Decreased brain and muscle ARNT-like protein 1 expression mediated the contribution of hyperandrogenism to insulin resistance in polycystic ovary syndrome

Figures

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  Fig. 1. Characteristics of DHEA-induced PCOS-like rat model. a Estrous cycles of control and DHEA rats in the last 8 days. E (estrum), P (proestrus), M (metestrus), D (diestrus). b HE staining of the ovaries of control and DHEA rats. * for CL, → for cystic follicles. c Body weight of control and DHEA rats over 4 weeks. d Ovarian weight of control and DHEA rats after 4-week treatment. e GTT and area under the curve of control and DHEA rats after 4-week treatment. f Serum leptin levels in control and DHEA rats after 4-week treatment. g Serum adiponectin levels in control and DHEA rats after 4-week treatment. h Serum testosterone levels in control and DHEA rats after 4-week treatment. i Serum LH and FSH levels as well as LH/FSH ratio in control and DHEA rats after 4-week treatment. N = 30 per group. * p < 0.05, ** p < 0.01

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  Fig. 2. Expression of circadian clock genes and insulin pathway genes in the liver tissue of control and DHEA rats. aBmal1, Clock, Per1, Per2, Cry1, and Cry2 mRNA expressions in the liver of control and DHEA rats. b P-AKT, T-AKT, and BMAL1 protein expressions in the liver of control and DHEA rats. Left, a representative western blot is shown. Middle, the immunoreactive bands for BMAL1 were quantified densitometrically. Right, the immunoreactive bands for AKT phosphorylation were quantified densitometrically. c mRNA expression of insulin pathway-associated genes including Sirt1, Nampt, Glut4, and Pparg in the liver of control and DHEA rats. N = 6 per time point and N = 30 for each group in total. * p < 0.05, ** p < 0.01

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  Fig. 3. Expression of circadian clock genes and insulin pathway genes in the adipose tissue of control and DHEA rats. aBmal1, Clock, Per1, Per2, Cry1, and Cry2 mRNA expressions in the adipose of control and DHEA rats. b P-AKT, T-AKT, and BMAL1 protein expressions in the adipose of control and DHEA rats. Left, a representative western blot is shown. Middle, the immunoreactive bands for BMAL1 were quantified densitometrically. Right, the immunoreactive bands for AKT phosphorylation were quantified densitometrically. c mRNA expression of insulin pathway-associated genes including Sirt1, Nampt, Glut4, and Pparg in the adipose of control and DHEA rats. N = 6 per time point and N = 30 for each group in total. * p < 0.05, ** p < 0.01

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  Fig. 4. Hyperandrogenism facilitated BMAL1-mediated insulin resistance through the NAMPT/NAD+/SIRT1 pathway in mature adipocytes. a The mature adipose cells were differentiated from 3 T3-L1 preadipocytes after oil red O staining. b Glucose uptake after NC siRNA, testosterone (10− 6 M for 24 h), Bmal1 siRNA, or Nampt siRNA treatment in mature adipocytes. Glucose uptake was measured after insulin stimulation (100 nM for 20 min). cBmal1, Sirt1, Nampt, Glut4, and Pparg mRNA expressions after knocking down Bmal1 in mature adipocytes. d Cellular NAD+ level after knocking down Bmal1 in mature adipocytes. eBmal1, Sirt1, Nampt, Glut4, and Pparg mRNA expressions after knocking down Sirt1 in mature adipocytes. fBmal1, Sirt1, Nampt, Glut4, and Pparg mRNA expressions after knocking down Nampt in mature adipocytes. gBmal1, Clock, Per1, Per2, Sirt1, and Nampt mRNA expressions after treatment with different doses of testosterone for 24 h in mature adipocytes. hBmal1, Nampt, Sirt1, Glut4, and Pparg mRNA expressions after Bmal1 overexpression and further incubation with testosterone (10− 6 M for 24 h) in mature adipocytes. * P < 0.05, ** P < 0.01, *** P < 0.001 against NC siRNA cells or against T0 0 M…