The Role of cAMP-PKA Pathway in Lactate-Induced Intramuscular Triglyceride Accumulation and Mitochondria Content Increase in Mice

The glycolytic product of exercise, lactate, has long been recognized to promote lipid accumulation by activation of G-protein-coupled receptor 81 (GPR81) and inhibition of the cyclic adenosine monophosphate-protein kinase A (cAMP –PKA) pathway in adipose tissue. Whether lactate causes a similar process in skeletal muscle is unclear. Lactate might also improve mitochondria content in skeletal muscle; however, the mechanism is not clarified either. In this study, using intramuscular injection of lactate to the gastrocnemius and intraperitoneal injection of forskolin (activator of cAMP-PKA pathway), we identified the role of the cAMP-PKA pathway in lactate-induced intramuscular triglyceride accumulation and mitochondrial content increase. The intramuscular triglyceride level in the gastrocnemius increased after 5weeks of lactate injection (p<0.05), and this effect was blocked by forskolin injection (p<0.05). Corresponding expression level changes of GPR81, P-PKA/PKA, P-CREB/cAMP-response element binding protein (CREB), and proteins related to lipid metabolism suggest that lactate could induce intramuscular triglyceride accumulation partly through the inhibition of the cAMP-PKA pathway. Meanwhile, the intramuscular expression of citrate synthase (CS) and the activity of CS increased after 5weeks of lactate injection (p<0.05), but the change of CS expression was not blocked by forskolin injection, suggesting other mechanisms might exist. Consequently, exploration for other potential mechanisms that might contribute to the lactate-induced mitochondria content increase was conducted. We found an increase in the contents of lactate-related metabolites in skeletal muscle mitochondria after acute lactate injection (the p-value of each analysis is less than 0.05). LHDA was also validated to exist in mitochondria in this study. These results provide a possibility for metabolism-related mechanisms of lactate-induced mitochondria content increase. Future study is needed to validate this hypothesis. In conclusion, lactate-induced intramuscular triglyceride accumulation is achieved by inhibition of lipolysis, and this process is regulated by the cAMP-PKA pathway. Promoted lipogenesis also contributes to lactate-induced triglyceride accumulation, and this process might also be regulated by the cAMP-PKA pathway. Lactate injection might increase mitochondria content and cAMP-PKA pathway might have a limited contribution, while other metabolism-related mechanisms might play a prominent role.

PL-017 High-fat overfeeding increases intramuscular triglyceride content and perilipin protein expression in human skeletal muscle

Objective High-fat high-calorie diets can induce whole body insulin resistance (IR) whilst increasing stores of intramuscular triglyceride (IMTG) contained within lipid droplets (LD). Perilipin (PLIN) proteins assist in IMTG storage. Synaptosomal-associated protein (SNAP23) may support LD growth and also direct IMTG-derived fatty acids (FA) to mitochondria for β-oxidation. The objectives of this study were: 1) to test the hypothesis that 7 days of high-fat overfeeding increases IMTG content to prevent lipid induced muscle IR and 2) identify changes in PLINs, SNAP23 and mitochondria content and colocalisation of PLINs with LD, and SNAP23 with LD and mitochondria. Methods Muscle biopsies were obtained from thevastus lateralisof thirteen healthy individuals (age: 23±1years, BMI: 24.4±0.7kg.m-2) before (0min) and during (30min) an oral glucose tolerance test (OGTT), pre and post 7-days consuming a high-fat (65% energy) high-calorie (+50% kcal) diet. IMTG, PLIN2, PLIN3, PLIN5, SNAP23 and mitochondria content were measured using (semi)-quantitative confocal immunofluorescence microscopy. PLIN2, PLIN3 and PLIN5 colocalisation to LD was measured using object-based colocalisation analyses. Pearson’s correlation coefficient quantified colocalisation between SNAP23 and plasma membrane (PM), mitochondria and LD. Phosphorylation of intermediates of the muscle insulin-signalling cascade (Akt and AS160) were measured at 0 and 30 min of the OGTT before and after the dietary intervention. Results Following overfeeding phosphorylation of Akt and AS160 in muscle was not impaired during the OGTT, however Matsuda index of whole-body insulin sensitivity decreased (-23%; P < 0.01). IMTG content increased in type I fibres (+100%; P < 0.001) due to both an increase in LD number (+43%; P < 0.001) and size (+44%; P< 0.001). Of the PLINs investigated, only PLIN3 content increased (+50%;P < 0.01) exclusively in type I fibres. PLIN2-associated LD increased (+80%; P < 0.01) in type I fibres only, whereas PLIN3 and PLIN5-associated LD were unaltered. SNAP23 and mitochondria content did not change, nor did the colocalisation of SNAP23 with the PM, mitochondria or LD. Conclusions Our data confirm the hypothesis that following high-fat overfeeding IMTG stores increased whilst activation of key muscle insulin signalling components were maintained. The increase in IMTG stores is likely supported by the concurrent increase in total PLIN3 content and a redistribution of existing stores of PLIN2 to the expanded LD pool in type I fibres. To confirm if increased IMTG storage protects muscle from IR future research should determine whether meal-derived FAs are directed to IMTG rather than ceramides and diacylglycerol.

Intramuscular triglyceride synthesis: importance in muscle lipid partitioning in humans

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.