Aims/hypotheses Ceramides and other sphingolipids comprise a family of lipid molecules that accumulate in skeletal muscle and promote insulin resistance. Chronic endurance exercise training decreases muscle ceramides and other sphingolipids, but less is known about the effects of a single bout of exercise. Methods We measured basal relationships and the effect of acute exercise (1.5 h at 50% V ⋅ O 2max ) and recovery on muscle sphingolipid content in obese volunteers, endurance trained athletes and individuals with type 2 diabetes. Results Muscle C18:0 ceramide (p = 0.029), dihydroceramide (p = 0.06) and glucosylceramide (p = 0.03) species were inversely related to insulin sensitivity without differences in total ceramide, dihydroceramide, and glucosylceramide concentration. Muscle C18:0 dihydroceramide correlated with markers of muscle inflammation (p = 0.04). Transcription of genes encoding sphingolipid synthesis enzymes was higher in athletes, suggesting an increased capacity for sphingolipid synthesis. The total concentration of muscle ceramides and sphingolipids increased during exercise and then decreased after recovery, during which time ceramide levels reduced to significantly below basal levels.Conclusions/interpretation These data suggest ceramide and other sphingolipids containing stearate (18:0) are uniquely related to insulin resistance in skeletal muscle. Recovery from an exercise bout decreased muscle ceramide concentration; this may represent a mechanism promoting the insulinsensitising effects of acute exercise.

Ceramides and sphingolipids are a family of lipid molecules that circulate in serum and accumulate in skeletal muscle, promoting insulin resistance. Plasma ceramide and dihydroceramide are related to insulin resistance, yet less is known regarding other ceramide and sphingolipid species. Despite its association with insulin sensitivity, chronic endurance exercise training does not change plasma ceramide and sphingolipid content, with little known regarding a single bout of exercise. We measured basal relationships and the effect of acute exercise (1.5 h at 50% V O2 max) and recovery on serum ceramide and sphingolipid content in sedentary obese individuals, endurancetrained athletes, and individuals with type 2 diabetes (T2D). Basal serum C18:0, C20:0, and C24:1 ceramide and C18:0 and total dihydroceramide were significantly higher in T2D and, along with C16:0 ceramide and C18:0 sphingomyelin, correlated positively with insulin resistance. Acute exercise significantly increased serum ceramide, glucosylceramide, and GM3 gangliosides, which largely decreased to basal values in recovery. Sphingosine 1-phosphate and sphingomyelin did not change during exercise but decreased below basal values in recovery. Serum C16:0 and C18:0 ceramide and C18:0 sphingomyelin, but not the total concentrations of either of them, were positively correlated with markers of muscle NF-B activation, suggesting that specific species activate intracellular inflammation. Interestingly, a subset of sphingomyelin species, notably C14:0, C22:3, and C24:4 species, was positively associated with insulin secretion and glucose tolerance. Together, these data show that unique ceramide and sphingolipid species associate with either protective or deleterious features for diabetes and could provide novel therapeutic targets for the future. insulin sensitivity; athlete's paradox; lipid composition; plasma biomarkers THE GLOBAL BURDEN OF DIABETES continues to rise, fueled by the even greater epidemic of prediabetes (9). Nevertheless, not all of those with prediabetes will develop diabetes (27), highlighting the need to identify nonglycemic markers of disease progression that may also prove useful as new targets for the treatment of diabetes itself. Advances in lipidomics have provided new tools to investigate processes that govern the development of diabetes and its complications beyond that of glucose itself. Serum ceramides, a family of sphingolipids involved in diverse and relevant metabolic processes, have received considerable attention in relation to diabetes, making them prime candidates for further examination. Unlike other biomarkers, discrete serum ceramide profiles are rendered across the spectrum of diabetogenesis, creating speculation that ceramides not only mark but contribute to the disease process (8, 17).Much of this speculation is derived from data generated from in vitro and animal experimentation. Human data to date are limited largely to associations between serum ceramide concentration and risk for both diabetes (15) and related comp...

Intramuscular triglyceride (IMTG) concentration is elevated in insulin-resistant individuals and was once thought to promote insulin resistance. However, endurance-trained athletes have equivalent concentration of IMTG compared with individuals with type 2 diabetes, and have very low risk of diabetes, termed the "athlete's paradox." We now know that IMTG synthesis is positively related to insulin sensitivity, but the exact mechanisms for this are unclear. To understand the relationship between IMTG synthesis and insulin sensitivity, we measured IMTG synthesis in obese control subjects, endurance-trained athletes, and individuals with type 2 diabetes during rest, exercise, and recovery. IMTG synthesis rates were positively related to insulin sensitivity, cytosolic accumulation of DAG, and decreased accumulation of C18:0 ceramide and glucosylceramide. Greater rates of IMTG synthesis in athletes were not explained by alterations in FFA concentration, DGAT1 mRNA expression, or protein content. IMTG synthesis during exercise in Ob and T2D indicate utilization as a fuel despite unchanged content, whereas IMTG concentration decreased during exercise in athletes. mRNA expression for genes involved in lipid desaturation and IMTG synthesis were increased after exercise and recovery. Further, in a subset of individuals, exercise decreased cytosolic and membrane di-saturated DAG content, which may help explain insulin sensitization after acute exercise. These data suggest IMTG synthesis rates may influence insulin sensitivity by altering intracellular lipid localization, and decreasing specific ceramide species that promote insulin resistance.

Several recent reports indicate that the balance of skeletal muscle phosphatidylcholine (PC) and phosphatidylethanolamine (PE) is a key determinant of muscle contractile function and metabolism. The purpose of this study was to determine relationships between skeletal muscle PC, PE and insulin sensitivity, and whether PC and PE are dynamically regulated in response to acute exercise in humans. Insulin sensitivity was measured via intravenous glucose tolerance in sedentary obese adults (OB; n = 14), individuals with type 2 diabetes (T2D; n = 15), and endurance-trained athletes (ATH; n = 15). Vastus lateralis muscle biopsies were obtained at rest, immediately after 90 min of cycle ergometry at 50% maximal oxygen consumption (V̇o2 max), and 2-h postexercise (recovery). Skeletal muscle PC and PE were measured via infusion-based mass spectrometry/mass spectrometry analysis. ATH had greater levels of muscle PC and PE compared with OB and T2D (P < 0.05), with total PC and PE positively relating to insulin sensitivity (both P < 0.05). Skeletal muscle PC:PE ratio was elevated in T2D compared with OB and ATH (P < 0.05), tended to be elevated in OB vs. ATH (P = 0.07), and was inversely related to insulin sensitivity among the entire cohort (r = -0.43, P = 0.01). Muscle PC and PE were altered by exercise, particularly after 2 h of recovery, in a highly group-specific manner. However, muscle PC:PE ratio remained unchanged in all groups. In summary, total muscle PC and PE are positively related to insulin sensitivity while PC:PE ratio is inversely related to insulin sensitivity in humans. A single session of exercise significantly alters skeletal muscle PC and PE levels, but not PC:PE ratio.

Different serum lipids predict intramuscular TAG accumulation in obese people vs athletes. The ability of serum lipidomics to predict intramuscular DAG and ceramide in insulin-resistant humans may prove a new biomarker to determine risk for diabetes.

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Circulating increases in branched chain amino acid (BCAA) levels have long been associated with type II diabetes and metabolic syndrome. Emerging data also suggest that impaired BCAA catabolism may play a role in heart failure progression. BCAA are catabolized via the branched chain ketoacid (BCKA) dehydrogenase enzyme complex (BCKDH). BCKD kinase (BCKDK) is a negative regulator of BCAA catabolism through its inhibitory phosphorylation of the BCKDHE1a subunit, and the phosphatase PPM1k dephosphorylates this same site to activate BCAA catabolism. Using an inhibitor of BCKDK (BT2), BCAA catabolism is increased in vivo. Here, we utilized metabolomics to evaluate the contribution of BCAA catabolism to substrate preference in heart and skeletal muscle. Surprisingly, BCKDK inhibition with BT2 had no effect on incorporation of glucose into TCA cycle intermediates in heart or skeletal muscle. Because others have recently shown that the primary site of BCAA catabolism is skeletal muscle, we knocked down BCKDK and PPM1k in human skeletal myocytes to further investigate how BCKDK loss or inhibition affects substrate utilization. Similar to our in vivo observations, knockdown of BCKDK and PPM1k had no effect on glucose and pyruvate utilization in a mitochondrial function assay. However, an increase in maximal respiration was observed after BCKDK knockdown when fatty acids were used. To evaluate the mechanisms underlying this increase we then performed RNAseq in these cells after BCKDK and PPM1K knockdown and observed changes in a number of genes that may explain these alterations in substrate utilization. Finally, we performed C13 BCAA metabolomics in human skeletal myocytes after BT2 treatment or knockdown of BCKDK and PPM1k. Using BT2, we observed a dose-responsive reduction in BCKA production from C13 BCAA by the muscle cells as expected; however, though BCKA production was increased after PPM1k was knocked down, we surprisingly did not observe a decrease in BCKA production after BCKDK knockdown. Collectively these data suggest that BCKDK inhibition may improve metabolism and cardiac function by altering substrate preference in skeletal myocytes.