An early, reversible cholesterolgenic etiology of diet-induced insulin resistance

Abstract

Objective: A buildup of skeletal muscle plasma membrane (PM) cholesterol content in mice occurs within 1 week of a Western-style high-fat diet and causes insulin resistance. The mechanism driving this cholesterol accumulation and insulin resistance is not known. Promising cell data implicate that the hexosamine biosynthesis pathway (HBP) triggers a cholesterolgenic response via increasing the transcriptional activity of Sp1. In this study we aimed to determine whether increased HBP/Sp1 activity represented a preventable cause of insulin resistance.

Methods: C57BL/6NJ mice were fed either a low-fat (LF, 10% kcal) or high-fat (HF, 45% kcal) diet for 1 week. During this 1-week diet the mice were treated daily with either saline or mithramycin-A (MTM), a specific Sp1/DNA-binding inhibitor. A series of metabolic and tissue analyses were then performed on these mice, as well as on mice with targeted skeletal muscle overexpression of the rate-limiting HBP enzyme glutamine-fructose-6-phosphate-amidotransferase (GFAT) that were maintained on a regular chow diet.

Results: Saline-treated mice fed this HF diet for 1 week did not have an increase in adiposity, lean mass, or body mass while displaying early insulin resistance. Consistent with an HBP/Sp1 cholesterolgenic response, Sp1 displayed increased O-GlcNAcylation and binding to the HMGCR promoter that increased HMGCR expression in skeletal muscle from saline-treated HF-fed mice. Skeletal muscle from these saline-treated HF-fed mice also showed a resultant elevation of PM cholesterol with an accompanying loss of cortical filamentous actin (F-actin) that is essential for insulin-stimulated glucose transport. Treating these mice daily with MTM during the 1-week HF diet fully prevented the diet-induced Sp1 cholesterolgenic response, loss of cortical F-actin, and development of insulin resistance. Similarly, increases in HMGCR expression and cholesterol were measured in muscle from GFAT transgenic mice compared to age- and weight-match wildtype littermate control mice. In the GFAT Tg mice we found that these increases were alleviated by MTM.

Conclusions: These data identify increased HBP/Sp1 activity as an early mechanism of diet-induced insulin resistance. Therapies targeting this mechanism may decelerate T2D development.

Keywords: Cholesterol; Insulin resistance; Membrane; Skeletal muscle.

Figure 1

Dietary and treatment intervention plan. Upon arrival to our facility, 2 wks before the diet and treatment intervention all mice were singly housed, given standard laboratory chow (SC) for 1 wk and then the low-fat (LF) diet for 1 wk to adapt to the modified diet. Following this 2-wk acclimation period, mice were either left on the LF diet (Groups 1 and 3, black line) or switched to the HF diet (Groups 2 and 4, red line). Mice were injected intraperitoneally with either saline (SAL, Groups 1 and 2, black) or 0.15 mg/kg mithramycin-A (MTM, Groups 3 and 4, green) daily during the 1-week LF or HF diet. All groups consisted of several cohorts of 6–9 mice for metabolic/tissue analyses performed immediately following the 1-wk diet/treatment period.

Figure 5

One week of HF feeding provokes O-linked glycosylation of Sp1 in skeletal muscle. Lysates from skeletal muscle from LF- and HF-fed mice were immunoprecipitated with an Sp1 antibody to detect O-linked glycosylation. (A) Eluted samples were immunoblotted (IB) with RL2 or Sp1 antibody (n = 3 per group). (B) Immunoprecipitates were subjected to enzymatic biotin labeling with a Click-iT kit and immunoblotted with streptavidin (Stp) antibody (n = 3 per group). Note Sp1 antibody labeling of the biotin-labeled O-GlcNAc did not yield interpretable immunoblots. Representable immunoblots are shown. Data represent means ± SEM. Difference between groups was analyzed using a two-tailed unpaired t test. ∗p < 0.05.

Figure 6

One week of HF feeding causes a cholesterolgenic response in skeletal muscle. (A) ChIP was performed on skeletal muscle lysates. Purified DNA and primers specific to the Sp1-binding sites in the promoter region of Hmgcr were used for qPCR (n = 4–5 per group). (B) Hmgcr mRNA expression (n = 5 per group). (C) Triad-enriched membrane cholesterol (n = 6 per group). (D) Two representative images of 1-mm sections of soleus muscle per group subjected to immunofluorescent labeling of F-actin. Note prior to imaging, all samples were de-identified to ensure an objective analysis. All images were taken in the same focal plane of the section and under identical microscopic parameters. (E) Immunofluorescence F-actin intensity/area for multiple (3–5) images captured in muscle sections per muscle per mouse (n = 3). Difference between groups was analyzed using a two-way ANOVA and Tukey's multiple comparison tests. ∗p < 0.05, ∗∗p < 0.005, ∗∗∗p < 0.0005, ∗∗∗∗p < 0.0001.

Figure 2

High-fat feeding for 1 week impaired glucose tolerance in saline-, but not MTM-, treated mice. (A–C) Body mass, lean mass, and adiposity following the 1-wk diet/treatment intervention for LF- (open symbols) and HF-fed (solid symbols) mice treated daily during the intervention with saline (black symbols) or MTM (green symbols) (n = 7 per group). (D) Average daily caloric intake during the 1-wk diet/treatment intervention (n = 6 per group). (E) Blood glucose measured after an intraperitoneal injection of glucose (2 g/kg, n = 5–6 per group). (F) Area under the curve (AUC). Data represent means ± SEM. Difference between groups was analyzed using a two-way ANOVA and Tukey's multiple comparison tests. ∗∗p < 0.005.

Figure 3

Daily MTM treatment prevented the development of insulin resistance in mice fed a HF diet for 1 week. (A) Arterial blood glucose, (B) plasma insulin, (C–D) Glucose infusion rate (GIR), (E) glucose disposal (R_d), (F) [¹⁴C]-2DG tissue glucose uptake (R_g), (G–H) glucose production (Endo R_a) and as %suppression from basal, and (I) tissue glycogen synthesis during the hyperinsulinemic-euglycemic clamp. Data represent means ± SEM from 7 to 9 mice per group. Difference between groups was analyzed using a two-way ANOVA and Tukey's multiple comparison tests. ∗p < 0.05, ∗∗p < 0.005, ∗∗∗p < 0.0005, ∗∗∗∗p < 0.0001.

Figure 4

Metabolic function and energy expenditure in mice during the diet/intervention. (A) Oxygen consumption (VO2), (B) carbon dioxide production (VCO2), (C) respiration (RER), (D) spontaneous physical activity (SPA), and (E–F) energy expenditure (EE). Data represent means ± SEM from 7 mice per group. (A–D) Difference between groups was analyzed using a two-way ANOVA and Tukey's multiple comparison tests. ∗p < 0.05, ∗∗p < 0.005, ∗∗∗p < 0.0005, ∗∗∗∗p < 0.0001 versus respective light group.

Figure 7

Overexpression of skeletal muscle GFAT causes membrane cholesterol accumulation and glucose intolerance in male mice fed standard laboratory chow. (A) Body mass of age-and weight-matched GFAT Tg and Wt littermate control mice (n = 6 per group), (B) Representative immunoblot of GFAT from skeletal muscle lysate, and (C) Skeletal muscle membrane cholesterol (n = 6 per group) from these mice. Data represent means ± SEM. Difference between groups was analyzed using a one-tailed unpaired t test. ∗p < 0.05.

Figure 8

Insulin-resistant GFAT Tg mice display an Sp1-mediated cholesterolgenic response. Age- and weight-matched male and female Tg and Wt littermate control mice fed standard laboratory chow were treated with either saline (+S) or MTM (+M) for 1 wk for determination of (A, B) Body mass (4–5 per group), (C) Skeletal muscle Hmgcr mRNA expression (n = 7–9 per group), and (D) Skeletal muscle membrane cholesterol (n = 4). Data represent means ± SEM. Difference between groups was analyzed using a one-way ANOVA and the Tukey's multiple comparison tests. ∗p < 0.05, ∗∗p < 0.005.