Regenerating 1 and 3b Gene Expression in the Pancreas of Type 2 Diabetic Goto-Kakizaki (GK) Rats

International audienceRegenerating (REG) proteins are associated with islet development, b-cell damage, diabetes and pancreatitis. Particularly, REG-1 and REG-3-beta are involved in cell growth/survival and/or inflammation and the Reg1 promoter contains interleukin-6 (IL-6)-responsive elements. We showed by transcriptome analysis that islets of Goto-Kakizaki (GK) rats, a model of spontaneous type 2 diabetes, overexpress Reg1, 3a, 3b and 3c, vs Wistar islets. Goto-Kakizaki rat islets also exhibit increased cytokine/chemokine expression/release, particularly IL-6. Here we analyzed Reg1 and Reg3b expression and REG-1 immuno-localization in the GK rat pancreas in relationship with inflammation. Isolated pancreatic islets and acinar tissue from male adult Wistar and diabetic GK rats were used for quantitative RT-PCR analysis. REG-1 immunohistochemistry was performed on paraffin sections with a monoclonal anti-rat REG-1 antibody. Islet cytokine/chemokine release was measured after 48 h-culture. Islet macrophage-positive area was quantified on cryostat sections using anti-CD68 and major histocompatibility complex (MHC) class II antibodies. Pancreatic exocrine-to-endocrine Reg1 and Reg3b mRNA ratios were markedly increased in Wistar vs GK rats. Conversely, both genes were upregulated in isolated GK rat islets. These findings were unexpected, because Reg genes are expressed in the pancreatic acinar tissue. However, we observed REG-1 protein labeling in acinar peri-ductal tissue close to islets and around large, often disorganized, GK rat islets, which may retain acinar cells due to their irregular shape. These large islets also showed peri-islet macrophage infiltration and increased release of various cytokines/ chemokines, particularly IL-6. Thus, IL-6 might potentially trigger acinar REG-1 expression and secretion in the vicinity of large diabetic GK rat islets. This increased acinar REG-1 expression might reflect an adaptive though unsuccessful response to deleterious microenvironment

Fetal pancreatic b-cell growth and insulin-like growth factors relationship in undernourished rats

Resumen del trabajo presentado al 38th EASD Annual Meeting of the European Association for the Study of Diabetes, celebrado en Budapest (Humgria) del 1 al 5 de septiembre de 2002.[Background and Aims] We have previously shown that Wistar fetuses from protein- caloric undernourished pregnant rats (U) at 21 days post coitum (dpc) exhibit increased β cell-mass. This alteration is correlated with increased insulinemia and total pancreatic insulin content, a pattern reminiscent to that reported in infants of mild diabetic mothers. Both Insulinlike Growth Factor (IGF)-I and -2 are essential players for growth and development during the fetal period. The aim of the present study was to investigate in the U fetuses at 21 dpc: 1) serum IGFs levels, 2) IGFs gene expression in the liver and pancreas, and 3) in vitro mitogenic effect of IGFs in isolated fetal islets using BrdU labelling index (LI). All values were compared to those in Wi star control fetuses (C). [Methods] Serum concentrations of IGF-I and IGF-2 were measured by radioimmuno assay and radioreceptor assay respectively. RNase protection assay was performed using RNA from pancreas and liver to evaluate IGFs or IGFBPs gene expression in both tissues. [Results] Similar serum IGF-I and-2levels were observed in U and C. IGF-I and IGF-2 mRNAs were detected in liver and pancreas of both C and U fetuses. Despite being decreased in the liver, IGF-l mRNA level was increased in U pancreases as compared to C. Concerning IGF-2 gene expression it was diminished in U pancreas while being normal in the liver as compared to C. No difference in IGFBP-I, -2 and -3 mRNA levels was detected in U liver when compared to C. However, gene expression of IGFBP-2 was increased and that of IGFBP-3 was decreased in U pancreases. Finally, the in vitro study showed a normal BrdU LI in U isolated fetal islets under basal conditions while it was found significantly increased twice in response to both IGF-I and IGF-2 (lOOng/ml) as compared to fetal C islets. [Conclusion] Our data suggest that in U fetuses at 21 dpc: I) the increased β-cell mass can be related to the stimulation of replicative β-cell response due to locally increased IGF-I in the pancreas 2) such IGF-I action is perhaps favored by an enhanced IGFBP-2 gene expression in pancreas, and 3) at variance with previous reports in several models of decreased intrapancreatic IGF-2 expression in fetuses, the low IGF-2 mRNA level as it is observed in the present U model does not correlate with a decreased β-cell growth.Peer Reviewe

Respective impact of inheritance and environmental programming on beta-cell mass and beta-cell function in the GK model

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Overexpression of regenerative, antioxidant and inflammatory genes in adult diabetic GK islets

National audienceIn the adult GK rat, a spontaneous model of type 2 diabetes, total pancreatic β-cell number is decreased by 60%. This alteration cannot be ascribed to increased β-cell apoptosis but is related to decreased β-cell replication. Moreover, the adult GK pancreas exhibits large islets disrupted by connective tissue. Our objective was therefore to identify genes possibly involved in the β-cell growth phenotype in adult diabetic GK rat. Differential gene expression was evaluated in islets of adult diabetic GK and normal Wistar (W) rats by high density oligonucleotide microarray. Total RNA was extracted from 16 week-old W and GK islets. Biotin-labeled cRNA probes were synthesized and hybridized to Affymetrix RG-U34A oligonucleotide microarrays containing approximately 7,000 rat genes. The arrays were scanned and expression values for the genes were determined using Affymetrix Microarray Suite 5.0 and Affymetrix Data Mining Tool 2.0. Expression pattern of reg in GK pancreas was determined by immunohistochemical analysis. No difference in expression levels of genes encoding growth factors for β-cells (GH, IGF1, PDGF, HGF, insulin) and the c-Myc transcription factor was found in Wistar and GK rats. By contrast, several reg-related genes were up-regulated in the GK islets (46-, 12- and 11-fold respectively for reg II, reg I and reg III genes). These results were confirmed by real-time PCR. Expression of several stress genes such as glutathione peroxidase (2.7-fold) , thioredoxine interating protein (5.4-fold) and heat shock protein 70 (2.3-fold) were increased in GK islets. Islets from GK rats also display increased expression of inflammatory genes such as lipocalin 2 (72.8-fold), decorin (4.1-fold) and annexins 1 and 2 genes (8.7- and 2.1-fold). To localize the reg-I protein in pancreas we stained sections of GK and W pancreases. Immunoreactivity was observed only in islets. Reg staining was colocalized with staining for insulin but did not seem restricted to the β-cells.Our data suggest that in GK islets 1) the increased expression of reg family genes whose involvement in β-cell regeneration has been proposed, may be an integral part of the β-cell growth phenotype of GK islets; 2) the lack of increased apoptosis of the β-cells may be related to activation of several stress genes that confer protection against β-cell death; 3) the increased expression of cytokine genes could be related to a local inflammatory response in GK islets; 4) the local inflammatory gene overexpression together with the cytokine environment in GK islets may have an influence on reg gene expression. Our working hypothesis is, that these expression changes reported for the first time in GK islets could represent an acquired adaptation in response to chronic hyperglycemia (glucotoxicity). To learn what genes are more directly involved in the pathology of the GK rats, we are currently analyzing the gene expression profile in young prediabetic GK rat

Restitution of defective glucose-stimulated insulin secretion in diabetic GK rat by acetylcholine uncovers paradoxical stimulatory effect of β-cell muscarinic receptor activation on cAMP production

Because acetylcholine (ACh) is a recognized potentiator of glucose-stimulated insulin release in the normal beta-cell, we have studied ACh's effect on islets of the Goto-Kakizaki (GK) rat, a spontaneous model of type 2 diabetes. We first verified that ACh was able to restore the insulin secretory glucose competence of the GK beta-cell. Then, we demonstrated that in GK islets 1) ACh elicited a first-phase insulin release at low glucose, whereas it had no effect in Wistar; 2) total phospholipase C activity, ACh-induced inositol phosphate production, and intracellular free calcium concentration ([Ca2+]i) elevation were normal; 3) ACh triggered insulin release, even in the presence of thapsigargin, which induced a reduction of the ACh-induced [Ca2+]i response (suggesting that ACh produces amplification signals that augment the efficacy of elevated [Ca2+]i on GK exocytosis); 4) inhibition of protein kinase C did not affect [Ca2+]i nor the insulin release responses to ACh; and 5) inhibition of cAMP-dependent protein kinases (PKAs), adenylyl cyclases, or cAMP generation, while not affecting the [Ca2+]i response, significantly lowered the insulinotropic response to ACh (at low and high glucose). In conclusion, ACh acts mainly through activation of the cAMP/PKA pathway to potently enhance Ca2+-stimulated insulin release in the GK beta-cell and, in doing so, normalizes its defective glucose responsiveness

Fetal insulin-like growth factor-2 production is impaired in the GK rat model of type 2 diabetes

At late fetal age (21.5 days postcoitum [dpc]), GK rats present a severely reduced β-cell mass compared with Wistar rats. This anomaly largely antedates the onset of hyperglycemia in GK rats. Thus, the β-cell mass deficit could represent the primary defect leading to type 2 diabetes in the adult. The aim of this work was to investigate, in GK fetuses at the end of fetal age (21.5 dpc), whether impaired availability of growth factors such as insulin, growth hormone, and IGFs and their IGF binding proteins (IGFBPs) could be instrumental in this anomaly. Although it confirms that GK fetuses are hypoinsulinemic despite enhanced plasma glucose level due to maternal hyperglycemia, the present study shows for the first time that IGF-2 expression in the liver and pancreas and IGF-2 serum levels are decreased in GK fetuses. Serum level as well as liver and pancreatic mRNA expression of IGFBP-2 were found to be normal in GK fetuses, whereas serum level and liver mRNA expression of IGFBP-1 were increased. Finally, we found that the maximal β-cell mitogenic response to IGFs in vitro is kept intact, therefore suggesting that the direct biological action of IGFs on fetal GK β-cells is not grossly impaired. In conclusion, in GK fetuses at 21.5 dpc, the defective IGF-2 production appears to be an early landmark in the pathological sequence leading to retardation of β-cell growth in the fetal GK rat.This work was supported by DGICYT (Direccion General de Investigacion, Ciencia, Tecnologia, Ministerio de Educacion, Ciencia, Spain; Grant PM 97-0017), by a grant from the Ministe`re de l’Education Nationale, de l’Enseignement Superieur et de la Recherche (no. 95-G-0103; program interministeriel “Aliment Demain”), and by the France/Spain program for Science (Cooperation Franco-Espagnole Center National de la Recherche Scientifique/Consejo Superior Investigaciones Cientificas; projects 5249 and 7963). S. Ramos was a recipient of a fellowship from Conserjeria de Educacion y Cultura from Comunidad Autonoma de Madrid.Peer Reviewe

Insulin-like growth factor 2 production is impaired in fetal GK rats

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Protein-caloric food restriction affects insulin-like growth factor-1 in fetal Wistar rat

We have previously shown that fetuses from protein-caloric undernourished pregnant rats (35% of control diet during the last week of pregnancy) at 21.5 d post coitum exhibit increased beta-cell mass. This alteration is correlated with increased insulinemia and total pancreatic insulin content, a pattern similar to that reported in infants of mild diabetic mothers. In this work, we investigated in undernourished fetuses: 1) whether availability of growth factors such as insulin, GH, and IGFs and their binding proteins (IGFBPs) could be implicated in this alteration, and 2) the beta-cell mitogenic response to IGFs in vitro. The results show that maternal undernutrition increases pancreatic IGF-I expression and islet IGF-I receptor content in undernourished fetuses, whereas hepatic IGF-I expression and serum IGF-I levels were decreased. No changes were observed in serum IGF-II, and its expression was diminished in undernourished pancreases and unchanged in the liver, compared with control fetuses. Serum levels and liver and pancreatic mRNA expression of IGFBP-1 were found to be normal in undernourished fetuses, whereas the serum concentration and abundance of IGFBP-2 mRNA in pancreas were increased. Finally, the beta-cell mitogenic response to IGFs in vitro was significantly increased in undernourished fetal islets, compared with controls. In conclusion, in undernourished fetuses the increased beta-cell mass can be related to the stimulation of replicative beta-cell response due to locally increased pancreatic IGF-I mRNA; this effect is perhaps potentiated or favored by the enhanced islet IGF-I receptor content and pancreatic IGFBP-2 gene expression.This work was supported by Grants BFI2001-2125 and BFI2002-00253 from Ministerio de Ciencia y Tecnología, Spain, and the France/Spain Program for Science (Cooperation Franco-Espagnole Centre National de la Recherche Scientifique/Consejo Superior Investigaciones Cientificas; projects 5249 and 7963).Peer Reviewe