shRNA-mediated down-regulation of Acsl1 reverses skeletal muscle insulin resistance in obese C57BL6/J mice

Abstract

Prolonged consumption of diet rich in fats is regarded as the major factor leading to the insulin resistance (IR) and type 2 diabetes (T2D). Emerging evidence link excessive accumulation of bioactive lipids such as diacylglycerol (DAG) and ceramide (Cer), with impairment of insulin signaling in skeletal muscle. Until recently, little has been known about the involvement of long-chain acyl-CoAs synthetases in the above mechanism. To examine possible role of long-chain acyl-coenzyme A synthetase 1 (Acsl1) (a major muscular ACSL isoform) in mediating HFD-induced IR we locally silenced Acsl1 in gastrocnemius of high-fat diet (HFD)-fed C57BL/6J mice through electroporation-delivered shRNA and compared it to non-silenced tissue within the same animal. Acsl1 down-regulation decreased the content of muscular long-chain acyl-CoA (LCACoA) and both the Cer (C18:1-Cer and C24:1-Cer) and DAG (C16:0/18:0-DAG, C16:0/18:2-DAG, C18:0/18:0-DAG) and simultaneously improved insulin sensitivity and glucose uptake as compared with non-silenced tissue. Acsl1 down-regulation decreased expression of mitochondrial β-oxidation enzymes, and the content of both the short-chain acylcarnitine (SCA-Car) and short-chain acyl-CoA (SCACoA) in muscle, pointing towards reduction of mitochondrial FA oxidation. The results indicate, that beneficial effects of Acsl1 partial ablation on muscular insulin sensitivity are connected with inhibition of Cer and DAG accumulation, and outweigh detrimental impact of decreased mitochondrial fatty acids metabolism in skeletal muscle of obese HFD-fed mice.

Fig 1. The effect of skeletal muscle Acsl1 partial ablation on the protein and gene expression of ACSL1 and muscular concentration of acyl-CoAs.

Panel (A1-A2)—mRNA expression of ACSL1; Panel (B1-B2)—protein expression of Acsl1; Panel (C1-C2) and (D1-D2)—the content of SCA-CoA and LCACoA, respectively. LFD_(+Acsl1)−gastrocnemius from LFD-fed mice, with intact Acsl1 expression (scrambled plasmid); HFD_(+Acsl1)—gastrocnemius from HFD-fed mice with intact Acsl1 expression (scrambled plasmid); HFD(-Acsl1)−contralateral hindlimb gastrocnemius from HFD-fed mice, with down-regulated Acsl1 expression (silencing shRNA plasmid). Panels A1 to D1 present effect of diet (LFD_(+Acsl1) and HFD_(+Acsl1) muscle). Panels A2 to D2 present effect of Acsl1 silencing within HFD-fed animals (HFD_(+Acsl1) vs HFD_(-Acsl1) muscle). Values are median ± interquartile range; n = 6 per group (mRNA and protein); n = 8 per group (other data). * -p ≤ 0.05; **- p ≤ 0.01.

Fig 2. The effect of skeletal muscle Acsl1 down-regulation on the expression of fatty acid transporters, CPT1B, mitochondrial β-oxidation proteins, ACC phosphorylation and the content of acyl-carnitines in the gastrocnemius of high-fat-diet-fed mice.

Panels (A1-C1; A2-C2)—protein expression of CD36, FABPpm, FATP; Panel (D1-D2)—protein expression of CPT1B; Panel (E1-E2)—phosphorylation state of ACC protein; Panels (F1-H1; F2-H2)–protein expression of mitochondrial β-oxidation trifunctional enzyme, very long- and medium-chain acyl-CoA dehydrogenases (HADHA, ACADVL and ACADM, respectively); Panels (I1-J1; I2-J2)—the content of short-chain (SCA-Car) and long-chain (LCA-Car) acyl-carnitines. LFD_(+Acsl1)−gastrocnemius from LFD-fed mice, with intact Acsl1 expression (scrambled plasmid); HFD_(+Acsl1)—gastrocnemius from HFD-fed mice with intact Acsl1 expression (scrambled plasmid); HFD(-Acsl1)−contralateral hindlimb gastrocnemius from HFD-fed mice, with down-regulated Acsl1 expression (silencing shRNA plasmid). Panels A1 to J1 present effect of diet (LFD_(+Acsl1) and HFD_(+Acsl1) muscle). Panels A2 to J2 present effect of Acsl1 silencing within HFD-fed animals (HFD_(+Acsl1) vs HFD_(-Acsl1) muscle).Values are median ± interquartile range; n = 6 per group (protein); n = 8 per group (other data). ns -p > 0.05; * -p ≤ 0.05; **- p ≤ 0.01.

Fig 3. Total lipid content in gastrocnemius in Acsl1-silenced mouse gastrocnemius.

Panel (A1-A2)—total content of triacylglycerols (TAG); Panel (B1-B2)—total content of diacylglycerols (DAG); Panel (C1-C2)—total content of ceramide (Cer). LFD_(+Acsl1)−gastrocnemius from LFD-fed mice, with intact Acsl1 expression (scrambled plasmid); HFD_(+Acsl1)—gastrocnemius from HFD-fed mice with intact Acsl1 expression (scrambled plasmid); HFD(-Acsl1)−contralateral hindlimb gastrocnemius from HFD-fed mice, with down-regulated Acsl1 expression (silencing shRNA plasmid). Panels A1 to C1 present the effect of diet (LFD_(+Acsl1) vs HFD_(+Acsl1) muscle). Panels A2 to C2 present effect of Acsl1 silencing within HFD-fed animals (HFD_(+Acsl1) vs HFD_(-Acsl1) muscle). Values are median ± interquartile range; n = 8 per group. **- p ≤ 0.01.

Fig 4. Insulin signaling pathway in Acsl1-silenced mouse gastrocnemius.

Panel (A1-A2)—insulin receptor phosphorylation (pIR Y972); Panel (B1-B2)—serine phosphorylation (pIRS-1 S1101) and Panel (C1-C2) tyrosine phosphorylation (pIRS-1 Y632) of insulin receptor substrate 1 (IRS-1); Panel (D1-D2)—protein expression of phosphoinositide 3-kinase (PI3K); Panel (E1-E2)—serine phosphorylation of Akt/ protein kinase B (pAKT S473); Panel (F1-F2)—threonine phosphorylation of Akt/ protein kinase B (pAKT T308); Panel (G1-G2)—serine phosphorylation of Akt/PKB 160kDa substrate (pAS160 S588). LFD_(+Acsl1)−gastrocnemius from LFD-fed mice, with intact Acsl1 expression (scrambled plasmid); HFD_(+Acsl1)—gastrocnemius from HFD-fed mice with intact Acsl1 expression (scrambled plasmid); HFD(-Acsl1)−contralateral hindlimb gastrocnemius from HFD-fed mice, with down-regulated Acsl1 expression (silencing shRNA plasmid). Panels A1 to F1 present the effect of diet (LFD_(+Acsl1) vs HFD_(+Acsl1) muscle). Panels A2 to F2 present effect of Acsl1 silencing within HFD-fed animals (HFD_(+Acsl1) vs HFD_(-Acsl1) muscle). Values are median ± interquartile range; n = 6 per group. * -p ≤ 0.05.

Fig 5. The effect of Acsl1 partial ablation on mouse gastrocnemius muscle insulin-stimulated glucose uptake.

Panel (A1-A2)—protein expression of glucotransporter 4 (GLUT4); Panel (B1-B2)—insulin-stimulated glucose uptake. LFD_(+Acsl1)−gastrocnemius from LFD-fed mice, with intact Acsl1 expression (scrambled plasmid); HFD_(+Acsl1)—gastrocnemius from HFD-fed mice with intact Acsl1 expression (scrambled plasmid); HFD(-Acsl1)−contralateral hindlimb gastrocnemius from HFD-fed mice, with down-regulated Acsl1 expression (silencing shRNA plasmid). Panels A1 and B1 present the effect of diet (LFD_(+Acsl1) vs HFD_(+Acsl1) muscle). Panels A2 and B2 present effect of Acsl1 silencing within HFD-fed animals (HFD_(+Acsl1) vs HFD_(-Acsl1) muscle). Values are median ± interquartile range; n = 6 per group. * -p ≤ 0.05.