C1q/TNF-related protein-12 (CTRP12), a novel adipokine that improves insulin sensitivity and glycemic control in mouse models of obesity and diabetes

Despite the prevalence of insulin resistance and type 2 diabetes mellitus, their underlying mechanisms remain incompletely understood. Many secreted endocrine factors and the intertissue cross-talk they mediate are known to be dysregulated in type 2 diabetes mellitus. Here, we describe CTRP12, a novel adipokine with anti-diabetic actions. The mRNA and circulating levels of CTRP12 were decreased in a mouse model of obesity, but its expression in adipocytes was increased by the anti-diabetic drug rosiglitazone. A modest rise in circulating levels of CTRP12 by recombinant protein administration was sufficient to lower blood glucose in wild-type, leptin-deficient ob/ob, and diet-induced obese mice. A short term elevation of serum CTRP12 by adenovirus-mediated expression improved glucose tolerance and insulin sensitivity, normalized hyperglycemia and hyperinsulinemia, and lowered postprandial insulin resistance in obese and diabetic mice. CTRP12 improves insulin sensitivity in part by enhancing insulin signaling in the liver and adipose tissue. Further, CTRP12 also acts in an insulin-independent manner; in cultured hepatocytes and adipocytes, CTRP12 directly activated the PI3K-Akt signaling pathway to suppress gluconeogenesis and promote glucose uptake, respectively. Collectively, these data establish CTRP12 as a novel metabolic regulator linking adipose tissue to whole body glucose homeostasis through insulin-dependent and independent mechanisms.     

Structure of Rpn10 and Its Interactions with Polyubiquitin Chains and the Proteasome Subunit Rpn12

Schizosaccharomyces pombe Rpn10 (SpRpn10) is a proteasomal ubiquitin (Ub) receptor located within the 19 S regulatory particle where it binds to subunits of both the base and lid subparticles. We have solved the structure of full-length SpRpn10 by determining the crystal structure of the von Willebrand factor type A domain and characterizing the full-length protein by NMR. We demonstrate that the single Ub-interacting motif (UIM) of SpRpn10 forms a 1:1 complex with Lys⁴⁸-linked diUb, which it binds selectively over monoUb and Lys⁶³-linked diUb. We further show that the SpRpn10 UIM binds to SpRpn12, a subunit of the lid subparticle, with an affinity comparable with Lys⁴⁸-linked diUb. This is the first observation of a UIM binding other than a Ub fold and suggests that SpRpn12 could modulate the activity of SpRpn10 as a proteasomal Ub receptor.     

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.     

C1q/TNF-related protein-9 promotes macrophage polarization and improves cardiac dysfunction after myocardial infarction

The timely regulation of inflammatory M1 macrophage polarization toward regenerative M2 macrophages suggests the possibility of immunotherapy after myocardial infarction (MI). C1q/TNF-related protein-9 (CTRP9) has anti-inflammatory effects and can ameliorate heart function in mice after long-term myocardial infarction. The role of CTRP9 in macrophage polarization remains completely unclear. This study determined whether CTRP9 can preserve post-MI early cardiac function through the regulation of macrophage polarization. In the present study, an adenovirus-delivered CTRP9 supplement promoted macrophage polarization at Day 3 post MI and improved cardiac function at Day 7 post MI. Pretreatment with gCTRP9 promoted the M1 to M2 polarization transition and attenuated inflammation after lipopolysaccharide + interferon-γ stimulation; the effects were partly abrogated by the adenosine monophosphate kinase (AMPK) inhibitor compound C and were obviously reinforced by pyrrolidine dithiocarbamate, a nuclear factor-κB (NF-κB) inhibitor. Meanwhile, CTPR9 markedly reduced the expression of toll-like receptor 4 (TLR4), myeloid differentiation factor 88 (MyD88), and NF-κB p65 phosphorylation by promoting AMPK phosphorylation in vivo and in vitro. Moreover, the competitive binding of gCTRP9 and LPS to the myeloid differentiation protein 2 (MD2)/TLR4 complex was associated with direct binding to MD2, thereby inhibiting the downstream signaling molecule MyD88. Taken together, we demonstrated that CTRP9 improved post-MI early cardiac function, at least in part, by modulating M1/M2 macrophage polarization, largely via the TLR4/MD2/MyD88 and AMPK-NF-κB pathways.     

CTRP5 ameliorates palmitate-induced apoptosis and insulin resistance through activation of AMPK and fatty acid oxidation

Lipotoxicity resulting from a high concentration of saturated fatty acids is closely linked to development of insulin resistance, as well as apoptosis in skeletal muscle. CTRP5, an adiponectin paralog, is known to activate AMPK and fatty acid oxidation; however, the effects of CTRP5 on palmitate-induced lipotoxicity in myocytes have not been investigated. We found that globular domain of CTRP5 (gCTRP5) prevented palmitate-induced apoptosis and insulin resistance in myocytes by inhibiting the activation of caspase-3, reactive oxygen species accumulation, and IRS-1 reduction. These beneficial effects of gCTRP5 are mainly attributed to an increase in fatty acid oxidation through phosphorylation of AMPK. These results provide a novel function of CTRP5, which may have preventive and therapeutic potential in management of obesity, insulin resistance, and type 2 diabetes mellitus.     

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.     

Osteopontin Is Cleaved at Multiple Sites Close to Its Integrin-binding Motifs in Milk and Is a Novel Substrate for Plasmin and Cathepsin D

Osteopontin (OPN) is a highly modified integrin-binding protein present in most tissues and body fluids where it has been implicated in numerous biological processes. A significant regulation of OPN function is mediated through phosphorylation and proteolytic processing. Proteolytic cleavage by thrombin and matrix metalloproteinases close to the integrin-binding Arg-Gly-Asp sequence modulates the function of OPN and its integrin binding properties. In this study, seven N-terminal OPN fragments originating from proteolytic cleavage have been characterized from human milk. Identification of the cleavage sites revealed that all fragments contained the Arg–Gly–Asp¹⁴⁵ sequence and were generated by cleavage of the Leu¹⁵¹–Arg¹⁵², Arg¹⁵²–Ser¹⁵³, Ser¹⁵³–Lys¹⁵⁴, Lys¹⁵⁴–Ser¹⁵⁵, Ser¹⁵⁵–Lys¹⁵⁶, Lys¹⁵⁶–Lys¹⁵⁷, or Phe¹⁵⁸–Arg¹⁵⁹ peptide bonds. Six cleavages cannot be ascribed to thrombin or matrix metalloproteinase activity, whereas the cleavage at Arg¹⁵²–Ser¹⁵³ matches thrombin specificity for OPN. The principal protease in milk, plasmin, hydrolyzed the same peptide bond as thrombin, but its main cleavage site was identified to be Lys¹⁵⁴–Ser¹⁵⁵. Another endogenous milk protease, cathepsin D, cleaved the Leu¹⁵¹–Arg¹⁵² bond. OPN fragments corresponding to plasmin activity were also identified in urine showing that plasmin cleavage of OPN is not restricted to milk. Plasmin, but not cathepsin D, cleavage of OPN increased cell adhesion mediated by the α_Vβ₃- or α₅β₁-integrins. Similar cellular adhesion was mediated by plasmin and thrombin-cleaved OPN showing that plasmin can be a potent regulator of OPN activity. These data show that OPN is highly susceptible to cleavage near its integrin-binding motifs, and the protein is a novel substrate for plasmin and cathepsin D.     

The Factor VII-activating Protease (FSAP) Enhances the Activity of Bone Morphogenetic Protein-2 (BMP-2)

Factor VII-activating protease (FSAP) is a circulating protease involved in the pathogenesis of atherosclerosis, calcification, and fibrotic processes. To understand how FSAP controls the balance of local growth factors, we have investigated its effect on the regulation of bone morphogenetic proteins (BMPs). BMP-2 is produced as a large pro-form and secreted as a mature heparin-binding growth factor after intracellular processing by pro-protein convertases (PCs). In this study, we discovered that FSAP enhances the biological activity of mature BMP-2 as well as its pro-form, as shown by osteogenic differentiation of C2C12 myoblasts. These findings were complemented by knockdown of FSAP in hepatocytes, which revealed BMP-2 processing by endogenous FSAP. N-terminal sequencing indicated that pro-BMP-2 was cleaved by FSAP at the canonical PC cleavage site, giving rise to mature BMP-2 (Arg²⁸²↓Gln²⁸³), as well as in the N-terminal heparin binding region of mature BMP-2, generating a truncated mature BMP-2 peptide (Arg²⁸⁹↓Lys²⁹⁰). Similarly, mature BMP-2 was also cleaved to a truncated peptide within its N-terminal region (Arg²⁸⁹↓Lys²⁹⁰). Plasmin exhibited a similar activity, but it was weaker compared with FSAP. Thrombin, Factor VIIa, Factor Xa, and activated protein C were not effective. These results were further supported by the observation that the mutation of the heparin binding region of BMP-2 inhibited the processing by FSAP but not by PC. Thus, the proteolysis and activation of pro-BMP-2 and mature BMP-2 by FSAP can regulate cell differentiation and calcification in vasculature and may explain why polymorphisms in the gene encoding for FSAP are related to vascular diseases.     

CTRP12 ameliorates atherosclerosis by promoting cholesterol efflux and inhibiting inflammatory response via the miR-155-5p/LXRα pathway

C1q tumor necrosis factor-related protein 12 (CTRP12), a conserved paralog of adiponectin, is closely associated with cardiovascular disease. However, little is known about its role in atherogenesis. The aim of this study was to examine the influence of CTRP12 on atherosclerosis and explore the underlying mechanisms. Our results showed that lentivirus-mediated CTRP12 overexpression inhibited lipid accumulation and inflammatory response in lipid-laden macrophages. Mechanistically, CTRP12 decreased miR-155-5p levels and then increased its target gene liver X receptor α (LXRα) expression, which increased ATP binding cassette transporter A1 (ABCA1)- and ABCG1-dependent cholesterol efflux and promoted macrophage polarization to the M2 phenotype. Injection of lentiviral vector expressing CTRP12 decreased atherosclerotic lesion area, elevated plasma high-density lipoprotein cholesterol levels, promoted reverse cholesterol transport (RCT), and alleviated inflammatory response in apolipoprotein E-deficient (apoE^−/−) mice fed a Western diet. Similar to the findings of in vitro experiments, CTRP12 overexpression diminished miR-155-5p levels but increased LXRα, ABCA1, and ABCG1 expression in the aortas of apoE^−/− mice. Taken together, these results suggest that CTRP12 protects against atherosclerosis by enhancing RCT efficiency and mitigating vascular inflammation via the miR-155-5p/LXRα pathway. Stimulating CTRP12 production could be a novel approach for reducing atherosclerosis.Subject terms: Non-coding RNAs, Cardiovascular diseases     

Demonstration of beta-lipotropin activating enzyme in porcine pituitary.

Porcine β-lipotropin was incubated with a crude porcine pituitary homogenate, and the main cleavage products of the hormone were isolated and identified. Our results gave evidence for the enzymic cleavage of the Lys⁴⁶-Met⁴⁷, Arg⁶⁰-Tyr⁶¹, Leu⁷⁷-Phe⁷⁸, and Lys⁷⁹-Asn⁸⁰ bonds of the β-lipotropin structure. The cleavage of the Arg⁶⁰-Tyr⁶¹ peptide bond was accompanied with the concomitant release of opiate activity in the first period of incubation, provided that bacitracin was present in the incubation mixture. The enzyme was differentiated from trypsin or plasmin and appears to be a specific intracellular protease involved in the biosynthesis of pituitary endorphins.     

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.     

The Polybasic Insertion in Autotaxin α Confers Specific Binding to Heparin and Cell Surface Heparan Sulfate Proteoglycans

Autotaxin (ATX) is a secreted lysophospholipase D that generates the lipid mediator lysophosphatidic acid (LPA), playing a key role in diverse physiological and pathological processes. ATX exists in distinct splice variants, but isoform-specific functions remain elusive. Here we characterize the ATXα isoform, which differs from the canonical form (ATXβ) in having a 52-residue polybasic insertion of unknown function in the catalytic domain. We find that the ATXα insertion is susceptible to cleavage by extracellular furin-like endoproteases, but cleaved ATXα remains structurally and functionally intact due to strong interactions within the catalytic domain. Through ELISA and surface plasmon resonance assays, we show that ATXα binds specifically to heparin with high affinity (K_d ∼10⁻⁸ m), whereas ATXβ does not; furthermore, heparin moderately enhanced the lysophospholipase D activity of ATXα. We further show that ATXα, but not ATXβ, binds abundantly to SKOV3 carcinoma cells. ATXα binding was abolished after treating the cells with heparinase III, but not after chondroitinase treatment. Thus, the ATXα insertion constitutes a cleavable heparin-binding domain that mediates interaction with heparan sulfate proteoglycans, thereby targeting LPA production to the plasma membrane.     

Histone Deacetylase 6 (HDAC6) Promotes the Pro-survival Activity of 14-3-3ζ via Deacetylation of Lysines within the 14-3-3ζ Binding Pocket

The phospho-binding protein 14-3-3ζ acts as a signaling hub controlling a network of interacting partners and oncogenic pathways. We show here that lysines within the 14-3-3ζ binding pocket and protein-protein interface can be modified by acetylation. The positive charge on two of these lysines, Lys⁴⁹ and Lys¹²⁰, is critical for coordinating 14-3-3ζ-phosphoprotein interactions. Through screening, we identified HDAC6 as the Lys⁴⁹/Lys¹²⁰ deacetylase. Inhibition of HDAC6 blocks 14-3-3ζ interactions with two well described interacting partners, Bad and AS160, which triggers their dephosphorylation at Ser¹¹² and Thr⁶⁴², respectively. Expression of an acetylation-refractory K49R/K120R mutant of 14-3-3ζ rescues both the HDAC6 inhibitor-induced loss of interaction and Ser¹¹²/Thr⁶⁴² phosphorylation. Furthermore, expression of the K49R/K120R mutant of 14-3-3ζ inhibits the cytotoxicity of HDAC6 inhibition. These data demonstrate a novel role for HDAC6 in controlling 14-3-3ζ binding activity.     

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.     

A Motif from Lys216 to Lys222 in Human BUB3 Protein Is a Nuclear Localization Signal and Critical for BUB3 Function in Mitotic Checkpoint

Human BUB3 is a key mitotic checkpoint factor that recognizes centromeric components and recruits other mitotic checkpoint molecules to the unattached kinetochore. The key amino acid residues responsible for its localization are not yet defined. In this study, we identified a motif from Lys²¹⁶ to Lys²²² in BUB3 as its nuclear localization signal. A BUB3 mutant with deletion of this motif (Del216–222) was found to localize to both the cytoplasm and the nucleus, distinct from the exclusively nuclear distribution of wild-type BUB3. Further analysis revealed that residues Glu²¹³, Lys²¹⁶, Lys²¹⁷, Lys²¹⁸, Tyr²¹⁹, and Phe²²¹, but not Lys²²², contribute to nuclear localization. Interestingly, the nuclear localization signal was also critical for the kinetochore localization of BUB3. The deletion mutant Del216–222 and a subtle mutant with four residue changes in this region (E213Q/K216E/K217E/K218E (QE)) did not localize to the kinetochore efficiently or mediate mitotic checkpoint arrest. Protein interaction data suggested that the QE mutant was able to interact with BUB1, MAD2, and BubR1 but that its association with the centromeric components CENP-A and KNL1 was impaired. A motif from Leu⁶¹ to Leu⁶⁵ in CENP-A was found to be involved in the association of BUB3 and CENP-A in cells; however, further assays suggested that CENP-A does not physically interact with BUB3 and does not affect BUB3 localization. Our findings help to dissect the mechanisms of BUB3 in mitotic checkpoint signaling.     

Proteolytic Processing Regulates Toll-like Receptor 3 Stability and Endosomal Localization

Toll-like receptors (TLRs) 3, 7, and 9 are innate immune receptors that recognize nucleic acids from pathogens in endosomes and initiate signaling transductions that lead to cytokine production. Activation of TLR9 for signaling requires proteolytic processing within the ectodomain by endosome-associated proteases. Whether TLR3 requires similar proteolytic processing to become competent for signaling remains unclear. Herein we report that human TLR3 is proteolytically processed to form two fragments in endosomes. Unc93b1 is required for processing by transporting TLR3 through the Golgi complex and to the endosomes. Proteolytic cleavage requires the eight-amino acid Loop1 within leucine-rich repeat 12 of the TLR3 ectodomain. Proteolytic cleavage is not required for TLR3 signaling in response to poly(I:C), although processing could modulate the degree of response toward viral double-stranded RNAs, especially in mouse cells. Both the full-length and cleaved fragments of TLR3 can bind poly(I:C) and are present in endosomes. However, although the full-length TLR3 has a half-life in HEK293T cells of 3 h, the cleaved fragments have half-lives in excess of 7 h. Inhibition of TLR3 cleavage by either treatment with cathepsin inhibitor or by a mutation in Loop1 decreased the abundance of TLR3 in endosomes targeted for lysosomal degradation.     

The Full-length Unprocessed Hedgehog Protein Is an Active Signaling Molecule

The hedgehog (HH) family of ligands plays an important instructional role in metazoan development. HH proteins are initially produced as ∼45-kDa full-length proteins, which undergo an intramolecular cleavage to generate an amino-terminal product that subsequently becomes cholesterol-modified (HH-Np). It is well accepted that this cholesterol-modified amino-terminal cleavage product is responsible for all HH-dependent signaling events. Contrary to this model we show here that full-length forms of HH proteins are able to traffic to the plasma membrane and participate directly in cell-cell signaling, both in vitro and in vivo. We were also able to rescue a Drosophila eye-specific hh loss of function phenotype by expressing a full-length form of hh that cannot be processed into HH-Np. These results suggest that in some physiological contexts full-length HH proteins may participate directly in HH signaling and that this novel activity of full-length HH may be evolutionarily conserved.