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Using ß-Cell Growth Factors to Enhance Human Pancreatic Islet Transplantation*

Division of Endocrinology and Metabolism, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15213

Address correspondence and requests for reprints to: Andrew F. Stewart, M.D., Chief, Endocrinology, University of Pittsburgh School of Medicine, BST E-1140, 3550 Terrace Street, Pittsburgh, Pennsylvania 15213. E-mail: stewart{at}msx.dept-med.pitt.edu * Supported by NIH

	Abstract

Top Abstract Introduction PTH-related protein (PTHrP) Hepatocyte growth factor (HGF) Placental lactogen (PL) Glucagon-like peptide-1 (GLP-1)... Betacellulin (BTC) Insulin-like growth factors... Reg proteins and islet... Conclusions Suggested Reading

 
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 ß 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 ß-cell proliferation, survival, and development and to take advantage of this new knowledge to enhance the availability, survival, and function of pancreatic ß 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.

	Introduction

Top Abstract Introduction PTH-related protein (PTHrP) Hepatocyte growth factor (HGF) Placental lactogen (PL) Glucagon-like peptide-1 (GLP-1)... Betacellulin (BTC) Insulin-like growth factors... Reg proteins and islet... Conclusions Suggested Reading

 
THE PROCESS OF human pancreatic ß-cell mass regulation (that is to say the integrated effects of ß-cell replication and regeneration, ß-cell death, and islet neogenesis) has become particularly relevant in the past several months as a result of exciting advances in the field of islet transplantation. Until recently, pancreatic islet transplantation as a treatment for diabetes mellitus in humans has been disappointing: the survival of transplanted islets in humans has been ephemeral, with 1-yr islet graft survival and insulin independence in the range of 8%. With the recognition that glucocorticoids and other immunosuppressive medications routinely used in pancreatic islet transplantation protocols induce insulin resistance and may also injure transplanted islets, a group of investigators in Edmonton, Canada, performed a series of human islet transplants in human type 1 diabetics. These exciting studies were described at the American Diabetes Association and The Endocrine Society meetings this summer and appeared in August 2000 in the New England Journal of Medicine. In what is now widely referred to as "the Edmonton study," survival of transplanted human islets into type 1 diabetic recipients was shown to be dramatically improved as a result of a modification of the immunosuppressive "cocktail" used to prevent autoimmune- and allograft-mediated destruction of transplanted islets. The Edmonton investigators used a glucocorticoid-free immunosuppression cocktail composed of low-dose tacrolimus, sirolimus, and an interleukin 2 receptor monoclonal antibody. The results were dramatic: seven of seven transplanted patients became insulin independent and remained so for a mean of 12 months.

This exciting news is not unaccompanied by bad news. The bad news is that for each of the transplanted diabetic recipients, the islets from two to four human pancreases were required to render them insulin independent. This means, for example, that for the estimated 16 million diabetics in the United States, of whom 10% are type 1 diabetics, there are insufficient pancreatic donors from whom one might obtain such enormous quantities of islets. At present, only a relative handful of pancreas donors become available each year. Thus, one important outcome of the Edmonton study is that it highlights the fact that if islet transplant is to become a widespread treatment of diabetes, new sources of pancreatic ß cells will need to be identified.

Such a source of ß cells might be through the induction of differentiation of embryonic or bone marrow stem cells, or of pancreatic ductular stem cells. It also may be that nonhuman islets (for example, porcine or primate) may be useful here. It also may be possible to develop continuously growing human ß-cell lines or to induce non-ß cells (e.g. liver cells or pituitary cells) to secrete insulin appropriately. In addition, efforts are underway in a number of laboratories to develop a mechanical artificial pancreas. Each of these strategies has pros and cons, and each is under development in multiple laboratories. However, these are future therapies. In contrast, the current feasibility of islet transplantation in humans has been documented by the Edmonton study, and efforts to increase human islet production are, therefore, active in many laboratories around the world.

So, the question naturally arises, "Is it possible to up-regulate pancreatic ß-cell mass and function?" Pancreatic islets were once viewed as terminally differentiated cells, incapable of proliferation. Studies performed in a number of laboratories over the years, however, have demonstrated that this is not the case. It has been demonstrated clearly that pancreatic ß cells can proliferate in response to a number of physiologic and pathophysiologic stimuli, including hyperglycemia, hypercaloric feeding, pregnancy, pancreatectomy, following the surgical correction of insulinoma-induced hypoglycemia, and, more recently, in response to genetic manipulations that induce insulin resistance, such as disruption of the insulin receptor or its signaling pathway. In addition to ß-cell proliferation within existing islets as a source of new ß cells, the development of completely new islets, so-called "islet neogenesis," also occurs in fetal and adult life. The precursor cells for these new pancreatic islets are believed to reside in the pancreatic duct, from the main duct down to its smallest branches, which serve to drain the pancreatic exocrine cell acinar clusters.

Conversely, it is also now quite evident that the total ß-cell mass of the pancreas can be regulated under physiologic and pathophysiologic circumstances by changes in the rate of cell death and/or apoptosis. Examples of settings in which the ß-cell death rate is accelerated include the induction of sustained hypoglycemia; remodeling of the maternal and neonatal islets postpartum; injury to the pancreatic ß cell induced by islet toxins such as streptozotocin, by cytokines such as interleukin 1, tumor necrosis factor , and interferon ; and in the autoimmune attack on ß cells that occurs in type 1 diabetes. Thus, in mature animals, including humans, there seems to be a continuous loss of mature ß cells through cell death, and this is balanced, under steady-state conditions, by concomitant processes of both proliferation of existing ß cells as well as islet neogenesis. This continuous turnover occurs in rodents at a rate such that 3% of ß cells are replaced every day.

Here, we will focus on peptide growth factors that may ultimately prove useful in augmenting islet mass in vitro before transplantation, or in vivo following transplantation. Space limitations preclude a detailed discussion of all the advances that are occurring in this area. We will, therefore, not address the very exciting and closely related area of signaling and cell cycle regulatory molecules, which may mediate the effects of islet growth factors and which may potentially serve as targets for orally active small molecules. Such molecules include p8, cdk4, T-antigen, Akt1/PKB, IRS-1, IRS-2, Bcl-2, and Bcl-x(L). Similarly, we will not address an equally important and exciting area, the role of islet-specific transcription factors such as IDX-1, Isl-1, PAX-4, PAX-6, Nkx 6.1, neurogenin-3, and others in inducing and sustaining the differentiated ß-cell phenotype, although it seems very clear that these molecules will be required partners or components, whether endogenous or introduced by gene transfer, in maintaining mature, highly efficient, and effective human islets. We will also pass over the ultimate need for successful and safe targeting vectors, which will be required for the successful delivery of these islet mass- enhancing factors into human islets in vivo, but, here again, progress is rapid, and there is reason for optimism. Instead, we will focus here on peptide growth factors that have been successfully deployed to augment ß-cell proliferation, mass, and function.

	PTH-related protein (PTHrP)

Top Abstract Introduction PTH-related protein (PTHrP) Hepatocyte growth factor (HGF) Placental lactogen (PL) Glucagon-like peptide-1 (GLP-1)... Betacellulin (BTC) Insulin-like growth factors... Reg proteins and islet... Conclusions Suggested Reading

 
PTHrP was discovered in the early 1980s as the factor responsible for humoral hypercalcemia of malignancy. Subsequent studies found PTHrP expression to be widespread in almost all tissues and organs of the body. One such tissue is the islet of Langerhans, in which all four endocrine cell types (, ß, , and pancreatic polypeptide cells) produce PTHrP. Not only is the peptide made in islets, but receptors for PTHrP also seem to be present on ß cells. To begin to evaluate the possible role of PTHrP in pancreatic islets, we prepared transgenic mice overexpressing PTHrP in the ß cells of islets using the rat insulin II promoter (RIP). These RIP-PTHrP mice displayed islet cell hyperplasia, significant hypoglycemia under both fasting and nonfasting conditions, as well as inappropriate hyperinsulinemia. Insulin expression was shown to be up-regulated both at the messenger RNA and protein level in whole pancreas of RIP-PTHrP mice.

The phenotype does not seem to result from a developmental effect of PTHrP on the pancreas, because transgenic mice at 1 week of age were normoglycemic and displayed normal islet mass despite expression of the PTHrP transgene. In contrast, a visible increase in islet mass (2-fold) was observed in RIP-PTHrP mice by 12 weeks of age, and increased further (3- to 4-fold) by 1 yr of age. Both an increase in the number of ß cells per islet as well as an increase in total islet number contribute to the enhancement of islet mass in these mice. The increase in islet mass in RIP-PTHrP transgenic mice does not seem to be a result of an increase in the proliferation rates of preexisting ß cells of the islet. Thus, the increased islet mass in RIP-PTHrP mice most likely results from a decrease in the normal rate of ß-cell turnover or apoptosis and/or enhanced neogenesis. In other cell types like chondrocytes, neuronal cells, and prostate carcinoma cells, PTHrP has been shown to have an antiapoptotic or protective effect against cell death. In line with this, ß cells of the RIP-PTHrP mice have also been shown to be more resistant to the cytotoxic effects of high doses of the diabetogenic agent streptozotocin (STZ): RIP-PTHrP transgenic mice remain relatively euglycemic unlike their normal littermates, which become severely diabetic following STZ injection. Histologically, the resistance to the diabetogenic effects of STZ seems to result, at least partially, from PTHrP-induced resistance to STZ-mediated ß-cell death.

These findings suggest that PTHrP may have potential in gene therapeutic strategies designed to increase ß-cell mass and function. Specifically, this peptide could prove to be valuable in improving islet transplant survival in type 1 diabetes.

	Hepatocyte growth factor (HGF)

Top Abstract Introduction PTH-related protein (PTHrP) Hepatocyte growth factor (HGF) Placental lactogen (PL) Glucagon-like peptide-1 (GLP-1)... Betacellulin (BTC) Insulin-like growth factors... Reg proteins and islet... Conclusions Suggested Reading

 
HGF is a mesenchyme-derived factor, originally identified as a circulating molecule implicated in liver regeneration after hepatic injury. Recent findings have demonstrated that HGF is a pleiotropic factor that promotes cell growth, cell motility, and morphogenesis in a wide variety of cells. To exert its actions, HGF binds with high affinity to a membrane-spanning tyrosine kinase receptor encoded by the proto-oncogene, c-met. The receptor, like the ligand, has a widespread distribution.

HGF and c-met have been observed in the pancreatic islet of several species, and several studies have demonstrated that HGF is a mitogen and an insulinotropic agent for fetal and adult islet cells in vitro. To determine whether local overexpression of HGF in the islet could result in an increase in islet mass and function in vivo, we developed transgenic mice overexpressing HGF in the islet under the control of the RIP. Like the RIP-PTHrP mice, RIP-HGF mice display a dramatic increase in islet mass, reflecting an increase both in islet size and ß-cell number. One of the mechanisms responsible for this increase in islet size is a marked increase in ß-cell proliferation. Associated with this increase in islet mass, RIP-HGF mice display lower blood glucose concentrations than their normal littermates under both fasting and nonfasting conditions, inappropriate hyperinsulinemia, and superior glucose tolerance compared with their normal siblings. In preliminary studies, islets isolated from these transgenic mice showed superior glucose-stimulated insulin secretion compared with the islets isolated from normal mice. The molecular mechanisms responsible for these effects are still under study.

Taken together, all these results suggest that HGF, like PTHrP, may be a candidate for future strategies aimed at the treatment of diabetes mellitus. Indeed, Nakano et al. have demonstrated that HGF ip administration ameliorates hyperglycemia in diabetic mice after transplantation of marginal quantities of pancreatic islets. These types of studies support to the potential for HGF in enhancing quantity, function, and survival of transplanted human islets.

	Placental lactogen (PL)

Top Abstract Introduction PTH-related protein (PTHrP) Hepatocyte growth factor (HGF) Placental lactogen (PL) Glucagon-like peptide-1 (GLP-1)... Betacellulin (BTC) Insulin-like growth factors... Reg proteins and islet... Conclusions Suggested Reading

 
ß cells in the pancreatic islet respond to GH, PRL, and PL with an increase in proliferation. PL has been implicated as the primary factor responsible for the enhanced islet mass and function that occur during pregnancy. PL interacts with receptors in the PRL/GH receptor family, stimulating the Jak-2/Stat-5 intracellular signaling pathway. These observations suggested that PL may have potential to increase ß-cell mass using gene transfer strategies in vivo in humans with diabetes.

Prior studies performed in vitro and over the short-term suggested that PL is more powerful as an islet mitogen than GH or PRL. To evaluate whether PL could stimulate the growth and function of islets in vivo over the long term, we prepared transgenic mice expressing mouse PL-1 under the control of the RIP. These RIP-mPL-1 mice, like the RIP-PTHrP and RIP-HGF mice described above, display islet hyperplasia, hypoglycemia, and inappropriate hyperinsulinemia. The increase in islet mass seen in the RIP-mPL-1 mice seems to be due principally to enhanced ß-cell proliferation and, to a lesser degree, to ß-cell hypertrophy.

These observations support the concept that PL may be important in vivo in sustaining islet growth and function during pregnancy in mammals. In addition, they support the potential for the use of PL in gene therapy and other strategies targeted at improving islet survival and function after transplantation.

	Glucagon-like peptide-1 (GLP-1) and exendin-4

Top Abstract Introduction PTH-related protein (PTHrP) Hepatocyte growth factor (HGF) Placental lactogen (PL) Glucagon-like peptide-1 (GLP-1)... Betacellulin (BTC) Insulin-like growth factors... Reg proteins and islet... Conclusions Suggested Reading

 
GLP-1 is generated from the proglucagon precursor by prohormone convertase cleavage and is secreted from intestinal L-cells into the circulation in response to oral glucose absorption. In rodents and diabetic humans, GLP-1 potentiates glucose-dependent insulin secretion, acting through GLP-1 receptors on the ß cell, which in turn act via adenylyl cyclase. The half-life of GLP-1 is measured in minutes, with rapid degradation in plasma resulting from the action of dipeptidyl peptidase-IV. This brief circulating half-life has made it difficult to augment islet mass and function using GLP-1. In contrast, exendin-4, isolated from the salivary glands of Gila monster lizards, is a potent structural analog of GLP-1, is resistant to dipeptidyl peptidase-IV action, and, therefore, has a longer circulating half-life. In mammals, it binds to the GLP-1 receptor on islets with similar affinity to GLP-1, but increases cAMP levels 3-fold higher than GLP-1 at equimolar concentrations, making it a more convenient agent for use in chronic animal studies.

Recently, exendin-4 administration to rats has been shown to increase both ß-cell and non-ß-cell mass in vivo. This was not due to hypertrophy, but rather through a 2-fold increase in the cell number of insulin- and glucagon-positive cells. Thus, it would seem that exendin-4 should belong in the potential armamentarium of agents that may prove useful in expanding islet mass and function over the long term in vivo.

	Betacellulin (BTC)

Top Abstract Introduction PTH-related protein (PTHrP) Hepatocyte growth factor (HGF) Placental lactogen (PL) Glucagon-like peptide-1 (GLP-1)... Betacellulin (BTC) Insulin-like growth factors... Reg proteins and islet... Conclusions Suggested Reading

 
The epidermal growth factor family consists of at least 15 members, including epidermal growth factor, transforming growth factor , and BTC. These factors activate a family of four receptor tyrosine kinases encoded by the erbB gene family. BTC, an 80 amino acid peptide initially identified as a factor in the conditioned medium of a mouse pancreatic ß-cell carcinoma cell line, is mitogenic for Balb/C 3T3 fibroblasts. Observations on the specific mitogenic action of BTC on the rodent insulinoma cell line INS-1 support a specific role for BTC in islet physiology. Whether it can increase islet mass and function in vivo remains undefined.

	Insulin-like growth factors (IGFs) I and II

Top Abstract Introduction PTH-related protein (PTHrP) Hepatocyte growth factor (HGF) Placental lactogen (PL) Glucagon-like peptide-1 (GLP-1)... Betacellulin (BTC) Insulin-like growth factors... Reg proteins and islet... Conclusions Suggested Reading

 
IGFs participate in the growth and function of almost every organ of the body. The stability, biological availability to tissues, and actions of IGFs are modulated by at least six IGF-binding proteins widely expressed in human and rat fetal tissues. Experiments with isolated islets from rat or human fetuses, or using established ß-cell lines, have shown that both IGF-I and -II can promote DNA synthesis in ß cells and reduce the rate of apoptosis in islets from neonatal rats. It has been suggested that IGF-I may play a prominent role in increasing the population of ß cells in the developing and regenerating pancreas. In the developing pancreas, IGF-II seems to be associated with the regulation of islet growth and differentiation and is much more abundant than IGF-I. In adults, IGF-I has been shown to colocalize with insulin in the ß cells of human and rat pancreas.

Devedjian et al. have prepared transgenic mice that overexpress IGF-II in the ß cell. These mice display increases in islet mass, hyperinsulinemia, and increased glucose-stimulated insulin secretion, but also display insulin resistance and frank diabetes. The authors argue that IGF-II drives islet proliferation and enhances islet function, but that sustained hyperinsulinemia also results in severe peripheral insulin resistance, which is unable to be overcome by the otherwise hyperfunctional islets. This would be surprising, given that the other transgenic models described above (RIP-PTHrP, RIP-mPL-1, RIP-HGF, and others) demonstrate lifelong hyperinsulinemia and hypoglycemia and do not develop diabetes. The explanation for these discrepant findings may lie in the observation that systemic secretion of IGF-II from the RIP-IGF-II islets occurs and could lead to insulin resistance. Alternatively, it is possible that the RIP-IGF-II transgene is expressed in liver, muscle, or other tissues that might independently lead to insulin resistance. Or perhaps mouse strain differences among the various RIP-growth factor mice are important. These results suggest that IGF-II may have some potential as an islet growth factor, but the accompanying diabetes is worrisome and requires explanation.

	Reg proteins and islet neogenesis-associated protein (INGAP)

Top Abstract Introduction PTH-related protein (PTHrP) Hepatocyte growth factor (HGF) Placental lactogen (PL) Glucagon-like peptide-1 (GLP-1)... Betacellulin (BTC) Insulin-like growth factors... Reg proteins and islet... Conclusions Suggested Reading

 
In 1988, Terazono et al. isolated a novel gene from a regenerating islet-derived complementary DNA (cDNA) library and suggested that Reg (regeneration-associated gene) might be involved in ß-cell regeneration after 90% pancreatectomy in the rat. Reg and Reg-related genes subsequently have been shown to constitute a multigene family. Reg was found to be expressed in regenerating islets but not in normal pancreatic islets or insulinomas. More recently, an apparent Reg receptor cDNA has been isolated from a rat islet cDNA library. The absence of Reg-I protein in the Reg-I protein knockout mice does not seem to induce any phenotypic abnormality. However, administration of recombinant rat Reg protein stimulated ß-cell replication in vitro and also increased the ß-cell mass in 90% pancreatectomized rats, as well as in non-obese diabetic mice, resulting in the amelioration of diabetes in both cases. Perhaps surprisingly, transgenic mice overexpressing the Reg-I gene in islet ß cells using the RIP did not show any islet enlargement analogous to that seen in the 90% pancreatectomized rats that received Reg I administration. Taken together, these results suggest a potential therapeutic role of Reg in diabetes, although more studies need to be done to clarify its role in normal pancreatic development and to elucidate whether its overexpression in the islet can ameliorate the hyperglycemia induced by pancreatectomy.

INGAP, with homology with the type III Reg proteins, was described in 1997. The INGAP gene is expressed in acinar cells but not in mouse or hamster islets. It has been observed that INGAP is mitogenic for pancreatic duct-derived cells, but not for ß cell-derived cells. Because duct cell proliferation seems to be prerequisite for islet neogenesis, it has been postulated this protein could be involved in islet neogenesis process. Despite the passage of 3 yr since the description of this peptide, no recent support for a physiologic or therapeutic role in has emerged. Whether it will prove useful or efficacious in expanding islet mass remains to be defined.

	Conclusions

Top Abstract Introduction PTH-related protein (PTHrP) Hepatocyte growth factor (HGF) Placental lactogen (PL) Glucagon-like peptide-1 (GLP-1)... Betacellulin (BTC) Insulin-like growth factors... Reg proteins and islet... Conclusions Suggested Reading

 
The Edmonton study on islet transplantation would seem to presage an increase in the study and eventual larger scale implementation of islet transplantation among patients with diabetes. Although the study of the regulation of proliferation, survival, and origins of pancreatic islets was well underway before the Edmonton report, this study has served to underscore the importance and relevance of the study of islet cell growth, survival, and development and to energize direct clinical application of this new-found knowledge. Over the coming years, one can anticipate that several separate but related research areas will expand, and converge on the ultimate clinical goal of allowing large-scale, widespread islet transplantation to become possible. Aficionados of miniature mechanical implantable closed loop glucose-sensing, insulin delivery systems will continue on their crusade to cure diabetes in this fashion. Transplant immunologists will continue on their crusade to develop host-friendly, islet-friendly immunosuppressive regimens. Gene therapists will continue on their crusade to develop specific, efficient, safe, nonimmunogenic, integrative viral or other gene delivery vectors to deliver immunologic and growth regulatory molecules of interest to the islet. Pancreatic developmental biologists and genomics experts will continue to identify new ß-cell transcription factors and developmental molecules, and ß-cell engineers will continue to try to use these molecules in driving the differentiation of undifferentiated stromal, embryonic, and/or pancreatic ductal cells toward a functional, glucose-sensing, insulin secretory phenotype.

For orthopedists, plastic surgeons, and ophthalmologists and their patients, there are tissue banks: bone banks, skin banks, and cornea banks. We envision a future in which tissue-typed, renewable banks of human ß cells or islets are available in large supply, waiting to be implanted in patients with diabetes, available for retransplant in cases where the original transplant has failed. We envision being able to expand the ß-cell population at will in vitro before islet transplant and in vivo after ß-cell transplant, using appropriate growth-regulatory, survival-regulatory molecules, in conjunction with appropriate regulative gene promoters and appropriate immunological strategies. Whichever of the strategies for islet transplantation ultimately proves most effective, it seems hard to imagine that there will not be a need to continue to drive their proliferation, expansion, and survival. For investigators working in the area of islet growth factor identification, characterization, signaling, and deployment, and for patients who will be on the receiving end of such technologies, these are exciting times, indeed.

	Suggested Reading

Top Abstract Introduction PTH-related protein (PTHrP) Hepatocyte growth factor (HGF) Placental lactogen (PL) Glucagon-like peptide-1 (GLP-1)... Betacellulin (BTC) Insulin-like growth factors... Reg proteins and islet... Conclusions Suggested Reading

 
The Edmonton study

1. Shapiro AMJ, Lakey JRT, Ryan EA, et al. 2000 Islet transplantation in seven patients with type 1 diabetes mellitus using a glucocorticoid-free immunosuppressive regimen. N Engl J Med. 343:230–238.

PTHrP

1. Gaich G, Orloff JJ, Atillasoy EJ, Burtis WJ, Ganz MB, Stewart AF. 1993 Amino-terminal PTH-related protein: specific binding and cytosolic calcium responses in rat insulinoma cells. Endocrinology. 132:1402–1409.

2. Drucker DJ, Asa SL, Henderson J, Goltzman D. 1989 The PTH-like peptide gene is expressed in the normal and neoplastic human endocrine pancreas. Mol Endocrinol. 3:1589–1595.

3. Vasavada RC, Cavaliere C, D’Ercole AJ, et al. 1996 Overexpression of PTH-related protein in the pancreatic islets of transgenic mice causes islet hyperplasia, hyperinsulinemia, and hypoglycemia. J Biol Chem. 271:1200–1208.

4. Porter SE, Sorenson RL, Dann P, García-Ocaña A, Stewart AF, Vasavada RC. 1998 Progressive pancreatic islet hyperplasia in the islet-targeted, PTH-related protein-overexpressing mouse. Endocrinology 139:3743–3751.

5. Vasavada RC, Cebrian A, García-Ocaña A, et al. PTH-related protein (PTHrP): in vivo inhibition of streptozotocin-induced ß cell death in transgenic mice. Proceedings of the 82nd Annual Meeting of The Endocrine Society, Toronto, Canada, 2000; p.76.

HGF

1. Zarnegar R, Michalopoulos, GK. 1995 The many faces of hepatocyte growth factor: from hepatopoiesis to hematopoiesis. J. Cell Biol. 129:1177–1180.

2. Otonkoski T, Cirulli V, Beattie GM, et al. 1996 A role for hepatocyte growth factor/scatter factor in fetal mesenchyme-induced pancreatic ß-cell growth. Endocrinology. 137:3131–3139.

3. Otonkoski T, Beattie GM, Rubin JS, Lopez AD, Baird A, Hayek A. 1994 Hepatocyte growth factor/Scatter factor has insulinotropic activity in human fetal pancreatic cells. Diabetes. 43:947–953.

4. García-Ocaña A, Takane K, Syed MA, Philbrick WM, Vasavada RC, Stewart AF. 2000 Hepatocyte growth factor overexpression in the islet of transgenic mice increases ß cell proliferation and induces hypoglycemia. J Biol. Chem. 275:1226–1232.

5. García-Ocaña A, Vasavada RC, Takane K, Reddy VT, Batt A, Stewart AF. 2000 Trangenic islets overexpressing hepatocyte growth factor (HGF) demonstrate superior glucose and insulin responses in vitro and in vivo compared with transgenic PTH-related protein (PTHrP), placental lactogen (PL), and normal islets. Diabetes. 49(Suppl 1):A43.

6. Nakano M, Yasunami Y, Maki T, et al. 2000 Hepatocyte growth factor is essential for amelioration of hyperglycemia in streptozotocin-induced diabetic mice receiving a marginal mass of intrahepatic islet grafts. Transplantation. 69:214–221.

PL

1. Sorenson RL, Brelje TC. 1997 Adaptation of islets of Langerhans to pregnancy: ß-cell growth, enhanced insulin secretion and the role of lactogenic hormones. Horm Metab Res. 29:301–307.

2. Sorenson RL, Johnson MG, Parsons JA, Sheridan JD. 1987 Decreased glucose stimulation threshold, enhanced insulin secretion, and increased ß cell coupling in islets of PRL-treated rats. Pancreas. 2:283–288.

3. Vasavada RC, García-Ocaña A, Zawalich WS, et al. 2000 Targeted expression of placental lactogen in the ß cells of transgenic mice results in ß cell proliferation, islet mass augmentation, and hypoglycemia. J Biol Chem. 275:15399–15406.

4. Takane K, García-Ocaña A, Vasavada RC, Batt A, Stewart AF. 2000 Viral gene delivery of placental lactogen and hepatocyte growth factor (HGF) enhances proliferation of isolated rodent and human islets. Diabetes. 49(Suppl 1):A53.

GLP-1/exendin-4

1. Drucker DJ. 1998 Perspectives in diabetes, glucagon-like peptides. Diabetes. 47:159–169.

2. Habener JF. 1993 The incretin notion and its relevance to diabetes. Endocrinol Metab Clin North Am. 22:775–794.

3. Young AA, Gedulin BR, Bhavsar S, et al. 1999 Glucose-lowering and insulin-sensitizing actions of exendin-4; studies in obese diabetic (ob/ob, db/db) mice, diabetic Fatty Zucker rats, and diabetic Rhesus monkeys (Macaca mulatta). Diabetes. 48:1026–1034.

4. Thorens B, Porret A, Buhler L, Deng S-P, Morel P, Widmann C. 1993 Cloning and functional expression of the human islet GLP-1 receptor; demonstration that exendin-4 is an agonist and exendin-(9–39) an antagonist of the receptor. Diabetes. 42:1678–1682.

5. Grieg NH, Holloway HW, De Ore KA, et al. 1999 Once daily injection of exendin-4 to diabetic mice achieves long-term beneficial effects on blood glucose concentrations. Diabetologia. 42:45–50.

6. Xu G, Stoffers DA, Habener JF, Bonner-Weir S. 1999 Exendin-4 stimulates both ß-cell replication and neogenesis, resulting in increased ß-cell mass and improved glucose tolerance in diabetic rats. Diabetes. 48:2270–2276.

7. Stoffers DA, Kieffer TJ, Hussain MA, et al. 2000 Insulinotropic glucagon-like peptide 1 agonists stimulate expression of homeodomain protein IDX-1 and increase islet size in mouse pancreas. Diabetes. 49:741–748.

BTC

1. Watanabe T, Shintani A, Nakata M, et al. 1994 Recombinant human betacellulin. Molecular structure, biological activities and receptor interaction. J Biol Chem. 269: 9966–9973.

2. Shing Y, Christofori G, Hanahan D, et al. 1993 Betacellulin: A mitogen from pancreatic ß cell tumors. Science. 259:1604–1607.

3. Riese DJ, Bermingham Y, van Raaij TM, Buckley S, Plowman GD, Stern DF. 1996 Betacellulin activates the epidermal growth factor receptor and erbB-4, and induces cellular response patterns distinct from those stimulated by epidermal growth factor or neuregulin-ß. Oncogene. 12:345–353.

4. Huotari M, Palgi J, Otonkoski T. 1998 Growth factor-mediated proliferation and differentiation of insulin-producing INS-1 and RINm5F cells: identification of betacellulin as a novel ß-cell mitogen. Endocrinology. 139:1494–1499.

5. Mashima H, Ohnishi H, Wakabayashi K, et al. 1996 Betacellulin and activin A coordinately convert amylase- secreting pancreatic AR42J cells into insulin-secreting cells. J Clin Invest. 97:1647–1654.

IGFs

1. Hogg J, Hill DJ, Han VKM. 1994 The ontogeny of insulin-like growth factor (IGF) and IGF binding protein gene expression in the rat pancreas. J Mol Endocrinol. 13:49–58.

2. Petrik J, Arany E, Mc Donald TJ, Hill DJ. 1998 Apoptosis in the pancreatic islet cells of the neonatal rat is associated with a reduced expression of insulin-like growth factor II that may act as a survival factor. Endocrinology. 139:2994–3004.

3. Smith FE, Rosen KM, Villa-Komaroff L, Weir GC, Bonner-Weir S. 1991 Enhanced insulin-like growth factor I gene expression in regenerating rat pancreas. Proc Natl Acad Sci USA. 88:6152–6156.

4. Rhodes CJ. 2000 IGF-I and GH postreceptor signaling mechanisms for pancreatic ß-cell replication. J Mol Endocrinol. 24:303–311.

5. Devedjian JC, George M, Casellas A, et al. 2000 Transgenic mice overexpressing insulin-like growth factor-II in ß cells develop type 2 diabetes. J Clin Invest. 105:731–740.

Reg proteins and INGAP

1. Narushima Y, Unno M, Nakagawara K, et al. 1997 Structure, chromosomal localization and expression of mouse genes encoding type III Reg, Reg III, Reg IIIß, Reg III. Gene. 185:159–168.

2. Kobayashi S, Akiyama T, Nata K, et al. 2000 Identification of a receptor for Reg (Regenerating Gene) protein, a pancreatic ß-cell regenerating factor. J Biol Chem. 275:10723–10726.

3. Rafaeloff R, Pittenger GL, Barlow SW, et al. 1997 Cloning and sequencing of the pancreatic islet neogenesis associated protein (INGAP) gene and its expression in islet neogenesis in hamsters. J Clin Invest. 99:2100–2109.

Received August 15, 2000.

Revised October 3, 2000.

Accepted October 23, 2000.

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