (1/10,000)
- symptoms: severe fasting hypoglycemia, but otherwise do well if prolonged fasting is avoided
- reason: alternate pathways of fatty acid oxidation, such as those in peroxisomes
- oxidation of odd-chain fatty acids
- oxidized normally until C5 acyl derivative is reached
- thiolytic cleavage with HS-CoA generates acetyl-CoA, propionyl-CoA
- propionyl-CoA enters metabolic sequence as succinyl-CoA (through methylmalonyl-CoA)
- -oxidation of unsaturated fatty acyl-CoA (SUPPLEMENTARY)
- unsaturated fatty acids occur in high concentrations in human depot fat
- two additional enzymes required for -oxidation
- first shifts cis double bond to trans double bond at the proper position
- second racemizes -hydroxy intermediate to the proper stereoisomer
formation and utilization of ketone bodies
- overview: ketone bodies
- examples:
acetone, acetoacetate (AcAc), and -hydroxybutyrate (OHB)
- synthesis:
response to extensive fasting, where fatty acids are major fuel (fasting, prolonged exercise)
- localization: liver mitochondria only
- regulation: acetyl-CoA accumulates because -oxidation is faster than TCA
- TCA allosterically slowed by NADH, citrate
- also allosterically slowed by high fatty acyl-CoA
- synthesis
- 2 acetyl-CoA acetoacetyl-CoA [ HS-CoA]
- acetyl-CoA + acetoacetyl-CoA -hydroxy--methylglutaryl-CoA (HMGCoA) [H2O HS-CoA]
- HMGCoA acetoacetate [ acetyl-CoA]
- acetoacetate acetone [ CO2]
- acetoacetate -hydroxybutyrate [-hydroxybutyrate dehydrogenase: NADH + H+ NAD+]
- utilization
- process
- AcAc and OHB transported from liver to extrahepatic tissues (primarily skeletal muscle, heart)
- -hydroxybutyrate dehydrogenase: in mitochondria, reversal of last reaction; converts OHB to AcAc
- transferase: acetoacetate + succinyl-CoA acetoacetyl-CoA + succinate
- acetoacetyl-CoA cleaved to acetyl-CoA (reversal of first reaction), entered into TCA cycle
- timing
- during prolonged starvation, brain adapts to use of ketone bodies as metabolic fuels
- skeletal muscle in well-conditioned athletes is more efficient at utilizing these compounds
- ketosis
- ketosis will result in net accumulation of H+, causing blood pH to fall (palmitate 4 acetoacetate- + 4 H+)
- ketosis: accumulation of ketone bodies in the blood
- ketonuria: accumulation of ketone bodies in the urine
- acidosis: lowering of blood pH
- starvation leads to ketoacidosis, though milder than what is seen in diabetis
ketone bodies and gluconeogenesis
- ketone bodies formed under same physiological conditions as what favors gluconeogenesis
- cannot be converted in net amounts to glucose: acetyl-CoA oxidized to two CO2 in one turn of the TCA cycle
summary: key regulatory events governing fat catabolism
- stored body fat catabolized in any fast lasting more than 4-6 hours
- initiated by drop in I/G in blood, which declines to minimum after 2-3 days of fasting
- leads to activation of lipolysis and the following:
- adipose: increased cAMP active PKA active hormone-sensitive lipase, increase in FA and glycerol
- liver takes up fatty acids, transports into mitochondria
- due to active CoA:carnitine acyl transferase (CCAT) resulting from low malonyl-CoA
- malonyl-CoA low because FA synthesis not taking place due to low insulin
- fatty acid oxidation keeps ATP/ADP and NADH/NAD+ ratios high in most tissues
- rate of fatty acid oxidation limited only by rate of electron transport
- this is governed by availability of NAD+ and ADP
- citrate synthase: inhibited in liver by FACoA, NADH, resulting in acetyl-CoA accumulation in mitochondria
- increase in mitochondrial acetyl-CoA gives ketone body synthesis by mass action
- (esterification):
2 FA-CoA + G3P L-phosphatidic acid (diglyceride-P) [ 2 CoASH]
- (hydrolysis):
L-phosphatidic acid diglyceride [H2O Pi]
- (esterification):
diglyceride + FA-CoA triglyceride [ CoASH]
- triglyceride transport: attached to VLDL, transported to target tissue through bloodstream
- triglyceride unloading
- hydrolysis by lipoprotein lipase, forming free FA and glycerol
- glycerol: transported back to liver
- FA: taken in by cells, resynthesized as triglycerides
- with enough triglyceride removal, VLDL LDL
- regulation: fatty acid biosynthesis
- acetyl-CoA carboxylase
- insulin
- allosteric: insulin-dependent kinase; phosphorylation and inactivation
- adaptive: IRE induces synthesis of acetyl-CoA carboxylase and other lipogenic enzymes
- glucagon
- allosteric: cAMP activates PKA; phosphorylation and inactivation at different site
- polyunsaturated fatty acids:
- allosteric: polyunsaturated fats suppress induction of lipogenic enzymes
- malonyl-CoA: accumulates when acetyl-CoA carboxylase is active
- function: inhibits CoA:carnitine acyl transferase
- logic: prevents oxidation of newly-synthesized fatty acyl-CoA, indirectly pushes towards FA esterification
- lipoprotein lipase: activated by insulin
- GLUT-4 transporter: activated by insulin
Notes: Lecture and Reading
overview of fat synthesis and storage in the fed state
- de novo fatty acid biosynthesis
- locations
- liver: major site
- adipose tissue: minor site
- liver
- glucose intake
- after a meal, concentration of glucose and insulin will be highest in the portal vein
- portal vein: flow from the gut to the liver (strategic for metabolism)
- GLUT-2: high Km transporter used to take glucose into the liver, even at high concentrations
- glucokinase: high Km glucose phosphorylation protein
- glucose that is not taken in is allowed to flow through the rest of the body
- fatty acid synthesis
- glucose is converted to glycogen, also metabolized in glycolysis to pyruvate
- pyruvate is converted to acetyl-CoA, some of which enters TCA and some of which enters FA synthesis
- requires NADPH for biosynthetic reactions
- NADPH derived partially from pentose phosphate pathway
- fatty acids esterified to triglycerides
- transport: triglycerides attached to VLDL and put into circulation
- adipose tissue
- storage: triglyceride is removed from VLDL, stored in the tissue
- synthesis: triglyceride can be made from the same reactions as used in the liver
- dietary absorption
- absorption: intestinal mucosa cells
- transport: packaged on lipoprotein particles called chylomicrons, transported to blood via lymph
- storage: removed from chylomicrons at adipose tissue, stored there
DE NOVO FATTY ACID SYNTHESIS AND TRIGLYCERIDE STORAGE
de novo synthesis of fatty acids
- overview: palmitic acid
- synthesis: cytoplasm
- 8 acetyl-CoA + 14 (NADPH + H+) + 7 ATP + H2O palmitic acid + 8 HSCoA + 14 NADP+ + 7 ADP + 7 Pi
- enzymes: acetyl-CoA carboxylase, fatty acid synthase
- other enzymes then modify palmitic acid (desaturation, elongation)
- acetyl-CoA
- source: glycolysis (cytoplasm), pyruvate dehydrogenase (mitochondrial matrix)
- because pyruvate DH occurs in the mitochondria, a transport system (the citrate shuttle) is necessary
- citrate shuttle
- each cycle
- transfers one acetyl-CoA from mitochondrial matrix to cytoplasm
- generates one NADPH from NADP+
- repetitions: palmitate synthesis
- 8 shuttles required to generate the 8 acetyl-CoA
- 8 NADPH are supplied by these shuttles, but 6 more are required
- other 6 NADPH come from pentose phosphate pathway
- citrate shuttle: provides cytosolic acetyl-CoA, NADPH
- citrate synthase:
oxaloacetate + acetyl-CoA --> citrate [H2O , HSCoA]
- (carrier protein, MC):
citratemitochondria citratecyt
- ATP citrate lyase:
citrate oxaloacetate + acetyl-CoA [HSCoA , ATP ADP + Pi]
- malate dehydrogenase:
oxaloacetate malate [NADH + H+ NAD+]
+
- NADP -dependent malate DH: malate pyruvate + CO2 [NADP+ NADPH + H+]
- (carrier protein, CM):
pyruvatecyt pyruvatemitochondria
- pyruvate carboxylase:
pyruvate + H2CO3 oxaloacetate [ATP ADP + Pi]
- net cost: 2 ATP
- pentose phosphate: remainder of required NADPH
- glucose 6-P 6-phosphogluconolactone [NADP+ NADPH + H+]
- 6-phosphogluconolactone 6-phosphogluconate [H2O ]
- 6-phosphgluconate ribulose 5-phosphate [ CO2, NADP+ NADPH + H+]
- net gain: 2 NADPH per glucose
- acetyl-CoA carboxylase: first committed step in fatty acid biosynthesis
- reaction:
H2CO3 + acetyl-CoA malonyl-CoA [biotin: ATP ADP + Pi]
- enzyme:
acetyl-CoA carboxylase
- mechanism: analogous to pyruvate carboxylase
- energetics: rate-limiting, first committed step in fatty acid biosynthesis
- fatty acid synthase
- net reaction: acetyl-CoA + 7 malonyl-CoA + 14 (NADPH + H+) + H2O
palmitic acid + 14 NADP+ + 7 CO2 + 8 HSCoA + 7 H2O
- mechanism: - malonyl acyl group of malonyl-CoA transferred to HS-ACP (acyl-carrier protein)
- acetyl group of acetyl-CoA transferred to another reactive sulfhydryl group on HS synthase
- acetyl group condenses with methylene group of malonyl-CoA, giving off CO2
- -keto group reduced to methylene group by three sequential reactions (rev. of -oxidation)
- ketone reduction to alcohol [NADPH + H+ NADP+]
- dehydration of alcohol to alkene [ H2O]
- alkene reduction to alkane [FADH2 FAD FADH2 (NADPH + H+ NADP+)]
- elongating chain transferred to HS synthase, malonyl-CoA attaches to ACP, and cycle restarts
- modification of palmitate
- palmitic acid: can be further elongated, desaturated by other enzymes
- must first be activated to palmitoyl-CoA before it can be modified or incorporated into a triglyceride
synthesis of triglycerides and storage
- synthesis
- precursor: must begin with glycerol 3-phosphate
- glycerol 3-P dehydrogenase (several tissues): dihydroxyacetone phosphate glycerol 3-phosphate
- glycerokinase (liver only): glycerol glycerol 3-phosphate
- glycerol 3-phosphate + 2 fatty acids L-phosphatidic acid + 2 HS-CoA
- L-phosphatidic acid: can be used in phospholipids
- intestinal cells
- absorption:
mucosal cells lining intestine
- esterification:
synthesis of triglycerides from hydrolysis products (ATP-requiring process)
- secretion:
triglycerides secreted from intestinal cells into blood
- blood stream
- transport:
triglycerides transported through blood on chylomicrons (CM)
- hydrolysis:
triglycerides hydrolyzed to glycerol, fatty acids
- target tissue
- absorption:
free fatty acids absorbed into target tissues
- esterification:
synthesis of triglycerides from hydrolysis products (ATP-requiring process)
- fat soluble vitamins: A, D, E, K
- vitamins A, D, E, and K are dissolved in fat; fat must be emulsified for vitamin absorption to occur
- vitamins incorporated into chylomicrons, typically reach liver in CMR
- deficiencies in fat absorption: lead to deficiencies in fat-soluble vitamins, essential fatty acids
Notes: Lecture and Reading
DIGESTION AND ABSORPTION OF DIETARY FAT
- lipids: overview
- lipids: class of biological molecules soluble in organic solvents but not water
- examples: triglycerides, phospholipids, sphingolipids, cholesterol, steroids, fat-soluble vitamins
absorption of dietary triglycerides
- emulsification
- purpose
- lipid enters the diet as large insoluble droplets that are incapable of absorption by intestinal epithelium
- emulsification increases solubility, making them better substrates for hydrolytic lipases
- micelles: amphipathic spherical aggretates with a hydrophobic core and hydrophilic surface
- detergent: amphipathic molecules capable of disrupting hydrophobic interactions, dispersing into mixed micelles
- biliary secretions: synthesized in liver, stored in gall bladder, secreted into duodenum during digestion
- bile acids: wedge-shaped steroid molecules with good detergent properties; returned to liver via portal vein
- phosphatidylcholine: component of the micelles
- cholesterol: component of the micelles
- pancreatic lipase
- origin:
synthesized in pancreas, secreted into duodenum
- function:
hydrolysis of emulsified triglyceride, typically at 1, 3 positions
- yields a 2-acyl monoglyceride
- fatty acid can migrate to 1 or 3 position for complete hydrolysis, though this is not necessary
- approximately 60% of ingested triglyceride is as 2-acyl monoglyceride
- absorption and reesterification
- absorption: fatty acids, monoglycerides absorbed by mucosal cells lining the intestine
- resynthesis: intestinal mucosa resynthesized triglycerides from the hydrolysis products
- fatty acids must be activated to fatty-acyl CoA, an ATP-requiring process (ATP AMP + PPi)
- fatty acyl-CoA derivatives used to synthesize triglycerides
2-acyl-monoglyceride + 2 FA-S-CoA triglyceride + 2 HS-CoA
- secretion into the blood
- transport in the blood is accomplished via a lipoprotein complex called a chylomicron
- chylomicron
- composition: apoproteins and other lipids such as cholesterol
- function: serves as transport vehicle for triglyceride in the blood
- distribution: once loaded, are secreted from mucosal cells into lymph, pass into venous blood via thoracic duct
- utilization of absorbed lipid
- can supply to numerous tissues, depending on the nutritional state of the animal
- process
- lipoprotein lipase: hydrolyzes triglycerides into glycerol and fatty acids (activated by insulin)
- fatty acids are taken in by the target, and the remaining glycerol is taken in by the liver
- chylomicron remnant (CMR): cholesterol-rich remains of chylomicron after triglyceride removal
- modification
- in the intestine, conjugated acids are hydrolyzed to regenerate the free carboxyl group, glycine, and taurine
- secondary bile acids: some primary bile acids modified by bacteria, resulting in loss of 7-hydroxy group
- enterohepatic circulation: primary means of getting rid of cholesterol
- bile: major components and molar ratio
- bile acids:
16
- phosphatidylcholine (lecithin):
3
- cholesterol:
1
- components are synthesized in the liver, stored in the gall bladder
- cholesterol and bile acids
- cholesterol is highly insoluble
- requires bile acids to emulsify the cholesterol and form mixed micelles with lecithin
- cholestasis: cholesterol proportion too high, resulting in insoluble gall stones and obstruction of the bile duct
- circulation
- circulated through intestine, absorbed by ileum, returned to liver via portal vein
- 15-30 g of circulation (2-4 g pool of bile acids, which circulates 5-10 times daily)
- reabsorption: both primary and secondary bile acids
- excretion
- bile acids: 0.2-0.6 g/day pass into feces, representing the major means of cholesterol excretion
- cholesterol: 1 g secreted into intestine; only about 0.5 g reabsorbed
- net steroid secretion: bile acids + cholesterol = approximately 1 g
- other components of the bile
- bilirubin: breakdown product of heme catabolism; transported to liver via albumin forexcretion
- biliary obstruction: can lead to accumulation of bilirubin in the liver, ultimately in the blood
- jaundice: accumulation of bilirubin or bilirubin diglucouronide in the blood, causing yellow discoloration of skin
- dietary absorption
- intestinal mucosa
- emulsification:
formation of mixed micelles from dietary cholesterol esters, free cholesterol
- hydrolysis:
cholesterol esterase of emulsified cholesterols, forming free cholesterol
- intestinal cells
- absorption:
mucosal cells of small intestine (free cholesterol only)
- esterification:
limited amounts of free cholesterol esterified
- bloodstream
- transport:
free cholesterol, CE incorporated into chylomicrons
triglycerides dumped into target tissues, forming CMR
- liver
- endocytosis:
CMR receptor-mediated endocytosis absorbs CMR
- metabolism:
cholesterol esters hydrolyzed, free cholesterol used in biosynthesis or storage
- homeostasis: input (diet, de novo) must be balanced against output (bile)
- transcriptional control: HMGCoA reductase and other lipogenic enzymes
- high cholesterol:
binds, inactivates steroid regulatory element (SRE) and binding protein (SREBP)
- high insulin:
binds, activates SREBP, increasing HMGCoA reductase and other lipogenic enzymes
- dietary control
- high cholesterol:
decreased HMGCoA reductase activity (sensed by CMR, LDL taken in by liver)
- low cholesterol:
increased HMGCoA reductase activity
- caloric intake:
increases insulin, circulating FA, which promote hepatic VLDL synthesis
- fatty acid ration:
increased polyunsaturated inhibits SRE-mediated events, causing cholesterol decrease
- cholesterol regulation: central role of liver
- regulates synthesis inversely with dietary cholesterol, caloric intake
- regulates excretion through synthesis of bile components (cholesterol, bile acids)
- regulates plasma lipoprotein (VLDL, nascent HDL) synthesis
- regulates uptake, catabolism of LDL and HDL
- liver transport to extrahepatic tissues
- VLDL catabolism: forms LDL, which can be taken in by numerous tissues
- classic LDL receptor-mediated endocytosis
- binding:
LDL binds high affinity receptor in clatharin-coated plasma membrane pits
- endocytosis:
- maturation:
- metabolism:
LDL is internalized
receptor returned to surface; endosome merges with lysosome, lipoprotiens degraded
biosynthesis: cell membranes, synthetic reactions
storage: esterification via ACAT, storage in lipid droplets
- regulation:
HMGCoA reductase: feedback inhibition by cholesterol internalized by this mechanism
LDL receptors: reduced in number by cholesterol feedback
ACAT: activated by excess cholesterol
- scavenger cell pathway: not well characterized; known to utilize another type of receptor
- active cells: macrophages, histiocytes (reticuloendothelial system); Kupffer cells (macrophages)
- atherosclerosis: foam cells in plaques derived from macrophages in the scavenger cell pathway
- most cholesterol is still removed using the classic LDL receptor-mediated endocytosis
- HDL function 1: surface-lipid acceptor in chylomicron, VLDL catabolism
- HDL: formed from nascent HDL precursors
- synthesis: may be directly synthesized by the liver
- catabolism: may form from surface coat of chylomicrons during chylomicron catabolism
- triglyceride removal from chylomicron, VLDL cores requires surface shrinking; done through HDL maturation
- lecithin:cholesterol acyl transferase (LCAT)
- function: esterifies cholesterol to prevent transfer back to VLDL, chylomicrons
- mechanism: transfers fatty acyl group from phospholipid (lecithin) to 3-OH of cholesterol, both at the surface
- HDL function 2: exchange of essential apoproteins between lipoprotein functions
- CM, VLDL: immature lipoproteins
- require transfer of specific apoproteins to, from HDL to complete metabolism and maturation
- HDL function 3: reverse cholesterol transport
- free cholesterol in membrane bilayers is exchangable
- LCAT: helps keep cholesterol locked into the lipoprotein
- nascent HDL, small spherical HDL: can remove excess cholesterol from tissue plasma membranes
- spherical HDL: transports the excess cholesterol ester to the liver for catabolism and excretion
- direct process: receptor-mediated endocytosis, catabolism of secondary lysosome
- indirect process: HDL transfer to VLDL, via cholesterol ester transfer protein
- cholesterol initially derived from excess extrahepatic free cholesterol
- metabolized to LDL before transferal to liver
Notes: Lecture and Reading
structure of cholesterol
- carbon backbone: 27C
- forms
- free form: unesterified, with alcohol group on C3; most common form in membranes
- esterified form: fatty acid chain ester-linked to C3; storage form, present as intracellular lipid droplets
- acyl-CoA:cholesterol acyl transferase (ACAT): catalyzes esterification of free cholesterol
sources of cholesterol
- dietary intake
- source: animal tissue, especially eggs, dairy products, and meat; less common in poultry, fish
- prevalence: varies with diet
- plants: lack cholesterol, but make ergosterol, an analog sterol
- synthesis
- source: de novo synthesis from acetyl-CoA
- prevalence: 60%, even in a high cholesterol diet (average: 300 mg via intake, 800 mg via synthesis)
synthesis of cholesterol
- distribution
- normal dietary intake: primary source is liver, though synthesis probably occurs in all tissues except RBCs
- low dietary intake: small intestine becomes a major producer in order to help with chylomicron synthesis
- much of the liver cholesterol is exported to the blood in lipoproteins, or converted to bile acids
- carbon source: acetyl-CoA
- all carbon in cholesterol is derived from acetyl-CoA
- initial steps: same as ketone body synthesis
- endocytosis of HDL
- key points
- dietary fat: drives chylomicron synthesis and cholesterol input (exogenous pathway)
- dietary CHO: drives VLDL synthesis (endogenous pathway)
- density
- triglycerides are relatively lighter than proteins
- removal of triglycerides causes a relative increase in density
transfer of cholesterol to extrahepatic (non-liver) tissues
- overview
- lipoproteins: form by which absorbed or synthesized cholesterol is returned to the liver
- VLDL: used to transport triglycerides from liver to adipose, giving off LDL
- LDL is the major source of cholesterol for non-hepatic tissues
- post absorptive state (12 h fast): 2/3 of total plasma cholesterol carried on LDL
- LDL carried to and metabolized by numerous tissue, including liver
- de novo synthesis inhibited as LDL cholesterol from LDL is taken up
- classic LDL receptor-mediated endocytosis
- binding
- LDL binds high affinity receptor localized in clatharin coat pits on plasma membrane
- internalized by endocytosis
- endosome maturation
- receptor returned to cell surface
- endosome fuses with lysosome
- lipoproteins degraded to monomeric constituents (cholesterol, fatty acids, amino acids, etc.)
- cytoplasmic free cholesterol: can be placed in cell membranes or used in other synthetic reactions
- excess free cholesterol
- esterified by acylCoA:cholesterol acyl transferase (ACAT) [FACoA + cholesterol chol. E + HSCoA]
- stored in lipid droplets
- regulation
- HMGCoA reductase: feedback inhibition by cholesterol internalized by this mechanism
- LDL receptors: reduced in number by cholesterol feedback
- ACAT: activated by excess cholesterol
- scavenger cell pathway
- plasma LDL
- has a half life of 12-24 hours
- up to 2/3 of LDL removed from plasma done via the classical LDL receptor pathway
- 1/3 or more of LDL removed via scavenger cell pathway
- scavenger cell pathway
- not well characterized; known to utilize another type of receptor
- active cells: macrophages, histiocytes (reticuloendothelial system); Kupffer cells (macrophages)
- atherosclerosis: foam cells in plaques derived from macrophages in the scavenger cell pathway
lipoprotein metabolism and the role of HDL
- overview: roles of HDL
- regulation: promotes catabolism of chylomicrons and VLDL
- reverse transport: carries excess cholesterol from extrahepatic tissues to the liver for excretion
- origin of HDL
- mature HDL: formed from nascent HDL precursors
- synthesis: may be directly synthesized by the liver
- catabolism: may form from surface coat of chylomicrons during chylomicron catabolism
- nascent HDL
- morphology: disc-shaped
- composition: phospholipids, cholesterol in a bilayer, complexed with apoproteins
- normally not observed in plasma in people with normal LCAT function
- lecithin:cholesterol acyl transferase (LCAT)
- function: conversion of nascent HDL to spherical HDL
- surface molecules (cholesterol, phospholipids, larger apoproteins) transferred to nascent HDL
- LCAT produces cholesterol esters that will not transfer back
- with accumulation, nascent HDL is converted to spherical HDL, with a cholesterol ester core
- mechanism
- transfers fatty acyl group from phospholipid (lecithin) to 3-OH of cholesterol, both at the surface
- cholesterol migrates to the interior of the lipoprotein particle
- role of HDL in lipid transfer during chylomicron and VLDL catabolism
- structure: chylomicrons, VLDL
- core: numerous triglycerides, smaller amount of cholesterol ester
- surface: apoproteins, phospholipid, cholesterol
- lipid transfer
- with triglyceride removal, core shrinks, and surface space must be reduced as well
- surface molecules transferred to HDL particles, with resultant increase in HDL size
- with increase in HDL surface particles, hydrophobic core must increase in size as well
- core molecules are esterified by lecitin:cholesterol acyl-transferase (LCAT)
- process: similar to that of conversion of nascent HDL to spherical HDL
- role of HDL in protein transfer during chylomicron and VLDL maturation
- CM
- chylomicron secreted by intestinal mucosa
- composition: triglyceride core, shell of phospholipid, cholesterol, and apoproteins
- apoproteins: AI, AII, AIV, B48
- CM
- generated from apoprotein exchange with HDL
- CM: receives apoCII, apoE
- spherical HDL: receives apoAI, apoAII
- apoprotein functions
- apoCII: activator of lipoprotein lipase
- apoE: protein recognized along with apoB by the hepatic CMR receptor
- VLDL
- lacks apoCII
- must receive apoCII from spherical HDL before VLDL can be catabolized by lipoprotein lipase
- role of HDL and LCAT in reverse cholesterol transport
- free cholesterol in membrane bilayers is exchangeable
- LCAT: helps keep cholesterol locked into the lipoprotein
- deficiencies: net transfer of cholesterol from free-cholesterol enriched plasma lipoproteins to membranes
- nascent HDL, small spherical HDL: can remove excess cholesterol from tissue plasma membranes
- cholesterolmembrane cholesterolHDL cholesterol esterHDL [LCAT]
- LCAT forces equilibrium to right, resulting in cholesterol removal
- spherical HDL: transports the excess cholesterol ester to the liver for catabolism and excretion
- direct process: receptor-mediated endocytosis, catabolism of secondary lysosome
- indirect process: transfer to VLDL
- transfer occurs via a plasma cholesterol ester transfer protein
- after conversion of VLDL to LDL, would be transferred to liver
deficiencies in lipoprotein metabolism
- dyslipidemia: major elevation or reduction of one or more of the major classes of lipid proteins
- hyperlipoproteinemias: most common
- originally classified based on whether triglyceride, cholesterol, or both are elevated
- now further typing based on the class of lipoproteins (e.g.chylomicrons, VLDL)
- hypercholesterolemias
- cause: usually by elevated LDL, VLDL, with the LDL having the highest cholesterol content
- elevated VLDL: would also cause elevated total plasma triglyceride
cholesterol and atherosclerosis
- atherosclerosis: accumulation in arterial tunica intima of plaque-like lipids which undergo calcification
- patterns
- strong correlation between circulating cholesterol / frequency of atherosclerosis, coronary heart disease (CHD)
- stronger correlation with LDL; inverse correlation with HDL
- clinical considerations
- high HDL seems to prevent atherosclerosis
- increase in HDL with decrease in LDL slows progression of atherosclerosis, sometimes even reversing it
- Tangier disease
- defect in ABC1 transporter (transport out of cholesterol)
- symptoms: yellow throat, caused by fatty/cholesterol accumulation
- discovered in 1961; cause discovered in 1999
TABLE: Function of Select Lipoproteins
apoprotein disease where role or function
AI
- major component of HDL
- activator of LCAT
B48 and
- major structural component of chylomicrons (B48),
B100
VLDL (B100), LDL (B100)
- protein recognized by LDL receptor
CII
- present on mature chylomicrons and VLDL
- activator lipoprotein lipase
CIII
- major component of chylomicrons, VLDL, and IDL
- prevents recognition of B and E rich lipoproteins by
hepatic CMR receptor and LDL receptor
E
- required with aboB on chylomicron remnant for
recognition by hepatic remnant receptor
TABLE: Lipoprotein Compositions
protein (%)
type
chylomicrons (ULDL)
very low density (VLDL)
low density (LDL)
high density (HDL)
2
10
20
46
defective
- abeta or hypobeta
- lipoproteinemia
- familial apoCII deficiency
cholesterol (%)
free
phospholipid
(%)
triglyceride
(%)
mol. wt.
ester
3
5
10
4
4
8
42
20
6
12
20
25
85
65
8
5
108 - 109
107 - 108
2-4 x 106
2-3 x 105
- glycolysis, pyruvate dehydrogenase: provides acetyl-CoA to TCA, maintains high ATP, acts in lipogenesis
- glycogenesis:
stores glucose as glycogen (primarily in liver and muscle)
- pentose phosphate pathway:
NADPH for fatty acid synthesis
- citrate shuttle:
cytoplasmic acetyl-CoA, NADPH for fatty acid synthesis
- fatty acid synthesis:
acetyl-CoA carboxylase, fatty acid synthase
regulation
- factors governing glucose oxidation
- glucose entry, phosphorylation: initial steps of glycolysis
- glucokinase (liver): synthesis induced by insulin
- GLUT-4: number in membrane increased by insulin
- phosphofructokinase: key control of glycolysis
- active due to high fructose 2,6-bisphosphate
- high I/G: low cAMP
- low cAMP: active PFK-2, inactive FBPase-2 (dephosphorylated)
- key control, promoting glycolysis despite high ATP (which would normally inhibit)
- pyruvate kinase: final step of glycolysis
- high I/G: low cAMP
- low cAMP: active pyruvate kinase (dephosphorylated)
- synthesis also induced by insulin
- pyruvate dehydrogenase: initial step in TCA cycle, forming acetyl-CoA
- active due to low acetyl-CoA (a feedback inhibitor)
- low acetyl-CoA due to low fatty acyl-CoA
- acetyl-CoA: funneled to TCA, rather than gluconeogenesis
- citrate synthase: active due to low mitochondrial fatty acyl-CoA
- increased fatty acid synthesis: high malonyl-CoA
- high malonyl-CoA: inhibits CoA:carnitine acyl transferase (CCAT)
- inhibited CCAT: low mitochondrial fatty-acyl-CoA
- low fatty acyl-CoA: active citrate synthase
- mitochondrial NADH
- with increased energy, mitochondrial NADH levels will rise
- this will cause inhibition of isocitrate dehydrogenase
- this results in an increase in citrate levels
- glucose 6-phosphate utilization
- glycogen: synthesized as an energy store
- glycogen synthase active, glycogen phosphorylase inactive (dephosphorylated)
- dephosphorylated form: due to low cAMP
- pentose phosphate pathway: used to provide NADPH to fatty acid biosynthesis
- NADPH: used in fatty acid biosynthesis, causing decline
- glucose 6-P dehydrogenase
- inhibited by NADPH; low NADPH thus causes increase in activity
- synthesis: induced by insulin
- fatty acid synthesis
- citrate shuttle: used to give a steady supply of acetyl-CoA to fatty acid biosynthesis
- citrate: accumulates in mitochondria due to slowed TCA cycle, spills into cytoplasm
- ATP citrate lyase: synthesis promoted by insulin
- NADP+-dependent malate DH: synthesis promoted by insulin
- pyruvate carboxylase: activated by high acetyl-CoA (usually always high enough to activate)
- acetyl-CoA carboxylase: key control point of fatty acid synthesis, resulting in increased malonyl-CoA
- activated by insulin-dependent phosphorylation
- activated by reversal of cAMP-dependent phosphorylation (constitutive phosphoprotein phosphatase)
- induced by high insulin
- fatty acid synthase: induced by insulin
- triglyceride synthesis and storage
- CCAT: functions to import fatty acids to mitochondria
- inhibited by high malonyl-CoA
- prevents oxidation, diverting fatty acids by mass action into triglyceride synthesis
- acetyl-CoA:
- fatty acyl-CoA
increased
increased
inhibits pyruvate DH
inhibits citrate synthase
- muscle:
branched chain (Val, Ile, Leu); exports Ala (taken by liver), Gln (taken by kidney, liver)
- kidney:
Gln (deamidinated to Glu, releasing NH3); Glu converted to Ala and exported
central reactions and role of glutamate
- amino acid transaminase: transamination
- reaction:
-AA + -ketoglutarate -keto acid + glutamate [PLP]
- enzyme:
amino acid transaminase
- mechanism: transamination (amino, keto groups essentially switched)
- energetics: reversal; requires pyridoxal phosphate as a cofactor
- use:
ALL transaminases use -ketoglutarate and glutamate as a substrate/product pair
- glutamate dehydrogenase: oxidative deamination
- reaction:
glutamate -ketoglutarate [H2O NH3; NAD+ NADH + H+]
- enzyme:
glutamate dehydrogenase
- mechanism: deamination, replacement with hydroxyl group; reduction to ketone
- energetics: reversible; often coupled to transaminations
- location:
mitochondrial matrix
- glutamate synthetase and glutaminase
- glutamine synthetase
- reaction: glutamate glutamine [NH3 ; ATP ADP + Pi]
- enzyme: glutamine synthetase
- glutaminase
- reaction: glutamine glutamate [H2O NH3]
- enzyme: glutaminase
- purpose: Gln can be used as a transport vehicle for NH3 in the blood, as in nitrogen excretion
- pyridoxine (vitamin B6)
- solubility:
water-soluble
- enzyme cofactor:
pyridoxal phosphate (PLP) or pyridoxamine phosphate
- distribution:
transaminase, dehydratase reactions
- glutamine as N-donor in purine, pyrimidine synthesis (SUPPLEMENTARY)
- Gln PRPP purines
- Gln carbamoyl-P [CP synthetase II] pyrimidines
47. Amino Acid Catabolism Excretion of Nitrogen as Urea and CSkeleton Metabolism
Study Guide
know the following:
- toxicity of ammonia
- 90 % of nitrogen excreted from body is from amino acid catabolism
- ammonia is toxic
- -ketoglutarate, a TCA cycle intermediate, is converted to Glu in the presence of excess ammonia
- on certain tissues, especially the brain, this can be devastating
- most excess ammonia is therefore converted to urea, a non-toxic product, prior to excretion
- ammonia generation
- transamination: Ala, Asp, others
- AA + -ketoglutarate -KA + glutamate [AA aminotransferase, PLP]
- glutamate -ketoglutarate [glutamate dehydrogenase: NAD+ NADH + H+; H2O NH3]
- nonoxidative dehydrogenation: Ser, Thr
- serine pyruvate [serine dehydratase, PLP: NH3]
- threonine propionyl-CoA [threonine dehydratase, PLP: NH3]
- deamidination: Gln, Asn
- glutamine glutamate [glutaminase: H2O NH3]
- malate DH:
malate oxaloacetate [NAD+ NADH + H+]
- aspartate aminotransferase:
oxaloacetate + glutamate aspartate aminotransferase + -ketoglutarate
- AA aminotransferase:
-AA + -ketoglutarate -keto acid + glutamate
- regulation of urea synthesis
- allosteric activation: N-acetylglutamate
- N-acetylglutamate: synthesized from glutamate, acetyl-CoA
- increases in dietary protein lead to increases in N-acetylglutamate (unknown mechanism)
- hormonal activation: glucagon
- glucagon increased cAMP synthesis of the enzymes of the urea cycle
- dietary amino acids: promote glucagon release from cells of the pancreas
- birth defects in urea cycle enzymes
- severe urea cycle deficiency: 1 / 30,000 newborn infants
- extent varies widely
- those with complete deficiencies may die from hyperammonemia after first protein feeding
- aggressive therapy must be begun immediately
entry of carbon chains into the central pathways of metabolism
- Phe: phenylalanine metabolism and phenylketonuria (PKU)
- normal metabolism
- phenylalanine hydroxylase: phenylalanine tyrosine
- transaminase:
tyrosine -keto acid
-keto acid fumarate, acetoacetate
- importance
- phenylalanine is an essential amino acid, tyrosine is not
- phenylalanine is the means by which tyrosine is generated
- defect: phenylketonuria
- etiology: defect in phenylalanine hydroxylase
- dietary phenylalanine accumulates
- metabolized to phenylpyruvate, then to phenyllactate and phenylacetate, which accumulate
- symptoms: neurological disorders, mental retardation
- frequency: carriers: 1/50 to 1/60
afflicted: 1/10,000
- screening: measurement of serum Phe levels after birth; required in Wisconsin screening programs
- treatment: affected infants placed on a synthetic low-phenylalanine formula, diet
- Val, Ile, and Leu: branched chain amino acid metabolism
- common path metabolism: initial reactions carried out by enzymes recognizing all three amino acids
- branched chain AA transaminase
- reaction:
AA + -ketoglutarate -keto acid + glutamate
- enzyme:
branched chain amino acid transaminase
- specificity:
Val, Ile, Leu
- branched chain -keto acid dehydrogenase
- reaction:
-keto acid R-CO-SCoA [HS-CoA CO2; NAD+ NADH + H+]
- enzyme:
branched chain -keto acid dehydrogenase
- specificity:
Val, Ile, Leu
- subsequent metabolism: glucogenic vs. ketogenic amino acids
- glucogenic: can give rise to glucose
- examples: Val, Ile
- give rise to propionyl-CoA, which can give a net conversion to glucose
- ketogenic: cannot be used to make glucose
- examples: Leu
- gives rise to acetyl-CoA, which cannot be used to make a net conversion to glucose
- maple syrup urine disease
- etiology: defect in branched chain -keto acid dehyrdogenase, causing keto acid accumulation
- symptoms: ketosis, neurological problems
- frequency: afflicted: 1/250,000
- screening: originally part of the Wisconsin screening program; no longer, due to low frequency
- B12:
anemia resulting from poor thymidilate synthetase, immature blood cell production
homocystinuria resulting from accumulation of homocysteine due to lack of B12
anemia resulting from secondary folate deficiency due to the folate trap
accumulation of methylmalonates resulting from lack of B12-dependent mutase reaction
Study Guide
part I know the following:
- primary electrolyte composition of body fluids
- intracellular anions:
K+, Mg2+
cations:
organic phosphates, proteins
- extracellular: anions:
Na+
cations:
Cl-, HCO3- fluid balance
- intracellular: cellular plasma membrane pumps (somewhat limited)
- extracellular: kidney (extensive control of total volume, ion composition)
- kidney function
- glomerular filtration: 125 mL/min (180 L/day), with composition similar to blood plasma minus proteins
- ionic reabsorption: 99 % of Na+, Cl-, HCO3-, glucose, amino acids reabsorbed
- water reabsorption: 99% of water reabsorbed, generating ~1L of hypertonic urine per day
- creatinine: generally not reabsorbed, thus allowing one to estimate GFR through creatinine clearance
part II know the following:
- acid-base balance: the mechanisms involved in maintaining H+ homeostasis and correction of imbalances
- balance: maintained only when H+ output equals H+ produced (~70 meq/day)
- input: can be altered significantly by diet and metabolism
- salts:
alkalinizing effect
net proton consumption in catabolism
- carboxylates:
alkalinizing effect
net proton consumption in catabolism
- amino groups:
acidifying effect
net proton production in catabolism
- inc. metabolism:
acidifying effect
H+ production in synthesizing lactate, ketone bodies
- body buffers
- types
- hemoglobin, plasma protein
- bicarbonate buffer
- phosphate buffer (minor in plasma, but important in urine)
- buffers can minimize the change in H+, but they do NOT correct the disturbance
- bicarbonate buffer
- Henderson-Hasselbach:
HA K a H A
pH pK a log
HA
H 2 CO 3 spontaneou
s H HCO 3
CO 2 (dissolved)
CO 2 (gas)
[HCO 3 ]
pH 6.1 log
(0.03 mM/mm Hg) (pCO 2 )
- HCO3 equilibrium:
- HCO3 buffer:
carbonic anhydrase
- simplifying assumptions
- at pH 7.4, all H2CO3 dissociates to HCO3
- at pH 7.4, all CO2 is dissolved
- [CO2(d)] = (0.03 mM/mm Hg)( p CO 2 )
- open system: CO2 term can be held constant by explusion from the equilibrium
- renal controls: kidney
- bicarbonate reabsorption
- Na+ brought in, H+ excreted to maintain electroneutrality
- H+ in lumen combines with HCO3- in lumen to make H2CO3
- carbonic anhydrase: breaks down H2CO3 into H2O + CO2
- CO2 comes into tubular cell, combines with H2O to make H2CO3 (carbonic anhydrase)
- H2CO3 breaks down into H+, HCO3- Na+/K+-ATPase: Na+ actively pumped into blood, HCO3 follows to maintain electroneutrality
- net reaction: HCO3- + Na+ (lumen) HCO3- + Na+ (blood)
- net excretion of H+
- fat:
15-20%
- inorganic:
7%
- carbohydrate:
<1%
- constituents of water
- intracellular:
60%
- interstitial, lymph: 20%
- dense CT, bone:
15%
- plasma:
7.5%
- balance
- intake: liquids imbibed, water ingested in foods, water produced during metabolism
- loss: skin, lungs, GI tract, kidneys
- electrolyte concentrations
- major cations and anions
- intracellular:
K+, Mg2+ organic phosphates, proteins
- extracellular:
Na+
Cl-, HCO3- electroneutrality
- total anions and cations in each compartment balance each other out
- membrane potential: charge imbalance is trivial compared to the total ion content
maintenance of water, electrolyte, and H+ balance
- fluid balance
- intracellular: cellular plasma membrane pumps (somewhat limited)
- extracellular: kidney (extensive control of total volume, ion composition
- nephron function
- glomerular filtrate
- filtration rate: 125 mL/min (180 L/day)
- composition: same as blood plasma, but with proteins filtered out
- reabsorption:
Na+, Cl 99.5 %
HCO399.9%
K+
93%
H2O
> 99%
- proximal transport
Na+:
actively reabsorbed, primarily in the proximal tubules
Cl-:
passively reabsorbed following Na+ (maintain electroneutrality)
water: passively reabsorbed following ions
others: reabsorbed in the proximal segments
- cystinuria: relatively common inherited metabolic defect in cysteine transporter
- distal transport
Na+:
actively reabsorbed (aldosterone: absorption)
water: passively reabsorbed (antidiuretic hormone: permeability)
- final urine
- volume:
0.6 1.2 L/day
- osmolarity:
3-4X plasma osmolarity
- composition:
urea:
major solute
NH4+, creatine, uric acid: additional secretion of nitrogen
sulfate:
protein catabolism
phosphate:
nucleic acid catabolism
organic bases:
ketone bodies, lactate
K +:
concentration usually higher in urine
Na+, Cl-:
concentration usually lower in urine
- pH:
much lower than plasma
- nitrogen excretion and creatine clearance
- nitrogen excretion in the average diet:
urea:
85 %
creatine:
4-5 %
NH4+:
2.5-3 %
uric acid:
1.5-2 %
others:
5%
- creatine
- daily excretion is relatively constant, varying only with muscle mass
- not reabsorbed from glomerular filtrate
- creatine clearance: can be used to index the glomerular filtration rate
- differences in metabolism in the renal cortex and renal medulla
- major consumer of energy: 360 kcal/day
- cortex: aerobic transport
- highly vascular, with blood providing necessary substrates for glucose transport
- metabolism uses glucose, fatty acids, and glutamine
- medulla: anaerobic transport
- less vascular, with poorer blood supply and resultantly poorer oxygen supply
- much of necessary glucose used may be produced by gluconeogenesis in the cortex
ACID BASE BALANCE
buffers
- Henderson-Hasselbach relationships
HA K a H A
pH pK a log
HA
pH 6.1 log
[HCO 3 ]
(0.03 mM/mm Hg) (pCO 2 )
CO2 production
- production due to metabolism: 15-20 mol/day
- determined by metabolic rate, carbon source used in metabolism
- must be balanced by the rate of alveolar ventilation
- balanced respiratory exchange of CO2: does not result in net change in H+ content
- rate of ventilation is finely regulated
- any change in pCO 2 will alter plasma pH
metabolic factors affecting H+ input
- diet: food and means of metabolism can affect H+ content of the body
- food catabolism
- acidic vinegar:
neutral:
complete combustion requires no net H+ change
- acetate salts:
alkalinizing:
complete combustion requires proton consumption
- citrate salts:
alkalinizing:
complete combustion requires proton consumption
- protein catabolism
- carboxylates:
alkalinizing:
complete combustion requires proton consumption
2 R - COO - H CO 2 RH
- amino groups:
acidifying:
2 R - NH 3 2R urea 2H
- incomplete metabolism: less efficient metabolism can give an acidifying effect
- anaerobic respiration: glucose 2 lactate + 2 H+
[lactate] may approach 5 mM during heavy work, causing a fall in pH
- ketone bodies:
palmitoyl residue 4 acetoacetate - + 4 H+
in diabetics, total acid load may approach 1 mol/day
renal reabsorption of HCO3- and excretion of H+
- acid base balance: kidney functions in regulation of [H+] in the blood
- regulated reabsorption of HCO3
- regulated excretion of H+
- reabsorption of bicarbonate as CO2: the HCO3-/CO2 cycle
- Na+ brought in, H+ excreted to maintain electroneutrality
- H+ in lumen combines with HCO3- in lumen to make H2CO3
- carbonic anhydrase: breaks down H2CO3 into H2O + CO2
- CO2 comes into tubular cell, combines with H2O to make H2CO3 (carbonic anhydrase)
- H2CO3 breaks down into H+, HCO3- Na+/K+-ATPase: Na+ actively pumped into blood, HCO3 follows to maintain electroneutrality
- net reaction: HCO3- + Na+ (lumen) HCO3- + Na+ (blood)
- net excretion of H+
- phosphate buffer: major urinary buffer, >50 % (2 Na+, HPO42-)
- CO2 from blood enters tubular cell, combines with H2O to make H2CO3 (carbonic anhydrase)
- H2CO3 breaks down into H+, HCO3- H+ excreted via H+ transporting ATPase, Na+ brought in to maintain electroneutrality
- H+ combines with HPO42- to make H2PO4-; Na+HPO4- is excreted
- Na+/K+-ATPase: Na+ actively pumped into blood, HCO3 follows to maintain electroneutrality
- net reaction: CO2 (lumen) HCO3- + Na+ (blood)
- metabolic example: lactate production
- glycogen is broken down into lactate, H+
- H+ combines with HCO3- to produce CO2 in the blood, which enters the kidney
- the proton is given off and excreted into the urine, regenerating bicarbonate, which reenters the blood
- excretion of NH3 as NH4+ in chronic acidosis
- ammonia is highly toxic, and must be excreted to avoid neurological problems
- for each NH3 excreted as urea, 1 H+ must be independently excreted
- cost of ammonia detoxificaiton is proton production
- during prolonged acidosis, liver and kidney adapt to secrete less urea, more NH4+
- this spares urinary buffers to handle other sources of acid
- glutamate/glutamine cycle
- glutamine synthetase:
Glu Gln
[NH3 ; ATP ADP + Pi]
- glutaminase:
Gln Glu
[H2O NH3]
- cycle is central to metabolism and ammonia transport
- ammonia transport
- liver: glutamine synthase used to make glutamine, which can safely transport ammonia through the blood
- kidney: glutaminase used to hydrolyze glutamine into glutamate
- ammonia: excreted into lumen, drawing a proton with it
- glutamate: used in gluconeogenesis
- adaptations: prolonged acidosis
- liver
- glutamine synthase:
induced
glutamine synthesis
- carbamoyl-P-synthetase I: inhibited
urea cycle
- glutaminase:
inhibited
glutamine metabolism
- kidney
- glutaminase:
induced
glutamine metabolism
- glutamate dehydrogenase: induced
gluconeogenesis
- PEP carboxykinase:
induced
gluconeogenesis
respiratory regulation of acid-base balance
- pH of the blood
- depends on ratio of [HCO3-]/[CO2(d)]
- pH can be altered by changing amount of dissolved CO2, as the lung does with degree of ventilation
- hypoventilation: slow, shallow breathing; decreases pH
- slow breathing causes increase in alveolar p CO 2
- diffusion of CO2 from blood will be slowed, so p CO 2 of the blood will increase
- hyperventilation: rapid, deep breathing
- rapid breathing causes decrease in alveolar p CO 2
- diffusion of CO2 from blood will be increased, so p CO 2 of the blood will decrease
- control of ventilation by the lung
- respiratory center of the brainstem: contain both [H+], p CO 2 sensors that moderate rate of ventilation
- peripheral sensors: similar but smaller effects on rate of ventilation
- carotid sinus: p CO 2 sensor that can alter ventilation
metabolic and respiratory disturbances of acid-base balance
- classification
- reasoning
- HCO3- / CO2 are the major blood buffer
- blood is a clinically accessible fluid
- when H+ is produced, HCO3- is lost (H+ + HCO3- CO2 exhaled) unless regenerated from CO2 in the kidney
- definitions
- disturbances: type
- acidosis: disturbance leading to an increase in [H+] of blood, where pH < 7.4 if not compensated
- alkalosis: disturbance leading to a decrease in [H+] of blood, where pH > 7.4 if not compensated
- disturbances: cause
- respiratory: disturbance caused by a change in plasma p CO 2
- metabolic: disturbance caused by a change in plasma [HCO3-]
- compensation
- compensation: correction of pH by alteration of the component not changed by the original disturbance
- kidney may work like hell to take in more HCO3- during metabolic acidosis, but this is not compensation
- metabolic disturbance respiratory compensation
- respiratory disturbance metabolic compensation
- metabolic acidosis
- metabolic acidosis: most common acid-base disturbance seen by a physician
- diagnosis: acidosis caused by decrease in plasma [HCO3-]
- examples
- diabetis: incomplete metabolism causes increased H+ input
- phenylketonuria: incomplete metabolism causes increased H+ input
- kidney malfunction: defect causes decreased H+ output
- compensation: increased ventilation causing decreased p CO 2 (limit of ~10 mm Hg)
- additional diagnostic features: anion gap
- blood ions can be cheaply determined
- on average, ([Na+] + [K+]) ([HCO3-] + [Cl-]) = ~10-20 mEq/L
- larger anion gap indicates presence of other fixed anions, consistent with metabolic disturbance
- anion sources
- PKU:
phenylpyruvate, phenyllactate
- B12:
L, D-methylmalonate
- biotin: pyruvate, lactate, propionate
[HR]
(reflection of hormone binding)
[H][R]
[H][R]
Kd
(reflection of ease with which hormone dissociates)
[HR]
Ka
- transporters:
- enzymes:
- proteins:
- small molecules:
- overview
- intracellular Ca2+ typically low (10-100 nM)
- in response to certain signals, Ca2+ can be increased, thereby acting as a second messenger
- many enzymes show a Kd for Ca2+ of ~1 m
- mechanisms of increasing Ca2+
- release of sequestered Ca2+ contained largely within the ER
- influx of extracellular Ca2+ (typical extracellular concentration: 1-10 m)
- hormonal effects which can be mediated by Ca2+ modulation
- hepatic glycogen degradation, gluconeogenesis: adrenaline (1 receptors):
- hormone secretory processes:
insulin release from pancreatic cells, among others
- muscle contraction, vasoconstriction:
oxytocin, adrenaline, angiotensin II
- mechanisms of Ca2+ action
- direct activation of Ca2+-dependent protein kinases, phospholipases
- calmodulin: Ca2+ binding protein that, upon activation, binds specific proteins to regulate activity
- Ca2+-calmodulin-activated protein kinases
- phosphorylase kinase
- phospholipase A2 (certain forms)
phospholipids
- the role of phospholipids in hormone action
- phospholipid metabolites: can act as intracellular, intercellular signaling molecules, activating phospholipases
- phospholipases
- PLA1: hydrolyzes ester bond of fatty acid at C1
- PLA2: hydrolyzes ester bond of fatty acid at C2
- PLC: hydrolyzes phosphodiester bond between glycerol, phosphate of head group
- PLD: hydrolyzes phosphodiester bond between phosphate and head group
- phospholipase regulatory molecules
- isoforms of PLA2:
Ca2+, Ca2+-calmodulin, phosphorylation, G-protein interaction
- isoforms of PLC:
heterotrimeric Gq, PLC-, tyrosine phosphorylation (by PLC-1, PLC-2)
- isoforms of PLD:
small MW G proteins
- PLA2 activation
- many hormone systems that activate Ca2+ mobilization result in enhanced PLA2 activity
- PLA2: linked to hormone-stimulated release of arachidonic acid
- arachidonic acid: precursor for eicosanoids
- eicosanids: FA derivatives that include prostaglandins, thromboxanes, leukotrienes
- eicosanids: mediate numerous processes including inflammation, smooth muscle contraction, clotting
- non-steroidal anti-inflammatory drugs (NSAIDs): inhibit production of eicosanoids
- phosphoinositide hydrolysis in hormone signal transduction
- phosphatidylinositol (PI): acts as a mediator in the cellular response of hormones that mobilize Ca 2+
- process
- phosphatidylinositol (PI) phosphatidylinositol-phosphate (PIP) [PI-4-kinase]
- phosphatidylinositol-phosphate (PIP) phosphatidylinositol 4,5-bisphosphate (PIP2) [PI-5-kinase]
- PIP2 diacylglycerol (DAG) + 1,4,5-trisphosphoinositol (IP 3) [PI-PLC]
- product functions
- DAG: activates isoforms of protein kinase C
- IP3: mobilizes Ca2+ from intracellular stores
- regulation: PI-PLC
- PI-PLC: phosphoinositide-specific phospholipase C
- Gq: G protein complex that regulates activity of PI-PLC
- summary
- hormonal signal causes activation of Gq protein complex
- this activates PI-PLC, which hydrolyzes PIP 2 to DAG and IP3
- DAG activates PKC, IP3 mobilizes Ca2+ from intracellular stores
metabolic regulation and homeostasis
- role of hormones in regulating homeostasis feedback controls
- overview
- homeostasis: maintained through combined efforts of endocrine, neural systems
- feedback control: level of one substance attenuates production or release of another substance or hormone
- feedback systems
- simple classical type
- mechanism:
controlled variable inhibits release from the gland that causes its release
- example:
insulin-glucose, parathyroid-hormone-Ca2+ levels
- complex-hormonogen type
- mechanism:
signal activates hormonogen, which effects a physiological change to cancel the signal
- example:
angiotensinogen-aldosterone
- hypothalamic-pituitary-target endocrine organ system
- mechanism:
hypothalamic signal secretes a releasing hormone, targeting anterior pituitary
anterior pituitary releases a tropic hormone, targeting tissue
tropic hormone feeds back on hypothalamus or anterior pituitary
- example:
CRF-ACTH-cortisol
- other factors influencing hormone levels
- transport of hormones
- hydrophobic hormones:
typically carried on plasma carrier proteins
- water-soluble hormones:
often present as free hormone, though carriers may facilitate/regulate as well
- albumin: general universal carrier of hormones
- metabolism of hormones
- modulation:
metabolism resulting in more, less, or inactive products, thus altering the response
- conversion:
metabolism resulting in hormones with different hormonal activity
- localization:
degradation typically in the liver, though other tissues can process as well
- insufficient response
- extent of a hormonal response can be limited by the physical capacity of a gland to secrete a hormone
- example: with high amounts of adipose tissue, pancreas cannot make enough insulin to meet demand
- interdependence of hormones and permissive effects
- hormonal response can be dependent on multiple signals with opposing effects
- decrease blood glucose:
insulin
- increase blood glucose:
glucagon, adrenaline
- permissive effect: the prior action of one hormone allows another hormone to exert its effects
- estrogen:
induces expression of progesterone receptor in the uterus
- progesterone:
only able to exert uterine effects with sufficient receptors
TABLE: Major Endocrine Glands Producing Polypeptide or AA-derived Hormones
endocrine gland
adenohypophysis
hormone
prolactin
ACTH
thyrotropin (TSH)
somatotropin (GH)
principal target
mammary gland
adrenal cortex
thyroid
general
ovary
testis
follicle-stimulating
hormone (FSH)
ovary
testis
neurohypophysis
pancreas
principal effect
proliferation, milk formation
synthesis, secretion of adrenal cortical steroids
synthesis, secretion of thyroxine and triiodothyronine
growth of bone and muscle
anabolic effect on Ca2+, phosphate, nitrogen metabolism
metabolism of CHO and lipid
elevation of muscle and cardiac glycogen
lutenization
progesterone secretion
development of interstitial tissue
secretion of testosterone
follicular development
with LH, secretion of estrogen and ovulation
- and -lipotropins
oxytocin
adipose cells
smooth muscle (esp. uterine)
vasopressin (ADH)
ejection of milk
water reabsorption
insulin
arterioles
general
glucagon
adrenal medulla
somatostatin
epinephrine,
norepinephrine
thyroid
thyroxine, triiodothyronine
parathyroids
atria of heart
calcitonin
parathormone
ANF
adipose tissue
liver
lipogenesis
glycogenolysis, gluconeogenesis
adipose tissue
pancreas, adenohypophysis
liver and muscle
adipose
general
release of lipid
metabolic rate, O2 consumption of tissues
general growth and metabolic activities
skeleton
skeleton, kidney, GI tract
kidney, vascular system, adrenal
cortex
Ca2+ metabolism
Ca2+, phosphate metabolism
Na+ excretion, blood volume, BP
hormone
testosterone
principal target
male accessory sex
principal effect
maturation, normal function
seminal vesicle
prostaglandins
general
smooth muscle, adipose,
platelets, brain, GI tract, etc.
ovary
estrone, estradiol
mammary glands
corpus luteum
progesterone
general
uterus
mammary glands
general
adrenal cortex
aldosterone
electrolyte metabolism
corticosterone, cortisol
insulin
- insulin chemistry
- structure: two polypeptide chains joined by two interchain disulfide bridges, with one intrachain bridge on A
- synthesis: produced in pancreatic cells
- synthesis: proinsulin synthesized on ribosomes of RER of cells
- transport: proinsluin transferred to Golgi, packaged into granules
- cleavage: proinsulin cleaved into insulin (90-95% efficiency)
- storage:
insulin is stored in granules as a Zn2+/insulin complex along with C-peptide
- secretion: upon proper signals (primarily high blood glucose), granules are secreted into the blood
- regulation
- process
- mechanism: ATP-dependent, Ca2+-dependent; cAMP modulates levels, but is not required
- degradation: promoted by thyroid hormones (T3, T4)
- factors
- blood glucose concentration: primary means of insulin modulation
- method of glucose intake: intravenous vs. oral
- intravenous: glucose gives a biphasic response at physiological concentrations; less potent than oral
- oral: insulin levels arise after glucose contacts the duodenum, before blood glucose has risen (anticipatory)
- amino acids (Arg, Leu): increase insulin secretion (also increases glucagon; does not significantly alter I/G)
- adrenaline: lowers insulin release via 2 (Gi-linked) receptor; conserves glucose for non insulin-dependents
- growth hormone (GH), glucocorticoids: enhance insulin secretion via islet hyperplasia (developmental)
- biological effects
- lowers blood glucose: stimulates muscle, adipose uptake
- stimulates glycolysis: induces glucokinase, pyruvate kinase expression
- inhibits gluconeogenesis: decreases availability of substrates, decreases activity/amount of enzymes
- inhibits adipose lipolysis: inhibits hormone-sensitive lipase (decreased cAMP, decreased PKA)
- stimulates liver, adipose lipogenesis: induces lipogenic enzymes (e.g. acetyl-SCoA carboxylase, FA synthase)
- stimulates general protein synthesis: facilitates, activates amino acids, decreases protein degradation
- promotes growth: very high levels of insulin
- signal transduction
- protein tyrosine kinase
- autophosphorylates tyrosine residues, which leads to phosphorylation of IRS-1, IRS-2
- phosphorylated IRS-1, IRS-2 serve as docking sites for multiple effector molecules
- G protein
- Gi protein activated, downregulating adenylate cyclase, reducing cAMP levels
- cAMP phosphodiesterase activated, further reducing cAMP levels
- disease states
- hypoinsulinism (diabetes mellitus): abnormality in the synthesis, secretion, and/or effect of insulin
- type I: poor insulin production leading to chronic high blood sugar
- type II: poor receptor response leading to chronic high blood sugar
- hyperinsulinism: excessive insulin production/overdose, leading to low blood sugar; much more dangerous
Notes: Lecture and Reading
TABLE: Overview of Major Pancreatic Hormones
hormone
site of synthesis
principal site of action
insulin
cells
muscle, liver
principal phenomena
utilization of CHO
stimulation of protein synthesis
glucagon
cells
adipose
liver
lipogenesis
glycogenolysis
somatostatin
cells
adipose
pancreatic islet cells
release of lipid
inhibition of insulin, glucagon secretion
insulin chemistry
- structure
- two polypeptide chains (A and B) joined by two interchain disulfide bridges; A intrachain disulfide bridge
- note: can be inactivated by proteolytic enzymes, and thus cannot be taken orally
- conservation across species
- animal insulin preparations are active in humans, though there can be antigenic problems
- porcine insulin: most closely related (substitution of alanine for threonine at B30)
- solubility
- aggregates in solution
- complexes with basic proteins (e.g. protamine) and divalent cations (e.g. Zn 2+)
- depot preparations: complexes of low solubility that dissociate slowly, giving a timed release
insulin biosynthesis, storage, and release
- hormones
- preproinsulin: translation product of insulin gene; cleaved into proinsulin
- proinsulin: insulin precursor with little insulin-like activity (2%), but partial immunoreactivity (50%) of insulin
- insulin: final hormone product secreted by the pancreatic cells
- key points in insulin biosynthesis
- synthesis:
proinsulin synthesized on ribosomes of RER of cells
- transport:
proinsluin transferred to Golgi, packaged into granules
- cleavage:
proinsulin cleaved into insulin (90-95% efficiency)
- storage:
insulin is stored in granules as a Zn2+/insulin complex along with C-peptide
- secretion:
upon proper signals (primarily high blood glucose), granules are secreted into the blood
regulation of insulin release
- overview
- requirements for insulin release
- triggering signal
(e.g. high blood glucose)
- energy source for ATP-dependent exocytosis (can be glucose)
- primary regulation: blood glucose concentrations
- triggering process: Ca2+-dependent
- intracellular cAMP
- high [cAMP] enhances insulin release
- low [cAMP] decreases insulin release
- many hormones that affect glucose levels do so by altering cAMP levels in pancreatic cells
- note: cAMP is not required for insulin release; it merely modulates the level
- biological half life
- half life: 5-15 minutes
- thyroid hormones (T3, T4): promote insulin degradation, decreasing its half life
- factors stimulating or inhibiting insulin secretion
- blood glucose concentration: primary means of insulin modulation
- method of glucose intake: intravenous vs. oral
- intravenous: glucose gives a biphasic response at physiological concentrations
- phase I: rapid discharge from cell granules, lasting 15-20 minutes before declining
- phase II: biosynthesis of insulin, peaking between 30-60 minutes
- oral: insulin levels arise after glucose contacts the duodenum, before blood glucose has risen
- blood glucose does not mediate the initial rise; instead, it is GI hormones (insulin-releasing polypeptides)
- anticipatory response: body preparation for a rise in glucose by prematurely releasing insulin
- comparison: the insulin response to oral glucose is much greater (~250 300 % of IV)
- amino acids (Arg, Leu): increase insulin (and glucagon) secretion
- mechanism
- stimulate release of GI factors that enhance insulin secretion from cells
- because they also stimulate glucagon release, I/G does not change markedly
- hormonal effects: logic of using protein to stimulate insulin secretion
- increased insulin: promotes protein synthesis
- increased glucagon: promotes gluconeogenesis
- note: amino acids cannot be stored, so their only other fate would be excretion
- epinephrine
- structure:
C6H4(OH)2-CH(OH)-CH2-NH-CH3
(OH: positions 3, 4)
- function:
principal catecholamine of energy metabolism
- catecholamine biosynthesis
- reactions
- tyrosine hydroxylase:
tyrosine dihydrophenylalanine (L-DOPA)
- DOPA decarboxylase:
L-DOPA dopamine
- dopamine -hydroxylase:
dopamine norepinephrine
- phenylethanolamine N-methyltransferase:
norepinephrine epinephrine
- key regulatory point
- enzyme: phenylethanolamine N-methyltransferase (PMNT)
- regulation: glucocorticoids (cortisol) induce synthesis of PMNT
- adrenaline storage and release
- localization: cytoplasmic granules of the chromaffin cells
- signal:
controlled by nerve stimulation (stress, hypotension, anoxia), profound hypoglycemia
- secretion:
acetylcholine: mobilizes Ca2+, promoting epinephrine release from granules
- adrenaline receptors
- 1 receptor: Gq-mediated PLC activation, used in smooth muscle contraction
- 2 receptor: Gi-mediated decrease in cAMP, suppressing insulin release from pancreatic cells
- receptors: Gs-mediated increase in cAMP, activation of PKA; similar glucagon effects, but works on muscle
- adrenaline biological effects
- non-metabolic
- -adrenergic stimulators: vasoconstriction
- -adrenergic stimulators: increased ventilation, blood flow, heart rate, and peripheral vessel dilation
- metabolic
- stimulates glycogenolysis:
- muscle: increased even at low adrenergic levels (: cAMP, active PKA)
- liver: increased at high levels of stress (: cAMP, active PKA)
- non-cAMP-dependent glycogen degradation (1: Gq, PLC, Ca2+ and DAG giving active PKC)
- stimulates lipolysis
- activates hormone-sensitive lipase (: Gs, cAMP, active PKA)
- glucocorticoids: permissive effect on hormone-sensitive lipase
- enhances gluconeogenesis: induces PEP carboxykinase
- inhibits insulin release: suppresses cAMP production in pancreatic cells (2: Gi, cAMP, inactive PKA)
- adrenaline regulation
- T3, T4: increase number, effectiveness (decrease Gi synthesis) of membrane receptors
- hyperthyroidism: -adrenergic hypersensitivity (G s effects relatively stronger)
- hypothyroidism: -adrenergic hyposensitivity (Gi effects relatively stronger)
- catabolism: monoamine oxidase (MAO), catechol-O-methyltransferase (COMT)
cortisol
- cortisol biosynthesis: synthesized in the adrenal cortex
- cortisol regulation
- transcriptional: primary regulation, under the control of the hypothalamus and pituitary glands
- release: enhanced during times of stress (e.g. starvation)
- timing: regulated on a circadian rhythm, peaking in the morning upon waking up
- cortisol biochemical effects
- by effect
- stimulates gluconeogenesis
- inhibits general protein synthesis, stimulates protein degradation
- inhibits uptake of amino acids by muscle, other peripheral tissues
- induces liver AA-catabolizing enzymes of gluconeogenic amino acids
- induces synthesis of several liver gluconeogenic enzymes
- permissive effect on lipolysis, glycogenolysis
- mechanism: not well understood, though at high concentrations, can lead to glycogen deposition in liver
- glycogen deposition mechanism: large increase in glucose 6-phosphate levels via its promotion of GNG
- inhibits insulin-induced glucose uptake in muscle, adipose tissues
- by nutrient
- carbohydrate:
- protein:
- fat:
gluconeogenesis
insulin uptake in peripheral tissues
degradation, synthesis in muscle
concentration of circulating AA
synthesis of gluconeogenic enzymes in liver
ureogenesis
peripheral lipolysis
circulating lipids
centralization of fat distribution (bringing it into the liver)
- stimulates gluconeogenesis
- inhibits general protein synthesis, stimulates protein degradation
- inhibits uptake of amino acids by muscle, other peripheral tissues
- induces liver AA-catabolizing enzymes of gluconeogenic amino acids
- induces synthesis of several liver gluconeogenic enzymes
- permissive effect on lipolysis, glycogenolysis
- mechanism: not well understood, though at high concentrations, can lead to glycogen deposition in liver
- glycogen deposition mechanism: large increase in glucose 6-phosphate levels via its promotion of GNG
- inhibits insulin-induced glucose uptake in muscle, adipose tissues
- summary: major metabolic actions of cortisol on CHO, protein, and fat
- carbohydrate:
gluconeogenesis
insulin uptake in peripheral tissues
- protein:
degradation, synthesis in muscle
concentration of circulating AA
synthesis of gluconeogenic enzymes in liver
ureogenesis
- fat:
peripheral lipolysis
circulating lipids
centralization of fat distribution (bringing it into the liver)
- liver
- adipose tissue
- skeletal muscle
- CNS
- red blood cells
review of metabolic profiles and functions of key cell types
- skeletal muscle: movement
- energy anabolism: reduced abilities
- lacks glucose 6-phosphatase, other key gluconeogenic enzymes
- lacks the pentose phosphate pathway
- lacks glycerol kinase (useful in triglyceride synthesis)
- energy catabolism: numerous sources
- hexoses
- fatty acids
- pyruvate
- ketone bodies
- metabolic activity
- O2 consumption
- resting: 30% of O2 consumption
- exercising: up to 80% of O2 consumption
- glycolysis: highly active
- TCA cycle: highly active
- glycogenolysis: activity dependent on need; cannot export glucose as does the liver
- protein catabolism
- during fasting, stress, skeletal muscle highly active in protein breakdown
- accounts for the majority of amino acids utilized by hepatic/renal gluconeogenesis
- differences in cardiac muscle
- function: must work continuously, and is more dependent on aerobic metabolism
- ultrastructure: higher levels of mitochondria, myoglobin
- liver: metabolic hub
- adaptability
- adapts rapidly to change in diet
- able to alter enzyme content quickly
- metabolic activity
- glycogen metabolism: serves as a readily accessible store of glucose
- gluconeogenesis: major site during short term fast
- fat synthesis: contains glycerol kinase (allowing it to synthesize fat without concurrent glycolysis)
- metabolite import and export: liver as a central hub of metabolism
- uptake: glucose without hormonal control, amino acids with hormonal facilitation
- export: glucose, fatty acids (as TG)
- adipose tissue: energy store
- function
- provides metabolic resiliency through efficient energy storage as triglyceride
- can provide fatty acids on demand
- metabolic activity: does very little work, and thus requires very little metabolism
- TCA: low
- glycogen storage: minimal
- gluconeogenesis: none
- fat synthesis: no glycerol kinase (requires glycolysis to provide glycerol phosphate)
- pentose phosphate: highly active, used in FA biosynthesis
- nervous tissue: makes you think and stuff
- metabolic substrates: depends largely on blood-brain barrier
- primary fuel: glucose, mannose
- secondary fuels: glycerol, lactate, butyrate, some amino acids
- fasting: ketone bodies (brain adapts through change in enzyme profile
- metabolic activity
high I/G
Gs inactivation leading to low cAMP, low PKA
insulin-dependent activation of cAMP phosphatase (lower cAMP, low PKA)
decreased glycogen phosphorylase
increased glycogen synthase
high I/G
activation of acetyl-CoA carboxylase (Gs and cAMP; also insulin-dependent kinase)
increased FA synthesis substrates
- I/G increases in glycolysis, decreases in gluconeogenesis
- insulin-dependent activation of PDH, high acetyl-CoA, giving high citrate
- insulin induction of lipogenic enzymes, pentose phosphate enzymes
- effects:
increased cytoplasmic acetyl-CoA, leading to malonyl-CoA and FA biosynthesis
- triglyceride synthesis and export from liver
- regulation:
high I/G
- mechanism:
fed: insulin activation of VLDL synthesis; FFA TG in adipose
stressed: glucocorticoid activation of VLDL synthesis; FFA CO2 in muscle
- effects:
decreased TG breakdown
increased TG synthesis by mass action
increased synthesis and secretion of VLDL
- protein synthesis
- regulation:
increased insulin, increased glucagon
- mechanism:
elevated blood AA stimulation of insulin, glucagon (even in absence of CHO)
- effects:
increased AA entry into muscle
increased protein synthesis in muscle, liver
- note: insulin is also permissive to growth hormone, which acts similarly to insulin
disease states: diabetes mellitus
- overview
- diabetes mellitus: deficiency in insulin synthesis, secretion, or effects leading to exaggerated catabolic state
- insulin activity: artificially low, leading to loss of glucose uptake in peripheral tissues
- glucagon: dominates, leading to very low I/G and the catabolic condition
- metabolic disturbances
- liver
- glycogen breakdown:
increased (despite high blood glucose)
- gluconeogenesis:
increased
- -oxidation:
increased, leading to elevated acetyl-CoA and ketosis
- muscle
- glycogen synthesis:
- glucose oxidation:
- protein synthesis:
- protein breakdown:
- adipose
- TG synthesis:
- TG breakdown:
- water, electrolyte balance
- hyperglycemia:
decreased
decreased
decreased
increased
decreased
increased
glycosuria, ketoacidosis, ketonuria
polydypsia
polyphagia
polyuria
frequent: major diagnostic tool for type I
insulin treatment
type II
insulin-resistant
adult onset
generally over 35
gradual: weeks to months
generally obese
present, even excessive, but ineffective
due to obesity
frequently none or mild
infrequent: insulin usually sufficient to
prevent lipolysis
necessary only in about 20-30% of
patients; diet alone can usually control
blood glucose
- hemoglobin A1c
- glycosylated Hb form
- higher than normal levels can be indicative of recent transient high blood glucose (e.g. binge eating)
SUPPLEMENTARY TABLE: Biochemican Indications and Their Causes in Diabetes Mellitus
indication
cause
hyperglycemia
decreased glucose uptake by peripheral tissues
increased hepatic glycogen mobilization
increased hepatic gluconeogenesis
glycosuria
glucose load exceeds capacity for reabsorption in renal tubule
ketoacidosis
increased -oxidation of adipose tissue fatty acids in liver, leading to elevated hepatic
acetyl-CoA concentrations and ketone body synthesis
ketonuria
ketone load exceeds capacity for reabsorption in renal tubule
hyperlactatemia
mobilization, metabolism of muscle glycogen to lactate, and lactate released as precursor of
gluconeogenesis (Cori cycle)
hyperlipidemia
free fatty acids derived from increased lipolysis in adipose tissue
hypertryglyceridemi
increased synthesis of triglyceride in liver, and increased VLDL synthesis
a
hypevolemia /
excessive loss of body water as urine due to glucose acting as an osmotic diuretic
hyperosmolarity
hyponatremia
loss of body sodium, as a result of glucose-induced osmotic diuresis
SUPPLEMENTARY TABLE: Clinical Symptoms and Their Causes in Diabetes Mellitus
symptom
polyuria
polydypsia
polyphagia
weight loss
tiredness
blurred vision
vomiting
hyperventilation
itching
cause
retention of glucose in renal tubule as glucose load exceeds absorptive capacity; glucose
acts as an osmotic diuretic, producing large volumes of urine
CNS-driven response to dehydration; may be mediated by angiotensin produced in
resopnse to hypovolemia
hunger stimulated by non-utilization of dietary glucose
increased catabolism of all metabolic fuel stores: muscle glycogen, protein, adipose TG
muscular weakness due to:
- proteolysis and mobilization of muscle protein
- reduced availability of metabolic substrate
dehydration of the lens, aqueous and vitreous humor; cataracts from sorbitol accumulation
CNS-driven response to ketones stimulating area postrema in floor of the fourth ventricle
respiratory compensation to metabolic acidosis (elevated plasma lactate, keto acids)
hyperosmotic blood leads to water loss from the skin
LHRH
GnRH
somatoliberin
GHRIH
LH, FSH
GH-RF
GH
PIF
dopamine
PRF
prolactin
melanocyte-stimulating hormone
release-inhibiting factor (melanostatin)
melanocyte-stimulating hormone
releasing factor (melanoliberin)
MIF
MSH
MRF
MSH
prolactin
general regulation
- general process
- releasing factors: secreted from hypothalamus as a result of extrahypothalamic or neural stimuli
- tropic hormones: release/synthesis in anterior pituitary as a result of releasing factors
- end organ hormones: final hormones produced in response to tropic hormones; generally feed back
- example: feedback control by the gonads
- extrahypothalamic stimuli (drugs, stress): stimulate hypothalamus
- hypothalamus: releases GnRH (releasing factor) into portal system
- anterior pituitary: releases FSH, LH (tropic hormones) into bloodstream
- gonads (target organ): produce gonadal steroids (end organ hormones), which cause the metabolic effect
- gonadal steroids: negatively feed back on anterior pituitary, hypothalamus
general overview of releasing factor mechanisms of action
- chemistry: most are peptides; PIF is dopamine (catecholamine)
- effects: primarily regulate tropic hormone secretion; secondarily regulate synthesis
- receptors: specific receptors for factors on the surface of specific cell types
- mechanism: changes in intracellular Ca2+, PI metabolism, and/or cAMP
- specific examples
- CRF:
enhances cAMP production in cells
- TRH, GnRH:
elevate intracellular Ca2+ via production of IP3 (release from ER) and DAG ( PKC)
- hypothalamic integration
- hypothalamus: organization allows processing of neurogenic signals from many sources
- changes in internal environment
- changes in external environment
- hormonal feedback signals
- pituitary hormones: levels directly, indirectly modified by hyopthalamus
- fluctuate wildly in response to diet, activity, stress, sleep, and other important factors
- half lives: 15-20 minutes
- neurogenic stimuli
- ACTH:
circadian, psychogenic, and stress-induced release
- TSH:
cold exposure in children; also somewhat in adolescence, adulthood
- gonadotropins:
regulated by numerous extrahypothalamic signals, including the pineal gland
- GH, gonadotropins: regulated by sleep-related precesses
posterior pituitary hormones: overview
- introduction
- posterior pituitary: distal component of a direct neurosecretory system beginning in the hypothalamus
- major hormones
- oxytocin: major hormone of ejection (babies, milk, vomit, sexual fluids)
- ADH (vasopressin): increases blood pressure through vasoconstrictive, anti-diuretic effects
- general chemistry: oxytocin and ADH
- structure:
- synthesis:
oxytocin
- synthesis:
- processing:
- secretion:
- effects:
- K+, H+:
excreted isoelectrically as a consequence of Na+ absorption
- renin-angiotensin system
- hormones
- renin: secreted by kidney JG cells in response to decreases in blood pressure, volume, or Na + levels
- angiotensinogen: circulating plasma 2 globulin produced by the liver
- angiotensin I: peptide cleavage product of angiotensinogen; not active
- angiotensin II: peptide cleavage product of angiotensin I; increases blood pressure
- most powerful pressor agent known; responsible for essential hypertension
- also acts on adrenal cortex to stimulate release of aldosterone (increases H2O reabsorption)
- mechanism: increased Ca2+ through Gq-linked stimulation of PI PLC (giving DAG, IP 3)
- angiotensin III: peptide cleavage product of angiotensin II; also active in aldosterone production
- process
- JG cells: sense decreased blood pressure, volume, or Na+ levels, secrete renin
- renin cleaves circulating angiotensinogen into angiotensin I
- angiotensin I is cleaved to angiotensin II
- enzyme: angiotensinase, angiotensin-converting enzyme, or ACE (primarily in the lung)
- this enzyme is the target of ACE inhibitors, which are intended to reduce blood pressure
- angiotensin II causes peripheral vasoconstriction, as well as synthesis of aldosterone
- aldosterone
- structure:
mineralcorticoid
- synthesis:
produced in the adrenal cortex
- function:
promotes the retention of Na+, excretion of K+ in the distal portion of the kidney
- mechanism: enters cell, forms steroid-receptor complex in the nucleus
regulates synthesis of specific transport proteins and/or enzymes necessary for Na + pump
- regulation: renin-angiotensin system: angiotensin II, angiotensin III stimulates aldosterone synthesis
Na+, K+: concentrations influence aldosterone synthesis
ACTH: permissive/supportive role
- electrolyte balance: maintained through actions of ADH, aldosterone
atrial natriuretic factor
- atrial natriuretic factor
- structure:
polypeptide hormone
- synthesis:
produced in the heart
- function:
works to lower blood pressure (opposes renin-angiotensin II-aldosterone system)
- mechanism: ANF-R1: coupled to guanylate cyclase ( cGMP), giving ANFs biological effects
ANF-R2: not coupled to guanylate cyclase; work to clear ANF from circulation
- effects:
Na+ uptake: increase GFR, inhibit tubular reabsorption of Na+
aldosterone: inhibits synthesis, release of aldosterone, partially by antagonism of angiotensin III
angiotensin II: antagonizes pressor effect
net effect: directly lowers blood pressure, indirectly reduces volume (through Na+ regulation)
selected hormonal disease states
- hypoaldosteronism (adrenal cortical insufficiency, Addisons disease)
- mechanism: excess salt loss, with osmotic reduction causing deficiency in ADH, resulting in water loss
insufficient Na+ reabsorption insufficient H+, K+ excretion
- result:
metabolic acidosis
- symptoms: dehydration, high blood pressure, acidosis
- hyperaldosteronism
- mechanism: excess salt reabsorption, with osmotic excess causing H2O retention and K+ and H+ loss
- result:
metabolic alkalosis
- diabetes mellitus
- mechanism: glucosuria: glucose exceeds renal threshold
ketonuria: ketone bodies exceed renal threshold
polyuria: excess glucose, ketone bodies act as osmotic diuretics, causing polyuria
- result:
major loss of water, Na+ via electroneutrality with ketone bodies
release of ADH to combat increased blood osmolarity, loss of body water
release of renin to combat Na+ loss, drop in blood volume/pressure
- symptoms: metabolic disturbances, ketoacidosis, and extensive alterations in water and electrolyte balance
circadian rhythms
- clinical:
defects in steroidogenesis are even more common than diabetes mellitus
growth hormone (GH, somatotropin)
- structure:
single polypeptide chain (191 amino acids), with high species specificity
- effects:
helps liberate fuels from energy rich tissues to promote growth of muscle, bone, cartilage
- stimulates skeletal growth
- stimulates chondrogenesis, osteogenesis via insulin-like growth factor I (IGF-I)
- increases collagen synthesis, calcification, and growth of the long bones at epiphyses
- note: will not enhance linear growth after puberty, but can lead to acromegaly
- stimulates general protein synthesis
- tissues: muscle, heart, bone, liver
- effects: AA uptake, mRNA synthesis, tRNA synthesis, protein synthesis
- increases muscle glycogen
- muscle: IGF-I mediated activation of glycogen synthesis
- liver: direct GH stimulation of glycogen breakdown
- note: GH is elevated during certain periods of low glucose, giving effects above
- increases plasma FFA
- stimulates lipolysis through GH-induced synthesis of hormone-sensitive lipase
- mechanism:
most effects mediated by insulin-like growth factors
- IGF-I: polypeptide with insulin-like metabolic effects
- mechanism: binds to cell-surface receptors, initiates insulin-like processes
- receptor: like insulin receptor, has intrinsic tyrosine kinase (Jak2)
- signal transduction
- GH binds 2 GH receptors, which activate intracellular tyrosine kinases (Jak2)
- Jak2 phosphorylates the signal transducers and activators of transcription (STATs)
- STATs dimerize, enter the nucleus, and modulate transcription
carriers: though water soluble, there are binding proteins for both GH and IGF
- regulation:
circadian: sleep-related release, unrelated to hypoglycemia, allowing for growth even in low I/G
neural: GRF and GIF (somatostatin), dependent on blood glucose (not primary regulation)
endocrine: activated response to T3, T4, cortisol
- clinical:
imbalances can cause gigantism, acromegaly, and dwarfism
prolactin (PRL, lactogenic hormone, luteotropic hormone, LtH)
- effects:
development of the mammary gland and milk production during lactation
- increased casein, lactalbumin, lactose synthetase, triglyceride synthesis
- mechanism:
similar to GH receptor: Jak2-mediated activation of STATs, modulation of transcription
- regulation:
neural: prolactin inhibitory factor (PIF, dopamine), the primary mode of regulation
neural: thyrotropin releasing factor (TRH), a moderate activator
endocrine: estrogen, giving a permissive effect
negative feedback: prolactin, distension increase release of inhibitory factor
Notes: Lecture and Reading
general considerations
- overview
- type:
polypeptides or glycoproteins (all water-soluble)
- size:
22 (MSH) to 190-200 (GH) amino acids
- targets:
most: small range of target tissues
GH:
broad range of target tissues
- processes:
metabolic regulation:
GH, ACTH, TSH
reproduction:
LH, FSH, prolactin
- grouping:
structural similarity
- polypeptide hormones (corticotropin-lipotropin group)
- included hormones: ACTH (adrenocorticotropic hormone)
MSH (melanocyte stimulating factor)
LPH (lipotropin)
- effects:
produced centrally during extreme stress, shock, and exercise via pro-opiocortin processing
- codeine, morphine, heroin: bind same receptors, mimic actions
glycoprotein hormones
- thyroid-stimulating hormone (TSH, thyrotropin)
- effects:
growth and function of the thyroid gland
- stimulates synthesis, release of the iodinated thyroid hormones
- iodinated thyroid hormones: thyroxine and triiodothyronine (T3 and T4)
- iodine, a large molecule, makes T3 and T4 quite hydrophobic
stimulates lipolysis in adipose tissue
- mechanism: Gs-mediated elevation of cAMP
- regulation: negative feedback: T3, T4
- primary means of regulation of release
- feedback occurs mostly at the anterior pituitary, rather than the hypothalamus
neural: thyrotropin releasing factor (TRF)
- clinical:
TSH has a long N-terminus, which can lead to autoimmune effects, constant activation
can lead to high T3 and T4, fever, goiter, Graves disease, high insulin metabolism
- follicle-stimulating hormone (FSH)
- effects:
stimulates growth and function of the gonads and accessory sex organs, induces steroid synthesis
female: enhances follicular maturation
increases androgen conversion to estrogens
upregulates FSH and LH receptor number in granulosa cells
helps initiate oogenesis
stimulates secretion of inhibin, which inhibits FSH release
male: stimulates spermatogenesis
induces synthesis of androgen-binding protein, inhibin
enhances development of seminiferous tubules
- mechanism: Gs-mediated increase in cAMP
- regulation: neural: gonadotropin-releasing hormone (GnRH)
estrogens: low levels inhibit, high levels stimulate release
inhibin: feeds back to inhibit release only of FSH, not LH
- lutenizing hormone (LH, interstitial cell stimulating hormone, ICSH)
- effects
female: with FSH, induces final follicular ripening, ovum release, corpus luteum formation
increases steroidogenesis by gonads, making estrogens
male: stimulates complete maturation of sperm
induces cholesterol to androgen conversion
- mechanism: Gs-mediated increase of cAMP, acting on cholesterol ester hydrolysis and 20-22 lyase
- regulation: neural: gonadotropin-releasing hormone (GnRH)
negative feedback: steroids feed back on anterior pituitary, hypothalamus
circadian rhythms
- clinical:
defects in steroidogenesis are even more common than diabetes mellitus
- human chorionic gonadotropin (HCG, a placental gonadotropin)
- structure:
similar to LH: glycoprotein of an and subunit
- subunit: identical to LH
- subunit: larger, contains more carbohydrate
- clinical:
measurement of subunit the basis for most pregnancy tests
growth hormone and prolactin
- growth hormone (GH, somatotropin)
- structure:
single polypeptide chain (191 amino acids)
high species specificity: only monkey, human GH actively promote growth in humans
- effects:
helps liberate fuels from energy rich tissues to promote growth of muscle, bone, cartilage
- stimulates skeletal growth
- stimulates chondrogenesis, osteogenesis via insulin-like growth factor I (IGF-I)
- increases collagen synthesis, calcification, and growth of the long bones at epiphyses
- note: will not enhance linear growth after puberty, but can lead to acromegaly
- stimulates general protein synthesis
- tissues: muscle, heart, bone, liver
- DHEA pathway
- 17-hydroxylase:
- 17-20 lyase:
- androstenedione pathway
- 17-hydroxylase:
- 17-20 lyase:
- vertical pathways
- aldosterone pathway
- 3-hydroxysteroid DH/isomerase:
- 21-hydroxylase:
- 11-hydroxylase:
- 18-hydroxylase DH:
- cortisol pathway
- 3-hydroxysteroid DH/isomerase:
- 21-hydroxylase:
- 11-hydroxylase:
- adrenal androgen path:
pregnenolone 17-OH-pregnenolone
17-OH-pregnenolone dehydroepiandrosterone (DHEA)
progesterone 17-OH-progesterone
17-OH-progesterone androstenedione
pregnenolone progesterone
progesterone dehydroxycorticosterone (MC)
dehydroxycorticosterone corticosterone (GC)
corticosterone aldosterone (MC)
- activated by angiotensin II
17-OH-pregnenolone 17-OH-progesterone
17-progesterone dexoycortisol
cortisol (GC)
dehydroepandrosterone (DHEA, C19) androstenedione (C19)
androstenedione testosterone
testosterone estradiol (trace)
- synthetic tendencies
- adrenal cortex: makes weak androgens through both pregenolone and progesterone (and thus higher DHEA)
- gonads: make androgens through progesterone (and thus very little DHEA
aldosterone (mineralcorticoids)
- function:
regulation of Na+ intake to maintain electrolyte and fluid balance
- synthesis:
zona glumerulosa of the adrenal gland
- effects:
distal renal tubule: increases reabsorption of Na+, excretion of K+ (enzyme synthesis)
skin, muscle, bone: decreases [Na+], increases [K+] in saliva, sweat, and GI contents
- mechanism:
alteration of synthesis (steroid-intracellular receptor complex)
- regulation:
permissive: ACTH (increased precursor synthesis; does NOT feed back on ACTH)
metabolite: angiotensin II, III, Na+, K+
- angiotensin II: enhances 22-lyase, 18 hydroxylase/DH (Gq release of Ca2+, activation of PKC)
cortisol (glucocorticoids)
- function:
energy metabolism, especially during fasting, CHO deprivation, and stress
- effects:
stimulates gluconeogenesis
- inhibits protein synthesis, inhibits peripheral AA uptake and activates liver AA uptake
- stimulates liver synthesis of gluconeogenic enzymes, esp. PEPCK
- exerts permissive effect on gluconeogenesis induced by glucagon, epinephrine
- high (pharmacological) doses: increased glucose, G6P, and thus glycogen storage in liver
permissive effect on lipolysis ( hormone-sensitive lipase, or HSL)
permissive effect on glycogenolysis (glucagon, adrenaline)
stimulates methylation of norepinephrine to epinephrine ( PNMT)
- PNMT: phenylethanolamine N-methyltransferase
- important enzyme in the adrenergic response
anti-inflammatory effect
- mechanism:
transcriptional: modulation of gene transcription (intracellular receptor-steroid complex)
post-transcriptional: mRNA stability
permissive: increased synthesis of protein kinase, downstream enzymes (e.g. HSL)
- regulation:
feedback controls: negative feedback on CRF, ACTH levels
circadian rhythms: highest in the morning, lowest before bedtime
stress: (trauma, infection) increases cortisol production
ACTH: stimulates glucocorticoid synthesis (activation of 20-22 lyase)
transport: bound to albumin, cortisol-binding globulin (CBG)
17-hydroxylase
low
elevated
low
elevated
low (lack of AII)
elevated
elevated
18-hydroxylase /DH
normal
normal
normal
normal
low
low
low
- numbering
- rings:
- numbers:
A, B, C, D (left to right)
1-10: loop around A, B
11-17: figure 8 around C, D
18:
-methyl group off 13
19:
-methyl group off 10
20, 21: first C of tail off -17; methyl group
22-26: linear chain off 20
27:
methy groups off 25
- stereochemical configuration
- C19 (methyl group off C10) determines or steroid
- C5, if it does not have a double bond, stereochemistry of H should be specified
- nomenclature
- gonane:
C17
core of structures of the gonads
- estrane:
C18
core of estrogens
- androstane:
C19
core of androgens
- pregnane:
C21
core of progestins: progesterone, aldosterone, cortisol
- cholane:
C24
core of bile acids
- cholestane:
C27
cholesterol
- substituents
- unsaturation:
-ane to -ene, -diene, -triene
(preceeded by appropriate numbers)
- hydroxyl group:
to -ol, -diol, -triol
(preceeded by appropriate numbers)
- carbonyl group:
to -one, -dione, -trione
(preceeded by appropriate numbers)
overview of adrenal steroidogenesis
- principal regulated step
- 20-22 lyase:
cholesterol pregnenolone
- ACTH is permissive
- reaction activated by angiotensin II]
- horizontal pathways
- DHEA pathway
- 17-hydroxylase: pregnenolone 17-OH-pregnenolone
- 17-20 lyase:
17-OH-pregnenolone dehydroepiandrosterone (DHEA)
- androstenedione pathway
- 17-hydroxylase: progesterone 17-OH-progesterone
- 17-20 lyase:
17-OH-progesterone androstenedione
- vertical pathways
- aldosterone pathway
- 3-hydroxysteroid DH/isomerase:
pregnenolone progesterone
- 21-hydroxylase:
progesterone dehydroxycorticosterone (MC)
- 11-hydroxylase:
dehydroxycorticosterone corticosterone (GC)
- 18-hydroxylase DH:
corticosterone aldosterone (MC)
- activated by angiotensin II
- cortisol pathway
- 3-hydroxysteroid DH/isomerase:
17-OH-pregnenolone 17-OH-progesterone
- 21-hydroxylase:
17-progesterone dexoycortisol
- 11-hydroxylase:
cortisol (GC)
- adrenal androgen path:
- enzymes
- horizontal
- 20-22 lyase: principal regulated step (activated by angiotensin II, with permissive effect by ACTH)
- 17-hydroxylase: from aldosterone pathway to cortisol pathway
- 17-20 lyase: from cortisol pathway to androgen pathway
- vertical
- 3-hydroxysteroid DH/isomerase: first step in aldosterone, cortisol pathways
- 21-hydroxylase: commits to either aldosterone or cortisol pathway; directly makes cortisol
- 18-hydroxylase DH: leads to aldosterone
- synthetic tendencies
- adrenal cortex
- weak androgens: makes weak androgens through both pregnenolone, progesterone
- tumor of adrenal cortex: excessive DHEA, excess testosterone
- gonads
- weak androgens: tends to use progesterone to make androgens
- tumor of gonads: excessive testosterone, very little DHEA
biological and metabolic effects of the adrenal cortical steroid hormones
- mineralcorticoids
- principal:
aldosterone
- function:
electrolyte and fluid regulation
- effects:
distal renal tubule: increases reabsorption of Na+, excretion of K+
- mechanism: induction of Na+ pump via intracellular receptor
skin, muscle, bone: decreases [Na+], increases [K+] in saliva, sweat, and GI contents
- glucocorticoids
- principal:
cortisol (hydrocortisone)
- function:
energy metabolism, especially during fasting, CHO deprivation, and stress
- effects:
stimulates gluconeogenesis
- inhibits protein synthesis, increasing the pool of AA available
- inhibits AA uptake in peripheral tissues
- stimulates AA uptake in liver
- stimulates liver synthesis of gluconeogenic enzymes, esp. PEPCK
- exerts permissive effect on gluconeogenesis induced by glucagon, epinephrine
- high (pharmacological) doses: increased glucose, G6P, and thus glycogen storage in liver
permissive effect on lipolysis ( hormone-sensitive lipase, HSL)
permissive effect on glycogenolysis (glucagon, adrenaline)
stimulates methylation of norepinephrine to epinephrine ( PNMT)
- PNMT: phenylethanolamine N-methyltransferase
- important enzyme in the adrenergic response
anti-inflammatory effect
others: erythropoiesis, gastric acid/pepsinogen, immunosupressants, cardiovascular effects
- mechanism: transcriptional: modulation of gene transcription in steroid fashion
post-transcriptional: mRNA stability
permissive: increased synthesis of protein kinase, downstream enzymes (e.g. HSL)
TABLE: Biological Activities (Relative to Cortisone) of Natural Corticosteroids in Adrenalectomized Rats
steroid
CHO activity
Na+ retention
anti-inflammatory activity
cortisone
100
100
100
cortisol
155
150
1,250
deoxycorticosterone
0
3,000
0
aldosterone
0
60,000
0
regulation of the adrenal cortical steroid hormones
- glucocorticoids (cortisol)
- feedback controls: negative feedback on CRF, ACTH levels
- circadian rhythms: highest in the morning, lowest before bedtime
- stress: (trauma, infection) increases cortisol production
- ACTH: stimulates glucocorticoid synthesis (activation of 20-22 lyase)
- storage: small amounts stored
- transport: bound to albumin, cortisol-binding globulin (CBG)
- CBG: increased by estrogens, thyroid hormones; decreased by nephrosis
17-hydroxylase
low
elevated
low
elevated
low (lack of AII)
elevated
elevated
18-hydroxylase /DH
normal
normal
normal
normal
low
low
low
- metabolism:
- effects:
- mechanism:
- regulation:
estrogens
- function:
- localization:
- chemistry:
- synthesis:
- metabolism:
- effects:
- mechanism:
- regulation:
female
- mechanism:
- regulation:
- clinical:
androgens
- function:
- localization:
- chemistry:
- synthesis:
- metabolism:
- effects:
- mechanism:
- regulation:
- clinical:
estrogens
- function:
- localization:
- chemistry:
- synthesis:
- metabolism:
- effects:
- mechanism:
- regulation:
- clinical:
serum [Pi]
kidney
Ca2+ reabsorption
reabsorption of Ca2+
Pi reabsorption
reabsorption of Pi
1-hydroxylase activity
bone
resorption of bone
resorption of bone
GI tract
no effect
absorption of Ca2+, Pi
(indirect: increased production
of 1,25-DHCC)
1,25-DHCC
Ca2+ reabsorption
Pi reabsorption
Ca2+ mobilization from bone
absorption of Ca2+, Pi
serum [Pi]
kidney
Ca2+ reabsorption
reabsorption of Ca2+
Pi reabsorption
reabsorption of Pi
1-hydroxylase activity
bone
resorption of bone
resorption of bone
GI tract
no effect
absorption of Ca2+, Pi
(indirect: increased production
of 1,25-DHCC)
1,25-DHCC
Ca2+ reabsorption
Pi reabsorption
Ca2+ mobilization from bone
absorption of Ca2+, Pi