Altered skeletal muscle lipase expression and activity contribute to insulin resistance in humans

Abstract

Objective: Insulin resistance is associated with elevated content of skeletal muscle lipids, including triacylglycerols (TAGs) and diacylglycerols (DAGs). DAGs are by-products of lipolysis consecutive to TAG hydrolysis by adipose triglyceride lipase (ATGL) and are subsequently hydrolyzed by hormone-sensitive lipase (HSL). We hypothesized that an imbalance of ATGL relative to HSL (expression or activity) may contribute to DAG accumulation and insulin resistance.

Research design and methods: We first measured lipase expression in vastus lateralis biopsies of young lean (n = 9), young obese (n = 9), and obese-matched type 2 diabetic (n = 8) subjects. We next investigated in vitro in human primary myotubes the impact of altered lipase expression/activity on lipid content and insulin signaling.

Results: Muscle ATGL protein was negatively associated with whole-body insulin sensitivity in our population (r = -0.55, P = 0.005), whereas muscle HSL protein was reduced in obese subjects. We next showed that adenovirus-mediated ATGL overexpression in human primary myotubes induced DAG and ceramide accumulation. ATGL overexpression reduced insulin-stimulated glycogen synthesis (-30%, P < 0.05) and disrupted insulin signaling at Ser1101 of the insulin receptor substrate-1 and downstream Akt activation at Ser473. These defects were fully rescued by nonselective protein kinase C inhibition or concomitant HSL overexpression to restore a proper lipolytic balance. We show that selective HSL inhibition induces DAG accumulation and insulin resistance.

Conclusions: Altogether, the data indicate that altered ATGL and HSL expression in skeletal muscle could promote DAG accumulation and disrupt insulin signaling and action. Targeting skeletal muscle lipases may constitute an interesting strategy to improve insulin sensitivity in obesity and type 2 diabetes.

FIG. 1.

Skeletal muscle lipase protein expression in lean, obese, and type 2 diabetic subjects. Quantitative bar graph of ATGL protein (A), HSL protein (B), HSL Ser660 phosphorylation (C), and the ratio HSL Ser660 phosphorylation to total HSL (D) in vastus lateralis samples of lean, obese, and type 2 diabetic subjects. E: Representative blots of lipases and the loading control GAPDH. F: Relationship between vastus lateralis ATGL protein expression and glucose disposal rate measured by euglycemic hyperinsulinemic clamp across individuals. White square, lean; black square, obese; open circle, type 2 diabetes. *P < 0.05 compared with obese; §P = 0.08 compared with lean.

FIG. 7.

Selective HSL inhibition disrupts insulin signaling. A: Total DAGs were measured in control myotubes and myotubes treated for 24 h with 1 μmol/L of the selective HSL inhibitor BAY (n = 6). B: Representative blots of Tyr1162 pIR, total IRalpha, Ser473 pAkt, total Akt, and GAPDH in the presence (+) or absence (−) of insulin in control myotubes and myotubes treated with BAY. Quantitative bar graphs of (C) Tyr612-IRS-1 phosphorylation (n = 4) and (D) Ser473 Akt phosphorylation (n = 4) in control myotubes and myotubes treated with the BAY compound. **P < 0.01; ***P < 0.001 vs. basal; #P < 0.05 vs. control insulin.

FIG. 2.

ATGL overexpression increases TAG hydrolysis in myotubes. A: Quantitative bar graph of ATGL protein during adenovirus-mediated ATGL overexpression vs. GFP control (n = 4). Insets are showing representative blots of two independent experiments. TAG hydrolase activity (B) and DAG hydrolase activity (C) were measured in control myotubes (GFP) and myotubes overexpressing ATGL (n = 4). Pulse-chase studies using [1-¹⁴C]oleate were performed to determine the kinetics of the different lipid pools in response to ATGL overexpression. The rate of incorporation of radiolabeled oleate into (D) TAG, (E) DAG, and (F) FFA was determined in control myotubes (GFP) and myotubes overexpressing ATGL. *P < 0.05; **P < 0.01 vs. GFP (n = 4).

FIG. 3.

Elevated ATGL expression promotes DAG and ceramide accumulation. Determination of (A) TAG, (B) DAG, and (C) ceramide content in control myotubes (GFP) and myotubes overexpressing ATGL. *P < 0.05 (n = 5).

FIG. 4.

Elevated ATGL expression disrupts insulin signaling and action. A: Glycogen synthesis was measured in the absence (open bars) or presence (black bars) of 100 nmol/L insulin in control myotubes (GFP) and myotubes overexpressing ATGL. *P < 0.05, **P < 0.01 vs. basal; #P < 0.05 vs. GFP insulin (n = 8 per group). B: Quantitative bar graph of basal Ser1101 IRS-1 phosphorylation (n = 4). ***P < 0.001 vs. GFP. C: Quantitative bar graph of Ser473 Akt phosphorylation (n = 11). *P < 0.05, ***P < 0.001 vs. basal; #P < 0.05 vs. Ad-GFP. D: Representative blots of Ser1101 pIRS-1, total IRS-1, Ser473 pAkt, total Akt, and the loading control GAPDH in the presence (+) or absence (−) of 100 nmol/L of insulin in control myotubes (GFP) and myotubes overexpressing ATGL.

FIG. 5.

ATGL-mediated insulin resistance involves PKC activation. A: Representative blots of Ser473 pAkt, total Akt, and GAPDH in the presence (+) or absence (−) of insulin and the nonselective PKC inhibitor calphostin C (1 μmol/L) in control myotubes (GFP) and myotubes overexpressing ATGL. B: Quantitative bar graph of insulin-stimulated Ser473 Akt phosphorylation (n = 3); insulin-stimulated Akt phosphorylation was calculated as the Δ between basal and insulin stimulation in each condition. *P < 0.05 vs. GFP; ##P < 0.01 vs. GFP calphostin C. C: Insulin-stimulated glycogen synthesis in myotubes expressing GFP and ATGL in the absence (control) or presence of calphostin C. Glycogen synthesis was expressed as the Δ change between glycogen synthesis under insulin stimulation and glycogen synthesis at baseline. *P < 0.05 vs. GFP (n = 4).

FIG. 6.

Rescue of ATGL-mediated insulin resistance by HSL. A: Representative blots of Ser473 pAkt, total Akt, and GAPDH in the presence (+) or absence (−) of insulin in control myotubes (GFP) and myotubes overexpressing ATGL alone (Ad-ATGL) or in combination with HSL (Ad-ATGL+Ad-HSL). B: Quantitative bar graph of insulin-stimulated Ser473 Akt phosphorylation (n = 4); insulin-stimulated Akt phosphorylation was calculated as the Δ between basal and insulin stimulation in each condition. *P < 0.05 vs. GFP. C: Insulin-stimulated glycogen synthesis was measured in control myotubes (GFP) and myotubes overexpressing ATGL alone (Ad-ATGL) or in combination with HSL (Ad-ATGL+Ad-HSL). *P < 0.05 vs. GFP (n = 6).