Effects of Simvastatin on Lipid Metabolism in Wild-Type Mice and Mice with Muscle PGC-1α Overexpression

Previous studies suggest that statins may disturb skeletal muscle lipid metabolism potentially causing lipotoxicity with insulin resistance. We investigated this possibility in wild-type mice (WT) and mice with skeletal muscle PGC-1α overexpression (PGC-1α OE mice). In WT mice, simvastatin had only minor effects on skeletal muscle lipid metabolism but reduced glucose uptake, indicating impaired insulin sensitivity. Muscle PGC-1α overexpression caused lipid droplet accumulation in skeletal muscle with increased expression of the fatty acid transporter CD36, fatty acid binding protein 4, perilipin 5 and CPT1b but without significant impairment of muscle glucose uptake. Simvastatin further increased the lipid droplet accumulation in PGC-1α OE mice and stimulated muscle glucose uptake. In conclusion, the impaired muscle glucose uptake in WT mice treated with simvastatin cannot be explained by lipotoxicity. PGC-1α OE mice are protected from lipotoxicity of fatty acids and triglycerides by increased the expression of FABP4, formation of lipid droplets and increased expression of CPT1b.

The RabGAPs TBC1D1 and TBC1D4 control uptake of long-chain fatty acids into skeletal muscle via fatty acid transporter SLC27A4/FATP4 Short title: RabGAPs in skeletal muscle lipid metabolism

The two closely related RabGTPase-activating proteins (RabGAPs) TBC1D1 and TBC1D4 play a crucial role in the regulation of GLUT4 translocation in response to insulin and contraction in skeletal muscle. In mice, deficiency in one or both RabGAPs leads to reduced insulin and contraction-stimulated glucose uptake, and to elevated fatty acid uptake and oxidation in both glycolytic and oxidative muscle fibers without altering mitochondrial copy number and the abundance of OXPHOS proteins. Here we present evidence for a novel mechanism of skeletal muscle lipid utilization involving the two RabGAPs and the fatty acid transporter SLC27A4/FATP4. Both RabGAPs control the uptake of saturated and unsaturated long-chain fatty acids (LCFAs) into skeletal muscle and knockdown of a subset of RabGAP substrates, <i>Rab8, Rab10 </i>or <i>Rab14, </i>decreased LCFA uptake into these cells. In skeletal muscle from <i>Tbc1d1/Tbc1d4</i> knockout animals, SLC27A4/FATP4 abundance was increased and depletion of SLC27A4/FATP4 but not FAT/CD36 completely abrogated the enhanced fatty acid oxidation in RabGAP-deficient skeletal muscle and cultivated C2C12 myotubes. Collectively, our data demonstrate that RabGAP-mediated control of skeletal muscle lipid metabolism converges with glucose metabolism at the level of downstream RabGTPases and involves regulated transport of LCFAs via SLC27A4/FATP4.

The RabGAPs TBC1D1 and TBC1D4 control uptake of long-chain fatty acids into skeletal muscle via fatty acid transporter SLC27A4/FATP4 Short title: RabGAPs in skeletal muscle lipid metabolism

The two closely related RabGTPase-activating proteins (RabGAPs) TBC1D1 and TBC1D4 play a crucial role in the regulation of GLUT4 translocation in response to insulin and contraction in skeletal muscle. In mice, deficiency in one or both RabGAPs leads to reduced insulin and contraction-stimulated glucose uptake, and to elevated fatty acid uptake and oxidation in both glycolytic and oxidative muscle fibers without altering mitochondrial copy number and the abundance of OXPHOS proteins. Here we present evidence for a novel mechanism of skeletal muscle lipid utilization involving the two RabGAPs and the fatty acid transporter SLC27A4/FATP4. Both RabGAPs control the uptake of saturated and unsaturated long-chain fatty acids (LCFAs) into skeletal muscle and knockdown of a subset of RabGAP substrates, <i>Rab8, Rab10 </i>or <i>Rab14, </i>decreased LCFA uptake into these cells. In skeletal muscle from <i>Tbc1d1/Tbc1d4</i> knockout animals, SLC27A4/FATP4 abundance was increased and depletion of SLC27A4/FATP4 but not FAT/CD36 completely abrogated the enhanced fatty acid oxidation in RabGAP-deficient skeletal muscle and cultivated C2C12 myotubes. Collectively, our data demonstrate that RabGAP-mediated control of skeletal muscle lipid metabolism converges with glucose metabolism at the level of downstream RabGTPases and involves regulated transport of LCFAs via SLC27A4/FATP4.

Relative lipid oxidation associates directly with mitochondrial fusion phenotype and mitochondria-sarcoplasmic reticulum interactions in human skeletal muscle

Disturbances in skeletal muscle lipid oxidation might induce ectopic fat deposition and lipotoxicity. Nevertheless, the cellular mechanisms that regulate skeletal muscle lipid oxidation have not been fully determined. We aimed to determine whether there was an association between relative whole body lipid oxidation and mitochondrial size or mitochondria-sarcoplasmic reticulum interactions in the skeletal muscle. Twelve healthy men were included [mean (standard deviation), 24.7 (1.5) yr old, 24.4 (2.6) kg/m2]. The respiratory quotient (RQ) was used to estimate relative lipid oxidation at rest and during exercise (50% maximal oxygen consumption, 600 kcal expended). A skeletal muscle biopsy was obtained from the vastus lateralis at rest. Transmission electron microscopy was used to determine mitochondrial size and mitochondria-sarcoplasmic reticulum interactions (≤50 nm of distance between organelles). Protein levels of fusion/fission regulators were measured in skeletal muscle by Western blot. Resting RQ and exercise RQ associated inversely with intermyofibrillar mitochondrial size ( r = −0.66 and r = −0.60, respectively, P < 0.05). Resting RQ also associated inversely with the percentage of intermyofibrillar mitochondria-sarcoplasmic reticulum interactions ( r = −0.62, P = 0.03). Finally, intermyofibrillar mitochondrial size associated inversely with lipid droplet density ( r = −0.66, P = 0.01) but directly with mitochondria fusion-to-fission ratio ( r = 0.61, P = 0.03). Our results show that whole body lipid oxidation is associated with skeletal muscle intermyofibrillar mitochondrial size, fusion phenotype, and mitochondria-sarcoplasmic-reticulum interactions in nondiabetic humans.

SUN-653 Bypassing Skeletal Muscle Lipid Handling Deficiencies as a Therapy for Metabolic Disease

Metabolic diseases and their serious sequelae such as non-alcoholic fatty liver disease (NAFLD) pose a substantial clinical burden. It is now well recognized that skeletal muscle is a major site for the metabolism of all major macronutrients, and derangements in these muscle processes significantly contribute to metabolic disease. Studies over the last 15 years have identified the transcription factor Krüppel-like factor 15 (KLF15) as an important regulator and effector of metabolic processes across various tissues, and furthermore, genome-wide studies have identified human KLF15 variants with increased body mass index and diabetes. Given the importance of skeletal muscle in maintaining metabolic homeostasis, we generated a skeletal muscle specific KLF15 knockout (K15-SKO) mouse to study the role of skeletal muscle KLF15 in regulating systemic metabolism. We found that this animal is prone to developing obesity and insulin resistance at baseline, a phenotype that is greatly exacerbated in response to high fat diet (HFD). Strikingly, K15-SKO mice show a propensity toward developing NAFLD, as demonstrated by increased micro- and macrovesicular steatosis, hepatocellular ballooning, increased hepatic fatty acid and triglyceride deposition, and elevated Cd36 expression. A potential cause of NAFLD is the accumulation of excess lipids and lipid intermediates due to defects in the lipid flux pathway in extrahepatic tissues. Indeed, we see defects in the expression of genes involved in the carnitine shuttle and a paucity of long-chain acylcarnitines in K15-SKO skeletal muscle. Furthermore, RNA sequencing of skeletal muscle from K15-SKO mice shows downregulation in a number of pathways involved in lipid handling. This indicates that KLF15 serves as a novel extrahepatic molecular regulator of hepatic health. It has been previously shown that a diet rich in short-chain fatty acids (SCFA) can bypass defects in lipid handling and ultimately improve metabolic health. To explore this therapeutic avenue, we gave K15-SKO mice either normal chow (NC) or a SCFA-rich diet for 7 weeks. We observed decreased weight gain and improved glucose homeostasis in SCFA-rich diet fed mice. In addition to being a preventative strategy, SCFA-rich diets may also serve as a potential therapy to rescue from metabolic disease. To this end, we gave K15-SKO mice HFD for 5 weeks followed by 7 weeks of either NC or SCFA-rich diet. We observed that providing SCFAs can improve metabolic health and ameliorate the phenotype seen due to defects in skeletal muscle lipid handling: mice given SCFA-rich diet following HFD had significantly decreased weight gain and improved insulin sensitivity. These studies demonstrate that skeletal muscle KLF15 serves as an important regulator of lipid flux and hepatic health, and that SCFA-rich diets are a promising candidate for metabolic disease resultant of impaired lipid handling.

SUN-582 Effect of the Combination of Conjugated Estrogens and Bazedoxifene on Muscle and Serum Lipidome in Obese Postmenopausal Women: A Placebo-Controlled Randomized Pilot Trial

Background and Objectives: Menopause is characterized by estrogen deficiency and predisposes women to weight gain and metabolic disturbances including lipid abnormalities. Orally-administered estrogens increase high-density lipoprotein (HDL) and triglycerides (TG) cholesterol and decreases low-density lipoprotein (LDL) cholesterol levels. The increase in serum TGs is not well understood. The objective of this study was to assess the effect of CE/BZA on serum and skeletal muscle lipid species in obese postmenopausal women. Methods: Randomized double-blind crossover pilot trial in 8 obese postmenopausal women (53± 3 years, BMI 35.7±3.2 kg/m2) assigned to 8 weeks of CE/BZA or placebo with 8 weeks washout in between. At the end of each 8-week treatment period, intrahepatic and skeletal muscle lipids were measured by proton magnetic resonance spectroscopy (1H-MRS) while serum and skeletal muscle lipidomics were assayed by ultrahigh performance liquid chromatography/mass spectrometry (UHPLC/MS). Results: No treatment differences were observed in intrahepatic lipid, soleus intramyocellular lipid (IMCL) or extramyocellular lipid (EMCL) as well as tibialis anterior IMCL or EMCL. The serum metabolome and lipidome comprised a total of 2002 biochemicals. Treatment with CE/BZA was associated with higher levels of diacylglycerols (DAGs) and triacylglycerols (TAGs) composed of long-chain saturated fatty acids (SFA, palmitic C16:0 and arachidic C20:0), monounsaturated FAs (MUFA, palmitoleic C16:1, oleic C18:1 and ecosenoic C20:1), and polyunsaturated FAs (PUFA, linoleic C18:2, arachidonic C20:4, eicosapentaenoic C20:5, and docosahexaenoic C22:6) compared to placebo (all p&lt;0.05). Treatment with CE/BZA was also associated with lower levels of several acylcarnitine species, which are markers of FA oxidation, including long-chain SFA (C14, C16 and C18), MUFA (C18:1 and C24:1) and PUFA (C18:2, C20:2 and C20:4). In addition, treatment with CE/BZA was associated with higher levels of phosphatidylcholines (PCs), phosphatidylinositols (PIs), phosphatidylethanolamines (PEs), sphingomyelins (SMs), and ceramides (CER), as well as lower levels of lysophophatidylcholines (LPCs). There were no treatment differences in carnitine or ketones levels. The skeletal muscle analysis comprised a total of 652 biochemicals, but unlike in serum, no significant treatment differences were observed in the skeletal muscle lipidome. Conclusions: Our lipidomic analysis supports a model in which CE/BZA (and likely all oral estrogens) increases hepatic de novo FA synthesis and esterification into TAGs for export into TAG-rich very low-density lipoproteins, as well as decreased FA oxidation, respectively. Although CE/BZA treatment inhibits FA oxidation, it is not associated with hepatic lipid accumulation as measured by MRS, or skeletal muscle lipid accumulation measured by MRS and lipidomics.

0295 Skeletal Muscle Diacylglycerol Accumulation and Impaired Insulin Sensitivity During Insufficient Sleep

Introduction Insufficient sleep impairs insulin sensitivity; however, the mechanism(s) by which this occurs are unknown. We previously reported an elevation in plasma free fatty acid concentration during insufficient sleep, suggesting dysregulated lipid metabolism. Lipid accumulation in muscle—specifically certain species of diacylglycerol (DAG)—is associated with impaired insulin sensitivity. We therefore tested the hypothesis that insufficient sleep leads to skeletal muscle DAG accumulation. Methods As part of an ongoing study, thirteen sedentary, healthy, lean adults (25.8±3.2y; 22.7±1.9kg/m2; 3F; mean±SD) participated in a controlled 6-day in-laboratory protocol with 9h in bed (habitual sleep) followed by 4 nights of 5h in bed (insufficient sleep), achieved by delaying bedtime by 4 hours. For one week prior to the study, participants maintained a 9h sleep schedule. Participants consumed energy balanced diets 3 days prior to and throughout the laboratory protocol. Insulin sensitivity was assessed using a hyperinsulinemic euglycemic clamp before and after insufficient sleep. Skeletal muscle biopsies of the vastus lateralis were taken immediately before each clamp. In a subset of subjects (n=10), quantitative lipidomic analyses using LC/MS/MS were performed on biopsied muscle tissue. Results Insulin sensitivity was impaired following insufficient sleep (10.7±1.5 vs 9.6±1.2 mg/kg/min, p&lt;0.05, mean±SEM). There were no changes in skeletal muscle concentration of total triglycerides (TAGs), nor specific TAG species. However, insufficient sleep tended to increase skeletal muscle accumulation of total 1,2-DAGs (p=0.13) and significantly increased specific saturated species of 1,2-DAG, including Di-C18:0 DAG (p&lt;0.05), previously implicated in insulin resistance. In contrast, 1,3-DAGs are not thought to impair insulin sensitivity and specific species were decreased or unchanged during insufficient sleep. Conclusion Preliminary findings suggest that skeletal muscle lipid accumulation of diacylglycerol species during insufficient sleep may be a contributing mechanism by which insufficient sleep dysregulates metabolic physiology. Support NIH K01DK110138, R03 DK118309, UL1 TR002535, and GCRC RR-00036

Skeletal muscle lipid droplets are resynthesized before being coated with perilipin proteins following prolonged exercise in elite male triathletes

Intramuscular triglycerides (IMTG) are a key substrate during prolonged exercise, but little is known about the rate of IMTG resynthesis in the postexercise period. We investigated the hypothesis that the distribution of the lipid droplet (LD)-associated perilipin (PLIN) proteins is linked to IMTG storage following exercise. Fourteen elite male triathletes (27 ± 1 yr, 66.5 ± 1.3 mL·kg−1·min−1) completed 4 h of moderate-intensity cycling. During the first 4 h of recovery, subjects received either carbohydrate or H2O, after which both groups received carbohydrate. Muscle biopsies collected pre- and postexercise and 4 and 24 h postexercise were analyzed using confocal immunofluorescence microscopy for fiber type-specific IMTG content and PLIN distribution with LDs. Exercise reduced IMTG content in type I fibers (−53%, P = 0.002), with no change in type IIa fibers. During the first 4 h of recovery, IMTG content increased in type I fibers ( P = 0.014), but was not increased more after 24 h, where it was similar to baseline levels in both conditions. During recovery the number of LDs labeled with PLIN2 (70%), PLIN3 (63%), and PLIN5 (62%; all P < 0.05) all increased in type I fibers. Importantly, the increase in LDs labeled with PLIN proteins only occurred at 24 h postexercise. In conclusion, IMTG resynthesis occurs rapidly in type I fibers following prolonged exercise in highly trained individuals. Furthermore, increases in IMTG content following exercise preceded an increase in the number of LDs labeled with PLIN proteins. These data, therefore, suggest that the PLIN proteins do not play a key role in postexercise IMTG resynthesis.