Adolfo Garcia-Ocaña

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.

OBJECTIVE: PKC-ζ activation is a key signaling event for growth factor–induced β-cell replication in vitro. However, the effect of direct PKC-ζ activation in the β-cell in vivo is unknown. In this study, we examined the effects of PKC-ζ activation in β-cell expansion and function in vivo in mice and the mechanisms associated with these effects. RESEARCH DESIGN AND METHODS: We characterized glucose homeostasis and β-cell phenotype of transgenic (TG) mice with constitutive activation of PKC-ζ in the β-cell. We also analyzed the expression and regulation of signaling pathways, G1/S cell cycle molecules, and β-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 β-cell proliferation, size, and mass compared with wild-type littermates. The increase in β-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-3β and phosphorylation/activation of mTOR, critical regulators of D-cyclin expression and β-cell proliferation, were enhanced in TG mouse islets, without changes in Akt phosphorylation status. Rapamycin treatment in vivo eliminated the increases in β-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 β-cell replication and expansion in vivo. CONCLUSIONS: PKC-ζ, through mTOR activation, modifies the expression pattern of β-cell cycle molecules leading to increased β-cell replication and mass with a concomitant enhancement in β-cell function. Approaches to enhance PKC-ζ activity may be of value as a therapeutic strategy for the treatment of diabetes.

Beta cell mass is reduced in T2DM, and unbiased genome-wide association studies link diabetes risk with cell cycle regulatory genes, including the p16 locus. In obesity, elevated free fatty acids (FFA) are independently associated with progression to T2DM. To test whether FFA restrict beta cell proliferation in vivo, C57BL/6j adult male mice were infused intravenously with saline (SAL), Liposyn II (LIP), 50% dextrose (GLU), or Liposyn II with 50% dextrose (L+G), all with heparin (2U/hr) and bromodeoxyuridine (BrdU; 10mg/hr), for 4 days. GLU and L+G infusions increased circulating BG and plasma insulin (p<0.001). LIP and L+G infusions increased plasma FFA (p<0.01). As previously shown, glucose infusion (GLU) increased beta cell proliferation (p<0.01 vs. SAL). Addition of Liposyn II (L+G) completely blocked glucose-induced beta cell proliferation (p<0.01 vs. GLU, p=ns vs. SAL). To determine whether the anti-proliferative effect of lipid was due to direct action of FFA on the beta cell, primary mouse islet cells were cultured in the presence of low (2mM) or high (15mM) glucose, with BSA control or a FFA mixture containing 7:2:1 linoleic [18:2]: oleic [18:1]: palmitic [16:0] acids (similar to Liposyn II FA components). Glucose-induced beta cell proliferation (p<0.01 for 2mM+BSA vs. 15mM+BSA) was blocked by addition of FFA (p<0.01 for 15mM+BSA vs. 15mM+FFA). In INS-1 cells, glucose-induced proliferation (p<0.01 for 2mM+BSA vs. 11mM+BSA) was also blocked by FFA (p<0.01 for 11mM+BSA vs. 11mM+FFA). In INS-1 cells the anti-proliferative effect of FFA was mediated by linoleic and palmitic acids. We hypothesized that FFA might prevent glucose-induced cyclin D2 protein stability or nuclear localization. However, in vivo, co-infusing lipid (L+G) altered neither the glucose-mediated increase in islet cyclin D2 protein nor its nuclear localization. In contrast, immunoblot showed that two cell cycle inhibitors downstream of D-cyclins, p16 and p18, were induced in L+G islets (p<0.01 for L+G vs. GLU for p16; p<0.05 for L+G vs. GLU for p18). In INS-1 cells, palmitic acid also induced p16 and p18 proteins (p<0.01 for p16; p<0.001 for p18). Remarkably, siRNA knockdown of either p16 or p18 rescued the anti-proliferative effects of palmitic acid in INS-1 cells. We conclude that FFA block glucose-induced beta cell proliferation by inducing p16 and p18, and we speculate that FFA may contribute to failure of beta cell mass expansion in T2DM. Sources of Research Support: NIH DK076562 and an ONCR pilot and feasibility award DK046204 (LCA); HL063767 (COD); and DK067351 and DK077096 (AGO). Nothing to Disclose: LCA, DAH, JLP, TR, MA, LCR, BZ, CPO, AG-O

Type 1 diabetes (T1D) is an autoimmune disease that results from islet immune infiltration and cellular cytotoxicity leading to selective and progressive destruction of beta cells. The non-obese diabetic (NOD) mouse model that spontaneously develops T1D shares multiple characteristics with the human disease, and can be employed to unravel clinically relevant and important information for the treatment of this disease. Low molecular weight (250mg/dl) compared with 8 out of 12 SAL-treated NOD mice. On the other hand, plasma insulin levels were increased in DS-treated mice compared with SAL-treated mice. Importantly, the rate of islets with severe insulitis was highly reduced in DS-treated NOD mice (41±17%, P<0.05) compared with SAL-treated NOD mice (78±12%). The diminished islet infiltration in DS-treated NOD mouse islets correlated with decreased beta cell apoptosis (0.27±0.14%, DS-treated vs. 10.5±0.1, SAL-treated) and increased beta cell mass (0.9±0.03 mg, DS-treated vs. 0.1±0.1 mg, SAL-treated), reaching similar levels to beta cell mass in non-diabetic NOD female mice at 10 weeks of age before DS treatment. These results clearly demonstrate that DS can decrease islet inflammation, protect beta cells and prevent T1D in NOD mice. Dextran sulfate can be a therapeutic tool for the treatment of T1D. Sources of Research Support: NIH/NIDDK Grant DK067351-06 and ADA Grant 1-10-BS-59 awarded to AG-O. Nothing to Disclose: SV, SE, AG-O

Human in vivo models of primary hyperparathyroidism (HPT), humoral hypercalcemia of malignancy (HHM), or lactational bone mobilization for more than 48 hours have not been described previously. We therefore developed 7‐day continuous‐infusion models using human parathyroid hormone(1–34) [hPTH(1–34)] and human parathyroid hormone–related protein(1–36) [hPTHrP(1–36)] in healthy human adult volunteers. Study subjects developed sustained mild increases in serum calcium (10.0 mg/dL), with marked suppression of endogenous PTH(1–84). The maximal tolerated infused doses over a 7‐day period (2 and 4 pmol/kg/h for PTH and PTHrP, respectively) were far lower than in prior, briefer human studies (8 to 28 pmol/kg/h). In contrast to prior reports using higher PTH and PTHrP doses, both 1,25‐dihydroxyvitamin D₃ [1,25(OH)₂D₃] and tubular maximum for phosphorus (TmP/GFR) remained unaltered with these low doses despite achievement of hypercalcemia and hypercalciuria. As expected, bone resorption increased rapidly and reversed promptly with cessation of the infusion. However, in contrast to events in primary HPT, bone formation was suppressed by 30% to 40% for the 7 days of the infusions. With cessation of PTH and PTHrP infusion, bone‐formation markers abruptly rebounded upward, confirming that bone formation is suppressed by continuous PTH or PTHrP infusion. These studies demonstrate that continuous exposure of the human skeleton to PTH or PTHrP in vivo recruits and activates the bone‐resorption program but causes sustained arrest in the osteoblast maturation program. These events would most closely mimic and model events in HHM. Although not a perfect model for lactation, the increase in resorption and the rebound increase in formation with cessation of the infusions are reminiscent of the maternal skeletal calcium mobilization and reversal that occur following lactation. The findings also highlight similarities and differences between the model and HPT. © 2011 American Society for Bone and Mineral Research

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.

A major goal in the treatment of diabetes is to induce β-cell growth while maintaining its function. Although numerous factors enhance rodent β-cell proliferation and function, there are relatively few studies in human islets. PTHrP and its receptor, parathyroid hormone-1 receptor (PTH1R), are both expressed in rodent β-cells. Furthermore, PTHrP enhances rodent β-cell growth and function both in vitro and in vivo. PTHrP is also made in human islets and its expression is increased in insulinomas. However, there is virtually nothing known regarding the effect of PTHrP in human β-cells. Based on the rodent studies, 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. This study shows that human islets indeed express PTH1R mRNA and protein, and that the receptor is made in human β-cells as shown by co-staining with insulin and PTH1R antibodies. Importantly, overexpression of PTHrP by adenoviral transduction caused a significant 3-fold increase in human β-cell proliferation. Furthermore, treatment with only the amino-terminus 1-36 peptide of PTHrP for 24 hours was sufficient to induce human β-cell replication, without causing significant changes in the expression of differentiation markers. Not only did PTHrP(1-36) peptide enhance proliferation, but significantly augmented glucose-stimulated insulin secretion, both at physiological (5 mM) and pathophysiological (22 mM) glucose concentrations by 36% and 67% respectively. Analysis of cell cycle molecules regulated by PTHrP(1-36) in human islets showed a significant and specific increase in the expression of the late G1/S cell cycle activator proteins cyclinE and cdk2. We further examined whether these cell cycle molecules by themselves could enhance human beta cell proliferation. Overexpression of cyclinE alone, but not cdk2, induced a 5-fold increase in human β-cell proliferation; and when both molecules were expressed simultaneously there is a further marked synergistic increase of 16-fold in replication. Thus, PTHrP(1-36) peptide enhances human β-cell proliferation as well as function, with associated upregulation of two specific cell cycle activators, which 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 post-menopausal osteoporosis. Sources of Research Support: Grants from the National Institutes of Health (DK078060 and DK072264 to RCV; DK055023 and DK07052-32 to AFS) and JDRF Research Awards (1-2008-46 to RCV; 1-2008-39 and 34-2008-630 to AFS). We are grateful to Drs. Irene Cozar-Castellano and Nathalie Taesch for sharing their expertise on the cell cycle; to Darinka Sipula, Taylor Rosa, Jeffrey Kleinberger and Fatimah Salim for superb technical assistance. We acknowledge the ICR and JDRF Basic Science Islet...

This is a particularly exciting time in the field of pancreatic islet growth, development, and survival. The recent publication of a study demonstrating that human pancreatic islet transplantation is both technically and immunologically feasible has highlighted the need for large supplies of pancreatic islets or pancreatic beta cells for larger-scale islet transplantation in patients with diabetes. This, together with a rapid expansion in the past several years of the understanding of mechanisms of islet growth, development, and survival, has accelerated and invigorated efforts to therapeutically harness the cellular mechanisms responsible for pancreatic beta-cell proliferation, survival, and development and to take advantage of this new knowledge to enhance the availability, survival, and function of pancreatic beta cells in human islet transplantation for diabetes mellitus. Here, we briefly review the confluence of events that have provided optimism and energy to the islet transplant field, and we focus on peptide growth factors that eventually may be deployed in the effort to augment islet mass and function in patients with diabetes.