No abstract

Melanocortin-3 and 4 receptors (MC3R and MC4R) can regulate energy homeostasis, but their respective roles especially the functions of MC3R need more exploration. Here Mc3r and Mc4r single and double knockout (DKO) rats were generated using CRISPR-Cas9 system. Metabolic phenotypes were examined and data were compared systematically. Mc3r KO rats displayed hypophagia and decreased body weight, while Mc4r KO and DKO exhibited hyperphagia and increased body weight. All three mutants showed increased white adipose tissue mass and adipocyte size. Interestingly, although Mc3r KO did not show a significant elevation in lipids as seen in Mc4r KO, DKO displayed even higher lipid levels than Mc4r KO. DKO also showed more severe glucose intolerance and hyperglycaemia than Mc4r KO. These data demonstrated MC3R deficiency caused a reduction of food intake and body weight, whereas at the same time exhibited additive effects on top of MC4R deficiency on lipid and glucose metabolism. This is the first phenotypic analysis and systematic comparison of Mc3r KO, Mc4r KO and DKO rats on a homogenous genetic background. These mutant rats will be important in defining the complicated signalling pathways of MC3R and MC4R. Both Mc4r KO and DKO are good models for obesity and diabetes research.

GPR54, Kisspeptin-1 receptor (KISS1R), a member of rhodopsin family, plays a critical role in puberty development and has been proposed to be involved in regulation of energy metabolism. This study aims to explore the function of GPR54 in adipogenesis, lipid metabolism, and obesity in addition to its effect through hormones. Results showed that when fed a high-fat diet, the weight growth of castrated or ovariectomized Gpr54−/− mice was significantly slower than that of WT control, together with a lower triglyceride concentration. The ratio of white adipose tissue was lower, and average size of adipocytes was smaller in Gpr54−/− mice. Meanwhile, there were less adipose tissue macrophages (ATMs), especially pro-inflammatory macrophages. Expression of inflammatory related genes also indicated that inflammatory response caused by obesity was not as drastic in Gpr54−/− mice as in WT mice. Liver triglyceride in Gpr54−/− mice was reduced, especially in female mice. On the other hand, oil drop formation was accelerated when hepatocytes were stimulated by kisspeptin-10 (Kp-10). Primary mesenchymal stem cells (MSCs) of Gpr54−/− mice were less likely to differentiate into adipocytes. When stimulated by Kp-10, 3T3-L1 cell differentiation into adipocytes was accelerated and triglyceride synthesis was significantly promoted. These data indicated that GPR54 could affect obesity development by promoting adipocyte differentiation and triglyceride accumulation. To further elucidate the mechanism, genes related to lipid metabolism were analyzed. The expression of genes involved in lipid synthesis including PPARγ, ACC1, ADIPO, and FAS was significantly changed in Gpr54−/− mice. Among them PPARγ which also participate in adipocyte differentiation displayed a marked reduction. Moreover, phosphorylation of ERK, which involved in GPR54 signaling, was significantly decreased in Gpr54−/− mice, suggesting that GPR54 may promote lipid synthesis and obesity development by activating MAP kinase pathway. Therefore, in addition to the involvement in hormone regulation, our study demonstrated that GPR54 directly participates in obesity development by promoting adipocyte differentiation and fat accumulation. This provided evidence of involvement of GPR54 in lipid metabolism, and revealed new potentials for the identification and development of novel drug targets for metabolic diseases.

CD4 T helper cells, especially T helper 17 (T17) cells, combined with immune regulatory network dysfunction, play key roles in autoimmune diseases including multiple sclerosis (MS). Betulinic acid (BA), a natural pentacyclic triterpenoid, has been reported to be involved in anti-inflammation, in particular having an inhibitory effect on proinflammatory cytokine interleukin 17 (IL-17) and interferon- (IFN-) production. In this study, we screened BA derivatives and found a BA derivative, SH479, that had a greater inhibitory effect on T17 differentiation. Our further analysis showed that SH479 had a greater inhibitory effect on T17 and T1, and a more stimulatory effect on regulatory T (Treg) cells. To evaluate the effects of SH479 on autoimmune diseases in vivo, we employed the extensively used MS mouse model experimental autoimmune encephalomyelitis (EAE). Our results showed that SH479 ameliorated clinical and histologic signs of EAE in both prevention and therapeutic protocols by regulating the T17/Treg balance. SH479 dose-dependently reduced splenic lymphocyte proinflammatory factors and increased anti-inflammatory factors. Moreover, SH479 specifically inhibited splenic lymphocyte viability from EAE mice but not normal splenic lymphocyte viability. At the molecular level, SH479 inhibited T17 differentiation by regulating signal transducer and activator of transcription-3 (STAT3) phosphorylation, DNA binding activity, and recruitment to the promoter in CD4 T cells. Furthermore, SH479 promoted the STAT5 signaling pathway and inhibited the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-B) signaling pathway. Together, our data demonstrated that SH479 ameliorated EAE by regulating the T17/Treg balance through inhibiting the STAT3 and NF-B pathways while activating the STAT5 pathway, suggesting that SH479 is a potential novel drug candidate for autoimmune diseases including MS.

GPR54 is highly expressed in the central nervous system and plays a crucial role in pubertal development. However, GRP54 is also expressed in the immune system, implying possible immunoregulatory functions. Here we investigated the role of GPR54 in T cell and immune tolerance. GPR54 deficiency led to an enlarged thymus, an increased number of thymocytes, and altered thymic micro-architecture starting around puberty, indicating GPR54 function in T-cell development through its regulatory effect on the gonadal system. However, flow cytometry revealed a significant reduction in the peripheral regulatory T cell population and a moderate decrease in CD4 single-positive thymocytes in prepubertal Gpr54 mice. These phenotypes were confirmed in chimeric mice with GPR54 deficient bone marrow-derived cells. In addition, we found elevated T cell activation in peripheral and thymic T cells in Gpr54 mice. When intact mice were immunized with myelin oligodendrocyte glycoprotein, a more severe experimental autoimmune encephalomyelitis (EAE) developed in the Gpr54 mice. Interestingly, aggravated EAE disease was also manifested in castrated and bone marrow chimeric Gpr54 mice compared to the respective wild-type control, suggesting a defect in self-tolerance resulting from GPR54 deletion through a mechanism that bypassed sex hormones. These findings demonstrate a novel role for GPR54 in regulating self-tolerant immunity in a sex hormone independent manner.