Augmenting muscle diacylglycerol and triacylglycerol content by blocking fatty acid oxidation does not impede insulin sensitivity

Abstract

A low fat oxidative capacity has been linked to muscle diacylglycerol (DAG) accumulation and insulin resistance. Alternatively, a low fat oxidation rate may stimulate glucose oxidation, thereby enhancing glucose disposal. Here, we investigated whether an ethyl-2-[6-(4-chlorophenoxy)hexyl]-oxirane-2-carboxylate (etomoxir)-induced inhibition of fat oxidation leads to muscle fat storage and insulin resistance. An intervention in healthy male subjects was combined with studies in human primary myotubes. Furthermore, muscle DAG and triacylglycerol (TAG), mitochondrial function, and insulin signaling were examined in etomoxir-treated C57bl6 mice. In humans, etomoxir administration increased glucose oxidation at the expense of fat oxidation. This effect was accompanied by an increased abundance of GLUT4 at the sarcolemma and a lowering of plasma glucose levels, indicative of improved glucose homeostasis. In mice, etomoxir injections resulted in accumulation of muscle TAG and DAG, yet improved insulin-stimulated GLUT4 translocation. Also in human myotubes, insulin signaling was improved by etomoxir, in the presence of increased intramyocellular lipid accumulation. These insulin-sensitizing effects in mice and human myotubes were accompanied by increased phosphorylation of AMP-activated protein kinase (AMPK). Our results show that a reduction in fat oxidation leading to accumulation of muscle DAG does not necessarily lead to insulin resistance, as the reduction in fat oxidation may activate AMPK.

Fig. 1.

The effect of etomoxir in healthy males on (A) Twenty-four hour glucose, fat, and protein oxidation rates (modified from ref. 33). (B) HOMA index. (C) Fat oxidation during a 2-h cycling protocol, represented as rates (Left) and area under the curve (in percentage, Right). (D) Blood glucose values during a 2-h cycling protocol. (E) Relative fraction of total GLUT4 associated with the sarcolemma and separated for type 1 and 2 muscle fibers. Data are expressed as mean + SEM (n = 10). *P < 0.05 etomoxir vs. placebo.

Fig. 2.

Effect of etomoxir on the respiratory rates of freshly isolated skeletal muscle mitochondria using a two-chamber oxygraph. Mitochondria were incubated in a respiration media containing malate. State-3 respiration (upon substrates and ADP) was achieved by addition of (A) palmitoyl CoA + carnitine, (B) palmitoyl-carnitine, and (C) pyruvate as different substrates. Maximal oxygen flux (state U) was obtained by titration of the chemical uncoupler FCCP in the presence of oligomycin. Values are expressed in nmol/mg mitochondrial protein/min (mean + SEM) (placebo n = 6, etomoxir n = 5). *P < 0.05 etomoxir vs. placebo.

Fig. 3.

Effect of etomoxir in mice on (A) IMCL levels and (B) total DAG levels in tibialis anterior muscle and saturated (SAT), monounsaturated (MUFA), and polyunsaturated (PUFA) fractions. Western blotting of the lipid droplet coating proteins (C) PLIN2 and (D) PLIN5 in tibialis anterior muscle of etomoxir-treated mice. Data are expressed as mean + SEM (placebo n = 6, etomoxir n = 5). *P < 0.05 etomoxir vs. placebo.

Fig. 4.

Effect of etomoxir on (A) whole-body glucose tolerance measured by an ipGTT (2 g/kg bw glucose) in mice. Effect of etomoxir on the expression of several proteins in the insulin signaling cascade was investigated in tibialis anterior muscle of mice. Western blotting of (B) pIRS1 (Ser307), (C) pAkt, and (D) GLUT4. (E) Phosphorylation of AMPK was examined by Western blotting in mouse tibialis anterior muscle. Human myotubes were incubated with 100 μM etomoxir and (F) IMCL accumulation is shown after 0, 3, and 6 h of etomoxir incubation. (G) Western blotting of pAkt in human myotubes incubated with etomoxir for 0, 3, and 6 h in the absence or presence of insulin. (H) pAMPK protein expression upon 0, 3, and 6 h of etomoxir incubation in human myotubes, measured by Western blotting. Representative Western blots are shown in Fig. S2. Data are expressed as mean + SEM (n = 10 per group for mice experiments, n = 6–8 per group for cell experiments) *P < 0.05 etomoxir vs. placebo.