PPARγ coactivator-1α contributes to exercise-induced regulation of intramuscular lipid droplet programming in mice and humans.

Intramuscular accumulation of triacylglycerol, in the form of lipid droplets (LD), has gained widespread attention as a hallmark of metabolic disease and insulin resistance. Paradoxically, LDs also amass in muscles of highly trained endurance athletes who are exquisitely insulin sensitive. Understanding the molecular mechanisms that mediate the expansion and appropriate metabolic control of LDs in the context of habitual physical activity could lead to new therapeutic opportunities. Herein, we show that acute exercise elicits robust upregulation of a broad program of genes involved in regulating LD assembly, morphology, localization, and mobilization. Prominent among these was perilipin-5, a scaffolding protein that affects the spatial and metabolic interactions between LD and their surrounding mitochondrial reticulum. Studies in transgenic mice and primary human skeletal myocytes established a key role for the exercise-responsive transcriptional coactivator PGC-1α in coordinating intramuscular LD programming with mitochondrial remodeling. Moreover, translational studies comparing physically active versus inactive humans identified a remarkably strong association between expression of intramuscular LD genes and enhanced insulin action in exercise-trained subjects. These results reveal an intimate molecular connection between intramuscular LD biology and mitochondrial metabolism that could prove relevant to the etiology and treatment of insulin resistance and other disorders of lipid imbalance.


Acute exercise, gene expression, and Western blotting (mouse)
Twelve-week-old male C57/Bl6j mice (n = 4) were habituated to the treadmill (3M/6M Columbus Instruments) for three days. Exercise began at 0900 h after food withdrawal at 0800 h. After a warm-up at 5 m/min, speed was increased to 10 m/min for 20 min, and then 1 m/min to a fi nal rate of 15 m/min, which was maintained for 1 h. Running was encouraged by gentle shock. Mice were anesthetized (100 mg/kg Nembutal) immediately postexercise or after 3 or 24 h of recovery. Control and MCK-PGC-1 ␣ transgenic mice were habituated but not exercised. TA muscles were excised and fl ash frozen. RT-PCR was performed using specifi c TaqMan assays (Applied Biosystems) on a Prism 7000. Western blot analysis ( 10 ) used the following antibodies: anti-PGC-1 ␣ (Chemicon or Calbiochem), anti-MLDP (Plin5/ OxPAT; American Research Products), or anti-ADRP (Plin2; Fitzgerald). Blots were normalized to total protein determined by Memcode Reversible Protein Stain (Thermo-Fisher Scientifi c).

Lipid analyses
Analysis of glycerolipid lipid synthesis in cultured cells and mouse muscle were as previously described ( 9,11 ). Quantitative analysis of total lipids and fatty acid composition in mouse TA muscles was performed using 10-20 mg of fl ash-frozen powdered tissue diluted 20-fold and homogenized in methanol. Diacylglycerols (DAG) and triacylglycerols (TAG) were extracted in methanol-chloroform and separated by thin-layer chromatography before analysis of the esterifi ed fatty acids by GC/MS ( 12 ).

Human skeletal muscle cells
Human skeletal myocytes were isolated, expanded, and differentiated as detailed in Ref. 11 . On differentiation day 4, cells were treated for 24 h with viruses encoding either ␤ -galactosidase or PGC-1 ␣ virus. Experimental treatments are described in the fi gure legends. During the pulse-chase experiments, 250 µl of medium was transferred to septum-sealed tubes and acidifi ed for assessment of oxidation products ( 14 CO 2 and 14 C-ASM) ( 11 ). Total RNA was isolated using Qiagen RNeasy kits and gene expression was evaluated using TaqMan RT-PCR. Fluorometric analysis of neutral lipid was performed on myotubes incubated with Adi-poRed as per the manufacturer's instructions (Cambrex). Recombinant adenoviruses encoding mouse PGC-1 ␣ and mouse perilipin 1 were kind gifts from Drs. Bruce Spiegelman and Andrew Greenberg , respectively. Adenoviruses were amplifi ed and purifi ed using published methods ( 13 ).

Human studies
Procedures were approved by the Institutional Medical Ethical Committee of Maastricht University. After providing informed written consent, a cohort of 19 lean, young endurance-trained (n = 9) and untrained (n = 10) male volunteers underwent physical examination and physiological assessment. Body composition and insulin sensitivity were evaluated by hydrostatic weighing and a 6 h euglycemic-hyperinsulinemic clamp, respectively. Trained subjects participated in endurance exercises three times a week for at least two consecutive years prior to study participation and had a VO 2 max above 55 ml kg Ϫ 1 min Ϫ 1 . Untrained subjects had a sedentary lifestyle and a VO 2 max below 45 ml kg Ϫ 1 min Ϫ 1 . Subjects were asked to refrain from exercise the last 48 h prior to the testing, and dietary intake was maintained during the last three days before each clamp with a standardized meal provided on the last day. Power calculations to determine subject numbers were correlates inversely with muscle insulin sensitivity and serves as a strong predictor of diabetes risk (3)(4)(5). It is noteworthy, however, that intramuscular lipid accumulation is not strictly a pathologic phenomenon. For example, lipid content is elevated in red compared with white skeletal muscles and increases in response to habitual exercise in both oxidative and glycolytic fi bers ( 6 ). Paradoxically, endurance-trained athletes have elevated IMTG content, in some cases exceeding that measured in type 2 diabetic subjects ( 3,6 ), but they retain exquisite insulin sensitivity. Despite decades of intense interest in skeletal muscle lipid metabolism, the precise molecular mechanisms that link oxidative fi ber type and contractile activity to increased IMTG content and enhanced control of lipid droplet metabolism have remained relatively understudied. Given the intimate spatial and functional relationship between muscle lipid droplets and the surrounding mitochondrial reticulum (shown herein and elsewhere) ( 7 ), we hypothesized that myocellular genesis of these two interconnected organelles might be regulated by a common molecular switch. We show here that exercise-induced activation of the mitochondrial program in skeletal muscle is indeed paralleled by transcriptional induction of a large set of genes that controls lipid droplet assembly as well as fatty acid fl ux in and out of the IMTG pool. A similar set of lipid droplet genes is likewise regulated by PPAR ␥ coactivator-1 ␣ (PGC-1 ␣ ), an exercise-responsive transcriptional coactivator known to play a major role in adaptive remodeling of muscle mitochondria. In aggregate, results of this study establish a novel role for PGC-1 ␣ in regulating skeletal muscle storage and turnover of IMTG, which in turn appears to impact mitochondrial fuel selection at multiple levels.

Animals
Studies were conducted in accordance with Duke University Institutional Animal Care and Use Committee using male and female C57/Bl6J mice or MCK-PGC-1 ␣ transgenic mice (kindly provided by Dr. Bruce Spiegelman , Harvard University) and littermate controls. Mice were housed in a temperature-controlled environment with a 12:12 h light/dark cycle and provided ad libitum access to Purina standard chow (SC) or high-fat diet (HF; Research Diets 45% fat) and water.

Transmission electron microscopy
Tibialis anterior (TA) muscles from female C57/Bl6J mice on either SC or HF diets and MCK-PGC-1 ␣ transgenic mice were removed in the fed state after Nembutal, immersed in 2% formaldehyde/2.5% glutaraldehyde, and processed using standard methods ( 8 ) at the Translational Core Facility at UNC-Chapel Hill.

Muscle incubations
Fatty acid oxidation and incorporation into glycerol lipids in mouse soleus and extensor digitorum longus (EDL) muscles was assessed as described ( 9 ). A similar buffer containing 5.5 mM glucose (2 µCi per ml [U- ( Fig. 1A , B ). This microstructural organization was maintained and even exaggerated in mouse TA muscles after several weeks of high-fat feeding ( Fig. 1C, D ). These images underscore the potential of muscle lipid droplets to modulate mitochondrial access to fatty acid fuel and to provide substrate during exercise ( 17 ). To assess the dynamic nature of IMTG in in the absence of contractile activity and/or other lipolytic stimuli, we performed a pulse-chase experiment by exposing isolated mouse soleus and EDL muscles to [1][2][3][4][5][6][7][8][9][10][11][12][13][14] C]oleate for 1 h, followed by a second hour of incubation without radioactive substrate. Predictably, rates of fatty acid incorporation into triacylglycerol was ‫ف‬ 3-fold greater in soleus than in EDL. During the chase period, [ 14 C-oleate]labeling of the IMTG pool decreased 43% and 52% in soleus and EDL, respectively, indicating a high turnover rate under these conditions ( Fig. 1E ). Recovery of 14 CO 2 , refl ecting oxidation of IMTG-derived fatty acids, was 1.75-fold greater in soleus compared with EDL, thus matching the relative abundance of TAG in these tissues ( Fig. 1F ). A second experiment sought to evaluate use of naturally deposited IMTG. To this end, we measured oxidation of [UL- 14 C]glucose in isolated muscles treated with 0 or 100 µM etomoxir, a potent inhibitor of carnitine palmitoyltransferase 1 and long-chain fatty acid oxidation ( Fig. 1G ). Exogenously supplied fatty acids were omitted from the incubation buffer; thus, effects of the inhibitor were attributed to metabolism of endogenous lipid. Addition of etomoxir increased rates of glucose oxidation 40% and 70% in soleus and EDL, respectively, implying that IMTG-derived fatty acids compete with glucose for use as an oxidative substrate ( 18 ), even in quiescent muscles from lean animals on a low-fat diet.
We next evaluated TA muscles from MCK-PGC-1 ␣ transgenic mice, which overexpress PGC-1 ␣ specifically in calculated based on the change in insulin sensitivity as measured by the euglycemic-hyperinsulinemic clamp.

Euglycemic-hyperinsulinemic clamp
A constant infusion of glucose tracer ([6,6-2H2] glucose) was initiated at t = Ϫ 60 min to determine insulin-stimulated glucose disposal in overnight-fasted subjects. At t = 0, the actual clamp procedure was started with a constant coinfusion of insulin (40 mU m Ϫ 2 min Ϫ 1 ) and glycerol combined with the infusion of a variable amount of a 20% glucose solution to maintain euglycemia at 5 mmol/l. In the basal period (t = Ϫ 30 to 0) and under steadystate clamp conditions (t = 330-360), blood was sampled and indirect calorimetry (ventilated hood) was performed. Biopsies from the vastus lateralis were obtained under local anesthesia (2% lidocaine) using the Bergstrom technique before and after the clamp. Subjects were instructed to keep a constant eating pattern and refrain from physical activity 48 h prior to the clamp. Nonoxidative glucose disposal (NOGD) was calculated as Rd minus carbohydrate oxidation. Metabolic fl exibility ( ⌬ RQ) was expressed as the change in respiratory quotient from the fasted state to the insulin-stimulated condition.

Human muscle biochemistry
Fresh cryosections (5 m) of skeletal muscle tissue were stained for intramyocellular lipid (IMCL) content by Oil Red O staining combined with fi ber-typing and immunolabeling of the basal membrane marker laminin to allow quantifi cation of IMCL as previously described ( 14 ). Total RNA was isolated from ‫ف‬ 20 mg of skeletal muscle tissue using acid phenol extraction and ethanol precipitation ( 15 ). Primers and probes were designed using Primer Express version 2.1 (Applied Biosystems), and sequences can be provided upon request. Real-time qRT-PCR ( 16 ) was performed as one-step reactions in Prism 7900 (Applied Biosystems) with GAPDH used as internal control. Tissues homogenates were prepared in RIPA-buffer [1% NP40, 0.5% SDS, 1 mM PMSF, 1 mm EDTA, Complete Inhibitor (Roche Diagnostics) in PBS, pH 7.4], and the 15,000 g supernatants were stored at Ϫ 80C before Western blot analysis using the following antibodies: ATGL (Cell Signaling Technology), CGI-58 (Novus Biologicals), PLIN2 (Progen Biotechnik), and PLIN5 (Progen Biotechnik). Protein contents were determined by immunoblotting with near-infrared detection (Odyssey, Licor). To adjust for intergel variation, the optical density of the band of interest per subject was normalized to the mean optical density of the complete gel.

Statistics
Statistical analyses were performed using JMP version 8.0 (SAS). Differences between groups were detected by unpaired ttests. A two-way ANOVA model for repeated measures was applied using trained and untrained as between-subject variables and basal and insulin-stimulated data as repeated within-subject variables. Gene expression and clinical data were correlated using linear regression analysis. To investigate the infl uence of training on insulin sensitivity and metabolic fl exibility, stepwise linear regression analyses for trained and untrained subjects were performed separately. Type I error rate was set a priori at P < 0.05.

RESULTS
Shown by transmission electron microscopy, lipid droplets are larger and more numerous in red compared with white regions of mouse quadriceps, and in both fi ber types the droplets are bordered by intermyofi brillar mitochondria overexpress mouse PGC-1 ␣ in primary human skeletal myotubes (HSkMC). rAd-mPGC-1 ␣ elicited a dose-dependent increase in protein abundance compared with low expression levels in HSkMC treated with the rAd-␤ -gal control virus ( Fig. 4A ). For subsequent experiments, a viral dose of 2.6 × 10 8 PFU/cm 2 was used to mimic the modest increase in muscle PGC-1 ␣ protein content achieved by exercise training ( 10,27 ). In addition to a classic panel of mitochondrial oxidative genes ( 10,27 ), rAd-mPGC-1 ␣ activated several mRNAs encoding proteins involved in lipid droplet assembly and metabolism ( Fig. 4B ). Likewise, staining with AdipoRed, a neutral lipid adherent fl uorometric dye, revealed higher lipid content in rAd-mPGC-1 ␣ -treated cells ( Fig. 4C ).
The impact of PGC-1 ␣ on myocyte lipid synthesis and turnover was evaluated using the pulse-chase protocol illustrated in Fig. 4D . HSkMC pretreated with ␤ -gal or PGC-1 ␣ adenovirus were exposed to [1-14 C]oleic acid for 24 h. During the pulse phase of the experiment, L-carnitine was omitted from the culture medium to disallow fat oxidation and limit confounding effects of enhanced oxidative capacity in the rAd-mPGC-1 ␣ group. Carnitine was added to chase medium to permit normal use of lipid substrate during this period. Analysis of cellular glycerolipids by TLC and autoradiography revealed a clear PGC-1 ␣ effect on labeling and turnover of TAG ( Fig. 4E, F ). During the 24 h pulse, [1-14 C]oleate incorporation into TAG was 1.5-fold greater in HSkMC treated with rAd-mPGC-1 ␣ compared with the control condition ( Fig. 4F ); whereas labeling of muscle ( 24 ). Similar to the exercise response, the MCK-PGC-1 ␣ mice had 5-fold higher mRNA levels of PGC-1 ␣ as well as a large subset of the lipid droplet genes compared with their nontransgenic (NT) counterparts ( Fig. 2C ). Interestingly, Plin5/OxPAT, which belongs to a family of lipid coat proteins ( 25 ), was the only perilipin member for which mRNA levels were upregulated in the MCK-PGC-1 ␣ mice. Western blot analyses confi rmed robust inductions of Plin5/OxPAT and ATGL protein expression in the transgenic mice (5.7-and 3-fold, respectively; Fig. 2D ). Despite no change in mRNA levels of Plin2/Adrp, abundance of this lipid droplet protein was increased 3.3-fold in the transgenic muscles, suggesting posttranscriptional regulation as previously described ( 26 ). Electron microscopy revealed a PGC-1 ␣ -associated increase in lipid droplet size and number ( Fig. 3A ), which was subsequently confi rmed by quantitative biochemical analysis of the contralateral TA muscles. PGC-1 ␣ overexpression increased total TAG and DAG content by 2.4-and 2.2-fold respectively ( Fig. 3B ), and it shifted the fatty acid composition of these glycerolipids toward an enrichment of the unsaturated 18:1 and 18:2 species ( Fig. 3C-F ). In aggregate, these data show that acute exercise activates the lipid droplet program, and they support a role for PGC-1 ␣ in mediating this response.
To determine whether PGC-1 ␣ -induced regulation of muscle lipid droplet metabolism occurs in a cell-autonomous manner independent of developmental and/or environmental factors, we used recombinant adenovirus to  strategies to perturb fatty acid traffi cking through the lipid droplet. Our initial approach exploited the fi rst-identifi ed member of the PAT-family, perilipin 1 (Plin1), an adipocyte-specifi c protein that promotes lipid droplet formation and antagonizes TAG hydrolysis in the absence of lipolytic stimuli ( 31,32 ). As expected, ectopic overexpression of Plin1 in HSkMC resulted in the sequestering of fatty acids in the lipid droplet compartment, evidenced by increased AdipoRed staining ( Fig. 5A ) and diminished glycerol release ( Fig. 5B ). Plin1-mediated disruption of lipid droplet turnover dampened HSkMC transcript levels of several classic targets of the peroxisome proliferator-activated receptor (PPAR) family of nuclear receptors, despite ample provision of PPAR-activating fatty acids ( Fig. 5C ). Likewise, treatment of HSkMC with the pharmacological lipase inhibitor diethylumbeliferyl phosphate (DEUP) ( 33 ) decreased mRNA expression of the same panel of PPAR-targeted genes ( Fig. 5D ). Thus, PGC-1 ␣ -mediated regulation of lipid droplet metabolism might contribute to adaptive remodeling of muscle mitochondria ( Fig. 5E ). DAG and phospholipid pools was either unchanged or only minimally affected ( Fig. 4G, H ). PGC-1 ␣ overexpression augmented loss of label from the TAG pool during the chase period, which corresponded with enhanced oxidation of endogenously-derived [1-14 C]fatty acid ( Fig. 4I ). Interestingly, glycerol release was lower in the rAd-mPGC-1 ␣ group at time 0 (pulse phase) and at 24 h, suggesting decreased lipolysis at those time points ( Fig. 4J ). We therefore surmised that under basal conditions (absence of lipolytic stimuli), PGC-1 ␣ overexpression might encourage myocyte reesterifi cation of glycerol and/or partially hydrolyzed TAG. Fitting with this presumption, rAd-mPGC-1 ␣ increased HSkMC mRNA expression of DGAT1 and glycerol kinase, both of which promote reesterifi cation and were likewise upregulated in MCK-PGC-1 ␣ transgenic mice ( Fig. 2B ).
Lastly, we sought to explore potential relationships between muscle expression of lipid droplet genes and To assess the translational relevance of the foregoing animal and cell-based studies, we next examined cross-sectional differences in expression of lipid droplet-associated proteins in muscle specimens biopsied from healthy young male subjects who were selected based on their levels of habitual physical activity. Detailed clinical and physiological characteristics of the study participants are provided in Table 1 . As expected, increased fi tness (VO 2 max) in the trained compared with untrained subjects (61.5 ± 3.7 versus 43.0 ± 3.6 ml/kg/min, P < 0.01) was accompanied by elevated IMTG content, evident in both type I and type II myofi bers ( Fig. 6A ), as well as a modest rise in muscle mRNA abundance of PGC-1 ␣ ( P = 0.059) and several genes evidenced by a 29% higher insulin-stimulated whole-body glucose disposal (Rd), which was principally attributable to oxidative glucose metabolism ( Fig. 6D, E ). Thus, the change in glucose oxidation in response to the clamp was 2-fold greater in trained than in untrained subjects whole-body glucose homeostasis as assessed by a hyperinsulinemic-euglycemic clamp. In the basal condition, energy expenditure, substrate oxidation rates (glucose and lipid), and respiratory quotient (RQ) were similar between groups. Insulin sensitivity was elevated in the trained subjects,  activation of the de novo lipogenesis pathway ( 41 ). Herein, we provide novel evidence that PGC-1 ␣ targets a number of genes involved in lipid droplet assembly and mobilization. Accordingly, heightened activity of PGC-1 ␣ in mouse TA muscles resulted in increased IMTG and DAG content, even when animals were fed a standard (low-fat) chow diet. Importantly, we also identifi ed a positive association between exercise training, PGC-1 ␣ abundance, and expression of lipid droplet genes in human skeletal muscle. When results of the animal, cell-based, and human studies are taken together, transcriptional regulation of the lipid droplet program was clearly manifest as functional changes in lipid droplet synthesis, turnover, and enhanced oxidation of TAG-derived fatty acids. Also noteworthy is that our experiments revealed a subset of lipid droplet-related genes that were upregulated by acute exercise but unchanged in muscles from the MCK-PGC1a transgenic mice. Thus, it appears that regulatory signals other than PGC-1 ␣ activation are required for the full effect of exercise on the lipid droplet program. Lipid droplets have gained increasing recognition as bona fi de organelles that participate in a diverse array of cellular functions. These particles are decorated and regulated by a complex and dynamic network of lipid droplet coat proteins that orchestrate droplet assembly, size, localization, and mobilization ( 42 ). Some of these proteins are permanent residents, whereas others appear to associate with the droplets in a transient manner, depending on the physiological setting. The best-studied members of this class of proteins belong to the PAT/perilipin family, which includes P lin 1, A DRP/Plin2, T IP47/Plin3, Plin 4, and Plin5 ( 43 ). In the present study, Plin5 clearly stood out as the most responsive lipid droplet target gene of PGC-1 ␣ .
Here and in another recent study ( 44 ), PLIN5 protein expression was found to be elevated in muscles of exercisetrained humans. The precise function of this protein remains unclear, but several lines of evidence point to a key role for Plin5 in governing the spatial and metabolic interactions between lipid intramuscular droplets and their surrounding mitochondrial reticulum (45)(46)(47)(48).
The recent spotlight on lipid droplets has led investigators to question whether and how lipid molecules generated during lipolysis are directed toward specifi c metabolic fates and/or signaling pathways. For example, emerging evidence suggests that lipid droplets per se provide an important and perhaps essential source of ligands for the PPAR family of transcription factors (28)(29)(30). Consistent with this paradigm, we found that disrupting IMTG mobilization via genetic or pharmacological maneuvers dampened (11.2 ± 1.5 versus 5.4 ± 1.4 µmol/kg/min, P < 0.05), whereas nonoxidative glucose disposal was similar between groups (31.8 ± 2.8 versus 29.1 ± 2.7 µmol/kg/min, P = 0.50; Fig. 6F ). Notably, the insulin-induced shift in wholebody substrate selection (from fat to glucose oxidation), commonly dubbed "metabolic fl exibility," was much more robust in the trained subjects than in their sedentary counterparts ( ⌬ RQ; 0.14 ± 0.05 versus 0.09 ± 0.06 AU; P < 0.05; Table 1 ).
A combination of univariate and stepwise linear regression analyses was used to examine interactions between fi tness level (VO 2 max), the lipid droplet program, and insulin action assessed by whole-body glucose disposal ( ⌬ Rd) or metabolic fl exibility ( ⌬ RQ) in trained compared with untrained subjects. Univariate factors that correlated signifi cantly ( P < 0.05) with ⌬ Rd (VO 2 max, along with ATGL and PLIN5 mRNA and protein abundance) and ⌬ RQ (VO 2 max, ATGL protein and mRNA expression, and SCD1 mRNA expression) were added to a stepwise linear regression analysis run within each group (trained versus untrained). Because G0S2 regulates ATGL activity ( 23 ) and was robustly induced by exercise in both mice and humans, we also included G0S2 mRNA expression in these models. In trained subjects, dominant predictors of ⌬ Rd included VO 2 max, PLIN5 mRNA and protein expression, along with ATGL and G0S2 mRNA abundance (model R 2 = 0.99, P < 0.05; Table 2 ). Predictors of ⌬ RQ included ATGL, SCD1, and G0S2 mRNA expression (model R 2 = 0.89, P < 0.05; Table 2 ). In the untrained subjects, only G0S2 mRNA levels predicted ⌬ Rd (model R 2 = 0.61, P < 0.05; Table 2 ) and ⌬ RQ (model R 2 = 0.49, P < 0.05; Table 2 ). In aggregate, these results build on evidence that exercise-induced adaptations in IMTG metabolism contribute positively to glucose homeostasis in trained subjects (34)(35)(36)(37). DISCUSSION PGC-1 ␣ is a promiscuous transcriptional coactivator that plays a prominent role in regulating mitochondrial genesis, oxidative metabolism, and muscle adaption to exercise ( 10,24,38,39 ). More recently, investigators suggested that PGC-1 ␣ might also promote intramuscular lipid storage ( 40,41 ). For example, gastrocnemius muscles from MCK-PGC-1 ␣ transgenic mice were found to have elevated TAG and DAG after high-fat feeding ( 39 ), whereas a subsequent report concluded that intramuscular lipid accumulation in these mice is secondary to specifi c when heavy reliance on fatty acid subsides to permit use of the newly available glucose. By contrast, in obese rodents and humans, this switch in metabolic currency fails, thereby refl ecting a state of "metabolic infl exibility" ( 36,(56)(57)(58). The molecular basis of this phenomenon remains murky, but poorly controlled mobilization and reesterification of IMTG-derived fatty acids could conceivably play a role. Accordingly, we speculate that the lipid droplets assembled in response to exercise training allow the muscles to better manage a heavy infl ux of fatty acid fuel and that precisely regulated traffi cking of substrate to and from IMTG contributes to optimal mitochondrial performance and metabolic fl exibility. This interpretation is consistent with our observation that insulin-stimulated substrate switching from fatty acid to glucose oxidation was markedly enhanced in trained subjects, despite elevated IMTG content. Moreover, stepwise regression analyses identifi ed ATGL, PLIN5, and G0S2 as strong predictors of insulin sensitivity and metabolic fl exibility, particularly in the trained subjects, suggesting that appropriate activation and termination of intramuscular lipolysis bears heavily on these traits. Although these correlative results are not defi nitive, they do point toward a new and provocative avenue for future investigations. expression of several PPAR target genes in primary HS-kMC. These fi ndings support a model in which PGC-1 ␣mediated enhancement of IMTG synthesis and turnover serves as a feed-forward mechanism to maintain the mitochondrial machinery that facilitates lipid catabolism ( Fig. 5E ). On a more conspicuous level, IMTG also serve as a potential source of oxidative substrate. Numerous studies have confi rmed the use of IMTG during various modes of exercise (reviewed in Refs. 49,50 ). Interestingly, the besttrained athletes tend to have the highest IMTG content prior to exercise and the greatest depletion at the end of the event ( 51,52 ), implying that training actually enhances muscle reliance on local lipids. By contrast, the extent to which mobilization and oxidation of IMTG occurs during rest is less clear and more controversial. Herein, we used isolated mouse soleus and EDL to show that both muscle types draw on endogenous lipid reserves under quiescent conditions, implying that IMTG also play a role in supporting resting energy expenditure. Whereas our fi ndings provide new insights into the molecular basis for IMTG accumulation in trained muscles, the question of why these lipids do not impinge upon glucose homeostasis in physically active individuals remains unanswered. Although the explanation for this "athlete's paradox" is probably complex at a molecular level, it is tempting to speculate that the lipid droplet proteome resident within trained muscles might permit tighter control over intracellular lipid traffi cking. For example, upregulation of triacylglycerol biosynthetic enzymes might facilitate rapid esterifi cation and/or reesterifi cation of fatty acids that are not needed as oxidative substrates. Conversely, the enhanced lipolytic potential conferred by elevated ATGL expression is likely to boost rates of IMTG mobilization when demand for fatty acid fuel rises. ATGL is a ubiquitously expressed lipase that selectively performs the fi rst step in TAG hydrolysis. Its catalytic activity increases dramatically in the presence of CGI-58, a coactivator that binds to both ATGL and the lipid droplet surface via distinct domains ( 53 ). ATGL also binds PLIN5 and G0S2, both of which modulate lipolysis by altering the interaction between ATGL and CGI-58. For instance, ectopic expression PLIN5 in CHO cells protected against ATGLmediated lipolysis in the basal state but enhanced lipolytic activity under adrenergically stimulated conditions ( 54 ). Another recent report showed that G0S2 acts as a potent insulin-inducible inhibitor of ATGL hydrolase activity, even in the presence of CGI-58 ( 55 ). Thus, the cadre of lipid droplet genes that are induced by exercise and PGC-1 ␣ exerts control over glycerolipid hydrolysis in both directions, possibly conferring enhanced regulatory capacity to rapidly modulate lipid mobilization according to metabolic demand.
In the context of this discussion, it is noteworthy that muscle fuel selection is disrupted in obese and insulin-resistant states ( 36,(56)(57)(58). Under healthy conditions, this tissue harbors robust capacity to adjust substrate preference in response to nutrient and hormonal cues. This versatility is classically evident during the transition between fasting and feeding (herein mimicked by the clamp condition),