Critical role of stearoyl-CoA desaturase-1 (SCD1) in the onset of diet-induced hepatic insulin resistance

Abstract

Stearoyl-CoA desaturase-1 (SCD1) catalyzes the synthesis of monounsaturated fatty acids from saturated fatty acids. Mice with a targeted disruption of Scd1 gene locus are lean and display increased insulin sensitivity. To examine whether Scd1 activity is required for the development of diet-induced hepatic insulin resistance, we used a sequence-specific antisense oligodeoxynucleotide (ASO) to lower hepatic Scd1 expression in rats and mice with diet-induced insulin resistance. Treatment of rats with Scd1 ASO markedly decreased liver Scd1 expression (approximately 80%) and total Scd activity (approximately 50%) compared with that in rats treated with scrambled ASO (control). Insulin clamp studies revealed severe hepatic insulin resistance in high-fat-fed rats and mice that was completely reversed by 5 days of treatment with Scd1 ASO. The latter treatment decreased glucose production (by approximately 75%), gluconeogenesis, and glycogenolysis. Downregulation of Scd1 also led to increased Akt phosphorylation and marked decreases in the expression of glucose-6-phosphatase (Glc-6-Pase) and phosphoenolpyruvate carboxykinase (PEPCK). Thus, Scd1 is required for the onset of diet-induced hepatic insulin resistance.

Figure 1. Systemic administration of Scd1 ASO downregulates liver Scd1 expression and activity in OF rats.

(A) Schematic of Scd1 role in lipid synthesis. CE, cholesteryl esters; PL, phospholipids; WE, wax esters. (B) Protocol for Scd1 ASO delivery (top panel). Rats received an i.p. injection of either a control ASO (SCR ASO) or Scd1 ASO (100 mg/kg of body weight) on day 0; on day 3, the animals were switched to a lard-enriched diet (HF) and received a second injection of ASOs; lastly, on day 5, an insulin clamp procedure was performed. The livers of OF animals treated with Scd1 ASO (black bars) displayed an 80% decrease in Scd1 mRNA and a 50% decrease in both Scd1 activity and C18 desaturation index. OF (gray bars) did not modify any of these parameters when compared with SC rats (white bars). (C) OF caused a decrease in hepatic LCFA CoA levels, which were restored to SC levels by Scd1 ASO treatment (top left panel). Liver Scd1 deficiency in OF animals (black bars) resulted in a 3.5-fold increase in liver TG (top right panel) when compared with SCR ASO OF animals (gray bars). JNK phosphorylation and activity were markedly increased in OF Scd1-deficient animals as compared with OF control animals, while OF (gray bars) did not have any effect when compared with SC animals (white bars). Wet wt, wet weight. *P < 0.05 versus SCR ASO OF.

Figure 2. Scd1 deficiency moderately decreases the expression of lipogenic genes in the liver.

(A and B) At the completion of insulin clamp studies, hepatic Scd1 deficiency in OF animals (black bars) did not significantly modify the levels of AMPK and FAS or the phosphorylation of AMPK and only modestly decreased ACC, p-ACC, and CYP7A1 in the liver when compared with SCR ASO OF (gray bars) and SC animals (white bars). (C and D) Under basal (overnight fast) conditions, hepatic Scd1 deficiency in OF animals (black bars) significantly decreased the expression of FAS, ACC, and CYP7A1 in the liver when compared with SCR ASO OF (gray bars) animals. *P < 0.05 versus SCR ASO OF.

Figure 3. Scd1 deficiency negates the effects of OF on hepatic glucose fluxes.

(A) Protocol for the hyperinsulinemic-pancreatic clamp procedure. (B) Glucose infusion rate, glucose uptake, and inhibition of glucose production (expressed as percentage inhibition from basal) during the clamp period in SC (white bars), control OF (gray bars), and Scd1-deficient OF animals (black bars). (C) Schematic of the major pathways and enzymatic reactions contributing to glucose production in the liver. [¹⁴C]-PEP, ¹⁴C-phosphoenolpyruvate; ³H/¹⁴C-UDPG, ³H/¹⁴C-UDP glucose; GK, glucokinase. (D) Glucose production, Glc-6-Pase flux, and glucose cycling during the clamp period in control SC (white bars), control OF (gray bars), and liver Scd1–deficient OF animals (black bars). (E) Rate of hepatic gluconeogenesis and glycogenolysis in control SC (white bars), control OF (gray bars), and liver Scd1–deficient OF animals (black bars). *P < 0.05, versus SCR ASO OF; n = 5–6 per condition.

Figure 4. Selective attenuation of liver Scd1 expression normalizes hepatic insulin action in OF rats.

(A) Protocol for the intraportal Scd1 ASO treatment (top panel). An indwelling catheter was placed into the portal vein, and 2 days later (day 0), the animals received a first intraportal injection of either SCR ASO or Scd1 ASO (25 mg/kg of body weight). On day 3, the rats were switched to a lard-enriched diet (HF) and received a second injection of ASOs. Lastly, on day 5, an insulin clamp procedure was performed (See Figure 2A). (B) Intraportal Scd1 ASO (black bar) decreased liver Scd1 mRNA when compared with SCR ASO (white bar; left panel). Selective attenuation of liver Scd1 (black bar) in OF rats improved hepatic insulin action when compared with SCR ASO (white bar) as shown by a significant decrease in glucose production (GP) (center panel) and a significant increase in insulin’s ability to inhibit GP (right panel). (C–F) To examine whether the decreased expression of SCD1 in adipose tissue is required for the effects of Scd1 ASO on GP, we compared subgroups of OF rats displaying similar decreases in liver SCD1 induced by either i.p. (C and D) or intraportal (E and F) ASO. (C) i.p. Scd1 ASO markedly suppressed Scd1 expression in liver and white fat. (E) Intraportal Scd1 ASO markedly decreased Scd1 expression in the liver but not in adipose tissue. (D and F) The 2 treatment regimens led to similar improvements in hepatic insulin action. *P < 0.05 versus SCR ASO OF; n = 5–6.

Figure 5. Scd1 deficiency normalizes hepatic insulin action in HF-fed mice.

(A) Protocol for Scd1 ASO treatment in mice. Male C57BL/6J mice were allocated to a HF diet for 3 weeks. Five days before the completion of the protocol (day 0), the mice received an i.p. injection of either a SCR ASO or Scd1 ASO (100 mg/kg of body weight); on day 3, the animals received a second ASO injection, and vascular catheters were implanted; finally, on day 5, the mice were subjected to an insulin clamp procedure. (B) Scd1 ASO (black bars) markedly decreased the hepatic levels of Scd1 mRNA and protein compared with SCR ASO (grey bars) in HF diet–fed mice. (C) Protocol for the insulin clamp procedure in mice. (D) The enzymatic (desaturase) activity of Scd1 was markedly increased by HF feeding in mice (gray versus white bars) and dramatically lowered by treatment with Scd1 ASO (black bars). The rapid attenuation of liver Scd1 expression and activity prevented the development of hepatic insulin resistance induced by HF feeding in mice but did not modify insulin action on glucose uptake. *P < 0.05 versus SCR ASO; n = 6–10.

Figure 6. Scd1 deficiency enhances hepatic insulin action in OF rats.

(A) Western blot analysis of Akt phosphorylation (Ser473) in the liver of SC control rats (SCR ASO SC) and in OF rats treated with either control (SCR ASO OF) or Scd1 ASO (Scd1 ASO OF). (B) OF (SCR ASO OF; gray bars) attenuated hepatic Akt phosphorylation at Ser473 and Thr308 as compared with SC diet (SCR ASO SC, white bars); Scd1 deficiency (Scd1 ASO OF, black bars) prevented the effect of OF and enhanced Akt phosphorylation 2- to 3-fold compared with animals on SC. (C) Liver expression of Pck1 and G6pc mRNA determined by Q-PCR. OF (gray bars) resulted in a marked increase in the hepatic expression of PEPCK and Glc-6-Pase when compared with an SC diet (white bars); ASO-mediated liver Scd1 deficiency (black bars) prevented the hepatic upregulation of these enzymes in response to OF and brought it down to levels below those of SC-fed animals. *P < 0.05 versus SCR ASO OF.

Figure 7. Scd1 deficiency enhances hepatic insulin signaling in OF rats.

(A) To investigate the effect of Scd1 deficiency on early insulin signaling, we injected fasted rats with an i.p. bolus of insulin and sampled the livers 5 minutes later. The acute administration of insulin failed to stimulate the phosphorylation of Irs1 (B and D) and its association with PI3K (B and E). However, treatment of OF rats with Scd1 ASO partly restored insulin responses (B, D, and E). (B) Western blot analysis of immunoprecipitated tyrosine-phosphorylated IRβ and Irs1, Irs1-associated p85α subunit of PI3K, and PTP1B in livers from control (SCR ASO) or Scd1 ASO–treated OF rats in the basal (–Ins) or insulin-stimulated state (+Ins). Densitometric analysis revealed a 35% decrease in PTP1B protein in Scd1 ASO– compared with SCR ASO–treated livers (data not shown). (C) Densitometric analysis showed that insulin-stimulated tyrosine phosphorylation of IRβ was modestly increased by Scd1 deficiency. (D) Densitometric analysis showed that insulin-stimulated tyrosine phosphorylation of Irs1 was markedly enhanced by Scd1 deficiency. (E) The recruitment of p85α PI3K to Irs1 was also enhanced in livers from Scd1-deficient (black bars) when compared with SCR ASO (white bars) OF rats. In C and D, left panels show basal and insulin-stimulated values while right panels show percentage increase from basal values. *P < 0.05 versus SCR ASO OF; n = 3–6 (average ± SEM). pY, phosphotyrosine.