Identification of a receptor for reg (regenerating gene) protein, a pancreatic beta-cell regeneration factor

Reg (regenerating gene) was isolated as a gene specifically expressed in regenerating islets (Terazono, K., Yamamoto, H., Takasawa, S., Shiga, K., Yonemura, Y., Tochino, Y., and Okamoto, H. (1988) J. Biol. Chem. 263, 2111-2114). Rat and human Reg gene products, Reg/REG proteins, have been demonstrated to stimulate islet beta-cell growth in vitro and in vivo and to ameliorate experimental diabetes. In the present study, we isolated a cDNA for the Reg protein receptor from a rat islet cDNA library. The cDNA encoded a cell surface 919-amino acid protein, and the cells into which the cDNA had been introduced bound Reg protein with high affinity. When the cDNA was introduced into RINm5F cells, a pancreatic beta-cell line that shows Reg-dependent growth, the transformants exhibited significant increases in the incorporation of 5'-bromo-2'-deoxyuridine as well as in the cell numbers in response to Reg protein. A homology search revealed that the cDNA is a homologue to a human multiple exostoses-like gene, the function of which has hitherto been unknown. These results strongly suggest that the receptor is encoded by the exostoses-like gene and mediates a growth signal of Reg protein for beta-cell regeneration.     

Expression of reg/PSP, a pancreatic exocrine gene: relationship to changes in islet beta-cell mass

A cDNA termed reg was recently isolated by differential screening of a library prepared from regenerating islets isolated from pancreatic remnants of rats subjected to 90% pancreatectomy and nicotinamide treatment. This led to speculation that this gene may be involved in expansion of beta-cell mass. In the current study we have measured reg expression after implantation and resection of a solid insulinoma tumor into rats, maneuvers known, respectively, to reduce and reexpand the volume of beta-cells in the islet. Animals with an implanted insulinoma tumor became profoundly hypoglycemic. Islet beta-cells declined from the normal 75% of total islet volume to less than 30%, in concert with a marked reduction in the reg mRNA level. Removal of the tumor resulted in a sharp increase in beta-cell replication, as measured by [3H]thymidine incorporation and a return to normal beta-cell volume within 4 days of tumor resection. This was associated with a transient induction in reg expression compared to that in tumor-bearing animals, effectively returning the amount of reg mRNA to the levels found in normal animals within 48 h; at later time points after tumor removal (3-7 days) reg expression declined, but then rose toward normal. In situ hybridization analysis localized the initial induction in reg mRNA expression to the exocrine pancreas. Continuous infusion of insulin into normal rats for 4 days, a maneuver that does not significantly reduce beta-cell mass, resulted in dramatically reduced insulin mRNA in islets, but no change in the levels of reg mRNA. We conclude that the diminution in pancreatic beta-cell mass caused by subcutaneous implantation of an insulinoma is associated with reduced reg gene expression and that the increase in beta-cell replication after resection of the tumor is preceded by return of reg gene expression toward normal.     

A Brief History of Pancreatic Reg: Implications as to its Clinical Importance

Although the pancreatic regenerating (reg) gene, was first isolated from a rat regenerating islets in 1988, its protein product was originally described in the 1970s. Reg proteins arise from a multigene family with three subtypes, and have a protein structure similar to calcium dependent lectins. Reg I and II have been implicated in control of pancreatic development and may play a role in maintenance of the beta-cell mass in the mature pancreas. Administration of reg I protein has been used in experimental animals as a therapy for surgically-induced diabetes mellitus. Reg I protein is also an inhibitor of calcium carbonate crystalization, important in maintaining the fluidity of pancreatic juice. The reg III gene, whose protein product is pancreatitis associated protein, is induced during pancreatic inflammation. Serum levels of reg III protein are a sensitive marker of severity of pancreatitis. It is an endogenous pancreatic factor that prevents the bacteria infection and scavenges oxygen-derived free radicals. Reg mRNA has been detected in non-pancreatic tissue such as the enterochromaffin-like cells of the stomach, neoplastic tissues of the colon, the small intestine, nervous system, liver tumors, and pituitary. Reg proteins are mitogens to intestinal epithelial cells, pancreatic ductal, beta cells, and Schwann cells, and are likely important to the overall integrity of the pancreas and gastrointestinal tract.     

A novel gene activated in regenerating islets

Administration of poly(ADP-ribose) synthetase inhibitors such as nicotinamide to 90% depancreatized rats induces regeneration of pancreatic islets, thereby ameliorating the surgical diabetes (Yonemura, Y., Takashima, T., Miwa, K., Miyazaki, I., Yamamoto, H., and Okamoto, H. (1984) Diabetes 33, 401-404). In screening the regenerating islet-derived cDNA library, we came across a novel gene encoding a 165-amino acid protein. The gene was expressed in regenerating islets but not in normal pancreatic islets, insulinomas, or regenerating liver. In 90% depancreatized and nicotinamide-injected rats, the expression of the gene was increased 1 month after the partial pancreatectomy and reached a peak 3 months after the operation. The increase in expression of the gene was temporally correlated with the increase in size of regenerating islets and the decrease in urinary glucose level. The gene was also found to be activated in hyperplastic islets of aurothioglucose-treated mice. Thus, the expression of the gene in both regenerating and hyperplastic islets suggests possible roles for this gene in replication, growth, and maturation of islet beta-cells. We also found that a human pancreas-derived cDNA library contained a homologue to the gene.     

Enhancement of mouse pancreatic regeneration and HIT-T15 cell proliferation with rat pancreatic extract

In this study, the effects of rat pancreatic extract (RPE) on regeneration of impaired mouse pancreas and proliferation of beta-cell line (HIT-T15) were investigated. RPE from the regenerating pancreas (2 days after 60% pancreatectomy) was treated to cure streptozotocin (STZ) induced diabetes in BALB/c mice. RPE-treated BALB/c mice for 21 consecutive days became euglycemic by day 30 and remained normoglycemic during a 150 day follow-up. Saline treated mice remained hyperglycemic sustained uncontrolled hyperglycemia. Islet neogenesis was observed in RPE-treated mice and confirmed by use of immunocytochemistry. Morphometric analysis of pancreas in reverted RPE-treated mice showed a new population of small islets compared with saline controls and an increased islet number. When HIT-T15 cells were treated with RPE, HIT-T15 cell proliferation and insulin secretion increased with increases in the RPE concentration. These results imply that RPE have the regeneration factors and help in the cure of diabetes.     

Expression of the protein tyrosine phosphatase-like protein IA-2 during pancreatic islet development.

A tyrosine phosphatase-like protein, IA-2, is a major autoantigen in Type 1 diabetes but its role in islet function is unclear. Tyrosine phosphorylation mediates regulation of cellular processes such as exocytosis, cell growth, and cell differentiation. To investigate the potential involvement of IA-2 in islet differentiation and insulin secretion, we analyzed by immunohistochemistry expression of IA-2 during islet development in fetal rats and during the maturation of insulin secretory responses after birth. In the fetus, IA-2 immunoreactivity was detected in primitive islets positive for insulin and glucagon at 12 days' gestation. Subsequently, IA-2 was only weakly detectable in the fetal pancreas. In neonatal rat, a progressive increase in IA-2 immunoreactivity was observed in islets from very low levels at 1 day of age to moderate labeling at 10 days. In the adult, relatively high levels of IA-2 were detected in islets, with heterogeneous expression in individual cells within each islet. IA-2 marks a population of endocrine cells that transiently appear early in pancreatic ontogeny. Islet IA-2 expression reappears after birth concomitant with the development of mature insulin secretory responses, consistent with a role for this protein in regulated hormone secretion.     

Diabetes and tumor formation in transgenic mice expressing Reg I

To examine the effect of overexpressed regenerating gene (Reg) I on pancreatic beta-cells, we generated transgenic mice expressing Reg I in islets (Reg-Tg mice). Three lines of Reg-Tg mice were established. In line-1 Reg-Tg mice, the expression level of Reg I mRNA in islets was 7 times higher than those in lines 2 and 3 of Reg-Tg mice, and line 1 mice developed diabetes by apoptosis of beta-cells, as well as various malignant tumors. In addition to the decrease in beta-cells, compensatory islet regeneration and proliferation of ductal epithelial cells were observed in line-1 Reg-Tg mice. Because Reg I protein was secreted primarily into pancreatic ducts from acinar cells, it may primarily stimulate the proliferation of ductal epithelial cells, and not beta-cells, and their differentiation into islets. Moreover, the tumor-promoting activity of Reg I protein should be considered for its possible clinical applications.     

神经元限制性沉默因子与胰岛素在成年小鼠胰腺中的表达

目的:观察神经元限制性沉默因子(NRSF)在正常成年小鼠胰腺组织中的表达情况。方法:以6～8周BALB/c小鼠胰腺为实验材料,制备冰冻切片,与地高辛标记的NRSF cDNA探针进行原位杂交,观察mRNA表达,并结合免疫组织化学方法检测NRSF和胰岛素的表达。结果:原位杂交显示,NRSF mRNA仅表达于胰腺组织外分泌部腺泡腺细胞中,胞浆呈蓝紫色,与免疫荧光组织化学检测NRSF蛋白表达的部位一致,而胰岛细胞中无NRSF mRNA及蛋白的表达。免疫酶组织化学染色显示,胰岛大部分细胞中表达胰岛素,胞浆染成黄棕色,而腺泡腺细胞则不表达胰岛素。结论:NRSF与胰岛素不存在共定位关系,即成年小鼠胰岛细胞不表达NRSF,而表达胰岛素。提示NRSF蛋白表达的消失可能是建立完全分化成熟、具有完好分泌反应的胰岛细胞所必需的。     

大鼠胰腺发育阶段Mesothelin的表达

目的:研究Mesothenlin在大鼠胰腺发育阶段的表达和细胞定位。方法:运用RT-PCR和Western Blot技术分别检测Mesothenlin在大鼠胰腺发育阶段的mRNA和蛋白表达水平;运用免疫荧光检测不同时期Mesothenlin在胰腺的组织细胞学定位。结果:RT-PCR结果显示E18.5 Mesothelin mRNA的表达水平显著增高,至P14达到高峰,成年较低。Western Blot结果显示其蛋白表达趋势与mRNA完全相同。免疫荧光结果显示在不同发育时期Mesothenlin与胰岛β细胞和间充质细胞共表达。结论:Mesothenlin在大鼠胚胎胰岛形成及生后结构重塑中出现显著性高表达,并表达于胰岛β细胞和间充质细胞。     

GPR40 gene expression in human pancreas and insulinoma

To assess gene expression of a membrane-bound G-protein-coupled fatty acid receptor, GPR40, in the human pancreas and islet cell tumors obtained at surgery were analyzed. The mRNA level of the GPR40 gene in isolated pancreatic islets was approximately 20-fold higher than that in the pancreas, and the level was comparable to or rather higher than that of the sulfonylurea receptor 1 gene, which is known to be expressed abundantly in human pancreatic beta cells. A large amount of GPR40 mRNA was detected in tissue extracts from two cases of insulinoma, whereas the expression was undetectable in glucagonoma or gastrinoma. The present study demonstrates that GPR40 mRNA is expressed predominantly in pancreatic islets in humans and that GPR40 mRNA is expressed solely in human insulinoma among islet cell tumors. These results indicate that GPR40 is probably expressed in pancreatic beta cells in the human pancreas.     

Characterization of the molecular mechanisms involved in the increased insulin secretion in rats with acute liver failure

To investigate the mechanism of hyperinsulinaemia in rats with acute liver failure induced by the administration of d-galactosamine (GalN), we focused on the role of polyprimidine tract-binding protein (PTB) in islet insulin synthesis. Recent reports indicate that PTB binds and stabilizes mRNA encoding insulin and insulin secretory granule proteins, including islet cell autoantigen 512 (ICA512), prohormone convertase 1/3 (PC1/3), and PC2. In the present study, glucose-stimulated insulin secretion was significantly increased in GalN-treated rats compared to controls. Levels of mRNA encoding insulin 1, ICA512, and PC1/3 were increased in the pancreatic islets of GalN-treated rats. This mRNA level elevation was not prevented by pretreatment with actinomycin D. When the PTB-binding site in insulin 1 mRNA was incubated with the islet cytosolic fraction, the RNA-protein complex level was increased in the cytosolic fraction obtained from GalN-treated rats compared to the level in control rats. The cytosolic fraction obtained from pancreatic islets obtained from GalN-treated rats had an increased PTB level compared to the levels obtained from the pancreatic islets of control rats. These findings suggest that, in rats with acute liver failure, cytosolic PTB binds and stabilizes mRNA encoding insulin and its secretory granule proteins.     

Miglitol (BAY m 1099) Treatment of Diabetic Hypothalamic-Dietary Obese Rats Improves Islet Response to Glucose

AXEN, KATHLEEN V., XUE LI, AND ANTHONY SCLAFANI. Miglitol (BAY m 1099) treatment of diabetic hypothalamic-dietary obese rats improves islet response to glucose. Obes Res. 1999;7:83–89. Objective : The well-absorbed α-glucosidase inhibitor, miglitol (BAY m 1099), was included in the diets of hypothalamic-dietary obese diabetic rats to investigate its ability to improve glycemia and thereby reverse glucotoxic effects on islet secretory response. Research Methods and Procedures : Female rats received bilateral electrolytic lesions of the ventromedial hypothalamus and were fed high-fat, sucrosesupplemented diets until hyperinsulinemia and hyperglycemia were observed after 3 hours of food deprivation (nonfed). Diabetic animals were assigned to miglitol-treated (40 mg/17 g of diet) or untreated groups for 3 weeks; pancreatic islets were isolated for incubation experiments. Results : No differences in food intake, body weights, or nonfed plasma glucose or insulin levels were seen between treated and untreated diabetic rats. Islets isolated from untreated diabetic rats showed elevated basal insulin release and no insulin secretory response to an elevation in glucose concentration. In contrast, islets obtained from miglitol-treated rats showed more normal basal release and a significant insulin secretory response to glucose. Incubation of islets, obtained from normal control rats or untreated diabetic rats, in media containing miglitol at levels estimated to exist in plasma of treated rats had no effect on islet insulin secretory responses to glucose. Discussion : Islet secretory response was improved despite continued hyperglycemia and severe insulin resistance. Miglitol treatment may improve islet sensitivity to glucose either through effects on islet metabolism requiring prolonged exposure or by improvement in postmeal glycemia, despite persistent hyperglycemia.     

Histidyl-proline diketopiperazine cyclo (His-Pro): identification and characterization in rat pancreatic islets

Measurements of cyclo (His-Pro) in the pancreas were carried out in the rat by a specific radioimmunoassay. Cyclo (His-Pro)-like immunoreactivity was identified in pancreatic islets with a mean concentration of 2023 pg/mg protein, 88-fold higher than that of the whole pancreas. Cyclo (His-Pro) immunoreactivity from pancreatic extracts was indistinguishable immunologically and chromatographically from synthetic cyclo (His-Pro). Insulin-induced hypoglycemia caused a significant, 53% decrease in pancreatic cyclo (His-Pro) concentrations, and FLA-63, a dopamine beta-oxidase inhibitor, also reduced islet cyclo (His-Pro) concentrations 51%. These data indicate that cyclo (His-Pro) is present in rat pancreatic islets and may play a potential role in modulating pancreatic responses to nutrient and pharmacologic stimuli.     

Co-expression and regulation of connexins 36 and 43 in cultured neonatal rat pancreatic islets

Fetal and neonatal pancreatic islets present a lower insulin secretory response as compared with adult islets. Prolonged culturing leads to an improvement of the glucose-induced insulin secretion response in neonatal pancreatic islets that may involve regulation of gap junction mediated cell communication. In this study, we investigated the effect of culturing neonatal islet cells for varying periods of time and with different glucose medium concentrations on the cellular expression of the endocrine pancreatic gap junction associated connexin (Cx) 36 and Cx43. We report here that the 7-d culture induced upregulation of the expression of these junctional proteins in neonatal islets in a time-dependent manner. A correlation was observed between the increased mRNA and protein expression of Cx36 and Cx43 and the increased insulin secretion following islet culturing. In addition, increasing glucose concentration within the culture medium induced a concentration-dependent enhancement of Cx36 islet expression, but not of Cx43 expression in cultured neonatal islets. In conclusion, we suggest that the regulation of gap junctional proteins by culture medium containing factors and glucose may be an important event for the maturation process of beta cells observed at in vitro conditions.     

Effect of a high-protein diet on the gene expression of a trypsin-sensitive, cholecystokinin-releasing peptide (monitor peptide) in the pancreas

The adaptation to a high protein diet of the concentration and mRNA level of a trypsin-sensitive, cholecystokinin-releasing peptide (monitor peptide), which was proposed to be the mediator of the cholecystokinin release in response to protein intake, was investigated in the rat pancreas. Adult rats were placed on one of two isocaloric diets. One group was fed a 22% casein diet (control diet) and the other a 64% casein diet (high-protein diet) for 14 days. In order to quantify the monitor peptide separately from pancreatic secretory trypsin inhibitor (PSTI-II), which is highly similar in its amino acid and mRNA nucleotide sequences to the monitor peptide but has less cholecystokinin-releasing activity, we used specific assay methods: HPLC was used for determining the monitor peptide concentration in zymogen granules and a synthetic oligonucleotide probe for determining the mRNA of the monitor peptide in the pancreas. The concentrations in the zymogen granules and the mRNA levels in the pancreas of the two peptides increased in parallel during the adaptation to the high protein diet, indicating that these two peptides were under the same control during the adaptation. The concentration and mRNA level of the monitor peptide, which were measured after 0, 3, and 14 days, increased throughout the experiment period, as did the concentration of trypsin. This suggested that the monitor peptide and trypsin may respond to similar signals during the adaptation to a high protein diet and that this apparent coordination may facilitate the adaptation of the pancreas to the diet.     

Podocyte-associated proteins FAT, alpha-actinin-4 and filtrin are expressed in Langerhans islets of the pancreas

Nephrin is a crucial podocyte molecule in the kidney glomerular filtration barrier and it is also expressed in Langerhans islet beta cells of the pancreas. Recently, genetic mapping of proteinuric kidney disease genes and animal models have revealed further important molecules for the kidney filtration function including alpha-actinin-4, podocin, FAT, and NEPH1. This study was addressed to explore the pancreatic expression of the podocyte molecules podocin, FAT, alpha-actinin-4, NEPH1, NEPH2, filtrin/NEPH3, synaptopodin and CD2 associated protein (CD2AP). The mRNA and protein expressions were studied by RT-PCR and immunoblotting, and localization in the pancreas was investigated by immunofluorescence. Of the nephrin-associated podocyte proteins, filtrin/NEPH3, FAT, and alpha-actinin-4 were found to be expressed in the pancreas at the gene and protein level and localized to Langerhans islets. Immunoreactivity with the podocin antibody was detected mostly in the exocrine pancreas. NEPH1 and synaptopodin expression was detected only at the mRNA level. Further studies are needed to unravel the functional role of these podocyte-associated molecules in the pancreatic Langerhans islets.