Rupangi Chhaya Vasavada

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; 1–11. ©2017 AACR.

Finding ways to stimulate the regeneration of endogenous pancreatic beta cells is an important goal in the treatment of diabetes. Parathyroid hormone-related protein (PTHrP), the full-length (1–139) and amino-terminal (1–36) peptides, enhance beta cell function, proliferation, and survival. Therefore, we hypothesize that PTHrP(1–36) has the potential to regenerate endogenous beta cells. The partial pancreatectomy (PPx) mouse model of beta cell injury was used to test this hypothesis. Male Balb/c mice underwent either sham-operation or PPx, and were subsequently injected with PTHrP(1–36) (160μg/kg) or vehicle (veh), for 7, 30, or 90 days. The four groups of mice, sham-veh, sham-PTHrP, PPx-veh, and PPx-PTHrP were assessed for PTHrP and receptor expression, and glucose and beta cell homeostasis. PTHrP-receptor, but not the ligand, was significantly up-regulated in islets from mice that underwent PPx compared to sham-operated mice. This suggests that exogenous PTHrP could further enhance beta cell regeneration after PPx. PTHrP did not significantly affect body weight, blood glucose, plasma insulin, or insulin sensitivity, in either sham or PPx mice. Glucose tolerance improved in the PPx-PTHrP versus PPx-veh mice only in the early stages of treatment. As hypothesized, there was a significant increase in beta cell proliferation in PPx-PTHrP mice at days 7 and 30; however, this was normalized by day 90, compared to PPx-veh mice. Enhanced beta cell proliferation translated to a marked increase in beta cell mass at day 90, in PPx-PTHrP versus PPx-veh mice. PTHrP(1–36) significantly enhances beta cell regeneration through increased beta cell proliferation and beta cell mass after PPx. Future studies will determine the potential of PTHrP to enhance functional beta cell mass in the setting of diabetes.

A major focus in the field of diabetes is to identify molecules that enhance endogenous β-cell regeneration. Parathyroid hormone-related protein (PTHrP) holds great promise in this regard. PTHrP enhances rodent β-cell function, proliferation, and survival in vitro, and in vivo in PTHrP-overexpressing transgenic mice. The amino-terminal 1-36 peptide is sufficient to produce these beneficial effects in vitro, in rodent and human β-cells. Current use of PTHrP(1-36) in clinical trials for the treatment of osteoporosis attests to the safety of this peptide for clinical use. This study is the first to examine the therapeutic regenerative potential of PTHrP(1-36) in a partial pancreatectomy (PPx) mouse model of β-cell injury. We have previously found that subcutaneous administration of PTHrP(1-36) at 80 or 160µg/kg markedly enhances β-cell proliferation and mass by 25 days in eight-week old normal male BalbC mice. To determine its effect on β-cell regeneration after injury, we injected PTHrP(1-36) (160µg/kg) or vehicle (veh) in partially (50%) pancreatectomized (PPx) or sham-operated BalbC male mice (n=5-6 mice/group). There were no changes in body weight, blood glucose, insulin levels, insulin sensitivity or incidence of hypercalcemia in the four groups of mice. As expected, PPx mice were glucose intolerant at 1 month compared to sham-operated mice. Importantly, at 1 week, PTHrP(1-36) significantly enhanced β-cell proliferation by 40% in PPx-PTHrP mice compared to the PPx-veh group. This increase in β-cell proliferation translated to a significant marked increase in β-cell mass at 3 months in the PPx-PTHrP (2.4±0.3 mg) versus PPx-veh mice (1.4±0.2 mg), causing a complete recovery of their β-cell mass, equivalent to the control sham-veh mice (2.6±0.3 mg). Interestingly, analysis of protein expression in islets from the four groups of mice after 1 week of treatment showed that PPx induces an increase in PTHrP receptor expression which is significant in the PPx-PTHrP group. Preliminary study of the cell-cycle proteins in islets from these treated mice suggests an increase in cyclin D2 and a decrease in p27 in PPx-PTHrP mice compared with PPx-veh mice. Our results clearly demonstrate the regenerative potential of PTHrP(1-36) to restore endogenous depleted β-cell mass without negatively impacting glucose homeostasis. These studies together with its effects on human β-cells makes PTHrP(1-36) a strong candidate for the future therapeutic treatment of diabetes. Sources of Research Support: Grants from the National Institutes of Health (DK078060 and DK072264) and JDRF Research Awards (1-2008-46). Disclosures: AFS: Member, Osteotrophin LLC. Nothing to Disclose: AM, HL, KW, NG, AO, CC, AGO, RCV

Osteoprotegerin (OPG), a soluble glycoprotein of the tumor necrosis factor receptor superfamily, was originally identified as a key regulator in bone metabolism. Recently, it has been shown that OPG is also expressed in the pancreas; enhances rodent β-cell survival against cytokines; and is significantly increased at the mRNA level in three different models of beta cell expansion, including pregnancy; suggesting a role for OPG in β-cell biology. However, nothing is known about the effects of OPG on human β-cells. Previously we found that lactogens protect β-cells against varied cell death inducers including glucolipotoxicity (GLT). To identify novel downstream targets of lactogens potentially involved in its pro-survival effects, we ran apoptosis gene expression arrays on normal (NL) and placental lactogen expressing transgenic (TG) mouse islets treated with and without GLT. OPG expression was increased in TG islets under control (22-fold) and GLT (9-fold) conditions compared to NL islets. A similar significant increase in OPG expression was seen using real time PCR, confirming these observations. Furthermore, OPG mRNA expression was increased in INS1 cells acutely treated with prolactin under control (6-fold) and GLT (1.5-fold) conditions. These results identify OPG as a novel downstream target of lactogens in b-cells. We next sought to determine if OPG had a direct effect on human β-cell survival. GLT-induced β-cell death in human islet cell cultures (n=3) treated with and without OPG (200ng) was measured by insulin and TUNEL co-staining. GLT caused a 3.5-fold increase in β-cell death which was significantly ameliorated by ~60% with OPG treatment, demonstrating a pro-survival effect of OPG on human β-cells. Based on the increase in OPG mRNA in rodent models of β-cell expansion, we analyzed the effect of OPG on human β-cell proliferation, by insulin- bromodeoxyuridine (BrdU) co-staining of human islet cell cultures (n=2-4) treated with different concentrations of OPG (25–200ng) in the presence of BrdU. There was a dose-dependent increase in human β-cell proliferation with a significant 2-fold increase at the highest OPG dose. Further studies to examine the effects and molecular mechanisms of OPG action are underway. Taken together, our studies reveal OPG to be a novel downstream target of lactogens, which independently enhances human β-cell proliferation and survival, making it one of the few factors with direct beneficial effects on the human β-cell. Sources of Research Support: DK078060 and DK072264. Nothing to Disclose: NGK, AO, AM, CC, RCV

Lactogens acting through the prolactin receptor (PRL-R) are essential for the maintenance of normal β-cell function, proliferation and mass; and also protect β-cells against distinct cell death inducers. The goal of this study was to examine whether lactogens are pertinent in the pathogenesis of type II diabetes, whether they protect β-cells against GLT, and to understand their mechanism of action. Despite numerous studies, the correlation between plasma PRL and obesity/diabetes in humans and rodents is unclear. We measured plasma PRL levels in leptin-deficient, obese, hyperglycemic C57Bl/6 ob/ob mice at ages 7-11 weeks. PRL levels were significantly lower in ob/ob mice (7.2±0.5ng/ml) compared to age-matched controls (17.3±2.0ng/ml). Furthermore, there was a 2-fold increase in PRL-R mRNA in islets from 11-week old ob/ob mice. Whether the changes in PRL/PRL-R levels contribute to the phenotype of these mice is under investigation. As GLT is a likely contributor to the reduction in β-cell mass in type II diabetes, we assessed the effect of lactogens on GLT-induced β-cell death. Primary β-cells from normal (NL) mice exposed to 25mM glucose/0.5mM palmitate showed a 2.6-fold increase in β-cell death which was completely inhibited in islets of transgenic (TG) mice, expressing mouse placental lactogen-1 (mPL-1) in β-cells. Importantly, in human islets, GLT induced a 3-fold increase in β-cell death which was attenuated to 1.8-fold with human PRL (p<0.02). To dissect the cellular mechanisms, INS-1 cells were treated with 0.25 and 0.5mM palmitate, resulting in a 15- and 21-fold increase in cell death which was significantly reduced to 10- and 9-fold respectively with PRL. Of the lactogen-activated signaling pathways, our studies with siRNA and dominant-negative mutant show that the JAK2/STAT5 pathway is critical in mediating the pro-survival effect of lactogens. Furthermore, the importance of lactogen-induced expression of the anti-apoptotic molecule, BclXL, was underscored when BclXL-siRNA eliminated the protective effect of PRL against GLT-mediated INS-1 cell death. Currently, analysis of gene expression in NL and mPL-1 TG mouse islets ± GLT, using apoptotic pathway gene arrays shows an increase in expression of distinct anti-apoptotic/anti-inflammatory genes in TG islets under basal and GLT conditions, which is being confirmed. Taken together these results highlight a potential role of lactogens in β-cell mass expansion in insulin resistance and type II diabetes. 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 acknowledge the ICR and JDRF Basic Science Islet Distribution Programs for supplying human islets. Nothing to Disclose: NGK, KW, XZ, RCV

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...

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.