The ER is responsible for calcium storage lipid
The ER is responsible for calcium storage, lipid and protein biosynthesis, and the folding, assembly, and maturation of proteins , and ER stress is one of the main causes in T2DM, which leads to β-cell apoptosis and dysfunction [, , ]. In this study, TG was used as an ER stress inducer. TG is a specific inhibitor of sarcoplasmic/endoplasmic reticulum Ca2+-ATPase (SERCA) pump, which pumps intracellular Ca2+ back into the ER, so TG treatment induces depletion of ER Ca2+. This causes the Ca2+-dependent chaperones to lose their activities, which in turns leads to misfolded protein accumulation and ER stress . During ER stress, misfolded proteins are accumulated in the ER lumen, which activates the UPR [6,22]. The UPR is regulated by different proteins localised to the ER membrane: PKR-like ER kinase (PERK), IRE1α and activating transcription factor 6α (ATF6α) [22,23]. Several studies have shown that chronic exposure to high bacteriological hyper-activates IRE1α, which spliced X-box binding protein 1 (XBP-1) mRNA, suppressed insulin biosynthesis, and activated pro-apoptotic pathways, leading to β-cell apoptosis [, , ]. Herein we showed that TG activated IRE1α pathway, and pre-treatment with O-1602 and Abn-CBD ameliorated this activation, so this suggested that GPR55 agonists protected from ER stress through IRE1α pathway. Furthermore, cell apoptosis in β-cells is one of the main concerns in T2DM, and it has been demonstrated that ameliorating ER stress could prevent from β-cell apoptosis [, , ,41]. In the present study, we demonstrated that O-1602 and Abn-CBD significantly improved the cell viability and reduced ER stress-induced apoptosis in MIN6 cells, and this effect was markedly prevented by GPR55 knockdown, confirming that this effect was through GPR55. In addition, O-1602 and Abn-CBD significantly reduced the cleavage of caspase-3, another marker of cell apoptosis. Similar effects were observed in Beta-TC-6 cells. Taken together, our findings suggested that the activation of GPR55 by O-1602 and Abn-CBD has a protective role against ER stress-induced apoptosis in pancreatic β-cells. Furthermore, we also examined the downstream pathways that were involved in this protection by O-1602 and Abn-CBD. It has been demonstrated that GPR55 ligands were able to induce CREB phosphorylation in GPR55 expressed-HEK293 cells, mouse and human islets, and cultured rat myotubes [20,24,42]. CREB is a transcription factor which binds to CRE, and regulates its downstream genes , and the activation of CREB was shown to increase cell proliferation and survival [, , ]. Here we also confirmed that O-1602 and Abn-CBD induced CREB phosphorylation in MIN6 cells, and the protection of ER stress-induced apoptosis by O-1602 and Abn-CBD was prevented by CREB knockdown. The phosphorylated-CREB binds to CRE, which recruits the co-activators, CREB binding protein (CBP), and activates its downstream gene transcription. Consistently, a study also showed that enhanced CREB-CBP-gene transcription increased β-cell proliferation and reduced β-cell apoptosis in mouse isolated islets . Furthermore, prevention of CREB degradation by a CREB proteasomal targeting sequence peptide protected from hyperglycaemia-induced apoptosis in INS-1E cells and human islets, and the insulin secretion and contents were also preserved by this peptide in human islets, so they suggested that preservation of CREB is essential for β-cell survival and maintenance of glucose sensing, insulin gene transcription and exocytosis . Therefore, CREB was suggested to be an emerging transcriptional element for the maintenance of glucose sensing, insulin secretion, and β-cell survival [26,, , ], and our results also confirmed its role in mediating β-cell survival by GPR55 agonists. It has been well recognised that CREB phosphorylation could be regulated by several kinaes including PKA, CaMKIV and Erk1/2 [, , ]. PKA is activated by cAMP, while CaMKIV and Erk1/2 are activated in response to the elevation of intracellular calcium concentration ([Ca2+]i) . GPR55 agonists were shown to increase cAMP production, as well as intracellular Ca2+ responses in a β-cell line, BRIN-BD11 . In this study, we found that O-1602 and Abn-CBD activated PKA, CaMKIV and Erk1/2, and these kinases contributed to CREB phosphorylation by O-1602 and Abn-CBD. Consistently, other studies also showed that GPR55 agonists significantly increased Erk1/2 phosphorylation in GPR55-HEK293 cells, cultured rat myotubes and human osteoclasts [20,24,49,50]. Taken together, we suggested that GPR55 agonists activated CaMKIV, PKA and Erk1/2 to induce CREB activation in MIN6 cells.