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

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.

et al. recently found that the gain-of-function mutation of Adcy3 gene had increased Adcy3 activity and cAMP production and consequently the mutant mice had significantly lower total body weights and fat mass compared to wild type controls after 12 weeks of HFD feeding 15 . It has been established that cAMP signaling, in addition to its involvement in regulating feeding behavior and leptin sensitivity in the hypothalamus 16,17 , also has a role in controlling adipose tissue development and function through regulating the expressions of genes related to adipogenesis, lipolysis, and thermogenesis [18][19][20] . There is also some evidence to suggest Adcy3 may play physiological roles in muscle and liver. For example, Griffin et al. found that Adcy3 is expressed in primary muscle cell and is associated with muscle cell migration and adhesion 21 . In addition, hepatic Adcy3 was reported to play a protective role in insulin resistance and obesity in mice with HFD-induced obesity 22 . The present study was aimed at determining whether Adcy3 functions in peripheral tissues related to metabolic disorders by regulating the development of adiposity and insulin resistance in mice.
Adcy3 +/− mice display impaired glucose homeostasis. The oral glucose tolerance test (OGTT) was performed 2 weeks prior to the end of the experimental period. Integrated plasma glucose concentration, as calculated by area under the curve (AUC), was increased in Adcy3 +/− mice relative to their WT counterparts on both chow (18% for male, p < 0.05; 13% for female) and HFD (26% for male, p < 0.05; 32% for female, p < 0.05; Fig. 3a,b,f,g). Fasting plasma glucose and insulin levels were measured at the end of the feeding period. Adcy3 +/− mice fed HFD had significantly higher fasting plasma concentrations of glucose (42% for male, p < 0.05; 69% for female, p < 0.05; Fig. 3c,h) and insulin (28% for male, p < 0.05; 38% for female, p < 0.05; Fig. 3d,i) compared with WT mice. Along with the plasma glucose and insulin levels, the HOMA-IR values indicated that insulin sensitivity was significantly decreased in Adcy3 +/− mice maintained on HFD (Fig. 3e,j).
The protein levels of phosphor-IR, phosphor-IRS1, phosphor-AKT, phosphor-GSK3β were significantly decreased in the epididymal adipose tissue of Adcy3 +/− mice relative to WT mice on both chow and HFD (Fig. 4a). The amount of GLUT4 found in the membrane fraction of the epididymal adipose tissue was significantly decreased, whereas the amount of total cellular GLUT4 remained the same in Adcy3 +/− mice when compared with WT mice (Fig. 4b). Similarly, the protein levels of phosphor-IRS1 and phosphor-AKT were significantly decreased in the liver and muscle of Adcy3 +/− mice relative to WT mice on both chow and HFD (Fig. 4c,d). The expression of the key gluconeogenesis enzymes, such as PEPCK and G6pase, were found to be significantly increased in the liver of Adcy3 +/− mice than in WT mice on both chow and HFD (Fig. 4e).

Insulin-stimulated GLUT4 translocation is decreased in Adcy3 deficient 3T3-L1 adipocytes.
To test whether Adcy3 affects the insulin-stimulated GLUT4 translocation from cytosol to plasma membrane, we transiently knocked down Adcy3 using siRNA in 3T3-L1 adipocytes and measured the membrane-bound GLUT4 in the presence of insulin by western blotting. Relative to scrambled siRNA-transfected controls, treatment with siRNA (Adcy3 SMARTpool) for 48 h achieved significant reductions of Adcy3 expression level in 3T3-L1 adipocytes (Fig. 4f). The loss of Adcy3 led to a significant decrease in the amount of plasma membrane GLUT4 after insulin stimulation (Fig. 4g).
Adcy3 expression in peripheral tissues of mice. HFD feeding significantly downregulated the mRNA expression of Adcy1, Adcy2, Adcy3, and Adcy8 in the epididymal adipose tissue of mice but had no effect on the expression of other Adcy isoforms (Fig. 5e). Adcy3 +/− mice showed significantly decreased total Adcy activity in the epididymal adipose tissue as compared to WT mice fed chow (− 31%, p < 0.01) and HFD (− 16%, p < 0.01) (Fig. 5a). Both mRNA and protein levels of Adcy3 were significantly reduced in the epididymal adipose tissue of Adcy3 +/− mice than in WT mice irrespective of diet (Fig. 5b,e). The protein level of Adcy3 was also significantly decreased in the liver and muscle of Adcy3 +/− mice relative to WT mice on both chow and HFD. (Fig. 5c,d).
Scientific RepoRts | 6:34179 | DOI: 10.1038/srep34179 Altered expression of molecules related to adipogenesis, fatty acid oxidation, and thermogenesis in Adcy3 +/− mice. The protein levels of PKA, phosphor-AMPK, phosphor-HSL, and phosphor-CREB were found to be significantly lower in the epididymal adipose tissue of Adcy3 +/− mice than in WT mice on both chow and HFD (Fig. 6b-d). The expression levels of PKA and phosphor-AMPK were also significantly decreased in the liver and muscle of Adcy3 +/− mice compared with WT mice irrespective of diet (Fig. 6e,f). The expression of genes encoding transcription factors, such as peroxisome proliferator-activated receptor γ2 (PPARγ2) and CCAAT/enhancer binding protein α (C/EBPα), and their targets, adipocyte fatty acid binding protein (aP2) and fatty acid synthase (FAS), were significantly increased in the epididymal adipose tissue of Adcy3 +/− mice relative to WT mice on both chow and HFD (Fig. 6a). The mRNA level of carnitine palmitoyl transferase 1 (CPT1) were significantly downregulated by Adcy3 haploinsufficiency in the epididymal adipose tissue of mice on both chow and HFD (Fig. 6h). Adcy3 haploinsufficiency significantly decreased the expression of thermogenic genes, such as (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.

Discussion
Adcy3 +/− mice (Adcy3 tm1Dgen /J) on a 129/OlaHsd background used in the present study were offspring from heterozygous intercross matings of mice originally obtained from the Jackson Laboratory and were found to be healthy and viable. In contrast, homozygous Adcy3 mice on a 129/OlaHsd background showed a postnatal lethal phenotype 23 , and other homozygous Adcy3 mice on a 129Sv/J background (Adcy3 tm1Drs ) struggled to survive, with an 80% fatality rate within 48 hours, and exhibited abnormal phenotypes, such as anosmia 8 , reduced male fertility 10 , and excretory dysfunction of kidney 11 . In the present study, the increased weight gain and adiposity (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. observed in the Adcy3 +/− mice were not associated with the increased consumption of food, thereby suggesting that Adcy3 haploinsufficiency does not promote hyperphagia. Consistent with our findings, Pitman et al. recently revealed that mice carrying a gain-of-function mutation in Adcy3 (Adcy3 Jll/+ ) were protected from diet-induced obesity due to increased oxygen consumption and physical activity without a change in food intake compared to their WT littermates during the 7 days of whole-body metabolic measurements 15 .
In the present study, all membrane-bound Adcy isoforms reported, from Adcy1 through Adcy9, were expressed in white adipose tissue (WAT) of mice (Fig. 5). Nevertheless, Adcy3 appears to be one of the major isoforms contributing to Adcy activity in WAT based on our observation that Adcy3 haploinsufficiency resulted in a significant decrease (-31%) in total Adcy activity in visceral adipose tissue of mice on chow. Chaudhry et al. reported that in brown adipose tissue of rats, the increase in Adcy activity corresponded to a selective upregulation of Adcy3 expression during the neonatal period when offspring are especially sensitive to environmental conditions and maintenance of body temperature 24 . Moreover, treatment of neonates with the sympathetic neurotoxin 6-hydroxydopamine abolished the perinatal increase in both Adcy activity and Adcy3 mRNA levels without affecting the expression of other Adcy isoforms 24 .
In the present study, the HFD-induced decrease in total Adcy activity observed in visceral adipose tissue of both WT and Adcy3 +/− mice was accompanied by downregulation of Adcy1, Adcy2, Adcy3, and Adcy8 mRNA expression. This finding is in line with previous reports indicating that expression of Adcy isoforms in a specific The cAMP signaling pathways are pivotal in regulation of adipose tissue development and function 27 . The accumulation of cAMP activates PKA in adipocytes of all colors and origins, thereby phosphorylating several proteins, such as CREB 20 , AMPK 28 , and HSL 29 (Fig. 7). Phosphor-CREB then activates the expression of PGC-1α , which induces the transcription of downstream thermogenic genes, including UCP1 20 . In parallel, phosphorylated AMPK inhibits preadipocyte differentiation by downregulating PPARγ and C/EBPα , which are the central regulators of adipogenesis and lipid storage in adipocytes 30 . Moreover, PKA-mediated phosphorylation induces translocation of the HSL to the surface of lipid droplets and enhances its catalytic activity 29 . The released free fatty acids are oxidized to fatty acyl-CoA which combines with CPT1 to enter mitochondria for its oxidation. In the present study, Adcy3 haploinsufficiency significantly decreased the protein levels of PKA, phosphor-CREB, phosphor-HSL, and phosphor-AMPK in WAT of mice fed HFD (Fig. 7). These results indicated that in WAT, Adcy3 might be associated with the regulation of cAMP-PKA-mediated signaling pathways that affect thermogenesis, fatty acid oxidation, and adipogenesis.
Here, Adcy3 haploinsufficiency reduced insulin sensitivity in mice, as demonstrated by as demonstrated by impairment of insulin signaling in WAT, liver, and muscle, along with increased AUC values and elevated fasting plasma glucose and insulin levels. This insulin resistance observed in Adcy3 +/− mice appears to be associated with decreased AMPK activity (Fig. 7). Reduced AMPK activity is known to decrease the phosphorylation of IRS-1 at Ser789, thus negatively regulating insulin signaling in adipocytes 31 . Moreover, Adcy3 +/− mice showed higher  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. plasma levels of proinflammatory cytokines, such as IL-6 and TNFα , relative to their WT littermates on both chow and HFD. It is well established that within adipose tissue, IL-6 and TNFα cause adipocyte insulin resistance through inactivation of both the insulin receptor and IRS-1, both of which result in diminished activation of phosphoinositol-3-kinase, the essential second messenger signal that governs most of metabolic effects associated with insulin 32,33 . Therefore, the increased plasma levels of proinflammatory cytokines could also be a contributing factor for the aggravated insulin resistance in Adcy3 +/− mice.
Possible application of Adcy3 as a target for insulin resistance and anti-obesity treatment has been implicated in a recent study by Liang et al. 22 revealing that hepatic Adcy3 is upregulated in mice with HFD-induced obesity by liraglutide, a glucagon-like peptide-1 analogue recently approved by the US Food and Drug Administration as an obesity treatment option. In conclusion, the Adcy3 heterozygous null mice displayed increased visceral adiposity in the absence of hyperphagia and developed abnormal metabolic features on both chow and HFD. We report for the first time that HFD decreased the Adcy3 expression in white adipose tissue, liver, and muscle and 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. Although the role of Adcy3 in humans has yet to be elucidated, our findings raise the possibility that Adcy3 activators may be useful agents for the prevention or treatment of obesity and associated metabolic complications.
Four-week-old male and female Adcy3 +/− or WT littermate mice were housed in a pathogen-free facility at 21 ± 2.0 °C, with 50 ± 5% relative humidity and a 12-h light/dark cycle. The mice were provided access to rodent chow and tap water ad libitum for 1 week. Thereafter, the mice (n = 8) were placed on chow or HFD for 17 weeks. The HFD consisted of 200 g fat/kg body weight (170 g of lard and 30 g of corn oil) and 1% (w/w) cholesterol (supplementary Table 1).
Food intake and body weight were measured daily and weekly, respectively. At the end of the experimental period, the mice were anesthetized with diethyl ether after they were fasted for 16 h. Blood samples were drawn from the inferior vena cava into an ethylene-diamine-tetra-acetic acid (EDTA)-coated tube, and the plasma samples were obtained by centrifuging the blood at 4,000 g for 15 min at 4 °C. The epididymal, retroperitoneal, mesenteric, perirenal, and subcutaneous fat-pads were dissected, removed, weighed, and immediately snap-frozen in liquid nitrogen and stored at -80 °C until further use. All animal experiments were performed in accordance with the Korea Food and Drug Administration guidelines. All experimental protocols were reviewed and approved by the Institutional Animal Care and Use Committee of the Yonsei Laboratory Animal Research Center (Permit no. 2011-0062).
Cell culture and transfection. 3T3-L1 fibroblasts obtained from American Type Culture Collection (Manassas, VA, USA) were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, penicillin (50 units/mL), and streptomycin (50 mg/mL). The cells were grown at 37 °C in a humidified 5% CO 2 atmosphere. We treated confluent cultures with 0.5 mM 3-isobutyl-1-methylxantine, 0.25 mM dexamethasone, and 10 mg/mL insulin to promote the differentiation of 3T3-L1 cells into adipocytes. After 2 days, the 3-isobutyl-1-methylxanthine and dexamethasone were removed, and insulin was continued for another 2 days. The growth medium was replenished at 2-day intervals until adipocyte differentiation. For Adcy3 knockdown, the differentiated adipocytes were treated with 50 nM of either control siRNA (nontargeting SMARTpool, catalog no. D-001810-10-05, Dharmacon, Lafayette, CO, USA) or siRNA against Adcy3 (On-TARGET plus SMART pool siRNA, catalog no. L-0588903-00-10, Dharmacon) on the ninth day of differentiation. Two days post-transfection, serum-starved cells were treated with 17 nM insulin for 15 min and then lysed for RNA or protein. The knockdown efficiency by siRNA was monitored by semiquantitative RT-PCR.
Biochemical analysis. Plasma content of total cholesterol (TC), triglyceride (TG), free fatty acid (FFA), and glucose were enzymatically determined using individual commercial kits (Bio-Clinical System, Gyeonggi-do, South Korea). Plasma concentration of insulin was measured using a commercially available mouse enzyme-linked immunosorbent assay (ELISA) kit (Millipore, Billerica, MA, USA). The homeostasis model assessment of basal insulin resistance (HOMA-IR) was calculated as [fasting plasma glucose × fasting plasma insulin/22.5] to assess insulin resistance. Hepatic lipids were extracted as described using the method developed by Folch et al. 34 . Triglyceride, cholesterol, and FFA levels in the hepatic lipid extracts were measured using the same enzymatic kits that were used for the plasma analyses.
Oral glucose tolerance test. The OGTT was performed 2 weeks before the end of the experimental period on 6 h-fasted mice by oral glucose administration (gavage with 2 g glucose/kg body weight). Blood glucose was measured from tail blood at times 0, 15, 30, 60, 90, and 120 min after glucose administration.
Adcy enzymatic assay. Fifty microliters of Adcy mixture [100 mM Tris-acetate (pH 7.4), 20 mM KCl, 10 mM MgCl 2 , 20 mM phosphoenolpyruvate, 100 μ g/mg pyruvate kinase, 2 mM ATP, 20 μ M guanosine triphosphate, 2 mM dithiothreitol, 0.4 mM bovine serum albumin, and 0.1 mM 3-isobutyl-1-methylxanthine] and 125 μ g of membrane fractions from epididymal adipose tissue were added into microcentrifugation tubes in duplicate and maintained at 4 °C. The reaction tubes were incubated in a water bath maintained at 37 °C for 30 min, and the reaction was terminated by heating at 95 °C for 5 min. The supernatant solution was stored after centrifugation at 10,000 g for 5 min at 4 °C. The cAMP assay was performed using the cAMP ELISA kit from Enzo Life Sciences International, Inc. (Plymouth Metting, PA, USA).
RNA extraction and semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR). Total RNA was isolated from the liver, epididymal, and subcutaneous adipose tissue of each mouse by using TRIzol (Invitrogen, Carlsbad, CA, USA) and was reverse-transcribed by using the Superscript II Kit (Invitrogen) according to the manufacturer instructions. The PCR was programmed as follows: 10 min at 94 °C, Scientific RepoRts | 6:34179 | DOI: 10.1038/srep34179 30-35 cycles of 94 °C for 30 s, 55 °C for 30 s, 72 °C for 1 min, and 10-min incubation at 72 °C. Then, 5 μ L of each PCR product was mixed with 1 μ L of 6-fold concentrated loading buffer and electrophoresed on a 2% agarose gel containing ethidium bromide. The band intensities were quantified using the Quantity One Analysis Software (Bio-Rad). The mRNA levels were normalized to that of the glyceraldehyde-3-phosphate dehydrogenase transcript. Sequences of all primers are listed in supplementary Table 2.
Statistics. Results on body weight gain and plasma biochemistries are presented as the mean ± standard error of mean (SEM) of eight mice in each group. The RT-PCR and western blot data are shown as the means ± SEM of three independent experiments (n = 2 or 3 per experiment) for each group, cumulatively including eight mice. An unpaired Student t-test analysis was used for all data comparisons between the Adcy3 +/− and WT control mice fed chow or HFD. All statistical analyses were performed with SPSS 21.0 software (IBM, Corp., Armonk, NY, USA), and significance was set at *P < 0.05, **P < 0.01 and ***P < 0.001.