antibodies against amp kinase ( ampk ) , phosporylated ampk ( pampk ) , acetyl coa carboxylase ( acc ) , phosporylated acc , akt , phosporylated akt , and tubulin were from cell signaling ; anti - oxphos was from mitosciences ; anti 
cd36-[hrp ] was purchased from novus biologicals , and human recombinant insulin ( novolin ) from novo nordisk canada .
this study was performed with the approval of the university of alberta animal policy and welfare committee .
experiments were carried out on male wild - type ( c57bl6 ) and cd36 knockout ( ko ) mice ( 19 ) maintained in a temperature - controlled room with a reversed 12-h light/12-h dark cycle .
mice were left relatively undisturbed for either 1214 or 5258 weeks of age with free access to water and standard rodent diet ( category no . 5001 ; labdiet ) . at 3234 weeks of age ,
a subset of mice was randomly divided into a low - fat diet group ( category no .
d12450b ; research diets ) and a high - fat diet group ( category no . d12492 ; research diets ) for a period of 12 weeks .
indirect calorimetry was performed using the comprehensive lab animal monitoring system ( oxymax / clams ; columbus instruments , colombus , oh ) . following an initial 24-h acclimatization period ,
mice were monitored every 13 min for 24 h to complete a 12-h dark ( active)/12-h light ( inactive ) cycle . the respiratory exchange ratio ( rer = vco2/vo2 )
was used to estimate the percent contribution of fat and carbohydrate to whole - body energy metabolism in mice in vivo .
total activity was calculated by adding z counts ( rearing or jumping ) to total counts associated with ambulatory movement and stereotypical behavior ( grooming and scratching ) . upon phospholipid digestion with phospholipase c ( 2 h at 30c ) and lipid
extraction , levels of triglycerides were determined in gastrocnemius muscle lysates by gas - liquid chromatography as previously described ( 20 ) .
identification and quantification of the major long - chain acyl - coa molecular species ( c16:0 , c18:0 , and c18:1 ) and c18-ceramides were performed by high - performance liquid chromatography as previously described ( 21 ) .
overnight - fasted mice were anesthetized with sodium pentobarbital , and gastrocnemius muscle was rapidly removed and freeze - clamped in liquid nitrogen and stored at 80c .
acylcarnitine measurements were made using flow - injection tandem mass spectrometry as previously described ( 14 ) , and organic acids were quantified as previously described ( 22 ) .
comparisons between groups were performed using unpaired student 's two - tailed t test or anova with a bonferroni post hoc test of pairwise comparisons where appropriate . a probability value of < 0.05 is considered significant . for further descriptions of the materials and methods , refer to the supplementary material available in an online appendix , available at http://diabetes.diabetesjournals.org/cgi/content/full/db09-1142/dc1 .
antibodies against amp kinase ( ampk ) , phosporylated ampk ( pampk ) , acetyl coa carboxylase ( acc ) , phosporylated acc , akt , phosporylated akt , and tubulin were from cell signaling ; anti - oxphos was from mitosciences ; anti 
cd36-[hrp ] was purchased from novus biologicals , and human recombinant insulin ( novolin ) from novo nordisk canada .
this study was performed with the approval of the university of alberta animal policy and welfare committee .
experiments were carried out on male wild - type ( c57bl6 ) and cd36 knockout ( ko ) mice ( 19 ) maintained in a temperature - controlled room with a reversed 12-h light/12-h dark cycle .
mice were left relatively undisturbed for either 1214 or 5258 weeks of age with free access to water and standard rodent diet ( category no . 5001 ; labdiet ) . at 3234 weeks of age ,
a subset of mice was randomly divided into a low - fat diet group ( category no .
d12450b ; research diets ) and a high - fat diet group ( category no .
indirect calorimetry was performed using the comprehensive lab animal monitoring system ( oxymax / clams ; columbus instruments , colombus , oh ) . following an initial 24-h acclimatization period ,
mice were monitored every 13 min for 24 h to complete a 12-h dark ( active)/12-h light ( inactive ) cycle .
the respiratory exchange ratio ( rer = vco2/vo2 ) was used to estimate the percent contribution of fat and carbohydrate to whole - body energy metabolism in mice in vivo .
total activity was calculated by adding z counts ( rearing or jumping ) to total counts associated with ambulatory movement and stereotypical behavior ( grooming and scratching ) .
upon phospholipid digestion with phospholipase c ( 2 h at 30c ) and lipid extraction , levels of triglycerides were determined in gastrocnemius muscle lysates by gas - liquid chromatography as previously described ( 20 ) .
identification and quantification of the major long - chain acyl - coa molecular species ( c16:0 , c18:0 , and c18:1 ) and c18-ceramides were performed by high - performance liquid chromatography as previously described ( 21 ) .
overnight - fasted mice were anesthetized with sodium pentobarbital , and gastrocnemius muscle was rapidly removed and freeze - clamped in liquid nitrogen and stored at 80c .
acylcarnitine measurements were made using flow - injection tandem mass spectrometry as previously described ( 14 ) , and organic acids were quantified as previously described ( 22 ) .
comparisons between groups were performed using unpaired student 's two - tailed t test or anova with a bonferroni post hoc test of pairwise comparisons where appropriate . a probability value of < 0.05 is considered significant . for further descriptions of the materials and methods , refer to the supplementary material available in an online appendix , available at http://diabetes.diabetesjournals.org/cgi/content/full/db09-1142/dc1 .
to determine whether an age - related decline in resting metabolic rate and energy expenditure might contribute to the development of insulin resistance , c57bl6 mice of 1214 ( young ) or 5258 ( middle - aged ) weeks of age were analyzed using indirect calorimetry . as mice aged and body weight was increased ( fig .
1a ) , substrate use was altered slightly , with reductions in rer indicating that middle - aged mice used more fatty acid throughout the day compared with young mice ( fig .
in addition , significant reductions in oxygen consumption ( vo2 ) ( fig . 1c ) and carbon dioxide production ( vco2 ) ( fig .
1d ) were observed during both the dark ( active ) and light ( inactive ) phases in middle - aged mice compared with young mice .
1e ) was decreased in middle - aged mice compared with young mice , whereas activity measurements were not significantly different between age - groups ( fig .
our data indicate that middle - aged mice have a lower metabolic rate than young mice and that this might increase susceptibility to weight gain , obesity , and metabolic disease .
body weight of young ( 1214 weeks of age ) and middle - aged ( 5258 weeks of age ) mice fed a standard laboratory diet ( a ) .
indirect calorimetry was performed to measure respiratory exchange ratio ( rer ) ( b ) , vo2 ( c ) , vco2 ( d ) , and heat production adjusted for bodyweight ( e ) ; total activity ( f ) was measured in both the dark ( active ) and light ( inactive ) phases .
p < 0.05 indicates comparisons performed between young and middle - aged mice in either dark or light phases ( mann - whitney u test ) .
two - way anova was performed for rer ( b ) and indicated the main effect for age ( p < 0.01 ) .
given that skeletal muscle metabolism contributes to whole - body basal metabolic rate , we addressed whether skeletal muscle metabolism was depressed in middle - aged mice by assessing the activities of two enzymes involved in regulating mitochondrial metabolism .
2a ) was elevated in the middle - aged compared with the young mice , and citrate synthase ( fig .
2b ) followed a similar upward trend , suggesting that -oxidation and tca cycle activity were not directly compromised in the middle - aged mice . since mitochondrial number ( fig .
2c ) or function did not appear to be altered at middle age , we next assessed whether peroxisome proliferator  activated receptor ( ppar) responsive genes or molecular signaling cascades known to regulate skeletal muscle fatty acid flux and fatty acid entry into the mitochondria were associated with this overall reduction in whole - body basal metabolic rate observed in middle age .
although a more comprehensive assessment of the multiple mediators of fatty acid utilization may reveal additional mechanisms , we did not observe any changes in protein levels of known ppar responsive genes involved in lipid metabolism , including malonyl - coa decarboxylase and acyl - coa synthetase 1 ( data not shown ) . as a result
, we also examined the energy - sensing kinase , ampk , which is known for its ability to govern energy metabolism ( 23 ) . in agreement with previous reports using older rodents ( 24,25 ) , levels of phosphorylated ampk
were significantly reduced in skeletal muscle of middle - aged mice compared with young mice ( fig .
moreover , the phosphorylation status of acetyl coa carboxylase ( acc ) , the downstream target of ampk that indirectly regulates fatty acid entry into the mitochondria and ultimately -oxidation , was significantly decreased in skeletal muscle from middle - aged mice ( fig .
although reduced ampk phosphorylation could be the result of impaired activity of upstream ampk kinases or increased ampk phosphatase activity , it is currently unknown which of these contributes to reduced ampk phosphorylation in our model .
however , consistent with decreased energy expenditure , increased adiposity , and reduced ampk activity in skeletal muscle from middle - aged mice , the levels of skeletal muscle triglycerides were significantly elevated in middle - aged mice compared with young mice ( fig .
2 g ) . given that lipid accumulation in skeletal muscle has been proposed in rodents ( 26 ) and humans ( 17,27,28 ) to be one of the primary causes of skeletal muscle insulin resistance , we next investigated whether glucose tolerance was impaired in middle - aged mice . despite elevated intramuscular triglycerides ( fig . 2 g ) and impaired basal and insulin - stimulated akt phosphorylation ( fig .
2h and i , respectively ) in skeletal muscle of middle - aged mice , whole - body glucose tolerance ( fig .
2k ) were not different in middle - aged compared with young mice , suggesting that age - induced alterations in skeletal muscle fatty acid handling and increased triglyceride storage precede the development of insulin resistance and metabolic disease .
alterations in fatty acid handling and reduced insulin signaling in skeletal muscle of middle - aged mice do not result in impaired whole - body glucose tolerance .
activity of two mitochondrial enzymes -had ( a ) and citrate synthase ( cs ) ( b ) was determined in gastrocnemius muscle from overnight fasted young ( 1214 weeks of age ) and middle - aged ( 5258 weeks of age ) mice .
immunoblot analysis using a total oxphos complex antibody cocktail was performed in gastrocnemius muscle lysates , and immunoblots were normalized against tubulin as a control for protein loading ( c ) .
phosphorylation status of ampk at threonine 172 ( d ) and acc at serine 79 ( f ) was detected using immunoblot analysis with phospho - specific antibodies .
immunoblots were quantified by densitometry and normalized against total protein levels of ampk ( e ) and acc ( f ) .
triglyceride ( tg ) levels ( g ) and phosphorylation status of akt ( h ) were determined in gastrocnemius muscle from overnight - fasted young and middle - aged mice .
gastrocnemius muscle was collected from a separate group of overnight - fasted young and middle - aged mice following intraperitoneal injection with either saline or human recombinant insulin ( 10 units / kg ) , and immunoblots were performed to detect phosphorylation status of akt at serine 473 in gastrocnemius muscle lysates ( i ) . glucose tolerance test ( j )
was performed in young (  ) and middle - aged (  ) mice following a 6-h fast .
serum insulin was detected in young and middle - aged mice after an overnight fast ( k ) .
p < 0.05 indicates comparisons performed between young and middle - aged mice ( mann - whitney u test ) .
since we speculated that middle - aged mice are more susceptible than young mice to the development of insulin resistance , young and middle - aged mice were subjected to high - fat feeding for 12 weeks . although young mice fed a high - fat diet displayed weight gain ( fig .
3a ) and signs of glucose intolerance compared with young mice fed a low - fat diet ( fig .
in contrast , middle - aged mice fed a high - fat diet showed significant weight gain ( body weight : low fat , 30.18  0.70 g , high fat , 55.14  1.56 g ; p < 0.05 ) and displayed dramatically elevated insulin levels ( fig .
in addition , whole - body glucose tolerance in the middle - aged mice fed a high - fat diet was significantly impaired compared with that in middle - aged mice fed a low - fat diet ( fig .
although activation of insulin signaling , as determined by the phosphorylation status of akt is not impaired in skeletal muscle of young ( fig .
3j ) mice fed a high - fat diet compared with that in mice fed a low - fat diet , this is likely due to elevated levels of circulating insulin ( fig .
3e and f ) observed in the respective high - fat groups . in support of the glucose tolerance data , homeostasis model assessment of insulin resistance values were significantly higher in the high - fat  fed middle - aged mice ( fig .
3k ) , suggesting that high - fat feeding induces more dramatic insulin resistance in middle - aged mice than in young mice .
interestingly , young mice on a high - fat diet were of the same weight as middle - aged mice on a low - fat diet ; yet , only the young mice on a high - fat diet had impaired glucose disposal ( data not shown ) .
although we can not discriminate between the effects of aging and increased adiposity because these variables coassociate , our findings do suggest that the increased weight gain associated with aging is not sufficient to alter whole - body glucose disposal and that other factors such as high - fat diet are likely involved . aging increases the susceptibility to the development of glucose intolerance and insulin resistance in mice fed a high - fat diet .
body weights ( a ) , fed and fasted blood glucose ( b ) , glucose tolerance test ( c ) , and phosphorylation status of akt ( d ) were measured in gastrocnemius muscle from young ( 1214 weeks of age ) mice following 12 weeks of high fat ( hf ) feeding .
serum insulin levels in the fasted ( e ) and fed ( f ) states obtained from young ( 1214 weeks of age ) and middle - aged ( 4852 weeks of age ) mice fed a low - fat ( lf ) and high - fat diet for 12 weeks .
fed and fasted blood glucose levels ( g ) , glucose tolerance test ( h ) , area under the curve ( auc ) for the glucose tolerance test ( i ) , and phosphorylation status of akt in gastrocnemius muscle ( j ) in middle - aged mice following 12 weeks of high - fat feeding .
homeostasis model assessment of insulin resistance ( homa - ir ) as a surrogate marker of insulin resistance in young and middle - aged mice fed a high - fat diet ( k ) .
immunoblot analysis using anti - cd36 antibody was performed in gastrocnemius muscle lysates from middle - aged mice fed a low- and high - fat diet , and immunoblots were quantified by densitometry and normalized against tubulin as a control for protein loading ( l ) .
triglyceride ( tg ) ( m ) , long - chain acyl - coa ( lccoa ) ( n ) , and c18 ceramide ( o ) levels in gastrocnemius muscle from overnight - fasted middle - aged mice fed a low- or high - fat diet .
two - way anova was performed to detect main effects of age , diet , and age  diet interactions on insulin levels ( e and f ) and homa - ir ( k ) .
significant effect of age , diet , and age  diet interaction ( p < 0.05 ) was observed for e , f , and k. * p < 0.05 indicates comparisons performed between young and middle - aged mice or between low- and high - fat fed mice in fed or fasted state ( mann - whitney u test ) . because high - fat diet  induced insulin resistance in young rodents is associated with an increased efficiency of fatty acid uptake into skeletal muscle ( 26 ) , protein expression of cd36 , a protein that facilitates fatty acid transport , was determined in skeletal muscle of mice fed a high - fat diet .
consistent with previous publications in young mice ( 26,29,30 ) , we observed a modest increase in cd36 protein expression in the muscle of young mice fed a high - fat diet , as well as an increase in intramuscular triglyceride levels ( data not shown ) . consistent with our hypothesis , cd36 expression was significantly elevated in skeletal muscle of middle - aged mice fed a high - fat diet compared with that in mice fed a low - fat diet ( fig .
increased cd36 protein expression , skeletal muscle triglyceride levels were significantly elevated in high - fat  fed middle - aged mice compared with those in low - fat  fed mice ( fig .
3 m ) , as were long - chain acyl - coa esters ( fig .
, these data suggest that increased cd36-mediated fatty acid transport may contribute to lipid accumulation and impaired insulin sensitivity in skeletal muscle of middle - aged mice fed a high - fat diet .
to investigate whether inhibition of fatty acid transport into the skeletal muscle could alter the observed responses of a middle - aged mouse to a high - fat diet , we utilized the cd36 ko mouse , which has skeletal muscle fatty acid uptake rates 4070% of those in wild - type mice ( 31,32 ) .
interestingly , there was a striking difference in weight gain between middle - aged wild - type and cd36 ko mice following 12 weeks of high - fat feeding ( fig .
4a ) , with middle - aged cd36 ko mice accumulating 51% less weight than the wild - type mice over the same period of time ( fig .
4d ) between groups could not account for this dramatic change in weight gain , indirect calorimetry indicated that energy expenditure ( fig .
4 g ) were significantly increased in middle - aged cd36 ko mice fed a high - fat diet compared with those in high - fat  fed middle - aged wild - type mice .
although this increased activity in the middle - aged cd36 ko mouse fed a high - fat diet could be attributed to the absence of obesity , heat production was also increased in middle - aged cd36 ko mice fed a low - fat diet compared with that in low - fat  fed middle - aged wild - type mice ( fig .
4h ) and in high - fat  fed middle - aged ko mice when normalized for body weight ( fig .
4i ) . protection of diet - induced obesity in middle - aged cd36 ko mice fed a high - fat ( hf ) diet for 12 weeks .
representative image of middle - aged ( 4852 weeks of age ) wild - type (  ) and cd36 ko (  ) mice fed a high - fat diet for 12 weeks ( a ) . weight gain ( b ) and food intake adjusted for body weight ( bw ) ( c ) in wild - type and ko mice fed a high - fat diet . respiratory exchange ratio ( rer ) ( d ) , oxygen consumption ( vo2 ) ( e ) , and carbon dioxide production ( vco2 ) ( f ) in both the dark ( active ) and light ( inactive ) phase following 12 weeks of high - fat feeding in wild - type and ko mice .
total activity for a complete dark / light cycle ( g ) , heat production ( h ) , and heat production adjusted for body weight ( i ) in wild - type and ko mice following 12 weeks of high - fat feeding .
values are the means  sem of n = 610 mice in each group . *
p < 0.05 indicates comparisons performed between the low - fat ( lf)-fed mice and between high - fat  fed mice ( mann - whitney u test [ b and g ] or anova with bonferroni post hoc test for pairwise comparison ) .
( a high - quality digital representation of this figure is available in the online issue . ) to gain a more comprehensive metabolic assessment of muscle metabolism in middle - aged wild - type and cd36 ko mice fed a low - fat or high - fat diet , we used mass spectrometry to measure a broad range of intermediary metabolites , including acylcarnitines of various chain lengths , organic acids , and amino acids
. acylcarnitines are by - products of fuel catabolism that respond to changes in substrate availability or flux limitations at specific mitochondrial enzymes ( 14,33,34 ) .
middle - aged cd36 ko mice fed a low - fat diet had elevated levels of acetyl - carnitine ( c2 ) , and -hydroxybutyryl - carnitine ( c4oh ) compared with their wild - type counterparts ( supplemental fig . 1a and supplemental table 1 ) . whereas several short - chain acylcarnitine species , including c2 and c4oh , as well as propionyl - carnitine ( c3 ) and succinyl - carnitine ( c4dc ) , tended to increase in response to a high - fat diet
, these same metabolites trended downward in cd36 ko mice fed a high - fat diet ( supplemental fig . 1a and supplemental table 1 ) .
in addition , several long - chain acylcarnitine species were reduced in muscle from wild - type mice fed a high - fat diet while at the same time levels of hydroxylated long - chain acylcarnitine ( lcoh ) species were increased , resulting in a robust increase in the long - chain  to  lcoh acylcarnitine ratio ( fig .
c ) . given that long - chain acylcarnitines accumulate when their production by mitochondrial carnitine palmitoyl transferase ( cpt)1 exceeds flux through -oxidation enzymes , such as long - chain acyl - coa dehydrogenase ( lcad ) and -had ( 35 ) , this pattern is consistent with a diet - induced shift in flux limitation from lcad to -had .
notably , levels of many long - chain and lcoh acylcarnitines were lower in the cd36 ko mice fed a high - fat diet than in the wild - type mice fed a high - fat diet ( fig . 5a ; supplemental table 1 ) .
the organic acids were less responsive to both diet and genotype , although subtle changes were detected in succinate , fumarate , and citrate levels ( supplemental fig .
muscle levels of amino acids were higher in wild - type mice fed a low - fat diet but were dramatically decreased following high - fat feeding . by comparison ,
amino acid levels remained unchanged in cd36 ko mice in response to a high - fat diet ( supplemental fig .
although these metabolite measurements do not fully characterize mitochondrial substrate flux , together the data suggest that ablation of cd36 not only alters baseline mitochondrial and intermediary metabolism but also significantly impacts the muscle response to lipid exposure . altered skeletal muscle lipid handling prevents development of insulin resistance in middle - aged cd36 ko mice fed a high - fat ( hf ) diet for 12 weeks .
acylcarnitine levels were measured in gastrocnemius muscle from overnight - fasted middle - aged ( 4852 weeks of age ) wild - type ( wt ) and cd36 ko mice fed a low- ( lf ) or high - fat diet for 12 weeks .
levels of individual long chain ( lc ) and lcoh species ( a ) , the sum total of all long - chain or lcoh species ( b ) , and the ratio of total lcoh  to  long - chain species ( c ) .
phosphorylation status of ampk ( thr172 ) ( d ) and acc ( ser 79 ) ( e ) was detected in gastrocnemius muscle using immunoblot analysis .
immunoblots were quantified by densitometry and normalized against total ampk ( d ) and acc ( e ) .
serum levels of free fatty acids ( ffas ) from high - fat  fed wild - type and cd36 ko mice were determined after 12 weeks of diet ( f ) .
intramuscular levels of triglyceride ( tg ) ( g ) , lccoa ( h ) , and c18 ceramide ( i ) were determined in gastrocnemius muscle obtained from wild - type and ko mice fed a high - fat diet for 12 weeks .
glucose tolerance testing ( j ) was performed in high - fat  fed wild - type and ko mice fasted for 6 h. fasted blood glucose ( k ) and serum insulin ( l ) levels obtained from middle - aged wild - type and ko mice fed a high - fat diet for 12 weeks .
insulin tolerance test with blood glucose levels expressed as percent change of blood glucose at time zero in middle - aged wild - type and ko mice fed a high - fat diet for 12 weeks ( m ) .
immunoblots were performed on gastrocnemius muscle isolated from middle - aged wild - type and ko mice following high - fat diet and phosphorylation status of akt measured and normalized to total akt levels ( n ) .
main effects of genotype , diet , and genotype  diet interactions on acylcarnitine levels ( a  c ) were detected by two - way anova . for simplicity ,
symbols indicate metabolites that were affected by genotype , diet , or a genotype - diet interaction .
detailed results of the statistical analysis for all acylcarnitine species are presented in supplemental table 1 .
* p < 0.05 indicates comparisons performed between low - fat  fed wild - type and low - fat  fed ko mice and between high - fat  fed wild - type and high - fat  fed ko mice ( mann - whitney u test or anova with bonferroni post hoc test [ j and m ] ) .
interestingly , despite the changes in muscle acylcarnitine levels , the activity of -had was not altered ( data not shown ) and middle - aged cd36 ko mice fed a high - fat diet were not protected from decreased levels of ampk or acc phosphorylation ( fig . 5d and e ) compared with young wild - type mice ( fig .
2d and f ) , suggesting that alternate mechanisms are responsible for the metabolic phenotype observed in cd36 ko mice .
similarly , absolute expression of ampk and acc in skeletal muscle from aged cd36 mice is not different between groups ( data not shown ) .
although serum free fatty acid levels were elevated in high - fat  fed cd36 ko mice ( fig .
5f ) , cd36 ablation resulted in a significant reduction in skeletal muscle triglycerides ( fig .
by contrast , ceramide levels remained similar between high - fat fed groups ( fig .
5i ) , suggesting that accumulation of lipid - derived intermediates other than ceramides , may contribute to impaired insulin sensitivity .
indeed , reduced intramuscular lipid accumulation in middle - aged cd36 ko mice was associated with both improved whole - body glucose tolerance ( fig .
5k ) compared to high - fat fed middle - aged wild - type mice . moreover , fasted insulin levels were significantly reduced ( fig .
5 m ) in high - fat fed middle - aged cd36 ko mice compared to high - fat fed middle - aged wild - type mice , suggesting that insulin sensitivity is restored by preventing lipid accumulation in skeletal muscle .
interestingly , despite improved glucose utilization and reduced plasma insulin levels in these mice , the phosphorylation status of akt was similar in skeletal muscle from middle - aged wild - type and cd36 ko mice on high - fat diet ( fig .
furthermore , we found no difference in glycogen content in livers from cd36 ko mice fed a low - fat or high - fat diet ( data not shown ) , suggesting that the improved glucose tolerance observed in these mice is the result of increased glucose uptake and/or glucose oxidation in skeletal muscle from cd36 ko mice and not alterations in hepatic glucose metabolism .
to determine whether an age - related decline in resting metabolic rate and energy expenditure might contribute to the development of insulin resistance , c57bl6 mice of 1214 ( young ) or 5258 ( middle - aged ) weeks of age were analyzed using indirect calorimetry . as mice aged and body weight was increased ( fig .
1a ) , substrate use was altered slightly , with reductions in rer indicating that middle - aged mice used more fatty acid throughout the day compared with young mice ( fig .
in addition , significant reductions in oxygen consumption ( vo2 ) ( fig . 1c ) and carbon dioxide production ( vco2 ) ( fig .
1d ) were observed during both the dark ( active ) and light ( inactive ) phases in middle - aged mice compared with young mice .
1e ) was decreased in middle - aged mice compared with young mice , whereas activity measurements were not significantly different between age - groups ( fig .
our data indicate that middle - aged mice have a lower metabolic rate than young mice and that this might increase susceptibility to weight gain , obesity , and metabolic disease .
body weight of young ( 1214 weeks of age ) and middle - aged ( 5258 weeks of age ) mice fed a standard laboratory diet ( a ) .
indirect calorimetry was performed to measure respiratory exchange ratio ( rer ) ( b ) , vo2 ( c ) , vco2 ( d ) , and heat production adjusted for bodyweight ( e ) ; total activity ( f ) was measured in both the dark ( active ) and light ( inactive ) phases .
p < 0.05 indicates comparisons performed between young and middle - aged mice in either dark or light phases ( mann - whitney u test ) .
two - way anova was performed for rer ( b ) and indicated the main effect for age ( p < 0.01 ) .
given that skeletal muscle metabolism contributes to whole - body basal metabolic rate , we addressed whether skeletal muscle metabolism was depressed in middle - aged mice by assessing the activities of two enzymes involved in regulating mitochondrial metabolism .
2a ) was elevated in the middle - aged compared with the young mice , and citrate synthase ( fig .
2b ) followed a similar upward trend , suggesting that -oxidation and tca cycle activity were not directly compromised in the middle - aged mice . since mitochondrial number ( fig .
2c ) or function did not appear to be altered at middle age , we next assessed whether peroxisome proliferator  activated receptor ( ppar) responsive genes or molecular signaling cascades known to regulate skeletal muscle fatty acid flux and fatty acid entry into the mitochondria were associated with this overall reduction in whole - body basal metabolic rate observed in middle age . although a more comprehensive assessment of the multiple mediators of fatty acid utilization may reveal additional mechanisms , we did not observe any changes in protein levels of known ppar responsive genes involved in lipid metabolism , including malonyl - coa decarboxylase and acyl - coa synthetase 1 ( data not shown ) . as a result , we also examined the energy - sensing kinase , ampk , which is known for its ability to govern energy metabolism ( 23 ) . in agreement with previous reports using older rodents ( 24,25 ) , levels of phosphorylated ampk
were significantly reduced in skeletal muscle of middle - aged mice compared with young mice ( fig .
moreover , the phosphorylation status of acetyl coa carboxylase ( acc ) , the downstream target of ampk that indirectly regulates fatty acid entry into the mitochondria and ultimately -oxidation , was significantly decreased in skeletal muscle from middle - aged mice ( fig .
although reduced ampk phosphorylation could be the result of impaired activity of upstream ampk kinases or increased ampk phosphatase activity , it is currently unknown which of these contributes to reduced ampk phosphorylation in our model .
however , consistent with decreased energy expenditure , increased adiposity , and reduced ampk activity in skeletal muscle from middle - aged mice , the levels of skeletal muscle triglycerides were significantly elevated in middle - aged mice compared with young mice ( fig
. 2 g ) . given that lipid accumulation in skeletal muscle has been proposed in rodents ( 26 ) and humans ( 17,27,28 ) to be one of the primary causes of skeletal muscle insulin resistance , we next investigated whether glucose tolerance was impaired in middle - aged mice . despite elevated intramuscular triglycerides ( fig . 2 g ) and impaired basal and insulin - stimulated akt phosphorylation ( fig .
2h and i , respectively ) in skeletal muscle of middle - aged mice , whole - body glucose tolerance ( fig .
2k ) were not different in middle - aged compared with young mice , suggesting that age - induced alterations in skeletal muscle fatty acid handling and increased triglyceride storage precede the development of insulin resistance and metabolic disease .
alterations in fatty acid handling and reduced insulin signaling in skeletal muscle of middle - aged mice do not result in impaired whole - body glucose tolerance .
activity of two mitochondrial enzymes -had ( a ) and citrate synthase ( cs ) ( b ) was determined in gastrocnemius muscle from overnight fasted young ( 1214 weeks of age ) and middle - aged ( 5258 weeks of age ) mice .
immunoblot analysis using a total oxphos complex antibody cocktail was performed in gastrocnemius muscle lysates , and immunoblots were normalized against tubulin as a control for protein loading ( c ) .
phosphorylation status of ampk at threonine 172 ( d ) and acc at serine 79 ( f ) was detected using immunoblot analysis with phospho - specific antibodies .
immunoblots were quantified by densitometry and normalized against total protein levels of ampk ( e ) and acc ( f ) .
triglyceride ( tg ) levels ( g ) and phosphorylation status of akt ( h ) were determined in gastrocnemius muscle from overnight - fasted young and middle - aged mice .
gastrocnemius muscle was collected from a separate group of overnight - fasted young and middle - aged mice following intraperitoneal injection with either saline or human recombinant insulin ( 10 units / kg ) , and immunoblots were performed to detect phosphorylation status of akt at serine 473 in gastrocnemius muscle lysates ( i ) .
glucose tolerance test ( j ) was performed in young (  ) and middle - aged (  ) mice following a 6-h fast .
serum insulin was detected in young and middle - aged mice after an overnight fast ( k ) .
p < 0.05 indicates comparisons performed between young and middle - aged mice ( mann - whitney u test ) .
since we speculated that middle - aged mice are more susceptible than young mice to the development of insulin resistance , young and middle - aged mice were subjected to high - fat feeding for 12 weeks .
3a ) and signs of glucose intolerance compared with young mice fed a low - fat diet ( fig .
in contrast , middle - aged mice fed a high - fat diet showed significant weight gain ( body weight : low fat , 30.18  0.70 g , high fat , 55.14  1.56 g ; p < 0.05 ) and displayed dramatically elevated insulin levels ( fig .
in addition , whole - body glucose tolerance in the middle - aged mice fed a high - fat diet was significantly impaired compared with that in middle - aged mice fed a low - fat diet ( fig .
although activation of insulin signaling , as determined by the phosphorylation status of akt is not impaired in skeletal muscle of young ( fig .
3j ) mice fed a high - fat diet compared with that in mice fed a low - fat diet , this is likely due to elevated levels of circulating insulin ( fig .
3e and f ) observed in the respective high - fat groups . in support of the glucose tolerance data ,
homeostasis model assessment of insulin resistance values were significantly higher in the high - fat  fed middle - aged mice ( fig .
3k ) , suggesting that high - fat feeding induces more dramatic insulin resistance in middle - aged mice than in young mice .
interestingly , young mice on a high - fat diet were of the same weight as middle - aged mice on a low - fat diet ; yet , only the young mice on a high - fat diet had impaired glucose disposal ( data not shown ) .
although we can not discriminate between the effects of aging and increased adiposity because these variables coassociate , our findings do suggest that the increased weight gain associated with aging is not sufficient to alter whole - body glucose disposal and that other factors such as high - fat diet are likely involved . aging increases the susceptibility to the development of glucose intolerance and insulin resistance in mice fed a high - fat diet .
body weights ( a ) , fed and fasted blood glucose ( b ) , glucose tolerance test ( c ) , and phosphorylation status of akt ( d ) were measured in gastrocnemius muscle from young ( 1214 weeks of age ) mice following 12 weeks of high fat ( hf ) feeding .
serum insulin levels in the fasted ( e ) and fed ( f ) states obtained from young ( 1214 weeks of age ) and middle - aged ( 4852 weeks of age ) mice fed a low - fat ( lf ) and high - fat diet for 12 weeks .
fed and fasted blood glucose levels ( g ) , glucose tolerance test ( h ) , area under the curve ( auc ) for the glucose tolerance test ( i ) , and phosphorylation status of akt in gastrocnemius muscle ( j ) in middle - aged mice following 12 weeks of high - fat feeding .
homeostasis model assessment of insulin resistance ( homa - ir ) as a surrogate marker of insulin resistance in young and middle - aged mice fed a high - fat diet ( k ) .
immunoblot analysis using anti - cd36 antibody was performed in gastrocnemius muscle lysates from middle - aged mice fed a low- and high - fat diet , and immunoblots were quantified by densitometry and normalized against tubulin as a control for protein loading ( l ) .
triglyceride ( tg ) ( m ) , long - chain acyl - coa ( lccoa ) ( n ) , and c18 ceramide ( o ) levels in gastrocnemius muscle from overnight - fasted middle - aged mice fed a low- or high - fat diet .
two - way anova was performed to detect main effects of age , diet , and age  diet interactions on insulin levels ( e and f ) and homa - ir ( k ) .
significant effect of age , diet , and age  diet interaction ( p < 0.05 ) was observed for e , f , and k. *
p < 0.05 indicates comparisons performed between young and middle - aged mice or between low- and high - fat fed mice in fed or fasted state ( mann - whitney u test ) . because high - fat diet  induced insulin resistance in young rodents is associated with an increased efficiency of fatty acid uptake into skeletal muscle ( 26 ) , protein expression of cd36 , a protein that facilitates fatty acid transport , was determined in skeletal muscle of mice fed a high - fat diet .
consistent with previous publications in young mice ( 26,29,30 ) , we observed a modest increase in cd36 protein expression in the muscle of young mice fed a high - fat diet , as well as an increase in intramuscular triglyceride levels ( data not shown ) .
consistent with our hypothesis , cd36 expression was significantly elevated in skeletal muscle of middle - aged mice fed a high - fat diet compared with that in mice fed a low - fat diet ( fig .
3l ) . in accordance with increased cd36 protein expression , skeletal muscle triglyceride levels were significantly elevated in high - fat  fed middle - aged mice compared with those in low - fat  fed mice ( fig .
3 m ) , as were long - chain acyl - coa esters ( fig .
together , these data suggest that increased cd36-mediated fatty acid transport may contribute to lipid accumulation and impaired insulin sensitivity in skeletal muscle of middle - aged mice fed a high - fat diet .
to investigate whether inhibition of fatty acid transport into the skeletal muscle could alter the observed responses of a middle - aged mouse to a high - fat diet , we utilized the cd36 ko mouse , which has skeletal muscle fatty acid uptake rates 4070% of those in wild - type mice ( 31,32 ) .
interestingly , there was a striking difference in weight gain between middle - aged wild - type and cd36 ko mice following 12 weeks of high - fat feeding ( fig .
4a ) , with middle - aged cd36 ko mice accumulating 51% less weight than the wild - type mice over the same period of time ( fig .
4d ) between groups could not account for this dramatic change in weight gain , indirect calorimetry indicated that energy expenditure ( fig .
4 g ) were significantly increased in middle - aged cd36 ko mice fed a high - fat diet compared with those in high - fat  fed middle - aged wild - type mice .
although this increased activity in the middle - aged cd36 ko mouse fed a high - fat diet could be attributed to the absence of obesity , heat production was also increased in middle - aged cd36 ko mice fed a low - fat diet compared with that in low - fat  fed middle - aged wild - type mice ( fig .
4h ) and in high - fat  fed middle - aged ko mice when normalized for body weight ( fig .
4i ) . protection of diet - induced obesity in middle - aged cd36 ko mice fed a high - fat ( hf ) diet for 12 weeks .
representative image of middle - aged ( 4852 weeks of age ) wild - type (  ) and cd36 ko (  ) mice fed a high - fat diet for 12 weeks ( a ) . weight gain ( b ) and food intake adjusted for body weight ( bw ) ( c ) in wild - type and ko mice fed a high - fat diet
. respiratory exchange ratio ( rer ) ( d ) , oxygen consumption ( vo2 ) ( e ) , and carbon dioxide production ( vco2 ) ( f ) in both the dark ( active ) and light ( inactive ) phase following 12 weeks of high - fat feeding in wild - type and ko mice .
total activity for a complete dark / light cycle ( g ) , heat production ( h ) , and heat production adjusted for body weight ( i ) in wild - type and ko mice following 12 weeks of high - fat feeding .
* p < 0.05 indicates comparisons performed between the low - fat ( lf)-fed mice and between high - fat  fed mice ( mann - whitney u test [ b and g ] or anova with bonferroni post hoc test for pairwise comparison ) .
( a high - quality digital representation of this figure is available in the online issue . )
to gain a more comprehensive metabolic assessment of muscle metabolism in middle - aged wild - type and cd36 ko mice fed a low - fat or high - fat diet , we used mass spectrometry to measure a broad range of intermediary metabolites , including acylcarnitines of various chain lengths , organic acids , and amino acids .
acylcarnitines are by - products of fuel catabolism that respond to changes in substrate availability or flux limitations at specific mitochondrial enzymes ( 14,33,34 ) .
middle - aged cd36 ko mice fed a low - fat diet had elevated levels of acetyl - carnitine ( c2 ) , and -hydroxybutyryl - carnitine ( c4oh ) compared with their wild - type counterparts ( supplemental fig . 1a and supplemental table 1 ) . whereas several short - chain acylcarnitine species , including c2 and c4oh , as well as propionyl - carnitine ( c3 ) and succinyl - carnitine ( c4dc ) , tended to increase in response to a high - fat diet , these same metabolites trended downward in cd36 ko mice fed a high - fat diet ( supplemental fig . 1a and supplemental table 1 ) .
in addition , several long - chain acylcarnitine species were reduced in muscle from wild - type mice fed a high - fat diet while at the same time levels of hydroxylated long - chain acylcarnitine ( lcoh ) species were increased , resulting in a robust increase in the long - chain  to  lcoh acylcarnitine ratio ( fig . 5a 
c ) . given that long - chain acylcarnitines accumulate when their production by mitochondrial carnitine palmitoyl transferase ( cpt)1 exceeds flux through -oxidation enzymes , such as long - chain acyl - coa dehydrogenase ( lcad ) and -had ( 35 ) , this pattern is consistent with a diet - induced shift in flux limitation from lcad to -had .
notably , levels of many long - chain and lcoh acylcarnitines were lower in the cd36 ko mice fed a high - fat diet than in the wild - type mice fed a high - fat diet ( fig .
the organic acids were less responsive to both diet and genotype , although subtle changes were detected in succinate , fumarate , and citrate levels ( supplemental fig .
muscle levels of amino acids were higher in wild - type mice fed a low - fat diet but were dramatically decreased following high - fat feeding . by comparison ,
amino acid levels remained unchanged in cd36 ko mice in response to a high - fat diet ( supplemental fig .
although these metabolite measurements do not fully characterize mitochondrial substrate flux , together the data suggest that ablation of cd36 not only alters baseline mitochondrial and intermediary metabolism but also significantly impacts the muscle response to lipid exposure .
altered skeletal muscle lipid handling prevents development of insulin resistance in middle - aged cd36 ko mice fed a high - fat ( hf ) diet for 12 weeks .
acylcarnitine levels were measured in gastrocnemius muscle from overnight - fasted middle - aged ( 4852 weeks of age ) wild - type ( wt ) and cd36 ko mice fed a low- ( lf ) or high - fat diet for 12 weeks .
levels of individual long chain ( lc ) and lcoh species ( a ) , the sum total of all long - chain or lcoh species ( b ) , and the ratio of total lcoh  to  long - chain species ( c ) .
phosphorylation status of ampk ( thr172 ) ( d ) and acc ( ser 79 ) ( e ) was detected in gastrocnemius muscle using immunoblot analysis .
immunoblots were quantified by densitometry and normalized against total ampk ( d ) and acc ( e ) .
serum levels of free fatty acids ( ffas ) from high - fat  fed wild - type and cd36 ko mice were determined after 12 weeks of diet ( f ) .
intramuscular levels of triglyceride ( tg ) ( g ) , lccoa ( h ) , and c18 ceramide ( i ) were determined in gastrocnemius muscle obtained from wild - type and ko mice fed a high - fat diet for 12 weeks .
glucose tolerance testing ( j ) was performed in high - fat  fed wild - type and ko mice fasted for 6 h. fasted blood glucose ( k ) and serum insulin ( l ) levels obtained from middle - aged wild - type and ko mice fed a high - fat diet for 12 weeks .
insulin tolerance test with blood glucose levels expressed as percent change of blood glucose at time zero in middle - aged wild - type and ko mice fed a high - fat diet for 12 weeks ( m ) .
immunoblots were performed on gastrocnemius muscle isolated from middle - aged wild - type and ko mice following high - fat diet and phosphorylation status of akt measured and normalized to total akt levels ( n ) .
main effects of genotype , diet , and genotype  diet interactions on acylcarnitine levels ( a  c ) were detected by two - way anova .
for simplicity , symbols indicate metabolites that were affected by genotype , diet , or a genotype - diet interaction .
detailed results of the statistical analysis for all acylcarnitine species are presented in supplemental table 1 .
* p < 0.05 indicates comparisons performed between low - fat  fed wild - type and low - fat  fed ko mice and between high - fat  fed wild - type and high - fat  fed ko mice ( mann - whitney u test or anova with bonferroni post hoc test [ j and m ] ) .
interestingly , despite the changes in muscle acylcarnitine levels , the activity of -had was not altered ( data not shown ) and middle - aged cd36 ko mice fed a high - fat diet were not protected from decreased levels of ampk or acc phosphorylation ( fig . 5d and e ) compared with young wild - type mice ( fig .
2d and f ) , suggesting that alternate mechanisms are responsible for the metabolic phenotype observed in cd36 ko mice . similarly ,
absolute expression of ampk and acc in skeletal muscle from aged cd36 mice is not different between groups ( data not shown ) .
although serum free fatty acid levels were elevated in high - fat  fed cd36 ko mice ( fig .
5f ) , cd36 ablation resulted in a significant reduction in skeletal muscle triglycerides ( fig .
by contrast , ceramide levels remained similar between high - fat fed groups ( fig .
5i ) , suggesting that accumulation of lipid - derived intermediates other than ceramides , may contribute to impaired insulin sensitivity .
indeed , reduced intramuscular lipid accumulation in middle - aged cd36 ko mice was associated with both improved whole - body glucose tolerance ( fig .
5k ) compared to high - fat fed middle - aged wild - type mice .
5 m ) in high - fat fed middle - aged cd36 ko mice compared to high - fat fed middle - aged wild - type mice , suggesting that insulin sensitivity is restored by preventing lipid accumulation in skeletal muscle .
interestingly , despite improved glucose utilization and reduced plasma insulin levels in these mice , the phosphorylation status of akt was similar in skeletal muscle from middle - aged wild - type and cd36 ko mice on high - fat diet ( fig .
furthermore , we found no difference in glycogen content in livers from cd36 ko mice fed a low - fat or high - fat diet ( data not shown ) , suggesting that the improved glucose tolerance observed in these mice is the result of increased glucose uptake and/or glucose oxidation in skeletal muscle from cd36 ko mice and not alterations in hepatic glucose metabolism .
consistent with previous reports ( 17 ) , our data show a significant decline in metabolic rate in middle - aged mice when compared to their young counterparts ( fig .
interestingly , although mitochondrial function was not directly assessed in this study , the decline in overall metabolic rate did not appear to stem from compromised mitochondrial function in skeletal muscle of middle - aged mice compared to young mice .
indeed , whole body rer was modestly decreased with aging and muscle activity of -had increased , suggesting there is a shift in substrate selection from carbohydrates toward fatty acids .
however , the age - associated reduction in overall metabolic rate did not appear to correlate with changes in maximal activities of mitochondrial enzymes in muscle from young and middle - aged mice ( fig .
2a and 2b ) , suggesting other factors may influence substrate oxidation in muscle , as well as overall metabolic rate .
consistent with this , the ampk / acc signaling axis was significantly reduced in skeletal muscle from middle - aged mice ( fig . 2d and 2f ) . still unclear
is whether reduced ampk phosphorylation in the older mice reflects a cause or consequence of reduced metabolic rate .
although results of a recent study suggest that acc - mediated shifts in fat oxidation per se do not impact whole body energy expenditure or susceptibility to diet - induced obesity ( 36 ) , ampk acts on a broad range of enzymatic and transcriptional targets that could affect energy balance via mechanisms other than substrate selection ( 25,37 ) . nonetheless , cd36 deficiency raised metabolic rate without activating ampk , indicating that other mechanisms are operative in this model ( see below ) . although akt phosphorylation is reduced in skeletal muscle from middle - aged mice compared to young mice ( fig .
2h and 2i ) whole - body glucose tolerance and plasma insulin levels remained normal ( fig .
2j and 2k ) , suggesting that impaired activation of insulin - signaling parameters in skeletal muscle precede overt changes in whole body glucose disposal and may increase the susceptibility of aged mice to diet - induced insulin resistance . to determine whether middle - aged mice indeed are more susceptible to developing insulin resistance in response to a high - fat diet compared to their younger counterparts , we subjected middle - aged mice to 12 weeks of a low - fat or high - fat diet .
as expected , middle - aged mice gained significantly more weight than young mice when fed a high - fat diet ( weight gain : middle - aged , 22.8  1.9 g versus young , 12.0  1.1 g ; p < 0.01 ) .
3c , 3h and 3i ) , high - fat diet - induced hyperinsulinemia was only observed in middle - aged mice ( fig . 3e and 3f ) , indicating that increased -cell insulin secretion was sufficient to offset overt hyperglycemia in this age - group ( fig .
3 g ) . given that prolonged hypersecretion of insulin by the -cells to compensate for peripheral insulin resistance can contribute to -cell failure ( 38 ) and potentially type 2 diabetes ( 39 ) , our data suggest that middle - aged mice have a heightened susceptibility to the development of insulin resistance .
however , this susceptibility might not be due to aging per se , since adiposity was also increased in the older mice . as our study design does not permit to discriminate between the effects of aging and adiposity on skeletal muscle metabolism and the development of insulin resistance , future experiments should be directed at conducting weight loss ( food restriction ) studies or exercise studies to determine if these 
nevertheless , as aging and increased adiposity co - associate our findings likely reflect the majority of middle - aged humans in the western world who are at risk of developing insulin resistance .
although intramuscular lipid accumulation associated with aging did not appear to result from mitochondrial dysfunction , we do show a significant twofold increase in cd36 protein expression in skeletal muscle of high - fat fed middle - aged mice compared to low - fat fed middle - aged mice ( fig .
3l ) , suggesting fatty acid transport into muscle exceeded the capacity for their oxidation .
although it is unknown what caused increased cd36 expression in our study , it may have resulted from high levels of plasma glucose levels that have been shown to regulate cd36 expression through both transcriptional and/or translational mechanisms in rodents and humans ( 40,41 ) . to determine whether reduced fatty acid transport and metabolism could rescue the high - fat diet - induced phenotype
consistent with our prediction , cd36 deficiency prevented the decline in metabolic rate and energy expenditure in middle - aged mice fed a high - fat diet as compared to age - matched wild - type mice ( fig . 4e and 4f ) . since food intake was similar between groups ( fig .
4c ) , we propose that increased energy expenditure in high - fat fed middle - aged cd36 ko mice contributes to their protection against diet - induced obesity ( fig .
4b ) . the blunted decline in metabolic rate in the middle - aged cd36 ko mice fed a high - fat diet ( fig .
4e and 4f ) was accompanied by alterations in muscle concentrations of several metabolic intermediates ( fig .
1 ) , but no improvement in ampk and acc phosphorylation ( fig . 5d and 5e , respectively ) .
the impact of the diet on muscle metabolites in this study differed to some extent as compared to a previous report ( 14 ) in which tissue specimens were harvested from younger animals in the fed state .
herein , tissues were collected after an overnight fast because we sought to evaluate a state of heightened fatty acid oxidation . under these conditions ,
the drop in long - chain acylcarnitines could reflect decreased fatty acid availability , lower cpt1 activity or increased long - chain acyl coa flux through lcad .
first , the high - fat diet increases rather than decreases lipid delivery to muscle .
second , of the two major products of cpt1 ( palmitoylcarnitine ( c16 ) and oleylcarnitine ( c18:1 ) , only the unsaturated species was reduced by the diet ( fig .
5a ) , suggesting upregulation of the isomerase enzyme that catalyzes conversion of the double bond ( 42 ) .
5c ) in response to chronic lipid exposure suggest a shift in flux limitation from the earlier to later steps in -oxidation .
lastly , the diet resulted in a robust drop in whole body rer , indicative of a systemic increase in fat oxidation .
notably , cd36 deficiency altered baseline levels of several muscle metabolites , and in general , tended to mitigate diet - induced changes in several acylcarnitine and amino acid species . this apparent resistance to diet - induced metabolic perturbations in the cd36 ko mice
might be directly related to a reduction in fat delivery and/or secondary to enhanced energy expenditure and insulin sensitivity .
although further work is necessary to fully understand the implications of these results , it is clear that loss of cd36 has a global impact on muscle fuel metabolism .
in addition , it is possible that other organs such as adipose tissue , liver and brain ( 43,44 ) are also involved in maintaining a high level of energy expenditure in the middle - aged cd36 ko mice .
overall , ablation of cd36 was associated with an improvement in whole - body glucose utilization and insulin sensitivity ( fig .
although the mechanisms responsible for this are not known , excessive intramuscular lipid accumulation induced by high - fat feeding was prevented in skeletal muscle of middle - aged cd36 ko mice ( fig . 5 g and 5h ) . whereas elevated intramuscular triglyceride levels were not associated with insulin resistance in young mice ( fig .
2j2k ) , preventing the more dramatic age- and diet - induced accumulation of intramuscular triglyceride and long - chain acyl coa levels in middle - aged cd36 ko mice correlated with improved whole - body insulin sensitivity .
although there is ample evidence indicating that cd36 ablation significantly reduces skeletal muscle fatty acid uptake ( 31,32 ) , it is also possible that the effects that we report using the cd36 ko mice are secondary to changes in fatty acid metabolism .
notwithstanding this later possibility , our data demonstrate that limiting cd36-mediated skeletal muscle fatty acid transport guards against whole - body and muscle insulin resistance in middle - aged mice fed a high - fat diet .
this finding suggests a potential therapeutic strategy for combating metabolic disease in the face of age - related abnormalities .
 objectivealthough advanced age is a risk factor for type 2 diabetes , a clear understanding of the changes that occur during middle age that contribute to the development of skeletal muscle insulin resistance is currently lacking . 
 therefore , we sought to investigate how middle age impacts skeletal muscle fatty acid handling and to determine how this contributes to the development of diet - induced insulin resistance.research design and methodswhole - body and skeletal muscle insulin resistance were studied in young and middle - aged wild - type and cd36 knockout ( ko ) mice fed either a standard or a high - fat diet for 12 weeks . 
 molecular signaling pathways , intramuscular triglycerides accumulation , and targeted metabolomics of in vivo mitochondrial substrate flux were also analyzed in the skeletal muscle of mice of all ages.resultsmiddle-aged mice fed a standard diet demonstrated an increase in intramuscular triglycerides without a concomitant increase in insulin resistance . 
 however , middle - aged mice fed a high - fat diet were more susceptible to the development of insulin resistance  a condition that could be prevented by limiting skeletal muscle fatty acid transport and excessive lipid accumulation in middle - aged cd36 ko mice.conclusionour data provide insight into the mechanisms by which aging becomes a risk factor for the development of insulin resistance . 
 our data also demonstrate that limiting skeletal muscle fatty acid transport is an effective approach for delaying the development of age - associated insulin resistance and metabolic disease during exposure to a high - fat diet . 
RESEARCH DESIGN AND METHODS
Reagents.
Mice.
Metabolic analysis in vivo.
Determination of skeletal muscle triglycerides, long-chain acyl-CoA, and ceramides.
Metabolic profiling.
Statistical analysis.
RESULTS
Age-induced slowing of metabolic rate increases susceptibility to metabolic disease.
Age-induced alterations in fatty acid handling and lipid accumulation in skeletal muscle precede the development of whole-body glucose intolerance.
Aging increases the sensitivity to diet-induced obesity and metabolic disease.
Ablation of CD36 protects against diet-induced obesity in middle-aged mice.
Alterations in muscle metabolites and lipid balance correspond with protection against diet-induced insulin resistance.
Ablation of CD36 and reduction in intramuscular lipid accumulation prevent the development of diet-induced insulin resistance.
DISCUSSION
Supplementary Material