Adenylyl cyclase 3 haploinsufficiency confers susceptibility to diet-induced obesity and insulin resistance in mice

Abstract

Adenylyl cyclase 3 (Adcy3), a member of the mammalian adenylyl cyclase family responsible for generating the second messenger cAMP, has long been known to play an essential role in olfactory signal transduction. Here, we demonstrated that Adcy3 heterozygous null mice displayed increased visceral adiposity in the absence of hyperphagia and developed abnormal metabolic features characterized by impaired insulin sensitivity, dyslipidemia, and increased plasma levels of proinflammatory cytokines on both chow and high-fat diet (HFD). Of note, HFD decreased the Adcy3 expression in white adipose tissue, liver, and muscle. We also report for the first time that Adcy3 haploinsufficiency resulted in reduced expression of genes involved in thermogenesis, fatty acid oxidation, and insulin signaling, with enhanced expression of genes related to adipogenesis in peripheral tissues of mice. In conclusion, these findings suggest that cAMP signals generated by Adcy3 in peripheral tissues may play a pivotal role in modulating obesity and insulin sensitivity.

Figure 1. Adcy3^+/− mice are prone to develop visceral adiposity.

(a,f) Changes in body weight during 17 weeks of feeding. (b,g) Cumulative body weight gain. (c,h) Daily food intake. (d,i) Food efficiency ratio (FER; bodyweight gain over the experimental period [g]/food intake over the experimental period [g]). (e,j) Visceral fat-pad weight. Data for both male (upper panels) and female (lower panels) mice are presented. Values are presented as means ± SEM (n = 8). Significant differences between groups are indicated by asterisks; *P < 0.05; **P < 0.01; ***P < 0.001.

Figure 2. Plasma and hepatic biochemical parameters in mice.

Plasma levels of (a,j) triglyceride, (b,k) total cholesterol, and (c,l) FFA. Hepatic contents of (d,m) triglyceride, (e,n) cholesterol, and (f,o) FFA. Plasma levels of (g,p) leptin, (h,q) IL-6, and (i,r) TNFα. Data for both male (upper panels) and female (lower panels) mice are presented. Values are presented as mean ± SEM (n = 8). Significant differences between groups are indicated by asterisks; *P < 0.05; **P < 0.01; ***P < 0.001.

Figure 3. Adcy3^+/− mice have impaired glucose tolerance and are less insulin sensitive.

(a,f) Oral glucose tolerance test. (b,g) Area under the curve. (c,h) Fasting plasma glucose levels. (d,i) Fasting insulin levels. (e,j) Homeostasis model assessment of basal insulin resistance. Data for both male (upper panels) and female (lower panels) mice are presented. Values are presented as means ± SEM (n = 8). Significant differences between groups are indicated by asterisks; *P < 0.05; **P < 0.01; ***P < 0.001.

Figure 4. Adcy3 haploinsufficiency led to impairment of insulin signaling.

(a,b) Protein levels of p-IR, IR, p-IRS1, IRS1, p-AKT, AKT, p-GSK3β, GSK3β, membrane GLUT4, and total GLUT4 in the epididymal adipose tissue. (c,d) Protein levels of p-IRS1, IRS1, p-AKT, and AKT in the (c) liver and (d) muscle. (e) mRNA levels of G6Pase and PEPCK in the liver of mice. (f) The knockdown efficiency by siRNA was monitored by semiquantitative RT-PCR. (g) Insulin-stimulated GLUT4 translocation in 3T3-L1 adipocytes treated with control siRNA or siRNA against Adcy3. The full-length blots/gels are presented in Supplementary Figs 1–7. Values are presented as means ± SEM (n = 8). Significant differences between groups are indicated by asterisks; *P < 0.05; **P < 0.01; ***P < 0.001.

Figure 5. Adcy3 expression in peripheral tissues of mice.

(a) cAMP concentrations in the epididymal adipose tissue of mice. (b–d) Protein level of Adcy3 in the (b) epididymal adipose tissue, (c) liver, and (d) muscle of mice. (e) mRNA levels of Adcy isoforms in the epididymal adipose tissue of mice. The full-length blots/gels are presented in Supplementary Figs 8–11. Values are presented as means ± SEM (n = 8). Significant differences between groups are indicated by asterisks; *P < 0.05; **P < 0.01; ***P < 0.001.

Figure 6. Adcy3^+/− mice display altered expression of genes related to adipogenesis, fatty acid oxidation, and thermogenesis.

(a) mRNA levels of PPARγ2, C/EBPα, and their target genes in the epididymal adipose tissues of mice. (b) Protein levels of p-AMPK and total AMPK in the epididymal adipose tissues of mice. (c) Protein levels of PKA, p-HSL, and total HSL in the epididymal adipose tissues of mice. (d) Protein levels of p-CREB and total CREB in the epididymal adipose tissues of mice. (e,f) Protein levels of PKA and p-AMPK in the (e) liver and (f) muscle of mice. (g,h) mRNA levels of thermogenic genes in the (g) epididymal and (h) subcutaneous adipose tissues of mice. (i) Gene expression of CPT1 in the epididymal adipose tissue of mice. The full-length blots/gels are presented in Supplementary Figs 12–20. Values are presented as means ± SEM (n = 8). Significant differences between groups are indicated by asterisks; *P < 0.05; **P < 0.01; ***P < 0.001.

Figure 7. Schematic presentation of the Adcy3-mediated signaling pathways related to adipogenesis, thermogenesis, fatty acid oxidation, and insulin resistance.