Pharmacological inhibition of glucosylceramide synthase enhances insulin sensitivity

Abstract

A growing body of evidence implicates ceramide and/or its glycosphingolipid metabolites in the pathogenesis of insulin resistance. We have developed a highly specific small molecule inhibitor of glucosylceramide synthase, an enzyme that catalyzes a necessary step in the conversion of ceramide to glycosphingolipids. In cultured 3T3-L1 adipocytes, the iminosugar derivative N-(5'-adamantane-1'-yl-methoxy)-pentyl-1-deoxynojirimycin (AMP-DNM) counteracted tumor necrosis factor-alpha-induced abnormalities in glycosphingolipid concentrations and concomitantly reversed abnormalities in insulin signal transduction. When administered to mice and rats, AMP-DNM significantly reduced glycosphingolipid but not ceramide concentrations in various tissues. Treatment of ob/ob mice with AMP-DNM normalized their elevated tissue glucosylceramide levels, markedly lowered circulating glucose levels, improved oral glucose tolerance, reduced A1C, and improved insulin sensitivity in muscle and liver. Similarly beneficial metabolic effects were seen in high fat-fed mice and ZDF rats. These findings provide further evidence that glycosphingolipid metabolites of ceramide may be involved in mediating the link between obesity and insulin resistance and that interference with glycosphingolipid biosynthesis might present a novel approach to the therapy of states of impaired insulin action such as type 2 diabetes.

FIG. 1

AMP-DNM reverses TNF-α-induced insulin resistance and surface expression of GM3 in 3T3-L1 adipocytes. Serum-starved 3T3-L1 adipocytes were treated with either vehicle/control, AMP-DNM (10 μmol/l), and/or TNF-α (0.6 nmol/l) for 24 h before stimulation with or without insulin (100 nmol/l for 5 min). A: Immunoblots of whole-cell lysates were performed in parallel as described in research design and methods. Representative blots are shown from one of three independent experiments. B: Cell surface expression of GM3 was determined on basal adipocytes (not stimulated with insulin) by fluorescence-activated cell sorter analysis. Values represent means ± SE of viable cells that stained positive for GM3. *P < 0.05; observed in four independent experiments.

FIG. 2

Improved insulin signaling in the liver of ob/ob mice treated with AMP-DNM. Animals were fed for 2 weeks with 0, 5, or 25 mg AMP-DNM/kg. To animals starved overnight, insulin (0.75 units/kg) was administered via the vena porta. Livers were collected after 5 min. Insulin receptor protein, and its autophosphorylation, and mTOR protein, and its serine phosphorylation, were visualized and quantified.

FIG. 3

Beneficial effects of iminosugar feeding on glucose metabolism in ob/ob mice. ob/ob mice were fed either a control diet (○ and □) or with diet providing a dose of 25 mg AMP-DNM · kg body wt⁻¹ · day⁻¹ (● and ■). Body weight (A) and blood glucose levels (B) were determined at indicated time points. During the clamp, glucose infusion rate (GIR) was monitored (C), the rate of glucose disposal [Rd(glc)], and the rate of endogenous hepatic glucose production [Ra(glc;endo)] were calculated (D). Data presented are means ± SD. Significant differences between both groups (*) are indicated. Statistical significances of differences were determined using Student’s t test, except in calculations over time where ANOVA with repeated measurements was used. P < 0.05 was considered statistically significant.

FIG. 4

Beneficial effects of iminosugar feeding in ZDF rats. ZDF rats (10 weeks old) were treated for 10 weeks with 0, 5, or 25 mg AMP-DNM · kg⁻¹ · day⁻¹ (n = 4 for each group). As a control, lean littermates were analyzed. Presented are means ± SD. A: Fasted blood glucose during treatment period. B: Nonfasted blood glucose during treatment period. C: Plasma insulin levels during treatment period. D: Oral glucose tolerance at week 2 (□) and week 9 (■) of treatment. E: A1C after 10 weeks of treatment. F: Relation of level of A1C (%) with hepatic glucosylceramide–to–ceramide ratio (GlcCer/Cer). Values are means ± SD. G: Pancreas glucagon immunostaining placebo-treated ZDF rats. H: Pancreas insulin immunostaining placebo-treated ZDF rats. I: Pancreas glucagon immunostaining 25 mg AMP-DNM–treated ZDF rats. J: Pancreas insulin immunostaining 25 mg AMP-DNM–treated ZDF rats. K: Pancreas glucagon immunostaining lean littermate. L: Pancreas insulin immunostaining lean littermate. ■, 0 mg AMP-DNM · kg⁻¹ · day⁻¹; ●, 5 mg AMP-DNM · kg⁻¹ · day⁻¹; ○, 25 mg AMP-DNM · kg⁻¹ · day⁻¹; ◇, lean rats.

FIG. 5

Beneficial effects of AMP-DNM on glucose homeostasis in diet-induced glucose-intolerant mice. Glucose-intolerant C57Bl/6J mice were obtained after a high-fat diet for 4 weeks. For 2 weeks, two groups of eight animals were administered, by daily oral gavage, 0, 5, and 25 mg AMP-DNM · kg⁻¹ · day⁻¹. Values represent means ± SD. A: Body weight. B: Fasted blood glucose. C: Oral glucose tolerance (expressed as AUC). D: Plasma insulin.