Phospho-BAD BH3 mimicry protects ß cells and restores functional ß cell mass in diabetes.

Strategies that simultaneously enhance the survival and glucose responsiveness of insulin-producing ß cells will greatly augment ß cell replacement therapies in type 1 diabetes (T1D). We show that genetic and pharmacologic mimetics of the phosphorylated BCL-2 homology 3 (BH3) domain of BAD impart ß-cell-autonomous protective effects in the face of stress stimuli relevant to ß cell demise in T1D. Importantly, these benefits translate into improved engraftment of donor islets in transplanted diabetic mice, increased ß cell viability in islet grafts, restoration of insulin release, and diabetes reversal. Survival of ß cells in this setting is not merely due to the inability of phospho-BAD to suppress prosurvival BCL-2 proteins but requires its activation of the glucose-metabolizing enzyme glucokinase. Thus, BAD phospho-BH3 mimetics may prove useful in the restoration of functional ß cell mass in diabetes.

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.

The NMR structure of human obestatin in membrane-like environments: insights into the structure-bioactivity relationship of obestatin.

The quest for therapeutic applications of obestatin involves, as a first step, the determination of its 3D solution structure and the relationship between this structure and the biological activity of obestatin. On this basis, we have employed a combination of circular dichroism (CD), nuclear magnetic resonance (NMR) spectroscopy, and modeling techniques to determine the solution structure of human obestatin (1). Other analogues, including human non-amidated obestatin (2) and the fragment peptides (6-23)-obestatin (3), (11-23)-obestatin (4), and (16-23)-obestatin (5) have also been scrutinized. These studies have been performed in a micellar environment to mimic the cell membrane (sodium dodecyl sulfate, SDS). Furthermore, structural-activity relationship studies have been performed by assessing the in vitro proliferative capabilities of these peptides in the human retinal pigmented epithelial cell line ARPE-19 (ERK1/2 and Akt phosphorylation, Ki67 expression, and cellular proliferation). Our findings emphasize the importance of both the primary structure (composition and size) and particular segments of the obestatin molecule that posses significant α-helical characteristics. Additionally, details of a species-specific role for obestatin have also been hypothesized by comparing human and mouse obestatins (1 and 6, respectively) at both the structural and bioactivity levels.

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.