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.

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.

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.

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 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.

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.

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.

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.

Activation of protein kinase C-ζ in pancreatic ß-cells in vivo improves glucose tolerance and induces ß-cell expansion via mTOR activation.

OBJECTIVE: PKC-ζ activation is a key signaling event for growth factor-induced ß-cell replication in vitro. However, the effect of direct PKC-ζ activation in the ß-cell in vivo is unknown. In this study, we examined the effects of PKC-ζ activation in ß-cell expansion and function in vivo in mice and the mechanisms associated with these effects. RESEARCH DESIGN AND METHODS: We characterized glucose homeostasis and ß-cell phenotype of transgenic (TG) mice with constitutive activation of PKC-ζ in the ß-cell. We also analyzed the expression and regulation of signaling pathways, G1/S cell cycle molecules, and ß-cell functional markers in TG and wild-type mouse islets. RESULTS: TG mice displayed increased plasma insulin, improved glucose tolerance, and enhanced insulin secretion with concomitant upregulation of islet insulin and glucokinase expression. In addition, TG mice displayed increased ß-cell proliferation, size, and mass compared with wild-type littermates. The increase in ß-cell proliferation was associated with upregulation of cyclins D1, D2, D3, and A and downregulation of p21. Phosphorylation of D-cyclins, known to initiate their rapid degradation, was reduced in TG mouse islets. Phosphorylation/inactivation of GSK-3ß and phosphorylation/activation of mTOR, critical regulators of D-cyclin expression and ß-cell proliferation, were enhanced in TG mouse islets, without changes in Akt phosphorylation status. Rapamycin treatment in vivo eliminated the increases in ß-cell proliferation, size, and mass; the upregulation of cyclins Ds and A in TG mice; and the improvement in glucose tolerance-identifying mTOR as a novel downstream mediator of PKC-ζ-induced ß-cell replication and expansion in vivo. CONCLUSIONS PKC:-ζ, through mTOR activation, modifies the expression pattern of ß-cell cycle molecules leading to increased ß-cell replication and mass with a concomitant enhancement in ß-cell function. Approaches to enhance PKC-ζ activity may be of value as a therapeutic strategy for the treatment of diabetes.

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.

Parathyroid hormone-related protein enhances human ß-cell proliferation and function with associated induction of cyclin-dependent kinase 2 and cyclin E expression.

OBJECTIVE: Inducing human ß-cell growth while enhancing function is a major goal in the treatment of diabetes. Parathyroid hormone-related protein (PTHrP) enhances rodent ß-cell growth and function through the parathyroid hormone-1 receptor (PTH1R). Based on this, we hypothesized that PTH1R is expressed in human ß-cells and that PTHrP has the potential to enhance human ß-cell proliferation and/or function. RESEARCH DESIGN AND METHODS: PTH1R expression, ß-cell proliferation, glucose-stimulated insulin secretion (GSIS), and expression of differentiation and cell-cycle genes were analyzed in human islets transduced with adenoviral PTHrP constructs or treated with PTHrP peptides. The effect of overexpression of late G1/S cell cycle molecules was also assessed on human ß-cell proliferation. RESULTS: We found that human ß-cells express PTH1R. More importantly, overexpression of PTHrP causes a significant approximately threefold increase in human ß-cell proliferation. Furthermore, the amino terminus PTHrP(1-36) peptide is sufficient to increase replication as well as expression of the late G1/S cell-cycle proteins cyclin E and cyclin-dependent kinase 2 (cdk2) in human islets. Notably, PTHrP(1-36) also enhances GSIS. Finally, overexpression of cyclin E alone, but not cdk2, augments human ß-cell proliferation, and when both molecules are expressed simultaneously there is a further marked synergistic increase in replication. CONCLUSIONS: PTHrP(1-36) peptide enhances human ß-cell proliferation as well as function, with associated upregulation of two specific cell-cycle activators that together can induce human ß-cell proliferation several fold. The future therapeutic potential of PTHrP(1-36) for the treatment of diabetes is especially relevant given the complementary therapeutic efficacy of PTHrP(1-36) in postmenopausal osteoporosis.

Novel proapoptotic effect of hepatocyte growth factor: synergy with palmitate to cause pancreatic {beta}-cell apoptosis.

Increasing evidence suggests that elevation of plasma fatty acids that often accompanies insulin resistance contributes to beta-cell insufficiency in obesity-related type 2 diabetes. Circulating levels of hepatocyte growth factor (HGF) are increased in humans with metabolic syndrome and obesity. HGF is known to protect beta-cells against streptozotocin and during islet engraftment. However, whether HGF is a beta-cell prosurvival factor in situations of excessive lipid supply has not been deciphered. Mice overexpressing HGF in the beta-cell [rat insulin type II promoter (RIP)-HGF transgenic mice] fed with standard chow display improved glucose homeostasis and increased beta-cell mass and proliferation compared with normal littermates. However, after 15 wk of high-fat feeding, glucose homeostasis and beta-cell expansion and proliferation are indistinguishable between normal and transgenic mice. Interestingly, RIP-HGF transgenic mouse beta-cells and normal beta-cells treated with HGF display increased sensitivity to palmitate-mediated apoptosis in vitro. Palmitate completely eliminates Akt and Bad phosphorylation in RIP-HGF transgenic mouse islets. HGF-overexpressing islets also show significantly decreased AMP-activated protein kinase-alpha and acetyl-coenzyme A carboxylase phosphorylation, diminished fatty acid oxidation, increased serine palmitoyltransferase expression, and enhanced ceramide formation compared with normal islets. Importantly, human islets overexpressing HGF also display increased beta-cell apoptosis in the presence of palmitate. Treatment of both mouse and human islet cells with the de novo ceramide synthesis inhibitors myriocin and fumonisin B1 abrogates beta-cell apoptosis induced by HGF and palmitate. Collectively, these studies indicate that HGF can be detrimental for beta-cell survival in an environment with excessive fatty acid supply.

Gestational diabetes mellitus resulting from impaired beta-cell compensation in the absence of FoxM1, a novel downstream effector of placental lactogen.

OBJECTIVE: The objectives of the study were to determine whether the cell cycle transcription factor, FoxM1, is required for glucose homeostasis and beta-cell mass expansion in maternal islets during pregnancy and whether FoxM1 is essential for placental lactogen (PL)-induced beta-cell proliferation. RESEARCH DESIGN AND METHODS: beta-Cell mass, beta-cell proliferation, and glucose homeostasis were assessed in virgin, pregnant, and postpartum mice with a pancreas-wide Foxm1 deletion (FoxM1(Deltapanc)). Wild-type islets were cultured with or without PL and examined for Foxm1 induction. Transgenic mice overexpressing PL in beta-cells were bred with FoxM1(Deltapanc) mice, and beta-cell proliferation was examined. RESULTS: Foxm1 was upregulated in maternal islets during pregnancy. In contrast to controls, beta-cell proliferation did not increase in pregnant FoxM1(Deltapanc) females. Mutant islets showed increased Menin and nuclear p27. FoxM1(Deltapanc) females developed gestational diabetes mellitus as pregnancy progressed. After parturition, euglycemia was restored in FoxM1(Deltapanc) females, but islet size was significantly reduced. Strikingly, beta-cell mass was normal in postpartum FoxM1(Deltapanc) pancreata due to a combination of increased beta-cell size and islet neogenesis. Evidence for neogenesis included increased number of endocrine clusters, increased proportion of smaller islets, and increased neurogenin 3 or insulin expression in cells adjacent to ducts. PL induced Foxm1 expression in cultured islets, and FoxM1 was essential for PL-mediated increases in beta-cell proliferation in vivo. CONCLUSIONS: FoxM1 is essential for beta-cell compensation during pregnancy. In the absence of increased beta-cell proliferation, neogenesis is induced in postpartum FoxM1(Deltapanc) pancreata. Our results suggest that FoxM1 functions downstream of PL to mediate its effects on beta-cell proliferation.

The retinoblastoma protein and its homolog p130 regulate the G1/S transition in pancreatic beta-cells.

OBJECTIVE: The retinoblastoma protein family (pRb, p130, p107) plays a central role in the regulation of cell cycle progression. Surprisingly, loss of pRb in the beta-cell has no discernible effect on cell cycle control. Therefore, we explored the effects of individual loss of either p130 or p107 in addition to the simultaneous loss of both pRb/p130 on the beta-cell. RESEARCH DESIGN AND METHODS: Adult mice deficient in either p130 or p107 or both pRb/p130 were examined for effects on beta-cell replication, function, and survival. The Cre-Lox system was also used to inactivate pRb in wild-type and p130-deficient beta-cells in vitro. RESULTS: In vivo loss of either p107 or p130 did not affect beta-cell replication or function. Combined pRb/p130 loss, however, resulted in dramatically accelerated proliferation as well as apoptotic cell death. Pancreas and beta-cell mass were significantly reduced in double mutants. Despite this, overall glucose tolerance was normal, except for mild postprandial hyperglycemia. Ex vivo, acute deletion of pRb in p130-deficient beta-cells also caused a striking increase in proliferation. The combined deletion of pRb/p130 upregulated islet expression of E2F2 but not E2F1. CONCLUSIONS: These studies define an essential role for the pocket proteins in controlling the G(1)/S transition in beta-cells. When deficient in both pRb and p130, beta-cells undergo unrestrained cell cycle reentry and activation of apoptosis. These studies underscore the central role of the pRb pathway in controlling beta-cell turnover and provide new cellular targets for beta-cell regeneration.

Lactogens promote beta cell survival through JAK2/STAT5 activation and Bcl-XL upregulation.

One of the goals in the treatment for diabetes is to enhance pancreatic beta cell function, proliferation, and survival. This study explores the role of lactogenic hormones, prolactin (PRL) and placental lactogen (PL), in beta cell survival. We have previously shown that transgenic mice expressing mouse placental lactogen-1 (mPL1) in beta cells under the rat insulin II promoter (RIP) are resistant to the diabetogenic and cytotoxic effects of streptozotocin (STZ) in vivo. The current study demonstrates that lactogens protect rat insulinoma (INS-1) cells and primary mouse beta cells against two distinct beta cell death inducers, STZ and dexamethasone (DEX), in vitro. Further, we identify the mechanism through which lactogens protect beta cells against DEX-induced death. The signaling pathway mediating this protective effect is the janus-activated-kinase-2/signal transducer and activator of transcription-5 (JAK2/STAT5) pathway. This is demonstrated in INS-1 cells and primary mouse beta cells using three separate approaches, pharmacological inhibitors, JAK2-specific siRNAs and a dominant-negative STAT5 mutant. Furthermore, lactogens specifically and significantly increase the anti-apoptotic protein Bcl-XL in insulinoma cells and mouse islets. Bcl-XL-specific siRNA significantly inhibits lactogen-mediated protection against DEX-induced beta cell death. We believe this is the first direct demonstration of lactogens mediating their protective effect through the JAK2/STAT5 pathway in the beta cell and through Bcl-XL in any cell type.