Overexpression of Parathyroid Hormone-Related Protein Inhibits Pancreatic β-Cell Death In Vivo and In Vitro

Abstract

Pancreatic ␤-cell survival is critical in the setting of diabetes as well as in islet transplantation. Transgenic mice overexpressing parathyroid hormone-related protein (PTHrP) targeted to ␤-cells using the rat insulin II promoter (RIP) display hyperinsulinemia, hypoglycemia, and islet hyperplasia, without a concomitant increase in ␤-cell proliferation rate or enlargement of individual ␤-cell size. Thus, the mechanism for increased ␤-cell mass is unknown. In this study, we demonstrated that ␤-cells of transgenic mice are resistant to the cytotoxic effects of streptozotocin (STZ) in vivo, as documented by a sixfold reduction in the rate of STZ-induced ␤-cell death in RIP-PTHrP mice relative to their normal siblings. The reduced cell death in transgenic mice is due neither to their increased islet mass nor to a decrease in their sensing of STZ, but rather results from PTHrP-induced resistance to ␤-cell death. This is also demonstrated in vitro by markedly reduced cell death rates observed in ␤-cells of transgenic mice compared with normal mice when cultured in the absence of serum and glucose or in the presence of STZ. Finally, we demonstrated that NH 2 -terminal PTHrP inhibits ␤-cell death. These findings support the concept that PTHrP overexpression increases islet mass in transgenic mice through inhibition of ␤-cell death. Diabetes 51:3003-3013, 2002 F ailure of ␤-cell survival is critical to the etiology of type 1 (1-3) and type 2 (4,5) diabetes, as well as in the setting of islet transplantation (6,7). Despite an enormous increase in our understanding of islet differentiation and development, there is sparse information regarding the factors and pathways that regulate growth, survival, and death of islet cells. A number of approaches have been taken, including development of knockout and transgenic mouse models, to address these questions. Of the several ␤-cell-targeted transgenic mouse models generated to date, only a handful have displayed an increase in islet mass without displaying a concomitant negative effect on islet structure or function (8 -12). The mechanisms through which the targeted proteins have brought about islet mass expansion are quite distinct. These mechanisms include accelerated replication of preexisting ␤-cells (9 -12), an increase in ␤-cell size (hypertrophy) (9,11,12), a reduction in the rate of ␤-cell death (13,14), and an augmentation of islet neogenesis (11,12,15).Parathyroid hormone-related protein (PTHrP), widely expressed in most tissues of the body in the fetus and adult, is also expressed in the four endocrine cell types of the islet and in pancreatic ductal cells (16). PTHrP receptors have been shown to be present in islets (17) and the ␤-cell line RINm5F (18). Transgenic mice overexpressing PTHrP as well as PTHrP-knockout mice have demonstrated critical roles for this peptide in the development and differentiation of many organs, including the skeleton (19,20), mammary gland (21), skin (22), teeth (23), vascular system (24), and others. To examine the consequences of PTHrP ...

Efficient gene delivery to pancreatic islets with ultrasonic microbubble destruction technology

“…Although transgene expression in pancreatic islets of rodents has been achieved by microinjection of fertilized embryos (24)(25)(26)(27)(28)(29)(30), safe and efficient in vivo targeting of genes to pancreatic islets has been an elusive goal. This paper describes a method for efficient gene delivery to the pancreatic islets, with detection of transgene-encoded proteins (luciferase, human insulin and Cpeptide, hexokinase I) for up to 3 weeks after transfection.…”

Protein Kinase C-ζ Activation Markedly Enhances β-Cell Proliferation

“…Single islet cell cultures were prepared by trypsinizing human or mouse islets for 8 -10 min at 37°C. Trypsinization was stopped with RPMI containing 5 mmol/l D-glucose, 10% FBS, 100 units/ml penicillin, and 100 g/ml streptomycin; cells were plated on 12-mm glass coverslips, placed in 24-well plates, and incubated at 37°C for 2 h to allow cells to attach to the glass surface (27). Islet cells were simultaneously transduced with purified adenovirus at a multiplicity of infection (MOI) of 100 plaque-forming units per cell or left uninfected.…”

Induction of β-Cell Proliferation and Retinoblastoma Protein Phosphorylation in Rat and Human Islets Using Adenovirus-Mediated Transfer of Cyclin-Dependent Kinase-4 and Cyclin D1

“…Cell death assay. ␤-Cell death was assessed using nuclear pyknosis as described in detail previously (20,22). Briefly, fresh sections were cut from the same blocks used for human islet immunohistochemistry described in Figs.…”