A brief glimpse at CTRP3 and CTRP9 in lipid metabolism and cardiovascular protection

Adipose tissue is now known to express and secrete numerous adipokines that not only regulate lipid metabolism but also function in a wide array of physiological or pathological processes. C1q tumor necrosis factor-related protein 3 (CTRP3, also known as CORS26/cartducin) and CTRP9, novel members of the adipokine family, have intersecting functions in the regulation of lipid metabolism and contribute to cardiovascular protection. Here, we focus on the novel advances concerning the roles of CTRP3 and CTRP9 in these processes and review the general mechanisms. This review should serve as a basis for the design of future experimental studies and may implicate these adipokines as future therapeutic targets.     

CTRP9: An emerging potential anti-aging molecule in brain

C1q/tumor necrosis factor (TNF)-related proteins (CTRPs) particularly CTRP9, have been established to be as adiponectin (APN) highly conserved paralogs which assemble several APN regulatory functions. Recently, growing body of evidences drawn significant attention to evaluate metabolic and cardiovascular effect of CTRP9. However, the potential role of CTRP9 in brain tissue has not yet fully illustrated. Here, we aimed to uncover latest advances regarding the CTRP9 related signaling pathways and during brain aging process.     

C1q/TNF-related Protein 4 (CTRP4) Is a Unique Secreted Protein with Two Tandem C1q Domains That Functions in the Hypothalamus to Modulate Food Intake and Body Weight

CTRP4 is a unique member of the C1q family, possessing two tandem globular C1q domains. Its physiological function is poorly defined. Here, we show that CTRP4 is an evolutionarily conserved, ∼34-kDa secretory protein expressed in the brain. In human, mouse, and zebrafish brain, CTRP4 expression begins early in development and is widespread in the central nervous system. Neurons, but not astrocytes, express and secrete CTRP4, and secreted proteins form higher-order oligomeric complexes. CTRP4 is also produced by peripheral tissues and circulates in blood. Its serum levels are increased in leptin-deficient obese (ob/ob) mice. Functional studies suggest that CTRP4 acts centrally to modulate energy metabolism. Refeeding following an overnight fast induced the expression of CTRP4 in the hypothalamus. Central administration of recombinant protein suppressed food intake and altered the whole-body energy balance in both chow-fed and high-fat diet-fed mice. Suppression of food intake by CTRP4 is correlated with a decreased expression of orexigenic neuropeptide (Npy and Agrp) genes in the hypothalamus. These results establish CTRP4 as a novel nutrient-responsive central regulator of food intake and energy balance.     

C1q/TNF-related protein-3 (CTRP3), a novel adipokine that regulates hepatic glucose output

Adipose tissue-derived adipokines play important roles in controlling systemic insulin sensitivity and energy balance. Our recent efforts to identify novel metabolic mediators produced by adipose tissue have led to the discovery of a highly conserved family of secreted proteins, designated as C1q/TNF-related proteins 1-10 (CTRP1 to -10). However, physiological functions regulated by CTRPs are largely unknown. Here we provide the first in vivo functional characterization of CTRP3. We show that circulating levels of CTRP3 are inversely correlated with leptin levels; CTRP3 increases with fasting, decreases in diet-induced obese mice with high leptin levels, and increases in leptin-deficient ob/ob mice. A modest 3-fold elevation of plasma CTRP3 levels by recombinant protein administration is sufficient to lower glucose levels in normal and insulin-resistant ob/ob mice, without altering insulin or adiponectin levels. The glucose-lowering effect in mice is linked to activation of the Akt signaling pathway in liver and a marked suppression of hepatic gluconeogenic gene expression. Consistent with its effects in mice, CTRP3 acts directly and independently of insulin to regulate gluconeogenesis in cultured hepatocytes. In humans, alternative splicing generates two circulating CTRP3 isoforms differing in size and glycosylation pattern. The two human proteins form hetero-oligomers, an association that does not require interdisulfide bond formation and appears to protect the longer isoform from proteolytic cleavage. Recombinant human CTRP3 also reduces glucose output in hepatocytes by suppressing gluconeogenic enzyme expression. This study provides the first functional evidence linking CTRP3 to hepatic glucose metabolism and establishes CTRP3 as a novel adipokine.     

Endopeptidase Cleavage Generates a Functionally Distinct Isoform of C1q/Tumor Necrosis Factor-related Protein-12 (CTRP12) with an Altered Oligomeric State and Signaling Specificity

Adipose tissue-derived adipokines are an important class of secreted metabolic regulators that mediate tissue cross-talk to control systemic energy balance. We recently described C1q/TNF-related protein-12 (CTRP12), a novel insulin-sensitizing adipokine that regulates glucose metabolism in liver and adipose tissue. However, the biochemical properties of CTRP12 and its naturally occurring cleaved isoform have not been characterized. Here, we show that CTRP12 is a secreted hormone subjected to multiple functionally relevant posttranslational modifications at highly conserved residues. For example, Asn³⁹ is glycosylated, whereas Cys⁸⁵ mediates the assembly of higher order oligomeric structure. Endopeptidase cleavage at Lys⁹¹ generates a cleaved globular gCTRP12 isoform, the expression of which is increased by insulin. PCSK3/furin was identified as the major proprotein convertase expressed by adipocytes that mediates the endogenous cleavage of CTRP12. Cleavage at Lys⁹¹ is context-dependent: mutation of the charged Arg⁹³ to Ala on the P2′ position enhanced cleavage, and triple mutations (K90A/K91A/R93A) abolished cleavage. Importantly, the two isoforms of CTRP12 differ in oligomeric structures and are functionally distinct. The full-length protein forms trimers and larger complexes, and the cleaved isoform consisted of predominantly dimers. Whereas full-length fCTRP12 strongly activated Akt signaling in H4IIE hepatocytes and 3T3-L1 adipocytes, gCTRP12 preferentially activated MAP kinase (ERK1/2 and p38 MAPK) signaling. Further, only fCTRP12 improved insulin-stimulated glucose uptake in adipocytes. These results reveal a novel mechanism controlling signaling specificity and function of a hormone via cleavage-dependent alteration in oligomeric state.     

Inhibition of CTRP6 prevented survival and migration in hepatocellular carcinoma through inactivating the AKT signaling pathway

C1qTNF-related proteins (CTRPs) are a member of the adiponectin paralogs family, which are implicated in regulation of various biological processes. Recently, CTRP6 was found upregulated in human hepatocellular carcinomas (HCC). However, the specific roles and molecular mechanisms of CTRP6 in HCC remain unclear. Here, we investigated the effects of CTRP6 on the vitality, apoptosis, migration, and invasion of HCC cells. Firstly, we measured the levels of CTRP6 in HCC tissues and cell lines. Our results showed that CTRP6 was markedly upregulated in HCC tissues and Hep3B cells. Then, the CTRP6 siRNA was transfected into Hep3B cells. Cell counting kit-8 (CCK-8) assay and flow cytometry analysis revealed that silencing CTRP6-induced cell viability inhibition, and apoptosis. The wound-healing and transwell assay showed that CTRP6 deficiency suppressed the migration and invasion of Hep3B cells. Meanwhile, the AKT phosphorylation level was reduced by CTRP6 silencing. Next, Hep3B cells were pretreated with insulin-like growth factor-1 (IGF-1) (an activator of AKT), and then transfected with CTRP6 siRNA, and the cell vitality, apoptosis, migration, and invasion were measured again. We found that all these alterations caused by CTRP6 inhibition could be reversed by IGF-1 treatment. Taken together, CTRP6 suppression blocked cell survival, migration, and invasion and promoted cell apoptosis through inactivating the AKT signaling pathway. Our findings present a novel potential molecular target for HCC therapy.     

CTRP3 promotes TNF-α-induced apoptosis and barrier dysfunction in salivary epithelial cells

BackgroundC1q/tumour necrosis factor-related protein 3 (CTRP3) plays important roles in metabolism and inflammatory responses in various cells and tissues. However, the expression and function of CTRP3 in salivary glands have not been explored.MethodsThe expression and distribution of CTRP3 were detected by western blot, polymerase chain reaction, immunohistochemical and immunofluorescence staining. The effects of CTRP3 on tumour necrosis factor (TNF)-α-induced apoptosis and barrier dysfunction were detected by flow cytometry, western blot, co-immunoprecipitation, and measurement of transepithelial resistance and paracellular tracer flux.ResultsCTRP3 was distributed in both acinar and ductal cells of human submandibular gland (SMG) and was primarily located in the ducts of rat and mouse SMGs. TNF-α increased the apoptotic rate, elevated expression of cleaved caspase 3 and cytochrome C, and reduced B cell lymphoma-2 (Bcl-2) levels in cultured human SMG tissue and SMG-C6 cells, and CTRP3 further enhanced TNF-α-induced apoptosis response. Additionally, CTRP3 aggravated TNF-α-increased paracellular permeability. Mechanistically, CTRP3 promoted TNF-α-enhanced TNF type I receptor (TNFR1) expression, inhibited the expression of cellular Fas-associated death domain (FADD)-like interleukin-1β converting enzyme inhibitory protein (c-FLIP), and increased the recruitment of FADD with receptor-interacting protein kinase 1 and caspase 8. Moreover, CTRP3 was significantly increased in the labial gland of Sjögren's syndrome patients and in the serum and SMG of nonobese diabetic mice.ConclusionsThese findings suggest that the salivary glands are a novel source of CTRP3 synthesis and secretion. CTRP3 might promote TNF-α-induced cell apoptosis through the TNFR1-mediated complex II pathway.     

CTRP1 protects against diet-induced hyperglycemia by enhancing glycolysis and fatty acid oxidation

Complement-C1q/tumor necrosis factor-α related protein 1 (CTRP1) is a 35-kDa glycoprotein that is secreted from various tissues. Although CTRP1 is highly increased in patients with type II diabetes and obesity, the metabolic roles of CTRP1 remain largely unknown. To unveil the physiological roles of CTRP1 in vivo, CTRP1 transgenic (TG) mice were challenged by a high-fat diet (HFD) and a high-sucrose drink (HS). Homeostatic model assessment-estimated insulin resistance values were decreased in HFD- or HS-fed CTRP1 TG mice compared with wild-type control mice. In this context, CTRP1 stimulated glucose uptake through the glucose transporter GLUT4 translocation to the plasma membrane and also increased glucose consumption by stimulating glycolysis. To analyze the roles of CTRP1 in lipid metabolism, acetyl-CoA carboxylase (ACC) and hormone-sensitive lipase levels were determined in CTRP1 TG mice, and the effect of CTRP1 on fatty acid oxidation was assessed in C2C12 myotubes. CTRP1 was found to inhibit ACC by phosphorylation and to stimulate fatty acid oxidation in C2C12 myotubes. Taken together, CTRP1 performs active catabolic roles in vivo. Therefore, CTRP1 seems to perform a defensive function against nutritional challenges.     

C1q/TNF‐related protein 9 inhibits the cholesterol‐induced Vascular smooth muscle cell phenotype switch and cell dysfunction by activating AMP‐dependent kinase

Vascular smooth muscle cells (VSMCs) switch to macrophage‐like cells after cholesterol loading, and this change may play an important role in the progression of atherosclerosis. C1q/TNF‐related protein 9 (CTRP9) is a recently discovered adipokine that has been shown to have beneficial effects on glucose metabolism and vascular function, particularly in regard to cardiovascular disease. The question of whether CTRP9 can protect VSMCs from cholesterol damage has not been addressed. In this study, the impact of CTRP9 on cholesterol‐damaged VSMCs was observed. Our data show that in cholesterol‐treated VSMCs, CTRP9 significantly reversed the cholesterol‐induced increases in pro‐inflammatory factor secretion, monocyte adhesion, cholesterol uptake and expression of the macrophage marker CD68. Meanwhile, CTRP9 prevented the cholesterol‐induced activation of the TLR4–MyD88–p65 pathway and upregulated the expression of proteins important for cholesterol efflux. Mechanistically, as siRNA‐induced selective gene ablation of AMPKα1 abolished these effects of CTRP9, we concluded that CTRP9 achieves these protective effects in VSMCs through the AMP‐dependent kinase (AMPK) pathway.     

CTRP9 protein protects against myocardial injury following ischemia-reperfusion through AMP-activated protein kinase (AMPK)-dependent mechanism

Ischemic heart disease is the major cause of death in Western countries. CTRP9 (C1q/TNF-related protein 9) is a fat-derived plasma protein that has salutary effects on glucose metabolism and vascular function. However, the functional role of CTRP9 in ischemic heart disease has not been clarified. Here, we examined the regulation of CTRP9 in response to acute cardiac injury and investigated whether CTRP9 modulates cardiac damage after ischemia and reperfusion. Myocardial ischemia-reperfusion injury resulted in reduced plasma CTRP9 levels and increased plasma free fatty acid levels, which were accompanied by a decrease in CTRP9 expression and an increase in NADPH oxidase component expression in fat tissue. Treatment of cultured adipocytes with palmitic acid or hydrogen peroxide reduced CTRP9 expression. Systemic administration of CTRP9 to wild-type mice, before the induction of ischemia or at the time of reperfusion, led to a reduction in myocardial infarct size following ischemia-reperfusion. Administration of CTRP9 also attenuated myocyte apoptosis in ischemic heart, which was accompanied by increased phosphorylation of AMP-activated protein kinase (AMPK). Treatment of cardiac myocytes with CTRP9 protein reduced apoptosis in response to hypoxia/reoxygenation and stimulated AMPK phosphorylation. Blockade of AMPK activity reversed the suppressive actions of CTRP9 on cardiomyocyte apoptosis. Knockdown of adiponectin receptor 1 diminished CTRP9-induced increases in AMPK phosphorylation and survival of cardiac myocytes. Our data suggest that CTRP9 protects against acute cardiac injury following ischemia-reperfusion via an AMPK-dependent mechanism.     

Late‐onset retinal degeneration pathology due to mutations in CTRP5 is mediated through HTRA1

Late‐onset retinal degeneration (L‐ORD) is an autosomal dominant macular degeneration characterized by the formation of sub‐retinal pigment epithelium (RPE) deposits and neuroretinal atrophy. L‐ORD results from mutations in the C1q‐tumor necrosis factor‐5 protein (CTRP5), encoded by the CTRP5/C1QTNF5 gene. To understand the mechanism underlying L‐ORD pathology, we used a human cDNA library yeast two‐hybrid screen to identify interacting partners of CTRP5. Additionally, we analyzed the Bruch's membrane/choroid (BM‐Ch) from wild‐type (Wt), heterozygous S163R Ctrp5 mutation knock‐in (Ctrp5^S163R/wt), and homozygous knock‐in (Ctrp5^S163R/S163R) mice using mass spectrometry. Both approaches showed an association between CTRP5 and HTRA1 via its C‐terminal PDZ‐binding motif, stimulation of the HTRA1 protease activity by CTRP5, and CTRP5 serving as an HTRA1 substrate. The S163R‐CTRP5 protein also binds to HTRA1 but is resistant to HTRA1‐mediated cleavage. Immunohistochemistry and proteomic analysis showed significant accumulation of CTRP5 and HTRA1 in BM‐Ch of Ctrp5^S163R/S163R and Ctrp5^S163R/wt mice compared with Wt. Additional extracellular matrix (ECM) components that are HTRA1 substrates also accumulated in these mice. These results implicate HTRA1 and its interaction with CTRP5 in L‐ORD pathology.     

CTRP3 protects against uric acid-induced endothelial injury by inhibiting inflammation and oxidase stress in rats

Hyperuricemia, which contributes to vascular endothelial damage, plays a key role in multiple cardiovascular diseases. This study was designed to investigate whether C1q/tumor necrosis factor (TNF)-related protein 3 (CTRP3) has a protective effect on endothelial damage induced by uric acid and its underlying mechanisms. Animal models of hyperuricemia were established in Sprague-Dawley (SD) rats through the consumption of 10% fructose water for 12 weeks. Then, the rats were given a single injection of Ad-CTRP3 or Ad-GFP. The animal experiments were ended two weeks later. In vitro, human umbilical vein endothelial cells (HUVECs) were first infected with Ad-CTRP3 or Ad-GFP. Then, the cells were stimulated with 10 mg/dL uric acid for 48 h after pretreatment with or without a Toll-like receptor 4 (TLR4)-specific inhibitor. Hyperuricemic rats showed disorganized intimal structures, increased endothelial apoptosis rates, increased inflammatory responses and oxidative stress, which were accompanied by reduced CTRP3 and elevated TLR4 protein levels in the thoracic aorta. In contrast, CTRP3 overexpression decreased TLR4 protein levels and ameliorated inflammatory responses and oxidative stress, thereby improving the morphology and apoptosis of the aortic endothelium in rats with hyperuricemia. Similarly, CTRP3 overexpression decreased TLR4-mediated inflammation, reduced oxidative stress, and rescued endothelial damage induced by uric acid in HUVECs. In conclusion, CTRP3 ameliorates uric acid-induced inflammation and oxidative stress, which in turn protects against endothelial injury, possibly by inhibiting TLR4-mediated inflammation and downregulating oxidative stress.     

The adiponectin paralog C1q/TNF-related protein 3 (CTRP3) stimulates testosterone production through the cAMP/PKA signaling pathway

CTRP3, a paralog of adiponectin, is a member of the C1q and tumor necrosis factor (TNF)-related protein (CTRP) superfamily. It is expressed at high levels in adipose tissue and has recently emerged as a novel adipokine. In the present study, we provide the first evidence for a physiological role of the new adipokine, CTRP3, in the reproductive system. CTRP3 was specifically expressed in interstitial Leydig cells, where testosterone is produced, in the adult mouse testis. CTRP3 increased testosterone production by TM3 mouse Leydig cells in a dose-dependent manner. The increased testosterone production was linked to upregulation of steroidogenic proteins expression, such as steroidogenic acute regulatory (StAR) protein and cholesterol side-chain cleavage cytochrome P450 (P450scc). Moreover, increases in intracellular cyclic AMP (cAMP) concentrations and the phosphorylation of cAMP-response element binding protein (CREB) in CTRP3-stimulated TM3 Leydig cells were observed. Inhibition of this signaling pathway by a specific protein kinase A (PKA) inhibitor, H89, blocked testosterone production in CTRP3-stimulated Leydig cells, suggesting that the stimulatory effect of CTRP3 on testosterone production is associated with activation of the cAMP/PKA signaling pathway. Thus, our results demonstrate a physiological role for CTRP3 in testicular steroidogenesis and provide novel insights in the intracellular mechanisms activated by this protein.     

The proregion of mouse BMP15 regulates the cooperative interactions of BMP15 and GDF9

Bone morphogenetic protein 15 (BMP15) and growth and differentiation factor 9 (GDF9) are secreted by the mammalian oocyte and are essential for ovarian follicular development, ovulation, and fertility. However, the secreted forms of the BMP15 and GDF9 proteins and the nature of cooperative molecular interactions between BMP15 and GDF9 previously reported have not been fully characterized. In this study, we found that recombinant mouse BMP15 and GDF9 are secreted as cleaved mature and proregion proteins, with BMP15 also secreted as uncleaved promature protein. Noncovalent interactions were identified between the mature and proregion proteins of each growth factor. Moreover, GDF9 mature protein was found to coimmunoprecipitate with the BMP15 proregion, suggestive of a heteromeric association between BMP15 and GDF9. Mouse GDF9 was found to exist mostly as a dimer of mature protein, in both the presence and absence of BMP15. In contrast, BMP15 formed mostly multimers of proregion and mature protein when combined with GDF9, providing further evidence for heteromeric interaction. Mouse BMP15 was found to act cooperatively with GDF9 in a rat granulosa cell thymidine incorporation bioassay and to signal through the BMPR2 and ACVR1B/TGFBR1/ACVR1C receptor-mediated pathways. Immunoneutralization experiments using GDF9 mature protein antibody indicated that these cooperative interactions are species specific. Additionally, immunoneutralization with proregion antibodies highlighted the involvement of the BMP15 proregion in BMP15/GDF9 cooperative interactions. Taken together, these findings support a novel hypothesis where the extracellular cooperative interactions of recombinant mouse BMP15 and GDF9 are multimeric, involving the proregion of BMP15, and may well be species specific.     

Knockdown of CTRP6 reduces the deposition of intramuscular and subcutaneous fat in pigs via different signaling pathways

The regulation of porcine subcutaneous (SC) and intramuscular (IM) fat deposition significantly affects pork quality and the lean meat percentage of the carcass, respectively. The adipokine C1q/tumor necrosis factor-related protein 6 (CTRP6), plays a significant role in regulating animal fat deposition. The purpose of this study was to understand the effects of CTRP6 gene knockdown in IM and SC adipocytes by RNA-seq analysis. A total of 1830 and 2936 differentially expressed genes (DEGs) were identified in SC and IM adipocytes, respectively. 844 were down- and 2092 were upregulated in SC adipocytes, while 648 were down- and 1182 were upregulated in IM adipocytes. Furthermore, 1778 DEGs were detected only in SC adipocytes, 672 DEGs only in IM adipocytes, and 1158 DEGs in both types of adipocytes. GO analysis indicated that DEGs involved in adipocyte differentiation were significantly enriched in both SC and IM adipocytes following treatment with CTRP6-siRNA. Moreover, KEGG pathway enrichment analysis revealed differences of metabolic regulation between IM and SC adipocytes. With CTRP6-silencing, the signaling pathways related to Ras and arachidonic acid metabolism were significantly enriched in IM adipocytes, while four other signaling pathways, encompassing the TNF, MAPK, p53 and adipokine pathway were specifically enriched in SC adipocytes. Interestingly, the effect of CTRP6-siRNA treatment was attenuated by the specific Ras activator ML-097 in IM adipocytes, while the specific p53 activator SJ-172550 had the corresponding effect in SC adipocytes. Altogether, we suggest that CTRP6 may be a differential regulator of the development and metabolism of IM and SC adipose tissues.     

Common silent mutations in all types of hereditary complement C1q deficiencies

Hereditary complete deficiency of complement component C1q is a rare genetic disorder that is associated with severe recurrent infections and a high prevalence of lupus-erythematosus-like symptoms. In the past, several single nucleotide polymorphisms have been identified in all three genes coding for the C1q A, B, and C chains. These point mutations which either lead to termination codons, frameshift, or amino acid exchanges were thought to be responsible for these defects as no other nonsense or missense mutations were found. As a result of the aberrations, either a nonfunctional C1q antigen is present or no C1q protein is detectable in the patients' sera. Screening 46 individuals from seven families with different forms of C1q deficiencies identified a homologous silent mutation at position Gly70 (GGG>GGA) of the C1q A gene of all 11 C1q-deficient patients. A high number of family members that were heterozygous for the coding mutations carried the silent mutation in the homozygous (18%) or heterozygous (36%) state. In addition to the Gly70 mutation in the A gene, another homozygous silent mutation (C gene at position Pro14, CCT>CCC) was detected in all C1q-deficient patients.