Increased hepatic Akt phosphorylation alleviated glucose intolerance and improved liver function in leptin-deficient mice

Abstract

Aim of the study: Akt is involved in upregulating the insulin-signaling pathways essential for maintaining glucose metabolism. Glycosphingolipids are involved in the pathogenesis of glucose intolerance and associated target organ injury. On the other hand, oral administration of b-glucosylceramide (GC) has been shown to alleviate insulin resistance. The present study aimed to determine the effects of oral administration of insulin and GC, separately and in combination, on Akt expression and the subsequent effect on metabolic syndrome characteristics in leptin-deficient mice.

Material and methods: Four groups of leptin-deficient ob/ob mice were orally administered for four weeks: vehicle, GC, short-acting insulin, and GC combined with insulin. Mice were followed for hepatic Akt expression and changes in tumor necrosis factor a (TNF-a) level, hyperlipidemia, and liver damage.

Results: In mice that received insulin or GC, particularly those that received both, the liver phosphorylation of Akt was significantly increased compared to those that received only vehicle. Serum TNF-a levels decreased in insulin-treated mice. These effects were associated with alleviating glucose intolerance and hyperlipidemia, as manifested by a significant glucose tolerance test improvement and reductions in serum triglyceride and cholesterol levels. Significant liver damage alleviation was noted by liver enzyme reductions in all treated groups, along with liver steatosis in the insulin-treated mice.

Conclusions: These data established the potential use of oral insulin administration with glycosphingolipids to alleviate glucose intolerance and associated liver damage and hyperlipidemia via increased Akt expression in the liver. The data support targeting Akt as a potent therapeutic target for metabolic syndrome.

Keywords: AKT; NAFLD; TNF-a; diabetes; steatosis.

Fig. 1

Phosphorylation of Akt in the liver following administration of insulin and GC. Four groups of leptin-deficient ob/ob mice were orally administered for four weeks: vehicle (group A), 2.5 mg/kg GC (group B), 2.5 IU of regular insulin (Humulin R, Lilly, group C), and the combination of both GC and insulin (group D). Phosphorylation of Akt in the liver was determined and compared with the actin control. A synergistic effect for the combination therapy was observed (Lanes D1-D3). Proteins were detected with phospho-specific Akt (Cell Signaling, USA, #9271); β-actin (Abcam, UK, sc-47778)

Fig. 2

Serum TNF-α levels following administration of insulin and GC. ELISA measured serum TNF-α levels in all mice in all groups. Oral administration of insulin was associated with a decrease in TNF-α serum levels (p = NS, for B vs. A; and p < 0.05, for C and D vs. A)

Fig. 3

Effects of oral administration of insulin and GC on glucose tolerance and hyperlipidemia. Mice in all experimental groups underwent a glucose tolerance test (GGT), and data are shown for areas under the curve. Following both oral insulin and oral GC, GGT decreased (A). A synergistic effect of the combination therapy with insulin and GC was observed (p ≤ 0.05 for B, C, and D vs. A; p < 0.01 for D vs. B and C). Both oral insulin and oral GC were associated with decreases in serum triglyceride (TG) levels (B, p < 0.05, for B vs. A) and with reductions in serum total cholesterol (C, p < 0.05, for B vs. A; and p < 0.005, for C and D vs. A). The changes were more significant in insulin-treated mice

Fig. 4

A) Changes in ALT serum levels following administration of insulin and GC. ALT serum levels were measured in all experimental groups. Decreases in insulin-treated and GC-treated mice were observed as early as the second week of therapy. At the end of four weeks of treatment, further reductions were observed, which were more significant in insulin-treated mice (p < 0.005, for B vs. A; and p < 0.001, for C and D vs. A). The hepatic triglyceride (TG) content was measured in all mice at the end of the four-week treatment period (B). A significant reduction in liver TGs was noted for insulin-treated mice (p = NS, for B vs. A; and p < 0.05, for C and D vs. A)