Ubiquitinated-Protein Aggregates Form in Pancreatic β-Cells During Diabetes-Induced Oxidative Stress and Are Regulated by Autophagy

Abstract

Diabetes-induced oxidative stress can lead to protein misfolding and degradation by the ubiquitin-proteasome system. This study examined protein ubiquitination in pancreatic sections from Zucker diabetic fatty rats. We observed large aggregates of ubiquitinated proteins (Ubproteins) in insulin-expressing ␤-cells and surrounding acinar cells. The formation of these aggregates was also observed in INS1 832/13 ␤-cells after exposure to high glucose (30 mmol/l) for 8 -72 h, allowing us to further characterize this phenotype. Oxidative stress induced by aminotriazole (ATZ) was sufficient to stimulate Ub-protein aggregate formation. Furthermore, the addition of the antioxidants N-acetyl cysteine (NAC) and taurine resulted in a significant decrease in formation of Ub-protein aggregates in high glucose. Puromycin, which induces defective ribosomal product (DRiP) formation was sufficient to induce Ub-protein aggregates in INS1 832/13 cells. However, cycloheximide (which blocks translation) did not impair Ub-protein aggregate formation at high glucose levels, suggesting that long-lived proteins are targeted to these structures. Clearance of Ub-protein aggregates was observed during recovery in normal medium (11 mmol/l glucose). Despite the fact that 20S proteasome was localized to Ub-protein aggregates, epoxomicin treatment did not affect clearance, indicating that the proteasome does not degrade proteins localized to these structures. The autophagy inhibitor 3MA blocked aggregate clearance during recovery and was sufficient to induce their formation in normal medium. Together, these findings demonstrate that diabetes-induced oxidative stress induces ubiquitination and storage of proteins into cytoplasmic aggregates that do not colocalize with insulin. Autophagy, not the proteasome, plays a key role in regulating their formation and degradation. To our knowledge, this is the first demonstration that autophagy acts as a defense to cellular damage incurred during diabetes. Diabetes 56:930 -939, 2007

Suppression of autophagy is protective in high glucose-induced cardiomyocyte injury

“…Given that glucose deprivation was able to induce autophagy, 26 it is not surprising that high glucose, a condition of nutrient excess, would inhibit autophagy in cardiomyocytes. As autophagy protected against diabetes-induced oxidative injury in pancreatic β-cells, 31 it was somewhat unexpected that the inhibited autophagy did not contribute to high glucose toxicity in the present study. Instead, this adaptive response constituted part of the mechanism that promotes cardiomyocyte survival during high glucose exposure.…”

“…30 This hypothesis has been confirmed by the observation in pancreatic β-cells that autophagy attenuates diabetes-induced oxidative stress and mediates the clearance of protein aggregates. 31 Also, β-cell-specific autophagy-deficient mice develop spontaneous diabetes, suggesting a necessary role for autophagy in maintaining normal β-cell function. 32,33 However, the functional role of autophagy in other diabetic organs remains unknown.…”

Fatty Acids Suppress Autophagic Turnover in β-Cells

“…Treatment of ␤-cells with FFAs and glucose has been previously associated with an increase in AP number (9,13,15), which was attributed to an increase in autophagic flux. Although recent advances have provided some evidence questioning this interpretation (18), this study is the first attempt to focus on the long term effect of FFAs on autophagic turnover.…”

“…Whether any of the diabetogenic factors such as nutrition and obesity impair autophagy is yet unclear. In various animal models (including mice fed high fat diet) (9, 14) and cellular models of diabetes (including glucotoxicity and lipotoxicity) (9,(13)(14)(15)(16), the number of APs has been reported to increase. Whether this increase reflects an increase or a decrease in autophagic turnover is unresolved.…”

Autophagy in adipose tissue and the beta cell: implications for obesity and diabetes

“…Reminiscent of the situation in adipose tissue, an increased number of autophagosomes and autophagolysosomes was observed in the beta cells of different models of type 2 diabetes, including ob/ob and db/db mice [75,76] and Akita mice [10]. In Zucker diabetic fatty rats and in insulinoma cells, hyperglycaemia and oxidative stress led to the accumulation of polyubiquitinated protein aggregates that were degraded by autophagy [77]. Importantly, beta cells of human type 2 diabetic patients showed a massive overload of autophagic vacuoles that associated with beta cell death, without nuclear condensation, which was referred to as autophagy-associated cell death [78].…”