Oleanolic acid reduces hyperglycemia beyond treatment period with Akt/FoxO1-induced suppression of hepatic gluconeogenesis in type 2 diabetic mice

The present study investigated the chronic efficacy of oleanolic acid (OA), a triterpenoid selected from our recent screening, on hyperglycemia in type-2 diabetic mice. C57BL/6J mice were fed a high-fat diet followed by low doses of streptozotocin to generate a type-2 diabetic model. OA (100 mg/kg/day) was administered orally for 2 weeks with its effects monitored for 6 weeks. High-fat feeding and streptozotocin generated a steady hyperglycemia (21.261.1 mM) but OA administration reversed the hyperglycemia by ,60%

Mice with a Disruption of the Imprinted Grb10 Gene Exhibit Altered Body Composition, Glucose Homeostasis, and Insulin Signaling during Postnatal Life▿

The Grb10 adapter protein is capable of interacting with a variety of receptor tyrosine kinases, including, notably, the insulin receptor. Biochemical and cell culture experiments have indicated that Grb10 might act as an inhibitor of insulin signaling. We have used mice with a disruption of the Grb10 gene (Grb10Δ2-4 mice) to assess whether Grb10 might influence insulin signaling and glucose homeostasis in vivo. Adult Grb10Δ2-4 mice were found to have improved whole-body glucose tolerance and insulin sensitivity, as well as increased muscle mass and reduced adiposity. Tissue-specific changes in insulin receptor tyrosine phosphorylation were consistent with a model in which Grb10, like the closely related Grb14 adapter protein, prevents specific protein tyrosine phosphatases from accessing phosphorylated tyrosines within the kinase activation loop. Furthermore, insulin-induced IRS-1 tyrosine phosphorylation was enhanced in Grb10Δ2-4 mutant animals, supporting a role for Grb10 in attenuation of signal transmission from the insulin receptor to IRS-1. We have previously shown that Grb10 strongly influences growth of the fetus and placenta. Thus, Grb10 forms a link between fetal growth and glucose-regulated metabolism in postnatal life and is a candidate for involvement in the process of fetal programming of adult metabolic health

Changes in Akt and FoxO1 in the liver 4 weeks after the removal of OA.

<p>Four weeks after the cessation of OA treatment, mice were sacrificed following a 5–7 hour fast. Liver samples were freeze-clamped and stored at −80°C for subsequent Western blotting analysis. Representative Western blot images of phosphorylated and total Akt and FoxO1(<i>A</i>). Quantification of p-Akt/GAPDH (<i>B</i>), t-Akt/GAPDH (<i>C</i>), p-/t-Akt (<i>D</i>), p-FoxO1/GAPDH (<i>E</i>), t-FoxO1/GAPDH (<i>F</i>) and p-/t-FoxO1 (<i>G</i>). * p<0.05 vs. CH; † p<0.05, †† p<0.01 vs. T2D-Veh, n = 6–8 per group.</p

Comparisons of blood glucose and ipGTT between pair-fed T2D-Veh and T2D-OA mice.

<p>Food intake (<i>A</i>), body weight (<i>B</i>) and basal blood glucose (<i>C</i>) were monitored throughout the pair-feeding study. ipGTT was performed in mice two weeks after the cessation of OA treatment, with an injection of glucose (1 g/kg, ip) after 5–7 hours of fasting. Blood glucose was monitored at 0, 15, 30, and 60 min following the glucose injection (<i>D</i>). ipGTT results were quantified by calculating the area under the blood glucose curve (AUC) and the incremental AUC (iAUC) (<i>E</i>). T2D-Veh, HF-fed mice with STZ injections; T2D-OA, HF-fed mice with STZ injections and OA treatment. † p<0.05, †† p<0.01 vs. T2D-Veh, n = 5–10 per group.</p

Changes in PEPCK and G6Pase expression in the liver 4 weeks after the removal of OA.

<p>Four weeks after the cessation of OA treatment, mice were sacrificed following a 5–7 hour fast. Liver samples were freeze-clamped and stored for subsequent analysis of PEPCK and G6Pase RNA expression. The expression levels of PEPCK and G6Pase mRNA relative to 18S (<i>A</i> and <i>B</i>). Correlation of p-Akt/GAPDH and FoxO1/GAPDH with G6Pase mRNA expression in T2D-Veh and T2D-OA groups by a best-fit regression analysis (<i>C</i>). * p<0.05 vs. CH, n = 6–8 per group. Proposed mechanism for the sustained reduction in hyperglycemia following the treatment of OA (<i>D</i>).</p

Effects of OA on insulin secretion and insulin in pancreatic β-cells.

<p>Four weeks after the cessation of OA treatment, fresh islets were isolated from T2D mice treated with or without OA, and insulin secretion in response to different glucose concentrations was measured (<i>A</i>). Pancreatic insulin content (<i>B</i>). β-cell area (expressed as a percentage of pancreatic area) (<i>C</i>). Representative images of immunohistochemical staining of β-cells (<i>D</i>). T2D, HF-fed mice with STZ injections; T2D-OA, HF-fed mice with STZ injections and OA treatment. n = 4–6 per group.</p

Changes in glucose flux into muscle, adipose tissue and liver 4 weeks after the removal of OA from the diet.

<p>An ipGTT (1 g/kg, ip) using 2-deoxy-D-[1,2-³H] glucose and D-[¹⁴C] glucose was performed 4 weeks after the cessation of OA treatment. At the end of the 40-min ipGTT, tissue samples were freeze-clamped immediately for the measurement of glucose uptake in quadriceps muscle (<i>A</i>) and epididymal fat (<i>B</i>), as well as the glucose incorporation into lipid (<i>C</i>) and glycogen (<i>D</i>) in the liver. * p<0.05, ** p<0.01 vs. CH, n = 6–12 per group.</p

Effects of OA on glucose tolerance and blood insulin.

<p>Studies were performed in mice two weeks after the removal of OA. ipGTT was performed with an injection of glucose (1 g/kg, <i>ip</i>) after 5–7 hours of fasting. Blood glucose was monitored at 0, 15, 30, and 60 min following the glucose injection (<i>A</i>). ipGTT results were quantified by calculating the area under the blood glucose curve (AUC) and the incremental AUC (iAUC) (<i>B</i>). Insulin levels throughout the ipGTT (<i>C</i>). The average value of blood insulin levels from 5 to 60 mins during the ipGTT (<i>D</i>). CH, normal chow fed mice; T2D-Veh, HF-fed mice with STZ injections; T2D-OA, HF-fed mice with STZ injections and OA treatment. ** p<0.01 vs. CH; †† p<0.01 vs. T2D-Veh, n = 5–8 per group.</p