Overexpression of AMPKalpha1 Ameliorates Fatty Liver in Hyperlipidemic Diabetic Rats

Abstract

5'-AMP-activated protein kinase (AMPK) is a heterotrimeric complex consisting of a catalytic (alpha) and two regulatory (beta and gamma) subunits. Two isoforms are known for catalytic subunit (alpha1, alpha2) and are encoded by different genes. To assess the metabolic effects of AMPKalpha1, we examined the effects of overexpression of adenoviral-mediated AMPKalpha1 in hyperlipidemic type 2 diabetic rats. The Otsuka Long-Evans Tokushima Fatty (OLETF) rat is an established animal model of type 2 diabetes that exhibits chronic and slowly progressive hyperglycemia and hyperlipidemia. Thirty five-week-old overt type 2 diabetic rats (n=10) were administered intravenously with Ad.AMPKalpha1. AMPK activity was measured by phosphorylation of acetyl CoA carboxlyase (ACC). To investigate the changes of gene expression related glucose and lipid metabolism, quantitative real-time PCR was performed with liver tissues. Overexpression of AMPKalpha1 showed that blood glucose concentration was decreased but that glucose tolerance was not completely recovered on 7th day after treatment. Plasma triglyceride concentration was decreased slightly, and hepatic triglyceride content was markedly reduced by decreasing expression of hepatic lipogenic genes. Overexpression of AMPKalpha1 markedly improved hepatic steatosis and it may have effective role for improving hepatic lipid metabolism in hyperlipidemic state.

Keywords: AMP-activated protein kinase (AMPK); AMPKα1 catalytic subunit; Adenovirus; Fatty liver; OLETF; Type 2 diabetes.

Fig. 1

Activity and expression of AMPK by Ad.AMPKα1 injection. (A) Non-radioactive AMPK activity assay. AMPK activity in the liver was measured on day 0 (0 day) and 7 (7 days) after Ad.AMPKα1 injection, and for the positive control with added AMPK recombinant protein (+AMPK). (B) Western blot analysis of phospho-ACC, phospho-AMPKα, AMPKα and myc-tagged AMPKα1 levels in liver on day 0 (0 day) and 7 (7 days) after Ad.AMPKα1 injection into OLETF rats. LETO, lean nondiabetic counterpart rats.

Fig. 2

Ad.AMPKα1 alters glucose metabolism. (A) Effect of Ad.AMPKα1 on glucose tolerance. Glucose tolerance was measured on days 7 after injection of Ad.lacZ (Ad.lacZ) or Ad.AMPKα1 (Ad.AMPKα1) into OLETF rats. LETO rats were employed as nondiabetic counterpart. (B) Response of insulin secretion to glucose loading. Plasma concentrations of insulin were measured on the same time of assessing blood glucose. (C) Quantitative RT-PCR analysis of enzymes related to gluconeogenesis and glycolysis. ^*Significantly different from Ad.lacZ-injected OLETF rats (p<0.05).

Fig. 3

Ad.AMPKα1 decreases lipid content in liver. (A) Sudan Black B stained liver cryostat section from normal LETO rats, Ad.lacZ-injected OLETF rats (Ad.lacZ) and Ad.AMPKα1-injected OLETF rats (Ad.AMPKα1). Magnification ×200. The liver of Ad.lacZ-injected OLETF rat shows accumulation of large fat globules (Black). The liver of Ad.AMPKα1-injected OLETF rat shows marked decrease of fat globules. (B) Quantitative RT-PCR analysis of enzymes related to lipid synthesis. (C) Western blot analysis of SCD1 and HMGCR in liver on day 7 after injection. ^*Significantly different from Ad.lacZ-injected OLETF rats (p<0.05).