Exchange protein directly activated by cAMP (Epac) 2a-knockout (KO) mice exhibit accelerated diet-induced obesity and are resistant to leptin-mediated adipostatic signaling from the hypothalamus to adipose tissue, with sustained food intake. However, the impact of Epac2a deficiency on hypothalamic regulation of sympathetic nervous activity (SNA) has not been elucidated. This study was performed to elucidate the response of Epac2a-KO mice to dexamethasone-induced muscle atrophy and acute cold stress. Compared to age-matched wild-type mice, Epac2a-KO mice showed higher energy expenditures and expression of myogenin and uncoupling protein-1 in skeletal muscle (SM) and brown adipose tissue (BAT), respectively. Epac2a-KO mice exhibited greater endurance to dexamethasone and cold stress. In wild-type mice, exogenous leptin mimicked the responses observed in Epac2a-KO mice. This suggests that leptin-mediated hypothalamic signaling toward SNA appears to be intact in these mice. Hence, the potentiated responses of SM and BAT may be due to their high plasma leptin levels.
Exchange protein directly activated by cAMP (Epac) is critical for cAMP signaling with cAMP-dependent protein kinases (1). Epac proteins are encoded by two distinct genes, yielding Epac1 and Epac2 (
Leptin is a critical hormone that links energy intake and storage to energy consumption (5). Non-obese humans and animals are responsive to exogenous leptin (6–8), which increases food satiety and energy consumption. Plasma leptin levels increase in proportion to adipose tissue mass to facilitate its role in the hypothalamus (9–11). The ARH responds to leptin and regulates the release of orexigenic and anorexigenic hormones (5). Appetite control by anorexigenic hormones is mediated by melanocortin 4 receptors (MC4R) (12). However, at the high leptin levels that are observed in the obese state, the hypothalamus becomes less responsive to exogenous leptin.
Hypothalamic leptin signaling elevates sympathetic nervous activity (SNA) to decrease body fat mass by increasing energy expenditure (EE), which is mediated by both MC4R-dependent and independent pathways (13–15). Obesity is associated with high blood pressure and hyperleptinemia, implying that leptin resistance does not influence the leptin-mediated hypothalamic activation of SNA (16). In addition, under leptin resistance, the effects of leptin on sympathetic tone in brown adipose tissue (BAT) were maintained by signaling from the dorsomedial hypothalamus (13).
The distribution of Epac2a in the distinct nuclei of the hypothalamus and its role in the leptin-mediated hypothalamic regulation of SNA has not been fully clarified. Thus, this study aimed to evaluate the possible changes in SNA in Epac2a-KO mice, by observing the response of peripheral tissues to dexamethasone-induced muscle atrophy and cold stress. Because the skeletal muscle and BAT possess plasmalemmal long-form leptin receptors (OB-Rb) (17) as well as sympathetic G-protein-coupled receptors, the results may be helpful for understanding whether leptin signaling in myocytes and brown adipocytes is affected under hypothalamic leptin resistance and hyperleptinemia.
Epac2a-KO mice appeared to be normal when compared to WT mice. We checked the protein expression of Epac2a in WT mice. Consistent with previous reports (3), the hypothalamus and heart expressed Epac2a but interscapular BAT (iBAT) and gastrocnemius muscle did not express Epac2a (Fig. 1A). Remarkably, Epac2a-KO mice had elevated fasting serum leptin levels (Fig. 1B), but food intake was not different from that of WT mice (Fig. 1C). No difference in rectal temperature was detected between the two groups (Fig. 1D). Oxygen consumption (VO2), carbon dioxide production (VCO2), and EE in KO mice were higher than those in WT mice (Fig. 1E), although the physical activity of KO mice was lower compared to WT mice (Fig. 1F). Myogenin levels were increased in the muscle of Epac2a-KO mice compared to the expression in WT mice (Fig. 1G). In addition, the cross-section area (CSA) of the muscle fibers was greater in KO mice. The average CSA of muscle fiber was 1546.6 ± 56.04 μm2 and 1808.9 ± 75.50 μm2 in WT and Epac2a-KO mice, respectively (
To observe the activity of BAT in Epac2a-KO mice, the mice were exposed to a cold environment (4°C) for 6 h. Epac2a-KO mice better-maintained body temperature in response to cold stress, and the decrease in body temperature induced by cold exposure was smaller than that in WT mice (Fig. 2A). The mass of iBAT and white adipose tissue at the thermoneutral condition was less in KO mice than in WT mice (Fig. 2B), consistent with the greater EE in KO mice. Nevertheless, the magnitude of cold stress-induced fat-mass decrease was similar between the two groups. Serum creatine kinase concentrations in the two groups were not different (data not shown), indicating that the shivering activity of the skeletal muscle did not differ. Comparison of the expression of thermogenesis-related proteins in iBAT showed greater cold stress-mediated elevation in the expression of uncoupling protein-1 (UCP-1), peroxisome proliferator-activated receptor gamma coactivator 1 alpha (PGC-1α), CCAAT-enhancer-binding proteins beta (C/EBPβ), carnitine palmitoyltransferase 1b (CPT1b), carnitine palmitoyltransferase 2 (CPT2), and peroxisome proliferator-activated receptor alpha (PPARα) in Epac2a-KO mice (Fig. 2C). Likewise, mRNA expression levels exhibited similar results to proteins (Fig. 2D). Remarkably, the expression of the UCP-1 protein in Epac2a-KO mice was higher than that in WT mice even at ambient temperature, and protein levels further increased during low-temperature exposure. After cold stress, we observed that the mRNA expression of cluster of differentiation 36 (Cd36), cytochrome c oxidase subunit VIIIb (Cox8b), and cytochrome complex (CytoC), which are associated with fatty acid uptake and mitochondrial oxidative metabolism, was higher in Epac2a-KO mice than in WT mice (Fig. 2E). To determine whether the hyperleptinemia observed in Epac2a-KO mice caused the increased cold endurance, leptin (5 μg/g) was administered intraperitoneally (i.p.) to WT mice, and then the mice were immediately exposed to a cold environment (4°C) for 6 h. Expectedly, the leptin-treated WT mice maintained a more stable body temperature than untreated WT mice (Fig. 2F).
The body weight of 7-week-old WT and Epac2a-KO mice did not differ (Fig. 3A). Dexamethasone treatment for 7 days (i.p.) significantly decreased the body weight of WT mice, but not that of Epac2a-KO mice. Accordingly, dexamethasone treatment reduced gastrocnemius muscle weight in WT mice, but not in Epac2a-KO mice. We then tested the functional performance of the gastrocnemius muscle with a treadmill exercise test. Treatment with dexamethasone significantly decreased the running time of the WT mice. Although dexamethasone treatment decreased the running time of Epac2a-KO mice, the decrease was less than that in WT mice (Fig. 3B). Consistent with the notion that dexamethasone damages skeletal muscle, serum creatine kinase levels in WT mice were elevated by dexamethasone treatment; however, the effect of dexamethasone in Epac2a-KO mice was minimal (Fig. 3C). Next, we evaluated the expression of proteins associated with protein synthesis and degradation in the gastrocnemius muscle with or without dexamethasone treatment (Fig. 3D). In WT mice, dexamethasone treatment significantly decreased the expression of p-AKT and p-mammalian target of rapamycin (p-mTOR), but increased the expression of p-eukaryotic initiation factor 4E (p-eIF-4E), atrogin-1, and muscle RING-finger protein (MuRF), suggesting increased protein degradation and decreased protein synthesis. Conversely, in Epac2a-KO mice, dexamethasone treatment increased the protein expression of p-AKT and p-mTOR, while the dexamethasone-mediated increase in p-eIF-4E, atrogin-1, and MuRF expression observed in WT mice was attenuated. Dexamethasone-induced body weight reduction in WT mice shown in Fig. 3A was not due to the decrease in fat mass (Fig. 3E). To elucidate the increase in SNA and leptin signaling in skeletal muscle, we measured glycogen content, glucose transporter type 4 (GLUT4) expression, and pSTAT3 expression from gastrocnemius muscle. Consistent with previous findings (18), glycogen content and protein level of GLUT4 were greater in KO mice treated with dexamethasone compared to WT mice treated with dexamethasone. Additionally, STAT3 phosphorylation was also higher in KO mice treated with dexamethasone, which is a direct signaling molecule for leptin. Furthermore, we observed more potentiated pPKA expression in iBAT and gastrocnemius muscle in Epac2a-KO mice (
WT mice were administered leptin (i.p.). After 45 min, an
The present study demonstrates that leptin-mediated activation of SNA through the hypothalamus is not impaired in Epac2a-KO mice. We confirmed that Epac2a expression was not detected in gastrocnemius muscle and BAT in WT mice (
In BAT, leptin was shown to increase glucose utilization through ObRb and the JAK/STAT pathway. It demonstrates that leptin increases the expression of some metabolic enzymes and PPAR activity in preadipocytes and BAT (17, 20). However, direct leptin action on UCP-1 expression in BAT was not observed, suggesting that thermogenesis in BAT is not directly associated with leptin receptor signaling. In fact, UCP-1 gene expression in BAT was induced by leptin, but through sympathetic innervation (21). Diet-induced obese mice, which are resistant to the anorectic effect of leptin but are hyperleptinemic, exhibited a higher iBAT temperature than mice fed a regular diet, whereas
In skeletal muscle, leptin directly exerts its action, which is like the action of insulin, by facilitating glycogen synthesis and glucose uptake, and promoting cell survival. Leptin deficiency (e.g.,
In conclusion, like arterial blood pressure, which is elevated in diet-induced obesity, hypothalamic leptin resistance in the absence of Epac2a cannot block leptin signaling to enhance sympathetic outflow. This suggests that Epac2a in the hypothalamus only affects leptin-mediated signaling for appetite control (22). The present results also suggest that leptin receptor-induced signaling in skeletal muscle and BAT might be normal in Epac2a-KO mice, unlike the leptin receptor signaling in the hypothalamus.
Detailed information is provided in the
This study was supported by a grant from the National Research Foundation of Korea (NRF) Grant funded by the Korean Government (MSIP) (No. 2014R1A5A2010008 and NRF-2013R1A2A2A01068220).
The authors have no conflicting interests.