Rupangi C. Vasavada

Hepatocyte Growth Factor Overexpression in the Islet of Transgenic Mice Increases Beta Cell Proliferation, Enhances Islet Mass, and Induces Mild Hypoglycemia

Hepatocyte growth factor (HGF) is produced in pancreatic mesenchyme-derived cells and in islet cells. In vitro, HGF increases the insulin content and proliferation of islets. To study the role of HGF in the islet in vivo, we have developed three lines of transgenic mice overexpressing mHGF using the rat insulin II promoter (RIP). Each RIP-HGF transgenic line displays clear expression of HGF mRNA and protein in the islet. RIP-mHGF mice are relatively hypoglycemic in post-prandial and fasting states compared with their normal littermates. They display inappropriate insulin production, striking overexpression of insulin mRNA in the islet, and a 2-fold increase in the insulin content in islet extracts. Importantly, beta cell replication rates in vivo are two to three times higher in RIP-HGF mice. This increase in proliferation results in a 2–3-fold increase in islet mass. Moreover, the islet number per pancreatic area was also increased by approximately 50%. Finally, RIP-mHGF mice show a dramatically attenuated response to the diabetogenic effects of streptozotocin. We conclude that the overexpression of HGF in the islet increases beta cell proliferation, islet number, beta cell mass, and total insulin production in vivo. These combined effects result in mild hypoglycemia and resistance to the diabetogenic effects of streptozotocin.

Overexpression of Parathyroid Hormone-related Protein in the Pancreatic Islets of Transgenic Mice Causes Islet Hyperplasia, Hyperinsulinemia, and Hypoglycemia

Parathyroid hormone-related protein (PTHrP) is produced by the pancreatic islet. It also has receptors on islet cells, suggesting that it may serve a paracrine or autocrine role within the islet. We have developed transgenic mice, which overexpress PTHrP in the islet through the use of the rat insulin II promoter (RIP). Glucose homeostasis in these mice is markedly abnormal; RIP-PTHrP mice are hypoglycemic in the post-prandial and fasting states and display inappropriate hyperinsulinemia. At the end of a 24-hour fast, blood glucose values are 49 mg/dl in RIP-PTHrP mice, as compared to 77 mg/dl in normal littermates; insulin concentrations at this time are 6.3 and 3.9 ng/ml, respectively. Islet perifusion studies failed to demonstrate abnormalities in insulin secretion. In contrast, quantitative islet histomorphometry demonstrates that the total islet number and total islet mass are 2-fold higher in RIP-PTHrP mice than in their normal littermates. PTHrP very likely plays a normal physiologic role within the pancreatic islet. This role is most likely paracrine or autocrine. PTHrP appears to regulate insulin secretion either directly or indirectly, through developmental or growth effects on islet mass. PTHrP may have a role as an agent that enhances islet mass and/or enhances insulin secretion.

Gestational diabetes mellitus resulting from impaired β-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 β-cell mass expansion in maternal islets during pregnancy and whether FoxM1 is essential for placental lactogen (PL)-induced β-cell proliferation. RESEARCH DESIGN AND METHODS β-Cell mass, β-cell proliferation, and glucose homeostasis were assessed in virgin, pregnant, and postpartum mice with a pancreas-wide Foxm1 deletion (FoxM1Δpanc). Wild-type islets were cultured with or without PL and examined for Foxm1 induction. Transgenic mice overexpressing PL in β-cells were bred with FoxM1Δpanc mice, and β-cell proliferation was examined. RESULTS Foxm1 was upregulated in maternal islets during pregnancy. In contrast to controls, β-cell proliferation did not increase in pregnant FoxM1Δpanc females. Mutant islets showed increased Menin and nuclear p27. FoxM1Δpanc females developed gestational diabetes mellitus as pregnancy progressed. After parturition, euglycemia was restored in FoxM1Δpanc females, but islet size was significantly reduced. Strikingly, β-cell mass was normal in postpartum FoxM1Δpanc pancreata due to a combination of increased β-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 β-cell proliferation in vivo. CONCLUSIONS FoxM1 is essential for β-cell compensation during pregnancy. In the absence of increased β-cell proliferation, neogenesis is induced in postpartum FoxM1Δpanc pancreata. Our results suggest that FoxM1 functions downstream of PL to mediate its effects on β-cell proliferation.

Protein kinase C-ζ activation markedly enhances β-cell proliferation : An essential role in growth factor-mediated β-cell mitogenesis

OBJECTIVE— Diabetes results from a deficiency of functional β-cells. Previous studies have identified hepatocyte growth factor (HGF) and parathyroid hormone–related protein (PTHrP) as two potent β-cell mitogens. The objective of this study is to determine 1) whether HGF and PTHrP have additive/synergistic effects on β-cell growth and proliferation; 2) the signaling pathways through which these growth factors mediate β-cell mitogenesis; and 3) whether activation of this/these signaling pathway(s) enhances human β-cell replication. RESEARCH DESIGN AND METHODS— We generated and phenotypically analyzed doubly transgenic mice overexpressing PTHrP and HGF in the β-cell. INS-1 and primary mouse and human islet cells were used to identify mitogenic signaling pathways activated by HGF and/or PTHrP. RESULTS— Combined overexpression of HGF and PTHrP in the β-cell of doubly transgenic mice did not result in additive/synergistic effects on β-cell growth and proliferation, suggesting potential cross-talk between signaling pathways activated by both growth factors. Examination of these signaling pathways in INS-1 cells revealed atypical protein kinase C (PKC) as a novel intracellular target activated by both HGF and PTHrP in β-cells. Knockdown of PKCζ, but not PKCι/λ, expression using specific small-interfering RNAs blocked growth factor–induced INS-1 cell proliferation. Furthermore, adenovirus-mediated delivery of kinase-dead PKCζ completely inhibited β-cell proliferation in primary islet cells overexpressing PTHrP and/or HGF. Finally, adenovirus-mediated delivery of constitutively active PKCζ in mouse and human primary islet cells significantly enhanced β-cell proliferation. CONCLUSIONS— PKCζ is essential for PTHrP- and HGF-induced β-cell proliferation. PKCζ activation could be useful in therapeutic strategies for expanding β-cell mass in vitro and in vivo.

Disruption of Hepatocyte Growth Factor/c-Met Signaling Enhances Pancreatic β-Cell Death and Accelerates the Onset of Diabetes

OBJECTIVE To determine the role of hepatocyte growth factor (HGF)/c-Met on β-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 β-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 β-cell survival against diabetogenic agents. Adult PancMet KO mice displayed normal glucose and β-cell homeostasis, indicating that pancreatic c-Met loss is not detrimental for β-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 β-cell mass compared with wild-type littermates. PancMet KO mice showed enhanced intraislet infiltration, islet nitric oxide (NO) and chemokine production, and β-cell apoptosis. c-Met-null β-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 β-cells against cytokines. CONCLUSIONS These results show that HGF/c-Met is critical for β-cell survival by attenuating NF-κB signaling and suggest that activation of the HGF/c-Met signaling pathway represents a novel strategy for enhancing β-cell protection.

Loss of HGF/c-Met Signaling in Pancreatic β-Cells Leads to Incomplete Maternal β-Cell Adaptation and Gestational Diabetes Mellitus

Hepatocyte growth factor (HGF) is a mitogen and insulinotropic agent for the β-cell. However, whether HGF/c-Met has a role in maternal β-cell adaptation during pregnancy is unknown. To address this issue, we characterized glucose and β-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 β-cell replication and increased β-cell apoptosis at gestational day (GD)15. The decreased β-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 β-cells were more sensitive to dexamethasone-induced cytotoxicity, whereas HGF protected human β-cells against dexamethasone in vitro. These detrimental alterations in β-cell proliferation and death led to incomplete maternal β-cell mass expansion in PancMet KO mice at GD19 and early postpartum periods. The decreased β-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 β-cell adaptation during pregnancy and that its absence/attenuation leads to gestational diabetes mellitus.

Diabetes mellitus—advances and challenges in human β-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 β cells, alone or in combination with insulin resistance. It logically follows that the replacement or regeneration of β 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 β cells using stem-cell strategies to transdifferentiate or reprogramme non-β cells into β cells or to discover small molecules or other compounds that can induce proliferation of human β 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 β-cell replication, the molecular mechanisms that regulate the cell cycle in human β cells, the upstream intracellular signalling pathways that connect them to cell surface receptors on β cells, the epigenetic mechanisms that control human β-cell proliferation and unbiased approaches for discovering novel molecules that can drive human β-cell proliferation. Finally, we discuss the potential and challenges of implementing strategies that replace or regenerate β cells.

ChREBP Mediates Glucose-Stimulated Pancreatic β-Cell Proliferation

Glucose stimulates rodent and human β-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 β-cells. We tested the hypothesis that ChREBP is required for glucose-stimulated β-cell proliferation. The relative expression of ChREBP was determined in liver and β-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 β-cells. Proliferation was measured by 5-bromo-2′-deoxyuridine incorporation, [3H]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 β-cells isolated from ChREBP−/− mice, in INS-1–derived 832/13 cells, and in primary rat and human β-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 β-cells, with concomitant increases in cyclin gene expression. In conclusion, ChREBP mediates glucose-stimulated proliferation in pancreatic β-cells.

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.

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

OBJECTIVE Inducing human β-cell growth while enhancing function is a major goal in the treatment of diabetes. Parathyroid hormone–related protein (PTHrP) enhances rodent β-cell growth and function through the parathyroid hormone-1 receptor (PTH1R). Based on this, we hypothesized that PTH1R is expressed in human β-cells and that PTHrP has the potential to enhance human β-cell proliferation and/or function. RESEARCH DESIGN AND METHODS PTH1R expression, β-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 β-cell proliferation. RESULTS We found that human β-cells express PTH1R. More importantly, overexpression of PTHrP causes a significant approximately threefold increase in human β-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 β-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 β-cell proliferation as well as function, with associated upregulation of two specific cell-cycle activators that together can induce human β-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.