Increasing Acyl CoA thioesterase activity alters phospholipid profile without effect on insulin action in skeletal muscle of rats

Abstract

Increased lipid metabolism in muscle is associated with insulin resistance and therefore, many strategies have been employed to alter fatty acid metabolism and study the impact on insulin action. Metabolism of fatty acid requires activation to fatty acyl CoA by Acyl CoA synthases (ACSL) and fatty acyl CoA can be hydrolysed by Acyl CoA thioesterases (Acot). Thioesterase activity is low in muscle, so we overexpressed Acot7 in muscle of chow and high-fat diet (HFD) rats and investigated effects on insulin action. Acot7 overexpression modified specific phosphatidylcholine and phosphatidylethanolamine species in tibialis muscle of chow rats to levels similar to those observed in control HFD muscle. The changes in phospholipid species did not alter glucose uptake in tibialis muscle under hyperinsulinaemic/euglycaemic clamped conditions. Acot7 overexpression in white extensor digitorum longus (EDL) muscle increased complete fatty acid oxidation ex-vivo but was not associated with any changes in glucose uptake in-vivo, however overexpression of Acot7 in red EDL reduced insulin-stimulated glucose uptake in-vivo which correlated with increased incomplete fatty acid oxidation ex-vivo. In summary, although overexpression of Acot7 in muscle altered some aspects of lipid profile and metabolism in muscle, this had no major effect on insulin-stimulated glucose uptake.

Figure 1

Overexpression of AAV9-tMCK-Acot7 and its effect on the opposing ACSL enzyme in tibialis muscle. Acot7 protein levels (A), thioesterase activity (B) ACSL1 protein level (C) Acyl-CoA synthase activity (D). Data is mean ± SEM, *p < 0.05, 2-way ANOVA, diet; ^†p < 0.01 2-way ANOVA, Acot7; ^#p < 0.01 post-hoc Holm and Sidak test, (n = 6).

Figure 2

Effect of AAV9-tMCK-Acot7 overexpression on lipid profile in tibialis muscle. Ceramide (Cer), cardiolipin (CL), diacylglyceride (DAG), triacylglyceride (TAG) (A); lysophosphatidylcholine (LPC), phosphatidylcholine (PC), ether-linked phosphatidylcholine (PC-O), phosphatidylglycerol (PG), phosphatidylserine (PS), (B) phosphatidylethanolamine (PE), ether-linked phosphatidylethanolamine (PE-O), sphingomyelin (SM) (C). Data is fold difference over control chow; *p < 0.05, 2-way ANOVA, diet; ^†p < 0.05, 2-way ANOVA, Acot7; ^#p < 0.05, post-hoc Holm and Sidak test for chow, ^§p < 0.05, post-hoc Holm and Sidak test for HFD animals (n = 6).

Figure 3

Effect of AAV9-tMCK-Acot7 overexpression on saturated, MUFA and PUFA content of phospholipids in tibialis muscle. Fatty acid composition of each lipid class as a percent of total fatty acid content of PC (phosphatidylcholine), PC-O (ether-linked phosphatidylcholine), PE (phosphatidylethanolamine), PE-O (ether-linked phorsphatidylethanolamine), PS (phosphatidylserine) lipid classes. Content of saturated fatty acid (SFA) (A) monounsaturated fatty acid (MUFA) (B) polyunsaturated fatty acid (PUFA) (C) are presented as the mean ± SEM for n = 6. *p < 0.05, 2-way ANOVA for effect of diet; ^†p < 0.05, 2-way ANOVA for effect of Acot7, ^#p < 0.05, post-hoc Holm and Sidak test for the effect of Acot7 overexpression within the Control Chow or HFD fed rats.

Figure 4

Effect of AAV9-tMCK-Acot7 on mitochondrial proteins in tibialis muscle. The dashed line indicates the level of the protein for control chow group. The protein levels of all other groups (control HFD, Acot7 overexpressing chow, Acot7 overexpressing HFD) have been normalised to control chow and shown as a fold difference. Data is mean ± SEM, n = 8. These animals received either AAV-GFP in control leg (n = 3) or saline injections (n = 5). No differences between the controls were observed.

Figure 5

Ex vivo effect of AAV9-tMCK-Acot7 on substrate oxidation in EDL (extensor digitorum longus) muscle strips. ¹⁴C-oleic acid incorporated into ¹⁴CO₂ (A), ¹⁴C-oleic acid incorporated into acid soluble metabolites (ASM) (B), total fatty acid oxidation (FAO) (C) ¹⁴C-glucose incorporated into ¹⁴CO₂ (D) in white and red EDL muscle strips. ^†p < 0.05, 2-way ANOVA, Acot7; ^#p < 0.05 post-hoc Holm and Sidak test. Data is mean ± SEM (n = 10–13).

Figure 6

In vivo effect of AAV9-tMCK-Acot7 on insulin stimulated glucose uptake in different muscle. Insulin stimulated glucose uptake (Rg’) in tibialis (A), white extensor digitorum longus (EDL) (B), red extensor digitorum longus (EDL) (C). ^§p = 0.05, *p < 0.01, 2-way ANOVA, diet; ^†p < 0.05, 2-way ANOVA, Acot7. Data is mean ± SEM (n = 6–8).