Decreased expression of endoplasmic reticulum chaperone GRP78 in liver of diabetic mice

doi:10.1016/j.bbrc.2011.11.118

Biochemical and Biophysical Research Communications

Volume 417, Issue 1, 6 January 2012, Pages 364-370

https://doi.org/10.1016/j.bbrc.2011.11.118 Get rights and content

Abstract

To identify molecular targets associated with the development of diabetes, we analyzed the hepatic proteome of obese diabetic db/db mice using electrophoresis on a high-resolution two-dimensional gel combined with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. By comparison between non-diabetic db/+ and diabetic db/db mice, six proteins and one protein were significantly decreased and increased in the diabetic mice, respectively. Among these proteins, two of the decreased proteins are involved in endoplasmic reticulum (ER) stress-related unfolded protein response, GRP78 and protein disulfide isomerase A3, and it was revealed that the decreased GRP78 expression in the liver of diabetic db/db mice is due to the reduction of GRP78 protein synthesis rather than RNA transcription. In addition, we found that the treatment of human hepatocyte HepG2 cells with oleic acid decreased the expression of GRP78, and attenuated the activation of AKT by insulin stimulation. These results suggest that decreased GRP78 expression may induce resistance to insulin by inhibiting the AKT activation, and plays an important role in the development of type 2 diabetes.

Highlights

► We analyzed the hepatic proteome of obese diabetic db/db mice. ► We revealed that the decreased GRP78 expression in the liver of diabetic db/db mice. ► It is due to the reduction of GRP78 protein synthesis rather than RNA transcription. ► GRP78 expression and AKT activation were decreased by the treatment with oleic acid. ► Decreased GRP78 expression may play an important role in the development of diabetes.

Introduction

Type 2 diabetes is one of the most prevalent and serious metabolic diseases globally, and peripheral insulin resistance and pancreatic β-cell dysfunction are the hallmarks of the disease [1], [2]. Normal pancreatic β-cells can compensate for insulin resistance by increasing insulin secretion, but insufficient compensation leads to the onset of glucose intolerance. Once hyperglycemia becomes apparent, the insulin resistance and dysfunction of β-cells progressively worsen. Insulin sensitizers are partially effective at improving glucose disposal in skeletal muscle and suppressing hepatic gluconeogenesis, and prevent the progression of diabetes. However, the pathophysiology of the insulin-resistant state is still uncertain, and more detailed knowledge of the mechanism that leads to insulin resistance is necessary to identify new targets for the development of anti-diabetic drugs.

The endoplasmic reticulum (ER) is a large membrane-enclosed cellular compartment in which secretory and membrane-bound proteins are synthesized and folded into their final three-dimensional structures [3]. Protein folding in ER lumen is facilitated by a number of molecular chaperones and folding enzymes, including glucose-regulated protein (GRP) 94, GRP78 and protein disulfide isomerase (PDI) [4]. In stressful conditions such as the accumulation of misfolded proteins, however, the capacity of these proteins becomes inadequate, and leads to a condition defined as ER stress. The cellular response to ER stress, referred to as unfolded protein response (UPR), results in activation of three linked signaling pathways emanating from three ER stress sensors: inositol-requiring protein 1α (IRE1α), PKR-like endoplasmic reticulum kinase (PERK) and activating transcription factor 6α (ATF6α) [5], [6]. The combined actions of these signaling cascades serve to reduce ER stress through induction of chaperone and attenuation of protein translation. Previous studies have shown that ER stress triggers reduced insulin action in adipocytes and hepatocytes [7], [8], [9], [10]. In addition, ER stress may account for altered insulin secretion from pancreatic β-cells [11], [12], and defect of UPR may play a dominant role in the development of type 2 diabetes.

Here we analyzed the hepatic proteome of obese diabetic db/db mice using electrophoresis on a high-resolution two-dimensional gel combined with matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry, and found that levels of two UPR-related proteins, GRP78 and PDI A3, were significantly decreased in the diabetic mice. In addition, we found that the treatment with oleic acid (OA) of human HepG2 cells decreased the expression of GRP78 and attenuated the activation of AKT, a serine/threonine kinase that regulates most of the metabolic actions of insulin. The significance of the altered expression of GRP78 during the development of diabetes is discussed.

Section snippets

Animals and preparation of hepatocytes

Male C57BLKS/J m⁺/Lepr^db (db/+) and C57BLKS/J Lepr^db/Lepr^db (db/db) mice were purchased from Jackson Laboratories (Bar Harbor, ME, USA) and housed with a 12-h light/dark cycle (lights off: 20:00–08:00) and ambient temperature regulated at 22 ± 2 °C. All mice were fed standard rodent chow and tap water ad libitum. Non-fasting blood glucose levels were determined weekly by the glucose oxidase method [13]. Data on body weights and plasma glucose levels over 14 weeks are summarized in Fig. 1A. For

Proteomic analysis of liver of diabetic db/db mice

To identify the proteins associated with the development of diabetes, we compared the expression profile of proteins in the liver of non-diabetic db/+ mice and diabetic db/db mice using 2-DE. Fig. 1B shows typical images of the gels. About 700 spots in each image were matched and quantified using PDQuest analyzing software, and six proteins and one protein were found to be expressed at decreased levels and in increased levels in diabetic db/db mice compared with those in non-diabetic db/+ mice,

Discussion

To identify the proteins associated with the development of diabetes, we conducted a proteomic analysis in the liver of db/db mice and found that levels of two UPR-related proteins, GRP78 and PDI A3, were significantly decreased in the diabetic mice. GRP78 is a member of the HSP70 family abundant in the lumen of ER, and plays important roles as a molecular chaperone that removes malfolded proteins in ER lumen. Overexpression of GRP78 has been shown to increase ER stress resistance and to have

Acknowledgments

This study was supported in part by a Grant-in-aid for the Open Research Center Program of Kyoto Pharmaceutical University from the Ministry of Education, Science, Culture and Sports of Japan.

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Decreased expression of endoplasmic reticulum chaperone GRP78 in liver of diabetic mice

Abstract

Highlights

Introduction

Section snippets

Animals and preparation of hepatocytes

Proteomic analysis of liver of diabetic db/db mice

Discussion

Acknowledgments

FEBS Lett.

Mutat. Res.

J. Biol. Chem.

J. Biol. Chem.

Cell Metab.

J. Biol. Chem.

Int. J. Biochem. Cell Biol.

Cellular mechanisms of insulin resistance

J. Clin. Invest.

Insulin signalling and the regulation of glucose and lipid metabolism

Nature

The mammalian unfolded protein response

Annu. Rev. Biochem.

Signal integration in the endoplasmic reticulum unfolded protein response

Nat. Rev. Mol. Cell Biol.

Endoplasmic reticulum stress links obesity, insulin action, and type 2 diabetes

Science

Oxidative stress, ER stress, and the JNK pathway in type 2 diabetes

J. Mol. Med.

The endoplasmic reticulum chaperone improves insulin resistance in type 2 diabetes

Diabetes

ER stress and SREBP-1 activation are implicated in beta-cell glucolipotoxicity

J. Cell Sci.

Determination of blood glucose using an oxidase–peroxidase system with a non-carcinogenic chromogen

J. Clin. Pathol.