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Impairment of the Ubiquitin-Proteasome Pathway Is a Downstream Endoplasmic Reticulum Stress Response Induced by Extracellular Human Islet Amyloid Polypeptide and Contributes to Pancreatic ß-Cell Apoptosis

¹ Endocrinology and Diabetes Unit, Laboratory of Experimental Diabetes, IDIBAPS (Institut d'Investigacions Biomèdiques August Pi i Sunyer), Hospital Clinic and University of Barcelona, Barcelona, Spain
² Institute of Diabetes, Sardà Farriol Foundation, Barcelona, Spain
³ Cambridge Institute for Medical Research, University of Cambridge, Department of Clinical Biochemistry, Wellcome Trust/MRC Building, Addenbrooke's Hospital, Cambridge, U.K
⁴ Laboratory of Cytomolecular Genetics, Department of Pathology, Haartman Institute, University of Helsinki and HUSLAB, Helsinki, Finland

Address correspondence and reprint requests to Dr. Anna Novials, Institute of Diabetes, Sardà Farriol Foundation, Pg. Bonanova, 69, 6th floor, 08017 Barcelona, Spain. E-mail: anovials{at}fsf.es

Abbreviations: ALLN, N-acetyl-Leu-Leu-norleucinal; EGFP, green fluorescent protein; ER, endoplasmic reticulum; FACS, fluorescence-activated cell sorting; FBS, fetal bovine serum; FITC, fluorescein isothiocyanate; hIAPP, human islet amyloid polypeptide; HSP, heat shock protein; IAPP, islet amyloid polypeptide; MODC, mouse ornithine decarboxylase; PI, propidium iodide; ROS, reactive oxygen species; RQ-PCR, real-time quantitative RT-PCR

OBJECTIVE—Human islet amyloid polypeptide (hIAPP) aggregation plays a major role in the development of islet amyloidosis in type 2 diabetes. It is known that extracellular hIAPP oligomers are toxic to pancreatic ß-cells and associated with apoptosis. We therefore investigated the molecular mechanism by which extracellular hIAPP mediates pancreatic ß-cell apoptosis.

RESEARCH DESIGN AND METHODS—MIN6 cells and primary cultures of human pancreatic islets were treated with freshly dissolved hIAPP peptide. Morphology of the cultures was evaluated by electron microscopy. Gene expression was analyzed by microarray, RT-PCR, and immunoblot. Calcium levels were measured in fura-2–loaded cells. Apoptosis was quantified by cytometry.

RESULTS—Increased expression of several heat shock proteins and activation of the spliced form of XBP-1, a transcription factor for overexpression of chaperones during endoplasmic reticulum (ER) stress, were detected together with morphological evidence of ER dysfunction. Intracellular calcium overload was detected in association with this process. Moreover, reduction in the proteasome activity, which was detected over time, contributed to the intracellular accumulation of ubiquitinated proteins, leading to a functional suppression of the ubiquitin-proteasome pathway. In addition, impairment of the proteasome function contributed to apoptosis, while, despite the presence of hIAPP, cell viability improved when a proteasome activator was overexpressed. The key cytotoxic events induced by extracellular hIAPP were also observed in treated human islets.

CONCLUSIONS—Our data suggest that ER stress responses are intracellular signaling mechanisms induced by extracellular hIAPP aggregation and that impairment of the ubiquitin-proteasome pathway is implicated in ER stress–mediated pancreatic ß-cell apoptosis.

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