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.
Background Soluble Resistance Related Calcium Binding Protein (sorcin) is a calcium (Ca2+) binding protein which has been shown to play a role in maintaining intracellular endoplasmic reticulum (ER) Ca2+ stores and lowering ER stress. Recently, our lab has demonstrated that sorcin expression was downregulated in the islets of Langerhans of mice fed a high-fat diet or in human islets incubated with the saturated fatty acid palmitate. We also showed that overexpression of sorcin under control of the rat insulin promoter (RIP7) in C57BL/6J mice, or whole body sorcin deletion in 129S1/SvImJ mice, improves or impairs insulin secretion and pancreatic β-cell function respectively. The mechanisms behind this beneficial role of sorcin in the pancreatic β-cell might depend on protection against lipotoxic endoplasmic reticulum (ER) stress through improved ER Ca2+ dynamics and activation of the Activating Transcription Factor 6 (ATF6) branch of the unfolded protein response (UPR). Whether sorcin is also implicated in hypothalamic ER stress during the progression of obesity is unknown. Aim To investigate a potential role of sorcin in hypothalamic ER stress, leptin resistance, hyperphagia and obesity. Methods Whole-body sorcin null mice, backcrossed onto the C57BL/6J genetic background, were used. Body weight, food intake and EchoMRI body composition were measured in vivo whereas qRT-PCR analysis of sorcin and ER stress markers expression were performed on the arcuate nucleus of the hypothalamus. Leptin signalling through STAT3 phosphorylation was measured by Western blots on sorcin-null HEK293 cells, engineered by CRISPR/Cas9, and transfected with leptin receptor (LepRb). Results Sorcin expression was not influenced in the arcuate nucleus (ARC) of the hypothalamus by diet-induced obesity. Whole-body sorcin ablation did not cause ARC ER stress nor changes in body weight, body composition or food intake in C57BL/6 male mice exposed to a high-fat, high-sugar diet. STAT3 phosphorylation (Y705) in response to leptin was not impaired in sorcin-null HEK293 cells. Conclusion In our model, whole body sorcin ablation did not increase hypothalamic ER stress nor influenced food intake or body weight.
Background: Soluble Resistance Related Calcium Binding Protein (sorcin) is a calcium (Ca2+) binding protein which has been shown to play a role in maintaining intracellular endoplasmic reticulum (ER) Ca2+ stores and lowering ER stress. Recently, our lab has demonstrated that sorcin expression was downregulated in the islets of Langerhans of mice fed a high-fat diet or in human islets incubated with the saturated fatty acid palmitate. We also showed that overexpression of sorcin under control of the rat insulin promoter (RIP7) in C57BL/6J mice, or whole body sorcin deletion in 129S1/SvImJ mice, improves or impairs insulin secretion and pancreatic β-cell function respectively. The mechanisms behind this beneficial role of sorcin in the pancreatic β-cell might depend on protection against lipotoxic endoplasmic reticulum (ER) stress through improved ER Ca2+ dynamics and activation of the Activating Transcription Factor 6 (ATF6) branch of the unfolded protein response (UPR). Whether sorcin is also implicated in hypothalamic ER stress during the progression of obesity is unknown. This could potentially contribute to the diminished satiety typically observed in overweight individuals. Aim: To investigate a potential role of sorcin in hypothalamic ER stress, leptin resistance, hyperphagia and obesity. Methods: Whole-body sorcin null mice, backcrossed onto the C57BL/6J genetic background, were used. Body weight, food intake and EchoMRI body composition were measured in vivo whereas qRT-PCR analysis of sorcin and ER stress markers expression were performed on the arcuate nucleus of the hypothalamus. Leptin signalling through STAT3 phosphorylation was measured by Western blots on sorcin-null HEK293 cells, engineered by CRISPR/Cas9, and transfected with leptin receptor (LepRb). Results: Sorcin expression was not influenced in the arcuate nucleus (ARC) of the hypothalamus by diet-induced obesity. Whole-body sorcin ablation did not cause ARC ER stress nor changes in body weight, body composition or food intake in C57BL/6 male mice exposed to a high-fat, high-sugar diet. STAT3 phosphorylation (Y705) in response to leptin was not impaired in sorcin-null HEK293 cells. Conclusion: In our model, whole body sorcin ablation did not increase hypothalamic ER stress nor influenced food intake or body weight.
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