Elsevier

Cellular Signalling

Volume 20, Issue 5, May 2008, Pages 907-915
Cellular Signalling

Crucial roles of neuronatin in insulin secretion and high glucose-induced apoptosis in pancreatic β-cells

https://doi.org/10.1016/j.cellsig.2008.01.005Get rights and content

Abstract

Neuronatin (Nnat) was initially identified as a selectively-expressed gene in neonatal brains, but its expression has been also identified in pancreatic β-cells. Therefore, to investigate the possible functions that Nnat may serve in pancreatic β-cells, two Nnat isotypes (α and β) were expressed using adenoviruses in murine MIN6N8 pancreatic β-cells, and the cellular fates and the effects of Nnat on insulin secretion, high glucose-induced apoptosis, and functional impairment were examined. Nnatα and Nnatβ were primarily localized in the endoplasmic reticulum (ER), and their expressions increased insulin secretion by increasing intracellular calcium levels. However, under chronic high glucose conditions, the Nnatβ to Nnatα ratio gradually increased in proportion to the length of exposure to high glucose levels. Moreover, adenovirally-expressed Nnatβ was inclined to form aggresome-like structures, and we found that Nnatβ aggregation inhibited the function of the proteasome. Therefore, when glucose is elevated, the expression of Nnatβ sensitizes MIN6N8 cells to high glucose stress, which in turn, causes ER stress. As a result, expression of Nnatβ increased hyperglycemia-induced apoptosis. In addition, the expression of Nnatβ under high glucose conditions decreased the expression of genes important for β-cell function, such as glucokinase (GCK), pancreas duodenum homeobox-1 (PDX-1), and insulin. Collectively, Nnat may play a critical factor in normal β-cell function, as well as in the pathogenesis of type 2 diabetes.

Introduction

Pancreatic β-cells secrete insulin, the only hormone in the human body that is capable of lowering blood glucose concentrations. Thus, pancreatic β-cell dysfunction and degeneration are hallmarks of both type 1 and type 2 diabetes. Several recent studies have shown that stress on the endoplasmic reticulum (ER) may contribute to these processes [1], [2]. In addition to storing cellular calcium, the ER is an organelle that functions in the folding and assembling of secretory and membrane proteins under the assistance of a vast array of specialized chaperones and enzymes. In order to maintain appropriate function, the ER exerts stringent quality control on its protein products. Any perturbation in ER function induces severe cellular damage. When the amount of unfolded and improperly folded proteins in the ER lumen exceeds the folding capacity of the ER, serious ER stress can occur. To survive ER stress, cells have self-protective, anti-stress mechanisms, termed the ER stress response [3]. However, malfunction of the ER stress response caused by aging, genetic mutations, or environmental factors can result in various diseases, including diabetes and neurogenerative disorders such as Alzheimer's disease and Parkinson's disease [4]. Pancreatic β-cells have highly developed ER that aids in the secretion of large amounts of insulin. Recent studies have revealed that β-cells are highly susceptible to ER stress, and that ER stress-mediated apoptosis in β-cells can be a cause of diabetes [5]. However, the protein that leads to ER stress under diabetic conditions remains unidentified.

Neuronatin (Nnat) was initially identified as a selectively-expressed gene in neonatal brains, and it was suggested that Nnat may be involved in neuronal cell differentiation during brain development [6]. Alternative splicing generates two Nnat isotypes. The alpha-form (Nnatα) is encoded by three exons, whereas the beta-form (Nnatβ) is encoded by the first and third exons only [7]. Although Nnat is primarily known to be expressed in the central nervous system, it is also expressed in non-neuronal tissues such as the pituitary glands, lungs, adrenal glands, uterus, skeletal muscles, ovaries, and pancreas [8]. It is particularly interesting that Nnat is highly expressed in the pancreas, including pancreatic β-cells. In addition, it was recently reported that knocking out the Nnat gene decreased insulin secretion in the presence of glucose [9]. This result demonstrated the possibility that Nnat may play critical roles in pancreatic β-cells. However, to date, the function of Nnat in the pancreas is largely unknown, and the role of Nnat in the pathogenesis of diabetes has never been reported.

To identify the role of Nnat in β-cells under both physiologic and pathologic conditions, we used adenoviruses to express both of the Nnat isotypes in mouse MIN6N8 pancreatic β-cells. The effects of Nnatα and Nnatβ expressions on insulin secretion, apoptosis induced by chronically elevated glucose levels, and functional impairment in MIN6N8 cells were examined.

Section snippets

Cell line and reagents

MIN6N8 insulinoma cells were kindly provided by Dr. M.S. Lee (Sungkyunkwan University, School of Medicine, Seoul, Republic of Korea), and were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 15% fetal bovine serum (FBS). Antibodies were obtained from the following commercial sources: anti-GRP78, GCK, PDX-1, insulin, CHOP, vimentin, caspase-12, and caspase-3 from Santa Cruz Biotechnology (Santa Cruz, CA), anti-p-eIF2α and p-JNK from Cell Signaling Technology (Beverly,

Nnatα and β are regulated by a proteasome pathway

Even though the differential expressions of Nnatα and Nnatβ during neonatal brain development has been reported, any differences in protein character and function remain unknown. First, to compare the cellular fates between Nnatα and Nnatβ in β-cells, we expressed each Nnat isoform with C-terminal YFP tags in MIN6N8 cells using adenovirus vectors. The expression of each Nnat isoform was confirmed by Western blotting. Results show that, while Nnatα was almost always detected at its expected

Discussion

To date, the putative functions of Nnat have been predicated upon two reports. In the first of these two reports, the expression of Nnat in pre-adipocytes was found to stimulate differentiation into mature adipocytes with early induction of adipogenic transcription factors [10]. In the other report, Nnat expression was found to be regulated by the BETA2 (NeuroD1) transcription factor, and knocking out the Nnat gene in β-cells caused a decrease in glucose-induced insulin secretion [9]. This

Acknowledgments

We thank Dr. Van-Anh Nguyen for reviewing the manuscript.

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