Insulin signaling in the hearts of uncoupling protein three knockout mice on a high fat diet

INTRODUCTION: People with diabetes are at high risk for cardiovascular disease; which is the major cause of death in diabetics. Current research suggests that excessive mitochondrial uncoupling is a potential mechanism for cardiac dysfunction in diabetic patients. Uncoupling protein 3 (UCP3) is an inner mitochondrial membrane protein that reduces the amount of reactive oxygen species (ROS) produced in the mitochondrial matrix by uncoupling the mitochondria. Mice lacking UCP3 (UCP3KO) have been shown to accumulate ROS and diacylglycerol in cardiac muscle. These two molecules have been shown to induce insulin resistance in skeletal muscle. The goal of my project was to determine if the absence of UCP3 in the heart will exacerbate diet-induced insulin resistance in the murine heart. MATERIALS AND METHODS: Whole heart homogenates were extracted from four groups of mice: (1) wild type (WT) fed normal chow (NC); (2) WT fed a high fat diet for 10 weeks (HF); (3) UCP3KO NC and (4) UCP3KO HF. Insulin signaling was examined using western-blotting to assess the phosphorylation status of key components of the insulin signaling pathway under non insulin and insulin-stimulated conditions in each group. Values form each group were analyzed using an ANOVA followed by a t test to determine any statistical significant differences between the groups. RESULTS: Phosphorylation of protein kinase B (Akt) (at serine 473 or threonine 308) and the downstream target glycogen synthase kinase 3 p at serine 9 was the same between all the groups. CONCLUSIONS: two major conclusions arise from this work; ( 1 ) 1 0 weeks of high fat diet does not affect insulin signaling in the heart and (2) despite increased ROS and DAG levels in the hearts of UCP3KO mice, insulin sensitivity was maintained. We speculate that UCP3KO mice have developed compensatory mechanisms that prevented the development of insulin resistance despite accumulation of compounds known to induce insulin resistance. These mechanisms remain to be determined in future studies.

INSULIN SIGNALING IN THE HEARTS OF UNCOUPLING PROTEIN THREE KNOCKOUT MICE ON A HIGH FAT DIET by Timothy James Tidwell A Senior Honors Thesis Submitted to the Faculty of the University of Utah in Partial Fulfillment of the Requirements for the Honors Degree of Bachelor of Science in Biology Approved: � E. Dale Abel Neil J. Supervisor Chair, Department of Biology �C.�b* ��\ ·ckers a�(!zd} Darryl Kropf M Departmental Honors Advisor Dean, Honors College May 2009 A BSTRACT ABSTRACT I N T R O D U C T I O N : People with diabetes diabetes are at cardiovascular INTRODUCTION: at high risk for cardiovascular h i c h is the major major cause of research of death in diabetics. Current Current research disease; w which suggests that mitochondrial uncoupling uncoupling is a potential mechanism m e c h a n i s m for suggests that excessive excessive mitochondrial cardiac dysfunction in diabetic diabetic patients. Uncoupling Uncoupling protein 3 (UCP3) inner cardiac dysfunction (UCP3) is an inner mitochondrial membrane m e m b r a n e protein that reduces reduces the amount amount of of reactive oxygen oxygen mitochondrial species (ROS) produced in the mitochondrial mitochondrial matrix matrix by uncoupling the species Mice lacking UCP3 UCP3 ((UCP3KO) U C P 3 K O ) have been shown s h o w n to accumulate accumulate mitochondria. Mice R O S and diacylglycerol molecules have been been cardiac muscle. These two molecules ROS diacylglycerol in cardiac s h o w n to induce induce insulin resistance in skeletal muscle. T h e goal of my project project was was The of my shown to determine if the absence a b s e n c e of of UCP3 U C P 3 in the heart heart will exacerbate exacerbate diet-induced diet-induced determine if MATERIALS A N D METHODS: M E T H O D S : Whole Whole insulin resistance in the murine heart. MATERIALS AND heart homogenates homogenates w e r e extracted from four groups g r o u p s of heart were of mice: (1) wild type (WT) fed normal chow c h o w (NC); (2) W T fed a high fat diet e e k s (HF); (3) UCP3KO UCP3KO WT diet for for 10 w weeks NC and (4) UCP3KO U C P 3 K O HF. Insulin signaling w as e x a m i n e d using western-blotting western-blotting was examined to assess assess the phosphorylation c o m p o n e n t s of of the insulin signaling signaling phosphorylation status of of key components pathway under insulin-stimulated conditions pathway under non insulin and insulin-stimulated conditions in each group. V a l u e s form each group group w e r e analyzed N O V A followed by a t test Values were analyzed using an A ANOVA test to determine any RESULTS: determine any statistical significant significant differences differences between the groups. RESULTS: Phosphorylation of of protein kinase B (Akt) (at serine 473 and 473 or or threonine threonine 308) and Phosphorylation ii ii synthase kinase 33 p~ at at serine serine 99 w was the downstream d o w n s t r e a m target gtycogen glycogen synthase a s the same same between all the groups. two major conclusions arise arise from this groups. CONCLUStONS: C O N C L U S I O N S : two major conclusions diet ddoes not affect affect insulin signaling signaling in the heart work; work; (1) ( 1 ) 110 0 weeks w e e k s of high fat diet o e s not heart ROS DAG hearts of and (2) despite increased increased R O S and D A G levels in the hearts of UCP3KO U C P 3 K O mice mice,, a s maintained. maintained. W e speculate C P 3 K O mice have have insulin sensitivity w was We speculate that that U UCP3KO d e v e l o p e d compensatory compensatory m e c h a n i s m s that insulin developed mechanisms that prevented prevented the development development of of insulin accumulation o m p o u n d s known to induce induce insulin insulin resistance despite accumu lation of of ccompounds resistance.. These T h e s e mechanisms m e c h a n i s m s remain to be determined studies. resistance determined in future studies. jii iii Table of of Contents Table Contents Introduction ................................................................................................... 11 Introduction Materials and and M Methods Materials e t h o d s .................................................................................. 6 Mouse generation ........................................................................................ 6 M o u s e generation Animal care and and diets diets .................................................................................. 6 A n i m a l care Metabolic chamber chamber ....................................................................................... 7 Metabolic Glucose tolerance tests tests ............................................................................... 7 G l u c o s e tolerance Ceramide DAG levels ........................................................................... 8 C e r a m i d e and D A G levels ROS measurements R OS m e a s u r e m e n t s .................................................................................... 8 Respiration and A ATP production in saponin-permeabilized saponin-permeabilized fibers ................ 8 Respiration T P production Western-blotting ........................................................................................... 9 Western-blotting Statistical methods methods ..................................................................................... 11 11 Results ......................................................................................................... 12 Body energy expenditure, food intake and activity activity ....................... 12 Body weights, energy Glucose tolerance tests ............................................................................. 14 G l u c o s e tolerance Respiration and A ATP saponin-permeabilized cardiac T P production in saponin-permeabilized cardiac fibers. fibers . 14 Diacylglycerol and ceramide levels ............................................................ 19 19 Reactive oxygen species ........................................................................... 19 Effect Effect of combined c o m b i n e d UCP3 U C P 3 deficiency deficiency and HFD on insulin signaling in the heart heart ................................................................................................................ 22 Discussion D i s c u s s i o n ................................................................................................... 26 Similar body weight, increased activity and enhanced e n h a n c e d mitochondrial coupling coupling ........................................................................................................... 26 iv iv Increased state 4 respiration with HFD occurs independently independently of UCP3 U C P 3 .. .. . 27 27 UCP3 U C P 3 reduces reactive oxygen species generation in the heart ....... ....... .. 27 27 Elevated diacylglycerol levels ..... ........ ......... .. ............. .. ............................ 28 28 Similar Similar glucose tolerance between WT W T and UCP3KO U C P 3 K O mice on HF ... ...... .. 29 29 Preserved insulin signaling in UCP3KO U C P 3 K O despite increased ROS R O S and DAG D A G 29 29 References R e f e r e n c e s ................................................................................................... 31 31 v 1 Introduction Introduction Diabetes, aa disease disease in which the body body is no longer longer able to make or properly respond to insulin, affects affects over properly over 20 million people in the United States. have a greater People with diabetes diabetes have greater risk for cardiovascular cardiovascular disease, and it is the number a u s e of of death in diabetics. Current Current research suggests suggests that number one ccause mitochondrial uncoupling uncoupling plays an important important role in the development development of cardiac mitochondrial of cardiac dysfunction dysfunction in humans h u m a n s and rodents with diabeties d i a b e t i e s1,2, ' '. 1 2 The heart is aa ddynamic that is constantly contacting and consuming T h e heart y n a m i c organ that constantly contacting consuming Mitochondrial A T P is the primary primary source of energy required required oxygen and energy. Mitochondrial ATP of energy for cardiomyocyte cardiomyocyte contractions. This This energy compounds energy is derived derived from reduced compounds such as N A D H and FADH2 series of proteins donate electrons electrons through a series of proteins NADH FADH2 which donate called the electron transport acceptor being being transport chain complex complex with the final electron acceptor o xygen. A s a result of protons are transported from the oxygen. As of this process, protons mitochondrial matrix matrix to the mitochondrial mitochondrial inter-membrane inter-membrane space. This mitochondrial This results in a gradient that T P synthase T P from A D P and and proton gradient that is used by A ATP synthase to produce A ATP ADP inorganic phosphate. In uncoupled mitochondria, the formation of proton of the proton inorganic gradient is no longer longer coupled with the production of T P . Instead, the protons are gradient of A ATP. translocated back to the mitochondrial matrix ATP translocated back matrix without without going through ATP synthase h i c h lowers the proton protonmotive d e c r e a s e s the rate of ATP synthase w which motive force and decreases of ATP process has been well studied and is believed to be production. The uncoupling uncoupling process mediated in part by proteins, located in the mitochondrial mitochondrial inner inner membrane, m e m b r a n e , called called uncoupling proteins proteins or or UCPs. UCPs. uncoupling 2 The discovered UCP was T h e first discovered UCP w a s UCP1, U C P 1 , which is abundant abundant in mammalian mammalian present in concentrations brown adipose adipose tissue and is present concentrations up to 10 % of of all 44 mitochondrial m e m b r a n e proteins thermogenesis . mitochondrial membrane proteins and is known to have have a role in thermogenesis T w o recently discovered orthologs of U C P 1 , namely namely UCP2 U C P 2 and UCP3, U C P 3 , are found found Two discovered orthologs of UCP1, mitochondria of of other heart other tissues beside brown adipose tissue such as heart in the mitochondria and skeletal muscle. At U C P 2 and UCP3 UCP3 w e r e thought thought to have a role in At first, UCP2 were tthermogenesis h e r m o g e n e s i s like UCP1, U C P 1 , however concentrations are so low low (0.1 ( 0 . 1% % to however their their concentrations 0.01%) o u l d not not be effective generating heat. In addition, proteins proteins effective in generating 0.0 1%) that that they w would similar U C P 2 and UCP3 U C P 3 have been found in ssome o m e fish and plants that that do not similar to UCP2 h u s it is believed that U C P 2 and UCP3 UCP3 d o not have thermogenic thermogenic properties. T Thus that UCP2 do have a significant t h e r m o g e n e s i s4 .. have significant function in thermogenesis 4 Other functions for UCP2 and UCP3 Other putative functions UCP3 include: (1) reduction of of the a m o u n t of (ROS) such as super super oxide and hydrogen hydrogen amount of reactive oxygen species (ROS) peroxide peroxide and (2) fatty acid and fatty acid peroxide export. Most Most scientific scientific e v i d e n c e supports U C P s prevent prevent cell damage d a m a g e by reducing the evidence supports the theory theory that that UCPs rate of R O S formation. ROS R O S is produced complex I through the FMN F M N region region of ROS produced at at complex w h e n there is a high ratio of N A D H to NAO+ N A D or transport when of NAOH or by reverse electron transport + w h e n the proton motive force is elevated and there is an increased amount a m o u n t of when 3 fully reduced ccoenzyme oenzyme Q h e mild uncoupling uncoupling ccaused a u s e d by UCPs U C P s lowers the Q3.. T The protonmotive force of inner mitochondrial mitochondrial membrane m e m b r a n e enough e n o u g h to reduce the protonmotive of the inner accumulation R O S within the mitochondrial mitochondrial matrix. If UCPs U C P s are inhibited by accumulation of of ROS G D P there increase in the membrane m e m b r a n e potential as well as an increase in GOP there is an increase 3 ROS generation under under certain conditions. In addition, UCP3 R O S generation U C P 3 knockout knockout mice have have 5 increased ROS increased R O S production in several tissues t i s s u e s ., 5 Recent studies have Recent studies have linked mitochondrial mitochondrial uncoupling to diabetes, diabetes. A study study in w women various mutant o m e n related various mutant haplotypes haplotypes of of the UCP2 U C P 2 I/ UCP3 U C P 3 region to an 6 Another of diabetes study found that that UCP3 expression levels increased risk of d i a b e t e s ., A n o t h e r study U C P 3 expression levels 6 7 were % lower of diabetics ., In contrast, overw e r e 50 % lower than controls controls in skeletal muscle muscle of over7 expression of was expression of UCP3 U C P 3 in skeletal muscle muscle w a s found to be protective protective from fat8 The induced insulin resistance ., T h e role of U C P 3 in the heart heart is unclear, unclear. In rats with induced of UCP3 8 U C P 3 mRNA m R N A increased by 9.4 of UCP3 9.4 fold induced type 1 diabetes, the expression of which could contribute contribute to d epressed A TP g e n e r a t i o n 9.. Finally, it has also been been depressed ATP generation 9 ssuggested u g g e s t e d that U C P 3 might might protect heart from lipid-induced lipid-induced oxidative stress stress that UCP3 protect the heart m o u n t of ROS . by reducing the aamount of ROS10. 1 0 The association of of obesity obesity with insulin resistance and type 2 diabetes T h e association diabetes is today's high-fat Western diet and increased sedentary well established, and today's high-fat W e s t e r n diet sedentary lifestyle have have m made worldwide epidemic a d e type 2 diabetes diabetes a worldwide e p i d e m i c 11,. Development D e v e l o p m e n t of 11 insulin resistance has been associated with alterations alterations in lipid metabolism metabolism and and increased intracellular intracellular lipid content content in skeletal muscle muscle and liver. Indeed, several several studies that accumulation accumulation of studies have have sshown h o w n that of intra-myocellular intra-myocellular triglycerides t r i g l y c e r i d e12,s 13,14,15 ' ' 1 2 1 3 1 4 , 1 5 or fatty acid metabolites of insulin or m e t a b o l i t e s16, '17, 18 has been implicated in the pathogenesis pathogenesis of insulin 1 6 1 7 , 1 8 resistance. Furthermore, studies studies in skeletal muscle muscle have sshown that dietary h o w n that dietary fat 18 directly reduce insulin signaling signaling through a ccascade can directly a s c a d e involving PKce P K C 0 , 19.. 1 8 , 1 9 4 The that are activated by T h e insulin signaling cascade is a series of of proteins proteins that insulin binding to insulin receptors on the plasma membrane. m e m b r a n e . As A s shown s h o w n in figure 1, activate a variety variety of 1 , activated insulin receptors then phosphorylate phosphorylate and activate insulin receptor substrate (IRS) proteins which then phosphorylates receptor substrate phosphorylates other other d o w n s t r e a m targets such as protein kinase B (Akt) (Akt).. One O n e result of of this downstream phosphorylation cascade cascade is recruitment recruitment of of a glucose glucose transporting protein transporting protein phosphorylation (GLUT4) to the plasma membrane and the stimulation of glucose uptake. Indeed, glucose uptake. (GLUT4) stimulation of state, GLUT4 G L U T 4 is stored in intracellular intracellular vesicles which which in a non insulin-stimulated state, membrane w h e n the cell is stimulated with are integrated into the plasma membrane when i n s u l i n . Akt is one of of the insulin"'. 20 DIABETES •f-FPA* many proteins proteins that are many that are insulin raceptof * FA U M « M insulin.. Its activated by insulin activation leads to the and TO j 4-IRS1 I Impaired fmoMr* LttgnaNngi phosphorylation and phosphorylation I fiiiiSMwfwIm I of glycogen glycogen inhibition of Figure 1. diet induced on I. Effects Effects of of high fat diet induced diabetes on synthase 3(3 (GSK3J3), synthase 3 ~ (GSK3P), insulin signaling and fatty fatty acid ac id metabolism. metabo lism. Diabetes results as illustrated in figure 11.. in aa decrease in phosphorylation of insulin signaling as we wellll phosphorylation of T h e inhibition of GSK3p GSK3p The an increase acid ox oxidation, and UCP3 as an increase in fatty fatty acid idation, ROS and affects the activity of affects e n z y m e s responsible for various cellular cellular functions including including,, storage of glucose enzymes of glucose of glycogen), glycogen), and an increase of of protein synthesis synthesis and cell growth. growth. (in the form of G S K 3 p may may al also play a rol role e v e l o p m e n t of of type 2 iabetes . T h e two so playa e in the ddevelopment 2 ddiabetes". The GSK3p 21 of GSK3p, G S K 3 p , glycogen glycogen synthase receptor substrate-1 (IRSmain targets of synthase and insulin receptor 5 1), are suppressed by G GSK3~. S K 3 p . GSK3~ GSK3(3 phosphorylates phosphorylates a serine residue of of IRS-1, IRS-1, which w h i c h has been show s h o w during in vitro vitro studies to decrease decrease the tyrosine tyrosine phosphorylation phosphorylation of of the insulin receptor receptor and IRS_141. I R S - 1 . Selective Selective inhibition of of GSK3~ GSK3P 4 1 22 Furthermore, increases glucose uptake and decreases decreases insulin resistance r e s i s t a n c e .. Furthermore, 22 diabetic diabetic patients show s h o w increased GSK3~ G S K 3 p activity23. activity . 23 We W e hypothesized that knockdown of of UCP3 U C P 3 in the heart heart might might promote fatinduced insulin resistance by inhibiting insulin signaling because because of of increased increased oxidative stress due d u e to an increase increase in ROS. R O S . To test this hypothesis hypothesis and develop a better better understanding understanding of of mechanisms m e c h a n i s m s that leads to fat-induced insulin resistance, resistance, w e e x a m i n e d insulin-stimulated insulin-stimulated Akt G S K 3 p phosphorylation phosphorylation in hearts from from we examined Akt and GSK3p U C P 3 K O mice and age-matched age-matched wild-types either normal chow c h o w (NC) or highhighUCP3KO wild-types fed either fat diet diet (HFO) (HFD) for 10 w e e k s . The The results show s h o w that UCP3KO U C P 3 K O mice on a high fat weeks. diet d o not not exhibit exhibit glucose intolerance and maintain phosphorylation phosphorylation on A K T and and AKT diet do glucose intolerance GSK3p. GSK3~. U C P 3 K O mice have a mechanism mechanism Based on these results it is likely that UCP3KO to prevent prevent the development d e v e l o p m e n t of of insulin resistance. 6 Materials and Materials and Methods Methods Mouse Mouse generation generation UCP3 were obtained obtained from the Lowell lab at Harvard Medical U C P 3 knockout knockout mice were Medical 24 24 described a replacement School and were w e r e generated as previously previously d e s c r i b e d .. Briefly, a replacement targeting vector a s prepared in which a segment of of the UCP3 UCP3 gene g e n e between between a segment vector w was codon, w a s removed and replaced with a PGKexons 22 and 3, including the start start codon, was a PGKNEO-poly(A) expression cassette. T h e targeting plasmid w a s linearized with Notl NEO-poly(A) The was and electroporated electroporated into J1 embryonic stem cells. J 1 embryonic Animal care Animal care and and diets diets Animals were day (between 77 AM A nimals w e r e studied in the random-fed state during the day AM and Care and Use a n d 1 PM) using protocols protocols approved by the Institutional Animal Care Use C o m m i t t e e of of the University University of Male wild-type U C P 3 K O mice (10 Committee of Utah. Male wild-type (WT) and UCP3KO w e e k s of of age) w e r e fed ad libitum either c h o w (NC) diet Harlan weeks were either aa normal chow diet (#8656, Harlan T e k l a d , Madison, Madison, W l ) or a high-sucrose diet D 1 2 4 5 1 , Research Research Teklad, WI) a high-fat high-fat (HF), high-sucrose diet ((012451, N e w Brunswick, NJ) for 10 weeks. w e e k s . The composition provided composition of of the diets diets is provided Diets, New in Table Table 1. 7 Table 1. Composition Composition of standard standard chow high fat diet Table of chow and and high fat diet N o r m a l chow: 24.5% 2 4 . 5 % calories from protein, 4 . 4 % calories from fat, and 54.5% 54.5% Normal 4.4% calories from carbohydrate Teklad, carbohydrate (#8656, Harlan T e k l a d , Madison, M a d i s o n , WI) Wl) High fat chow: (012451, ( D 1 2 4 5 1 , Research Diets, New New Brunswick, NJ), which provided provided 2 0 % calories calories from protein (200 gm% g m % casein), 4 5 % calories gm% 20% 45% calories from fat (178 gm% lard, 25 ggm% 35% carbohydrate [50% m % soybean s o y b e a n oil), and 3 5 % of of calories calories from carbohydrate [ 5 0 % sucrose] sucrose] (73 ggm% gm% m % corn starch, 100 100 gm% g m % maltodextrin, and 173 g m % sucrose) sucrose) Metabolic chamber Metabolic chamber Indirect was four-chamber Oxymax Indirect calorimetry calorimetry w a s performed with a four-chamber O x y m a x system system ( C o l u m b u s Instruments, Columbus, C o l u m b u s , OH). Animals e r e allowed to adapt adapt to the (Columbus Animals w were metabolic chamber 02, V C 0 2 , heat heat production, food and water metabolic chamber for for 44 h and then V V02, VC02, intake, a n d movement movement w e r e measured measured every minutes for d a y s from from for 33 days and were every 15 minutes individually housed mice. A v e r a g e d data from 6 x p r e s s e d as individually Averaged 6 p.m. to 66 a.m. are eexpressed day values. night values values and data data from 66 a.m. to 66 p.m. are expressed expressed as as day Glucose Glucose tolerance tolerance tests tests After a 6 h fast mice w were weighed glucose (1 After ere w e i g h e d and glucose (1 g/kg body body weight) was was administered intra-peritoneally to nonsedated nonsedated animals. Tail vein blood (3 IJL) uL) was was administered intra-peritoneally s a m p l e d at 0 - 1 2 0 min for (Glucometer Elite, Bayer Bayer Corp., 0-120 for glucose glucose determination determination (Glucometer sampled Tarrytown, NY, USA). 8 Ceramide and and DAG levels Ceramide DAG levels whole were extracted using methods described Total lipids from w h o l e hearts w e r e extracted described 5 previousll extracts w was determined using a p r e v i o u s l y .. Ceramide C e r a m i d e content content in the extracts a s determined 25 diacylglycerol assay (ceramide:DAG assay) (Amersham (Amersham radiometric diacylglycerol assay kit (ceramide:DAG Biosciences) according manufacturer's instructions. Biosciences) according to the manufacturer's ROS measurements ROS measurements The amount was fluorescence assay. A non T he a m o u n t of of ROS ROS w a s measured m e a s u r e d using a fluorescence non fluorescent ccompound fluorescent o m p o u n d (H2DCFDA, ( H 2 D C F D A , C400 C 4 0 0 Molecular Molecular Probes) is oxidized by ROS ROS into aa highly fluorescent 2',T-dichlorofluoorescein highly fluorescent 2',7'-dichlorofluoorescein (DCF) ccompound. ompound. A A fluorescent was fluorescent dye that is insensitive insensitive to ROS R O S (C369, Molecular Molecular Probes) w a s used for normalization. Tissue Tissue w was containing 50 mmol/L a s homogenized h o m o g e n i z e d in a buffer buffer containing mmol/L p h o s p h a t e buffer, 1 mmol/L mmol/L EDTA, 0.5 mmol/L PMSF, P M S F , 1 I-ImollL umol/L pepstatin, 80 phosphate inhibitor (pH 7.4) 7.4) and then centrifuged at 900g for 15 minutes minutes at mg/L trypsin inhibitor 4 ° C . Approximately ug of of homogenized h o m o g e n i z e d tissue w a s mixed with enough e n o u g h dye dye was 4°C. Approximately 100 I-Ig a n d homogenization homogenization buffer buffer to create a final dye umol/L. The The and dye concentration of of 25 I-Imol/L. preparation w a s then incubated incubated at 37°C preparation was 3rC for 30 min and read at two time points (0 and 30 min) using a microplate microplate fluorescence reader (Bio-Tek (Bio-Tek Instruments) Instruments) at an fluorescence reader 8 5 nm n m and emission C 3 6 9 and 50 excitation of of 4485 emission of of 530 nm, at at sensitivity sensitivity 25 for C369 C400. for C400. Respiration and and ATP A TP production production in saponin-permeabi/ized fibers Respiration in saponin-permeabilized fibers Mitochondrial respiratory parameters were Mitochondrial respiratory parameters w e r e studied in saponin-permeabilized saponin-permeabilized cardiac and soleus fibers cardiac f i b e r s1,2 ' using 20 I-ImollL umol/L palmitoyl-carnitine palmitoyl-carnitine (PC) with 2 1 2 9 mmoliL for cardiac f.!moliL rotenone m m o l / L malate for cardiac fibers and 5 mmoliL mmol/L succinate succinate and 10 |nmol/L rotenone for soleus D P (State 2 or V0), of A AOP or VO), soleus fibers. Basal respiration before the addition of maximally ADP-stimulated mmol/L) respiration (State 3 or V A D P ) , and and maximally AOP-stimulated (1 mmol/L) VAOP), of oligomycin (1 IJg/mL), ug/mL), which inhibits A T P synthase synthase respiration in the presence of ATP activity (State 4 or VOIigo) T P concentration a s measured measured ATP concentration w was activity VOligo) were were determined. A bioluminescence assay assay using the Enliten Luciferase/Luciferin Luciferase/Luciferin Reagent Reagent by a bioluminescence ( P r o m e g a , Madison, M a d i s o n , WI). Wl). (Promega, Western-blotting Western-blotting Frozen w whole were h o l e hearts hearts w e r e homogenized homogenized in a lysis buffer buffer [0.05 moliL mol/L sodium sodium pyrophosphate, sodium fluoride, 0.03 0.03 M HEPES, 1 p y r o p h o s p h a t e , 0.1 mol/L sodium H E P E S , 0.001 moliL mol/L EOTA, EDTA, 1 % Triton X-1 00, X-10 0 , protease inhibitor (Roche) and phosphatase inhibitor cocktail 1 + 2 (Roche)) The (Roche)] and a n d incubated on ice for 1 hr. T h e samples were then centrifuged at 9000 supernatant w was 9000 rpm at 4°C for 15 minutes. The resulting supernatant a s then used for the western of 20-60 ug of a s run using S D S - P A G E (10 % % 20-60 f.!g of protein w was SOS-PAGE western blots. A A quantity quantity of acylamide PVDF membrane m e m b r a n e (Immobilon-FL (Immobilon-FL acylamide gel) and then transferred to a PVOF Transfer M e m b r a n e , Millipore, Billerica, MA). The T h e membrane membrane w a s then blocked blocked Transfer Membrane, was milk in PBS for 1 hour. Primary Primary and secondary e r e mixed mixed using 5 % milk secondary antibodies antibodies w were m e m b r a n e for 1 hour. hour. in PBS with 0.01 % tween 20 and incubated with the membrane S e c o n d a r y antibodies w e r e then revealed using an Odyssey Odyssey infrared scanner. scanner. Secondary were (LI-COR Bioscience, Lincoln, NE) and the florescence of of the secondary antibody (LI-COR secondary antibody w a s then quantified Odyssey software. The T h e names n a m e s and the was quantified using the provided Odyssey conditions of of antibodies used are ssummarized u m m a r i z e d in Table Table 2. 10 Table and conditions primary and Table 2. Names. Names, references references and conditions of of primary and secondary secondary antibodies antibodies Primary Primary antibodies: antibodies: Phospho 473 CS-9275, P h o s p h o -Akt - A k t serine 4 7 3 (Cell Signaling, C S - 9 2 7 5 , Danvers Danvers MA). 1/1000 dilution in PBS with 0.1 % % tween 20. Incubation overnight overnight at +4° C Phospo-Akt threonine 308 (Cell Signaling, C CS-9271, Phospo-Akt threonine S - 9 2 7 1 , Danvers Danvers MA). 1/1000 dilution in PBS with 0.1 % % tween 20. Incubation overnight overnight at +4° C Akt (Upstate Total Akt (Upstate 07-416, Millipore, Billerica MA). 1/1000 dilution in PBS with 0.1 % % tween 20. Incubation overnight overnight at +4° C Phospho-GSK3b CS-9336, P h o s p h o - G S K 3 b Serine 9 (Cell Signaling, C S - 9 3 3 6 , Danvers Danvers MA) 1/1000 dilution in PBS with 0.1 % overnight at +4° C % tween 20. Incubation overnight Total GSK3 G S K 3 a/J3 a/p (Santa Cruz Cruz Biotechnology Biotechnology ssc-7291, c - 7 2 9 1 , Santa Santa Cruz Cruz CA) CA) 1/1000 dilution in PBS with 0.1 % % tween 20. Incubation overnight overnight at +4° C S e c o n d a r y antibodies: antibodies: Secondary Anti-Mouse-AlexaFluor 8 0 (Invitrogen A 2 1 0 5 8 , Carlsbad CA) 1/10000 dilution in Anti-Mouse-AlexaFluor 6680 A21058, PBS with 0.1 % % tween 20. Incubation: 1 hour hour at room temperature. Anti-Rabbit-AlexaFluor A21109, Carlsbad CA) 1/10000 dilution in Anti-Rabbit-AlexaFluor 680 (Invitrogen A 2 1 1 0 9 , Carlsbad % tween 20. Incubation: 1 hour hour at PBS with 0.1 % at room temperature. Anti-Mouse A n t i - M o u s e Licor Licor 800 (WVR ( V W R RL-611-132-122, LI-COR LI-COR Bioscience, Lincoln NE) 11 1/5000 dilution in PBS with 0.1 0.1 % tween 20. Incubation Incubation:: 1 hour hour at room room temperature temperature.. Anti-Rabbit Licor 800 (VWR Anti-Rabbit ( V W R RL-611-132-121 R L - 6 1 1 - 1 3 2 - 1 2 1 , LI-COR LI-COR Bioscience, Bioscience, Lincoln NE) NE) 0.1 % tween 20. Incubation 1/5000 dilution in PBS with 0.1 Incubation:: 1 hour hour at room room temperature. Statistical Statistical methods methods Data are mean±SEM. ANOVA m e a n ± S E M . Data were analyzed using a 1-way 1-way A N O V A followed by a t test. Significant differences differences were w e r e accepted at P < 0.05. 0.05. 12 Results Results Body food intake intake and and activity Body weights, weights, energy energy expenditure, expenditure, food activity Wild type and UCP3KO diet (HFD) similar body U C P 3 K 0 mice fed a high fat diet (HFD) had similar we expenditure w were w e iights g h t s (Figure 2). The T h e overall energy energy intake and expenditure e r e monitored monitored using a metabolic metabolic chamber chamber to see if there are any differences T and and using are any diffe rences between W WT (Figure 33 A), A), carbon UCP3KO when U C P 3 K O mice w h e n fed a HFD. H F D . Oxygen O x y g e n cco o nnsumed s u m e d (V02) ( V 0 2 ) (Figure carbon dioxide exhaled 3 C), C), food (Figure e x h a l e d ,, (VC02) ( V C 0 2 ) (Figure 3 B) heat heat produced (Figure (Figure 3 (Figure 3 E) and wate lly different waterr intake (Figure 3 D) we w e rre e not statistica statistically different between the contrast, UCP3KO U C P 3 K O mice had increased m o v e m e n t as u g g e s t e d by groups. In contrast, increased movement as ssuggested the increase in activity when w h e n compa c o m p a rred e d to W T mice (Figure 3 F-H). F-H). WT mice (Figure OWTHF • WTHF -- 40 C> 35 CI) 30 J:: 25 C> 'iii 20 3: 15 10 0 5 ID 0 I:lIUCP3KOHF HUCP3KOHF ,., " w e i g h t s in W T and Figure 2. Body weights WT UCP3KO U C P 3 K O mice mice after 10 w wee e e kks s of high diet. fat diet. .... -:-711. ~"to '" SSt.. ..§.4ttt 0 3... .... :>2'" • 2.50 A 70eo o WTday _6000 ~U C P 3 K O day oWf night ~SOOO '" ~4000 = -=-1010 N fil UCP3KO night B 1.20 2.50 .-1 ,00 2.00 '"1!O.1O " .iO ~ ~ 0' 0 ~ .40 :>1000 0.20 8z 2.00 • ... ... ~.50 x SOt g..tO • 1-301 ... • . 50 0.00 ~40' ,.. • 100 ~ "l! ~ . SO iii.oo F. 3':0.50 0.00 0.00 ". ... ".m ... 700 ~ D ~4fO 1'! ';300 .... '" • 100 ** H JOO ",50 - -a2GO ~1S0 ..• 110 Figure 3. metabolism, food and water WT and UCP3KO HFdiet Figure 3. Whole Wh ole body energy en ergy metabolism. water intakes and an d activity in wr UCP3KO mice fed a HF diet for 10 weeks, A) Volume of oxygen consumed expressed as mL ml_ per kg per per hour. B) Volume of carbon dioxide exhaled intake E) food exhaled expressed expressed as mL per per kg per per hour h ou r C) heat heat produced produ ced expressed as Kcal per per hour. h ou r. D) Water water intake intake in take F)Total F) Total activity activity in thex th ex direction di rection expressed expressed in arbitrary arbitrary units. u nits. G) Total activity activity in the the yy direction expressed expressed in wr arbitrary arbitrary units.H)Total u nits.H) Total activity activity in thez th e z direction direction expressed expressed in arbitrary arbitrary units.** u nits. ~ P P < 0.05 0.05 compared compared to to WT night 14 Glucose tolerance tolerance tests tests Glucose U C P 3 K O mice on a h o l e body body glucose glucose To determine determine if UCP3KO a have fat diet diet develop develop w whole glucose tolerance tolerance tests w e r e performed. If the mice are glucose glucose intolerance, glucose were intolerance it could mean that they resistant and that the heart heart intolerance they are also insulin resistant muscle are not not taking up glucose glucose at a normal rate. WT W T mice and UCP3KO UCP3KO and muscle H F D had similar similar responses responses to glucose mice on a HFD glucose at all time points (Figure 4). Respiration and A TP production production in saponin-permeabi/ized cardiac Respiration and ATP in saponin-permeabilized cardiac fibers fibers To determine determine if lack lack of UCP3 affects mitochondrial coupling w e examined examined of UCP3 affects mitochondrial we o x y g e n consumption c o n s u m p t i o n and A T P production saponin-permeabilized cardiac oxygen ATP production in saponin-permeabilized fibers. T h e basal respiration without D P (state 2), the maximal ADP A D P stimulated The without A ADP T P synthase ATP synthase with oligomycin oligomycin (state 4) (state 3) and respiration while blocking A w e r e determined. d e t e r m i n e d . State e r e not statistically statistically different different were State 2 and State 33 respiration w were b e t w e e n the groups. Both W T and UCP3KO U C P 3 K O on HF exhibited a significant a significant between WT increase in State 4 b s e n c e of ATP increase 4 respiration which is respiration in the aabsence of ATP synthase (Figure 5 A ) . Interestingly, while state 3 respiration w a s not not affected affected synthase (Figure A). was a b s e n c e of of UCP3, UCP3, A T P production w a s slightly slightly increased in UCP3KO U C P 3 K O on by the absence ATP was increase in A T P / O ratios between UCP3KO U C P 3 K O in HF, which resulted in aa significant significant increase ATP/O these (Figure 5 B-C). T h e s e data suggest suggest that (1) diet-induced diet-induced elevation elevation these animals animals (Figure These in State State 4 respiration occurred independently independently of of UCP3 UCP3 because b e c a u s e WT W T and and U C P 3 K O mice showed s h o w e d similar increases in state 4 UCP3KO similar increases 4 respiration and that that (2) mitochondria from UCP3KO U C P 3 K O mice ffed-HFD e d - H F D are more coupled because because they mitochondria cconsume o n s u m e less oxygen per per A T P produced. To see if the results w e r e unique unique to the ATP were 15 heart, were heart, samples samples w e r e taken from skeletal muscle (soleus) (soleus) and the experiments experiments were w e r e repeated on mitochondria mitochondria isolated from the muscles. muscles. Mitochondria Mitochondria from from soleus similar respiration pattern (figure 6) soleus showed s h o w e d a similar 6).. 16 Glucose lole re nce tolerence -o- WTHF ==- 350 "0300 0,250 .s200 ~ 150 0100 g 50 (!) 0 .J-_-.--.---.-~~ O -I 1 1 1 1 1 o0 5 15 30 60 120 120 _UCP3KO HF Time (min) Figure 4. a HFD 4. Glucose Glucose tolerance tests tests in WT W T and UCP3KO U C P 3 K O mice fed a H F D for 10 weeks. weeks. 17 25 A oWTNC .UCP3KONC 20 ~ "0 IIIUCP3KOHF - 15 "0 10 01 r<lWTHF E c .€ E .s N 0 > 5 o VO VADP Voligo 80 5 C1 60 4 '2 ~ '0 E 40 fl.. 20 * i "E .s 0 3 ~ 2 a: !;i 0 0 Figure 5. Mitochondrial consumption from mitochondria Mitochondrial oxygen consumption mitochondria isolated from hearts of WT mice fed fed aa normal normal cchow a HFD for 10 weeks. A) VVa (state 2). V W T and and UCP3KO U C P 3 K O mice h o w or or a HFD for 10 w e e k s . A) (state 2). V ADP DP 0 A * Voligo, (state 4). 8) ATP ATP/O (state 3). Voiigo, B) A T P production C) A T P / O ratios. Data are mean±SEM. mean±SEM. * P < + 0.05 vs. VS. NC. PP << 0.05 0.05 vs. VS. W WT HF. 0.05 T HF. + 18 aWTNC • UCP3KONC OWTMF BUCP3MOMF • v. vo B 4. 40 ~ ~ = E .~E ^o "0 i VADP VADP Vollgo Voligo C + 3 3 * i 3. 30 • 2 • 2 2. 20 • E ccz ...a.i— ,. ~ ~ o ~ a. ~ 11 • 10 • • L-L-_ • L--'-_ Figure 6. Respiration Respiration in in soleus soleus muscle. A) Vo (state 2). D P (state (state Va (state 2). VVADP (state 3). 3) . V V.i ii" ,, (state A 0 go 4) 8) B) AlP A T P production C) C) AlP A T P dividing by oxygen o n s u m e d ..•* P < 0.05 when oxygen cconsumed 0.05 when c o m p a r e d to WT W T NC. + P < 0.05 0.05 when w h e n cco om p a r e d to WT W T HF # P < 0.05 when compared mpared 0.05 when c o m p a r e d to UCP3KO U C P 3 K O NC compared 19 Diacylglycerol and ceramide ceramide levels levels Diacylglycerol and A c c u m u l a t i o n of of fatty acid intermediates intermediates plays an important Accumulation important role in the pathogenesis verify if similar pathogenesis of of insulin resistance in skeletal muscle. To verify m e c h a n i s m s lead to insulin resistance in cardiac e determined ceramide mechanisms cardiac tissue, w we determined ceramide and diacylglycerol T and UCP3KO U C P 3 K O fed either either a WT diacylglycerol (DAG) levels in hearts from W h o w or or a H F D for 10 w eeks. A s shown Figure 7A, HFD resulted in a normal cchow a HFD weeks. As shown in Figure slight concentration that a s similar T and slight increase in ceramide concentration that w was similar between W WT U C P 3 K O mice. In contrast, DAG D A G levels w ere 2 higher in the hearts of were 2 fold higher UCP3KO U C P 3 K O mice on HF ccompared o m p a r e d to W T mice on HF (Figure 7B). WT (Figure 78). UCP3KO Reactive oxygen Reactive oxygen species species As important role in reducing ROS generation in A s UCP3 U C P 3 was w a s sshown h o w n to play an important R O S generation many we that UCP3KO hearts will have higher many tissues, w e hypothesized hypothesized that U C P 3 K O hearts higher levels of R O S compared c o m p a r e d to W T . Both W T and UCP3KO U C P 3 K O mice on a H F D sshowed h o w e d an ROS WT. WT a HFD increase when increase in ROS R O S production w h e n compared c o m p a r e d to NC-fed animals. However, However, U C P 3 K O mice on HF sshowed h o w e d even greater greater amounts amounts of of ROS R O S generation generation when when UCP3KO c o m p a r e d to W T mice fed the same compared WT same diet diet (Figure 8). 20 - 60 A 500 B ++ .* C1 E o [40 0;400 oUCP3KONC E . ==o E Cl <I: 200 OWTNC E 300 a. ." '" ~20 ~ o 100 o .1-1...Figure 7. Ceramide and DAG D A G levels levels in hearts from WT W T and UCP3KO U C P 3 K O mice mice fed H F D for 10 w e e k s . A) Concentration x p r e s s e d as pmol NC or HFD weeks. A) Concen tration of of is ceramide ceramide eexpressed pmol of ce ramide per tissue . B) Concentration Concentration of of diacylglycerol ceramide per mg of tissue. diacylglycerol is expressed expressed as *. Data are are mean±SEM. < 0.005 VS. NC. pmol pmol of of DAG D A G per per mg mg of of tissue. tissue. Data mean±SEM. * * P P < 0.005 vs. NC. ++ PP < < 0.005 0.005 vs. vs. WT W T HF HF.. + + 21 250 + O WTNC _UCP3KO Ne C WT HF 200 o 0 0 UCP3KO HF ~ 1<> 150 ..,en ~ 0 ~ 100 () 50 ~ o ~ Figure 8. Reactive oxygen species (ROS) measured the hearts (ROS) generation m e a s u r e d in the olWT HFD for lor 10 w weeks. of W T and UCP3KO U C P 3 K O led fed either NC or HFD e e k s . Data are 0.05 vs. vs. W WT HF . mean±SEM. m e a n ± S E M . "P * * P < 0.005 0.005 vs. NC. +p P << 0.05 T HF. + 22 Effect on insulin Effect of of combined combined UCP3 UCP3 deficiency deficiency and and HFD HFD on insulin signaling signaling in the the heart heart To determine if HFD impairs insulin signaling and to assess the role of UCP3 To determine if H F D impairs insulin signaling and to assess the role of U C P 3 in the process, we performed immuno-blotting using either WT NC and WT HF in the process, w e performed immuno-blotting using either W T NC and W T HF mice or UCP3KO NC and UCP3KO HF mice. We used the phosphorylation state mice or U C P 3 K O NC and U C P 3 K O HF mice. W e used the phosphorylation state of proteins in the insulin signaling cascade as a measure of insulin resistance. of proteins in the insulin signaling c a s c a d e as a measure of insulin resistance. Figure 9 shows representative blots from mice fed a high fat diet, separate Figure 9 s h o w s representative blots from mice fed a high fat diet, separate primary and secondary antibodies were used for total Akt (Figure 9 A) and primary and secondary antibodies w e r e used for total Akt (Figure 9 A) and phospo-Akt (Figure 9 B). Fluorescence from the bands on the western blots was phospo-Akt (Figure 9 B). Fluorescence from the bands on the western blots w a s quantified data obtained was quantified independently independently of of the the images images and and the the data obtained w a s put put into into graph graph form (Figures 10-11). First, we determined Akt phosphorylation in hearts that form (Figures 10-11). First, w e determined Akt phosphorylation in hearts that were perfused with or without insulin. WT HF mice had significantly lower levels w e r e perfused with or without insulin. W T HF mice had significantly lower levels of Akt phosphorylation compared to mice fed a normal chow diet (Figure 10 A). of Akt phosphorylation compared to mice fed a normal c h o w diet (Figure 10 A). In not have statistically significant In contrast, contrast, UCP3KO U C P 3 K O mice mice did did not have statistically significant different different levels levels of of Akt the diet Akt phosphorylation phosphorylation regardless regardless of of the diet (Figure (Figure 10 10 B). B). Next Next to to determine determine the the impact of UCP3 impact of U C P 3 deficiency deficiency on on insulin insulin signaling, signaling, phosphorylation phosphorylation state state of of various various the insulin signaling cascade were proteins proteins in in the insulin signaling cascade was w a s examined. e x a m i n e d . Western W e s t e r n blots blots w e r e set set and UCP3KO a normal up up to to compared c o m p a r e d WT W T and U C P 3 K O directly directly on on either either a a high high fat fat diet diet or or a normal diet diet in in the the presence presence and and absence a b s e n c e of of insulin. insulin. Akt Akt phosphorylation phosphorylation (both (both on on serine serine 473 GSK3~ (Figure 11 4 7 3 (Figure (Figure 11 11 A-B) A-B) and and threonine threonine 308 308 (Figure (Figure 11 11 C-D), C-D), and and GSK3(3 (Figure 11 E-F) The results E-F) phosphorylation phosphorylation were w e r e determined. determined. The results showed showed no no differences differences in in Akt Akt or G GSK3~ or S K 3 p phosphorylation phosphorylation between between the the groups. groups. 23 A Total AKT B - 100KDa =~ - SOKDa,= - 55 KDa'= Figure 9. Representative blots comparing wr W T and UCP3KO U C P 3 K O mice on a high fat diet with or without witho ut insulin. A) Total Akt B) Phospo-Akt Phospo-Akt on serine 473. 473. 24 B B A WT 5 UCP3KO UCP3K0 4 cUCP3KONC •UCP3K0NC cWTNC QWTNC M (0 ""QI 4 IWTNC WTNC insulin «~ .. ~ 2 'Z QI (D I/) oUCP3KOHF •UCP3K0HF DWTHF QWTHF 3 cr:::: IUCP3KONC •UCP3K0NC insulin 3 QWTHF ~WTHF insulin HUCP3K0HF flUCP3KO HF insulin 2 ( o~- o~- NC NC HF HF NC NC HF HF Figure Figure 10. Effect Effect of of HFD H F D on insulin signaling in the heart. A) Ratio of phosphorylated Akt over phosphorylated (on serine 473) Akt over total Akt in WT W T mice either either on a normal normal diet or or a high fat diet with or or without diet without insulin (arbitrary units). 8) B) Ratio of phosphorylated Akt in UCP3KO phosphorylated Akt over over total Akt U C P 3 K O mice either either on a normal diet or a high fat diet with or without without insulin (arbitrary (arbitrary units). Data are mean±SEM. mean±SEM. **P < 0.05 0.05 vs. NC. 25 B HF m 7 6 ~ 5 -WTinsulin 4 3 2 1 DUCP3KO ....... Q) c: '- Q) V) +-' ~ « A DWT ",UCP3KO insulin 0 5 4 3 2 1 0 8 7 m 6 Q) c: 5 c: 4 0 Q) '3 ..c +-' 2 +-' ~ « 1 0 C 9 8 7 6 5 4 3 2 1 0 D 4 E 3 F 00 0 0') C1J c: 3 "- C1J UJ 2 CQ. M ~ 1 NC 2 1 UJ C) 0 0 Figure of insulin signaling proteins in the hearts hearts of WT Figure 11. 1 1 . Representative Representative blots of of W T and U C P 3 K O mice fed NC or e e k s . A) Akt 7 3 , high fat feeding. B) Akt Akt UCP3KO or HF for 10 w weeks. Akt serine serine 4473, 7 3 , normal chow c h o w feeding. C) Akt threonine Akt threonine threonine 308, high fat feeding D) Akt Akt threonine serine 4473, c h o w feeding. E) G S K 3 p serine 9, high fat feeding F) GSK3(3 308, normal chow GSK3~ GSK3~ serine 9, c h o w feeding. normal chow 26 Discussion Discussion Uncoupling similar body weight, oxygen Uncoupling protein 3 knockout knockout mice have have similar oxygen consumption, and water intake but higher c o n s u m p t i o n , carbon dioxide exhaled, food intake a n d water higher activity compared activity c o m p a r e d to wild type mice. Despite having higher higher concentrations concentrations of diacylglycerol and on a high fat diet diet sshowed similar glucose diacylglycerol a n d ROS, R O S , UCP3KO mice on h o w e d similar glucose tolerance tests. T These signaling in hearts aass tolerance h e s e animals maintained intact intact insulin signaling the ability ability of insulin to phosphorylate downstream ssuggested u g g e s t e d by the phosphorylate d o w n s t r e a m targets such as Akt and GSK3p. Similar and enhanced enhanced mitochondrial Similar body body weight, weight, increased increased activity activity and mitochondrial coupling coupling on a HF HF exhibit exhibit similar UCP3KO mice on similar body weights weights despite despite similar metabolic parameters parameters (0 (O22 consumed c o n s u m e d CO2 x h a l e d , food a n d water nd CO 2 eexhaled, and water intake) aand metabolic h e increase increase in activity lower body weight weight activity should result in a lower increased activity. T The are more efficient efficient at converting food into energy. T he unless UCP3KO mice are The similar might be sustained by increase increase coupling in the the similar body weights might mitochondria respiration show s h o w that mitochondria. Data from the the mitochondria that UCP3KO HF mitochondria than W T HF mice as sshow h o w by the the higher higher mice have more coupled mitochondria WT A T P to oxygen o x y g e n ratio. T a s true for the h o l e body body and a n d not just ATP Too confirm that that this w was the w whole h e ssame a m e tests were mitochondria isolated from tthe he the heart, tthe were performed on on mitochondria the results from the muscle confirm that soleus muscle. Indeed, the the soleus soleus muscle that the the UCP3KO mice produced more more A T P than W T mice which w o u l d allow h e m to WT would allow tthem ATP have increased increased activity activity a n d maintain the a m e body body weight have and the ssame weight as wild type mice. 27 Increased with HFD occurs independently independently of UCP3 Increased state state 4 respiration respiration with HFD occurs of UCP3 W e demonstrated demonstrated that HFD increased state 4 We that HFD 4 respiration (respiration in the a b s e n c e of of A T P synthesis) T and UCP3KO U C P 3 K 0 mice. Other proteins the absence ATP synthesis) similarly similarly in W WT Other proteins beside UCP3 U C P 3 that that use the proton gradient gradient to transport molecules (pyruvate, fatty beside transport molecules A T P etc) in and out mitochondria could be responsible for the acids, ATP out of of the mitochondria uncoupling observed. O n e of h e m , adenine uncoupling One of tthem, adenine nucleotide nucleotide translocator translocator (ANT), has s h o w n to be active in the heart heart and has the ability ability to uncouple uncouple been shown N T is an inner inner mitochondrial membrane m e m b r a n e protein that expressed that is expressed mitochondria. A ANT heart that exchanges A T P and A D P between the inner inner m e m b r a n e space space in the heart that exchanges ATP ADP membrane a n d the mitochondrial mitochondrial matrix matrix using the proton gradient energy source. and gradient as an energy have shown that N T promotes u n c o u p l i n g36 and that Previous studies have that A ANT promotes mild uncoupling 36 basal proton conductance conductance is dependent d e p e n d e n t on A N T 37.. It has has also s h o w that ANT also been show that in 3 7 heart fibers with mild A T P synthase heart ATP synthase inhibition that that there is an increase increase in direct A T P translocation from the mitochondria mitochondria to the cytosol c y t o s o l42.. T h u s , it is possible possible that that ATP Thus, 42 UCP3 d o e s not playa play a significant significant role in state 44 respiration uncoupling. UCP3 does UCP3 reduces oxygen species in the the heart heart UCP3 reduces reactive reactive oxygen species generation generation in UCP3KO of ROS which is U C P 3 K O mice mice had had increased increased levels levels of R O S which is consistent consistent with with previous theories that the mitochondria to reduce reduce the previous theories that UCP3 U C P 3 may may uncouple uncouple the mitochondria to the 242 4 , 2 7 amount of ROS formed by the mitochondrial a m o u n t of R O S formed by reducing reducing the mitochondrial membrane m e m b r a n e potential p o t e n t i a l , 27,' a n d 2 28. 8 and . It that an an increase increase in associated with with insulin It has has been been hypothesized hypothesized that in ROS R O S is is associated insulin 29 resistance and other of diabetes diabetes such such as as cardiovascular cardiovascular disease resistance and other complications complications of disease , 29, 30 1 — — 28 In an over over fed state, w where caloric intake exceeds exceeds energy energy expenditure, h e r e caloric 31 increase in the amount mitochondrial NADH N A D H and ROS R O S formation formation . there is an increase amount of of mitochondrial 31 T h e cell may ccompensate o m p e n s a t e for m o u n t of of nutrients nutrients in the The for this by decreasing decreasing the aamount 32 developing insulin resistance to prevent prevent oxidative a m a g e .. It is cell, thus developing oxidative ddamage 32 u n k n o w n at this time why U C P 3 K O mice do h o w signs resistance unknown why UCP3KO do not sshow signs of of insulin resistance despite higher levels of ROS. O n e possibility possibility could be that UCP3KO U C P 3 K O mice mice have have of ROS. One despite higher levels d e v e l o p e d compensatory protective mechanisms m e c h a n i s m s to detoxify detoxify ROS. R O S . For For example, example, developed compensatory protective interest to investigate antioxidant it will be of of interest investigate the level and the function of of antioxidant m echanisms. mechanisms. Elevated diacylglycerol diacylglycerol levels Elevated levels UCP3KO diacylglycerol than W WT U C P 3 K O mice on HF have higher higher levels of of diacylglycerol T fed HFD. T h e ccause a u s e of D A G is unknown studies unknown at this point. Other Other studies HFD. The of this increase increase in DAG cconducted o n d u c t e d on skeletal muscle of of UCK3KO U C K 3 K O mice have sshown h o w n that UCP3KO U C P 3 K O mice mice have similar fat oxidation oxidation rates24, have similar r a t e s 32.. Furthermore, levels of of skeletal intramuscular intramuscular 2 4 , 3 2 26 32 and higher h i g h e r in triacylglycerol (IMTG) have been reported to be both lower lower triacylglycerol (IMTG) 32 26 skeletal muscle muscle UCP3KO U C P 3 K O mice. Further Further research is needed needed to clarify if the mice mice in the present present study have higher higher levels of of fat oxidation or or IMTG IMTG in the heart. Due Due study have of DAG D A G it w a s hypothesized to the elevated elevated levels levels of was hypothesized that that insulin signaling will be impaired. 33 D A G is a known activator activator of P K C 9 which can playa play a role in insulin DAG of PKce 3 3 of IRS-1 at serine 1101 which inhibits the insulin resistance by phosphorylation phosphorylation of signaling ccascade a s c a d e 34.. In this regards, PKCe P K C 9 knockout knockout mice are protected from a 3 4 29 high fat diet diet induced insulin resistance 35 . In the present present study, PKCe PKC9 phosphorylation was studies should focus on PKC8 phosphorylation w a s not not investigated. Future studies PKC9 activation and its effects activation effects on the insulin signaling cascade. cascade. Similar glucose WT and and UCP3KO UCP3KO mice HF Similar glucose tolerance tolerance between between WT mice on on HF Contrary our hypothesis hypothesis that that elevated DAG would Contrary to our D A G and ROS ROS w o u l d increase increase insulin resistance, UCP3KO similar glucose glucose tolerance as WT U C P 3 K O mice sshowed h o w e d similar W T mice when when Other studies studies showed tolerance in UCP3KO fed the HFD. Other s h o w e d improved glucose glucose tolerance UCP3KO mice on HF HF compared WT study were c o m p a r e d to W T mice on HF26. H F . However, However, the mice in that that study were 2 6 b a c k c r o s s e d 10 generation C57BL/6 background h e r e a s our our mice are on a backcrossed generation to C57BLl6 background w whereas mixed background. b a c k g r o u n d . The different genetic background may may be responsible for the different genetic differences glucose tolerance differences in glucose tolerance tests. Preserved insulin insulin signaling signaling in UCP3KO despite increased increased ROS ROS and Preserved UCP3KO despite and DAG DAG Western of the phosphorylation of various various proteins including AKT W e s t e r n blotting of phosphorylation state of AKT and GSK3~ significant differences differences between UCP3KO a nd G S K 3 P revealed no significant U C P 3 K O mice and WT WT that 10 w weeks slight effect effect on Akt mice. It is interesting to note that e e k s of of HFD H F D had a slight phosphorylation in W WT This is consistent work from phosphorylation T mice. This consistent with a recent published work our laboratory w where we that up to 5 w weeks of HFD, Akt phosphorylation our laboratory here w e sshowed h o w e d that e e k s of H F D , Akt phosphorylation a was we w a s preserved in the heare h e a r t .. However, w e expected e x p e c t e d to see altered insulin insulin 38 signaling in UCP3KO U C P 3 K O due R O S and DAG D A G levels. In contrast, due to the increased ROS these mice insulin-stimulated Akt S K 3 phosphorylation. These These mice maintained maintained insulin-stimulated Akt and G GSK3 not in agreement agreement with prior prior studies showing that U C P 3 K O mice were were results are not that UCP3KO 24 Again, protected from glucose glucose intolerance intolerance and insulin rresistance e s i s t a n c e , 26, 39.. A g a i n , it is 2 4 , 2 6 , 3 9 30 possible that the discrepancies discrepancies came from the differences differences in the genetic genetic of the HFD. HFD. background of of the mice but also the duration and composition of phosphorylation of Akt does does necessarily necessarily imply imply intact insulin insulin Finally, preserved phosphorylation of Akt 40 a l . two weeks w e e k s of of HF feeding in C57B/6 C 5 7 B / 6 mice mice Wright et al. response. In the study by Wright 4 0 phosphorylation but resulted in a significant significant reduction in GLUT4 GLUT4 did not alter alter Akt Akt phosphorylation translocation in the heart. Thus, further characterize further studies are needed to characterize G L U T 4 translocation in the UCP3KO U C P 3 K O mice after after HFD. HFD. GLUT4 In conclusion our that uncoupling our results show s h o w that uncoupling protein 3 knockout knockout mice do not develop develop insulin resistance in the heart heart despite the accumulation of of ROS R O S and and DAG, D A G , the cause c a u s e of of which is completely completely unknown. One O n e area that has been been researched little and could be responsible for the protection is the altered fatty U C P 3 K O mice. As A s of of yet there is no direct acid metabolism exhibited by UCP3KO D A G , triglycerides and UCP3 UCP3 but it is clear clear that there is a connection between DAG, difference way UCP3KO U C P 3 K O mice metabolize metabolize and store fats. More research research difference in the way needs to be conducted to further further understand this phenomenon p h e n o m e n o n and if it is needs m e c h a n i s m for responsible for protecting the mice from insulin resistance. If the mechanism protecting was w a s know k n o w it could be researched as a potential treatment treatment for insulin insulin resistance and diabetes. 31 References References 1. S, S Sena S, O'Neill Tathireddy P, Young Abel 1. Boudina Boudina S, e n a S, O'Neill BT, BT, Tathireddy P, Y o u n g ME, ME, A b e l ED. ED. Reduced Reduced mitochondrial oxidative capacity and increased mitochondrial oxidative capacity and increased mitochondrial mitochondrial uncoupling uncoupling impair myocardial energetics in in obesity. obesity. Circulation impair myocardial energetics Circulation 2005;112:2686-2695. 2005;112:2686-2695. 2. Boudina S, S, Sena Sena S, S, Theobald Theobald H, Sheng X, W Wright XX, et 2. Boudina H, S h e n g X, r i g h t JJJ, J , Hu Hu XX, et al. al. mitochondria energetics in in the the heart mitochondria energetics heart in in obesity-related obesity-related diabetes: diabetes: direct direct evidence for increased and activation evidence for increased uncoupled uncoupled respiration respiration and activation of of uncoupling uncoupling proteins. Diabetes 2007;56:2457-2466 proteins. Diabetes 2007;56:2457-2466 3. produce reactive species. Biochem 3. Murphy Murphy MP. M P . How H o w mitochondria mitochondria produce reactive oxygen oxygen species. B i o c h e m J. J. 2009 2 0 0 9 Jan Jan 1;417(1):1-13. 1 ;417(1 ):1-13. T. Physiological functions of of the the mitochondrial 4.. Brand, 4 Brand, M., M., & & Esteves, Esteves, T. Physiological functions mitochondrial uncoupling 2005 2, 85-93. uncoupling proteins proteins UCP2 U C P 2 and and UCP3. U C P 3 . Cell Cell Metabolism Metabolism 2 0 0 5 ,, 2, 85-93. 5. J. Mitochondrial uncoupling protein and its 5. Nabben, N a b b e n , M., M., & & Hoeks, Hoeks, J. Mitochondrial uncoupling protein 3 3 and its role role in in cardiacand skeletal muscle metabolism. Behav 22007, cardiac- and skeletal muscle metabolism. Physiol Physiol Behav 0 0 7 , November N o v e m b e r 28, 28, Epub Epub ahead a h e a d of of print. print. 6. Hsu, Y., Niu, T., S Song, Y., Tinker, Tinker, L., Kuller, L., variants in 6. H s u , Y., Niu, T., o n g , Y., L , Kuller, L, & & S, S, L. L. Genetic Genetic variants in the the UCP2-UCP3 gene cluster and and risk of diabetes the W Women's UCP2-UCP3 g e n e cluster risk of diabetes in in the o m e n ' s Health Health Initiative Observational Study. Study. Diabetes Diabetes 22008, Initiative Observational 0 0 8 , 57 5 7 (4), (4), 1101-7. 1101-7. 7. Chan, C.,, & proteins: role 7. C han, C & Harper, Harper, M. M. Uncoupling Uncoupling proteins: role in in insulin insulin resistance resistance and and Diabetes Review 2006, 2 insulin insufficiency. insulin insufficiency. Current Current Diabetes Review 2006, 2 ((3),271-83. 3 ) , 271-83. 8. C Choi, C.,, Fillmore, J., J., Kim, JJ., Z., Kim, S., Collier, E., et al. a!. hoi, C . , Liu, Z., Overexpression of of uncoupling Overexpression uncoupling protein 3 in skeletal muscle muscle protects against against fat-induced insulin resistance. J Clin Clin Invest Invest 2007, 117 7 7 7 ((7), 7 ) , 1995-2003 1995-2003 32 C 9. Hidaka, S., Kakuma, Yoshimatsu, K a k u m a , T., Y o s h i m a t s u , H., Sakino, H., Fukuchi, S., & Sakata, T. Streptozotocin treatment upregulates uncoupling uncoupling protein 3 expression Streptozotocin treatment upregulates expression in the rat heart. Diabetes 48 (2), 430-5. Diabetes 1999, 48 lO.N a b b e n , M., & Hoeks, J. Mitochondrial Mitochondrial uncoupling uncoupling protein 3 and its role in 10. Nabben, cardiac- and skeletal muscle Behav 2007, muscle metabolism. Physiol Physiol Behav 2 0 0 7 , November N o v e m b e r 28, Epub ahead a h e a d of of print. Epub 11 . Z i m m e t P, Alberti KG, K G , Shaw S h a w J. Global and societal implications implications of 11. Zimmet of the diabetes epidemic. Nature. 2001 Dec diabetes Dec 13;414(6865):782-7. 13;414(6865):782-7. McGarry JJD. D. W h a t if Minkowski Minkowski had been ageusic? n alternative What ageusic? A An alternative angle on 12. McGarry diabetes. Science. 1992 1992 Oct Oct 30;258(5083):766-70. 30;258(5083):766-70. 13. Pan DA, Lillioja S, Kriketos A AD, D , Milner Milner MR, Baur Baur LA, Bogardus Bogardus C, Jenkins Jenkins A B , Storlien LH. L H . Skeletal muscle muscle triglyceride levels are inversely AB, triglyceride levels inversely related to 1997 Jun;46(6):983-8. insulin action. Diabetes. 1997 14. Perseghin G, Scifo P, De Cobelli F, Pagliato E, Battezzati A, Arcelloni C, Vanzulli A, Testolin G, Pozza G, Del Maschio Maschio A, Luzi L. Intramyocellular Intramyocellular triglyceride umans: a of in vivo insulin resistance in hhumans: triglyceride content content is a determinant determinant of 1H-13C nuclear magnetic magnetic resonance resonance spectroscopy offspring of 1 H-13C nuclear spectroscopy assessment assessment in offspring type 2 diabetic diabetic parents. Diabetes. 1999 1999 Aug;48(8):1600-6. 15. Krssak M, Falk Petersen K, Dresner SM, 15.Krssak Dresner A, DiPietro L, Vogel S M , Rothman R o t h m a n DL, R o d e n M, Shulman S h u l m a n GI. G l . Intramyocellular Intramyocellular lipid concentrations concentrations are correlated correlated Roden with insulin sensitivity 1H N M R spectroscopy H NMR spectroscopy study. sensitivity in humans: a 1 Diabetologia. 1999 Jan;42(1): 113-6. 33 16. Kim JK, Fillmore JJJ, J , Chen C h e n Y, Yu C, Moore Moore IK, Pypaert Pypaert M, Lutz EP, Kako Y, Velez-Carrasco W,, Goldberg IJ, Breslow Shulman V elez-Carrasco W Breslow JL, S h u l m a n GI. G l . Tissue-specific Tissue-specific overexpression of lipoprotein lipase causes tissue-specific tissue-specific insulin resistance. overexpression of Proc Natl A c a d Sci USA. U S A . 2001 Jun 19;98(13):7522-7. 19;98(13):7522-7. Epub 2001 Jun 5. Acad 17.Samuel VT, AJ, 17. S a m u e l V T , Liu ZX, Qu Q u X, Elder Elder BD, Bilz S, Befroy Befroy 0, D, Romanelli A J , Shulman Shulman GI. of hepatic G l . Mechanism M e c h a n i s m of hepatic insulin resistance in non-alcoholic non-alcoholic fatty liver disease. J Bioi 2004 Jul 30;279(31):32345-53. Epub 2004 2004 May Biol Chem. C h e m . 2004 May 27. 27. Zhang 0, Z Zong Wang 18. Yu C, Chen C h e n Y, Cline GW, GW, Z h a n g D, o n g H, W a n g Y, Bergeron R, Kim JK, Cushman SW, Atcheson White MF, Kraegen EW, Shulman Cushman S W , Cooney Cooney GJ, GJ, A t c h e s o n B, White Shulman of insulin receptor GI. G l . Mechanism M e c h a n i s m by which fatty acids inhibit insulin activation of receptor substrate-1 (IRS-1 )-associated phosphatidylinositol 3-kinase activity in )-associated phosphatidylinositol 3-kinase activity muscle. J Bioi Chem. Biol C h e m . 2002 Dec Dec 27;277(52):50230-6. Epub 2002 Nov Nov 14. 19. Dresner Dresner A, Laurent Laurent 0, D, Marcucci M, Griffin ME, M E , Dufour Dufour S, Cline GW, G W , Slezak Slezak LA, A Andersen n d e r s e n OK, DK, Hundal RS, Rothman DL, Petersen KF, Shulman Shulman GI. Gl. Effects glucose transport transport and IRS-1-associated Effects of of free fatty acids on glucose IRS-1-associated phosphatidylinositol phosphatidylinositol 3-kinase 3-kinase activity. J Clin Invest. 1999 1999 Jan;103(2):253-9. Jan;103(2):253-9. between insulin signaling 20. Watson, 20. W a t s o n , R. T., & & Pessin, J. E. Bridging the GAP G A P between signaling Sci 2006, 31, 31,215-222. and GLUT4 G L U T 4 translocation. Trends Trends Biochem. Biochem. Sci2006, 215-222. 21. Doble, & Woodgett, J. R. GSK-3: of the trade for a multi-tasking 21.D o b l e , B. W., & G S K - 3 : tricks of multi-tasking kinase. Journal Journal of of Cell Cell Science Science 2003, 116, 116, 1175-1186. 22. Dokken Sioniger JA, Henriksen EJ. A Acute selective glycogen glycogen synthase 22.D o k k e n BB, Sloniger c u t e selective synthase e n h a n c e s insulin signaling in prediabetic prediabetic insulin-resistant insulin-resistant kinase-3 inhibition enhances 34 rat skeletal muscle. A Am m J Physiol Endocrinol Metab. 2005 2 0 0 5 Jun;288(6):E1188Jun;288(6):E11880 0 5 Jan 25. 94. Epub 22005 O, Eldar-Finkelman Eldar-Finkelman H. T h e role of 23. Kaidanovich 0, The of glycogen synthase synthase kinase-3 in Expert Opin Ther 2002 insulin resistance and type 2 diabetes. Expert Ther Targets. 2002 O ct;6(5):555-61. Oct;6(5):555-61. 24. Vidal-Puig A AJ, Zhang Boss 0, Szczepanik A, J , Grujic Grujic D, Z h a n g CY, Hagen T, Boss O, Ido Y, Szczepanik W a d e J, Mootha Mootha V, Cortright Cortright R, Muoio D M , Lowell BB. Energy Energy metabolism metabolism in Wade DM, knockout mice J. BioI. Biol. Chem., V o l . 275, 2 7 5 , Issue Issue 221, 1, uncoupling protein 3 gene knockout Vol. 1 6 2 5 8 - 1 6 2 6 6 , May May 26, 2000 2000 16258-16266, 25. C h a v e z JA, Knotts TA, W a n g LP, Li G, Dobrowsky Dobrowsky RT, Florant Florant GL, Summers Summers 25.Chavez Wang S A role for ceramide, but but not diacylglycerol, in the antagonism of insulin insulin SA antagonism of Biol C hem. 2 0 0 3 Mar Mar signal transduction by saturated fatty acids. J Bioi Chem. 2003 2 1 ;;278(12):10297-303. 2 7 8 ( 1 2 ) : 1 0 2 9 7 - 3 0 3 . Epub 2003 Jan 13. 21 26. Costford, 2 6.C o s t f o r d , S. R., Chaudhry, S. N., Salkhordeh, M., Harper, M. E. (2006) Effects of overexpression of of uncoupling of the Presence, absence a b s e n c e and overexpression uncoupling protein-3 protein-3 metabolism in congenic m . J. Physiol. Endocrinol Endocrinol on adiposity adiposity and fuel metabolism congenic mice. A Am. Metab. 2 9 0 , E 1 3 0 4 - E 1 3 1 2 Metab.290,E1304-E1312 27. Echtay, A JJ., 27.E c h t a y , K. S., Esteves, T. C., C , Pakay, J. L., L , JJekabsons, e k a b s o n s , M. B., Lambert, A. ., Portero-Otin, M., Pamplona, R., Vidal-Puig, A. A J., W Wang, a n g , S., Roebuck, S. JJ., ., Brand, M. D. A signalling role for for 4-hydroxy-2-nonenal 4-hydroxy-2-nonenal in regulation of mitochondrial uncoupling. EMBO E M B O J. 2 0 0 3 22,4103-4110 22,4103-4110 mitochondrial 2003 28. Echtay, Cadenas, 28.E c h t a y , K. S., Roussel, D., St-Pierre, J., Jekabsons, M. B., C a d e n a s , S., A, Harper, J. A., A, Roebuck, S. JJ.,. , Morrison, A., A, Pickering, S., et al Stuart, J. A., 35 Superoxide uncoupling proteins. Nature 2002 S u p e r o x i d e activates activates mitochondrial mitochondrial uncoupling Nature 2 0 0 2 415,96415,96- 99 29. Goldstein BJ, Mahadev Wu M a h a d e v K, W u X. Redox paradox: insulin action is facilitated insulin-stimulated reactive oxygen species multiple potential signaling signaling by insulin-stimulated species with multiple targets. Diabetes. 2005 eb;54(2):311-21. 2005 FFeb;54(2):311-21. oxidative stress the pathogenic pathogenic mechanism 30. Ceriello A, Motz 30.Ceriello Motz E. Is oxidative m e c h a n i s m underlying underlying insulin resistance, diabetes, and cardiovascular cardiovascular disease? disease? T The h e ccommon o m m o n soil hypothesis Arterioscler T Thromb Vasc 2004 May;24(5):816-23. hypothesis revisited. Arterioscler hromb V a s e BioI. Biol. 2004 May;24(5):816-23. 0 0 4 Feb 19. Epub 22004 31. Maddux W,, Lawrence JC Jr., Goldfine Goldfine A AL, Goldfine 10, Evans JL. 31.M a d d u x BA, See W Lawrence JC L , Goldfine ID, Evans muscle Protection against against oxidative stress-induced stress-induced insulin resistance resistance in rat L6 muscle micromolar concentrations concentrations of 0 0 1 ; 50: 404404cells by micromolar of a-lipoic acid. Diabetes. Diabetes. 22001; 4 10 410 32. Joris Wim Gert Schaartb, Jodil Joris Hoeksa, H o e k s a , Matthijs Matthijs K.C. Hesselinkb, W i m Sluiterc, Gert Claphame, Wim Sarisa and Willemsa, Alastair Alastair Morrissond, John C. C laphame, W i m H.M. Sarisa Patrick high-fat feeding on intramuscular Patrick Schrauwen. S c h r a u w e n . The T h e effect effect of of high-fat intramuscular lipid and lipid peroxidation peroxidation levels levels in UCP3-ablated UCP3-ablated mice. FEBS F E B S Lett. 2 0 0 6 Feb Feb 2006 20;580(5):1371-5. Epub 2006 2006 Jan Yu GW, Zhang 0, Z Zong Wang 33. Y u C, Chen C h e n Y, Cline G W, Z h a n g D, o n g H, W a n g Y, Bergeron R, Kim JK, C u s h m a n SW, S W , Cooney C o o n e y GJ, GJ, A t c h e s o n B, White MF, M F , Kraegen EW, Shulman Shulman Cushman Atcheson G l . Mechanism M e c h a n i s m by w h i c h fatty acids inhibit insulin activation of receptor GI. which acids inhibit of insulin receptor (IRS-1 )-associated phosphatidylinositol substrate-1 (IRS-1)-associated phosphatidylinositol 3-kinase 3-kinase activity activity in Biol C hem. 2 0 0 2 Dec Dec 27;277(52):50230-6. Epub 2 0 0 2 Nov Nov 14. muscle. J Bioi Chem. 2002 2002 36 34. Yu Li, Timothy Timothy J. Soos, Xinghai Li, Jiong W Wu, u , Matthew Matthew DeGennaro, D e G e n n a r o , Xiaojian Xiaojian Sun||, Dan R. Littman, Morris J. Birnbaum, and Roberto D. Polakiewicz. Polakiewicz. Sunil. Phosphorylating IRS1 at Protein Kinase C 08 Inhibits Insulin Signaling Signaling by Phosphorylating SeM 1 0 1 .. . . BioI. Biol. C hem., V ol. 2 7 9 , Issue 4 4 , 4 5 3 0 4 - 4 5 3 0 7 , October 2 9 , 2004 Chem., Vol. 279, 44,45304-45307, October 29,2004 Ser1101 Fillmore JJJ, J , Sunshine M J , Albrecht Higashimori T, Kim DW, D W , Liu 35. Kim JK, Fillmore Sunshine MJ, Albrecht B, Higashimori ZX, Soos Soos T W , O'Brien R , Littman DR, Shulman S h u l m a n GI. G l . PKC-theta PKC-theta GW, O'Brien W WR, TJJ,, Cline G knockout mice mice are protected from fat-induced insulin resistance. J Clin Invest. Invest. knockout 2 0 0 4 Sep;114(6):823-7. Sep;114(6):823-7. 2004 36.Arvier 36. Arvier M, Lagoutte Lagoutte L, Johnson Johnson G, Dumas D u m a s JJF, F , Sion B, Grizard G, Malthiery Malthiery Y, Simard G, Ritz P. A Adenine translocator promotes d e n i n e nucleotide nucleotide translocator promotes oxidative oxidative after dexamethasone phosphorylation phosphorylation and mild uncoupling in mitochondria mitochondria after dexamethasone treatment. A m J Physiol Endocrinol Metab. 2007 Nov;293(5):E1320-4. Epub Epub Am 2 0 0 7 Aug A u g 14 2007 Wallace MD, 37. Brand M D , Pakay Pakay JL, Ocloo A, Kokoszka Kokoszka J, W a l l a c e DC, Brookes Brookes PS, Cornwall EJ. T h e basal proton conductance of mitochondria mitochondria depends d e p e n d s on The conductance of a d e n i n e nucleotide nucleotide translocase 0 0 5 Dec Dec 1 1;392(Pt adenine translocase content. Biochem J. 22005 ;392(Pt 2):353-62. 38. Boudina Sena S, O'Neill O'Neill BT, Zaha VG, 0, W Wright Boudina S, Bugger Bugger H, Sena Zaha V G , IIkun llkun O, r i g h t JJ, JJ, Mazumder M a z u m d e r PK, Palfreyman Palfreyman E, Tidwell T JJ,, Theobald H, Khalimonchuk Khalimonchuk 0, O, W a y m e n t B, S h e n g X, Rodnick Rodnick KJ, Centini R, Chen C h e n D, Litwin S E, W e i m e r BE, Wayment Sheng SE, Weimer A b e l ED. Contribution of of impaired myocardial insulin signaling to Abel mitochondrial dysfunction oxidative stress 2009 mitochondrial dysfunction and oxidative stress in the heart. Circulation. 2009 Mar 10;119(9):1272-83. 10;119(9):1272-83. Epub 2009 Feb 23 Mar 37 39.Gong, 3 9 . G o n g , D. W., Monemdjou, M o n e m d j o u , S., Gavrilova, 0., O., Leon, L. R., Marcus-Samuels, M a r c u s - S a m u e l s , B., C h o u , C. J., Everett, C., C , Kozak, L. P., Li, C., C , Deng, C., C , et al (2000) Chou, (2000) Lack of obesity and normal response to fasting and thyroid hormone hormone in mice lacking lacking obesity uncoupling protein-3. J. BioI. Biol. Chem. C h e m . 275,16251-16257 275,16251-16257 uncoupling 4 0 . Wright J J , Kim J, Buchanan B u c h a n a n J, Boudina Boudina S, Sena S, Bakirtzi K, IIkun llkun 0, O, 40. Wright JJ, Theobald T h e o b a l d HA, Cooksey C o o k s e y RC, Kandror Kandror KV, Abel ED. Mechanisms M e c h a n i s m s for increased increased myocardial fatty acid utilization following short-term high-fat high-fat feeding. myocardial Cardiovasc Res. 2 0 0 9 Feb 4. [Epub ahead of Cardiovasc 2009 of print] of insulin receptor 441. 1 . Eldar-Finkelman Eldar-Finkelman H and Krebs EG. Phosphorylation Phosphorylation of receptor substrate impairs insulin action. Proc Proc Natl substrate 1 by glycogen synthase synthase kinase 3 impairs A c a d Sci USA USA 1997 1997 94: 9660-9664. 9660-9664. Acad 42. Joubert F, Mateo 4 2 . Joubert Mateo P, Gillet Gillet B, Beloeil JC, Mazet Mazet JL, Hoerter Hoerter JA. CK C K flux or direct T P transfer: versatility versatility of of energy transfer pathways pathways evidenced by NMR NMR direct A ATP energy transfer 0 0 4 Jan-Feb;256-257(1-2):43-58 Jan-Feb;256-257(1-2):43-58 in the perfused heart. Mol Cell Biochem. 22004