GRP75 mediates endoplasmic reticulum–mitochondria coupling during palmitate-induced pancreatic β-cell apoptosis

Shweta Tiwary,Arun Nandwani,Rukshar Khan,Malabika Datta

doi:10.1016/j.jbc.2021.101368

Abstract

The endoplasmic reticulum (ER) and mitochondria are structurally connected with each other at specific sites termed mitochondria-associated membranes (MAMs). These physical links are composed of several tethering proteins and are important during varied cellular processes, such as calcium homeostasis, lipid metabolism and transport, membrane biogenesis, and organelle remodeling. However, the attributes of specific tethering proteins in these cellular functions remain debatable. Here, we present data to show that one such tether protein, glucose regulated protein 75 (GRP75), is essential in increasing ER–mitochondria contact during palmitate-induced apoptosis in pancreatic insulinoma cells. We demonstrate that palmitate increased GRP75 levels in mouse and rat pancreatic insulinoma cells as well as in mouse primary islet cells. This was associated with increased mitochondrial Ca2+ transfer, impaired mitochondrial membrane potential, increased ROS production, and enhanced physical coupling between the ER and mitochondria. Interestingly, GRP75 inhibition prevented these palmitate-induced cellular aberrations. Additionally, GRP75 overexpression alone was sufficient to impair mitochondrial membrane potential, increase mitochondrial Ca2+ levels and ROS generation, augment ER–mitochondria contact, and induce apoptosis in these cells. In vivo injection of palmitate induced hyperglycemia and hypertriglyceridemia, as well as impaired glucose and insulin tolerance in mice. These animals also exhibited elevated GRP75 levels accompanied by enhanced apoptosis within the pancreatic islets. Our findings suggest that GRP75 is critical in mediating palmitate-induced ER–mitochondrial interaction leading to apoptosis in pancreatic islet cells.

Highlights

Eukaryotic cells possess a complex network among various intracellular compartments and organelles, in addition to each executing their specific own functions
Palmitate-induced apoptosis is often preceded by induction of endoplasmic reticulum (ER) stress, and our data demonstrated that palmitate (0.4 mM) increased the protein levels of the ER stress markers, binding immunoglobulin protein (BiP) and C/EBP homologous protein (CHOP) (Fig. 2A)
While incubation of MIN6 cells with an ER stress inducer, tunicamycin (5 μg/ml; 8 h) induced ER stress within the cells as evidenced by increased protein levels of BiP and CHOP, there was no significant change in the protein levels of glucose regulated protein 75 (GRP75) (Fig. 2B), suggesting that induction of GRP75 levels can be achieved without the mediation of ER stress

Summary

Results

We had previously shown that during pancreatic β-cell apoptosis, there is ER and mitochondria dysfunction accompanied by increased transfer of calcium from the ER to mitochondria [29, 30]. As in the in-situ PLA, overexpression of GRP75 alone was adequate enough to promote significant decrease in ER–mitochondria proximity (Fig. 7D) These data substantiate the role of GRP75 in mediating ER– mitochondria interactions during palmitate-induced cellular dysfunction in MIN6 cells. When evaluated in a transgenic diabetic (db/db) mice model, it was observed that GRP75 levels are significantly increased in the pancreatic tissues of these mice as compared with normal (db/+) mice (Fig. 9G) All these data suggest that GRP75 plays a potential critical mediatory role in pancreatic islet apoptosis mediated by palmitate, in vitro and in vivo

Discussion

Experimental procedures

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