Induction of islet B-cell regeneration in partially pancreatectomized rats by poly(ADP-ribose) synthetase inhibitors

Summary In order to clarify the mechanism of the prevention of diabetes mellitus developing after subtotal pancreatectomy, we examined regenerative activities of islet B-cells in 90% pancreatectomized and poly (ADP-ribose) synthetase inhibitor-treated rats by using autoradiographic and stathmokinetic techniques. Thirty days after 90% pancreatectomy, islets of rats without 3-aminobenzamide treatment were decreased in number, small in size and had irregular contour. Degranulation of the B-cells and fibrotic degeneration were frequently encountered. On the contrary, islets of remaining pancreas in rats receiving 3-aminobenzamide were increased in number, and their diameters ranged from 0.3–0.6 mm, being about two fold larger than those of normal rats. The labeling index of³H-thymidine autoradiography and the mitotic indices in islet B-cells were increased in a temporally correlated manner in the 3-aminobenzamide treated rats. The mitotic indices of the 3-aminobenzamide-treated group on the 5th, 7th, 10th and 15th days were significantly larger than those in the control group. These results indicate that poly (ADP-ribose) synthetase inhibitors can induce self-replication or regeneration of B-cells in partially pancreatectomized rats.     

Effects of poly (ADP-ribose) synthetase inhibitor on B-cells of a canine pancreas after massive pancreatectomy.

Nicotinamide, a poly (ADP-ribose) synthetase inhibitor, was administered intravenously to massive pancreatectomized dogs (NA group), whereas saline was administered intravenously to massive pancreatectomized dogs (saline group). Stathmokinetic studies were done to clarify the regenerating activities of B-cells. Nicotinamide significantly increased mitotic indices of B-cells after pancreatectomy (p less than 0.01). On the second day after pancreatectomy, mitotic index of B-cells in the NA group (2.00 +/- 0.20%) was higher than that in the saline group (0.25 +/- 0.10%). Though insulin content of pancreatic tissue decreased 2 d after pancreatectomy in the both groups, recovery of insulin content was significantly more immediate and more sufficient in the NA group. Intravenous glucose tolerance tests showed a significantly greater increase of insulin release on the 31st and 62nd day after pancreatectomy, and better glucose tolerance on the 62nd day in the NA group compared with the saline group. These results are considered to be attributed to the regenerating and protecting effects of nicotinamide on B-cells after pancreatectomy, and suggest a clinical value of nicotinamide also in man, especially for Sandmeyer's diabetes.     

Insulitis and islet-cell antibody formation in rats with experimentally reduced beta-cell mass

Summary We studied the effect of severe reduction of beta-cell mass by 90% pancreatectomy on the immune tolerance to the endocrine pancreas. Four months after subtotal pancreatectomy all LEW.Han rats had developed mononuclear infiltration of islets and 9 of 14 rats were positive for islet-cell antibodies. Electron microscopy revealed lymphocytic invasion of endocrine tissue, lysis of beta cells and phagocytotic macrophages. None of these changes were seen 2 weeks after 90% pancreatectomy or 4 months after 10% pancreatectomy. Weekly substitution of islet antigens in the form of a homogenate of 100 islets into 90% pancreatectomized LEW.Han rats almost completely prevented the development of insulitis and autoantibodies. The dependence of insulitis on T cells was shown when 90% pancreatectomy in LEW.rnu rats (i.e., the congenic athymic nude strain), did not result in islet infiltration. The exocrine tissue remained normal in all experimental groups. During the observation period insulitis was not associated with overt diabetes but was accompanied by substantial enlargement of islets and of beta-cell mass, as shown by morphometry. Suppression of islet inflammation by injection of islet antigens abolished beta-cell regeneration, despite continuing metabolic stress in rats with 90% pancreatectomy. The findings indicate induction of islet autoimmunity in response to 90% but not to 10% pancreatectomy. We conclude that severe reduction of the islet-antigen mass allows the development of T-cell-dependent islet autoimmunity which indicates a loss of immune tolerance. In addition, the data suggest the existence of islet-antigen autoreactive immune cells in rats not genetically predisposed to autoimmune diabetes. Finally, we conclude that selective beta-cell regeneration occurs in association with insulitis.Abbreviations PX Pancreatectomy - NOD non-obese diabetic - Ag antigen - ICA islet-cell antibodies - IDDM insulin-dependent diabetes mellitus     

Promotion of beta-cell regeneration by betacellulin in ninety percent-pancreatectomized rats.

Betacellulin is thought to promote growth and differentiation of pancreatic beta-cells. We investigated the effect of betacellulin on regeneration of pancreatic beta-cells in 90%-pancreatectomized rats. Ninety percent pancreatectomy was performed in male Wistar rats and betacellulin (0.5 microg/g body weight) or saline was administered daily for 10 d starting immediately after pancreatectomy. In pancreatectomized rats, the morning-fed plasma glucose was significantly lower and the plasma insulin concentration was significantly higher in betacellulin-treated rats than those in control rats for up to 4 wk. Thirty days after pancreatectomy, a glucose tolerance test was performed. Betacellulin reduced the plasma glucose response to ip glucose loading. In control rats, the plasma insulin concentration was significantly lower and did not respond to glucose. In contrast, the plasma insulin concentration increased slightly but significantly in betacellulin-treated rats. Thirty days after pancreatectomy, the beta-cell mass was greater and the insulin content was significantly higher in betacellulin-treated rats than those in control rats. The numbers of islet cell-like cluster and bromodeoxy uridine/insulin double- positive cells in both islet cell-like cluster and islets were significantly higher in betacellulin-treated rats. These results indicate that administration of betacellulin improves glucose metabolism by promoting beta-cell regeneration in 90%-pancreatectomized rats.     

Protective effect of 3-aminobenzamide,an inhibitor of poly (ADP-ribose) synthetase,against streptozotocin-induced diabetes

Summary The addition of 3-aminobenzamide (a potent inhibitor of poly(ADP-ribose)synthetase) into the incubation medium, prevents streptozotocin-induced inhibition of glucose-stimulated insulin release from isolated islets [control 142±14U·islet^–1·h^–1; streptozotocin (0.5mg/ml) 31±8; 3-aminobenzamide (l.0 mg/ml) 96±11; streptozotocin plus 3-aminobenzamide 122±19]. In vivo, intraperitoneal 3-aminobenzamide 300 mg/kg body weight prevents the appearance of overt diabetes in streptozotocin-treated rats. These protective effects of 3-aminobenzamide are dose-dependent and are similar to those exerted by nicotinamide. Taking into account that poly ADP-ribosylation is involved in the repair of damaged DNA, the protection exerted by 3-aminobenzamide against the diabetogenic effect of streptozotocin strongly supports the view that this acute effect may be a major consequence of the activation of DNA repair mechanisms in islet cells.     

Long-term effects of exposure of pancreatic islets to nicotinamide in vitro on DNA synthesis,metabolism and B-cell function

Summary Using³H-thymidine labeling techniques, we found that rates of DNA synthesis in islet cells doubled when mouse pancreatic islets were cultured for 1 week with 10 mmol/1 nicotinamide, a potent poly(ADP-ribose) synthetase inhibitor. Culture with nicotinamide partially inhibited glucose-stimulated insulin release, whereas the islet insulin content and rate of (pro)insulin biosynthesis remained unchanged. Long-term exposure to nicotinamide decreased glucose oxidation and ATP content in the islets. The findings support the view that poly(ADP-ribose)synthetase inhibitors stimulate islet cell replication, but may be accompanied by significant inhibitory effects on islet cell function.     

Expression of reg protein in rat regenerating islets and its co-localization with insulin in the Beta cell secretory granules

Summary Regenerating islets can be induced by the administration of poly(ADP-ribose) synthetase inhibitors to 90% depancreatized rats. In screening a regenerating isletderived cDNA library, we previously isolated a novel gene, reg (regenerating gene), which encodes a 165-amino acid protein with a 21-amino acid signal sequence. In the present study, we have examined the expression and localization of reg protein in the regenerating islets by immunocytochemical techniques using a monoclonal antibody against a recombinant rat reg protein of 144 amino acids without the signal sequence. Light microscopy examination showed strong immunoreactivity for reg protein in the regenerating islets of the rats at two weeks and two months after the 90% pancreatectomy, whereas reg protein was almost undetectable in normal rat islets or in the islets of the rats one year after the pancreatectomy. Almost all the reg protein-positive cells were stained for insulin. By applying the immunogold technique at the ultrastructural level, it was demonstrated that both reg protein and insulin occur in the central granular core of the regenerating Beta cell secretory granules. These results suggest that reg protein is synthesized in and secreted from the regenerating Beta cells and that its expression is closely associated with Beta-cell regeneration.     

Induction of murine teratocarcinoma cell differentiation by suppression of poly(ADP-ribose) synthesis.

Poly(ADP-ribose) synthesizing activity in mouse teratocarcinoma EC-A1 cells decreased markedly during differentiation induced by retinoic acid; the activities assayed in permeabilized cells decreased to 25% and 10% of the activity of control (uninduced cells) 2 and 3 days, respectively, after the addition of 0.1 microM retinoic acid to the culture medium. This change preceded changes in morphology and DNA synthesis, which became prominent after 4 days. The decrease in poly(ADP-ribose) synthesizing activity appeared to be caused by a diminution of the synthetase protein and not by a decrease in its catalytic activity, because the full activity disclosed by DNase I treatment decreased in parallel, albeit at about 20 times higher levels. When 8 mM 3-aminobenzamide or 10 mM nicotinamide, specific inhibitors of poly(ADP-ribose) synthetase, was added to the culture medium, the cells underwent differentiation after 7-9 days. An analogue, 3-aminobenzoic acid, which is not inhibitory to the synthetase, induced differentiation much less efficiently than did 3-aminobenzamide, and the effect of 3-aminobenzoic acid appeared to be ascribable to its potent cytotoxicity. Immunohistochemical analysis using anti-poly(ADP-ribose) antibody confirmed the marked reduction in poly(ADP-ribose) synthesizing activity in nuclei of the cells treated with retinoic acid or 3-aminobenzamide but not with 3-aminobenzoic acid. These results suggest that a decrease in poly(ADP-ribose) synthesis triggers differentiation of teratocarcinoma cells.     

Effects of streptozotocin exposure in vitro on the replication and repair of DNA in fetal rat pancreatic islet cells

It was recently proposed that the role for poly(ADP-ribose) synthetase during DNA repair was exerted via a depletion of cellular NAD in order to slow down energy-requiring processes in the cell, notably DNA replication. This would enable the cell to more efficiently repair damaged DNA. In the present study fetal rat pancreatic islet cells were exposed to 2.2 mM streptozotocin (SZ). Using [3H]thymidine labeling and autoradiographic techniques, it was found that cellular nuclear silver grain counts, an index of DNA repair synthesis, were doubled after SZ exposure. However, autoradiographically measured DNA replication remained unaffected. Cellular NAD + NADH contents were reduced by 60% in the SZ-treated islets. Estimates of the poly(ADP-ribose) synthetase activity showed that this was doubled in islets exposed to SZ. Immediately after the SZ treatment the islets exhibited a 35% reduction in insulin secretion in response to 16.7 mM glucose. Taken together, the present findings do not favor the suggested role for poly(ADP-ribose) synthetase during DNA repair. Rather we observed an increased activity of this enzyme, a lowered cellular NAD + NADH content, and an increased rate of DNA repair synthesis concomitant with an unchanged DNA replicative rate in the SZ-exposed islets.     

Mechanisms of streptozotocin- and alloxan-induced damage in rat B cells

Summary In studies to evaluate possible inhibitors of the B-cell toxin, streptozotocin, the superoxide scavenger, superoxide dismutase, did not prevent or reduce the toxic effects of streptozotocin as determined by loss of insulin secretion from rat pancreatic B cells in monolayer culture. However, 1,1-dimethyl urea, a scavenger of the hydroxyl radical, did afford significant protection. Both scavengers diminished the cytotoxic effects of alloxan. The inhibitors of poly (ADP-ribose) synthetase, 3-aminobenzamide and nicotinamide, also were effective in attenuating alloxan- and streptozotocin-induced B-cell toxicity. Tests of the hydroxyl-scavenging ability of the three streptozotocin antagonists revealed that 3-aminobenzamide, nicotinamide and 1,1-dimethyl urea were effective scavengers of this free radical. Conversely, 1,1-dimethyl urea, although not as potent as 3-aminobenzamide or nicotinamide, was found to inhibit poly (ADP-ribose) synthetase. These data indicate that these chemicals most likely attenuate alloxan-induced toxicity by scavenging the hydroxyl radical and diminish streptozotocin-induced toxicity by inactivation of the poly (ADP-ribose) system.     

Protection of nonobese diabetic mice from autoimmune diabetes by reduction of islet mass before insulitis.

Nonobese diabetic mice spontaneously develop diabetes that is caused by autoimmune cell-mediated destruction of pancreatic beta cells. Here we report that surgical removal of 90% of pancreatic tissue before onset of insulitis induced a long-term diabetes-free condition in nonobese diabetic mice. Pancreatectomy after development of moderate insulitis had no effect on the course of diabetes. The effect of pancreatectomy was abrogated with subsequent development of diabetes by infusion of islet-cell-specific T lymphocytes and by transplantation of pancreatic islets. Lymphocytes from pancreatectomized diabetes-free mice exhibited low response to islet cells but responded normally to alloantigens. These results suggest that the islet cell mass plays a critical role in development of autoimmune diabetes.     

Mitotic activity and DNA synthesis of rat islet cells following partial pancreatectomy.

The regenerative potential of the adult rat pancreas following a 50% pancreatectomy was investigated. Blood glucose values and body weight were unchanged by the procedure. The mitotic index (MI) of alpha, beta, and exocrine cells was examined in formalin-fixed sections of the pancreas stained for hormones and 5-bromo-2'-deoxyuridine (BrDU) after treating the animals with 2.5 mg/kg colchicine and/or 50 mg/kg BrDU prior to sacrifice. The MI was unchanged within the control group. For pancreatectomized versus control animals, in beta cells after 5 days the MI was 0.84% versus 0.41% (p less than 0.05), while 7 days postoperatively it was 1.04% versus 0.57% (p less than 0.01); in alpha cells, the MI was 0.32% versus 0.17% (p less than 0.05) at 5 days and 0.47% versus 0.24% (p less than 0.05) at 7 days. The MI of exocrine cells rose transiently on day 3 postoperatively to 0.74% in the pancreatectomized group but remained low at 0.15% (p less than 0.01) in the control group. The mean ratio of BrDU + ve beta cells was 1.19% in the pancreatectomy group and 0.45% in the control group on day 5 postoperatively (p = 0.01). A highly significant (r = 0.97; p less than 0.001) correlation existed between BrDU + ve and mitotic cells. There was no difference between the two groups in the area occupied by insulin-positive cells in pancreatic sections, nor in the number of beta cells per islet.(ABSTRACT TRUNCATED AT 250 WORDS)     

Accumulation of 10-kilobase DNA replication intermediates in cells treated with 3-aminobenzamide.

During eukaryotic DNA synthesis there is formation of, in addition to Okazaki fragments, discrete 10-kilobase (kb) DNA replication intermediates. We have investigated the ligation of 10-kb DNA replication intermediates to high molecular weight DNA, using the drug 3-aminobenzamide, an inhibitor of poly(ADP-ribose) synthetase. In human melanoma cells treated with this inhibitor, there is an accumulation of 10-kb DNA. In contrast, in cells treated with aphidicolin, which inhibits DNA polymerase alpha, there is continued ligation of 10-kb DNA to high molecular weight DNA. Furthermore, using sequential treatment with aphidicolin and 3-aminobenzamide, one can observe the conversion of radiolabeled Okazaki fragments into 10-kb intermediates. The 10-kb DNA pieces are, however, not ligated to high molecular weight DNA in the presence of 3-aminobenzamide. Our results imply that functioning poly(ADP-ribose) synthetase is necessary for the ligation process.     

Increased islet DNA synthesis and glucose-derived lipid and amino acid production in association with beta-cell hyperproliferation in normoglycaemic 60 % pancreatectomy rats

Aims/hypothesis: Glycaemia does not change following a 60 % pancreatectomy in rats because of enhanced beta-cell function and proliferation (so-called beta-cell adaptation). We previously studied these rats 4 weeks after surgery and showed hypersensitization of glucose-induced insulin secretion because of increased glucokinase activity. In this study of 60 % pancreatectomy rats 5 days after surgery, when beta-cell proliferation increased threefold, we investigated whether increases in glucose metabolism enhance the production of glucose-derived lipid, amino acids and DNA. Methods: Isolated islets from 60 % pancreatectomy and sham-operated control rats 5 days or 4 weeks after surgery were studied. Results: Five days after 60 % pancreatectomy surgery, islet glucose phosphorylation increased threefold, but overall glucose usage increased only twofold. The glucose-6-phosphate (G6P) concentration thus doubled, resulting in a sixfold increase in G6P metabolism through the pentose phosphate shunt (PPS). The pentose phosphate shunt generates ribose-5-phosphate for nucleotide synthesis, and DNA synthesis doubled in the partial pancreatectomy islets. In contrast, partial pancreatectomy rats 4 weeks after surgery had a smaller increase in glucokinase activity and their islet glucose-6-phosphate concentration and pentose phosphate shunt activity were equal to that of the control rats. DNA synthesis and beta-cell proliferation, based on BrdU incorporation were close to normal. Another consequence of the heightened glucose metabolism in the 5-day partial pancreatectomy islets was twofold increase in production of glucose-derived lipid and the amino acids, alanine and glutamate. Conclusions/interpretation: The enhanced glucokinase activity in 60 % pancreatectomy rats supports the compensatory beta-cell hyperproliferation by increasing production of glucose-derived DNA, lipids and amino acids. [Diabetologia (2001) 44: 1026–1033] Received: 8 February 2001 and in revised form: 12 April 2001     

Enhanced insulin-like growth factor I gene expression in regenerating rat pancreas.

Insulin-like growth factor I (IGF-I) mRNA expression was studied after 90% partial pancreatectomy in the rat to determine whether IGF-I was associated with pancreatic regeneration. The level of IGF-I mRNA was maximally increased (4-fold above control value) 3 days after pancreatectomy, but thereafter gradually decreased, returning to control levels by 14 days after surgery. By in situ hybridization, IGF-I mRNA in both pancreatectomized and sham-operated rats was localized to capillary endothelial cells, indicating that this is the site of IGF-I expression in the normal rat pancreas. However, enhanced IGF-I mRNA expression was localized to focal areas of regeneration unique to pancreatectomized rats. In these areas, epithelial cells of proliferating ductules and individual connective tissue cells expressed IGF-I, suggesting that IGF-I may play an important role in the growth or differentiation of pancreatic tissue.     

Role of prolactin versus growth hormone on islet B-cell proliferation in vitro: implications for pregnancy

This study investigated the effects of homologous rat PRL (rPRL) and rat GH (rGH) on islet growth as indicated by modifications in insulin secretion, islet cell proliferation, and islet volume with neonatal and adult rat islets in vitro. The number of proliferating cells was determined by immunocytochemical staining for 5-bromo-2'-deoxyuridine (BrdU) incorporated into replicating DNA during the final 24 h of culture. When neonatal rat islets were examined by laser scanning confocal microscopy, more than 90% of the BrdU-labeled nuclei were observed in B-cells with insulin immunoreactivity. In neonatal rat islets, rPRL was much more effective than rGH in increasing insulin secretion (3.7-fold vs. 1.4-fold) during the 10 days of culture. The number of BrdU-labeled nuclei per islet was increased from 2.9 +/- 0.4 (n = 77) in control islets to 47.3 +/- 2.7 (n = 95) with rPRL (16.3-fold) and 9.7 +/- 0.8 (n = 84) with rGH (3.3-fold). The effects of rPRL and rGH on both insulin secretion and BrdU labeling were approximately additive. After 10 days, an increased average islet volume was only observed with rPRL. When followed for 36 days, the total amount of islet tissue was unchanged for control islets (1.1-fold), more than doubled with rPRL (2.5-fold), and only slightly increased with rGH (1.4-fold). From the observed rates of islet cell proliferation and increases in islet volumes, doubling times of 23-24 days for rPRL and 89-91 days for rGH can be estimated. Of other proposed islet growth factors, cholecystokinin, epidermal growth factor, platelet-derived growth factor, and 2-aminoisobutyric acid, an increase in insulin secretion and islet cell proliferation was only observed with cholecystokinin in neonatal rat islets. However, both effects were less than 20% of those observed with rPRL. In adult rat islets, rPRL was also more effective than rGH in increasing insulin secretion (1.6-fold vs. 1.2-fold) during the 9 days of culture. The number of BrdU-labeled nuclei per islet was increased from 2.7 +/- 0.5 (n = 96) in control islets to 9.5 +/- 0.6 (n = 175) with rPRL (3.5-fold). In contrast to the neonatal islets, rGH had no effect on the number of BrdU-labeled nuclei per islet in adult rat islets (2.4 +/- 0.3, n = 194).(ABSTRACT TRUNCATED AT 400 WORDS)     

The Reg gene family and Reg proteins: with special attention to the regeneration of pancreatic β-cells

Pancreatic β-cells of the islets of Langerhans are the only cells that produce insulin in humans, as well as in most animals, but they have been thought to have a limited capacity for regeneration, which is a predisposing factor for the development of diabetes mellitus. Strategies for influencing the replication and growth of the β-cell mass are therefore important for the prevention and/or treatment of diabetes. We have established a model for islet regeneration in 90% depancreatized rats by the administration of poly(ADP-ribose) synthetase inhibitors such as nicotinamide. The regenerating islets in the remaining pancreas of poly(ADP-ribose) synthetase inhibitor-treated rats were markedly enlarged and consisted largely of insulin-producing β-cells, preventing the development of diabetes mellitus that would otherwise be caused by the 90% pancreatectomy. In screening the regenerating islet-derived cDNA library, we found a novel gene and named it Reg (i.e.,regenerating gene). The rate Reg cDNA had a single open reading frame that encoded a 165-amino acid protein with a 21-amino acid signal peptide. We also isolated the human REG cDNA which encoded a 166-amino acid protein with a 22-amino acid signal peptide. The amino acid sequence of human REG protein has 68% homology to that of rat Reg protein. Recombinant rat Reg protein without the signal peptide, produced in yeast, stimulated β-cell replication and increased the β-cell mass in the residual pancreas of 90% depancreatized rats, ameliorating the surgical diabetes. Recombinant human REG protein also induced an expansion of the β-cell mass in non-obese diabetic (NOD) mice, resulting in the amelioration of diabetes. These results, as well as several other lines of evidence, indicate that Reg protein is a growth factor for pancreatic β-cells and also suggest that the administration of Reg protein and/or activation of the Reg gene can be used as a potential therapeutic approach for diabetes mellitus. We have further isolated several Reg and Reg-related genes from human, rat and mouse, and revealed that they constitute a multigene family, the Reg gene family. Based on the primary structures of proteins encoded by the Reg gene family, we have grouped the members of the family into three subclasses, type I, II, and III. Type I Reg (Reg I) encodes a β-cell growth factor, Reg I protein, as mentioned above. Some of the type III Reg (Reg III) have recently been suggested to play roles in the regeneration of cells other than pancreatic β-cells, such as neuronal cells and epithelial cells in the alimentary tract.     

Human beta cells are exceedingly resistant to streptozotocin in vivo

Streptozotocin (STZ) causes beta cell death in rodents via the mechanism of DNA damage precipitating poly(ADP-ribose) synthetase activation followed by lethal nicotinamide adenine dinucleotide depletion. It is unclear whether humans are susceptible to this mechanism. Islets were isolated from STZ-sensitive (CD1 mice and Lewis rats) and resistant [fish (tilapia)] species and from man and then were transplanted into diabetic nude mice under the kidney capsule. Normoglycemic recipients with normal glucose tolerance tests on d 30 were injected with increasing iv doses of STZ and their plasma glucose levels followed for 5 d; glucose tolerance tests were repeated on nondiabetic mice. Mice were then killed; grafts and native pancreata were examined. Based upon three criteria (i.e. nonfasting plasma glucose levels, glucose tolerance tests, and islet histology), the following observations were made: 1) Recipients of rat islets were resistant to 25 mg/kg but were uniformly diabetic at doses of 50 or 75 mg/kg. 2) Recipients of mouse islets were resistant to 75 mg/kg but were uniformly diabetic at 150 or 200 mg/kg. 3) Recipients of the fish islets were resistant to 300, 400, and 450 mg/kg. 4) Recipients of human islets were resistant to 100, 200, 300, 400, and 450 mg/kg. The results in recipient mice bearing long-term rat, mouse, or fish islet grafts were the same as previously published dose-response data for each donor species. We extrapolate from our results based on human islet grafts in mice that human beta cells are exceedingly resistant to STZ.     

Hypersecretion of islet amyloid polypeptide from pancreatic islets of ventromedial hypothalamic-lesioned rats and obese Zucker rats

To investigate the possible role of islet amyloid polypeptide (IAPP) in the development of type 2 diabetes mellitus, we examined the IAPP content and secretion in pancreatic islets isolated from ventromedial hypothalamic (VMH)-lesioned rats and genetically obese Zucker rats, using a specific RIA for IAPP. Obesity and hyperinsulinemia were observed in rats 21 days after VMH lesioning. IAPP content was increased in the islets of VMH-lesioned rats compared with findings in the sham-operated controls (100.9 +/- 6.6 vs. 72.8 +/- 3.85 fmol/islet; P less than 0.01). Isolated islets of VMH-lesioned rats secreted larger amounts of IAPP in the presence of 2.8 mM and 16.7 mM glucose (2.99 +/- 0.98 and 11.2 +/- 1.29 fmol.islet(-1).3 h-1) than was noted in sham-operated rats (ND and 6.65 +/- 0.78 fmol.islet(-1).3 h-1). In the obese Zucker rats, aged 14 weeks, IAPP concentrations in the islets were elevated compared with lean rats (133.3 +/- 10.6 vs. 84.4 +/- 8.5 fmol/islet; P less than 0.01). The isolated islets secreted larger amounts of IAPP in response to 2.8 mM and 16.7 mM glucose (2.83 +/- 0.88 and 15.81 +/- 1.35 fmol.islet(-1).3 h-1) than did those from lean control rats (0.36 +/- 0.19 and 12.49 +/- 1.20 fmol.islet(-1).3 h-1). These results strongly suggest that overproduction and hypersecretion of IAPP occur in animals with obesity and hyperinsulinemia.