Junctophilin 3 Plays A Critical Role In Insulin Secretion From Islet β Cells

Diabetes is a metabolic disorder characterized by hyperglycaemia. Oral medications and insulin injections treat but do not cure diabetes. The pathogenesis of diabetes is complicated, thus understanding the underlying mechanisms in the insulin secretion process contributes to the treatment of diabetes.Insulin secretion from pancreatic β-cells is caused by glucose-stimulated ATP increase and Ca2+ entry via voltage-dependent Ca2+ channels (VGCC). Glucose increases insulin secretion by increasing Ca2+ through providing ATP in the face of energy-consuming processes triggered by Ca2+ influx through voltage-gated Ca2+ channels (VGCC). Glucose-stimulated insulin secretion (GSIS) is the principal mechanism of insulin secretion. The mechanism involved in triggering GSIS is defined as the KATP channel-dependent pathway and -independent pathway. Defective mitochondrial function results in impaired GSIS and may contribute to the development of type 2 diabetes. Therefore finding new targets in regulting GSIS process may contributes to diabetes treatment.Insulin secretion is found to be associated with alterations in intracellular Ca2+ handling of both rodent and human pancreatic β cells. Ryanodine receptor (RyR), inositol 1,4,5-trisphosphate receptor (IP3R), sarco-endoplasmic reticulum Ca2+-ATPase (SERCA), and Store-operated Ca2+ entry (SOCE) are closely linked with insulin release. Junctophilins (JPs) contributed to the formation of junctional membrane complexes (JMCs) by spanning endoplasmic reticulum (ER) and interacting with the plasma membrane. JMCs exist in all excitable cells and are the structural foundation for functional crosstalk between ion channels on membranes. Considering rapidly increased cytosolic Ca2+ concentration through a combination of Ca2+ store release and extracellular Ca2+ influx plays key roles in β cells, we speculate that JP subtypes might exist in β cells, thereby take an essential role in JMC formation and insulin release. To date, however, whether JPs expressed in β cells and contribute to modulating insulin release has not yet been described. ER and mitochondria could be connected with Mitofusin 1 (Mfh1) and Mitofusin 2 (Mfh2), thereby facilitates the Ca2+ shuttling between these two organellae. In the Jph2 knockout myocytes, mitochondria showed abnormal structures, indicated that JMCs may also regulate the connection between ER and mitochondria. In the first section, we detected the expressions of JP subtypes in human and mouse β cells, and explored the role of JP in the insulin secretion function of mouse β cells as well as the underlying mechanisms.In the second section (addendum), we explored the role of mitochondrial metabolism related signal in the regenerative medicine of diabetes. Embryonic stem (ES) cells can differentiate into insulin-positive cells, offers innovative approach to promote adult stem cell differentiation, screen anti-diabetes drugs, supply donor β cell sources for cell transplantation and reveal mechanisms for induced pluripotent stem (iPS) cell researches.Peroxisome proliferator-activated receptors (PPARs) are nuclear receptors that play key roles in fatty acid catabolism, mitochondrial metabolism and cell differentiation. PPARs functioning as the sensor in fatty acid oxidation facilitate ES cell differentiation by promoting mitochondrial oxidative phosphorylation. Our previous work showed PPARβ activation was related to the differentiation of INS+ cells from mouse ES cells accompanied by normal mitochondrial membrane potential (ΔΨm) formation, however the GSIS function of the induced cells as well as the underlying mechanisms remained unclear. Here we investigated the role of PPARP in the GSIS function of induced INS+ cells, and explored the mechanisms in the differentiation. Pancreatic and duodenal homeobox-1 (Pdx-1) is essential for functionalβ cell generation. Forkhead box protein 01 (Foxo1) and Glycogen synthase kinase-3β (Gsk3β) are a negative regulator of Pdx-1. To date, the association between PPARβ activation and these molecules in ES cell-derived INS+ cell differentiation has not been releaved. In the second section, we explored the role of PPARβ activation in the differentiation of mouse and human ES cells into INS+ cells as well as the underlying mechanisms.1. Junctophilin3 takes a critical role in islet β cell insulin secretion function.Here, we investigated the expression of JPs in human and mouse β cells, and explored the mechanisms in GSIS of mouse islet β cells. Our results demonstrated that JP3 expresses in mouse and human pancreatic β cells. In mouse islets, JP3 maintains GSIS and contributes to PM-ER-mitochondrial axis integrity. Knockdown of Jp3 by siRNA in mouse islets impaired GSIS function, coupled with decreased mitochondrial ΔΨm, ATP production and IP3R mediated mitochondrial Ca2+ transient, indicating that the impaired GSIS function was associated with damaged mitochondrial function. Jp3 deficiency selectively decreased RyR-mediated Ca2+ transient, which thereby inhibited the nuclear translocation of Pgc-la and Mfh2 expression. Moverover, Pgc-la and PPARβ existed in the nucleus of human β cells, thus we inferred that they may modulate the expression of Mfn2, mitochondrial function as well as the insulin secretion. We also demonstrated that JP3 binded to RyR2 directly in human pancreatic tissues, indicating that JP3 may involve in the KATP-indenpent pathway in GSIS function. As a result, knockdown of Jp3 disturbed ER-mitochondria tethering, reduced ATP production, thereby impaired GSIS function.2. PPARβ activation promotes functional insulin-positive cell differentiation (addendum)Here, we explored the role of PPARβ activation in the differentiation of mouse and human ES cells into INS+ cells. Our results indicated that PPARP activation increased the number induced INS+ cells and the GSIS function. During mouse ES cell differentiation, PPARP activation specifically promoted β like cells by upregulating Pdx-1 expression. Therefore, we next investigated the roles of two Pdx-1 negative regulators, Foxol and Gsk3β in PPARβ-mediated differentiation. As a result, although both regulators were modulated by PPARβ, Gsk3β did not affect INS+ cell differentiation. In contrast, Foxol is a major signaling molecule involved in PPARβ/Pdx-1-promoted functional INS+ cell generation. PPARβ activated the PI3K/Akt signaling pathway, thereby increased cytosolic p-Foxol, which lead to the decreased nuclear Foxol. Most importantly, we further revealed that PPARβ activation also exhibited its promoting effect on human ES cell-derived INS+ cell differentiation and insulin secretion via the same signaling pathway in mouse ES cell differentiation.Conclusion:1. JP3 expresses in human and mouse β cells. It maintained the ultrastructure of plasma membrane-ER-mitochondria and regulated the insulin secretion in mouse islet (3 cells. In mouse islets, JP3 maintains GSIS and contributes to PM-ER-mitochondrial axis integrity. Jp3 deficiency resulted in the damage of GSIS accompanied by energetic deficit due to decreasing in Mfn2 expression and Pgc-1α nuclear translocation, suggesting a novel role of JP3 in regulating GSIS. In human pancreas, JP3 binded to RyR2 directly in human pancreatic tissues, suggesting that JP3 may involve in the K.ATP-indenpent pathway in GSIS function as well. Pgc-1α and PPARβ existed in the nucleus of human β cells, indiacting that they may modulate the expression of Mfn2, mitochondrial function as well as the insulin secretion.2. PPARp activation facilitated the human and mouse ES cells-derived INS+ cell differentiation and amplified GSIS of induced cells by regulating the key factor Pdx-1. The p-Foxo1/Foxo1 status modulated by PI3K/Akt activation contributes to PPARβ-mediated INS+ cell differentiation and GSIS.