BIOCHEMICAL PAPER TASKS II

METABOLISM OF POIKILOTHERMIC HERBIVORS

(TURTLE)

Group 6

Member :
1. Sidiq Kurniawan 22820138
2. April Fiana Nasution 22820147
3. Dhiyaa Ryien Murniyadi 22820148
4. Ni Kadek Galuh Feby Utami 22820149
5. Azmi Khalid Amrulloh 22820150

FACULTY OF VETERINARY MEDICINE

WIJAYA KUSUMA UNIVERSITY, SURABAYA
SURABAYA 2022
 DISCUSSION

Reptiles are bloody vertebrates _ cold (Poikilothermic) that can adapt temperature
body with environment surrounding . Reptiles don't can arrange internal temperature proper
animal bleeding mammal _ heat (Homoiothermic) so they depending on the environment
around For can arrange temperature body them . Sunbathe below _ ray sun is effort reptile in
warm yourself and improve metabolism body , meanwhile For cool temperature body , reptile
usually move to shady place _ or move to area waters (Taylor and O'Shea, 2004). Body
reptile covered by scales composed of keratin and flat or prickly . Function scale from body
reptile is For arrange water circulation that allows reptiles to survive spared from threat
dehydration moment Far from water areas (McDiarmid et al., 2012). Reptiles don't own ear
external and hair nor fur . By and large reptile is animal carnivore . Types of turtles and some
type lizard like an iguana is herbivore , while the chameleon is type reptile eater insect or
insectivores (O'Shea and Halliday, 2001). System reproduction reptile is oviparous and
partially oviviparous , examples in kind boa constrictor (Boa constrictor) which is one type
snake with reproduction oviviparous (Goin and Goin, 1971).

Classification reptile according to Halliday et al ., (1993) as following :

Kingdom : Animalia
Phylum : Chordata
Sub- Phylum : Vertebrata
Class : Reptilia
Order: Squamata, Testudinata , Crocodilia and Rhynchocephalia

Testudinata Order consists from type turtles and tortoises . The most ancient type of
reptiles is turtles , tortoises and terrapins, they are has live on earth since 200 years ago in the
mesozoic era . By and large turtle move slow and have the shell that makes they more easy
For recognized from type reptile other . Type of turtle classified into two groups based on
method interesting neck turtle to in body that is pleurodira (side-necked turtles) and
cryptodira (hidden-necked turtles). Member from second group This capable interesting head
, limbs and tail to in shell For protect self from predators. Group pleurodira generally occupy
many aquatic habitats _ spread over the area tropical Asia, Australia to South America.
Example from group pleurodira is type turtle This can move head and neck For folded to side
between caparace and plastron. Example group pleurodira is type turtle red- breasted turtle (
Emydura many subgloboses ). found in Papua and its surroundings .
Group cryptodir in general normal found in terrestrial habitats , freshwater habitats
and marine habitats . Types of turtles in groups cryptodira own ability For interesting part
neck until head they to in shell . Ability For folding and pulling part neck until head to in
shell in groups pleurodira nor cryptodir in general used For defense To use protect self from
predators. Example species from group cryptodira is turtle Muzzle pig ( Carettochelys
insculpta ). The order testudinata which is spread all over the world consists of from about
260 types of 75 genera and 13 families (McDiarmid, 2012). While in Indonesia found 48
species from 8 families namely Cheloniidae, Dermochelyidae , Trionychidae , Geomydidae ,
Carettochelydae , Testudinidae, Emydidae and Chelidae (Iskandar, 2000).
Freshwater turtle _ omnivore ( Emydura macquarii macquarii ), the is species tortoise
_ _ neck common in Australia , observed on site the . The biota data shows source excess food
_ PFAS food thresholds and likelihood is mechanism key For high PFAS level For enter
population turtle . With thus , investigation beginning to PFAS bioaccumulation and uses tool
ecosurveillance omics based for evaluate impact metabolic from PFAS improvements have
been done . In accordance with limitation permission dynamics population , only four captured
turtle _ from location collision and one turtle from location background rear ( control )
secondary .

Metabolites , lipids and proteins are extracted of the serum collected use method
extraction one pot ( This process offer chance For maximizing information biological from
One sample , omit need For gather a number of samples per groove work . kindly short , 100
L of serum combined with 450 L of methanol cold (−20 °C): ethanol (50%v/v; LiChrosolv ®,
Merck, Darmstadt, Germany), and divortex for 2 minutes . Samples were centrifuged
(Centrifuge 5430R, Eppendorf, Hamburg, Germany) at 14,000rcf at 4°C for 5 minutes
Metabolites metabolism carbon center measured on a coupled Agilent Infinity Flex II UHPLC
to Agilent 6470 Triple Quadrupole Mass Spectrometer ( QQQ -MS) following Sartan (2016)
and Gyawali et al . (2021); lipids and metabolites that are not targeted analyzed on a coupled
Agilent Infinity Flex II UHPLC with Agilent 6546 Quadrupole Time-of-Flight Mass
Spectrometer ( QToF -MS) following Syah et al . ( 2021) and Beale et al . (2021); turtle serum
protein extracted and analyzed through proteomics based on SWATH-MS on a modified
SCIEX 6600 TripleToF -MS from Bose et al . ( 2020) and Bose et al . (2021). Information
Addition serve details more carry on about reported extraction , analysis , and QAQC from
metabolites , lipids, and proteins.

Biochemistry _ functional of the turtle serum analyzed produce 133 metabolites

metabolism carbon center , 210 annotated polar metabolites , 332 annotated lipids , and 102
annotated proteins . Metabolites metabolism carbon center is source energy main from all
organism live ( Eylemet al., 2021), whereas collected polar metabolites through analysis QToF
represent metabolites watery addition related . Lipid fraction represents non-polar complement
( Ebshiana et al ., 2015). From the amount of these , 32 metabolites carbon central and 64 polar
metabolites significant increase or discharged ( i.e. , FC≥ 2.0) in group impact with refers to
the sample control , represents various class metabolites like metabolism purines ( eg ,
allantoin, tamarind urate , and adenosine ), metabolism leucine-isoleucine-valine ( eg , 3-
hydroxyisovaleric acid , ketoleucine , isoleucine , and valine ), guanine nucleotides ( eg , dGTP
, dGDP , guanosine diphosphate , and acid adenylsuccinate ), nucleotides pyrimidines ( eg ,
dTDP , CDP, and dCTP ), nucleotides uracil ( eg , uridine diphosphate ) triphosphate
triphosphate ), among others. For lipids, 115 compounds found increase or reduce in a manner
significant in the group impact with refers to the sample control , which represents class of
lipids such as glycerophosphocholine (n =48), sphingomyelin (n =20), triacylglycerol ceramide
(n = 10), and glycerophosphoethanolamine (n = 4). Supplementary Image . S1, S2 and S3
deliver summary from all metabolites metabolism carbon significant centers and annotated
lipids . plot mountain fiery ( fig . 3) summarizing change significant fold _ in the measured
biochemical data . Different metabolites and lipids in a manner significant This furthermore
become target analysis impact pathway (Fig. 4), in which metabolism pyrimidine (P- value
<0.001), metabolism purines (P- value <0.001), metabolism glycerophospholipids (P- value =
0.021), metabolism sour linoleic (P- value = 0.031), and biosynthesis valine-leucinisoleucine
(P- value = 0.031) was considered enriched and exposed impact in a manner significant .
Important line other enriched _ but No identified in a manner significant disturbed including
metabolism glutamate (P- value = 0.0561), metabolism starch and sucrose (P- value = 0.064),
metabolism galactose (P- value = 0.104), and glutathione metabolism (P- value = 0.130).
Temporary track This No meet the P-threshold assigned value (P- value ≤ 0.05), them of course
represent track with Enough
activity _ enzyme related BCAA metabolism and gene expression in muscle turtle
during period hunger and gifts Eat period short showed onARA . 2. Rules change activity
BCAT and ALT enzymes are consistent with KLF15 mRNA, but consistent BCAT mRNA
expression with change activity enzymes , and ALT respectively gradually increase . Compared
to with gift normal eating , activity BCAT and ALT enzymes increase in a manner significant
in muscle after 3 days starvation (P<0.05 ), but No There is significant difference _ in gene
expression (P>0.05). After starving for 7 days , the activity and expression of the BCAT and
ALT genes increased in a manner significantly , and ALT activity reached its maximum level
. (P<0.05). Activity BCAT enzyme and mRNA levels continued increase and both reach level
maximum after 10 days starvation (P<0.05), while ALT mRNA also increased but activity A
little decreased (P<0.05). Then after starving within 15 days , BCAT gene activity and
expression began decrease but Still more tall from level ate normally (P<0.05), while ALT
increased in a manner significant and expressive reach mark highest (P<0.05). In addition , the
activity and expression of BCAT and ALT genes decreased in a manner significant after
refeeding for 7 days , everything return to normal feeding levels except ALT mRNA level
(P>0.05).
content of BCAA and Ala in muscle turtle during period starvation and term refeeding short
showed in Table 3 and ARA. 3. BCAA content shows downward trend _ first and then increase
during starvation (Fig. 3A), whereas alanine (Ala) otherwise (Fig. 3B). Compared to with gift
eat normally, the content of BCAA and Ala is not There is change significant in muscle after
3 days starving (P>0.05 ). After starving for 7 days , BCAA content starts reduce in a manner
significantly in muscle , while Ala increased (P<0.05). Then continued BCAA content
decreased and reached its lowest level after 10 days starvation , while Ala reached the highest
level

activity _ metabolism chain amino acids branch related BCAT enzymes , ALT start
increase in muscle turtle after starving for 3 days compared to with gift eat normally. After
starving for 7 days , indicator growth as FBW, VSI and HSI show trend significant decrease .
_ KLF15 mRNA expression and protein levels ranging regulated , and enzyme gene activity
and expression related BCAA metabolism BCAT, ALT increases in a manner significant .
Temporary That's it , the BCAA content starts reduce in a manner significant along with Ala
increase . This is possible Because KLF15 expression is regulated to up and facilitating mRNA
and BCAT and ALT activity , so that increase capacity catabolism chain amino acids branch
and lower
P enyu increase intake food and capacity metabolism muscle after starving period short
and refeeding, which keeps balance metabolism and energy homeostasis . Findings This show
that KLF15 is role regulator important metabolism chain amino acids branch in the muscle
turtle .
 Improvement _ Ala content is caused Because sour glutamate produced by
decomposition _ chain amino acids branch , and modified to be Ala below action ALT enzymes
. Reported that expression overexpression of KLF15 in muscle skeleton and cardiomyocytes
increase BCAT2 promoter activity and reduce BCAA concentrations in cell (Shimizu et al .,
2011; Yoshikawa et al ., 2009), which is similar with results obtained _ in study this . In
addition , KLF15, BCAT and ALT expression remained increase in muscle after 10 days
starvation , and expression of KLF15 and BCAT mRNA levels reached mark highest . Likewise
activity _ BCAT enzymes also achieve highest after 10 days starve , but ALT has decreased .
Currently , the breakdown of BCAAs continues continues and reaches its lowest level . On the
other hand , Ala 's content reaches its highest level . this result show that turtle shell Chinese
software exploits chain amino acids branch from muscle For maintain energy homeostasis in
body in condition starving . studies have show that BCAA catabolism is necessary provide
substrate carbon For gluconeogenesis heart For maintain normal euglycemia in circumstances
fasting. Gray et al., 2007). Besides that , the KLF15 is capable Inhibit lipogenesis and promote
gluconeogenesis with increase regulation enzyme critical deep BCAA decomposition
circumstances fasting , so provide substrate gluconeogenic For heart ( Teshigawara et al .,
2005). Then , KLF15 and BCAT gene expression and activity enzyme both of them decrease
in a manner significant when starving be extended up to 15 days , while ALT increases in a
manner significant . Whereas more BCAA content tall from normal feed , and Ala decreased
in a manner gradually . This is possible Because chain amino acids branch increase
decomposition For provide energy for body after starving . However , BCAAs are not can
unraveled become muscle when reach level certain , and in gradually return to balanced level
. _ this _ in accordance with Takeuchi's opinion et al . (2016), who considers that KLF15
participates in regulation various track metabolism body in condition starving . It promotes
gluconeogenesis and provide energy For maintain activity physiology and homeostasis body
(Takeuchi et al ., 2016). In conjunction with this , level intestinal and hepatic protein turnover
increase with extension hunger , and intake clean precursor gluconeogenicity is also increased
in the liver , showed enhancement gluconeogenesis (de Blaauw et al ., 1996).

BIBLIOGRAPHY
Beale, DJ, Shah, R., Karpe, AV, Hillyer, KE, McAuley, AJ, Au, GG, Marsh, GA, Vasan, SS,
2021 Metabolic profile of the weasel model without symptom SARS-CoV-2 infection .
Metabolites 11, 327.

Bose, U., Broadbent, JA, Byrne, K., Blundell, MJ, Howitt, CA, Colgrave , ML, 2020. Multiple
proteins barley hordein-null line analysis revealed storage protein synthesis and
mechanism compensation . J. Agriculture . Food Chemistry . 68, 5763–5775.
Bose, U., Broadbent, JA, Juhász , A., Karnaneedi , S., Johnston, EB, Stockwell, S., Byrne, K.,
Limviphuvadh , V., Maurer-Stroh, S., Lopata, AL, Colgrave , ML, 2021. Protein extraction
protocol for measurement optimal proteome and quantitation arginine kinase from Acheta
domesticus cricket For evaluation security food . Food Chemistry . 348, 129110.
de Blaauw, I., Deutz, NE, Von Meyenfeldt , MF, 1996. Metabolism in vivo amino acids from
the gut and liver during starving short and long . I. J. Physiol . 270, G298–G306. De
Simone, R., Vissicchio, F., Mingarelli , C., De Nuccio, C., Visentin, S., Ajmone -Cat,

Eylem, CC, Reçber , T., Waris, M., Kdanr , S., Nemutlu , E., 2021.The most advanced GC-MS
application approach For investigate metabolism carbon center . microchemistry . J.
106892.

Gray, S., Feinberg, MW, Hull, S., Kuo, CT, Watanabe, M., Sen-Banerjee, S., DePina, A.,
Haspel, R., Jain, MK, 2002. Krüppel-like factor KLF15 regulates glucose transporters
sensitive GLUT4. J. Biol . Chemistry 277, 34322–34328

Gyawali, P., Karpe, A.V., Hillyer, KE, Nguyen, TV, Hewitt, J., Beale, DJ, 2021. A multi-
approach platform metabolomics for identify possible biomarkers for contamination feces
humans in GreenshellTM shellfish (Perna canaliculus). sci. Total Environment . 771.

Dynamic MRM databases and methods metabolomics agilent . agile Overview technical
technology , no publication . 5991–6482EN.
Shimizu, N., Yoshikawa, N., Ito, N., Maruyama, T., Suzuki, Y., Takeda, S., Nakae, J., Tagata,
Y., Nishitani, S., Takehana, K. , 2011. Crosstalk between receptor glucocorticoids and the
sensory nutrition of mTOR in muscle framework . Cell Metab . 13, 170-182.
Takeuchi, Y., Yahagi, N., Aita, Y., Murayama, Y., Sawada, Y., Piao, X., Toya, N., Oya, Y.,
Shikama, A., Takarada , A. , 2016. KLF15 makes it possible transition fast between
lipogenesis and gluconeogenesis during fasting . Cell Representative . 16, 2373–2386.

Teshigawara , K., Ogawa, W., Mori, T., Matsuki, Y., Watanabe, E., Hiramatsu, R., Inoue, H.,
Miyake, K., Sakaue, H., Kasuga, M. , 2005. The role of factors like Krüppel 15 in PEPCK
gene expression in the liver . Biochemistry . Biophysic . Res. communion . 327, 920–926.
Uchida, S.,
Tanaka, Y., Ito, H., Saitoh -Ohara, F., Inazawa , J., Yokoyama, KK, Sasaki, S.,