CTRP9 alleviates diet induced obesity through increasing lipolysis mediated by enhancing autophagy-initiation complex formation.

Recently, emerging evidence has suggested that obesity become a prevalent health threat worldwide. Reportedly, CTRP9 can ameliorate HFD induced obesity. However, the molecular mechanism underlying the role of CTRP9 in obesity remains elusive. In this study, we reported its major function in the regulation of lipolysis. First, we found that the expression of CTRP9 was decreased in mature adipocytes and white adipose tissue of obese mice. Then, we showed that overexpression adipose tissue CTRP9 alleviated diet-induced obesity and adipocytes hypertrophy, improved glucose intolerance and raised energy expenditure. Moreover, CTRP9 increased the lipolysis in vitro and vivo. Additionally, we determined that CTRP9 enhanced autophagy flux in adipocytes. Intriguingly, knock down Beclin1 by SiRNA abolished the effect of CTRP9 on lipolysis. Mechanically, CTRP9 enhanced the expression of SNX26. We demonstrated that SNX26 was a component of the ATG14L-Beclin1-VPS34 complex and enhanced the assembly of the autophagy-initiation complex. Collectively, our results suggested that CTRP9 alleviated diet induced obesity through enhancing lipolysis mediated by autophagy-initiation complex formation.

C1q/tumour necrosis factor-related protein-9 aggravates lipopolysaccharide-induced inflammation via promoting NLRP3 inflammasome activation.

The NLRP3 inflammasome plays a vital role in inflammation by increasing the maturation of interleukin-1ß (IL-1ß) and promoting pyroptosis. Given that C1q/tumour necrosis factor-related protein-9 (CTRP9) has been shown to be involved in diverse inflammatory diseases, we sought to assess the underlying impact of CTRP9 on NLRP3 inflammasome activation. In vitro, macrophages isolated from murine peritonea were stimulated with exogenous CTRP9, followed by lipopolysaccharide (LPS) and adenosine 5'-triphosphate (ATP). We demonstrated that CTRP9 markedly augmented the activation of the NLRP3 inflammasome, as shown by increased mature IL-1ß secretion, triggering ASC speck formation and promoting pyroptosis. Mechanistically, CTRP9 increased the levels of NADPH oxidase 2 (NOX2)-derived reactive oxygen species (ROS). Suppressing ROS with N-acetylcysteine (NAC) or interfering with NOX2 by small interfering RNA weakened the promoting effect of CTRP9 on the NLRP3 inflammasome. Furthermore, NLRP3 inflammasome activation, pyroptosis and secretion of mature IL-1ß were significantly decreased in macrophages from CTRP9-KO mice compared to those from WT mice with the same treatment. In vivo, we established a sepsis model by intraperitoneal injection of LPS into WT and CTRP9-KO mice. CTRP9 knockout improved the survival rates of the septic mice and attenuated NLRP3 inflammasome-mediated inflammation. In conclusion, our study indicates that CTRP9 aggravates LPS-induced inflammation by promoting NLRP3 inflammasome activation via the NOX2/ROS pathway. CTRP9 could be a promising target for NLRP3 inflammasome-driven inflammatory diseases.

CTRP9 alleviates foam cells apoptosis by enhancing cholesterol efflux.

The apoptosis of foam cells leads to instability of atherosclerotic plaques. This study was designed to explore the protective role of CTRP9 in foam cell apoptosis. In our experiment, CTRP9 alleviated foam cell apoptosis. Meanwhile, CTRP9 upregulated the expression of proteins important for cholesterol efflux, such as LXRα, CYP27A1, ABCG1 and ABCA1, and improved cholesterol efflux in foam cells. Moreover, CTRP9 inhibited Wnt3a and ß-catenin expression and ß-catenin nuclear translocation in foam cells. In addition, adenovirus overexpression of Wnt3a abolished the effect of CTRP9 on macrophage apoptosis. Mechanistically, the AMPK inhibitor abolished the effect of CTRP9 on foam cell apoptosis, and downregulation of AdipoR1 by siRNA abrogated the activation of AMPK and the effect of CTRP9 on foam cell apoptosis. We concluded that CTRP9 achieved these protective effects on foam cells through the AdipoR1/AMPK pathway.

CTRP9 induces macrophages polarization into M1 phenotype through activating JNK pathway and enhances VSMCs apoptosis in macrophages and VSMCs co-culture system.

Inflammation plays a critical role in the development of atherosclerosis (AS), which has been identified as a major predisposing factor for stroke. Macrophages and VSMCs are associated with plaque formation and progression. Macrophages can dynamically change into two main functional phenotypes, namely M1 and M2, they can produce either pro-inflammatory or anti-inflammatory factors which may affect the outcome of inflammation. As a member of CTRPs family, CTRP9 has been reported play important protective roles in the cardiovascular system. However, whether CTRP9 can regulate macrophage activation status in inflammatory responses and have effect on VSMCs behaviors in co-culture system have not been fully investigated. In the present study, using peritoneal macrophages treated with CTRP9, we found that CTRP9 facilitated macrophages towards M1 phenotype, promoted TNF-α secretion and MMPs expression. CTRP9 showed synergistic effect with LPS in inducing M1 macrophages. In macrophages-VSMCs co-culture system, apoptosis and down-regulated proliferation of VSMCs were accelerated with CTRP9-treated macrophages. Then we attempted to explore the underlying molecular mechanisms of CTRP9 resulting in M1 activation. The c-Jun NH2-terminal kinases (JNK) are members of the mitogen activated protein kinases (MAPK) family, plays a central role in the cell stress response, with outcomes ranging from cell death to cell proliferation and survival. We found JNK expression was upregulated following CTRP9 stimulation, and inhibiting JNK phosphorylation level was associated with decreased expression of M1 markers and TNF-α concentration. Moreover, VSMCs apoptosis were ameliorated after inhibition of JNK. These results suggested that CTRP9 may promote macrophage towards M1 activation status through JNK signaling pathway activation.

Globular CTRP9 protects cardiomyocytes from palmitic acid-induced oxidative stress by enhancing autophagic flux.

BACKGROUND: Oxidative stress in cardiac myocytes is an important pathogenesis of cardiac lipotoxicity. Autophagy is a cellular self-digestion process that can selectively remove damaged organelles under oxidative stress, and thus presents a potential therapeutic target against cardiac lipotoxicity. Globular CTRP9 (gCTRP9) is a newly identified adiponectin paralog with established metabolic regulatory properties. The aim of this work is to investigate whether autophagy participates the protection effects of gCTRP9 in neonatal rat cardiac myocytes (NRCMs) under oxidative stress and the underlying mechanism. RESULTS: NRCMs were treated with PA of various concentrations for indicated time period. Our results showed that PA enhanced intracellular ROS accumulation, decreased mitochondrial membrane potential (Δψm) and increased activation of caspases 3. These changes suggested lipotoxicity due to excessive PA. In addition, PA was observed to impair autophagic flux in NRCMs and impaired autophagosome clearance induced by PA contributes to cardiomyocyte death. Besides, we found that gCTRP9 increased the ratio of LC3II/I and the expression of ATG5 which was vital to the formation of autophagosomes and decreased the level of P62, suggesting enhanced autophagic flux in the absence or presence of PA. The result was further confirmed by the methods of infection with LC3-mRFP-GFP lentivirus and blockage of autophagosome-lysosome fusion by BafA1. Moreover, gCTRP9 reestablished the loss of mitochondrial membrane potential, suppressed ROS generation, and reduced PA -induced myocyte death. However, the protective effect of gCTRP9 on the cardiac lipotoxicity was partly abolished by blockade of autophagy by autophagy-related 5 (ATG5) siRNA, indicating that the effect of gCTRP9 on cell survival is critically mediated through regulation of autophagy. CONCLUSION: Autophagy induction by gCTRP9 could be utilized as a potential therapeutic strategy against oxidative stress-mediated damage in cardiomyocytes.

CTRP9 knockout exaggerates lipotoxicity in cardiac myocytes and high-fat diet-induced cardiac hypertrophy through inhibiting the LKB1/AMPK pathway.

CTRP9 has been reported to regulate lipid metabolism and exert cardioprotective effects, yet its role in high-fat diet (HFD)-induced cardiac lipotoxicity and the underlying mechanisms remain unclear. In the current study, we established HFD-induced obesity model in wild-type (WT) or CTRP9 knockout (CTRP9-KO) mice and palmitate-induced lipotoxicity model in neonatal rat cardiac myocytes (NRCMs) to investigate the effects of CTRP9 on cardiac lipotoxicity. Our results demonstrated that the HFD-fed CTRP9-KO mice accentuated cardiac hypertrophy, fibrosis, endoplasmic reticulum (ER) stress-initiated apoptosis and oxidative stress compared with the HFD-fed WT mice. In vitro, CTRP9 treatment markedly alleviated palmitate-induced oxidative stress and ER stress-induced apoptosis in NRCMs in a dose-dependent manner. Phosphorylated AMPK at Thr172 was reduced, and phosphorylated mammalian target of rapamycin (mTOR) was strengthened in the heart of the HFD-fed CTRP9-KO mice compared with the HFD-fed control mice. In vitro, AMPK inhibitor compound C significantly abolished the effects of CTRP9 on the inhibition of the apoptotic pathway in palmitate-treated NRCMs. In a further mechanistic study, CTRP9 enhanced expression of phosphorylated LKB1 at Ser428 and promoted LKB1 cytoplasmic localization. Besides, silencing of LKB1 gene by lentivirus significantly prohibited activation of AMPK by CTRP9 and partially eliminated the protective effect of CTRP9 on the cardiac lipotoxicity. These results indicate that CTRP9 exerted anti-myocardial lipotoxicity properties and inhibited cardiac hypertrophy probably through the LKB1/AMPK signalling pathway.

CTRP9 induces iNOS expression through JAK2/STAT3 pathway in Raw 264.7 and peritoneal macrophages.

The C1q tumor necrosis factor (TNF)-related proteins 9 (CTRP9), an adipocyte-derived cytokine, affects a number of physiological processes, including immune function and inflammation. We investigated whether CTRP9 affects the expression of inflammation-related genes in Raw 264.7 and peritoneal macrophages. The CTRP9-induced expression of iNOS increased in a time- and dose-dependent manner. LPS and CTRP9 promote the expression of iNOS jointly in Raw 264.7 and peritoneal macrophages. CTRP9 induced the phosphorylation of JAK2 and STAT3 in Raw 264.7 and peritoneal macrophages. VX509 (JAK2 inhibitor) reduced the CTRP9-induced iNOS protein production. In addition, the CTRP9-induced phosphorylation of JAK2 and STAT3 was dramatically reduced by VX509. Collectively, these results suggest that JAK2/STAT3 signaling is involved in the CTRP9-induced expression of iNOS.