Abstract
The ubiquitin/proteasome system (UPS), a major cellular protein degradation machinery, plays key roles in the regulation of many cell functions. Glucotoxicity mediated by chronic hyperglycaemia is detrimental to the function and survival of pancreatic beta cells. The aim of our study was to determine whether proteasome dysfunction could be involved in beta cell apoptosis in glucotoxic conditions, and to evaluate whether such a dysfunction might be pharmacologically corrected. Therefore, UPS activity was measured in GK rats islets, INS-1E beta cells or human islets after high glucose and/or UPS inhibitor exposure. Immunoblotting was used to quantify polyubiquitinated proteins, endoplasmic reticulum (ER) stress through CHOP expression, and apoptosis through the cleavage of PARP and caspase-3, whereas total cell death was detected through histone-associated DNA fragments measurement. In vitro, we found that chronic exposure of INS-1E cells to high glucose concentrations significantly decreases the three proteasome activities by 20% and leads to caspase-3-dependent apoptosis. We showed that pharmacological blockade of UPS activity by 20% leads to apoptosis in a same way. Indeed, ER stress was involved in both conditions. These results were confirmed in human islets, and proteasome activities were also decreased in hyperglycemic GK rats islets. Moreover, we observed that a high glucose treatment hypersensitized beta cells to the apoptotic effect of proteasome inhibitors. Noteworthily, the decreased proteasome activity can be corrected with Exendin-4, which also protected against glucotoxicity-induced apoptosis. Taken together, our findings reveal an important role of proteasome activity in high glucose-induced beta cell apoptosis, potentially linking ER stress and glucotoxicity. These proteasome dysfunctions can be reversed by a GLP-1 analog. Thus, UPS may be a potent target to treat deleterious metabolic conditions leading to type 2 diabetes.
Highlights
Type 2 diabetes is characterized by chronic hyperglycemia caused by an impaired function and survival of insulin producing pancreatic beta cells and an insulin resistance of peripheral tissues [1]
Our results clearly show that proteasome activity is a key determinant of the survival/apoptosis balance in beta cells, that could be part of the glucotoxicity mechanisms involved in diabetes pathophysiology
We confirm that endoplasmic reticulum (ER) stress, as evidenced by the two fold increase in CHOP expression (Figures 1E and 1F), is involved in the increased apoptosis observed in beta cells submitted to high glucose
Summary
Type 2 diabetes is characterized by chronic hyperglycemia caused by an impaired function and survival of insulin producing pancreatic beta cells and an insulin resistance of peripheral tissues [1]. Glucose is the main regulator of beta cell functional mass, it can be deleterious when present in excessive amounts during prolonged exposure [2]. Such a glucotoxicity is responsible for apoptosis of beta cells [3], mainly through inducing an oxidative stress, an endoplasmic reticulum (ER) stress, and an inflammatory reaction. The ubiquitin/proteasome system (UPS) is one of the major degradation pathways for maintaining protein homeostasis. It degrades misfolded, oxidized or damaged proteins, and regulates proteins involved in many cellular processes, such as signal transduction, cell cycle regulation, cell death, and gene transcription [4,5]. The 20S proteasome is a cylindrical structure made of two outer rings of asubunits and two inner heptameric rings of b-subunits that carry the proteolytic activities, classified as caspase-like (b1), trypsin-like (b2), and chymotrypsin-like (b5), which cleave after acidic, basic and hydrophobic amino acids, respectively [6]
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