Insulin-Regulated Srebp-1c and Pck1 mRNA Expression in Primary Hepatocytes from Zucker Fatty but Not Lean Rats Is Affected by Feeding Conditions

Yan Zhang,Wei Chen,Yang Li,Rui Li,Guoxun Chen,Yuebin Ge,Nai Sum Wong

doi:10.1371/journal.pone.0021342

Yan Zhang, Wei Chen + Show 5 more

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https://doi.org/10.1371/journal.pone.0021342

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Abstract

Insulin regulates the transcription of genes for hepatic glucose and lipid metabolism. We hypothesized that this action may be impaired in hepatocytes from insulin resistant animals. Primary hepatocytes from insulin sensitive Zucker lean (ZL) and insulin resistant Zucker fatty (ZF) rats in ad libitum or after an overnight fasting were isolated, cultured and treated with insulin and other compounds for analysis of gene expression using real-time PCR. The mRNA levels of one insulin-induced (Srebp-1c) and one insulin-suppressed (Pck1) genes in response to insulin, glucagon, and compactin treatments in hepatocytes from ad libitum ZL and ZF rats were analyzed. Additionally, the effects of insulin and T1317 on their levels in hepatocytes from ad libitum or fasted ZL or ZF rats were compared. The mRNA levels of Srebp-1c, Fas, and Scd1, but not that of Insr, Gck and Pck1, were higher in freshly isolated hepatocytes from ad libitum ZF than that from ZL rats. These patterns of Srebp-1c and Pck1 mRNA levels remained in primary hepatocyte cultured in vitro. Insulin's ability to regulate Srebp-1c and Pck1 expression was diminished in hepatocytes from ad libitum ZF, but not ZL rats. Glucagon or compactin suppressed Srebp-1c mRNA expression in lean, but not fatty hepatocytes. However, glucagon induced Pck1 mRNA expression similarly in hepatocytes from ad libitum ZL and ZF rats. Insulin caused the same dose-dependent increase of Akt phosphorylation in hepatocytes from ad libitum ZL and ZF rats. It synergized with T1317 to induce Srebp-1c, and suppressed Pck1 mRNA levels in hepatocytes from fasted, but not that from ad libitum ZF rats. We demonstrated that insulin was unable to regulate its downstream genes' mRNA expression in hepatocytes from ad libitum ZF rats. This impairment can be partially restored in hepatocytes from ZF rats after an overnight fasting, a phenomenon that deserves further investigation.

Highlights

The increased rate of metabolic diseases, such as obesity, diabetes and cardiovascular disease, has become a major public health concern [1,2]
To analyze the mRNA levels of hepatic insulin-regulated genes, primary hepatocytes were isolated from Zucker lean (ZL) and Zucker fatty (ZF) rats in ad libitum condition
The mRNA levels of two genes for glucose metabolism, Gck (Fig. 1E) and Pck1 (Fig. 1F), in the freshly isolated primary hepatocytes of ZF rats were similar to those of ZL rats. All these results demonstrated that ZF rat hepatocytes had significantly higher mRNA levels of Srebp-1c, fatty acid synthase (Fas), and stearoyl-CoA desaturase 1 (Scd1) than ZL rat hepatocytes did, but not that of Insr, Gck, and Pck1

Summary

Introduction

The increased rate of metabolic diseases, such as obesity, diabetes and cardiovascular disease, has become a major public health concern [1,2]. Insulin regulates the expression of a variety of genes responsible for glycolysis, glycogenesis and lipogenesis, and inhibits gluconeogenesis [7]. This insulin-regulated hepatic gene expression, at least in part, is responsible for glucose and lipid homeostasis [8,9]. Insulin increases the expression of glucokinase gene (Gck) [10,11], the enzyme responsible for the first step of hepatic glycolysis. It suppresses the expression of the cytosolic form of phosphoenolpyruvate carboxykinase (Pck1)

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