Circulating betatrophin is associated with insulin resistance in humans: cross-sectional and interventional studiesin vivoandin vitro

Figures

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  Figure 1. Circulating betatrophin levels and ROC curve analysis in the study cohort (A) Circulating betatrophin levels in normal and PCOS women with IR (vs. controls: ** P < 0.01). (B) Circulating betatrophin levels according to BMI (normal-weight: BMI < 25kg/m2 and overweight/obese: BMI >25 kg/m2, vs. normal weight: **P < 0.01). (C) Prevalence of elevated PCOS in different quartiles of betatrophin: quartile 1, <0.33μg/L; quartile 2, 0.33-0.49μg/L; Quartile 3, 0.49-0.62μg/L; Quartile 4, >0.62μg/L (vs. quartile 1: *P <0.05, **P <0.01). (D) ROC curve analyses for the prediction of IR according to the betatrophin levels. (E) Circulating betatrophin levels in both IR and healthy women during EHC (vs. controls: ∆P<0.01, #P<0.05; vs. 0 minutes: *P <0.01). (F) Circulating betatrophin levels in PCOS women with IR pre- and post-metformin treatment (vs. baseline: **P<0.01). (G) Circulating betatrophin levels during EHC pre- and post-metformin treatment (vs. baseline: ∆P < 0.01, #P < 0.05; vs. 0 min: *P < 0.01).

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  Figure 2. Expression of betatrophin at mRNA and protein levels in mice (A) Distribution of betatrophin mRNA expression in insulin target tissues from C57BL/6J mice (n=5). Betatrophin mRNA expression in liver (B) and fat (C) of SD- and HFD-fed mice. Betatrophin protein expression in liver (D) and fat (E) of SD- and HFD-fed mice (n = 5/group). SD, standard chow diet; HFD, high-fat diet; Data are presented as the mean ± SD. * P < 0.05, ** P < 0.01 vs. SD.

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  Figure 3. The effects of insulin, metformin and rosiglitazone on the expression of betatrophin in mouse hepatocytes and the hepatocyte/macrophage co-culture systems (A-B) Dose- dependent effects of insulin on betatrophin mRNA (A) and protein(B) expression in mouse hepatocytes. (C-D) Dose-dependent effects of metforminin the presence of 100 nM insulin on betatrophin mRNA (C) and protein (D)expression in mouse hepatocytes. (E-F) Dose- dependent effects of rosiglitazone in the presence of 100 nM insulin on betatrophin mRNA (E) and protein (F) expression in mouse hepatocytes. (G-H) Betatrophin mRNA (G) and protein (H) expression in cell lysates from the hepatocyte/macrophage co-culture systems with or without rosiglitazone treatment in the presence of 100 nM insulin. (I-J) Betatrophin mRNA (I) and protein (J) expression in cell lysates from the hepatocyte/macrophage co-culture systems with or without metformin treatment in the presence of 100 nM insulin. Hep, hepatocytes; Mac, macrophage; Ros, rosiglitazone. Data are presented as the means ± SE. * P < 0.05, **P < 0.01.

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  Figure 4. The effects of betatrophinexpression on insulin signaling in hepatocytes with insulin stimulation Primary hepatocytes were infected with or without recombinant adenovirus vectors encoding betatrophin (A-C) or shRNA specific (D-F) or for betatrophin control green fluorescent protein (Ad-GFP) for 48 h, followed with 100 nmol/L insulin stimulation for 10 min. Western blot was conducted to examine the phosphorylation levels of InsR (A and D), AKT (B and E) and IRS-1 (C and F). Data are presented as the means ± SD. * P < 0.05, ** P < 0.01 vs. control.

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  Figure 5. Effects of serum from normal and IR women on insulin signaling and betatrophin Mouse primary hepatocytes were treated by serum from normal subjects, IR women and IR women after 3 or 6 months of metformin treatment for 24hr. (A) Phosphorylation levels of insulin receptor (InsR). (B) Phosphorylation levels of Akt kinase (Akt). (C) Betatrophin protein expression. Each experiment was performed in three replicates. Control, saline; Normal, normal women serum; Data are shown as the means ± SD. * P <0.05, ** P<0.01.