Marie-Noëlle Gangnerau

Islet Inflammation and Fibrosis in a Spontaneous Model of Type 2 Diabetes, the GK Rat

The molecular pathways leading to islet fibrosis in diabetes are unknown. Therefore, we studied gene expression in islets of 4-month-old Goto-Kakizaki (GK) and Wistar control rats. Of 71 genes found to be overexpressed in GK islets, 24% belong to extracellular matrix (ECM)/cell adhesion and 34% to inflammatory/immune response families. Based on gene data, we selected several antibodies to study fibrosis development during progression of hyperglycemia by immunohistochemistry. One-month-old GK and Wistar islets appeared to be similar. Two-month-old GK islets were strongly heterogenous in terms of ECM accumulation compared with Wistar islets. GK islet vascularization, labeled by von Willebrand factor, was altered after 1 month of mild hyperglycemia. Numerous macrophages (major histocompatibility complex class II+ and CD68+) and granulocytes were found in/around GK islets. These data demonstrate that marked inflammatory reaction accompanies GK islet fibrosis and suggest that islet alterations in this nonobese model of type 2 diabetes develop in a way reminiscent of microangiopathy.

Glucose metabolism and β-cell mass in adult offspring of rats protein and/or energy restricted during the last week of pregnancy

An association between low birth weight and later impaired glucose tolerance was recently demonstrated in several human populations. Although fetal malnutrition is probably involved, the biological bases of such a relationship are not yet clear, and animal studies on the matter are scarce. The present study was aimed to identify, in adult (8-wk) female offspring, the effects of reduced protein and/or energy intake strictly limited to the last week of pregnancy. Thus we have tested three protocols of gestational malnutrition: a low-protein isocaloric diet (5 instead of 15%), with pair feeding to the mothers receiving the control diet; a restricted diet (50% of the control diet); and a low-protein restricted diet (50% of low-protein diet). Only the low-protein diet protocols, independent of total energy intake, led to a lower birth weight. The adult offspring female rats in the three deprived groups exhibited no decrease in body weight and no major impairment in glucose tolerance, glucose utilization, or glucose production (basal state and hyperinsulinemic clamp studies). However, pancreatic insulin content and β-cell mass were significantly decreased in the low-protein isocaloric diet group compared with the two energy-restricted groups. Such impairment of β-cell mass development induced by protein deficiency limited to the last part of intrauterine life could represent a situation predisposing to impaired glucose tolerance.

Impaired pancreatic beta cell function in the fetal GK rat. Impact of diabetic inheritance.

The Goto-Kakisaki (GK) rat is a genetic model of non-insulin-dependent diabetes. At 21.5 d of age we found that GK fetuses had an increased plasma glucose concentration, a decreased plasma insulin level, and a reduced pancreatic beta cell mass. To investigate the beta cell function during fetal life we used a hyperglycemic clamp protocol applied to the mothers, which allowed us to obtain a steady-state hyperglycemia in the corresponding fetuses. At variance, with Wistar (W) fetuses, plasma insulin concentration in GK fetuses did not rise in response to hyperglycemia. In contrast, GK fetal pancreas released insulin in response to glucose in vitro to the same extent as W fetal pancreas. Such a discrepancy between the in vivo and in vitro results suggests that the lack of pancreatic reactivity to glucose as seen in vivo is extrinsic to the fetal GK beta cell. Finally, the importance of gestational hyperglycemia was investigated by performing crosses between GK and W rats. Fetuses issued from crosses between W mother and GK father or GK mother and W father had a beta cell mass close to normal values and were still able to increase their plasma insulin levels in response to hyperglycemia in vivo. Our data suggest that hyperglycemia in utero does not influence the severity of the decrease of the beta cell mass or the lack of the insulin secretory response to glucose in the fetal GK rat. Moreover they indicate that conjunction of GK genes originating from both parents is necessary in order for these defects to be fully expressed.

Regenerating 1 and 3b gene expression in the pancreas of type 2 diabetic Goto-Kakizaki (GK) rats.

Regenerating (REG) proteins are associated with islet development, β-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, 3α, 3β and 3γ, vs Wistar islets. Goto-Kakizaki rat islets also exhibit increased cytokine/chemokine expression/release, particularly IL-6. Here we analyzed Reg1 and Reg3β 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 Reg3β 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.

Is defective pancreatic beta-cell mass environmentally programmed in Goto-Kakizaki rat model of type 2 diabetes?: insights from crossbreeding studies during suckling period.

Objective: The Goto-Kakizaki (GK) rat is a spontaneous model of type 2 diabetes with a well established pathological pancreatic beta-cell development. Hyperglycemia experienced during early postnatal life contributes to the programming of endocrine pancreas. We have analyzed the consequences of hyperglycemic versus euglycemic suckling period for the pancreatic beta-cell mass and the in vivo glucose tolerance and insulin secretion in 4-week-old unweaned control Wistar (W), diabetic GK, and in offspring issued from crosses between normoglycemic W and diabetic GK rats. Methods: Mother/father crosses yielded offspring designated as follows: W/W, GK/GK, W/GK, and GK/W. In vivo glucose tolerance and insulin secretion tests were performed on males 4 weeks after birth, that is, just before weaning. Beta-cell mass was determined by immunohistochemistry and morphometry. Results: Four-week-old W/GK and GK/W rats are normoglycemic, normoinsulinemic, and display a similarly small beta-cell mass. Both W/GK and GK/W rats exhibit in vivo glucose intolerance and defective insulin secretion in response to glucose. Conclusions: Our data obtained from crossbreeding studies during suckling period suggest that the defective pancreatic beta-cell mass is not environmentally programmed in the GK model of type 2 diabetes. Rather, they support the hypothesis that the beta-cell mass defect in the GK is linked to genetic determinism.