Abstract
Calcium homeostasis is essential for maintaining the viability and function of pancreatic β cells and plays a key role in preventing the development of diabetes. Decreased levels of ATPase sarcoplasmic/endoplasmic reticulum Ca2+-transporting 2 (ATP2a2), the main calcium pump in β cells, are often found in individuals with diabetes and in diabetic animal models. However, the regulators of ATP2a2 and the molecular mechanisms responsible for controlling ATP2a2 activity remain unclear. Etoposide-induced protein 2.4 (Ei24) is also down-regulated in β cells of diabetic individuals, whereas the effect of decreased Ei24 level on β-cell function is not clarified. Here, using Cre-LoxP and CRISPR/Cas9-based genomic knockout (KO) approaches to generate pancreatic β cell-specific Ei24 KO mice and pancreatic β-cell lines, we found that Ei24 regulates ATP2a2 activity. Specifically, we observed that Ei24 binds to ATP2a2 through Ei24 residues 293-299, which we named here the ATP2a2-interacting region (AIR). Loss of Ei24 inactivated ATP2a2, disrupted calcium homeostasis, and deactivated the calcium/calmodulin-dependent protein kinase kinase 2 (CAMKK2)-AMP-activated protein kinase (AMPK) pathway. Elevation of calcium concentration in the endoplasmic reticulum or agonist-induced AMPK activation rescued pancreatic β-cell survival and improved glucose tolerance of Ei24 KO mice. Our findings indicate that targeting the Ei24-ATP2a2 interaction to increase ATP2a2 activity can protect pancreatic β cells and improve glucose homeostasis in diabetic models, suggesting that Ei24 could potentially serve as a target to prevent or manage diabetes.
Highlights
Calcium homeostasis is essential for maintaining the viability and function of pancreatic  cells and plays a key role in preventing the development of diabetes
Our findings indicate that targeting the Etoposide-induced protein 2.4 (Ei24) –ATPase sarcoplasmic/endoplasmic reticulum Ca2؉-transporting 2 (ATP2a2) interaction to increase ATP2a2 activity can protect pancreatic  cells and improve glucose homeostasis in diabetic models, suggesting that Ei24 could potentially serve as a target to prevent or manage diabetes
The results showed that loss of Ei24 leads to glucose intolerance, decreased insulin biosynthesis, impaired -cell cytosolic calcium homeostasis, reduced ER calcium levels, and increased apoptosis
Summary
To test the possible function of Ei24 in pancreatic -cell function under a pathological condition, we first examined the levels of Ei24 in HFD-fed mice, ob/ob mice, and GK rats. The results showed that the insulin release returned to the basal level (Fig. 1K) This suggested that the impaired glucose tolerance in KO mice may be primarily caused by insufficient GSIS. Increased protein levels of cleaved poly(ADP-ribose) polymerase (c-PARP) and cleaved caspase-3 (c-caspase-3), which mark apoptotic cascades within cells, were observed in the islets of the KO mice (Fig. 2I) These findings may account for the lack of -cell mass as well as the decreased insulin production. The insulin tolerance test that was administered on day 15 indicated that the insulin sensitivity did not significantly change (Fig. 7, C and D), further suggesting the protective role of CDN1163 on  cells These results strongly emphasize a pivotal role for ATP2a2 activity, which is modulated by Ei24 in the maintenance of calcium homeostasis and -cell functions
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