the close association between the growing epidemics of obesity and type 2 diabetes continues to fuel interest in understanding how alterations in lipid metabolism contribute to the development of insulin resistance .
an excess of fatty acids beyond the cells energy requirements results in the accumulation of triacylglycerol within lipid droplets , and the deposition of lipids in tissues other than adipose tissue is a feature of obesity and type 2 diabetes .
skeletal muscle serves as an important site for glucose disposal after a meal   and it has been known for many years that the accumulation of intramyocellular triacylglycerol predicts insulin resistance in muscle , independent of adiposity [ 25 ] .
however , the association is not clear cut because highly trained endurance athletes store as much triacylglycerol in their muscles as patients with type 2 diabetes , yet display remarkable insulin sensitivity .
thus , the matching of fatty acid flux from the lipid droplet with mitochondrial demand for fatty acid substrate may be an important regulatory process to avoid defects in insulin action . both skeletal and cardiac muscle store significant amounts of triacylglycerol within intracellular lipid droplets , which are located in close proximity to the mitochondria and endoplasmic reticulum .
intracellular triacylglycerol is an important energy substrate in skeletal   and cardiac muscle   and triacylglycerol turnover is relatively high in muscles , which ensures a continuous substrate supply to buffer changes in systemic free fatty acid levels .
however , an oversupply of fatty acids , beyond the energetic needs of the myocyte , eventually causes insulin resistance via a diverse range of mechanisms including the accumulation of lipid metabolites , maladaptive proinflammatory signaling and alterations in membrane physical properties . in this context
, an inability to mobilize fatty acids from triacylglycerol   and/or increase triacylglycerol breakdown in skeletal muscle , without an increase in energy demand , can result in excessive intracellular lipid accumulation and insulin resistance .
the understanding of lipid droplet biology and the regulation of intracellular lipid fluxes have expanded in recent years with the discovery of perilipin ( plin ) family members .
plin1 was first discovered as a lipid droplet - associated protein in white adipocytes and is a critical regulator of lipid metabolism , and more recently plin2 and plin5 have been shown to play major roles in liver and cardiac metabolism , respectively [ 1821 ] . while plin5 is highly expressed in skeletal muscle , its role in the control of intramyocellular lipid trafficking , substrate utilization and insulin action is not well understood .
mild overexpression of plin5 via dna electrotransfer into the glycolytic muscle of rats increased triacylglycerol accumulation , which is consistent with the general consensus that plin5 plays an important role in lipid droplet accumulation .
while plin5 increased triacylglycerol accumulation , this appeared to have no effect on skeletal muscle insulin action . intriguingly , and perhaps paradoxically , the same authors have shown that plin5 may also localize to the mitochondria , suggesting that plin5 might direct fatty acids from the lipid droplet to the mitochondria for oxidation . while these short - term overexpression models have provided useful insights into plin5 function in muscle , the physiological significance of plin5 deletion on skeletal muscle and systemic metabolic control
, we tested the hypothesis that plin5 plays an important role in regulating skeletal muscle fatty acid metabolism , and in turn , in regulating insulin action .
we have generated plin5 deficient mice and show that plin5 regulates skeletal muscle triacylglycerol flux , whole - body substrate metabolism and insulin action in a tissue - specific context .
a targeted vector containing plin5 ( nm_001013706.2 ; figure  1a ) interrupted in exon 5 , 6 , 7 was generated by the trans - nih knock - out mouse project ( komp ) and obtained from the komp repository ( project i d csd40589 , es clone epd0301_4_e04 ) .
cells were microinjected into the blastocyl cavity and injected blastocysts were transferred to the uteri of pseudopregnant cd1 female mice . to test for germline transmission
mice were confirmed to contain the plin5 disrupted allele by southern blot analysis of kpni digested genomic tail dna ( figure  1b ) .
mice were fed a low fat ( 5% energy from fat ) or high - fat diet ( 43% energy from fat , specialty feeds , wa , australia ) and had free access to water .
all experiments were performed in 4  h fasted mice commencing at 1100  h unless otherwise stated .
whole body metabolism was measured in metabolic cages ( columbus instruments , columbus , oh ) .
mice were housed individually for 48  h for assessment of oxygen uptake , carbon dioxide production and physical activity .
whole body fat and lean mass were measured by dual energy x - ray absorptiometry ( lunar pixi # 51042 , piximus , wi , usa ) .
running experiments were performed on an eco 3/6 treadmill ( columbus instruments , columbus , oh ) as described in ref . .
to determine maximal running speed , mice ran at 10  m / min on a 5% grade for 2  min and the speed was increased by 2  m / min until exhaustion .
mice were anaesthetized with isoflurane and kept under sedation while the soleus muscle was excised .
fatty acid   and glucose metabolism were assessed by radiometric methods as described in ref . .
lipids were extracted   and triacylglycerol content determined using the tg - gpo - pap reagent ( cat .
diacylglycerol and ceramide content was determined by an [ p]atp - linked enzymatic method as previously described in ref . .
mice were intraperitoneally injected with d - glucose ( 2  g / kg mass ) .
tail blood was collected and blood glucose was determined using a glucometer ( accu - chek , roche , mannheim , germany ) .
mice were anaesthetized and a catheter was inserted into the jugular vein as previously described .
following five days recovery , mice were fasted for 4  h prior to beginning the clamp .
blood glucose was equilibrated with a 90  min continuous infusion of [ 3-h]-glucose ( 0.05 
hyperinsulinemic - euglycemic clamp commenced with the increase of [ 3-h]-glucose tracer to 2  ci / min and insulin infusion ( 4  mu / kg / min ) .
exogenous glucose ( 25% w / v ) was infused to maintain basal blood glucose levels , which averaged 8.1    0.3  mm .
tail blood samples were collected and blood glucose was measured every 5  min . after 120  min , a 10  ci bolus of 2-deoxy - d-[1-c ] glucose ( 2-[c ] dg ; perkin elmer ) was administered to assess tissue - specific glucose uptake ( rg ) . at the conclusion of the clamp
, mice were anaesthetized with an intravenous injection of sodium pentobarbitone ( 100  mg / kg iv ) and tissues rapidly dissected and snap frozen in liquid nitrogen for analysis of 2-[c ] dg uptake .
whole body and tissue specific insulin sensitivity was assessed in anaesthetized ( 2% , isoflurane ) mice as described in ref . .
briefly , 2-[1,2-h ] deoxyglucose ( 10  ci ) was injected intravenously with 0.5  u / kg insulin .
blood was taken from the tip of the tail at 2 , 5 , 10 , 15 and 20  min after injection .
animals were sacrificed by exsanguination with tissue removed quickly and tissue specific glucose clearance was determined from total and phosphorylated 2-[1,2-h ] deoxyglucose .
whole blood was collected into edta tubes then centrifuged for 3  min at 8000    g. plasma free fatty acids and triacylglycerol were determined by enzymatic colorimetric assays ( wako chemicals , wako , va , usa ) . high density lipoproteins ( hdl ) and
very ( vldl ) and low density lipoproteins ( ldl ) were assessed by elisa ( ab65390 , abcam , cambridge , uk ) .
plasma -hydroxybutyrate was analyzed by a colorimetric assay ( 700190 , cayman chemicals , ann arbor , mi , usa ) .
tissues were rapidly frozen in melting isopentane after being coated in oct tissue - tek ( sakura finetek , the netherlands ) .
serial 10  m sections were cut at  20  c on to superfrost ultra plus glass slides .
slides were fixed in bouin 's solution with 0.1% triton x-100 ( sigma  aldrich ) for 30  min .
sections were stained for lipid droplets with freshly made , filtered oil red o ( sigma  aldrich ) made in 60% triethylphosphate ( sigma  aldrich ) .
-galactosidase expression was identified using commercial reagents ( mir 2600 , mirus bio , madison , wi , usa ) .
sections were blocked in pbs with 1% bsa for 1  h then incubated overnight with plin5 antibody ( cat .
gp31 , progen biotechnik , germany , 0.02  mg / ml ) while mitochondrial staining was performed using the mitochondrial antibody directed against the oxidative phosphorylation complexes i  v ( total oxphos cat .
primary antibodies were substituted for concentration matched guinea pig serum for plin5 ( antibodies australia ) , or oxphos cocktail pre - absorbed 4:1 overnight with mouse igg1 and igg2a ( dako , denmark ) .
immunohistochemistry images were analyzed for colocalization with imagej ( nih ) by using manders ' colocalization coefficient , m2 , which provides a fraction of the colocalizing objects in the images .
total rna was extracted , reverse transcribed and cdna used for qrt - pcr as described in ref .  .
stain - free images were collected after transfer for loading control ( chemidoc mp and imagelab software version 4.1 , bio - rad laboratories , nsw , australia ) .
thiobarbituric acid reactive substances ( tbars ) was measured in skeletal muscle lysates by using a colorimetric assay for the formation of malondialdehyde ( 10009055 , cayman chemicals , ann arbor , mi , usa ) .
recombinant ha - tagged murine plin5 was phosphorylated by camp - dependent protein kinase ( pka ) as described in ref .   and the location of the ha - plin5 was confirmed by immunoblot .
myoblasts were differentiated to myotubes by switching to 3% horse serum medium and were left to differentiate for five days prior to experiments . fatty acid metabolism ( radiometric methodology ) was assessed as described in ref .   and mitochondrial function using the seahorse xf analyzer .
statistical analysis was performed by one - way or two - way analysis of variance ( anova ) with repeated measures , and specific differences were located using a bonferroni post  hoc test .
interbreeding of heterozygous mice for the plin5 null allele yielded mice that were homozygous for each of the two alleles .
the absence of the plin5 allele was confirmed by southern blotting ( figure  1b ) and the absence of plin5 mrna was confirmed by pcr ( figure  1c ) .
the absence of plin5 protein was confirmed with immunohistochemistry of skeletal muscle ( figure  1d ) .
a secondary confirmation was the presence of the lacz in the hearts of plin5 mice , as detected by -galactosidase staining ( figure  1d ) .
triacylglycerol content was reduced by 52% in the hearts of plin5 mice ( figure  1e ) and coincided with a marked reduction of lipid droplets stained by oro ( figure  1f ) .
this is consistent with the prominent reduction in cardiac lipid content reported in another line of plin5 mice .
there was no such reduction in triacylglycerol content in the liver or mixed skeletal muscle ( figure  1f ) .
interestingly , triacylglycerol content was lower in red quadriceps ( oxidative ) muscle and tended ( 122% , p  <  0.08 ) to be higher in white quadriceps ( glycolytic ) muscle of plin5 compared with plin5 mice ( figure  1f )
. this may reflect the greater capacity of oxidative fibers to oxidize triglyceride - derived fatty acids compared with glycolytic fibers .
while the deletion of one plin gene can induce compensatory increases in the other plin genes , the expression of plin 2 , 3 and 4 were not altered in plin5 mice ( figure  1 g ) , nor were there changes in the expression of prominent lipases or co - activator proteins associated with triacylglycerol lipolysis ( figure  1 g , h ) .
plin5 and plin5 mice had similar body weights during development ( figure  2a ) and dexa examination revealed no difference in lean and fat mass ( figure  2b ) .
this was supported by post mortem examination showing no difference in epididymal fat , heart and liver mass , indicating that the absence of plin5 had no effect on adiposity and gross morphology ( table  1 ) .
neither food intake ( figure  2c ) nor energy expenditure ( vo2 ) was different between genotypes ( figure  2d ) .
however , the respiratory exchange ratio was increased in plin5 mice ( figure  2e ) , demonstrating an 11% decrease in whole - body fatty acid oxidation and a reciprocal increase in carbohydrate oxidation .
these changes can not be attributed to increased physical activity ( figure  2f ) .
fasting muscle and liver glycogen ( figure  2 g ) , plasma glucose and insulin concentrations were not different between groups ; neither were plasma free fatty acids , triacylglycerols and hdl or vldl / ldl cholesterol levels ( table  1 ) .
plasma  - hydroxybutyrate , a marker of liver fatty acid oxidation , was not altered by genotype ( table  1 ) .
based on the high abundance of plin5 in skeletal muscle   and the quantitative importance of skeletal muscle in regulating whole - body substrate metabolism , we next examined the effects of plin5 deletion on skeletal muscle fatty acid metabolism in isolated soleus muscle .
total fatty acid uptake , fatty acid oxidation and the incorporation of fatty acids into tri- and diacylglycerol were not different between the two genotypes ( figure  3a  d ) .
 - adrenergic activation of pka is a major regulator of triglyceride metabolism in adipocytes and most likely oxidative skeletal muscle , although pka action in muscle requires further confirmation .
we first performed in  vitro kinase assays to demonstrate that plin5 is a substrate for protein kinase a ( figure  3e ) , then we examined fatty acid metabolism in response to forskolin , an activator of camp / pka signaling .
forskolin reduced fatty acid uptake , oxidation and storage ; however , these effects were not impacted by plin5 deletion .
thus , plin5 deletion does not affect the uptake , oxidation or esterification of extracellular - derived fatty acids in muscle under basal and pka - stimulated conditions .
further studies in isolated soleus muscle revealed no difference in glucose oxidation between plin5 and plin5 mice ( 104    5 vs. 105    5  nmol / mg tissue / h , respectively .
plin5 interacts with atgl to inhibit spontaneous and  - adrenergic stimulated lipolysis , suggesting that plin5 is more likely to regulate fatty acid flux from endogenous triacylglycerol stored in lipid droplets rather than modulate the oxidation of extracellular - derived fatty acids .
we performed pulse - chase experiments on primary myotubes generated from the skeletal muscle of wild - type and plin5 mice to evaluate triacylglycerol metabolism .
plin5 mrna expression was retained in myotubes generated from plin5 mice and was absent in plin5 myotubes ( figure  3f ) .
intracellular triacylglycerol was pre - labeled with [ 1-c ] oleate for 4  h , then incubated in medium without radiolabeled fatty acids for 4  h , with or without isoproterenol .
the oxidation of triacylglycerol - derived fatty acids was increased in plin5 myotubes ( genotype effect , p  =  0.009 , figure  3 g ) and this coincided with increased depletion of the c labeled triacylglycerol in plin5 myotubes ( genotype effect , p  =  0.02 , figure  3h ) .
consistent with this notion , 24  h fasted wild - type mice stored significant amounts of lipid in their skeletal muscle whereas the skeletal muscle of plin5 mice were devoid of marked lipid droplet staining ( figure  3i ) , indicating increased intramyocelluar lipolysis and demonstrating an inability to expand the intramyocellular triacylglycerol pool .
intramyocellular lipid droplet biology and mitochondrial biogenesis / metabolism are linked through shared molecular signals   and increasing intramyocellular triacylglycerol flux is proposed to enhance mitochondrial biogenesis , most likely via peroxisome proliferator - activated receptor ( ppar ) mediated transcriptional reprogramming .
oxygen consumption rate was not different under basal or maximally stimulated conditions in plin5 vs. plin5 myotubes ( figure  4a  d ) . in line with these in  vitro observations , markers of mitochondrial content and function were conserved in skeletal muscle in  vivo .
the expression of genes related to fatty acid uptake and storage ( cd36 , dgat1 ) , fatty acid  oxidation ( cpt1b , mcad , pdk4 ) and oxidative phosphorylation ( acox , atp synthase , nrf1 , ppargc1a , tfam , ucp2 ) were similar in the mixed quadriceps muscle between genotypes ( figure  4e ) .
in support of these observations , the maximal activities of the mitochondrial enzymes citrate synthase ( cs ) and  - hydroxyacyl coa dehydrogenase ( -had ) were not affected in plin5 mice ( figure  4f ) and the maximal running capacity of plin5 mice was not different from plin5 littermates during the graded exercise test ( figure  4 g ) .
others have suggested that plin5 regulates intracellular lipid fluxes by physically recruiting mitochondria to the ld surface .
we detected no difference in the mitochondria to lipid droplet contact within skeletal muscle of plin5 and plin5 mice using confocal microscopy ( figure  4h , i ) .
skeletal muscle triacylglycerol accumulation is associated with insulin resistance   yet , paradoxically ; decreasing intramyocellular triacylglycerol turnover can improve insulin action .
therefore , we next asked whether plin5 deletion would impact glucose tolerance and insulin action .
glucose tolerance was improved in plin5 mice compared to wild - type mice as indicated by an attenuated blood glucose excursion after glucose administration ( figure  5a ) .
plasma insulin increased ( p  =  0.07 , main effect ) mildly after glucose administration and was not different between genotypes ( p  =  0.26 ) ( figure  5b ) .
this suggests improved glucose effectiveness or enhanced insulin sensitivity in plin5 mice . to assess whole body and tissue - specific insulin action , we performed hyperinsulinemic euglycemic clamps on plin5 and wild - type mice .
mice were clamped at 8.1    0.3  mm glucose , 398    38  pm insulin ( no  differences between genotypes ) and steady - state gir was achieved in both groups ( figure  5c ) .
the glucose infusion rate required to maintain euglycemia was 15% lower in the plin5 mice ( p  =  0.08 ) , indicating a strong trend towards impaired whole - body insulin action ( figure  5d ) .
hepatic glucose production was 41% lower ( p  =  0.08 ) in plin5 vs. plin5 mice during the clamp ( figure  5e ) .
this was associated with reduced expression of the gluconeogenic genes g6pc and pepck in livers from plin5 mice ( figure  5f ) , suggestive of enhanced hepatic insulin sensitivity . the glucose disposal rate ( gdr ) during the clamp
was reduced by 22% in plin5 mice ( figure  5 g ) and this was associated with reduced 2-deoxyglucose uptake into mixed skeletal muscle ( 34% , p  =  0.08 , figure  5h ) and reduced muscle glycogen content after the clamp ( figure  5i ) .
glucose uptake into the heart ( figure  5h ) was not different between genotypes and glucose uptake was reduced in white adipose tissue of plin5 mice ( 40% , figure  5h ) , although this is a quantitatively small contribution to systemic glucose disposal .
taken together , these data show that plin5 mice become insulin resistant due to reduced insulin - stimulated glucose disposal in skeletal muscle and adipose tissue , but maintain insulin sensitivity in the liver .
ceramide was increased by 53% in the skeletal muscle of plin5 compared with plin5 mice , whereas diacylglycerol was not different ( figure  5j ) .
lipidomic analysis of cultured myotubes supported this observation : ceramide ( figure  5k , l ) , dihydro- and hexosyl - ceramides and sphingomyelin ( figure  5l ) were increased in plin5 myotubes , whereas other lipid types such as phospholipids and sterols were mostly unchanged ( figure  5 m ) .
there was no evidence for inflammation ( jnk phosphorylation , degradation of ib , tnf and il-6 mrna expression ) , er stress ( phosphorylated gadd34 and perk ) or oxidative stress ( thiobarbituric acid reactive substances ) in the skeletal muscle of plin5 mice compared with wild - type mice ( data not shown ) . because obesity impacts lipid metabolism and insulin action , we next examined the metabolic phenotype of plin5 mice that were fed a high - fat diet for eight weeks .
high - fat feeding increased body mass ( figure  6a ) and fat mass ( figure  6b ) in mice of both genotypes .
whole - body energy expenditure ( figure  6c ) , rer ( figure  6d ) and daily physical activity ( figure  6e ) were not different between plin5 and plin5 mice .
there was no difference in skeletal muscle fatty acid oxidation ( figure  6f ) or incorporation into triacylglycerol ( figure  6 g ) between genotypes and lipid droplet density , diacylglycerol and ceramide levels were similarly unaffected ( data not shown ) . fasting blood glucose ( figure  6h , p  =  0.07 ) and plasma insulin ( figure  6i ) were not different between phenotypes .
glucose tolerance was markedly better in high - fat fed plin5 compared with plin5 mice ( figure  6j ) .
indeed , high - fat fed plin5 mice had comparable glucose tolerance to low - fat fed plin5 mice ( figure  6k ) .
differences in plasma insulin levels did not explain the improved glucose tolerance ( figure  6l ) .
neither whole body deoxyglucose clearance ( p  =  0.30 ) nor tissue - specific clearance into the key sites of glucose disposal was different between genotypes ( figure  6 m , n ) , suggesting insulin - independent mechanisms may be responsible for the improved glucose tolerance i.e. glucose effectiveness ( mass action of glucose ) .
lipid metabolism and insulin action are intimately linked   and lipid droplets stored within tissues are critical for controlling intracellular lipid flux . in this regard ,
the highly conserved perilipin family of proteins reside on the surface of lipid droplets and are important for lipid droplet formation , lipolytic regulation during times of energetic demand and protection against lipotoxicity when cells are exposed to an excess of fatty acids .
plin5 is unique among this family because it is highly expressed in oxidative , glucoregulatory tissues such as skeletal muscle , is localized to multiple intracellular locations including the lipid droplet , endoplasmic reticulum , mitochondria and the cytosol   and may regulate a dynamic interaction between the lipid droplet and mitochondria that is proposed to be relevant to the etiology and treatment of insulin resistance .
uncertainty surrounds the metabolic roles of plin5 ( discussed below ) , partly because most cell systems used to date poorly reflect the metabolic requirements of the major oxidative tissues in  vivo . to address this question
, we generated plin5 mice and examined lipid metabolism and insulin action in skeletal muscle , a major site for fatty acid and glucose disposal .
the controversy surrounding plin5 's involvement in metabolism arises from the apparently conflicting observations in immortalized cell lines that plin5 promotes both fatty acid oxidation and storage   and either increases or decreases lipolysis .
the prevailing views with respect to lipolysis are : ( 1 ) atgl and cgi-58 interact separately with plin5 on lipid droplets to facilitate their interaction and drive lipolysis ; or ( 2 ) plin5 and cgi-58 independently recruit atgl to lipid droplets with opposite effects , cgi-58-atgl interactions drive lipolysis while plin5 binding to atgl inhibits lipolysis . in the current study , we show that plin5 does not influence fatty acid transport into myotubes , fatty acid oxidation or fatty acid esterification into glycerolipids .
however , plin5 ablation increases intramyocellular lipolysis and invokes a remarkable decrease in muscle lipid droplet content in fasted , but not fed mice .
hence , our data agree with the interpretation that plin5 provides a barrier function on lipid droplets to prevent excessive lipolysis .
these novel findings in skeletal muscle mostly agree with the previously described role of plin5 in the heart .
plin5 ablation reduced lipid droplet size , which was due to increased atgl - mediated tag lipolysis and , independently , an increased capacity for fa oxidation in cardiomyocytes . on the other hand , cardiac - specific overexpression of plin5 inhibits atgl - mediated lipolysis resulting in increased lipid droplet size , myocardial steatosis , mild impairments in oxidative metabolism and modest cardiac dysfunction .
together , these studies support the view that plin5 is a highly abundant protein in muscle that is required to repress intramyocellular lipolysis .
this effect is most prominent during prolonged nutrient deprivation , where fatty acid substrate from both intramyocellular and adipose tissue triacylglycerol is enhanced .
hence , plin5 may protect myocytes from unnecessary depletion of intramyocellular fuels and/or control intracellular fatty acid flux to prevent lipotoxic outcomes .
mitochondrial dysfunction in skeletal muscle is proposed to contribute to reduced fatty acid oxidation , lipid deposition and the development of insulin resistance in obese individuals .
indeed , such observations have provided the impetus for developing therapeutics aimed at enhancing mitochondrial function and fatty acid oxidation to treat obesity ( e.g. acetyl coa carboxylase inhibitors , ampk activators ) . while this rationale is probably oversimplified
, emerging evidence indicates that increasing fatty acid flux from lipid droplets can induce mitochondrial biogenesis , most likely via a ppar - mediated program , and in turn improve cell function [ 4345,58 ] . while we demonstrated that fatty acid oxidation from intramyocellular lipid was increased with plin5 deletion , mitochondrial content and function
were not enhanced in either isolated myotubes or intact skeletal muscle ; exogenous fatty acid and glucose oxidation were not enhanced in  vitro and whole - body fatty acid oxidation was actually reduced by 11% in plin5 mice . the brain and skeletal muscle account for 45% of the daily energy expenditure ,
therefore in the absence of increased glucose oxidation in these tissues , other tissues are most likely accounting for the decrease in systemic fatty acid oxidation and reciprocal increase in glucose oxidation .
while the effects of plin5 overexpression in skeletal muscle are equivocal , overexpression of plin5 in cardiac muscle reduces intracellular lipolysis and is associated with reduced mrna contents of oxidative phosphorylation proteins , decreased mitochondrial enzyme activities and mildly impaired mitochondrial respiration . aside from tissue specific actions , the reasons for this apparent discrepancy are unclear .
it is possible that the increase in intramyocellular lipolysis with plin5 deletion was insufficient to drive a ppar transcriptional program .
this possibility is supported by the similarity in pgc1 mrna and pgc1 target genes between plin5 and control mice shown here , and further supported by a recent report in muscle - specific atgl null and overexpressing mice showing a dissociation of intramyocellular lipolysis , mitochondrial biogenesis and fatty acid oxidation . alternatively , plin5 may modulate the cells molecular program via direct transcription / co - activator functions , which may be functionally redundant in plin5 mice . while intramyocellular triacylglycerol accumulation is linked to insulin resistance , emerging data now link the etiology of insulin resistance to the uncoupling of intramyocellular lipolysis from the cellular demand for fatty acids . in this context
, we proposed that the increase in muscle lipolysis combined with no additional demand for fatty acid substrate would reduce insulin sensitivity in plin5 mice .
indeed , our results show that plin5 mice have reduced skeletal muscle insulin sensitivity , as demonstrated during hyperinsulinemic euglycemic clamps .
this was associated with ceramide accumulation , which is a negative regulator of insulin signal transduction .
other complex ceramides and sphingomyelin were increased , while phospholipids , sterol lipids and diacylglycerol were unchanged with plin5 deletion . hence , fatty acids derived from accelerated intracellular lipolysis appear to be preferentially directed into sphingolipids , which in turn cause insulin resistance .
this interpretation is consistent with a recent human study demonstrating the inverse : increased plin5 content , decreased muscle ceramide ( not diacylglycerol ) and improved insulin sensitivity in the skeletal muscle of endurance trained compared with obese men .
the reduction in skeletal muscle insulin sensitivity in plin5 mice is unlikely to be due to impaired blood flow and insulin delivery , secondary to impaired cardiac function , because impairments in the cardiac function of plin5 mice is not apparent until 3038 weeks of age , and we assessed insulin sensitivity in mice aged 16 weeks when cardiac function is normal .
in contrast to muscle , both hepatic glucose production and the insulin sensitive gluconeogenic gene expression were lower in plin5 mice during the insulin clamp conditions , suggestive of improved hepatic insulin sensitivity .
moreover , plin5 mice have normal fasting glucose and insulin and improved glucose tolerance compared with wild - type mice following glucose administration .
this highlights the important role of liver in maintaining glycemia under basal and post - prandial conditions ( i.e. gtt )   and suggests that the improved hepatic insulin action compensates for the reduced peripheral insulin sensitivity in plin5 mice .
it is important to note that progressive declines in muscle insulin sensitivity have no impact on glucose tolerance in c57bl/6 mice   and that glucose effectiveness is quantitatively more important than insulin sensitivity in the determination of glucose tolerance .
the contribution of glucose effectiveness over insulin sensitivity is more pronounced with intraperitoneal glucose administration , which bypasses the gut and precludes an incretin effect , thus resulting in minimal insulin secretion .
the uncoupling of muscle and liver insulin sensitivity is not without precedence either ; plin1 mice present with a very similar phenotype , although the factors governing these responses in each genotype are unlikely to be conserved given the unique tissue expression patterns of plin1 ( adipose ) and plin5 ( muscle , liver ) .
understanding the cell - specific mechanisms that mediate the dichotomy in insulin action is beyond the scope of these studies and future work will address this intriguing question .
plin5 plays a protective role in regulating lipid homeostasis in several tissues [ 2022 ] , including skeletal muscle , and plin5 abundance is a predictor of insulin sensitivity in humans .
we tested whether plin5 deletion would exacerbate the insulin resistance induced by high - fat feeding , through an inability to appropriately sequester lipids into lipid droplets .
energy balance , substrate partitioning and muscle lipid metabolism and storage were mostly unperturbed in plin5 compared with plin5 mice with high fat feeding . despite these similarities , plin5 mice had lower blood glucose and enhanced glucose tolerance , which was not explained by hyperinsulinemia or enhanced insulin sensitivity in peripheral tissues .
thus , while plin5 acts to sequester triacylglycerol in the lipid droplet and prevent excessive lipolysis under standard dietary conditions , the substantial increase in lipid flux associated with high - fat feeding overwhelms the  capacity to store incoming fatty acid as triacylglycerol , resulting in  an increase in lipotoxic intermediates . under these conditions , the  contribution of plin5 deletion and intramyocellular lipolysis to  sphingolipid accumulation is likely to be negligible , and insulin  resistance ensues irrespective of plin5 deletion .
interestingly , plin5 mice are protected from high - fat feeding induced glucose intolerance , suggesting that blocking plin5 may be a mechanism to improve hepatic glucose metabolism . though at this point we can not define the precise mechanisms mediating this improvement , future studies in mice with liver - specific ablation of plin5 should help delineate how this occurs . taken together , our studies show that plin5 suppresses skeletal muscle lipolysis , particularly during prolonged nutrient deprivation , does not influence mitochondrial reprogramming and is required for the maintenance of insulin sensitivity in skeletal muscle by preventing ceramide production .
others have shown that plin5 expression is increased by ppar agonists , fasting , fatty acids and endurance exercise training ; situations characterized by increased lipid flux in skeletal muscle .
hence , our data support the hypothesis that plin5 is required to match lipolysis of intramyocellular triacylglycerol to metabolic demands , which helps to maintain insulin sensitivity in skeletal muscle .
 defective control of lipid metabolism leading to lipotoxicity causes insulin resistance in skeletal muscle , a major factor leading to diabetes . here , we demonstrate that perilipin ( plin ) 5 is required to couple intramyocellular triacylglycerol lipolysis with the metabolic demand for fatty acids . 
 plin5 ablation depleted triacylglycerol stores but increased sphingolipids including ceramide , hydroxylceramides and sphingomyelin . 
 we generated perilipin 5 ( plin5)/ mice to determine the functional significance of plin5 in metabolic control and insulin action . 
 loss of plin5 had no effect on body weight , feeding or adiposity but increased whole - body carbohydrate oxidation . 
 plin5/ mice developed skeletal muscle insulin resistance , which was associated with ceramide accumulation . 
 liver insulin sensitivity was improved in plin5/ mice , indicating tissue - specific effects of plin5 on insulin action . 
 we conclude that plin5 plays a critical role in coordinating skeletal muscle triacylglycerol metabolism , which impacts sphingolipid metabolism , and is requisite for the maintenance of skeletal muscle insulin action . 
Introduction
Methods
Results
Discussion
Author contributions