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 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

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

BMTs activate AMPK through CaMKK and not through intracellular Ca²⁺ changes.

<p>Serum-starved L6 myotubes were treated with either vehicle (V) alone, BMT-17 at 0.1 µM, 1 µM or 10 µM for 30 min before lysis; 10 µg of lysate was then analysed by Western blot (<b>A</b>) and quantified by densitometry (<b>B</b>). A representative blot is shown. Serum-starved L6 myotubes were pre-treated with either 150 µM EGTA-AM (+) or diluent (-) for 15 min before then being treated with 10 µM BMT-17 or vehicle for a further 30 min before lysis. AMPK was then isolated from 50 µg lysate by pan-AMPKβ immunoprecipitation and assessed by AMPK <i>in vitro</i> kinase assay (<b>C</b>). Data are means relative to untreated vehicle control (V) ± SEM from 5 independent experiments. *p<0.05, **p<0.01 to V by one-way ANOVA.</p

Incubation with elevated glucose levels or leucine increases muscle glycogen content at 30 min and 1h.

<p>Glycogen content was measured in EDL muscles incubated for 30 or 60 min in media containing 5.5 or 25 mM glucose (A) or 5.5mM glucose with or without 100μM leucine (B). Since no changes were found between 5.5mM glucose at 30 and 60min, only 30min values are shown. Results are means <u>+</u> SE (n = 4–6). *P <0.05 compared to incubation with 5.5 mM glucose.</p

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

BMTs do not directly activate AMPK in isolated AMPK complexes.

<p>AMPK complexes were isolated from untreated 18 hr serum-starved L6 myotube lysates by immunoprecipitation using a pan-AMPKβ antibody. The isolated AMPK was then incubated <i>in vitro</i> with either vehicle (V) alone, 10 µM BMT-17, BMT-1 or Abbott compound (A-769662) in the presence or absence of 200 µM AMP in AMPK assay buffer for 10 min at 30°C. The <i>in vitro</i> kinase assay was then initiated with the addition of 200 µM ³²P-Mg.ATP and assayed for a further 10 min at 30°C. Incorporation of the ³²P into the substrate peptide was then assessed by beta scintillation counting. Data are means ± SEM for 5 separate experiments. **p<0.01 for compound effect, ^##p<0.01 for AMP effects by two-way ANOVA. No interaction between treatment groups was apparent.</p

Inhibition of glucose-induced mTOR/p70S6K phosphorylation does not affect AMPK phosphorylation.

<p>EDL were preincubated in the presence of rapamycin (100μM) for 30 min and then with 5.5 or 25 mM glucose for 1hr. Muscle lysates were analyzed for P-AMPK Thr^<b>172</b> (A), P-mTOR Ser^<b>2448</b> (B) and AMPK Ser^{<b>485/491</b>} (C) by western blot. Results show quantification of western blots by densitometry. Results are means ± SE (n = 5). *, p<0.05 compared to values for 5.5 mM glucose.</p

Incubation with elevated glucose levels or leucine or and <i>in vivo</i> glucose infusion increases PP2A activity at 2h (<i>ex vivo</i>) or 8h (<i>in vivo</i>).

<p>EDL muscles incubated for 30, 60 and 120 min in media containing 5.5 or 25 mM glucose (A) or 5.5mM glucose with or without 100μM leucine (C) and red gastrocnemius muscles from rats infused with glucose for 0, 3, 5, or 8h (B) were analyzed for PP2A activity as described in the methods section. Results are means <u>+</u> SE (n = 4–6). *P <0.05 compared to 30 min incubation with 5.5 mM glucose (A) and (C) or the 0h group for infusions (B).</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