BACKGROUND: This study aimed to elucidate the molecular mechanism through which C1q/tumor necrosis factor (TNF)-related protein 9 (CTRP9) acts in the formation and differentiation of brown adipose tissue (BAT).
METHODS: Adenovirus particles encoding CTRP9 and green fluorescent protein were inoculated into the scapula of C57BL/6J mice and fed a high-fat diet for 8 weeks; the body weight, lipid droplet morphology, glucose tolerance, insulin tolerance, and protein expression levels were analyzed. In addition, CTRP9 adenovirus was transfected into brown preadipocytes, and differentiation was induced to identify the effect of CTRP9 overexpression on adipocyte differentiation.
RESULTS: CTRP9 overexpression significantly increased the weight gain of mice. Additionally, the CTRP9 overexpression group exhibited significantly increased adipose tissue weight and glucose clearance rates and decreased insulin sensitivity and serum triglyceride levels compared to the control group. Furthermore, CTRP9 overexpression significantly upregulated the adipose triglyceride lipase (ATGL) and perilipin 1 protein expression levels in BAT. The cell experiment results confirmed that CTRP9 overexpression significantly inhibited the adipogenesis of brown adipocytes as evidenced by the downregulation of uncoupling protein 1, beta-3 adrenergic receptor, ATGL, and hormone-sensitive lipase mRNA levels and the significant suppression of uncoupling protein 1, ATGL, and perilipin 1 protein levels in brown adipocytes.
CONCLUSIONS: The finding of this study demonstrated that CTRP9 promotes lipolysis by upregulating ATGL expression in vivo and inhibits the differentiation of brown preadipocytes in vitro.

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.

UNASSIGNED: The senescence of endothelial cells is of great importance involving in atherosclerosis (AS) development. Recent studies have proved the protective role of mesenchymal stem cell-derived extracellular vesicles in AS, herein, we further desired to unvei their potential regulatory mechanisms in endothelial cell senescence.
UNASSIGNED: Senescence induced by H2O2 in primary mouse aortic endothelial cells (MAECs) was evaluated by SA-β-gal staining. Targeted molecule expression was detected by qRT-PCR and Western blotting. The biological functions of MAECs were determined by CCK-8, flow cytometry, transwell, and tube formation assays. Oxidative injury was assessed by LDH, total and lipid ROS, LPO and MDA levels. The proliferation of adipose-derived mesenchymal stem cell (ADSCs) was analyzed by EdU assay. Effect of ADSCs-derived extracellular vesicles (ADSC-EVs) on AS was investigated in HFD-fed ApoE-/- mice.
UNASSIGNED: miR-674-5p was up-regulated, while C1q/TNF-related protein 9 (CTRP9) was down-regulated in H2O2-induced senescent MAECs. CTRP9 was demonstrated as a target gene of miR-674-5p. miR-674-5p inhibition restrained senescence, oxidative stress, promoted proliferation, migration, and angiogenesis of H2O2-stimulated MAECs via enhancing CTRP9 expression. Moreover, treatment with ADSC-EVs inhibited H2O2-induced senescence and dysfunction of MAECs through regulating miR-674-5p/CTRP9 axis. In the in vivo AS mouse model, ADSC-EVs combination with miR-674-5p silencing slowed down AS progression via up-regulation of CTRP9.
UNASSIGNED: ADSC-EVs repressed endothelial cell senescence and improved dysfunction via promotion of CTRP9 expression upon miR-674-5p deficiency during AS progression, which might provide vital evidence for ADSC-EVs as a promising therapy for AS.

Cholesterol efflux from foam cells in atherosclerotic plaques is crucial for reverse cholesterol transport (RCT), an important antiatherogenic event. ATP-binding cassette (ABC) transporters, ABCA1 and ABCG1, are key receptors in the cholesterol efflux pathway. C1q/tumor necrosis factor-related protein-9 (CTRP9) is a newly discovered adipokine and exhibits an atheroprotective activity. However, the role of CTRP9 in RCT still remains unknown. In this work, we investigated the effect of subcutaneous administration of CTRP9 protein on RCT and atherosclerotic lesion formation in ApoE-/- mice fed with a high-fat diet. CTRP9-dependent regulation of cholesterol efflux and ABC transporters in RAW 264.7 foam cells was determined. Our results showed that CTRP9 protein decreased atherosclerotic lesions, increased cholesterol efflux, and upregulated liver ABCA1 and ABCG1 expression in ApoE-/- mice. CTRP9 treatment dose-dependently increased mRNA and protein expression of ABCA1, ABCG1, and LXR-α in RAW 264.7 foam cells. Moreover, the expression and phosphorylation of AMPK was potentiated upon CTRP9 treatment. Notably, CTRP9-induced cholesterol efflux and upregulation of ABCA, ABCG1, and LXR-α were impaired when AMPK was knocked down. AMPK depletion restored cholesterol accumulation in CTRP9-treated RAW 264.7 cells. Taken together, subcutaneous injection is an effective novel delivery route for CTRP9 protein, and exogenous CTRP9 can facilitate cholesterol efflux and promote RCT in an animal model of atherosclerosis. The atheroprotective activity of CTRP9 is mediated through the activation of AMPK signaling.

BACKGROUND: Atherosclerosis (AS) is commonly regarded as a key driver accounted for the leading causes of morbidity and mortality among cardiovascular and cerebrovascular diseases. A growing body of evidence indicates that autophagy in macrophages involved in AS might be a potential therapeutic target. C1q/TNF-related protein 9 (CTRP9) has been proven to delay the progression of cardiovascular diseases. However, the relations between CTRP9 and Sirt1, as well as their effects on macrophages autophagy have not been fully explored.
METHODS: Macrophages were differentiated from mononuclear cells collected from peripheral blood samples of healthy donors. The in vitro AS models were constructed by ox-LDL treatment. Cell viability was determined by CCK-8 assay. Immunofluorescence assay of LC3 was implemented for evaluating autophagy activity. Oil Red O staining was performed for lipid accumulation detection. ELISA, cholesterol concentration assay and cholesterol efflux analysis were conducted using commercial kits. Cycloheximide assay was implemented for revealing protein stability. RT-qPCR was used for mRNA expression detection, and western blotting was performed for protein level monitoring.
RESULTS: CTRP9 attenuated impaired cell viability, autophagy inhibition and increased lipid accumulation induced by ox-LDL. Moreover, CTRP9 maintained Sirt1 protein level through enhancing its stability through de-ubiquitination, which was mediated by upregulated USP22 level. CRTP9 exerted its protective role in promoting autophagy and reducing lipid accumulation through the USP22/Sirt1 axis.
CONCLUSIONS: Collectively, CTRP9 alleviates lipid accumulation and facilitated the macrophages autophagy by upregulating USP22 level and maintaining Sirt1 protein expression, thereby exerting a protective role in AS progression in vitro.

Regular exercise is recommended as an important component of therapy for cardiovascular diseases in clinical practice. However, there are still major challenges in prescribing an optimized exercise regimen to individual patients with established cardiac disease. Here, we tested the effects of different exercise doses on cardiac function in mice with established myocardial infarction (MI). Exercise was introduced to mice with MI after 4 weeks of surgery. Low-dose exercise (15 min/day for 8 weeks) improved mortality and cardiac function by increasing 44.39% of ejection fractions while inhibiting fibrosis by decreasing 37.74% of distant region. Unlike higher doses of exercise, low-dose exercise consecutively upregulated cardiac expression of C1q complement/tumor necrosis factor-associated protein 9 (CTRP9) during exercise (>1.5-fold). Cardiac-specific knockdown of CTRP9 abolished the protective effects of low-dose exercise against established MI, while cardiac-specific overexpression of CTRP9 protected the heart against established MI. Mechanistically, low-dose exercise upregulated the transcription factor nuclear receptor subfamily 2 group F member 2 by increasing circulating insulin-like growth factor 1 (IGF-1), therefore, upregulating cardiac CTRP9 expression. These results suggest that low-dose exercise protects the heart against established MI via IGF-1-upregulated CTRP9 and may contribute to the development of optimized exercise prescriptions for patients with MI.

Atherosclerosis is a huge threaten to the human health, C1q/TNF-related protein 9 (CTRP9) has been previously reported possessing vascular protective functions. Our study is aimed to reveal the mechanism of the regulative effects of CTRP9 on the foam cell formation.
Primary human macrophages were isolated from human monocytes donated by healthy volunteers. CCK-8 assay was performed for determining the cell viability. Oil Red O staining was employed for measuring the lipid accumulation. Cholesterol ester and cholesterol concentration were detected by commercial kits for evaluating the intracellular cholesterol. Ubiquitination assay was performed to reveal the ubiquitination level of CD36, cycloheximide assay was applied for determining the half-life of CD36 protein. Quantitative real-time PCR and western blot assays were performed for detecting the mRNA and protein expression. Pre-treatment with CTRP9 in primary human macrophages markedly suppressed the cholesterol accumulation concentration after oxidized low-density lipoprotein treatment. CD36 was significantly increased after oxidized low-density lipoprotein exposure while was reduced by CTRP9 treatment. Up-regulation of CD36 significantly reversed the CTRP9-mediated protective effects in foam cells. The differential expression levels of several deubiquitinating enzymes preliminarily indicated that USP11 was obviously decreased after CTRP9 treatment. USP11 knockdown decreased the CD36 protein expression and pre-treatment with 10 μg/mL MG132 significantly maintained the CD36 level from USP11 knock down. Up-regulation of CD36 reversed the alterations on the cholesterol metabolism caused by CTRP9 or USP11 knockdown.
CTRP9 regulates the USP11/CD36 axis to protect the macrophages form transforming into foam cells by suppressing intracellular lipid and cholesterol accumulation, which is a potential therapeutic agent for atherosclerosis.

According to growing research, C1q/TNF-Related Protein-9 (CTRP9) appears to be linked to type 2 diabetes mellitus (T2DM). But the literature on circulating levels of CTRP9 in patients with T2DM has been contradictory.
This is a systematic review and meta-analysis to reassess the circulating level of CTRP9 in patients with T2DM, with and without complications.
Relevant studies published until October 31, 2021, were identified from the PubMed, Embase, Web of Science, Cochrane Library, WanFang, CNKI, VIP, and CBM databases. Participants with age ≥18 years with clinically diagnosed T2DM were included. Sex and diabetes complications were not restricted. The data were extracted by 2 reviewers independently using a standard data collection form.
Analysis demonstrated significantly lower circulating levels of CTRP9 in patients with T2DM than in patients without diabetes (standardized mean difference [SMD] = -1.36; 95% CI -1.78 to -0.93; P < .001), I2 = 97.5%, P < .001). Furthermore, the circulating level of CTRP9 in patients with T2DM-related complications was lower than that in patients with T2DM without complications, regardless of macrovascular complications or microvascular complications (SMD = -1.062; 95% CI -1.466 to -0.658; P < .001, I2 = 91.3%, P < .001). Subgroup analyses revealed that factors such as body mass index, T2DM duration, and fasting blood glucose were the sources of heterogeneity (P = .047, P = .034, and P = .07, respectively).
The present systematic review and meta-analysis found CTRP9 levels were lower in T2DM patients with or without complications. However, since this was a meta-analysis of most observational studies, these findings still need to be verified by further studies with a large sample size.

C1q/tumor necrosis factor-related protein-9 (CTRP9) is linked to diverse pathological conditions via the effects on cell apoptosis, inflammatory response, and oxidative stress. However, its functional relevance in ischemic brain injury is not well determined. The present work aimed to evaluate the role of CTRP9 in ischemia/reperfusion-associated neuronal injury using an in vitro model. The cultured cortical neurons were subjected to oxygen-glucose deprivation/reoxygenation (OGD/R) to simulate ischemia/reperfusion in vitro. CTRP9 level was lowered in cultured neurons exposed to OGD/R. Neurons with overexpressed CTRP9 were resistant to OGD/R-elicited injuries, including neuronal apoptosis, oxidative stress, and pro-inflammatory response. Mechanism research revealed that CTRP9 could boost the activation of the nuclear factor erythroid 2-related factor (Nrf2) pathway associated with modulation of the Akt-glycogen synthase kinase-3β (GSK-3β) axis. CTRP9 regulated the transduction of the Akt-GSK-3β-Nrf2 cascade via adiponectin receptor 1 (AdipoR1). Restraining Nrf2 could diminish CTRP9-mediated neuroprotective effects in OGD/R-injured neurons. Altogether, these results confirmed that CTRP9 exerts a protective effect on OGD/R-injured neurons by modulating Akt-GSK-3β-Nrf2 cascade via AdipoR1. This work suggests a possible link between CTRP9 and ischemic brain injury.

BACKGROUND: C1q/TNF-related protein 9 (CTRP9) and adiponectin (APN) have beneficial metabolic regulatory and vasoprotective effects. This study explored alteration of CTRP9 and APN multimers during onset of ischemic stroke and development, to provide novel clinical and experimental basis for recognition and prevention of ischemic stroke.
METHODS: There were 269 patients with ischemic stroke and 182 control subjects included in this study. Serum levels of CTRP9 and APN multimers in different disease stages were measured.
RESULTS: Serum CTRP9, total APN (tAPN), and high-molecular weight (HMW) APN decreased gradually in stage I (acute stage, within 72 h of onset) of ischemic stroke and increased during stage III (11th day to one month) and stage IV (1 month after), compared to control. In the non-hyperlipidemia group, serum CTRP9, tAPN, and HMW were decreased in ischemic stroke patients compared to control (P < 0.05). Serum CTRP9 is closely related to serum tAPN and HMW (r = 0.992, 0.991). Serum CTRP9 are protective against ischemic stroke (OR = 0.400, 95% CI 0.197-0.810, P < 0.05).
CONCLUSIONS: Lower serum CTRP9, tAPN, LMW, and HMW are significantly associated with increased ischemic stroke risk in non-hyperlipidemia subjects. CTRP9, tAPN, and HMW isoforms may be valuable clinical indicators for patients with ischemic stroke.