Impaired mitochondrial fat oxidation induces adaptive remodeling of muscle metabolism

Shawna E. Wicksa,1, Bolormaa Vandanmagsara,1, Kimberly R. Hayniea, Scott E. Fullerb, Jaycob D. Warfela, Jacqueline M. Stephensb, Miao Wangc, Xianlin Hanc, Jingying Zhangd, Robert C. Nolande, and Randall L. Mynatta,d,2

aGene Nutrient Interactions Laboratory, Pennington Biomedical Research Center, Baton Rouge, LA 70808; bAdipocyte Biology Laboratory, Pennington Biomedical Research Center, Baton Rouge, LA 70808; cSanford–Burnham Medical Research Institute, Orlando, FL 32827; dTransgenic Core Facility, Pennington Biomedical Research Center, Baton Rouge, LA 70808; and eSkeletal Muscle Metabolism Laboratory, Pennington Biomedical Research Center, Baton Rouge, LA 70808

Edited by Michael Roden, University of Düsseldorf, Düsseldorf, Germany, and accepted by the Editorial Board May 12, 2015 (received for review September 26, 2014) The correlations between intramyocellular lipid (IMCL), decreased However, dyslipidemia and insulin resistance have been ob- fatty acid oxidation (FAO), and insulin resistance have led to the served (15, 16). There have been a handful of studies that have hypothesis that impaired FAO causes accumulation of lipotoxic used pharmacological inhibition of Cpt1 to target insulin re- intermediates that inhibit muscle insulin signaling. Using a skeletal sistance. The results are inconsistent and, in some cases, com- muscle-specific carnitine palmitoyltransferase-1 KO model, we pletely contradictory due to species/model differences and methods show that prolonged and severe mitochondrial FAO inhibition of assessing insulin sensitivity (17–23). These studies also lack tissue results in increased carbohydrate utilization, along with reduced specificity and are generally of short duration and/or initiated after physical activity; increased circulating nonesterified fatty acids; and development of insulin resistance/diabetes. The lack of tissue increased IMCLs, diacylglycerols, and ceramides. Perhaps more impor- specificity is most problematic. Decreased hepatic gluconeogenesis tantly, inhibition of mitochondrial FAO also initiates a local, adaptive as a result of liver Cpt1 inhibition may have effects on whole-body response in muscle that invokes mitochondrial biogenesis, compen- insulin response independent of muscle (18, 22). Furthermore, satory peroxisomal fat oxidation, and catabolism. Loss of Cpt1 inhibition is associated with both cardiac hypertrophy and its major fuel source (lipid) induces an energy deprivation response in hepatic steatosis (24–26). There have been attempts to inhibit the muscle coordinated by signaling through AMP-activated protein muscle isoform, Cpt1b (23), specifically; however, long-term risks kinase (AMPK) and peroxisome proliferator-activated receptor gamma α of cardiac hypertrophy and mortality (27) suggest that pharmaco- coactivator 1-alpha (PGC1 ) to maintain energy supply for locomotion logical strategies must target specifically skeletal muscle. and survival. At the whole-body level, these adaptations result in re- Two of the studies have directly examined the impact of Cpt1 sistance to obesity. inhibition on the link between decreased FAO, increased IMCL, and lipotoxicity-induced insulin resistance. Using the muscle- carnitine palmitoyltransferase | muscle | fatty acid | lipid | carbohydrate selective Cpt1b inhibitor, oxfenicine, Keung et al. (23) showed decreased diacylglycerol (DAG) and ceramide levels following onsiderable evidence suggests that when oversupply of di- short-term treatment of obese mice. In contrast, Timmers et al. Cetary fat exceeds the storage capacity of adipose tissue, ec- (21) showed increased IMCL and DAG accumulation follow- “ topic lipids accumulate in skeletal muscle, leading to metabolic ing 8 d of treatment with the nonselective global Cpt1 in- ” stress that induces insulin resistance. One prevailing theory is hibitor, etomoxir. These studies revealed short-term benefits in that impaired skeletal muscle fatty acid oxidation (FAO) (1–4) leads to cytosolic accumulation of lipotoxic intermediates that Significance are directly linked to defects in insulin signaling (5–11). Recent findings counter this premise because they have shown that models of insulin resistance consistently exhibit enhanced (not Many theories regarding the causes of insulin resistance in skel- reduced) FAO, as demonstrated by elevated incomplete β-oxi- etal muscle center on the ability of muscle to oxidize fat, with dation and accumulation of excess lipid-derived acylcarnitines evidence supporting either decreased or increased fatty acid ox- (12, 13). Supporting evidence was obtained using a whole-body idation (FAO) as causal to insulin resistance. Inhibition of fatty genetic approach to elevate levels of the endogenous carnitine acid transport into mitochondria specifically in mouse muscle palmitoyltransferase-1 (Cpt1) inhibitor, malonyl-CoA (12). These results in a rather remarkable phenotype. Despite an accumula- studies have led to the contrasting theory that lipid overload and tion of lipids in muscle, insulin sensitivity is maintained. The incomplete FAO within the mitochondria accelerate the pro- muscle responds to decreased FAO by adapting muscle metabo- gression of insulin resistance (14). Faced with a plethora of studies lism to use other fuel sources, and by an increased reliance upon supporting both hypotheses, a crucial question remains: “Is in- peroxisomal fat oxidation. There is also an increase in mito- hibition of mitochondrial FAO in skeletal muscle sufficient to chondrial biogenesis. At the whole-body level, the mice seem to initiate development of insulin resistance?” enter an energy conservation mode with reduced activity, energy Cpt1 is essential for long-chain acyl-CoA transport into the expenditure, and resistance to diet-induced obesity. mitochondria, and lies at the nexus of both the lipotoxicity and the mitochondrial overload hypotheses. If decreased FAO is a Author contributions: S.E.W., B.V., R.C.N., and R.L.M. designed research; S.E.W., B.V., K.R.H., S.E.F., J.D.W., M.W., X.H., J.Z., R.C.N., and R.L.M. performed research; J.M.S., M.W., root cause of lipotoxicity, then muscle-specific ablation of Cpt1b and X.H. contributed new reagents/analytic tools; S.E.W., B.V., M.W., X.H., R.C.N., and R.L.M. activity should lead to impaired FAO, intramyocellular lipid analyzed data; and S.E.W., B.V., R.C.N., and R.L.M. wrote the paper. (IMCL) accumulation, and insulin resistance. In stark contrast, The authors declare no conflict of interest. the mitochondrial overload hypothesis suggests that decreased This article is a PNAS Direct Submission. M.R. is a guest editor invited by the Editorial Cpt1b activity would preserve insulin sensitivity by preventing Board. unbalanced overfueling of β-oxidation. 1S.E.W. and B.V. contributed equally to this work. Characterization of the rare genetic disorders of Cpt1 and 2To whom correspondence should be addressed. Email: [email protected]. Cpt2 has focused almost exclusively on the functional, rather This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. than metabolic, implications of sustained decrements to FAO. 1073/pnas.1418560112/-/DCSupplemental.

E3300–E3309 | PNAS | Published online June 8, 2015 www.pnas.org/cgi/doi/10.1073/pnas.1418560112 Downloaded by guest on September 23, 2021 treatment of insulin resistance but contradictory conclusions mice reach maximum body weight by 12–13 wk of age (Fig. 1D). PNAS PLUS with respect to the role of lipotoxicity. In combination with the The difference in body mass is mainly attributed to changes in fat studies by Conti et al. (22) using the liver-specific Cpt1 inhibitor, mass. Control mice continue to accrue fat, whereas the fat mass − − teglicar, it is clearly important to understand the detailed mecha- of Cpt1bm / mice is held at postpuberty levels (Fig. 1E). Im- nisms underlying the impact of tissue-specific and isoform-selective portantly, lean mass is similarly maintained at young adult levels − − Cpt1 inhibition to develop therapeutic inhibitors with maximum in Cpt1bm / mice, resulting in a slight difference from control metabolic benefits and minimal side effects. littermates by 20 wk of age (Fig. 1F). On the whole, the impact of prolonged inhibition of fatty acid To determine if food intake was causing the differences in fat mass, transport into skeletal muscle mitochondria has not been stud- food intake and body weight were measured over the period when − − ied. To address this issue directly, we created mice with skeletal the fat mass between the control and Cpt1bm / mice is diverging. − − − − muscle-specific deficiency of Cpt1b (Cpt1bm / ). Body weight and fat mass become significantly less in Cpt1bm / mice compared with control mice (Fig. 2 A and B) well before food − − Results intake becomes significantly less in Cpt1bm / mice compared To determine the effects of impaired skeletal muscle mito- with control mice (Fig. 2C). Fat-free mass does not become sig- − − chondrial FAO, we targeted the muscle-specific isoform, Cpt1b. nificantly less in Cpt1bm / mice compared with control mice until We crossed mice bearing floxed alleles of Cpt1b to Mlc1f-Cre about 100 d of age (Fig. 2D). Overall, these small differences in transgenic mice for specific ablation of Cpt1b in skeletal muscle, daily food intake lead to a cumulative difference in food intake of − − but not cardiac tissue (28). Details of the Cpt1b targeting con- almost −15 g in the Cpt1bm / mice over the 8-wk period (Fig. 2F), − − struct and crossing scheme to generate floxed mice are shown in whereas body weight and fat mass were about 3 g less in Cpt1bm / − − Fig. S1. Cpt1bm / mice have normal levels of Cpt1b expression mice compared with control mice over the 8-wk period (Fig. 2E). in the heart, but there is near-complete knockdown of both To see if there were direct effects in adipose tissue, we mea- expression and Cpt activity in skeletal muscle (Fig. 1 A and B). sured markers of mitochondrial number, lipolysis, β-oxidation, − − Mitochondrial FAO in Cpt1bm / mice is decreased significantly adipocyte beiging/browning, and inflammation at 10 wk of age to levels comparable to control mitochondria treated with the (just before divergence of fat pad mass) and at 16 wk (a few inhibitor etomoxir (Fig. 1C). weeks after divergence of fat pad mass) in inguinal and epidid- ymal fat depots. There were no significant effects observed in the Impairment of Mitochondrial FAO in Muscle Attenuates Development epididymal fat depot. In inguinal adipose tissue, Pnpla2/Atgl − − − − of Adiposity. Cpt1bm / mice and control mice were fed a mod- trended higher in 10-wk-old Cpt1bm / mice but was significantly − − erate fat (25% kcal) breeder chow diet. Cpt1bm / mice are in- higher by 16 wk. The β-oxidation , 3-hydroxyacyl–CoA distinguishable from controls at weaning and continue to gain dehydrogenase, was significantly elevated at 10 and 16 wk in − − − − weight normally until 8–10 wk of age. Unexpectedly, Cpt1bm / Cpt1bm / mice. Citrate synthase (Cs) trended higher in 10-wk-old

AB60 C 80 150 fl/fl fl/fl Cpt1b Cpt1b m-/- m- /- Cpt1b 60 Cpt1b fl/fl 40 100 Cpt1b +M-CoA * * * ** ** ** ** * 40

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Cpt activity Cpt 20 mRNA (A.U.) mRNA * (nmol/mg protein) Palmitate oxidation Palmitate 0 0 0 0 2 4 6 8 10 CO (nmol B E B E Time (min) fl/fl m- /- Heart EDL LiverBrainiWAT Cpt1b Cpt1b GastrocSoleus Kidney

D fl/fl E F fl/fl Cpt1b fl/fl Cpt1b 50 m- /- 20 Cpt1b 30 m- /- Cpt1b m- /- Cpt1b * * Cpt1b * ** * PHYSIOLOGY 40 *** * 15 * * * * * ** * * * * * * * 20 30 * * * 10 * * 20 * FFM (g)FFM 10 5 * 10 Fat mass (g) * Body Mass (g) 0 0 0 5 9 13 17 21 25 29 33 5 9 13 17 21 25 29 33 5 9 13 17 21 25 29 33 Age (w eeks) Age (weeks) Age (weeks)

Fig. 1. Mice with skeletal muscle-specific Cpt1b deficiency have impaired mitochondrial FAO in muscle and decreased whole-body adiposity. (A) Tissue- − − specific deletion in Cpt1bm / mice was confirmed by qRT-PCR analysis of Cpt1b mRNA (n = 5–7). Gastroc, gastrocnemius; iWAT; inguinal white adipose tissue. (B) Skeletal muscle effects were confirmed in isolated mitochondria by Cpt activity assay. Cpt1bm−/− mitochondria were identical to normal mitochondria treated with the Cpt1 inhibitor malonyl-CoA (M-CoA). (C) Impaired FAO was shown in isolated mitochondria using the [1-14C]palmitate-radiolabeled assay. B, basal conditions; E, treated with the Cpt1 inhibitor etomoxir. Ablation of Cpt1b decreases weight gain (D) and fat accumulation (E), with maintenance of lean mass (F)(n = 8–10 per group), in mice on a 25% fat chow diet. *P < 0.05 or **P < 0.01, genotype effect detected by ANOVA for multiple groups or by Student’s t test. Results shown are representative of multiple independent experiments, and are expressed as mean ± SEM.

Wicks et al. PNAS | Published online June 8, 2015 | E3301 Downloaded by guest on September 23, 2021 fl/fl A 30 Cpt1b B fl/fl C 250 m-/- Cpt1b * Cpt1b m-/- ** 28 4 Cpt1b 200 **** ** * ** * * * * ** * *** * ****** * * ** 26 * ** *** 150 *** *** 3 * 24 100 Cpt1bfl/fl Weight (g) Weight Fat Mass (g) 50 m-/- 22 2 (g) Food Intake Cpt1b 20 0 60 70 80 90 100 110 120 50 60 70 80 90 100 110 120 60 70 80 90 100 110 120 Days Days Days DEF 22 fl/fl fl/fl Cpt1b 5 Cpt1b 220 fl/fl Cpt1bm-/- m-/- Cpt1b Cpt1b m-/- 20 4 210 Cpt1b * * * 3 18 * 200

FFM (g) 2 * 16 1 190 Food Intake (g) 14 (g) gain Weight 0 180 50 60 70 80 90 100 110 120 Days

− − Fig. 2. Cumulative food intake is altered by skeletal muscle-specific ablation of Cpt1b. (A) Body weight diverges in Cpt1bm / mice by 70 d of age. Fat mass is significantly less by 63 d of age (B), but cumulative food intake is not significantly reduced until 88 d of age (C). (D) Significant divergence of lean mass does not occur until 100 d of age. Cumulative (56–119 d of age) weight gain (E) and food intake (F) are significantly reduced in Cpt1bm−/− mice. Data are expressed as mean ± SEM (n = 9–10 per group). *P < 0.05 by Student’s t test.

− − Cpt1bm / mice but was significantly higher by 16 wk (Fig. and B). Analysis of covariance (ANCOVA) suggests that the S2A). Ucp-1, Cidea, and Elovl3 were all significantly elevated in genotype effect on energy expenditure results from the reduced − − − − Cpt1bm / mice at 10 and 16 wk (Fig. S2B). TNF-α mRNA levels activity of Cpt1bm / mice (Fig. 3C). In addition, the respiratory − − were not different at 10 wk of age but TNF-α mRNA levels were exchange ratio (RER) is significantly higher in Cpt1bm / mice, − − significantly less in Cpt1bm / mice by 16 wk (Fig. S2C). suggesting increased carbohydrate oxidation (Fig. 3D). Given the up-regulation of markers for FAO and uncoupling in adipose tissue, one might expect increased energy expenditure Cpt1bm−/− Mice Exhibit Moderate Myodegeneration and Impaired − − − − to account for decreased weight gain. However, Cpt1bm / mice Exercise Performance. Given that the Cpt1bm / mice are hypo- have overall reduced energy expenditure and activity (Fig. 3 A active, we checked for muscle damage and detected increased

B A Cpt1bfl/fl 1000 m-/- fl/fl 5500 Cpt1b Cpt1b 800 Cpt1bm-/- 5000 600 4500

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(ml/hr/kg FFM) (ml/hr/kg 200 2 3500 V0 3000 0 D C 0.95 110 Cpt1bfl/fl Cpt1bm-/- 100 0.90

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80 V02= FFM + FM + group VO2 Actual 0.80 group effect p=0.0002 70 V02= FFM + FM + activity + group group effect p=0.1608 60 LLLLDDD 60 70 80 90 100 110 VO2 Predicted P<.0001 RSq=0.63 RMSE=5.6911

Fig. 3. Abrogation of Cpt1b decreases activity but increases carbohydrate use. Indirect calorimetry shows decreased energy expenditure (A) and activity level − − (B) in Cpt1bm / mice (n = 7–8 at 5 mo of age). (C) ANCOVA analysis suggests that the genotype effect on energy expenditure is due to the decreased activity − − − − of Cpt1bm / mice. Red symbols indicate Cpt1bfl/fl, and black symbols indicate Cpt1bm / .(D) RER values are higher, reflecting increased glucose utilization. D,

dark period; FFM, fat-free mass; FM, fat mass; L, light period; VO2, consumption.

E3302 | www.pnas.org/cgi/doi/10.1073/pnas.1418560112 Wicks et al. Downloaded by guest on September 23, 2021 − − PNAS PLUS myoglobin in serum of Cpt1bm / mice (Fig. 4A). H&E and muscle-specific Cpt1b depletion successfully recapitulates a phosphotungstic acid-hematoxylin (PTAH) staining shows multi- model of FAO impairment and lipid accumulation. focal degeneration and loss of cross-striations in some fibers by 3 mo of age (Fig. 4B). Mild functional implications appear to Impaired Muscle Mitochondrial FAO Does Not Inhibit Insulin Response. m−/− manifest as mice age. We initially used rotarod performance, a Fasting insulin and glucose are significantly lower in Cpt1b mice than control mice at 2 mo of age, when there is no difference measure of neuromuscular coordination and muscle function, to m−/− test for impairments, and found no difference in young (12-wk- in body composition between Cpt1b mice and controls (Fig. 6 m−/− A and B). Insulin values in control mice increase with age, but old) Cpt1b mice (Fig. 4C). We next used computerized gait m−/− analysis to probe for more subtle alterations. No differences in gait Cpt1b mice maintain low insulin values with age (Fig. 6A), − − were detected in young Cpt1bm / mice, although mild gait dis- which is in agreement with less body fat. Although plasma NEFA turbanceswereapparentby1yofage(Fig.4D). Despite the and TAGs and intramyocellular DAGs and ceramides are con- decrement in mitochondrial FAO, treadmill-tested endurance is sidered classic hallmarks of insulin resistance, there is no differ- − − only moderately decreased in Cpt1bm / mice at both young (12- ence in response to a bolus of insulin. Insulin tolerance tests (ITTs) show an equivalent insulin response in weight-matched wk-old) and old (1-y-old) ages (Fig. 4E). − − Cpt1bm / and control 10- to 12-wk-old mice (Fig. 6C) and 18- m−/− m−/− Impaired Muscle Mitochondrial FAO Increases Both Systemic and to 20-wk-old Cpt1b mice (Fig. 6D). The older Cpt1b IMCLs. Plasma nonesterified fatty acid (NEFA) and triacylglyc- mice have lower baseline glucose values, but the slopes after erols/triglycerides (TAGs) are significantly elevated, both prior insulin administration are essentially the same. Because results (2–3 mo) and subsequent (12 mo) to weight divergence (Fig. from the ITT studies provide a picture of whole-body glucose handling in response to insulin, we elected to perform studies 5A). Bodipy staining of gastrocnemius reveals marked IMCL − − using isolated muscle strips to test the direct impact of Cpt1b accumulation in Cpt1bm / mice (Fig. 5B), which is visible as deficiency on insulin responsiveness. Consistent with the whole- large lipid droplets by EM (Fig. 5C). We next examined dif- body ITT results, basal and insulin-stimulated glucose uptake in ferent lipid species in soleus and extensor digitorum longus m−/− isolated EDL and soleus muscle from Cpt1b mice was not sig- (EDL) muscle. Intramuscular TAGs are more than tripled in m−/− nificantly different from control mice (Fig. S3). Consistent with the Cpt1b soleus but are not statistically elevated in EDL (Fig. whole-body and isolated muscle results, insulin-stimulated pAkt 5D). Several of the DAG species were significantly higher in m−/− fl/fl − − (Ser473) is similar between Cpt1b and Cpt1b mice after in- Cpt1bm / soleus (Fig. 5E). Likewise, most of the DAG species − − jection of an insulin bolus (Fig. 6E). Likewise, insulin-stimulated were significantly elevated in Cpt1bm / EDL (Fig. 5E). Total − − translocation of Glut4 to the plasma membrane is similar in both m / − − ceramides are approximately doubled in Cpt1b soleus, with genotypes (Fig. 6F). Despite high levels of DAG in Cpt1bm / the C16, C18, and C18:1 species all being significantly increased muscle, membrane localization of protein kinase C theta (PKCΘ) (Fig. 5F), whereas C16 is significantly increased in EDL (Fig. is not significantly increased (Fig. 6G). Taken together, these data − − 5G). Lastly, of fatty acid transport (Cd36, strongly suggest that insulin signaling is intact in Cpt1bm / mice. Fatp1), fatty acid binding (Fabp3), and lipid droplet forming (Plin5) proteins is coordinately increased in all fiber types ex- Cpt1bm−/− Mice Have Increased Carbohydrate Utilization. A consis- amined (soleus, EDL, red quadriceps, and white quadriceps; Fig. tent hallmark of studies that have found beneficial effects of 5H and Table S1). Taken together, we can conclude that skeletal acute Cpt1 inhibition is a natural shift in substrate selection that

A 2-3 month 12 month B Cpt1bfl/fl Cpt1bm-/- Cpt1bfl/fl Cpt1bm-/- 800 800 * * 600 600

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5 Duration (min) Latency to fall (s) 0 0 fl/fl m-/- 0 0 Cpt1bfl/fl Cpt1bm-/- Cpt1b Cpt1b Cpt1bfl/fl Cpt1bm-/- fl/fl m-/- fl/fl m-/- Distance from midline (mm) Cpt1b Cpt1b Cpt1b Cpt1b

− − Fig. 4. Cpt1b depletion causes mild impairment of muscle function. (A) Serum levels of myoglobin are elevated in Cpt1bm / mice (n = 8–12). (B) H&E and PTAH staining of gastrocnemius shows multifocal myodegeneration and loss of cross-striations in some fibers (in 3-mo-old mice). *Sites of myodegeneration. (Scale − − bars: 50 μm.) (C) Rotarod performance is normal in 3-mo-old mice. (D) Computerized gait analysis reveals mild impairment in rear leg extension in 1-y-old Cpt1bm / mice. (E) Treadmill endurance is significantly decreased in Cpt1bm−/− mice. *P < 0.05 by Student’s t test or ANOVA (n = 8–12 for behavioral assays).

Wicks et al. PNAS | Published online June 8, 2015 | E3303 Downloaded by guest on September 23, 2021 ABC D 2.5 60 Cpt1bfl/fl

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1.0 40 20 /- 0.5 TAG (mg/dL) 20 m-/- NEFA (mmol/L) m-

0.0 0 protein) (ng/mg TAG 2-3 12 0 Cpt1b 2-3 12 Cpt1b Soleus EDL Age (month) Age (month) E 1.0 Soleus 0.4 EDL * * fl/fl Cpt1bfl/fl * Cpt1b 0.8 m-/- * Cpt1bm-/- 0.3 * * Cpt1b 0.6 * * 0.2 * 0.4 * 0.1 * * 0.2 * * *

DAG (nmol/mg protein) (nmol/mg DAG 0.0

DAG (nmol/mg protein) 0.0 0 :0 3 0:4 : 16:0 D16:0 D18:2 D18 0-18:3 :0-18:1 1-20:1 :2-20:4 D D18:2 D18:0 2-20:4 8 16:0-18 18:1-18:216:1-20:1 18: 18:1-20:4 D16: D16 D16:0-18: D18:1-18:2 D1 D18:1-2 D18:0-20:4 D D16:0-18:1D16:0-18:0 D D D D18:0-20:4 :1, D16: D18 D18:1, D FG H fl/fl EDL fl/fl 2.5 fl/fl 40 Soleus Cpt1b 200 Cpt1b Cpt1b ** m-/- m-/- Cpt1bm-/- ** Cpt1b Cpt1b 2.0 30 * 150 * 1.5 * 20 100 ** 1.0

10 50 0.5

* ** change) (Fold mRNA 0 0.0 0 Ceramides (ng/mg protein) Ceramides (ng/mg protein) (ng/mg Ceramides C16 C18 C18:1 C24 C24:1 C16 C18 C18:1 C24 C24:1 Cd36 Fatp1 Plin5 Fabp3

Fig. 5. Skeletal muscle-specific Cpt1b ablation alters local and systemic lipid homeostasis. (A) Plasma NEFA and TAG levels are increased in both young and aged Cpt1bm−/− mice (n = 8–12). (B) Bodipy staining of gastrocnemius muscle shows IMCL accumulation (representative images, n = 4). (Scale bar: 100 μm.) (C) EM imaging of soleus shows enlarged lipid droplets. (Scale bar: 10 μm.) (D) IMCL accumulation is accompanied by increased levels of TAGs in soleus, but not in EDL (n = 4 per group). (E) Individual DAG species are also increased in soleus and EDL (n = 8–10 per group). Individual ceramide species are also increased in − − soleus (F) and EDL (G)(n = 4 per group). (H) Expression of fatty acid transport, binding, and lipid droplet proteins is increased in Cpt1bm / muscle. *P < 0.05 or **P < 0.01, differences detected by Student’s t test. Data are shown as mean ± SEM. Plasma was collected from mice that had been fasted for 4 h.

favors glucose utilization. Although we did not observe any im- regulation of energy and lipid metabolism, mitochondrial bio- provements or detriments to insulin-stimulated glucose uptake genesis, and function. Phosphorylation of the α-subunit of − − − − in Cpt1bm / mice, glucose utilization appears to be enhanced. AMPK at Thr-172 is significantly increased in Cpt1bm / − − Blood glucose levels are significantly lower in the Cpt1bm / muscle (Fig. 8A). We found robust increases in expression of mice compared with controls during an i.p. glucose tolerance test the AMPK target peroxisome proliferator-activated receptor in both 10- to 12-wk-old mice and 18- to 20-wk-old mice (Fig. 7 A gamma coactivator 1-alpha (Pgc1α) in both red and white mus- and B). Likewise, we detected increased pyruvate dehydrogenase cles without changes in Pgc1β or PPARα mRNA (Fig. 8B and (PDH) subunit-Pdha1 mRNA expression (Fig. 7C), increased Table S1). PGC1α is known as a master regulator of mitochon- PDH activity (Fig. 7D), and increased complete oxidation of drial biogenesis. Consistent with AMPK/PGC1α activation, EM pyruvate via the tricarboxylic acid (TCA) cycle in muscle ho- in soleus muscle reveals large bands of mitochondria surround- − − mogenates (Fig. 7E). The increased carbohydrate oxidation is ing enlarged lipid droplets in Cpt1bm / mice (Fig. S4A). Mito- further supported at the whole-body level, because the RER is chondria appear larger, with denser cristae. Measurement of − − higher in Cpt1bm / mice (Fig. 3D). Furthermore, glycogen mitochondrial copy number (as the ratio of DNA copy number levels are significantly reduced in red quadriceps but only mod- of the mitochondrial , Cytb and Cox2, to the nuclear genes, erately reduced in white quadriceps (Fig. 7F). Taken together, glucagon and β-globin) shows a 50% increase in mitochondrial − − these data indicate that there is normal insulin action and in- content in Cpt1bm / muscle compared with control mice (Fig. − − creased glycolytic flux in Cpt1bm / mouse muscle. 8C). Additionally, we found a coordinate increase in expression of genes associated with citric acid cycle and oxidative phos- Impaired Mitochondrial FAO Leads to Mitochondrial Biogenesis. To phorylation of mitochondria, including Cs, Ndufs8 (complex I), − − gain further insight into how impaired mitochondrial FAO af- Cox5a (complex IV), and SdhB (complex II) in Cpt1bm / fects skeletal muscle metabolism in a chronic setting, we exam- muscle (Fig. 8D and Table S1). Expression of genes related to ined the activation of the energy sensor AMP-activated protein mitochondrial FAO, such as Hadha, Ech1, Cpt2, and Cact,is − − kinase (AMPK) and mRNA expression of genes involved in the significantly up-regulated in muscle of Cpt1bm / mice compared

E3304 | www.pnas.org/cgi/doi/10.1073/pnas.1418560112 Wicks et al. Downloaded by guest on September 23, 2021 PNAS PLUS AB15 C D 250 250 250 Cpt1bfl/fl 200 m-/- * * ** 200 Cpt1b 200 * 10 * 150 150 ** 150 100 5 100 100 Cpt1bfl/fl m-/-

Insulin (ng/ml) Cpt1b *** (mg/dl) Glucose 50 50 50 Blood glucose (mg/dl) glucose Blood 0 0 (mg/dl) glucose Blood 0 0 2 12 2 12 0 20 40 60 0 20 40 60 Age (months) Age (months) Minutes post insulin Minutes post insulin EF G pAkt (Ser473) Glut4 PKCΘ Akt Caveolin- Caveolin-1 PBS Ins PBS Ins 1 GAPDH Cpt1bfl/fl Cpt1bm-/- PBS Ins PBS Ins Cpt1bfl/fl Cpt1bm-/- m- Cpt1bfl/fl Cpt1b 1.5 1.0 /- 0.8 1.0 2 fl/fl 0.6 fl/fl Cpt1bfl/fl Ins Cpt1b Ins Cpt1b A.U. A.U. /Caveolin-1) Ratio 0.4 m-/- Ratio m-/- m-/- 0.5 θ Cpt1b A.U. 1 Cpt1b Ins Cpt1b Ins Ratio 0.2

(p-Akt/t-Akt) 0 0.0 0.0 (Glut4/Caveolin-1) (PKC

Fig. 6. Depletion of Cpt1b in skeletal muscle does not impair whole-body insulin response or muscle insulin signaling. Blood glucose (A) and insulin levels (B) are decreased in Cpt1bm−/− mice after a 4-h fast. There is no difference in response to an ITT in Cpt1bm−/− mice (n = 8–10) at 10–12 wk (C)or18–20 wk (D). (E) Insulin (Ins)-stimulated phosphorylation of Akt is unchanged in Cpt1bm−/− and Cpt1bfl/fl muscle (n = 4). (F) Similarly, insulin-stimulated translocation of − − Glut4 to the membrane is unaffected by lack of Cpt1b (n = 3). (G) Membrane-associated levels of PKCΘ are not increased in Cpt1bm / mice (n = 4). Data are shown as mean ± SEM. *P < 0.05 or **P < 0.01, Student’s t test. A.U., arbitrary units.

with control mice (Fig. 8E). Additionally, a proteomic analysis of signal promoting mobilization and subsequent utilization of − − muscle of Cpt1bm / mice compared with control mice confirmed amino acids as metabolic fuel. much of the mRNA analysis in Table S1, including a number of − − mitochondrial proteins up-regulated in Cpt1bm / mice (Table 1). Impaired Mitochondrial FAO Leads to Compensatory Peroxisomal Furthermore, upstream pathway analysis predicts activation of FAO. In addition to measuring FAO in mitochondria, we mea- α sured FAO in the whole-muscle homogenate. FAO in whole- PGC1 (Fig. 8F). Importantly, despite lifelong deficiency of the m−/− mitochondrial enzyme Cpt1b, isolated mitochondria respire nor- muscle homogenate is only slightly reduced in Cpt1b muscle mally (Fig. S4B). Collectively, the EM, quantitative RT-PCR (qRT- compared with control muscle (Fig. 9E). Peroxisomes are ca- PCR), mitochondrial gene copy number, proteomics, and mi- pable of processing a variety of long-chain and very-long-chain tochondrial function studies indicate adaptive biogenesis of fatty acids, and tend to be up-regulated in response to elevated − − fatty acids, such as the environment in Cpt1b-deficient mice. functional mitochondria in skeletal muscle of Cpt1bm / mice. Expression of peroxisomal FAO genes, including Acox1, Peci, Decr2, Pmp70, Pex19, and the medium-chain carnitine O-octa- Metabolic Remodeling Leads to Alternative Substrate Utilization by m−/− Mitochondria. To probe further the implications of decreased noyltransferase, Crot, is up-regulated in muscles from Cpt1b mice (Fig. 9F and Table S1). We used extracellular flux analysis mitochondrial FAO on metabolism within the muscle, we used m−/− muscle homogenates to examine oxidation of other substrates. to confirm that Cpt1b mitochondria are unable to oxidize long-chain acyl-CoA esters but can respirate on long-chain Oxidation of the amino acid Leu is significantly increased in PHYSIOLOGY m−/− acylcarnitines, which could theoretically be supplied by peroxi- Cpt1b muscle homogenate (Fig. 9A). Gene expression of the somes (Fig. S4C). To confirm changes to peroxisomal function, Bcat2 and Bckdha is elevated (Fig. 9B), and metab- we measured oxidation of lignoceric acid (C24:0), which requires olomics analysis reveals significant elevations in muscle levels of peroxisomes to be catabolized (29). Results show that both total nearly all amino acids tested (Fig. 9 C and D). This effect ap- m−/− and partial lignoceric acid oxidation is increased in Cpt1b pears to be confined to muscle, because only Gly and citrulline homogenates (Fig. 9G). Taken together, the data strongly sug- − − are elevated in serum (Fig. S5). Furthermore, propionylcarnitine gest that Cpt1bm / mice partially compensate for reduced mi- (C3) and succinylcarnitine (C4-DC) acylcarnitines are signifi- − − tochondrial FAO by induction of peroxisomal FAO. cantly increased in Cpt1bm / muscle (Fig. S6). Production of C3 is commonly used as an indicator of amino acid catabolism, Discussion because the breakdown of Val, Leu, Ile, and Met provides a There have been decades of conflicting literature regarding significant source of propionyl-CoA. Additionally, propionyl- pharmacological inhibition of Cpt for treatment of insulin re- CoA enters the TCA cycle after it is carboxylated to form suc- sistance. Given this ongoing confusing literature and the con- cinyl-CoA. As such, the increase in C4-DC and C3 in the present trasting theories of the role of impaired vs. increased muscle study is quite likely a result of increased amino acid catabolism in FAO on development of insulin resistance, we developed a − − Cpt1bm / muscle. Globally, these analyses provide compelling skeletal muscle-specific model of Cpt1b deficiency. As many evidence that Cpt1b deficiency in muscle induces a localized would expect, inhibition of mitochondrial FAO causes the

Wicks et al. PNAS | Published online June 8, 2015 | E3305 Downloaded by guest on September 23, 2021 A B 400 * 500 * 300 * 400 * 300 * 200 200 fl/fl fl/fl 100 Cpt1b Cpt1b m-/- 100 Cpt1b Cpt1bm-/-

Blood glucose (mg/dl) glucose Blood 0 0 0 20 40 60 (mg/dl) glucose Blood 0 20 40 60 Minutes post glucose Minutes post glucose C D E F 10 400 150 * * * 400 8 Cpt1bfl/fl

A.U.) 300

3 300 m-/- 6 100 Cpt1b 200 4 200 50 **

100 Ox. Pyruvate C

2 Activity PDH 100 14 (mg/kg tissue) mRNA (x10 mRNA 3- Glycogen content Glycogen (nmol/mg protein/hr) (nmol/mg 0 protein/hr) (nmol/mg 0 0 Pdha1 0 R.Q. W.Q.

Fig. 7. Glucose utilization is increased by abrogation of Cpt1b. Glucose disposal capacity is significantly improved in Cpt1bm−/− mice, as measured by a glucose tolerance test, at both 10–12 wk (A) and 18–20 wk (B)(n = 8–10). (C) Expression of Pdha1 is increased. Both PDH (assayed with [1-14C]pyruvate) (D)and complete pyruvate oxidation (Ox.) (measured with 3-[14C]pyruvate) (E) are significantly increased (n = 3–6, representative of multiple repeated experiments). (F) Glycogen content is decreased, suggesting reliance on glucose utilization, in red quadriceps (R.Q.), but not in white quadriceps (W.Q.) (n = 4). *P < 0.05 or **P < 0.01, differences detected by Student’s ttest.

muscle to rely more heavily on carbohydrate for fuel. Enhanced examined related to fatty acid uptake and transport, lipolysis, − − glucose disposal and elevated pyruvate oxidation as a result of carnitine/acylcarnitine shuttle, and β-oxidation in Cpt1bm / restricted FAO are consistent with substrate competition models muscle (Table S1). However, the long-chain acyl-CoAs cannot between glucose and fatty acids and the of enter the mitochondria, leading to a massive accumulation of hexokinase and PDH regulating both glucose uptake and oxi- IMCL. It has been previously suggested that IMCL, specifically dation (14, 30). However, as with any homeostatic system, per- TAGs, are not detrimental, per se, but that it is rather the ac- turbations in one component cause compensatory adaptations. cumulation of lipotoxic DAGs and ceramides that impairs insulin Our results suggest that impaired skeletal muscle mitochondrial signaling. However, our results clearly show substantial increases FAO has complex effects that go beyond the regulation of glu- in both DAGs and ceramides, and yet no detriments to insulin cose uptake and oxidation. signaling. This result is at first counterintuitive; however, other These adaptations are vital to sustain energy supply to muscle reports have found similar increases in lipotoxic species without and are the result of several interrelated pathways. We suggest induction of insulin resistance, including the well-known ath- that an energy deficit signal is induced that elicits a suite of ef- lete’s paradox (31–34). Elevated DAGs have been found in en- fects, including mitochondrial biogenesis, compensatory oxida- durance athletes with high insulin sensitivity (35). In these tion of fat by peroxisomes, and the use of amino acids for fuel. studies, insulin sensitivity was associated both with specific DAG To our knowledge, this study is the first report of enhanced species and with higher oxidative capacity (35), suggesting that peroxisomal FAO in muscle following decreased mitochondrial alterations to nutrient and energy balance may play a role in FAO, and the implications of prolonged reliance on peroxisomal determining the response to a lipotoxic environment. As a result, fat catabolism are unknown. one possible explanation for our findings is that activation of Another adaptation that appears to be directly affected by energy/nutrient deprivation pathways is capable of overriding the reduced mitochondrial FAO is alterations in the cellular re- traditional lipid-induced decrements in insulin-stimulated glu- sponse to accumulation of lipotoxic species. The loss of mi- cose uptake as a means to rescue energy production; however, tochondrial FAO induces up-regulation of virtually every gene this possibility remains untested. It is also possible that as yet

− − Table 1. Quantitative proteomics results from Cpt1bm / muscle No. of Proteins significantly Process overrepresented (+)or P value of GO biological identified up-regulated Expected value in underrepresented statistical process proteins in Cpt1bm−/− Cpt1bm−/− (−) in Cpt1bm−/− overrepresentation test

Respiratory electron 44 22 6.37 + 2.65E-05 transport chain Generation of precursor 46 22 6.66 + 5.71E-05 metabolites and energy Oxidative phosphorylation 33 18 4.78 + 1.39E-04 Cellular process 161 10 23.32 − 5.08E-02 Primary metabolic process 216 17 31.29 − 6.65E-02

− − Quantitative proteomics results from Cpt1bm / muscle were evaluated using the Panther statistical overrepresentation test for (GO) Biological Processes (www.pantherdb.org, version 8.0). The top five identified pathways are shown.

E3306 | www.pnas.org/cgi/doi/10.1073/pnas.1418560112 Wicks et al. Downloaded by guest on September 23, 2021 A B C PNAS PLUS pAMPK 15 fl/fl 2.0 AMPK Cpt1b ** Cpt1bfl/fl *** m-/- * GAPDH Cpt1b 1.5 Cpt1bm-/- A.U.) 10 Cpt1bfl/fl Cpt1bm-/- 2 1.5 1.0 * 1.0 5 fl/fl 0.5

Cpt1b change) (Fold A.U. Ratio 0.5 Cpt1bm-/- (x10 mRNA *

Mitochondrial number Mitochondrial 0.0 0 Cytb/ Cox2/ (p-AMPK/AMPK) 0.0 Pgc1α Pgc1β Pparα Pparγ β-globin glucagon D EF 30 * ** fl/fl 15 Cpt1b Cpt1bfl/fl m-/- Cpt1b Cpt1bm-/- A.U.) A.U.)

3 * 20 3 10 ** ** ** 10 5 ** * mRNA (x10 mRNA (x10 0 0 Cs Ndufs8 Sdhb Cox5a Hadha Ech1 Cpt2 Cact

− − Fig. 8. PGC1α pathway leads to mitochondrial biogenesis in Cpt1bm / muscle. (A) Phosphorylation of AMPKα at Thr172 is increased by FAO deficiency. − − (B) Gene expression of PGC1α and PPARγ, but not PGC1β or PPARα mRNA, is increased in Cpt1bm / muscle. Mitochondrial proliferation is induced, as shown by increased mtDNA copy number (quadriceps, n = 11) (C), coordinate up-regulation of mitochondrial gene expression (D), and increased expression of mitochondrial FAO genes (E). (F) Proteomics analysis (n = 7–8) detected significant overrepresentation of mitochondrial genes, and mechanistic network analysis predicts activation of a PGC1α signaling pathway. *P < 0.05 or **P < 0.01, differences detected by Student’s t test. Data are shown as mean ± SEM.

− − unknown differences between specific species of DAGs and mass between control and Cpt1bm / mice implicate decreased ceramides and their subcellular locations are responsible for the food intake as a partial contributor. The finding that inhibition of discrepancy (33, 36). Recent studies in humans have correlated mitochondrial FAO in muscle increases oxidative and uncou- both individual DAG species, particularly saturated DAG, and pling capacity in inguinal fat suggests that energy may be dissi- their subcellular localization to insulin resistance in humans (37, pated as heat. Elucidating the exact mechanisms that prevent − − 38). In whole-muscle homogenates, we observe increases and a gain of fat mass in Cpt1bm / mice could have wide-ranging few decreases in saturated, unsaturated, and mixed DAG species implications in the obesity field. − − in the Cpt1bm / muscle. Nevertheless, whether or not altered distribution of lipotoxic species plays a role, our results argue Materials and Methods strongly that impaired mitochondrial FAO, even coupled with Animal Studies. Animal studies were conducted at Pennington Biomedical chronic cellular lipid oversupply, is not sufficient to induce Research Center’s American Association for the Accreditation of Laboratory insulin resistance. Animal Care-approved facility, with mice receiving a standard chow diet, In summary, to our knowledge, the first and almost foregone composed of 20% (wt/wt) protein, 25% (wt/wt) fat, and 55% (wt/wt) car- results from decreased transport of fatty acids into the mito- bohydrate (Purina Rodent Chow no. 5015; Purina Mills), and were approved by the Institutional Animal Care and Use Committee.

chondria are impaired mitochondrial FAO and the concomitant PHYSIOLOGY increase in carbohydrate oxidation. More important are the Animal Procedures. Body composition was measured using a Bruker NMR observed adaptations in muscle. To our knowledge, these results Minispec (Bruker Corporation). Food intake was measured as described by provide the first observation in which decreased mitochondrial Kruger et al. (39), with slight modifications. Plasma collections were per- FAO elicits a signal indicating a shortage of lipid in the muscle formed by submandibular bleed. Glucose tolerance tests were performed causing the up-regulation of genes governing fatty acid uptake, following a 4-h fast by i.p. injection of 20% D-glucose (40 mg of glucose per transport, storage, and oxidation, yet the lipids cannot enter the mouse). ITTs were done in the fed state, using an i.p. dose of 0.04 units (U) mitochondria. However, the increased lipids are uncoupled to per mouse. For insulin signaling studies, mice were fasted overnight (∼16 h) their usual association with the interference in the insulin sig- and given an i.p. injection of insulin (1.0 U/kg of body weight) or saline, and naling cascade. Understanding these uncoupling mechanisms tissues were collected 10 min later. Tissue processing and isolated muscle may be important in the treatment of type 2 diabetes. One could experiments are described in SI Materials and Methods. Indirect calorimetry conclude that the increased number of mitochondria and oxi- was done in a 16-chamber Oxymax system (Columbus Instruments) as de- scribed by Albarado et al. (40). Behavioral studies were performed with dative capacity offset the lipid accumulation similar to the ath- ’ slight modifications from the method of Wicks et al. (41). Endurance was lete s paradox, but because the mitochondria have reduced FAO measured following three habituation sessions; speed was increased from capacity, there may be other unexplored explanations. Perhaps · −1 · −1 − − 6mmin by 0.5 m min at 5-min intervals until exhaustion. the most striking phenotype is that Cpt1bm / mice do not gain or lose fat mass beyond 10–12 wk of age. Careful measurements Metabolic Profiling. ELISA kits were used for measurement of insulin (Ultra of food intake and body composition during the divergence in fat Sensitive Mouse Insulin ELISA kit; Crystal Chem, Inc.), NEFA, and TAG (NEFA

Wicks et al. PNAS | Published online June 8, 2015 | E3307 Downloaded by guest on September 23, 2021 B C 15 * A fl/fl 4 * 50 Cpt1b fl/fl * m-/- * Cpt1b * Cpt1b m-/- * 3 40 Cpt1b 10

A.U.) * 2 30 * * 2 5 *

20 acids Amino * * (nmol/mg protein) (nmol/mg * 1 10 mRNA (x10 mRNA (nmol/g tissue/h) 0 Leucine Oxidation Leucine 0 et 0 Cit Bckdha Bcat2 Val Thr M His Phe Tyr Asp Trp Orn/Asn D 400 E Cpt1bfl/fl 15 m-/- 300 * Cpt1b 10 200 *

/mg protein/hr /mg ** 2 5 Amino acids Amino 100 * * * (nmol/mg protein) (nmol/mg *

* Palmitate Oxidation 0

(nmol CO 0 B E B E rg fl/fl m-/- Gly Ala Ser Pro A Glu Cpt1b Cpt1b Leu/IleLys/Gln F 30 G fl/fl ** 200 Cpt1b ** 150 * Cpt1bm-/- *

A.U.) 150 2 20 100 100 10 50 ** 50 mRNA (x10 (pmol/g tissue/h) ** (pmol/g tissue/h) 0 0 0 Acox1 Peci Decr2 Pmp70 Cpt1bfl/fl Cpt1bm-/- Cpt1bfl/fl Cpt1bm-/- Lignocerate Oxidation- Total Oxidation- Lignocerate Lignocerate Oxidation- Partial Oxidation- Lignocerate

− − Fig. 9. Cpt1b deficiency in skeletal muscle remodels energy metabolism, leading to compensatory amino acid catabolism and peroxisomal FAO. Cpt1bm / muscles use amino acids as an alternative energy source, as shown by enhanced Leu oxidation in muscle homogenates (A) and increased expression of genes involved in amino acid metabolism (B). Leu oxidation was measured from [U-14C]Leu in gastrocnemius homogenate (n = 4). (C and D) Consistent with amino acid catabolism in Cpt1bm−/− muscle, levels of nearly all amino acids are elevated. (E) Although mitochondrial FAO is decreased, there is no reduction in FAO in quadriceps muscle homogenates (measured with [1-14C]palmitate; n = 3, representative of multiple independent experiments). B, basal; E, treated with the Cpt1 inhibitor etomoxir. (F) Up-regulation of peroxisomal activity is suggested by increased expression of peroxisomal FAO genes (n = 5–7). (G) Increased peroxisomal capacity for FAO is shown by enhanced oxidation of lignoceric acid (C24:0), which can only be oxidized by peroxisomes. Lignoceric acid (partial and total) oxidation was measured from [1-14C]lignoceric acid in gastrocnemius homogenate (n = 4). *P < 0.05 or **P < 0.01, differences detected by Student’s t test. Data are shown as mean ± SEM.

and triglyceride-H; Wako Diagnostics). Isolation of lipid and quantification (100 μM) was measured over the course of 2 h in reaction media (pH 7.8) con-

of TAGs and ceramides were performed as described by Obanda et al. (42). sisting of the following: 100 mM sucrose, 10 mM Tris·HCl, 10 mM K2HPO4, DAGs were measured as described by Wang et al. (43). Glycogen was mea- 80 mM KCl, 1 mM MgCl2·6H2O, 2 mM L-carnitine, 0.2 mM malate, 0.08 mM sured using a commercial kit (65620; Abcam) according to the manu- pyridoxal phosphate, 0.4 mM α-ketoglutarate, 1 mM DTT, 0.1 mM nicotin- facturer’s instructions. Measurement of acylcarnitines, amino acids, and amide-adenine dinucleotide, 2 mM ATP, and 0.05 mM CoA. proteomics is described in SI Materials and Methods. CPT Activity Assay. CPT activity was assayed using isolated mitochondria qRT-PCR. Total RNA from mouse tissue was isolated using an RNeasy Mini Kit (120 μg per well) according to principles outlined by Bieber et al. (47). Mito- (Qiagen) and DNase digested. cDNA synthesized with the iScript cDNA syn- chondrial assays are described in SI Materials and Methods. thesis kit was used for qRT-PCR with the SYBR Green system (Bio-Rad). DNA was isolated using a DNA/RNA Mini Kit (Qiagen). Primer details are provided Immunofluorescence and Bodipy Staining. Gastrocnemius muscles were frozen in Table S2. in a mixture of Optimal Cutting Temperature Compound (Sakura) and Trag- acanth (Spectrum). Sectioned samples were fixed in 4% paraformaldehyde Substrate Oxidation Assays. Red quadriceps or mixed gastrocnemius muscle solution (Invitrogen), incubated in a 1:100 dilution of Bodipy in PBS (Invitrogen), homogenates were prepared as previously described (44). Mitochondrial and mounted in Citifluor (Ted Pella, Inc.). PTAH and H&E staining was per- isolation was performed as described by Frezza et al. (45); the IBm2 buffer formed by standard protocols in formalin-fixed tissues at the Louisiana State was modified to 70 mM sucrose, 200 mM mannitol, 5 mM EGTA/Tris (pH 7.4), University-AgCenter Histology Core. and 10 mM Tris·HCl (pH 7.4). Fatty acid and pyruvate oxidation was assessed using whole-muscle homogenates or isolated skeletal muscle mitochondria EM. EM was performed at the Socolofsky Microscopy Center at Louisiana State as in the study by Hulver et al. (46); etomoxir was used at a final concen- University. Materials were fixed in 2% (wt/vol) glutaraldehyde/1% (wt/vol) tration of 100 μM. Peroxisomal FAO was measured from [1-14C]lignoceric formaldehyde and then postfixed in 2% (wt/vol) osmium tetroxide, en bloc 14 14 acid (20 μM) as previously shown (44). Capture of CO2 from [U- C]Leu stained in 0.5% uranyl acetate, and embedded in Epon-NMA. Ultrathin sections

E3308 | www.pnas.org/cgi/doi/10.1073/pnas.1418560112 Wicks et al. Downloaded by guest on September 23, 2021 (70 nm) were mounted on collodion-coated copper grids, stained with Reynolds of Medicine for the use of the Duke Proteomics Core Facility, and Drs. PNAS PLUS lead citrate, and imaged with a JEOL 100CX transmission electron microscope. Matthew Foster and Arthur Moseley for performing the proteomic analysis. Dr. Nobuko Wakamoto (LSU-Agricultural Center Histology Department) is also acknowledged for assistance with muscle pathology staining and Statistical Analysis. Data were analyzed by t test, ANOVA/repeated measures interpretation. This work used Pennington Biomedical Research Center core ANOVA, with Bonferroni posttests using GraphPad Prism 5 software. ANCOVA facilities (Proteomics and Metabolomics, Genomics, Cell Biology and Bio- analysis was performed using JMP software from SAS. The P value was set at imaging, Transgenic and Animal Phenotyping, and Animal Metabolism and <0.05 a priori. Behavior) that are supported, in part, by Center of Biomedical Research Excellence (COBRE) (NIH Grant 8P20-GM103528) and Nutrition Obesity Re- ACKNOWLEDGMENTS. We thank Tamra Mendoza, Estrellita Bermudez, search Center (NORC) (NIH Grant 2P30-DK072476) center grants from the Steven Bond, Dieyun Ding, Jeffrey Covington, Sudip Bajpeyi, Krisztian National Institutes of Health. This research was supported by American Di- Stadler, David Burke, Ryan Grant, and Eric Ravussin for critical advice, abetes Association Grant 1-10-BS-129 and NIH Grant R01DK089641 (to R.L.M.). reagents, and/or technical assistance. We also thank the Socolofsky Mi- S.E.W. is supported by Fellowship T32 AT004094, and received a pilot and croscopy Center, the Department of Agricultural Chemistry, and the feasibility grant from NORC (NIH Grant 2P30-DK072476). R.C.N. is supported W. A. Callegari Environmental Center at Louisiana State University (LSU) for as a project primary investigator on the COBRE grant (NIH Grant 9P20- measurement of lipid species and EM. We thank the Duke University School GM103528) and as primary investigator on NIH Grant R01DK103860.

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