Bromodomain Protein Inhibition Protects ß-Cells from Cytokine-Induced Death and Dysfunction via Antagonism of NF-κB Pathway.

Cytokine-induced ß-cell apoptosis is a major pathogenic mechanism in type 1 diabetes (T1D). Despite significant advances in understanding its underlying mechanisms, few drugs have been translated to protect ß-cells in T1D. Epigenetic modulators such as bromodomain-containing BET (bromo- and extra-terminal) proteins are important regulators of immune responses. Pre-clinical studies have demonstrated a protective effect of BET inhibitors in an NOD (non-obese diabetes) mouse model of T1D. However, the effect of BET protein inhibition on ß-cell function in response to cytokines is unknown. Here, we demonstrate that I-BET, a BET protein inhibitor, protected ß-cells from cytokine-induced dysfunction and death. In vivo administration of I-BET to mice exposed to low-dose STZ (streptozotocin), a model of T1D, significantly reduced ß-cell apoptosis, suggesting a cytoprotective function. Mechanistically, I-BET treatment inhibited cytokine-induced NF-kB signaling and enhanced FOXO1-mediated anti-oxidant response in ß-cells. RNA-Seq analysis revealed that I-BET treatment also suppressed pathways involved in apoptosis while maintaining the expression of genes critical for ß-cell function, such as Pdx1 and Ins1. Taken together, this study demonstrates that I-BET is effective in protecting ß-cells from cytokine-induced dysfunction and apoptosis, and targeting BET proteins could have potential therapeutic value in preserving ß-cell functional mass in T1D.

The Clock-modulatory Activity of Nobiletin Suppresses Adipogenesis Via Wnt Signaling.

The circadian clock machinery exerts transcriptional control to modulate adipogenesis and its disruption leads to the development of obesity. Here, we report that Nobiletin, a circadian clock amplitude-enhancing molecule, displays antiadipogenic properties via activation of Wnt signaling pathway that is dependent on its clock modulation. Nobiletin augmented clock oscillatory amplitude with period lengthening in the adipogenic mesenchymal precursor cells and preadipocytes, accompanied by an induction of Bmal1 and clock components within the negative feedback arm. Consistent with its clock-modulatory activity, Nobiletin strongly inhibited the lineage commitment and terminal differentiation of adipogenic progenitors. Mechanistically, we show that Nobiletin induced the reactivation of Wnt signaling during adipogenesis via transcriptional up-regulation of key components within this pathway. Furthermore, Nobiletin administration in mice markedly reduced adipocyte hypertrophy, leading to a significant loss of fat mass and reduction of body weight. Last, Nobiletin inhibited the differentiation of primary preadipocytes, and this effect was dependent on a functional clock regulation. Collectively, our findings uncover a novel activity of Nobiletin in suppressing adipocyte development in a clock-dependent manner, implicating its potential application in countering obesity and associated metabolic consequences.

The clock-modulatory activity of Nobiletin suppresses adipogenesis via Wnt signaling.

The circadian clock machinery exerts transcriptional control to modulate adipogenesis and its disruption leads to the development of obesity. Here we report that Nobiletin, a clock amplitude-enhancing molecule, displays anti-adipogenic properties via activating a clock-controlled Wnt signaling pathway that suppresses adipocyte differentiation. Nobiletin augmented clock oscillation with period length shortening in the adipogenic mesenchymal precursor cells and preadipocytes, accompanied by an induction of Bmal1 and core clock components. Consistent with its circadian clock-modulatory activity, Nobiletin inhibited the lineage commitment and terminal differentiation of adipogenic progenitors. Mechanistically, we show that Nobiletin induced the re-activation of Wnt signaling during adipogenic differentiation via transcriptional up-regulation of key components of this pathway. Furthermore, Nobiletin administration in mice markedly reduced adipocyte hypertrophy, leading to a significant loss of fat mass and body weight reduction. Lastly, Nobiletin inhibited the maturation of primary preadipocytes and this effect was dependent on a functional clock regulation. Collectively, our findings uncover a novel activity of Nobiletin in suppressing adipocyte development, implicating its potential therapeutic application in countering obesity and its associated metabolic consequences.

VGLL4 and MENIN function as TEAD1 corepressors to block pancreatic ß cell proliferation.

TEAD1 and the mammalian Hippo pathway regulate cellular proliferation and function, though their regulatory function in ß cells remains poorly characterized. In this study, we demonstrate that while ß cell-specific TEAD1 deletion results in a cell-autonomous increase of ß cell proliferation, ß cell-specific deletion of its canonical coactivators, YAP and TAZ, does not affect proliferation, suggesting the involvement of other cofactors. Using an improved split-GFP system and yeast two-hybrid platform, we identify VGLL4 and MENIN as TEAD1 corepressors in ß cells. We show that VGLL4 and MENIN bind to TEAD1 and repress the expression of target genes, including FZD7 and CCN2, which leads to an inhibition of ß cell proliferation. In conclusion, we demonstrate that TEAD1 plays a critical role in ß cell proliferation and identify VGLL4 and MENIN as TEAD1 corepressors in ß cells. We propose that these could be targeted to augment proliferation in ß cells for reversing diabetes.

TEAD1 regulates cell proliferation through a pocket-independent transcription repression mechanism.

The Hippo-TEAD pathway regulates cellular proliferation and function. The existing paradigm is that TEAD co-activators, YAP and TAZ, and co-repressor, VGLL4, bind to the pocket region of TEAD1 to enable transcriptional activation or repressive function. Here we demonstrate a pocket-independent transcription repression mechanism whereby TEAD1 controls cell proliferation in both non-malignant mature differentiated cells and in malignant cell models. TEAD1 overexpression can repress tumor cell proliferation in distinct cancer cell lines. In pancreatic ß cells, conditional knockout of TEAD1 led to a cell-autonomous increase in proliferation. Genome-wide analysis of TEAD1 functional targets via transcriptomic profiling and cistromic analysis revealed distinct modes of target genes, with one class of targets directly repressed by TEAD1. We further demonstrate that TEAD1 controls target gene transcription in a motif-dependent and orientation-independent manner. Mechanistically, we show that TEAD1 has a pocket region-independent, direct repressive function via interfering with RNA polymerase II (POLII) binding to target promoters. Our study reveals that TEAD1 target genes constitute a mutually restricted regulatory loop to control cell proliferation and uncovers a novel direct repression mechanism involved in its transcriptional control that could be leveraged in future studies to modulate cell proliferation in tumors and potentially enhance the proliferation of normal mature cells.

Dual pancreatic adrenergic and dopaminergic signaling as a therapeutic target of bromocriptine.

Bromocriptine is approved as a diabetes therapy, yet its therapeutic mechanisms remain unclear. Though bromocriptine's actions have been mainly attributed to the stimulation of brain dopamine D2 receptors (D2R), bromocriptine also targets the pancreas. Here, we employ bromocriptine as a tool to elucidate the roles of catecholamine signaling in regulating pancreatic hormone secretion. In ß-cells, bromocriptine acts on D2R and α2A-adrenergic receptor (α2A-AR) to reduce glucose-stimulated insulin secretion (GSIS). Moreover, in α-cells, bromocriptine acts via D2R to reduce glucagon secretion. α2A-AR activation by bromocriptine recruits an ensemble of G proteins with no ß-arrestin2 recruitment. In contrast, D2R recruits G proteins and ß-arrestin2 upon bromocriptine stimulation, demonstrating receptor-specific signaling. Docking studies reveal distinct bromocriptine binding to α2A-AR versus D2R, providing a structural basis for bromocriptine's dual actions on ß-cell α2A-AR and D2R. Together, joint dopaminergic and adrenergic receptor actions on α-cell and ß-cell hormone release provide a new therapeutic mechanism to improve dysglycemia.

Dopamine regulates pancreatic glucagon and insulin secretion via adrenergic and dopaminergic receptors.

Dopamine (DA) and norepinephrine (NE) are catecholamines primarily studied in the central nervous system that also act in the pancreas as peripheral regulators of metabolism. Pancreatic catecholamine signaling has also been increasingly implicated as a mechanism responsible for the metabolic disturbances produced by antipsychotic drugs (APDs). Critically, however, the mechanisms by which catecholamines modulate pancreatic hormone release are not completely understood. We show that human and mouse pancreatic α- and ß-cells express the catecholamine biosynthetic and signaling machinery, and that α-cells synthesize DA de novo. This locally-produced pancreatic DA signals via both α- and ß-cell adrenergic and dopaminergic receptors with different affinities to regulate glucagon and insulin release. Significantly, we show DA functions as a biased agonist at α2A-adrenergic receptors, preferentially signaling via the canonical G protein-mediated pathway. Our findings highlight the interplay between DA and NE signaling as a novel form of regulation to modulate pancreatic hormone release. Lastly, pharmacological blockade of DA D2-like receptors in human islets with APDs significantly raises insulin and glucagon release. This offers a new mechanism where APDs act directly on islet α- and ß-cell targets to produce metabolic disturbances.

Light-Dark Patterns Mirroring Shift Work Accelerate Atherosclerosis and Promote Vulnerable Lesion Phenotypes.

Background Despite compelling epidemiological evidence that circadian disruption inherent to long-term shift work enhances atherosclerosis progression and vascular events, the underlying mechanisms remain poorly understood. A challenge to the use of mouse models for mechanistic and interventional studies involving light-dark patterns is that the spectral and absolute sensitivities of the murine and human circadian systems are very different, and light stimuli in nocturnal mice should be scaled to represent the sensitivities of the human circadian system. Methods and Results We used calibrated devices to deliver to low-density lipoprotein receptor knockout mice light-dark patterns representative of that experienced by humans working day shifts or rotating shift schedules. Mice under day shifts were maintained under regular 12 hours of light and 12 hours of dark cycles. Mice under rotating shift schedules were subjected for 11 weeks to reversed light-dark patterns 4 days in a row per week, followed by 3 days of regular light-dark patterns. In both protocols the light phases consisted of monochromatic green light at an irradiance of 4 µW/cm2. We found that the shift work paradigm disrupts the foam cell's molecular clock and increases endoplasmic reticulum stress and apoptosis. Lesions of mice under rotating shift schedules were larger and contained less prostabilizing fibrillar collagen and significantly increased areas of necrosis. Conclusions Low-density lipoprotein receptor knockout mice under light-dark patterns analogous to that experienced by rotating shift workers develop larger and more vulnerable plaques and may represent a valuable model for further mechanistic and/or interventional studies against the deleterious vascular effects of rotating shift work.

Chronic circadian shift leads to adipose tissue inflammation and fibrosis.

The circadian clock exerts temporal coordination of metabolic pathways. Clock disruption is intimately linked with the development of obesity and insulin resistance, and our previous studies found that the essential clock transcription activator, Brain and Muscle Arnt-like 1 (Bmal1), is a key regulator of adipogenesis. However, the metabolic consequences of chronic shiftwork on adipose tissues have not been clearly defined. Here, using an environmental lighting-induced clock disruption that mimics rotating shiftwork schedule, we show that chronic clock dysregulation for 6 months in mice resulted in striking adipocyte hypertrophy with adipose tissue inflammation and fibrosis. Both visceral and subcutaneous depots display enlarged adipocyte with prominent crown-like structures indicative of macrophage infiltration together with evidence of extracellular matrix remodeling. Global transcriptomic analyses of these fat depots revealed that shiftwork resulted in up-regulations of inflammatory, adipogenic and angiogenic pathways with disruption of normal time-of-the-day-dependent regulation. These changes in adipose tissues are associated with impaired insulin signaling in mice subjected to shiftwork, together with suppression of the mTOR signaling pathway. Taken together, our study identified the significant adipose depot dysfunctions induced by chronic shiftwork regimen that may underlie the link between circadian misalignment and insulin resistance.

Tead1 is essential for mitochondrial function in cardiomyocytes.

Mitochondrial dysfunction occurs in most forms of heart failure. We have previously reported that Tead1, the transcriptional effector of Hippo pathway, is critical for maintaining adult cardiomyocyte function, and its deletion in adult heart results in lethal acute dilated cardiomyopathy. Growing lines of evidence indicate that Hippo pathway plays a role in regulating mitochondrial function, although its role in cardiomyocytes is unknown. Here, we show that Tead1 plays a critical role in regulating mitochondrial OXPHOS in cardiomyocytes. Assessment of mitochondrial bioenergetics in isolated mitochondria from adult hearts showed that loss of Tead1 led to a significant decrease in respiratory rates, with both palmitoylcarnitine and pyruvate/malate substrates, and was associated with reduced electron transport chain complex I activity and expression. Transcriptomic analysis from Tead1-knockout myocardium revealed genes encoding oxidative phosphorylation, TCA cycle, and fatty acid oxidation proteins as the top differentially enriched gene sets. Ex vivo loss of function of Tead1 in primary cardiomyocytes also showed diminished aerobic respiration and maximal mitochondrial oxygen consumption capacity, demonstrating that Tead1 regulation of OXPHOS in cardiomyocytes is cell autonomous. Taken together, our data demonstrate that Tead1 is a crucial transcriptional node that is a cell-autonomous regulator, a large network of mitochondrial function and biogenesis related genes essential for maintaining mitochondrial function and adult cardiomyocyte homeostasis.NEW & NOTEWORTHY Mitochondrial dysfunction constitutes an important aspect of heart failure etiopathogenesis and progression. However, the molecular mechanisms are still largely unknown. Growing lines of evidence indicate that Hippo-Tead pathway plays a role in cellular bioenergetics. This study reveals the novel role of Tead1, the downstream transcriptional effector of Hippo pathway, as a novel regulator of mitochondrial oxidative phosphorylation and in vivo cardiomyocyte energy metabolism, thus providing a potential therapeutic target for modulating mitochondrial function and enhancing cytoprotection of cardiomyocytes.

The Nuclear Receptor and Clock Repressor Rev-erbα Suppresses Myogenesis.

Rev-erbα is a ligand-dependent nuclear receptor and a key repressor of the molecular clock transcription network. Accumulating evidence indicate that the circadian clock machinery governs diverse biological processes in skeletal muscle, including muscle growth, repair and mass maintenance. The physiological function of Rev-erbα in myogenic regulation remains largely unknown. Here we show that Rev-erbα exerts cell-autonomous inhibitory effects on proliferation and differentiation of myogenic precursor cells, and these actions concertedly inhibit muscle regeneration in vivo. Mechanistic studies reveal Rev-erbα direct transcriptional control of two major myogenic mechanisms, proliferative pathway and the Wnt signaling cascade. Consistent with this finding, primary myoblasts lacking Rev-erbα display significantly enhanced proliferative growth and myogenic progression. Furthermore, pharmacological activation of Rev-erbα activity attenuates, whereas its inhibition by an antagonist promotes these processes. Notably, upon muscle injury, the loss-of-function of Rev-erbα in vivo augmented satellite cell proliferative expansion and regenerative progression during regeneration. Collectively, our study identifies Rev-erbα as a novel inhibitory regulator of myogenic progenitor cell properties that suppresses postnatal myogenesis. Pharmacological interventions to dampen Rev-erbα activity may have potential utilities to enhance regenerative capacity in muscle diseases.

Tead1 is required for perinatal cardiomyocyte proliferation.

Adult heart size is determined predominantly by the cardiomyocyte number and size. The cardiomyocyte number is determined primarily in the embryonic and perinatal period, as adult cardiomyocyte proliferation is restricted in comparison to that seen during the perinatal period. Recent evidence has implicated the mammalian Hippo kinase pathway as being critical in cardiomyocyte proliferation. Though the transcription factor, Tead1, is the canonical downstream transcriptional factor of the hippo kinase pathway in cardiomyocytes, the specific role of Tead1 in cardiomyocyte proliferation in the perinatal period has not been determined. Here, we report the generation of a cardiomyocyte specific perinatal deletion of Tead1, using Myh6-Cre deletor mice (Tead1-cKO). Perinatal Tead1 deletion was lethal by postnatal day 9 in Tead1-cKO mice due to dilated cardiomyopathy. Tead1-deficient cardiomyocytes have significantly decreased proliferation during the immediate postnatal period, when proliferation rate is normally high. Deletion of Tead1 in HL-1 cardiac cell line confirmed that cell-autonomous Tead1 function is required for normal cardiomyocyte proliferation. This was secondary to significant decrease in levels of many proteins, in vivo, that normally promote cell cycle in cardiomyocytes. Taken together this demonstrates the non-redundant critical requirement for Tead1 in regulating cell cycle proteins and proliferation in cardiomyocytes in the perinatal heart.

UBE3A Suppresses Overnutrition-Induced Expression of the Steatosis Target Genes of MLL4 by Degrading MLL4.

Regulation of the protein stability of epigenetic regulators remains ill-defined despite its potential applicability in epigenetic therapies. The histone H3-lysine 4-methyltransferase MLL4 is an epigenetic transcriptional coactivator that directs overnutrition-induced obesity and fatty liver formation, and Mll4+/- mice are resistant to both. Here we show that the E3 ubiquitin ligase UBE3A targets MLL4 for degradation, thereby suppressing high-fat diet (HFD)-induced expression of the hepatic steatosis target genes of MLL4. In contrast to Mll4+/- mice, Ube3a+/- mice are hypersensitive to HFD-induced obesity and fatty liver development. Ube3a+/-;Mll4+/- mice lose this hypersensitivity, supporting roles of increased MLL4 levels in both phenotypes of Ube3a+/- mice. Correspondingly, our comparative studies with wild-type, Ube3a+/- and Ube3a-/- and UBE3A-overexpressing transgenic mouse livers demonstrate an inverse correlation of UBE3A protein levels with MLL4 protein levels, expression of the steatosis target genes of MLL4, and their decoration by H3-lysine 4-monomethylation, a surrogate marker for the epigenetic action of MLL4. Conclusion: UBE3A indirectly exerts an epigenetic regulation of obesity and steatosis by degrading MLL4. This UBE3A-MLL4 regulatory axis provides a potential therapeutic venue for treating various MLL4-directed pathogeneses, including obesity and hepatic steatosis.

Tead1 is required for maintaining adult cardiomyocyte function, and its loss results in lethal dilated cardiomyopathy.

Heart disease remains the leading cause of death worldwide, highlighting a pressing need to identify novel regulators of cardiomyocyte (CM) function that could be therapeutically targeted. The mammalian Hippo/Tead pathway is critical in embryonic cardiac development and perinatal CM proliferation. However, the requirement of Tead1, the transcriptional effector of this pathway, in the adult heart is unknown. Here, we show that tamoxifen-inducible adult CM-specific Tead1 ablation led to lethal acute-onset dilated cardiomyopathy, associated with impairment in excitation-contraction coupling. Mechanistically, we demonstrate Tead1 is a cell-autonomous, direct transcriptional activator of SERCA2a and SR-associated protein phosphatase 1 regulatory subunit, Inhibitor-1 (I-1). Thus, Tead1 deletion led to a decrease in SERCA2a and I-1 transcripts and protein, with a consequent increase in PP1-activity, resulting in accumulation of dephosphorylated phospholamban (Pln) and decreased SERCA2a activity. Global transcriptomal analysis in Tead1-deleted hearts revealed significant changes in mitochondrial and sarcomere-related pathways. Additional studies demonstrated there was a trend for correlation between protein levels of TEAD1 and I-1, and phosphorylation of PLN, in human nonfailing and failing hearts. Furthermore, TEAD1 activity was required to maintain PLN phosphorylation and expression of SERCA2a and I-1 in human induced pluripotent stem cell-derived (iPS-derived) CMs. To our knowledge, taken together, this demonstrates a nonredundant, novel role of Tead1 in maintaining normal adult heart function.

Novel Function of Rev-erbα in Promoting Brown Adipogenesis.

Brown adipose tissue is a major thermogenic organ that plays a key role in maintenance of body temperature and whole-body energy homeostasis. Rev-erbα, a ligand-dependent nuclear receptor and transcription repressor of the molecular clock, has been implicated in the regulation of adipogenesis. However, whether Rev-erbα participates in brown fat formation is not known. Here we show that Rev-erbα is a key regulator of brown adipose tissue development by promoting brown adipogenesis. Genetic ablation of Rev-erbα in mice severely impairs embryonic and neonatal brown fat formation accompanied by loss of brown identity. This defect is due to a cell-autonomous function of Rev-erbα in brown adipocyte lineage commitment and terminal differentiation, as demonstrated by genetic loss- and gain-of-function studies in mesenchymal precursors and brown preadipocytes. Moreover, pharmacological activation of Rev-erbα activity promotes, whereas its inhibition suppresses brown adipocyte differentiation. Mechanistic investigations reveal that Rev-erbα represses key components of the TGF-ß cascade, an inhibitory pathway of brown fat development. Collectively, our findings delineate a novel role of Rev-erbα in driving brown adipocyte development, and provide experimental evidence that pharmacological interventions of Rev-erbα may offer new avenues for the treatment of obesity and related metabolic disorders.

PD-L1-driven tolerance protects neurogenin3-induced islet neogenesis to reverse established type 1 diabetes in NOD mice.

A breakdown in self-tolerance underlies autoimmune destruction of ß-cells and type 1 diabetes. A cure by restoring ß-cell mass is limited by the availability of transplantable ß-cells and the need for chronic immunosuppression. Evidence indicates that inhibiting costimulation through the PD-1/PD-L1 pathway is central to immune tolerance. We therefore tested whether induction of islet neogenesis in the liver, protected by PD-L1-driven tolerance, reverses diabetes in NOD mice. We demonstrated a robust induction of neo-islets in the liver of diabetic NOD mice by gene transfer of Neurogenin3, the islet-defining factor, along with betacellulin, an islet growth factor. These neo-islets expressed all the major pancreatic hormones and transcription factors. However, an enduring restoration of glucose-stimulated insulin secretion and euglycemia occurs only when tolerance is also induced by the targeted overexpression of PD-L1 in the neo-islets, which results in inhibition of proliferation and increased apoptosis of infiltrating CD4(+) T cells. Further analysis revealed an inhibition of cytokine production from lymphocytes isolated from the liver but not from the spleen of treated mice, indicating that treatment did not result in generalized immunosuppression. This treatment strategy leads to persistence of functional neo-islets that resist autoimmune destruction and consequently an enduring reversal of diabetes in NOD mice.

Bmal1 and ß-cell clock are required for adaptation to circadian disruption, and their loss of function leads to oxidative stress-induced ß-cell failure in mice.

Circadian disruption has deleterious effects on metabolism. Global deletion of Bmal1, a core clock gene, results in ß-cell dysfunction and diabetes. However, it is unknown if this is due to loss of cell-autonomous function of Bmal1 in ß cells. To address this, we generated mice with ß-cell clock disruption by deleting Bmal1 in ß cells (ß-Bmal1(-/-)). ß-Bmal1(-/-) mice develop diabetes due to loss of glucose-stimulated insulin secretion (GSIS). This loss of GSIS is due to the accumulation of reactive oxygen species (ROS) and consequent mitochondrial uncoupling, as it is fully rescued by scavenging of the ROS or by inhibition of uncoupling protein 2. The expression of the master antioxidant regulatory factor Nrf2 (nuclear factor erythroid 2-related factor 2) and its targets, Sesn2, Prdx3, Gclc, and Gclm, was decreased in ß-Bmal1(-/-) islets, which may contribute to the observed increase in ROS accumulation. In addition, by chromatin immunoprecipitation experiments, we show that Nrf2 is a direct transcriptional target of Bmal1. Interestingly, simulation of shift work-induced circadian misalignment in mice recapitulates many of the defects seen in Bmal1-deficient islets. Thus, the cell-autonomous function of Bmal1 is required for normal ß-cell function by mitigating oxidative stress and serves to preserve ß-cell function in the face of circadian misalignment.

Effects of CaMKII-mediated phosphorylation of ryanodine receptor type 2 on islet calcium handling, insulin secretion, and glucose tolerance.

Altered insulin secretion contributes to the pathogenesis of type 2 diabetes. This alteration is correlated with altered intracellular Ca(2+)-handling in pancreatic ß cells. Insulin secretion is triggered by elevation in cytoplasmic Ca(2+) concentration ([Ca(2+)]cyt) of ß cells. This elevation in [Ca(2+)]cyt leads to activation of Ca(2+)/calmodulin-dependent protein kinase II (CAMKII), which, in turn, controls multiple aspects of insulin secretion. CaMKII is known to phosphorylate ryanodine receptor 2 (RyR2), an intracellular Ca(2+)-release channel implicated in Ca(2+)-dependent steps of insulin secretion. Our data show that RyR2 is CaMKII phosphorylated in a pancreatic ß-cell line in a glucose-sensitive manner. However, it is not clear whether any change in CaMKII-mediated phosphorylation underlies abnormal RyR2 function in ß cells and whether such a change contributes to alterations in insulin secretion. Therefore, knock-in mice with a mutation in RyR2 that mimics its constitutive CaMKII phosphorylation, RyR2-S2814D, were studied. This mutation led to a gain-of-function defect in RyR2 indicated by increased basal RyR2-mediated Ca(2+) leak in islets of these mice. This chronic in vivo defect in RyR2 resulted in basal hyperinsulinemia. In addition, S2814D mice also developed glucose intolerance, impaired glucose-stimulated insulin secretion and lowered [Ca(2+)]cyt transients, which are hallmarks of pre-diabetes. The glucose-sensitive Ca(2+) pool in islets from S2814D mice was also reduced. These observations were supported by immunohistochemical analyses of islets in diabetic human and mouse pancreata that revealed significantly enhanced CaMKII phosphorylation of RyR2 in type 2 diabetes. Together, these studies implicate that the chronic gain-of-function defect in RyR2 due to CaMKII hyperphosphorylation is a novel mechanism that contributes to pathogenesis of type 2 diabetes.