Overexpression of carnitine palmitoyltransferase-1 in skeletal muscle is sufficient to enhance fatty acid oxidation and improve high-fat diet-induced insulin resistance

Abstract

Objective: Skeletal muscle insulin resistance is associated with lipid accumulation, but whether insulin resistance is due to reduced or enhanced flux of long-chain fatty acids into the mitochondria is both controversial and unclear. We hypothesized that skeletal muscle-specific overexpression of the muscle isoform of carnitine palmitoyltransferase 1 (CPT1), the enzyme that controls the entry of long-chain fatty acyl CoA into mitochondria, would enhance rates of fatty acid oxidation and improve insulin action in muscle in high-fat diet insulin-resistant rats.

Research design and methods: Rats were fed a standard (chow) or high-fat diet for 4 weeks. After 3 weeks, in vivo electrotransfer was used to overexpress the muscle isoform of CPT1 in the distal hindlimb muscles (tibialis anterior and extensor digitorum longus [EDL]). Skeletal muscle insulin action was examined in vivo during a hyperinsulinemic-euglycemic clamp.

Results: In vivo electrotransfer produced a physiologically relevant increase of approximately 20% in enzyme activity; and although the high-fat diet produced insulin resistance in the sham-treated muscle, insulin action was improved in the CPT1-overexpressing muscle. This improvement was associated with a reduction in triacylglycerol content, the membrane-to-cytosolic ratio of diacylglycerol, and protein kinase C theta activity. Importantly, overexpression of CPT1 did not affect markers of mitochondrial capacity or function, nor did it alter skeletal muscle acylcarnitine profiles irrespective of diet.

Conclusions: Our data provide clear evidence that a physiological increase in the capacity of long-chain fatty acyl CoA entry into mitochondria is sufficient to ameliorate lipid-induced insulin resistance in muscle.

FIG. 1.

CPT1 overexpression increases CPT1 activity and fatty acid oxidation and reduces fatty acid incorporation into TAG. Representative immunoblots and quantification of CPT1 protein expression in tibialis anterior (A), representative immunoblots and quantification of CPT1 protein expression in EDL muscle (B), CPT1 activity in isolated mitochondria from tibialis anterior muscles (C), fatty acid oxidation rate in isolated EDL muscles ex vivo (D), and fatty acid incorporation into TAG in isolated EDL muscles ex vivo (E) from a leg electroporated with an empty vector (▪) or CPT1 vector (□) in animals placed either on a standard chow diet or high-fat diet for 4 weeks. Data are means ± SE; n = 8. *P < 0.05 main effect for CPT1 expression; †P < 0.05 main effect for diet; #P < 0.05 vs. high-fat diet control leg.

FIG. 2.

Muscle acylcarnitine levels and the ratio of fatty acid oxidation to CO₂ and ASMs in overnight fasted animals. Acylcarnitine levels in tibialis anterior muscle (A) and the ratio of fatty acid oxidation to CO₂ and ASMs (B) in EDL muscle from a leg electroporated with an empty vector or CPT1 vector, in animals placed either on a standard chow diet or high-fat diet (HFD) for 4 weeks. Data are means ± SE; n = 8.

FIG. 3.

CPT1 overexpression protects against high-fat feeding–induced decrements in insulin action and signaling in skeletal muscle ex vivo. Rates of basal and insulin-stimulated 2-deoxyglucose uptake in isolated EDL muscle from a leg electroporated with an empty vector (▪) or CPT1 vector (□), in animals placed on a high-fat diet for 4 weeks (A); representative immunoblots and quantification of phosphorylation of (Tyr⁶¹²)/total IRS1 (B); and phosphorylation (Ser⁴⁷³)/total Akt (C) in isolated EDL muscle obtained from a leg electroporated with an empty vector or CPT1 vector, in animals placed on a high fat-diet for 4 weeks and then incubated in the presence (Insulin) or absence (Basal) of insulin. Dashed line represents the level of insulin-mediated glucose uptake or phosphorylation observed in the empty vector leg from chow-fed animals. Data are means ± SE; n = 7. *P < 0.05 vs. high-fat diet control leg under insulin-stimulated conditions.

FIG. 4.

CPT1 overexpression protects against high-fat feeding–induced decrements in insulin action and signaling in skeletal muscle in vivo. 2-Deoxyglucose uptake (Rg′) (A) and representative immunoblots and quantification of phosphorylation (Ser⁴⁷³)/total Akt (B) in tibialis anterior muscle obtained from a leg electroporated with an empty vector or CPT1 vector, in animals placed on a high-fat diet for 4 weeks and then underwent a hyperinsulinemic-euglycemic clamp for 120 min. Dashed line represents the level of insulin-mediated glucose uptake or Akt phosphorylation observed in the empty vector leg from chow-fed animals. Data are means ± SE; n = 7. †P < 0.05 main effect for diet; #P < 0.05 vs. high-fat diet control leg.

FIG. 5.

CPT1 overexpression decreases TAG accumulation and the membrane-to-cytosolic ratio of DAG in skeletal muscle. TAG content (A) and the membrane-to-cytosol ratio of DAG (B) and ceramide (C) in tibialis anterior muscle obtained from a leg electroporated with an empty vector (▪) or CPT1 vector (□), in animals placed either on a standard chow diet or high-fat diet for 4 weeks. Data are means ± SE; n = 5–7. *P < 0.05 main effect for CPT1 expression; †P < 0.05 main effect for diet; #P < 0.05 vs. high-fat diet control leg.

FIG. 6.

CPT1 overexpression protects against high-fat feeding–induced decrements in serine phosphorylation of IRS1 and activation of PKCθ and JNK in skeletal muscle. Representative immunoblots and quantification of phosphorylation (Ser³⁰⁷)/total IRS1 (A), membrane-to-cytosol ratio of PKCθ (B), and phosphorylation (Thr¹⁸³/Tyr¹⁸⁵)/total JNK (C) in tibialis anterior muscle obtained from a leg electroporated with an empty vector (▪) or CPT1 vector (□), in animals placed either on a standard chow diet or high-fat diet for 4 weeks. Data are means ± SE; n = 5–7. *P < 0.05 main effect for CPT1 expression; †P < 0.05 main effect for diet; #P < 0.05 vs. high-fat diet control leg.