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The sarcoendoplasmic reticulum (ER) Ca2+ ATPase 2 (SERCA2) pump is a P-type ATPase tasked with the maintenance of ER Ca2+ stores. Whereas β-cell SERCA2 expression is reduced in diabetes, the role of SERCA2 in the regulation of whole-body glucose homeostasis has remained uncharacterized. To this end, SERCA2 heterozygous mice (S2HET) were challenged with a high-fat diet (HFD) containing 45% of kilocalories from fat. After 16 weeks of the HFD, S2HET mice were hyperglycemic and glucose intolerant, but adiposity and insulin sensitivity were not different between HFD-fed S2HET mice and HFD-fed wild-type controls. Consistent with a defect in β-cell function, insulin secretion, glucose-induced cytosolic Ca2+ mobilization, and the onset of steady-state glucose-induced Ca2+ oscillations were impaired in HFD-fed S2HET islets. Moreover, HFD-fed S2HET mice exhibited reduced β-cell mass and proliferation, altered insulin production and proinsulin processing, and increased islet ER stress and death. In contrast, SERCA2 activation with a small molecule allosteric activator increased ER Ca2+ storage and rescued tunicamycin-induced β-cell death. In aggregate, these data suggest a critical role for SERCA2 and the regulation of ER Ca2+ homeostasis in the β-cell compensatory response to diet-induced obesity.

We previously identified intersectin, a multiple EH and SH3 domain-containing protein, as a component of the endocytic machinery. Overexpression of the SH3 domains of intersectin blocks transferrin receptor endocytosis, possibly by disrupting targeting of accessory proteins of clathrin-coated pit formation. More recently, we identified mammalian Sos, a guanine-nucleotide exchange factor for Ras, as an intersectin SH3 domainbinding partner. We now demonstrate that overexpression of intersectin's SH3 domains blocks activation of Ras and MAP kinase in various cell lines. Several studies suggest that activation of MAP kinase downstream of multiple receptor types is dependent on endocytosis. Thus, the dominant-negative effect of the SH3 domains on Ras/MAP kinase activation may be indirectly mediated through a block in endocytosis. Consistent with this idea, incubating cells at 4°C or with phenylarsine oxide, treatments previously established to inhibit EGF receptor endocytosis, blocks EGF-dependent activation of MAP kinase. However, under these conditions, Ras activity is unaffected and overexpression of the SH3 domains of intersectin is still able to block Ras activation. Thus, intersectin SH3 domain overexpression can effect EGF-mediated MAP kinase activation directly through a block in Ras, consistent with a functional role for intersectin in Ras activation. Src homology 3 (SH3)1 domains are 50-to 70-amino acid protein modules that mediate protein-protein interactions through binding to specific proline-rich peptide sequences. The recruitment to the plasma membrane of the Ras guanine-nucleotide exchange factor, mammalian son-of-sevenless (mSos), via the actions of the adaptor protein Grb2, is a well characterized example of a functional role for SH3 domain-mediated interactions (1-6). Through its SH3 domains, Grb2 interacts with proline-rich sequences in mSos, and through its SH2 domain, Grb2 interacts with SHC or activated growth factor receptors, thereby recruiting mSos to the membrane where it can activate Ras (7,8). Activated Ras initiates a phosphorylation cascade culminating in phosphorylation and activation of the p42/p44 MAP kinases (Erk-1 and Erk-2).SH3 domain-mediated protein-protein interactions also function broadly in vesicular trafficking, particularly in clathrin-mediated endocytosis (9). For example, several laboratories have recently identified and cloned a novel protein, variously referred to as intersectin (frog and man) (10, 11), Dap160 (fruit fly) (12), Ese (mouse) (13), and EHSH (rat) (14), which contains two Eps15 homology (EH) domains and four or five tandem SH3 domains. Through its SH3 domains, intersectin interacts with the endocytic enzyme dynamin, and through its EH domains, intersectin binds the epsins (10), which are recently identified EH domain-interacting proteins that localize to clathrin-coated pits (15). Intersectin is also localized to clathrin-coated pits in neurons and non-neuronal cells (16), and it has been proposed that the protein functions as a molecular scaffold in clat...

Edited by Jeffrey E. Pessin Alterations in endoplasmic reticulum (ER) calcium (Ca 2؉) levels diminish insulin secretion and reduce ␤-cell survival in both major forms of diabetes. The mechanisms responsible for ER Ca 2؉ loss in ␤ cells remain incompletely understood. Moreover, a specific role for either ryanodine receptor (RyR) or inositol 1,4,5-triphosphate receptor (IP 3 R) dysfunction in the pathophysiology of diabetes remains largely untested. To this end, here we applied intracellular and ER Ca 2؉ imaging techniques in INS-1 ␤ cells and isolated islets to determine whether diabetogenic stressors alter RyR or IP 3 R function. Our results revealed that the RyR is sensitive mainly to ER stress-induced dysfunction, whereas cytokine stress specifically alters IP 3 R activity. Consistent with this observation, pharmacological inhibition of the RyR with ryanodine and inhibition of the IP 3 R with xestospongin C prevented ER Ca 2؉ loss under ER and cytokine stress conditions, respectively. However, RyR blockade distinctly prevented ␤-cell death, propagation of the unfolded protein response (UPR), and dysfunctional glucose-induced Ca 2؉ oscillations in tunicamycin-treated INS-1 ␤ cells and mouse islets and Akita islets. Monitoring at the single-cell level revealed that ER stress acutely increases the frequency of intracellular Ca 2؉ transients that depend on both ER Ca 2؉ leakage from the RyR and plasma membrane depolarization. Collectively, these findings indicate that RyR dysfunction shapes ER Ca 2؉ dynamics in ␤ cells and regulates both UPR activation and cell death, suggesting that RyR-mediated loss of ER Ca 2؉ may be an early pathogenic event in diabetes. This work was supported by National Institutes of Health Grants R01 DK093954 and UC4 DK 104166 (to C. E-M.); Department of Veterans Affairs Merit Award I01BX001733 (to C. E-M.); and Sigma Beta Sorority, Ball Brothers Foundation, and George and Frances Ball Foundation gifts (to C. E-M.).