1

PROGRAMME OF MASTER EDUCATION

(BIOLOGY)

FACULTY OF SCIENCE AND MATHEMATICS

SBF 6014
ANIMAL METABOLISM AND ADAPTATION

ARTICLE DISCUSSION

LECTURER: DR WONG CHEE FAH

CHEN SOON KIONG M20131000468

30 JANUARY 2014
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Article Discussion 1



The article entitled Mitochondrial K
ATP

Channels In Skeletal Muscle: Are Protein Kinase C
And G, And Nitiric Oxide Synthase Involved In The Fatigue Process?

The authors included several group of researchers which are:
Elizabeth Snchez-Duarte, Xchitl Trujillo and Miguel Huerta from research Enrico
Stefani Boonfanti, University Center for Medical Research, University pf Colima,
Colima, Mexico ,
Mnica Ortiz-Mesina, Christian Corts-Rojo and Salvador Manzo-valos from Institute
of Chemical-Biological Research,
Alfredo Saavedra-Molina and Roco Montoya-Prez from Coordination General
Postgraduate Studies University Michoacana of San Nicolas de Hidalgo.

These research paper have been published in the Dove Press Journal: Open access Animal
Physiology on 24 October 2012.













3

Abstract

Slow-twitch skeletal muscle, in contrast to fast-twitch muscle is more resistant to fatigue.
This paper discussed the role of mitochondrial K
ATP

channels on muscle fatigue tension. They
used an in-vitro model to study the effect diazoxide and its combination with several inhibitors
of the signaling routes in the fatigue process assessed on slow-twitch skeletal muscle fibers of
the chicken. They investigated the action of activators and inhibitors of the mitochondrial K
ATP

channels on fatigued slow skeletal muscle, studying twitch and fatigue tension after inducing the
muscle fatigue by continuous electrical stimulation.

The results showed that diazoxide (30M) increased post-fatigue twitch tension by about
60% with respect to the fatigue condition (p<0.05). In addition, diazoxide increased post-
fatigued tension (103.38% 7.97% in peak tension, and 1.63.82% 11.42% in total tension, n=4;
p<0.05 respectively). mitochondrial K
ATP
channels with diazoxide significantly reduced muscle
fatigue.

From the study, authors concluded that diazoxide activate the mitochondrial K
ATP
to
reduce the fatigue effect by increasing the post fatigue tension, same as reported in previous
study, but at much lower concentration (30M). Despite the findings, the authors discovered
there were lack of direct interactions between the inhibitors (protein kinase C, staurosporine;
protein kinase G, KT5823; and nitric oxide synthase, metil N
G
-Nitro-L-arginine ester, L-NAME)
and inhibition of muscle fatigue tension during mitochondrial K
ATP

activiation.

This result suggest a possible role of mitochondrial K
ATP
channels in the fatigue process,
yet the lack of interaction between inhibitors of signaling routes and mitochondrial K
ATP

activation is due to type of skeletal muscle fibers, the stimulation protocols and the use of birds
instead of rats in this study.

Keywords: mitochondrial K
ATP
channels, diazoxide, fatigue, skeletal muscle


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Problem

The problem is clearly stated. Authors of this article plan to provide a clearer
understanding of the function of mitochondrial K
ATP
channel activator (diazoxide) in suppressing
the muscle fatigue. In addition, authors also want to determine the role of other inhibitors
relating to fatigue signaling routes such as protein kinase C, staurosporine; protein kinase G,
KT5823; and nitric oxide synthase, metil N
G
-Nitro-L-arginine ester, L-NAME in during fatigue
tension.

Through the experiment, authors want to determine the participation of mitochondrial
K
ATP
in fatigue, stress using the in vitro model of avian (bird) slow skeletal muscle, and compare
their results with previous study regarding the roles of activators and inhibitors in mitochondrial
K
ATP
channel.


Hypothesis

The hypothesis for this research are:

H
0
1 : There is no significant effect of diazoxide in the total and maximal tension of twitches
in fatigued muscle fibers
H
0
2 : There is no significant effect of 5-hydroxydecanoate on twitch tension in fatigued slow
skeletal muscle fibers.
H
0
3 : There is no significant effect of diazoxide and 5-hydroxydecanoate on tension in slow
muscle fibers.
H
0
4 : There is no significant effect of diazoxide and staurosprine on tension in slow muscle
fibers.
H
0
5 : There is no significant effect of diazoxide and KT5823 on tension in slow muscle fibers.
H
0
6 : There is no significant effect of diazoxide and staurosprine L-NAME on post-fatigue
tension in slow muscle fibers

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Key Terms

The key terms were define well allowing easy understanding on the topic. Authors have difine
fatigue as reduced physical power to carry out activities or the difficulties to maintain ATP
production. The authors cited skeletal muscle as key term since their experiment is related to the
ATP production in avian skeletal muscle.

In addition, authors used mitochondrial ATP-sensitive potassium channels as the third
key word to indicate the main purpose of this study, as well as explaining the role the channels
play in several cellular functions by detecting the intracellular ATP/ADP relationship


Review of Literature

The sources cited in this study is comprehensive with the uses of 42 references. Authors
of this article do focused on comparing their results with several references. Although no bias on
the references used, authors of this article however do focused on several specific articles for
discussion on their experimental findings. For example, authors compared their findings on the
concentration of diazoxide in increased post-fatigue twitch tension which is much lesser than
Garcia et al. (2009).

In addition, authors also mentioned that no effect on post-fatigue tension even with
higher dosage (500 M) of 5-hydroxydecanoate. The author using several journals as reference,
including Garlid et al. (1997), Hanley et al. (2005) and Garcia et al. (2009) to conclude that an
intercellular mediator is necessary to activate the effect of 5-hydroxydecanoate and guarantee the
mitochondrial K
ATP
activation.

The references used by authors are most related and up to date for the topic of their study.
The references used by authors falled between the year of 1969 and 2012. Journal from the year
1969 was used since the article is related to the properties and structures of fast and slow muscle
in the chicken which very related to the specimen they used in the study. The recent articles used
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by author in the introduction part include the article written by Andrade et al. (2011) which
discussed the post-fatigue tension in slow skeletal muscle fibers of the chicken. The author also
refer to the protocols used by Andrade et al. (2011) to produce twitches to establish a fatigue
protocol in the slow latissimus dorsi muscle.

In fact, authors using most of this references as comparison to their findings, providing
futher discussion relating to the effects of KATP channel inhibitors on muscle fatigue. Author
suggested further studies to have better understand the mechanisms involving mitochondrial
K
ATP

channels and slow muscle fibers fatigue.


Design and procedures

Authors carried out experiments to test their hypothesis. They carry out an ethical dissection on
the chicken to obtain anterior latissimus dorsi muscle, which composed of slow muscle fibers.

According to the methodology, chickens were anersthetized with chloroform at first, in
order to minimize animal pain and distress thus allows extraction of fresh muscle tissues. The
introduction of fatigue stress onto the muscle tissues was conducted by referring to protocols
from Andrade et al. (2011). Authors used different electrical stimulation frequencies to produce
twitches, enabling them to establish a fatigue protocol.

Subsequently, the effects of chemicals (activators and inhibitors) on the signaling routes
were conducted through the used of normal Ginsborg saline buffer with pH adjusted to 7.4.
Finally, all data were analyzed through statistical analysis.

It was an original study which carried further study by using different type of inhibitors
and different type of skeletal muscle. Although there were similarities in the principles and
methodology as compared to other previous studies, yet the authors had emphasized that this
study as they are using the slow muscle fibers of avian rather than heart and fast skeletal muscle
of the rat (Garlid et al., 1997 and Montoya-Perez et al., 2010).
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Authors had used statistical approaches in analyzing their experimental data. Both
pClamp 9.2 software (Axon Instruments) and Sigmaplot 10.0 software (Systat Software Inc.
Erkrath, Germany). were used to analyze data and plot the graph. Comparison of means was
done using the Students t-test, accepting a significant effect when P was ,0.05.

The flow of methodology used was in proper arrangement, starting from dissecting of
muscle tissues, which followed by electrical stimulation for establishing fatigue and chemical
reactions, eventually ended with statistical analysis using computer software. No pilot study
were conducted, as authors had many related good references before they conducted the similar
experimental procedures. In adidition, the authors of this article only aim to provide further
discussion on the effect of protein linase C and G, nitiric oxide synthase involvement in the
fatifue process of avian slow skeletal muscle.


Variables

Variables involved in this study are:
Control variables: diazoxide (activators) and inhibitor
Reponse variables: tension in g
Constant variables: chicken slow muscle fibers


Data Analysis and presentation

Authors carried out data analysis using the Clamfit subroutine of the pClamp 9.2 software where
as Sigmaplot 10.0 software is used to elaborate graphs. Student T-test is then used to compare
the mean from the results obtained.

The data was quantitative. All the data were recorded in form of normalized form,
dividing a large findings into much smaller fractions, allowing easier comparison between them.
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In other words, authors had divided their results into several parts but all of this parts are
correlated to each other.

For example, at first authors discussed the effect of diazoxide and 5-hydroxydecanoate on
the fatigued slow skeletal muscle fibers seperately before continue to discussed the effect of
adding 5-hydroxydecanoate into diazoxide on fatigued slow skeletal muscle fibers.

The authors have successfully conducted a experiment that enable them to explain the
role of protein kinase C And G, nitiric oxide synthase in relationship to mitochondrial K
ATP

channels during the fatigue process of avian slow skeletal muscle.


Weakness

From the results, the authors explain the main problems they do not obtain similar results as
compare to previous studies. They discovered that using different type of skeletal muscle fibers
(they used chicken or avian slow muscle instead of heart or fast skeletal muscle like in a rat)
would have different impact on the outcome of experiment.


Conclusions and Implications

In their conclusion, authors of this articles stated there must be more future research into the
topics as their findings are slightly different that other previous study. The emphasized that it is
important to clarify the effect of mitochondrial K
ATP
channel inhibitors on muscle fatigue as
most findings are still contradict with each other. More importantly, authors also mentioned the
important of studying the pharmacological properties of mitochondrial K
ATP
channel in avian
muscle as it could lead to more discovery relating to cellular mechanisms involved in slow
muscle fiber fatigue.

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The conclusion was short, brief but stated the important point of this study, which authors
would like to see more research into the slow muscle fiber fatigue. The conclusion is related to
the original purpose of this study, which contributing to the futher understanding on the effect of
activators and inhibitors of mitochondrial K
ATP
channel found in avian slow muscle fibers.


Overall Assessment

Overall, this is a highly specific article which related to the mitochondrial K
ATP
channel
mechanisms in slow muscle fiber of avian (chicken). The authors had used several key terms in
their article, including fatigue, slow skeletal muscle, mitochondria, ATP, diazoxide and 5-
hydroxydecanoate to guide readers in understanding their research.

The article was clearly divided into five main parts starting from introduction, materials
and methods, results, discussion and references. The abstract is easy to understand as authors
have also divided into four main parts, namely the background of study, methods, results and
conclusion. Although clearly divided, I found out the abstract of this article is quite hard to
understand as authors were using many technical terms specific to topic related with
mitochondrial ATP -dependent potassium channels and muscle tissue fibers of animals.
Nonetheless, the materials and methods used were easily understood as authors describe in
details the protocols they used in the experiment. For example, authors explained the purpose of
selecting anterior latissimus dorsi muscle of chicken as the specimen for this study because of
the nature of this tissue that composed only the slow muscle fibers. Furthermore, authors also
able to provide clear explaination on the electrical stimulation procedure to introduce a fatigue
situation onto the slow skeletal muscle. Beside providing the reference they used (Andrade et al.,
2011), authors also provide important parameters including supramaximal twitches at 300 ms
and 0.2 Hz (Grass S-48 stimulator and SIU-5B stimuli isolation unit) to induce bundle
contractions.

Nonetheless, through the comprehensive discussion, I understand that during the
development of fatigue, ATP production will be ceased or reduced. The biochemical reactions
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that happen to alters the production of ATP will determine how far the muscle become fatigued.
Authors also stated, diazoxide would enhanced the activity of ATP channels, increasing the post-
fatigue tension, whereas activation of mitochondrial K
ATP
channels is inhibited by 5-
hydroxydecanoate.

Other activators that induce the opening of mitochondrial K
ATP
channels include protein
kinase C and protein kinase G which function in a way similar to that achieved by the such as
diazoxide or cromakalim in heart, liver, and brain mitochondria.

Although there were some dissimilarities between the results from this study to other
previous literatures, the authors had mentioned that it is mostly due to the different in vitro
model of fatigue (chickens slow skeletal muscle in contrasts to heart and fast skeletal of rat) as
well as different modes of stimulation in introducing fatigue. Finally, authors also mentioned that
there are still lots more to be done in order to fully understand the mechanisms of mitochondrial
K
ATP
channels in relation to slow skeletal muscle fibers.













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Vocabulary

1. Diazoxide = potassium channel activator, which causes local relaxation
in smooth muscle by increasing membrane permeability to potassium ions.
2. Dislocation = Displacement of a body part, especially the temporary
displacement of a bone from its normal position.
3. Dissection = Process of disassembling and observing something to determine
its internal structure and as an aid to discerning the functions and relationships of its
components.
4. Decapitation = Separation of the head from the body. behead.
5. Staurosporine = A selective inhibitor for protein kinase C
6. Agonist = A chemical that binds to some receptor of a cell and triggers a
response by that cell.
7. Ischemic condition = Condition restriction in blood supply to tissues, causing a
shortage of oxygen and glucose needed for cellular metabolism.
8. 5-hydroxydecanoate = inhibitor of the mitoK
ATP
channels




REFERENCES
Ortiz-mesina, M., Corts-rojo, C., Manzo-valos, S., Saavedra-molina, A., & Montoya-prez, R.
(2012). Mitochondrial K ATP channels in skeletal muscle: are protein kinases C and G ,
and nitric oxide synthase involved in the fatigue process?, 2128.





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Article Discussion 2


The article entitled Are Tropical Small Mammals Physiologically Vunerable to
Arrhenius Effects and Climate Change?

The authors included
Barry G. Lovegrove, Cindy Canale and Danielle Levesque from School of Life
Sciences, University of KwaZulu-Natal, Private Bag X01, Scottsville 3209, South
Africa,
Gerhard Fluch and Thomas Ruf from Department of Integrative Biology and
Evolution, Institute of Wildlife Ecology, University of Veterinary Medicine, Vienna
1160, Austria;
Milada ehkov-Petr from Zoo Decin, Pastyrska stena, Z izkova 15, Decn
40502, Czech Republic; and Tarsius, o.s., Na Pesine 256, Decn 405 05, Czech
Republic
These research papers have been published in the Chicago Journal by The University of
Chicago Press on 10 January 2013. The articles source is from Physiological and Biochemical
Zoology.












13

Abstract

This article is to study relationship between ambient temperature (T
a
) and metabolic rate (MR) in
four tropical aboreal mammals which is affected directly by global warming through the
increases in global average air temperature and the increase in the frequency and intensity of
heatwave.

Authors found that basal metabolic rates (BMR) and ambient (T
a
) are associated with
unpredictable climate and resource availability rather than high thermal extremes in tropical and
semitropical mammals (Lovegrove, 2003; Withers et al. 2006). Hence, skin temperature and core
body temperature have been collected and studied from four tropical small arboreal mammals
which are Philipine tarsiers (Tarsius syrichta), the greater hedgehog tenrec (Setifer setosus), the
golden-brown mouse lemur (Microcebus ravelobensis) and the reddish-gray mouse lemur
(Microcebus griseorufus)

Results showed that the maximum daytime rest phase skin temperature (T
skin
) of all
species are ~2
0
3
0
C higher than the maximum active phase T
skin.
the collar-mounted

telemeters
were not influenced by T
a
at night. The results also showed that tarsiers was most vulnerable to
sustained heat stress. Arrhenius effect caused the increased in metabolic rates during climate
change.

Keywords: Arrhenius effects, ambient temperature, metabolic rate, skin temperature









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Problem

The problem is clearly stated. Authors of this article want to study the relationship between
ambient temperature (T
a
) and metabolic rate (MR) in four tropical aboreal mammals which is
affected directly by global warming through the increases in global average air temperature and
the increase in the frequency and intensity of heatwave. In addition, authors also want to
determine the Arrhenius effects affected by global warming.

The problem is very important because very few data have been measured in free-ranging
aboreal small mammals at high T
a.
Besides, arboreality is important also because some small
arboreal mammals do not exploit the cooler, thermally buffered underground realms during the
day.

The purpose of study is to determine the Arhenius effect on the four aboreal mammals.
Through the experiment, authors determine the relationship between ambient temperature (T
b
)
and metabolic rate (MR) by obtained skin temperatures (T
skin
) from free ranging tarsiers (Tarsius
syrichta) and mouse lemur(Microcebus griseorufus), core body temperature (T
b
) from the greater
hedgehog tenrec (Setifer setosus) and the gray brown mouse lemur (Microcebus ravelobensis)
The results are then compared with previous study.


Hypothesis

The hypothesis involved in this research are:

H
0
1 : There is no significant correlation between the maximum day T
a
and the maximum
day T
skin
.
H
0
2 : There is no significant relationship between putative vulnerability and Arrhenius
effects.
H
0
3 : There is no significant correlation between heat stress with climate change.
15

H
0
4 : There is no significant correlation between the minimum night T
a
and the minimum
night T
skin
.
H
0
5 : There is no significant correlation between the daily maximum T
a
and the daily
maximum T
skin
.
H
0
6 : There is no significant differences between the maximum day T
skin
and the
maximum night T
skin
.


Key Terms

The key terms did not define well in the abstract. Authors should define arrhenius effect as a key
term to allow easy understanding on the topic by reader. Beside, the authors should cited skin
temperature and ambient temperature as key terms since their experiment is related to the study
of those temperature. In addition, metabolic rate becomes third key term as authors study the
changes of metabolic rates during climate change.


Review of Literature

The sources cited in this study is comprehensive with the uses of 70 references. The references
used by authors falled between the year of 1972 and 2012. However, the references are mostly
up to date and related the topic of their study. Journal from the year 1972 was used since the
article is related to the energy metabolism and evaporative water loss as well as effects of burrow
temperature and and water vapour pressure of squirrel.

Authors do refered surgical procedure from Lovergrove (2009). Authors also focused on
comparing their results with several references. Authors do focused on several specific articles
for discussion on their experimental findings. For example, authors compared the study of tarsier
with Gursky (1998) finding. Other references included journals from Kobbe et al. (2011), Blanco
and Rahalinarivo, (2010) and Dausmann (2012). There is no bias on the references used. For
example, authors compared their findings on the small tropical mammals, tarsiers may be
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vulnerable to sustained heat stress with climate change which is differ from the finding of
Sherwood and Huber (2010).


Design and procedures

Experimental research methodology was used. Authors carried out experiments to test their
hypothesis. They carry out tests on the four tropical small mammals to obtain their ambient
temperature and skin temperature as well as Arrhenius effect during climate change. From the
methodology, four tropical mammals were studied.

It was an original study which carried further study by using different type of mammals.
Although there were similarities in the principles and methodology as compared to other
previous studies, yet these studies still important as a reference for other researchers who like to
continue the research as well.

The T
skin
was measured using custom-constructed programmable data loggers based on
digitally readable temperature sensors. Besides, the environment data were obtained for each day
from the US Naval Observatory website. The procedures were well structured. The procedure
started from collecting of data from four tropical mammals, obtained the arrhenius effect, and
then analyzed the data. Data collected were then analyzed by using SPSS, version 18 and
compared to an value of 0.05. No pilot study was conducted, as the authors had many related
good references before they conducted the similar experimental procedures.








17

Variables

Variables involved in this study are:
Control variables: arrhenius effect
Reponse variables: skin temperature (T
skin
), core body temperature or ambient
temperature (T
b
)
Constant variables: four types of species


Data Analysis and presentation

Authors carried out data analysis using the SPSS version 18 and compared to an value of 0.05
statistically. The data was quantitative.

The results have been discuss one by one at discussion stage. For example, for the study
of vulnerability to arrhenius effects, the daytime T
skin
tarsiers was influenced by daytime T
a.

Through the study of water availability, authors come out with the conclusion that tropical
heterothermy may be the reduction of the amount water required important to maintain water
balance at normathermy.


Weakness

During the inquiry, authors have been hashing out the weakness and problems. Authors
explained that they are lack of information about the importance of water availability and relative
humidity associated with the ability of small arboreal mammals to cope with global warming.

Beside, authors also mentioned the mechanistic understanding of the metabolic responses
to hyperthermia under different water vapor pressure environment is very poor in small tropical
mammals.

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In addition, authors stated that there is shortage of relative humidity data collected at the
same time with free-ranging physiological data which limit the capacity of climate change
purposely to estimate the gradient between temperature, T
w
and T
b
in microclimates and
microclimates.


Conclusions and Implications

In the conclusion, Authors stated that The conclusion is related to the original purpose of this
study.

Accordingg to authors based on the study of vulnerability to arrhenius effects, the
daytime T
skin
tarsiers was influenced by daytime T
a.
Through the study of water availability,
authors come out with the conclusion that tropical heterothermy may be the reduction of the
amount water required important to maintain water balance at normathermy. The results and
conclusions of the research could be the key to identifying the effects of Arrhenius through the
ambient temperature and temperature during climate change.

Authors suggested that hyperthemic torpor at high T
a
can offset Arrhenius effects.
Arrhenius effects associated with climate change can theoretically impose energetic costs that
can affect fitness.


Overall Assessment

Overall assessment, the research paper is quite hard to understand possibly it need person
who is expertise in this field. The article was clearly divided into five main parts starting from
introduction, materials and methods, results, discussion and references. The abstract is easy to
understand.

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The materials and methods used were easily understood as authors describe in details the
protocols they used in the experiment. For example, authors stated that published data about of
free-ranging T
b
and T
skin
in tropical small mmamals have been measured and reevaluated.

Through the discussion, all the hypothesis have been observed and the results are
compared with the previous literatures. Authors mentioned that further study still need to fully
understand whether any difference physiologically between a torpid , small tropical endotherm in
term of its thermal response to high Ta to a simlar sized tropical ectotherm.




Vocabulary

1. Arboreal animals =Animals living in trees; arboreous
2. Normothermic = A state of normal body temperature
3. Hyperthermia = Unusually high body temperature
4. Torpor = The condition in hibernating poikilotherms during winter when
body temperature drops in a parallel relation to ambient environmental temperatures
5. Enthalpy = measure of the heat content of a chemical or physical system; it
is a quantity derived from the heat and work relations studied in thermodynamics
6. Basoendotherms = mammals with low temperature


REFERENCES

Wu, G., Bazer, F. W., Burghardt, R. C., Johnson, G. a, Kim, S. W., Knabe, D. a, Spencer, T.
E. (2011). Proline and hydroxyproline metabolism: implications for animal and human
nutrition. Amino acids, 40(4), 105363. doi:10.1007/s00726-010-0715-z