Defective IGF-2 and IGF-1R protein expressions in pancreatic rudiment precede beta cell mass anomaly in GK rat

International audienceThe Goto-Kakizaki (GK) rat is a spontaneous model of type 2 diabetes with a defective betacell mass detectable in late fetal age. To determine the involvement of IGF-2 in this defect, we evaluated during GK pancreatic organogenesis: 1) the GK beta-cell development on embryonic day (E) 13.5 and E18.5; 2) IGF-2 and IGF-1 receptor (IGF-1R) pancreatic protein expressions at E13.5 and E18.5; 3) the in vitro development of E13.5 GK pancreatic rudiment; 4) the effect of IGF-2 addition on beta-cell mass in pancreatic rudiment in vitro. GK rats were obtained from our local colony. Wistar (W) rats were used as control. Beta-cell quantitative analyses were determined by immunohistochemistry and morphometry at E13.5, E18.5 and in pancreatic rudiments after culture. IGF-2 and IGF-1R pancreatic protein expressions were evaluated using Western blot analyses at E13.5 and E18.5. For culture experiments, dorsal pancreatic rudiments were dissected at E13.5, separated from its surrounding mesenchyme and cultured for 7 days onto three-dimensional collagen gel at 5.5 mM of glucose. Recombinant IGF-2 (100 ng/ml) was added every day to the culture medium. To label cells in the S phase, BrdU was added to the culture medium one hour before the end of the culture. Apoptosis in pancreatic rudiment was evaluated using the ApopTag kit. The number of beta-cell was normal in GK pancreatic rudiment at E13.5. However, pancreatic beta-cell mass was decreased by 74% in E18.5 GK as compared to W control at the same age. 2) IGF-2 and IGF-1R protein expressions were respectively decreased by 27% and 42% in E13.5 GK pancreatic rudiments as compared to W controls. Again, IGF-2 and IGF-1R pancreatic protein expressions were decreased (by 58% and 34% respectively) in GK at E18.5 as compared to W values. 3) After 7 days of culture, GK pancreatic rudiments exhibited a beta-cell number reduced by 78%. This beta-cell defect was similar to that observed in E18.5 GK. After culture, the total pancreatic-cell and the beta-cell proliferation rates in GK pancreatic rudiment were decreased respectively by 27% and by 40% as compared to W values. Moreover, the apoptotic rate of total cells was increased by 42% in GK pancreatic rudiment after culture. 4) The daily addition of IGF-2 to GK pancreatic rudiment increased the beta-cell number by 123%, the total cell proliferation rate by 55%, and the beta-cell proliferation rate by 111% as compared to GK pancreatic rudiment cultured without IGF-2. Taken together these data show that while beta-cell mass is already decreased at E18.5, the differentiation of the first beta cells is in fact normal in E13.5 GK pancreas. Thus, a defective IGF-2 and IGF-1R protein expressions within the GK pancreatic rudiment represent a primary anomaly. The isolated GK pancreatic rudiment as maintained in vitro under the conditions here described, mimicks the GK beta-cell deficiency as observed in vivo. This in vitro approach was useful to highlight the effect of an IGF-2 supplementation on GK beta-cell growth. Moreover, it also suggests that, IGF signaling pathway seems to be functional in GK beta cells in response to IGF-2

Defective IGF-2 and IGF-1R protein expressions in pancreatic rudiment precede beta cell mass anomaly in GK rat

International audienceThe Goto-Kakizaki (GK) rat is a spontaneous model of type 2 diabetes with a defective betacell mass detectable in late fetal age. To determine the involvement of IGF-2 in this defect, we evaluated during GK pancreatic organogenesis: 1) the GK beta-cell development on embryonic day (E) 13.5 and E18.5; 2) IGF-2 and IGF-1 receptor (IGF-1R) pancreatic protein expressions at E13.5 and E18.5; 3) the in vitro development of E13.5 GK pancreatic rudiment; 4) the effect of IGF-2 addition on beta-cell mass in pancreatic rudiment in vitro. GK rats were obtained from our local colony. Wistar (W) rats were used as control. Beta-cell quantitative analyses were determined by immunohistochemistry and morphometry at E13.5, E18.5 and in pancreatic rudiments after culture. IGF-2 and IGF-1R pancreatic protein expressions were evaluated using Western blot analyses at E13.5 and E18.5. For culture experiments, dorsal pancreatic rudiments were dissected at E13.5, separated from its surrounding mesenchyme and cultured for 7 days onto three-dimensional collagen gel at 5.5 mM of glucose. Recombinant IGF-2 (100 ng/ml) was added every day to the culture medium. To label cells in the S phase, BrdU was added to the culture medium one hour before the end of the culture. Apoptosis in pancreatic rudiment was evaluated using the ApopTag kit. The number of beta-cell was normal in GK pancreatic rudiment at E13.5. However, pancreatic beta-cell mass was decreased by 74% in E18.5 GK as compared to W control at the same age. 2) IGF-2 and IGF-1R protein expressions were respectively decreased by 27% and 42% in E13.5 GK pancreatic rudiments as compared to W controls. Again, IGF-2 and IGF-1R pancreatic protein expressions were decreased (by 58% and 34% respectively) in GK at E18.5 as compared to W values. 3) After 7 days of culture, GK pancreatic rudiments exhibited a beta-cell number reduced by 78%. This beta-cell defect was similar to that observed in E18.5 GK. After culture, the total pancreatic-cell and the beta-cell proliferation rates in GK pancreatic rudiment were decreased respectively by 27% and by 40% as compared to W values. Moreover, the apoptotic rate of total cells was increased by 42% in GK pancreatic rudiment after culture. 4) The daily addition of IGF-2 to GK pancreatic rudiment increased the beta-cell number by 123%, the total cell proliferation rate by 55%, and the beta-cell proliferation rate by 111% as compared to GK pancreatic rudiment cultured without IGF-2. Taken together these data show that while beta-cell mass is already decreased at E18.5, the differentiation of the first beta cells is in fact normal in E13.5 GK pancreas. Thus, a defective IGF-2 and IGF-1R protein expressions within the GK pancreatic rudiment represent a primary anomaly. The isolated GK pancreatic rudiment as maintained in vitro under the conditions here described, mimicks the GK beta-cell deficiency as observed in vivo. This in vitro approach was useful to highlight the effect of an IGF-2 supplementation on GK beta-cell growth. Moreover, it also suggests that, IGF signaling pathway seems to be functional in GK beta cells in response to IGF-2

Insulin production and glucose metabolism in isolated pancreatic islets of rats with NIDDM

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Metabolic and secretory response to glucose by islets from rats with non-insulin-dependent diabetes

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Defective IGF2 and IGF1R protein production in embryonic pancreas precedes beta cell mass anomaly in the Goto-Kakizaki rat model of type 2 diabetes

International audienceThe Goto-Kakizaki (GK) rat is a spontaneous model of type 2 diabetes. Defective beta cell mass detectable in late fetal age precedes the onset of hyperglycaemia. Our hypothesis was that an embryonic IGF production deficiency might be involved in beta cell mass anomaly in the diabetic GK rat. To test this, we evaluated during pancreatic organogenesis: (1) the beta cell development in GK rats on embryonic day (E) 13.5 and E18.5; (2) IGF2 and IGF1 receptor (IGF1R) pancreatic protein production on E13.5 and E18.5; (3) the in vitro development of GK pancreatic rudiment on E13.5; and (4) the in vitro effect of IGF2 addition on beta cell mass. Beta cell quantitative analyses were determined by immunohistochemistry and morphometry. IGF2 and IGF1R pancreatic protein production was evaluated using western blot analyses. Dorsal pancreatic rudiments were dissected on E13.5, separated from surrounding mesenchyme and cultured for 7 days without or with recombinant IGF2. While beta cell mass was already decreased on E18.5, the differentiation of the first beta cells was in fact normal in E13.5 GK pancreas. Moreover, defective IGF2 and IGF1R protein production was detected in GK pancreatic rudiment as early as E13.5. The isolated GK pancreatic rudiment as maintained in vitro mimics the GK beta cell deficiency observed in vivo. This last approach enabled us to show that GK beta cells were fully responsive to IGF2 as far as their net growth is concerned. In diabetic GK rat, defective IGF2 and IGF1R protein production in embryonic pancreas precedes beta cell mass anomaly. IGF2 supplementation expands the pool of beta cells