Characterisation of the effect of diet composition on C57BL/6 mice.

<p>(A) Effect of Chow, HCD and HFD on body weight over 12 weeks (n = 5 per group). (B) Effect of Chow, HCD and HFD on percentage body fat over 12 weeks, as assessed by DEXA scan (n = 5 per group). (C) RQ and (D) Energy expenditure from a representative day and night. Data from CLAMS metabolic cages after 12 weeks on diet (n = 4 per group). (E) Food intake and (F) Energy intake averaged over 12 weeks (n = 5 per group). Results are expressed as mean ± SEM. Statistical analysis was by two-way repeated measures ANOVA followed by Dunnett’s post-hoc test (A and B) or a one-way ANOVA followed by Dunnett’s post-hoc test (C, D and E). (*: p<0.05, ***: p<0.001) (a: p<0.05 vs Chow group, b: p<0.05 vs HCD group)</p

Dietary Macronutrient Composition Directs ChREBP Isoform Expression and Glucose Metabolism in Mice

<div><p>Carbohydrate response element binding protein (ChREBP) is a lipogenic transcription factor that is thought to be involved in the development of hepatic steatosis and insulin resistance. Increased ChREBP expression in liver results in increased hepatic steatosis, and the isoform ChREBPβ in adipose tissue can predict insulin sensitivity in obese humans. As ChREBP is activated by glucose, it was postulated that the composition of diet would regulate ChREBP isoform expression in metabolically relevant tissues.</p><p>We compared the effects of diets with high complex carbohydrate, high fat, or a normal chow on ChREBP expression and metabolic parameters in C57BL/6 mice. We found that diets high in fat decrease ChREBP expression in adipose tissue, but isocaloric diets high in carbohydrate have no effect. Interestingly, this decrease in adipose ChREBP was associated with increased inflammatory markers. In the same animals a high carbohydrate diet induced a robust increase in hepatic ChREBPβ expression (≈2-fold; p = 0.0002), but little detectable change in the more abundant ChREBPα transcript. This change was accompanied by increased expression of target genes liver pyruvate kinase (p<0.0001), acetyl-CoA carboxylase (p = 0.0191) and stearoyl-CoA desaturase-1 (p = 0.0045). This increase in ChREBP expression was associated with increased hepatic steatosis, despite no changes in body weight or body fat when compared to chow-fed mice. Unexpectedly, mice fed a high carbohydrate diet displayed enhanced sensitivity to exogenous insulin, despite having mild glucose intolerance and increased liver steatosis.</p><p>In summary, we have shown the composition of diet can selectively regulate ChREBP isoform expression in a tissue specific manner. Furthermore, we have shown a high complex carbohydrate diet selectively increases hepatic ChREBPβ expression, which associates with hepatic steatosis but not insulin resistance. In contrast, a high fat diet reduces adipose ChREBP, which associates with inflammation and insulin resistance.</p></div

Effect of diet on inflammatory gene expression and serum inflammatory markers.

<p>Effect of diet on gene expression in liver (A), WAT (B) and ileum (C) and serum resistin (D) and PAI-1 (E). N = 5 per group. For gene expression, results were normalized to expression of the housekeeping gene 36B4, and then shown as fold change versus the chow control mice. Results are expressed as mean ± SEM. Statistical analysis was by two-way ANOVA (A, B, C) or one-way ANOVA (D, E) followed by Dunnett’s post-hoc test. (*: p<0.05, **: p<0.01, ***: p<0.001, ****: p<0.0001 versus chow fed mice)</p

Effect of diet on sensitivity to exogenous insulin in C57BL/6 mice.

<p>(A) Serum non-esteried fatty acid (NEFA) levels 10 minutes after a saline or insulin injection (5U/mouse) (n = 3–4 per group). (B) Pancreatic insulin content after 12 weeks on diet (n = 13–14). Western blotting for Akt, p-Ser473-Akt in liver (C), WAT (E) and gastrocnemius muscle (G) of mice after a 4 hour fast followed by either a saline or insulin injection (5U/mouse) 10 minutes before sacrifice. Band density was analysed using Image J and pAkt/Akt ratio was calculated and graphed using chow/saline as control (D, F, H). Results are expressed as mean ± SEM. Statistical analysis was by two-way ANOVA followed by Dunnett’s post-hoc test (A, D, F, H). (*: p<0.05, **: p<0.01 vs sham controls) (a: p<0.01 vs sham controls, b: p<0.01 vs HFD insulin)</p

Effect of diet on ChREBP gene expression and the lipogenic pathway.

<p>Effect of diet on gene expression in liver (A), skeletal muscle (B), white adipose tissue (C) and ileum (D). N = 5 per group. Results were normalized to expression of the housekeeping gene 36B4, and then shown as fold change versus the chow control mice. Results are expressed as mean ± SEM. Statistical analysis was by two-way ANOVA followed by Dunnett’s post-hoc test. (*: p<0.05, **: p<0.01, ***: p<0.001, ****: p<0.0001 versus chow fed mice)</p

A high-calorie, high-carbohydrate diet results in glucose intolerance but improved insulin sensitivity.

<p>(A) Glucose tolerance test (GTT), (B) Area under the curve (AUC) of GTT, (C) Insulin tolerance test (ITT), and (D) AUC of ITT. Tests performed after 12–14 weeks on diet. (E) GTT and (F) ITT percentage change after 2 weeks on diet. (G) Serum insulin. Statistical analysis was by two-way repeated measures ANOVA followed by Dunnett’s post-hoc test (A, C, E and F), one-way ANOVA followed by Dunnett’s post-hoc test (B, D), or two-way ANOVA followed by Dunnett’s post-hoc test (G). (**: p<0.01, ***: p<0.001, ****: p<0.0001) (a: p<0.05 vs Chow group, b: p<0.05 vs HCD group, c: p<0.05 HCD vs chow, d: p<0.05 HFD vs chow)</p

Primer sequences for RT-qPCR

<p>Primer sequences for RT-qPCR</p

α-MSH (100 nM) stimulated TBC1D1 S237 and T596 phosphorylation in WT and AMPK KD mice.

<p>TBC1D1 S237 and T596 phosphorylation sites were measured in soleus muscle using WB as described (n indicated in the individual bars). Phosphorylation of TBC1D1 S237 and T596 is normalized to total TBC1D1. Findings are shown as representative immunoblots and pooled data is quantified in bar graphs as arbitrary units. 2-way RM ANOVA was used to calculate statistical significance (*p < 0.05, **p < 0.01, ***p < 0.001 vs. vehicle). ^# indicates a significant effect of genotype (^#p < 0.05, ^##p < 0.01, ^###p < 0.001).</p

α-MSH (100 nM) stimulated 2-Deoxy Glucose uptake and TBC1D1 S237, T596 and S700 phosphorylation +/- H89 in dissected soleus explants from WT mice.

<p>A: Soleus muscle was dissected, stimulated and 2-DG was measured as described (n indicated in the individual bars). B: Phosphorylation of TBC1D1 was measured in soleus muscle using WB as described. TBC1D1 S237, T596 and S700 phosphorylation is normalized to total TBC1D1. Findings are shown as a representative immunoblot and pooled data quantified in bar graphs as arbitrary units. 2-way RM ANOVA was used to calculate statistical significance (*p < 0.05, **p < 0.01, ***p < 0.001 vs. vehicle). ^# indicates a significant effect of genotype (^#p < 0.05, ^##p < 0.01, ^###p < 0.001). Data generated in the experiment are only obtained from experiment day 4.</p

α-MSH stimulated 2-Deoxy Glucose uptake in soleus and extensor digitorum longus (EDL) muscle explants.

<p>3.A: soleus explants from WT and AMPK KD mice were stimulated with α-MSH (100 nM). Data is presented as the mean ± SEM of pooled data from a series of experiments (see individual bars). 3.B: EDL explants from WT and AMPK KD mice were stimulated with α-MSH (100 nM). 3.C: Phosphorylation of Akt was measured in soleus explants after α-MSH-stimulation (100 nM). Data is presented as the mean ± SEM. 2-way RM ANOVA was used to calculate statistical significance (*p < 0.05, **p < 0.01, ***p < 0.001 vs. vehicle). ^# indicates a significant effect of genotype (^#p < 0.05, ^##p < 0.01, ^###p < 0.001).</p