Reg1 and Reg3β expression in the pancreas of adult diabetic Goto-Kakizaki rats

International audienceReg protein expression is associated with islet development, β-cell damage, diabetes and pancreatitis. We previously reported that islets of 4-month-old (4-mo) Goto-Kakizaki (GK) rats, a spontaneous model of type 2 diabetes, overexpress Reg1, 3α, 3β and 3γ vs age-matched Wistar control islets. Reg1 and 3β are involved in cell growth/survival control and inflammation, respectively. Diabetic GK islets also exhibit progressive inflammation reaction, consisting of high CCL2 (MCP-1), CCL3 (MIP-1α), CXCL-1 (murine IL-8 analog) and IL-6 expression/release and mostly peri-islet mac rophage infiltration. Importantly, Reg1 gene promoters contain IL-6-responsive elements. Here we analyzed in greater detail the pancreatic expression of Reg1 and Reg3β in diabetic GK rats. Isolated pancreatic islets and acinar (exocrine) tissue from male Wistar and GK rats were used for quantitative RT-PCR analysis, normalized with GAPDH. Islet IL-6, CCL2, CCL3 and CXCL1 release was measured by LuminexTM technology after a 48h islet culture on collagen. Pancreatic Reg immunohistochemistry (IHC) was performed on paraffin sections with a rabbit anti-human polyclonal antibody (Ab) and a mouse anti-rat monoclonal anti-Reg1 Ab. Macrophage infiltration was detected on cryostat sections using CD68 and MHC class II antibodies. Islet macrophage+ area was quantified and expressed as % of corresponding islet area. Statistical analyses used the Student’s t-test for unpaired data. The exocrine/endocrine ratio of Reg mRNA expression in 4-mo normoglycemic Wistar and diabetic GK rats was: 1) Reg1: 41.3±2.4 and 5.0±1.5, respectively (n=3 different isolations/group, p<0.005); 2) Reg3β: 74.2±17.0 and 7.0±4.9, respectively (n=3, p<0.02). Furthermore, Reg1 and 3β were overexpressed in GK vs Wistar islets (x11.2±1.4, p<0.005 and 77.9±16.3, p<0.01, respectively, n=3 in both cases). Next, the polyclonal Reg Ab stained most islet insulin+ cells in 4-mo Wistar pancreas but much less so in GK pancreas. By contrast, the monoclonal anti-Reg1 Ab stained just a few peri-islet exocrine cells in Wistar pancreas but many more in GK rats, particularly around large islets. Since IL-6 stimulates Reg1 expression, we compared cytokine/chemokine release by small and large 2.5-mo Wistar and GK islets. Large GK islets released significantly more IL-6 and CCL3 than large Wistar islets (x4.7±0.9, p<0.02 and 2.7±0.2, p<0.005, respectively, n=3 in both cases). Concomitantly, CD68+ and MHC II+ peri-islet macrophage infiltration correlated with islet size in 2.5-mo GK rats (r=0.57 and r=0.95, respectively). Reg1 and 3β genes are strongly expressed in the exocrine pancreas of control rats. They are markedly overexpressed in islets of diabetic GK vs control Wistar rats, probably reflecting an adaptive stress response. While the polyclonal Ab mainly stains islet insulin+ cells, Reg1 protein is the hallmark of acinar tissue around large GK islets. Its peri-insular localization might result from increased islet release of IL-6 (a factor stimulating Reg1 expression) and is similar to that of macrophage infiltration

Reg1 and Reg3β expression in the pancreas of adult diabetic Goto-Kakizaki (GK) rats

Reg protein expression is associated with islet development, β-cell damage, diabetes and pancreatitis. We previously reported that islets of 4-month-old (4-mo) GK rats, a spontaneous model of type 2 diabetes, overexpress Reg1, 3α, 3β and 3γ vs age-matched Wistar control islets. Reg1 and 3β are involved in cell growth/survival control and inflammation, respectively. Diabetic GK islets also exhibit progressive inflammation reaction, consisting of high CCL2 (MCP-1), CCL3 (MIP-1α), CXCL-1 (murine IL-8 analog) and IL-6 expression/release and mostly peri-islet macrophage infiltration. Importantly, Reg1 gene promoters contain IL-6-responsive elements. Here we analyzed in greater detail the pancreatic expression of Reg1 and Reg3β in diabetic GK rats. Isolated pancreatic islets and acinar tissue from male Wistar and GK rats were used for quantitative RT-PCR analysis. Islet IL-6, CCL2, CCL3 and CXCL1 release was measured by LuminexTM technology after a 48h islet culture on collagen. Pancreatic Reg immunohistochemistry (IHC) was performed on paraffin sections with a rabbit anti-human polyclonal antibody (Ab) and a mouse anti-rat monoclonal anti-Reg1 Ab. Macrophage infiltration was detected on cryostat sections using CD68 and MHC class II antibodies. Islet macrophage+ area was quantified and expressed as % of corresponding islet area. Statistical analyses used the Student’s t-test for unpaired data. The exocrine/endocrine ratio of Reg mRNA expression in 4-mo normoglycemic Wistar and diabetic GK rats was: 1) Reg1: 41.3±2.4 and 5.0±1.5, respectively (n=3 different isolations/group, p<0.005); 2) Reg3β: 74.2±17.0 and 7.0±4.9, respectively (n=3, p<0.02). Furthermore, Reg1 and 3β were overexpressed in GK vs Wistar islets (x11.2±1.4, p<0.005 and 77.9±16.3, p<0.01, respectively, n=3 in both cases). Next, the polyclonal Reg Ab stained most islet insulin+ cells in 4-mo Wistar pancreas but much less so in GK pancreas. By contrast, the monoclonal anti-Reg1 Ab stained just a few peri-islet exocrine cells in Wistar pancreas but many more in GK rats, particularly around large islets. Since IL-6 stimulates Reg1 expression, we compared cytokine/chemokine release by small and large 2.5-mo Wistar and GK islets. Large GK islets released significantly more IL-6 and CCL3 than large Wistar islets (x4.7±0.9, p<0.02 and 2.7±0.2, p<0.005, respectively, n=3 in both cases). Concomitantly, CD68+ and MHC II+ peri-islet macrophage infiltration correlated with islet size in 2.5-mo GK rats (r=0.57 and r=0.95, respectively). Reg1 and 3β genes are strongly expressed in the exocrine pancreas of control rats. The two genes are markedly overexpressed in islets of diabetic GK vs control Wistar rats, probably reflecting an adaptive stress response. While the polyclonal Ab mainly stains islet insulin+ cells, Reg1 protein is the hallmark of acinar tissue around large GK islets. Its peri-insular localization might result from increased islet release of IL-6 (a factor stimulating Reg1 expression) and is similar to that of macrophage infiltration

Defective pancreas vascularisation and downregulation of soluble epoxyde hydrolase gene expression in various organs in Goto-Kakizaki (GK) foetuses

International audienceWe identified islet inflammation associated with microangiopathy in diabetic GK rats (a spontaneous model of T2D). Signs of islet microangiopathy are also present in prediabetic 1-week-old GK neonates (1-w-GK), together with islet oxidative stress (OS). In addition, GK rats, particularly females, show hypercholesterolemia. Both hyperglycemia and hypercholesterolemia trigger OS and have deleterious effects on vessels. Both also increase placental inflammation, which takes place even during normal pregnancy. Moreover, there is evidence for linking dysmetabolic conditions during pregnancy to hypertension, cardiovascular disease and diabetes in the offspring. This probably occurs via altered foetal programming. Because endothelial signals are essential during islet development, vascular alterations might act during foetal life to trigger T2D. In E21 GK foetuses, β-cell mass is decreased by 80%. Our aims were to evaluate blood glucose and lipids, pancreas vascularisation and gene expression in various organs in E21 GK foetuses, as compared to Wistar controls. glycemia and lipid assays were done on sera; 2) expression of 7 genes, selected on the basis of previous studies conducted in 1-w- Wistar and GK islets, were evaluated by quantitative RT-PCR in umbilical cord, placenta, liver and pancreas; 3) pancreatic vascularisation was quantified after immunohistochemistry for nestin, a recognized endothelial marker. systemic glucose, cholesterol and cholesterol/HDL ratio were significantly higher in GK foetuses, with no difference in triglycerides, FFA and HDL levels; 2) while in 1-w-GK islets, we found a significant upregulation of genes encoding inflammatory molecules, such as caspase-1 (x9.0), IL-18 (x3.9), IL-15 (x1.4) and CXCL-1 (x3.9), the pancreatic expression of the 4 genes was variable from one E21 GK RNA preparation to another, being however more often upregulated. The same trend toward upregulation of inflammatory genes was observed in the placenta, while it was less marked in liver and umbilical cord; the expression of 2 genes encoding proangiogenic factors, neuropilin-1 and neuropeptide Y, which was significantly downregulated in 1-w-GK islets (x0.6 and x0.1, respectively), was not decreased in most E21 GK organs; nevertheless, gene encoding soluble epoxyde hydrolase (sEH), which inhibits the catabolism of epoxyeicosatrienoic acids (EETs), was found to be downregulated by 70-90 % in E21 GK foetal organs; sEH, by increasing levels of EETs, would augment their vasodilatator, antiinflammatory, antioxidative and proangiogenic effects; 3) pancreatic vascularisation was decreased by 60 % in E21 GK foetuses. E21 GK foetuses are hyperglycemic and hypercholesterolemic. Their reduced β-cell mass is associated with decreased pancreatic vasculari sation, which might participate to T2D pathophysiology. The strong underexpression of sEH in various GK foetal organs suggests a very early defence mechanism in GK rats, probably linked to maternal OS induced by carbohydrate and/or lipid disturbances

The prediabetic period is characterized by islet microangiopathy in the Goto-Kakizaki rat, a spontaneous model of type 2 diabetes

International audienceEndothelial signals are essential during pancreas development. Goto-Kakizaki (GK) rats have a 70% decrease in β-cell mass at E18, while it is similar at E13 to that of Wistar control rats. Because adult diabetic GK rats are hypercholesterolemic, we hypothesized that maternal hypercholesterolemia together with maternal/fetal hyperglycemia might alter perinatal pancreas development through islet microangiopathy (atherosclerosis) programming. Our aims were to: 1) demonstrate alterations of vascularisation in islets from 1-week-old GK rats, during the so-called prediabetic period; 2) measure systemic levels of lipids and various cytokines and chemokines around birth. Sera were obtained from 1-week-old male Wistar and GK rats (except otherwise stated) for radioimmunoassays; 2) Islets from these rats were hand-picked after collagenase digestion; 3) Molecular approaches included Affymetrix gene array and low density arrays targeted at endothelium, angiogenesis and cardiovascular disease biomarkers (OligoGEArrayRat). Gene expression was confirmed by qRT-PCR. Results: Affymetrix gene array (230A) showed differential expression of several genes which might be involved in inflammation in GK versus Wistar islets. Notably, the gene encoding epoxide hydrolase, the enzyme responsible for epoxyeicosatrienoic acids (EETs) conversion was decreased by 90%. EETs have anti-inflammatory and protective vascular effects. We then analysed expression of genes present on the 3 targeted arrays mentioned above (128 different genes/array). We obtained 20 up- and 8 down-regulated genes. Among the up-regulated genes were those encoding: caspase 1 (IL-1-converting enzyme, x10) that cleaves IL-1 and IL-18 into their active forms; various cytokines/chemokines involved in macrophage/granulocyte movements: CXCL2 (MIP-2α, x4.1), CXCL-1 (chemokine KC, x3.9), IL-15 (x1.4) and IL- 18 (x3.7); also fibronectin 1 (x2.3), ICAM-1 (x2.6), VEGFR3 (x2), carboxypeptidase 2 (TAFI, x1.9) and angiotensinogen (x1.9). These 3 groups have pro-atherosclerotic effects. Among the down-regulated genes were those encoding: osteoproteogerin (x0.4), neuropilin 1 (x0.6), platelet-derived growth factor (x0.6), coagulation factor III (tissue factor, x0.4) and the neuropeptide Y (x0.1) that acts as vascular mitogen. These data suggest that angiogenesis is deficient in neonatal GK islets. Concomitantly, prediabetic 1-week old-GK rats had lower leptin and higher circulating cholesterol/HDL ratio, MCP-1 and MIP-1α levels than Wistar rats. The latter data might reflect systemic vascular disturbances in GK neonates. Soon after birth many pro-atherosclerotic genes were upregulated in GK islets, while pro-angiogenic genes were downregulated. These data highlight early deficient islet vascularisation that might be responsible for the GK β-cell mass defect and therefore may be of potential therapeutic value

Hypercholesterolaemia, signs of islet microangiopathy and altered angiogenesis precede onset of type 2 diabetes in the Goto-Kakizaki (GK) rat

International audienceThe adult non-obese Goto–Kakizaki (GK) rat model of type 2 diabetes, particularly females, carries in addition to hyperglycaemia a genetic predisposition towards dyslipidaemia, including hypercholesterolaemia. As cholesterol-induced atherosclerosis may be programmed in utero, we looked for signs of perinatal lipid alterations and islet microangiopathy. We hypothesise that such alterations contribute towards defective pancreas/islet vascularisation that might, in turn, lead to decreased beta cell mass. Accordingly, we also evaluated islet inflammation and endothelial activation in both prediabetic and diabetic animals. Blood, liver and pancreas were collected from embryonic day (E)21 fetuses, 7-day-old prediabetic neonates and 2.5-month-old diabetic GK rats and Wistar controls for analysis/quantification of: (1) systemic variables, particularly lipids; (2) cholesterol-linked hepatic enzyme mRNA expression and/or activity; (3) pancreas (fetuses) or collagenase-isolated islet (neonates/adults) gene expression using Oligo GEArray microarrays targeted at rat endothelium, cardiovascular disease biomarkers and angiogenesis, and/or RT-PCR; and (4) pancreas endothelial immunochemistry: nestin (fetuses) or von Willebrand factor (neonates). Systemic and hepatic cholesterol anomalies already exist in GK fetuses and neonates. Hyperglycaemic GK fetuses exhibit a similar percentage decrease in total pancreas and islet vascularisation and beta cell mass. Normoglycaemic GK neonates show systemic inflammation, signs of islet pre-microangiopathy, disturbed angiogenesis, collapsed vascularisation and altered pancreas development. Concomitantly, GK neonates exhibit elevated defence mechanisms. These data suggest an autoinflammatory disease, triggered by in utero programming of cholesterol-induced islet microangiopathy interacting with chronic hyperglycaemia in GK rats. During the perinatal period, GK rats show also a marked deficient islet vascularisation in conjunction with decreased beta cell mass