Remodeling lipid metabolism and improving insulin responsiveness in human primary myotubes

Abstract

Objective: Disturbances in lipid metabolism are strongly associated with insulin resistance and type 2 diabetes (T2D). We hypothesized that activation of cAMP/PKA and calcium signaling pathways in cultured human myotubes would provide further insight into regulation of lipid storage, lipolysis, lipid oxidation and insulin responsiveness.

Methods: Human myoblasts were isolated from vastus lateralis, purified, cultured and differentiated into myotubes. All cells were incubated with palmitate during differentiation. Treatment cells were pulsed 1 hour each day with forskolin and ionomycin (PFI) during the final 3 days of differentiation to activate the cAMP/PKA and calcium signaling pathways. Control cells were not pulsed (control). Mitochondrial content, (14)C lipid oxidation and storage were measured, as well as lipolysis and insulin-stimulated glycogen storage. Myotubes were stained for lipids and gene expression measured.

Results: PFI increased oxidation of oleate and palmitate to CO(2) (p<0.001), isoproterenol-stimulated lipolysis (p = 0.01), triacylglycerol (TAG) storage (p<0.05) and mitochondrial DNA copy number (p = 0.01) and related enzyme activities. Candidate gene and microarray analysis revealed increased expression of genes involved in lipolysis, TAG synthesis and mitochondrial biogenesis. PFI increased the organization of lipid droplets along the myofibrillar apparatus. These changes in lipid metabolism were associated with an increase in insulin-mediated glycogen storage (p<0.001).

Conclusions: Activation of cAMP/PKA and calcium signaling pathways in myotubes induces a remodeling of lipid droplets and functional changes in lipid metabolism. These results provide a novel pharmacological approach to promote lipid metabolism and improve insulin responsiveness in myotubes, which may be of therapeutic importance for obesity and type 2 diabetes.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1. Fatty acid oxidation.

Cultured myotubes were incubated with 100 µM of [1-14C] oleate (A) or [1-14C] palmitate (B) for 3 h, and radiolabel incorporation into CO2 was determined as a measure of complete fat oxidation (FAO). Radiolabel incorporation into acid soluble molecules (ASM) was measured to assess incomplete FAO. The ratio of CO2 to ASM was calculated for oleate (C) and palmitate (D). Data are expressed as mean ± SEM of 4 separate experiments. ** p<0.01, *** p<0.001 versus control.

Figure 2. Mitochondrial content and enzymes activity.

(A) Mitochondrial content determined by mtDNA copy number. (B) Mitochondrial content determined by Mitotracker Green FM. (C) β-hydroxyacyl-CoA dehydrogenase (β-HAD) activity adjusted for protein content. (D) Citrate synthase (CS) activity adjusted for protein content. Data are expressed as mean ± SEM of 4 separate experiments. ** p

Figure 3. Confocal microscopy of lipid droplets.

Lipid droplets were stained with Bodipy 493/503 (green) and nuclei were stained with DAPI (blue). Note the increase in the organization of lipid droplets along the myofibrillar apparatus in panels B (20×) and D (40×) upon PFI treatment.

Figure 4. Lipid storage and lipolytic capacity.

(A) Cultured myotubes were incubated with 100 µM [1-14C] oleate for 3 h, then the cells were harvested and total lipid extracted. Total lipid synthesis was measured as incorporation of radiolabel into total cellular lipids. (B) Lipid intermediates species such as phospholipids (PL), diacylglycerol (DAG), and triacylglycerol (TAG) rates of synthesis were measured by thin-layer chromatography. (C) Lipolysis measured as glycerol release was determined by subtracting baseline values to values during the stimulation with 1 µM of isoproterenol (a non-selective β-adrenergic agonist). The graph represents the changes in lipolysis at baseline in response to isoproterenol in control and PFI conditions. Data are expressed as mean ± SEM of 4 separate experiments. * p<0.05, ** p<0.01 versus control.

Figure 5. Glucose metabolism and insulin sensitivity.

(A) Incorporation of [U-14C] glucose into glycogen was measured in either the absence (open bar) or the presence (black bar) of 100 nM of insulin. ** p<0.01 versus baseline, † p<0.05 versus control. (B) Insulin-stimulated glycogen storage was measured by the change in glycogen storage at baseline in response to insulin and used as a marker of insulin responsiveness in this model. Data are expressed as mean ± SEM of 4 separate experiments. ** p<0.01 versus control.