LGR4 is essential for maintaining ß-cell homeostasis through suppression of RANK.

Pancreatic ß-cell stress contributes to diabetes progression. This study demonstrates that Leucine-rich repeat-containing G-protein-coupled-receptor-4 (LGR4) is critical for maintaining ß-cell health and is modulated by stressors. In vitro , Lgr4 knockdown decreases proliferation and survival in rodent ß-cells, while overexpression protects against cytokine-induced cell death in rodent and human ß-cells. Mechanistically, LGR4 suppresses Receptor Activator of Nuclear Factor Kappa B (NFκB) (RANK) and its subsequent activation of NFκB to protect ß-cells. ß-cell-specific Lgr4 -conditional knockout (cko) mice exhibit normal glucose homeostasis but increased ß-cell death in both sexes and decreased proliferation only in females. Male Lgr4 cko mice under stress display reduced ß-cell proliferation and a further increase in ß-cell death. Upon aging, both male and female Lgr4 cko mice display impaired ß-cell homeostasis, however, only female mice are glucose intolerant with decreased plasma insulin. We show that LGR4 is required for maintaining ß-cell health under basal and stress-induced conditions, through suppression of RANK. Teaser: LGR4 receptor is critical for maintaining ß-cell health under basal and stressed conditions, through suppression of RANK.

RANKL/RANK is required for cytokine-induced beta cell death; osteoprotegerin, a RANKL inhibitor, reverses rodent type 1 diabetes.

Treatment for type 1 diabetes (T1D) requires stimulation of functional ß cell regeneration and survival under stress. Previously, we showed that inhibition of the RANKL/RANK [receptor activator of nuclear factor kappa Β (NF-κB) ligand] pathway, by osteoprotegerin and the anti-osteoporotic drug denosumab, induces rodent and human ß cell proliferation. We demonstrate that the RANK pathway mediates cytokine-induced rodent and human ß cell death through RANK-TRAF6 interaction and induction of NF-κB activation. Osteoprotegerin and denosumab protected ß cells against this cytotoxicity. In human immune cells, osteoprotegerin and denosumab reduce proinflammatory cytokines in activated T-cells by inhibiting RANKL-induced activation of monocytes. In vivo, osteoprotegerin reversed recent-onset T1D in nonobese diabetic/Ltj mice, reduced insulitis, improved glucose homeostasis, and increased plasma insulin, ß cell proliferation, and mass in these mice. Serum from T1D subjects induced human ß cell death and dysfunction, but not α cell death. Osteoprotegerin and denosumab reduced T1D serum-induced ß cell cytotoxicity and dysfunction. Inhibiting RANKL/RANK could have therapeutic potential.

LGR4, a G Protein-Coupled Receptor With a Systemic Role: From Development to Metabolic Regulation.

Leucine-rich repeat-containing G protein-coupled receptor 4 (LGR4/GPR48), a member of the GPCR (G protein-coupled receptors) superfamily, subfamily B, is a common intestinal crypt stem cell marker. It binds R-spondins/Norrin as classical ligands and plays a crucial role in Wnt signaling potentiation. Interaction between LGR4 and R-spondins initiates many Wnt-driven developmental processes, e.g., kidney, eye, or reproductive tract formation, as well as intestinal crypt (Paneth) stem cell pool maintenance. Besides the well-described role of LGR4 in development, several novel functions of this receptor have recently been discovered. In this context, LGR4 was indicated to participate in TGFß and NFκB signaling regulation in hematopoietic precursors and intestinal cells, respectively, and found to be a new, alternative receptor for RANKL (Receptor Activator of NF kappa B Ligand) in bone cells. LGR4 inhibits the process of osteoclast differentiation, by antagonizing the interaction between RANK (Receptor Activator of NF kappa B) and its ligand-RANKL. It is also known to trigger anti-inflammatory responses in different tissues (liver, intestine, cardiac cells, and skin), serve as a sensor of the circadian clock in the liver, regulate adipogenesis and energy expenditure in adipose tissue and skeletal muscles, respectively. The extracellular domain of LGR4 (LGR4-ECD) has emerged as a potential new therapeutic for osteoporosis and cancer. LGR4 integrates different signaling pathways and regulates various cellular processes vital for maintaining whole-body homeostasis. Yet, the role of LGR4 in many cell types (e.g. pancreatic beta cells) and diseases (e.g., diabetes) remains to be elucidated. Considering the broad spectrum of LGR4 actions, this review aims to discuss both canonical and novel roles of LGR4, with emphasis on emerging research directions focused on this receptor.

Lactogens Reduce Endoplasmic Reticulum Stress-Induced Rodent and Human ß-Cell Death and Diabetes Incidence in Akita Mice.

Diabetes occurs due to a loss of functional ß-cells, resulting from ß-cell death and dysfunction. Lactogens protect rodent and human ß-cells in vitro and in vivo against triggers of ß-cell cytotoxicity relevant to diabetes, many of which converge onto a common pathway of endoplasmic reticulum (ER) stress. However, whether lactogens modulate the ER stress pathway is unknown. This study examines whether lactogens can protect ß-cells against ER stress and mitigate diabetes incidence in Akita (Ak) mice, a rodent model of ER stress-induced diabetes, akin to neonatal diabetes in humans. We show that lactogens protect INS-1 cells, primary rodent and human ß-cells in vitro against two distinct ER stressors, tunicamycin and thapsigargin, through activation of the JAK2/STAT5 pathway. Lactogens mitigate expression of proapoptotic molecules in the ER stress pathway that are induced by chronic ER stress in INS-1 cells and rodent islets. Transgenic expression of placental lactogen in ß-cells of Ak mice drastically reduces the severe hyperglycemia, diabetes incidence, hypoinsulinemia, ß-cell death, and loss of ß-cell mass observed in Ak littermates. These are the first studies in any cell type demonstrating that lactogens modulate the ER stress pathway, causing enhanced ß-cell survival and reduced diabetes incidence in the face of chronic ER stress.

Tissue-specific deletion of the retinoblastoma protein in the pancreatic beta-cell has limited effects on beta-cell replication, mass, and function.

Animal studies show that G(1/S) regulatory molecules (D-cyclins, cdk-4, p18, p21, p27) are critical for normal regulation of beta-cell proliferation, mass, and function. The retinoblastoma protein, pRb, is positioned at the very end of a cascade of these regulatory proteins and is considered the final checkpoint molecule that maintains beta-cell cycle arrest. Logically, removal of pRb from the beta-cell should result in unrestrained beta-cell replication, increased beta-cell mass, and insulin-mediated hypoglycemia. Because global loss of both pRb alleles is embryonic lethal, this hypothesis has not been tested in beta-cells. We developed two types of conditional knockout (CKO) mice in which both alleles of the pRb gene were inactivated specifically in beta-cells. Surprisingly, although the pRb gene was efficiently recombined in beta-cells of both CKO models, changes in beta-cell mass, beta-cell replication rates, insulin concentrations, and blood glucose levels were limited or absent. Other pRb family members, p107 and p130, were not substantially upregulated. In contrast to dogma, the pRb protein is not essential to maintain cell cycle arrest in the pancreatic beta-cell. This may reflect fundamental inaccuracies in models of beta-cell cycle control or complementation for pRb by undefined proteins.

CDK4/6 Inhibition on Glucose and Pancreatic Beta Cell Homeostasis in Young and Aged Rats.

Genetic deletion of cyclin-dependent kinase 4 (Cdk4) is associated with pancreatic beta cell loss and glucose dysregulation in rodents. Palbociclib, one of the first selective CDK4/6 inhibitors approved for the treatment of advanced breast cancer, is currently being investigated as an adjuvant treatment in patients with early-stage breast cancer and in a variety of cancers covering a wide-range of patient populations. Hence, longer chronic toxicity studies were necessary to further examine its safety profile. The effects of different doses and duration of palbociclib administration on glucose and beta cell homeostasis in young (two months) versus aged (12 months) rats was compared. Glucose dysregulation, due to pancreatic beta cell degeneration, was observed in young rats administered the highest dose of palbociclib for 6 months. Abnormal pancreatic islet histology and activation of the endoplasmic reticulum stress response in beta cells were detected after shorter administration with high-dose palbociclib in young rats. To test the hypothesis that palbociclib-associated inhibition of beta cell proliferation will more profoundly affect younger animals that have not achieved replicative quiescence, we administered high-dose palbociclib to aged rats for 6 months. In contrast to the young rats, despite equivalent exposures to palbociclib, no evidence of impaired glucose tolerance, hypoinsulinemia, beta cell vacuolization, or beta cell loss was seen in aged rats. Palbociclib administration induces beta cell failure in young but not aged rats.Implications: Although adult humans receiving palbociclib have not displayed detectable adverse effects on glucose metabolism, the risk of beta cell failure in children remains unexplored. Mol Cancer Res; 15(11); 1531-41. ©2017 AACR.

Growth factors and beta cell replication.

Recent studies have demonstrated that human islet allograft transplantation can be a successful therapeutic option in the treatment of patients with Type I diabetes. However, this impressive recent advance is accompanied by a very important constraint. There is a critical paucity of pancreatic islets or pancreatic beta cells for islet transplantation to become a large-scale therapeutic option in patients with diabetes. This has prompted many laboratories around the world to invigorate their efforts in finding ways for increasing the availability of beta cells or beta cell surrogates that potentially could be transplanted into patients with diabetes. The number of studies analyzing the mechanisms that govern beta cell proliferation and growth in physiological and pathological conditions has increased exponentially during the last decade. These studies exploring the role of growth factors, intracellular signaling molecules and cell cycle regulators constitute the substrate for future strategies aimed at expanding human beta cells in vitro and/or in vivo after transplantation. In this review, we describe the current knowledge on the effects of several beta cell growth factors that have been shown to increase beta cell proliferation and expand beta cell mass in vitro and/or in vivo and that they could be potentially deployed in an effort to increase the number of patients transplanted with islets. Furthermore, we also analyze in this review recent studies deciphering the relevance of these specific islet growth factors as physiological and pathophysiological regulators of beta cell proliferation and islet growth.

Targeted inactivation of hepatocyte growth factor receptor c-met in beta-cells leads to defective insulin secretion and GLUT-2 downregulation without alteration of beta-cell mass.

Overexpression of hepatocyte growth factor (HGF) in the beta-cell of transgenic mice enhances beta-cell proliferation, survival, and function. In the current studies, we have used conditional ablation of the c-met gene to uncover the physiological role of HGF in beta-cell growth and function. Mice in which c-met is inactivated in the beta-cell (MetCKO mice) display normal body weight, blood glucose, and plasma insulin compared with control littermates. In contrast, MetCKO mice displayed significantly diminished glucose tolerance and reduced plasma insulin after a glucose challenge in vivo. This impaired glucose tolerance in MetCKO mice was not caused by insulin resistance because sensitivity to exogenous insulin was similar in both groups. Importantly, in vitro glucose-stimulated insulin secretion in MetCKO islets was decreased by approximately 50% at high glucose concentrations compared with control islets. Furthermore, whereas insulin and glucokinase expression in MetCKO islets were normal, GLUT-2 expression was decreased by approximately 50%. These changes in beta-cell function in MetCKO mice were not accompanied by changes in total beta-cell mass, islet morphology, islet cell composition, and beta-cell proliferation. Interestingly, however, MetCKO mice display an increased number of small islets, mainly single and doublet beta-cells. We conclude that HGF/c-met signaling in the beta-cell is not essential for beta-cell growth, but it is essential for normal glucose-dependent insulin secretion.

PKCζ Is Essential for Pancreatic ß-Cell Replication During Insulin Resistance by Regulating mTOR and Cyclin-D2.

Adaptive ß-cell replication occurs in response to increased metabolic demand during insulin resistance. The intracellular mediators of this compensatory response are poorly defined and their identification could provide significant targets for ß-cell regeneration therapies. Here we show that glucose and insulin in vitro and insulin resistance in vivo activate protein kinase C ζ (PKCζ) in pancreatic islets and ß-cells. PKCζ is required for glucose- and glucokinase activator-induced proliferation of rodent and human ß-cells in vitro. Furthermore, either kinase-dead PKCζ expression (KD-PKCζ) or disruption of PKCζ in mouse ß-cells blocks compensatory ß-cell replication when acute hyperglycemia/hyperinsulinemia is induced. Importantly, KD-PKCζ inhibits insulin resistance-mediated mammalian target of rapamycin (mTOR) activation and cyclin-D2 upregulation independent of Akt activation. In summary, PKCζ activation is key for early compensatory ß-cell replication in insulin resistance by regulating the downstream signals mTOR and cyclin-D2. This suggests that alterations in PKCζ expression or activity might contribute to inadequate ß-cell mass expansion and ß-cell failure leading to type 2 diabetes.

Characterization of mice doubly transgenic for parathyroid hormone-related protein and murine placental lactogen: a novel role for placental lactogen in pancreatic beta-cell survival.

Transgenic overexpression of either parathyroid hormone-related peptide (PTHrP) or mouse placental lactogen type 1 (mPL1) in pancreatic beta-cells, using the rat insulin II promoter (RIP), results in islet hyperplasia either through prolonged beta-cell survival or through increased beta-cell proliferation and hypertrophy, respectively. For determining whether the two proteins might exert complementary, additive, or synergistic effects on islet mass and function when simultaneously overexpressed in beta-cells in vivo, RIP-PTHrP and RIP-mPL1 mice were crossed to generate mice doubly transgenic for PTHrP and mPL1. These double-transgenic mice displayed marked islet hyperplasia (threefold), hypoglycemia, increased beta-cell proliferation (threefold), and resistance to the diabetogenic and cytotoxic effects of streptozotocin compared with their normal siblings. Although the phenotype of the double-transgenic mice was neither additive nor synergistic relative to their single-transgenic counterparts, it was indeed complementary, yielding the maximal salutary phenotypic features of both individual transgenes. Finally, mPL1, for the first time, was shown to exert a protective effect on the survival of beta-cells, placing it among the few proteins that can improve function and proliferation and prolong the survival of beta-cells. Placental lactogen 1 is an attractive target for future therapeutic strategies in diabetes.

Overexpression of parathyroid hormone-related protein inhibits pancreatic beta-cell death in vivo and in vitro.

Pancreatic beta-cell survival is critical in the setting of diabetes as well as in islet transplantation. Transgenic mice overexpressing parathyroid hormone-related protein (PTHrP) targeted to beta-cells using the rat insulin II promoter (RIP) display hyperinsulinemia, hypoglycemia, and islet hyperplasia, without a concomitant increase in beta-cell proliferation rate or enlargement of individual beta-cell size. Thus, the mechanism for increased beta-cell mass is unknown. In this study, we demonstrated that beta-cells of transgenic mice are resistant to the cytotoxic effects of streptozotocin (STZ) in vivo, as documented by a sixfold reduction in the rate of STZ-induced beta-cell death in RIP-PTHrP mice relative to their normal siblings. The reduced cell death in transgenic mice is due neither to their increased islet mass nor to a decrease in their sensing of STZ, but rather results from PTHrP-induced resistance to beta-cell death. This is also demonstrated in vitro by markedly reduced cell death rates observed in beta-cells of transgenic mice compared with normal mice when cultured in the absence of serum and glucose or in the presence of STZ. Finally, we demonstrated that NH(2)-terminal PTHrP inhibits beta-cell death. These findings support the concept that PTHrP overexpression increases islet mass in transgenic mice through inhibition of beta-cell death.

Osteoprotegerin and Denosumab Stimulate Human Beta Cell Proliferation through Inhibition of the Receptor Activator of NF-κB Ligand Pathway.

Diabetes results from a reduction of pancreatic ß-cells. Stimulating replication could normalize ß-cell mass. However, adult human ß-cells are recalcitrant to proliferation. We identified osteoprotegerin, a bone-related decoy receptor, as a ß-cell mitogen. Osteoprotegerin was induced by and required for lactogen-mediated rodent ß-cell replication. Osteoprotegerin enhanced ß-cell proliferation in young, aged, and diabetic mice. This resulted in increased ß-cell mass in young mice and significantly delayed hyperglycemia in diabetic mice. Osteoprotegerin stimulated replication of adult human ß-cells, without causing dedifferentiation. Mechanistically, osteoprotegerin induced human and rodent ß-cell replication by modulating CREB and GSK3 pathways, through binding Receptor Activator of NF-κB (RANK) Ligand (RANKL), a brake in ß-cell proliferation. Denosumab, an FDA-approved osteoporosis drug, and RANKL-specific antibody induced human ß-cell proliferation in vitro, and in vivo, in humanized mice. Thus, osteoprotegerin and Denosumab prevent RANKL/RANK interaction to stimulate ß-cell replication, highlighting the potential for repurposing an osteoporosis drug to treat diabetes.

Human ß-cell proliferation and intracellular signaling: part 3.

This is the third in a series of Perspectives on intracellular signaling pathways coupled to proliferation in pancreatic ß-cells. We contrast the large knowledge base in rodent ß-cells with the more limited human database. With the increasing incidence of type 1 diabetes and the recognition that type 2 diabetes is also due in part to a deficiency of functioning ß-cells, there is great urgency to identify therapeutic approaches to expand human ß-cell numbers. Therapeutic approaches might include stem cell differentiation, transdifferentiation, or expansion of cadaver islets or residual endogenous ß-cells. In these Perspectives, we focus on ß-cell proliferation. Past Perspectives reviewed fundamental cell cycle regulation and its upstream regulation by insulin/IGF signaling via phosphatidylinositol-3 kinase/mammalian target of rapamycin signaling, glucose, glycogen synthase kinase-3 and liver kinase B1, protein kinase Cζ, calcium-calcineurin-nuclear factor of activated T cells, epidermal growth factor/platelet-derived growth factor family members, Wnt/ß-catenin, leptin, and estrogen and progesterone. Here, we emphasize Janus kinase/signal transducers and activators of transcription, Ras/Raf/extracellular signal-related kinase, cadherins and integrins, G-protein-coupled receptors, and transforming growth factor ß signaling. We hope these three Perspectives will serve to introduce these pathways to new researchers and will encourage additional investigators to focus on understanding how to harness key intracellular signaling pathways for therapeutic human ß-cell regeneration for diabetes.

Diabetes mellitus--advances and challenges in human ß-cell proliferation.

The treatment of diabetes mellitus represents one of the greatest medical challenges of our era. Diabetes results from a deficiency or functional impairment of insulin-producing ß cells, alone or in combination with insulin resistance. It logically follows that the replacement or regeneration of ß cells should reverse the progression of diabetes and, indeed, this seems to be the case in humans and rodents. This concept has prompted attempts in many laboratories to create new human ß cells using stem-cell strategies to transdifferentiate or reprogramme non-ß cells into ß cells or to discover small molecules or other compounds that can induce proliferation of human ß cells. This latter approach has shown promise, but has also proven particularly challenging to implement. In this Review, we discuss the physiology of normal human ß-cell replication, the molecular mechanisms that regulate the cell cycle in human ß cells, the upstream intracellular signalling pathways that connect them to cell surface receptors on ß cells, the epigenetic mechanisms that control human ß-cell proliferation and unbiased approaches for discovering novel molecules that can drive human ß-cell proliferation. Finally, we discuss the potential and challenges of implementing strategies that replace or regenerate ß cells.

Hepatocyte growth factor gene therapy for islet transplantation.

Recent clinical studies have documented that human islet transplantation has the potential to replace pancreatic endocrine function in patients with type 1 diabetes. These studies have also highlighted an enormous shortage of human islets that impedes the use of islet transplantation in clinical practice on a larger scale. To address this problem, one potential approach is to use islet growth factors to increase beta cell replication, to improve beta cell function and to enhance beta cell survival. In that context, transgenic mice overexpressing hepatocyte growth factor (HGF) in the pancreatic beta cell display increased beta cell proliferation, function and survival. More importantly, HGF-overexpressing transgenic mouse islets markedly improve transplant performance in severe combined immunodeficiency (SCID) mice and reduce the number of islets required for successful islet transplantation. Recently, adenoviral-mediated gene transfer of HGF into normal rodent islets has confirmed the beneficial effects of HGF in improving islet transplant outcomes in two marginal mass islet transplant models in rodents: islet transplant under the kidney capsule in SCID mice; and portal islet allograft transplantation in rats treated with the Edmonton immunosuppressive regimen. These studies suggest that ex vivo HGF gene therapy has the potential to reduce the number of human islets required for successful islet transplantation.

Hepatocyte growth factor/c-Met signaling is required for ß-cell regeneration.

Hepatocyte growth factor (HGF) is a mitogen required for ß-cell replication during pregnancy. To determine whether HGF/c-Met signaling is required for ß-cell regeneration, we characterized mice with pancreatic deletion of the HGF receptor, c-Met (PancMet KO mice), in two models of reduced ß-cell mass and regeneration: multiple low-dose streptozotocin (MLDS) and partial pancreatectomy (Ppx). We also analyzed whether HGF administration could accelerate ß-cell regeneration in wild-type (WT) mice after Ppx. Mouse islets obtained 7 days post-Ppx displayed significantly increased c-Met, suggesting a potential role for HGF/c-Met in ß-cell proliferation in situations of reduced ß-cell mass. Indeed, adult PancMet KO mice displayed markedly reduced ß-cell replication compared with WT mice 7 days post-Ppx. Similarly, ß-cell proliferation was decreased in PancMet KO mice in the MLDS mouse model. The decrease in ß-cell proliferation post-Ppx correlated with a striking decrease in D-cyclin levels. Importantly, PancMet KO mice showed significantly diminished ß-cell mass, decreased glucose tolerance, and impaired insulin secretion compared with WT mice 28 days post-Ppx. Conversely, HGF administration in WT Ppx mice further accelerated ß-cell regeneration. These results indicate that HGF/c-Met signaling is critical for ß-cell proliferation in situations of diminished ß-cell mass and suggest that activation of this pathway can enhance ß-cell regeneration.

Hepatocyte growth factor ameliorates hyperglycemia and corrects ß-cell mass in IRS2-deficient mice.

Insulin resistance, when combined with decreased ß-cell mass and relative insufficient insulin secretion, leads to type 2 diabetes. Mice lacking the IRS2 gene (IRS2(-/-) mice) develop diabetes due to uncompensated insulin resistance and ß-cell failure. Hepatocyte growth factor (HGF) activates the phosphatidylinositol 3-kinase/Akt signaling pathway in ß-cells without recruitment of IRS1 or IRS2 and increases ß-cell proliferation, survival, mass, and function when overexpressed in ß-cells of transgenic (TG) mice. We therefore hypothesized that HGF may protect against ß-cell failure in IRS2 deficiency. For that purpose, we cross-bred TG mice overexpressing HGF in ß-cells with IRS2 knockout (KO) mice. Glucose homeostasis analysis revealed significantly reduced hyperglycemia, compensatory hyperinsulinemia, and improved glucose tolerance in TG/KO mice compared with those in KO mice in the context of similar insulin resistance. HGF overexpression also increased glucose-stimulated insulin secretion in IRS2(-/-) islets. To determine whether this glucose homeostasis improvement correlated with alterations in ß-cells, we measured ß-cell mass, proliferation, and death in these mice. ß-Cell proliferation was increased and death was decreased in TG/KO mice compared with those in KO mice. As a result, ß-cell mass was significantly increased in TG/KO mice compared with that in KO mice, reaching levels similar to those in wild-type mice. Analysis of the intracellular targets involved in ß-cell failure in IRS2 deficiency showed Pdx-1 up-regulation, Akt/FoxO1 phosphorylation, and p27 down-regulation in TG/KO mouse islets. Taken together, these results indicate that HGF can compensate for IRS2 deficiency and subsequent insulin resistance by normalizing ß-cell mass and increasing circulating insulin. HGF may be of value as a therapeutic agent against ß-cell failure.

Loss of HGF/c-Met signaling in pancreatic ß-cells leads to incomplete maternal ß-cell adaptation and gestational diabetes mellitus.

Hepatocyte growth factor (HGF) is a mitogen and insulinotropic agent for the ß-cell. However, whether HGF/c-Met has a role in maternal ß-cell adaptation during pregnancy is unknown. To address this issue, we characterized glucose and ß-cell homeostasis in pregnant mice lacking c-Met in the pancreas (PancMet KO mice). Circulating HGF and islet c-Met and HGF expression were increased in pregnant mice. Importantly, PancMet KO mice displayed decreased ß-cell replication and increased ß-cell apoptosis at gestational day (GD)15. The decreased ß-cell replication was associated with reductions in islet prolactin receptor levels, STAT5 nuclear localization and forkhead box M1 mRNA, and upregulation of p27. Furthermore, PancMet KO mouse ß-cells were more sensitive to dexamethasone-induced cytotoxicity, whereas HGF protected human ß-cells against dexamethasone in vitro. These detrimental alterations in ß-cell proliferation and death led to incomplete maternal ß-cell mass expansion in PancMet KO mice at GD19 and early postpartum periods. The decreased ß-cell mass was accompanied by increased blood glucose, decreased plasma insulin, and impaired glucose tolerance. PancMet KO mouse islets failed to upregulate GLUT2 and pancreatic duodenal homeobox-1 mRNA, insulin content, and glucose-stimulated insulin secretion during gestation. These studies indicate that HGF/c-Met signaling is essential for maternal ß-cell adaptation during pregnancy and that its absence/attenuation leads to gestational diabetes mellitus.

ChREBP mediates glucose-stimulated pancreatic ß-cell proliferation.

Glucose stimulates rodent and human ß-cell replication, but the intracellular signaling mechanisms are poorly understood. Carbohydrate response element-binding protein (ChREBP) is a lipogenic glucose-sensing transcription factor with unknown functions in pancreatic ß-cells. We tested the hypothesis that ChREBP is required for glucose-stimulated ß-cell proliferation. The relative expression of ChREBP was determined in liver and ß-cells using quantitative RT-PCR (qRT-PCR), immunoblotting, and immunohistochemistry. Loss- and gain-of-function studies were performed using small interfering RNA and genetic deletion of ChREBP and adenoviral overexpression of ChREBP in rodent and human ß-cells. Proliferation was measured by 5-bromo-2'-deoxyuridine incorporation, [(3)H]thymidine incorporation, and fluorescence-activated cell sorter analysis. In addition, the expression of cell cycle regulatory genes was measured by qRT-PCR and immunoblotting. ChREBP expression was comparable with liver in mouse pancreata and in rat and human islets. Depletion of ChREBP decreased glucose-stimulated proliferation in ß-cells isolated from ChREBP(-/-) mice, in INS-1-derived 832/13 cells, and in primary rat and human ß-cells. Furthermore, depletion of ChREBP decreased the glucose-stimulated expression of cell cycle accelerators. Overexpression of ChREBP amplified glucose-stimulated proliferation in rat and human ß-cells, with concomitant increases in cyclin gene expression. In conclusion, ChREBP mediates glucose-stimulated proliferation in pancreatic ß-cells.

Disruption of hepatocyte growth factor/c-Met signaling enhances pancreatic beta-cell death and accelerates the onset of diabetes.

OBJECTIVE: To determine the role of hepatocyte growth factor (HGF)/c-Met on ß-cell survival in diabetogenic conditions in vivo and in response to cytokines in vitro. RESEARCH DESIGN AND METHODS: We generated pancreas-specific c-Met-null (PancMet KO) mice and characterized their response to diabetes induced by multiple low-dose streptozotocin (MLDS) administration. We also analyzed the effect of HGF/c-Met signaling in vitro on cytokine-induced ß-cell death in mouse and human islets, specifically examining the role of nuclear factor (NF)-κB. RESULTS: Islets exposed in vitro to cytokines or from MLDS-treated mice displayed significantly increased HGF and c-Met levels, suggesting a potential role for HGF/c-Met in ß-cell survival against diabetogenic agents. Adult PancMet KO mice displayed normal glucose and ß-cell homeostasis, indicating that pancreatic c-Met loss is not detrimental for ß-cell growth and function under basal conditions. However, PancMet KO mice were more susceptible to MLDS-induced diabetes. They displayed higher blood glucose levels, marked hypoinsulinemia, and reduced ß-cell mass compared with wild-type littermates. PancMet KO mice showed enhanced intraislet infiltration, islet nitric oxide (NO) and chemokine production, and ß-cell apoptosis. c-Met-null ß-cells were more sensitive to cytokine-induced cell death in vitro, an effect mediated by NF-κB activation and NO production. Conversely, HGF treatment decreased p65/NF-κB activation and fully protected mouse and, more important, human ß-cells against cytokines. CONCLUSIONS: These results show that HGF/c-Met is critical for ß-cell survival by attenuating NF-κB signaling and suggest that activation of the HGF/c-Met signaling pathway represents a novel strategy for enhancing ß-cell protection.