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

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

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

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

Effects of OA on triglyceride levels in plasma and liver during and after OA treatment.

<p>Lipid accumulation in the liver and plasma was assessed both during OA treatment (at week 2) and 4 weeks after the cessation of OA treatment. Mice were euthanized following a 5–7 hour fast and samples of plasma (<i>A</i>) and liver (<i>B</i>) were collected for the measurement of triglyceride levels. 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

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 blood glucose, food intake and body weight in T2D and T1D mice over time.

<p>HF-fed mice with STZ injections were treated with (T2D-OA) or without (T2D-Veh) OA in the diet for two weeks, at the end of which OA was removed from the diet. Blood glucose, food intake and body weight were monitored between 14:00 and 16:00 once a week (<i>A, B</i> and <i>C</i>). CH-fed mice with STZ injections were treated with (T1D-OA) or without (T1D-Veh) OA in the diet for two weeks, at the end of which OA was removed from the diet. Effects of OA on hyperglycemia in T1D mice (D). CH, normal chow fed mice. Data are expressed as means ± SE. † p<0.05, †† p<0.01 vs. T2D-Veh group, n = 11–16 per group.</p