Sorcin is a calcium-binding protein involved in maintaining endoplasmic reticulum (ER) Ca 2+ stores. We have previously shown that overexpressing sorcin under the rat insulin promoter was protective against high-fat diet-induced pancreatic beta-cell dysfunction in vivo. Activating transcription factor 6 (ATF6) is a key mediator of the unfolded protein response (UPR) that provides cellular protection during the progression of ER stress. Here, using nonexcitable HEK293 cells, we show that sorcin overexpression increased ATF6 signalling, whereas sorcin knock out caused a reduction in ATF6 transcriptional activity and increased ER stress. Altogether, our data suggest that sorcin downregulation during lipotoxic stress may prevent full ATF6 activation and a normal UPR during the progression of obesity and insulin resistance.

Word count: 5222List of abbreviations: ATF6, activating transcription factor 6; BiP, binding immunoglobulin protein; CHOP, C/EBP homologue protein; ChREBP, Carbohydrate Responsive Element Binding Protein; DTT, dithiothreitol; ER, endoplasmic reticulum; FFA, free fatty acids; GRP78, glucose-regulated protein 78; HFD, high fat diet; MEF, mouse embryonic fibroblast; NEFA, nonesterified fatty acid; NFAT, nuclear factor of activated T-cells; RIP, rat insulin promoter; RyR, ryanodine receptor; SERCA, sarco/endoplasmic reticulum Ca 2+ -ATPase; UPR, unfolded protein response.Sorcin activates ATF6 despite lowering ER stress for bioRxiv 2 ABSTRACT Levels of the transcription factor ATF6, a key mediator of the unfolded protein response, that provides cellular protection during the progression endoplasmic reticulum (ER) stress, are markedly reduced in the pancreatic islet of patients with type 2 diabetes and in rodent models of the disease, including ob/ob and high fat-fed mice. Sorcin (gene name SRI) is a calcium (Ca 2+ ) binding protein involved in maintaining ER Ca 2+ homeostasis.We have previously shown that overexpressing sorcin under the rat insulin promoter in transgenic mice was protective against high fat diet-induced pancreatic beta cell dysfunction, namely preserving intracellular Ca 2+ homeostasis and glucose-stimulated insulin secretion during lipotoxic stress.Additionally, sorcin overexpression was apparently activating ATF6 signalling in MIN6 cells despite lowering ER stress.Here, in order to investigate further the relationship between sorcin and ATF6, we describe changes in sorcin expression during ER and lipotoxic stress and changes in ATF6 signalling after sorcin overexpression or inactivation, both in excitable and non-excitable cells.Sorcin mRNA levels were significantly increased in response to the ER stress-inducing agents thapsigargin and tunicamycin, but not by palmitate. On the contrary, palmitate caused a significant decrease in sorcin expression as assessed by both qRT-PCR and Western blotting despite inducing ER stress. Moreover, palmitate prevented the increase in sorcin expression induced by thapsigargin. In addition, sorcin overexpression significantly increased ATF6 transcriptional activity, whereas sorcin inactivation decreased ATF6 signalling. Finally, sorcin overexpression increased levels of ATF6 immunoreactivity and FRET imaging experiments following ER stress induction by thapsigargin showed a direct sorcin-ATF6 interaction.Altogether, our data suggest that sorcin down-regulation during lipotoxicity may prevent full ATF6 activation and a normal UPR during the progression of obesity and insulin resistance, contributing to beta cell failure and type 2 diabetes.