Potential Role Of Endoplasmic Reticulum Redox Changes In Endoplasmic Reticulum Stress And Impaired Protein Folding In Obesity-Associated Insulin Resistance

Abstract

Endoplasmic reticulum (ER) stress plays an important role in the pathogenesis of obesity-related inflammation and insulin resistance in adipose tissue. However, the mechanisms responsible for induction of ER stress are presently unclear. Proper ER redox state is crucial for oxidative protein folding and secretion and impaired protein folding in ER leads to induction of unfolded protein response and ER stress. However, while ER redox state is more oxidizing compared to the rest of the cell, its regulation is poorly understood. In order to determine the effects of ER redox state on development of ER stress and insulin resistance, several fluorescence-based sensors have been developed. However, these sensors have yielded results that are inconsistent with each other and with earlier non-fluorescence-based studies. In this study we attempted to develop and characterize a sensitive tool to study the ER redox state in adipocytes in real-time by targeting a new generation of redox-sensitive green fluorescent protein (roGFP) to ER. The roGFP1-iL sensor targeted to the ER is termed ‘eroGFP1-iL’ by convention. The ER-targeting eroGFP1-iL construct contains the signal peptide from adiponectin and the ER retention motif KDEL and has a midpoint reduction potential of -229 mV in vitro in oxidized and reduced lipoic acid. Despite having a midpoint reduction potential that is 50 mV higher than the previously determined midpoint reduction potential of the ER, eroGFP1-iL was found capable of detecting both oxidizing and reducing changes in the ER. In an attempt to determine the mechanisms by which roGFP1-iL detects oxidizing changes, we found that, first, glutathione mediated the formation of disulfide-bonded roGFP1-iL dimers with an intermediate excitation fluorescence spectrum resembling a mixture of oxidized and reduced monomers. Second, glutathione facilitated dimerization of roGFP1-iL, which in effect shifted the equilibrium from oxidized monomers to dimers, thereby increasing the molecule’s reduction potential compared with a dithiol redox buffer like lipoic acid. From this study, we concluded that the glutathione redox couple in ER significantly raised the reduction potential of roGFP1-iL in vivo by facilitating its dimerization while preserving its ratiometric nature, which makes it suitable for monitoring oxidizing and reducing changes in ER with high reliability in real-time. The ability of roGFP1-iL to detect both oxidizing and reducing changes in ER and its dynamic response in glutathione redox buffer between approximately -190 and -130 mV in vitro suggest a range of ER redox potential consistent with those determined by earlier approaches that did not involve fluorescent sensors. Our primary aim in developing eroGFP1-iL as a redox-sensing tool was to be able to assess whether redox changes represent an early initiator of ER stress in obesity-induced reduction in high molecular weight (HMW) adiponectin in circulation. Hypoxia is a known mediator of redox changes. We found that oligomerization of HMW adiponectin was impaired in the hypoxic conditions observed in differentiated fat cells. The redox-active antioxidant ascorbate was found capable of reversing hypoxia-induced ER stress. Lastly, we demonstrated that changes in ER redox condition is associated with ER stress response and is implicated in the mechanism of action of the insulin-sensitizing agent troglitazone and desensitizing agent palmitate. Using the redox sensing property of eroGFP1-iL, palmitate was found to be an effective modulator of redox changes in the ER and troglitazone was found to cause oxidizing changes in the ER. The action of palmitate in causing aberrant ER redox conditions was associated with aberrant HMW adiponectin multimerization. Palmitate-induced ER stress was ameliorated by troglitazone. Taken together, the data suggest a potential role of ER redox changes in ER stress and impaired protein folding in adipocytes.