Adipsin inhibits Irak2 mitochondrial translocation and improves fatty acid ß-oxidation to alleviate diabetic cardiomyopathy.

BACKGROUND: Diabetic cardiomyopathy (DCM) causes the myocardium to rely on fatty acid ß-oxidation for energy. The accumulation of intracellular lipids and fatty acids in the myocardium usually results in lipotoxicity, which impairs myocardial function. Adipsin may play an important protective role in the pathogenesis of DCM. The aim of this study is to investigate the regulatory effect of Adipsin on DCM lipotoxicity and its molecular mechanism. METHODS: A high-fat diet (HFD)-induced type 2 diabetes mellitus model was constructed in mice with adipose tissue-specific overexpression of Adipsin (Adipsin-Tg). Liquid chromatography-tandem mass spectrometry (LC-MS/MS), glutathione-S-transferase (GST) pull-down technique, Co-immunoprecipitation (Co-IP) and immunofluorescence colocalization analyses were used to investigate the molecules which can directly interact with Adipsin. The immunocolloidal gold method was also used to detect the interaction between Adipsin and its downstream modulator. RESULTS: The expression of Adipsin was significantly downregulated in the HFD-induced DCM model (P < 0.05). Adipose tissue-specific overexpression of Adipsin significantly improved cardiac function and alleviated cardiac remodeling in DCM (P < 0.05). Adipsin overexpression also alleviated mitochondrial oxidative phosphorylation function in diabetic stress (P < 0.05). LC-MS/MS analysis, GST pull-down technique and Co-IP studies revealed that interleukin-1 receptor-associated kinase-like 2 (Irak2) was a downstream regulator of Adipsin. Immunofluorescence analysis also revealed that Adipsin was co-localized with Irak2 in cardiomyocytes. Immunocolloidal gold electron microscopy and Western blotting analysis indicated that Adipsin inhibited the mitochondrial translocation of Irak2 in DCM, thus dampening the interaction between Irak2 and prohibitin (Phb)-optic atrophy protein 1 (Opa1) on mitochondria and improving the structural integrity and function of mitochondria (P < 0.05). Interestingly, in the presence of Irak2 knockdown, Adipsin overexpression did not further alleviate myocardial mitochondrial destruction and cardiac dysfunction, suggesting a downstream role of Irak2 in Adipsin-induced responses (P < 0.05). Consistent with these findings, overexpression of Adipsin after Irak2 knockdown did not further reduce the accumulation of lipids and their metabolites in the cardiac myocardium, nor did it enhance the oxidation capacity of cardiomyocytes expose to palmitate (PA) (P < 0.05). These results indicated that Irak2 may be a downstream regulator of Adipsin. CONCLUSIONS: Adipsin improves fatty acid ß-oxidation and alleviates mitochondrial injury in DCM. The mechanism is related to Irak2 interaction and inhibition of Irak2 mitochondrial translocation.

Adipsin alleviates cardiac microvascular injury in diabetic cardiomyopathy through Csk-dependent signaling mechanism.

BACKGROUND: Microvascular complications are associated with an overtly increased risk of adverse outcomes in patients with diabetes including coronary microvascular injury which manifested as disruption of adherens junctions between cardiac microvascular endothelial cells (CMECs). However, particular mechanism leading to diabetic coronary microvascular hyperpermeability remains elusive. METHODS: Experimental diabetes was induced in mice with adipose tissue-specific Adipsin overexpression (AdipsinLSL/LSL-Cre) and their respective control (AdipsinLSL/LSL). In addition, cultured CMECs were subjected to high glucose/palmitic acid (HG + PA) treatment to simulate diabetes for a mechanistic approach. RESULTS: The results showed that Adipsin overexpression significantly reduced cardiac microvascular permeability, preserved coronary microvascular integrity, and increased coronary microvascular density. Adipsin overexpression also attenuated cardiac dysfunction in diabetic mice. E/A ratio, an indicator of cardiac diastolic function, was improved by Adipsin. Adipsin overexpression retarded left ventricular adverse remodeling, enhanced LVEF, and improved cardiac systolic function. Adipsin-enriched exosomes were taken up by CMECs, inhibited CMECs apoptosis, and increased CMECs proliferation under HG + PA treatment. Adipsin-enriched exosomes also accelerated wound healing, rescued cell migration defects, and promoted tube formation in response to HG + PA challenge. Furthermore, Adipsin-enriched exosomes maintained adherens junctions at endothelial cell borders and reversed endothelial hyperpermeability disrupted by HG + PA insult. Mechanistically, Adipsin blocked HG + PA-induced Src phosphorylation (Tyr416), VE-cadherin phosphorylation (Tyr685 and Tyr731), and VE-cadherin internalization, thus maintaining CMECs adherens junctions integrity. LC-MS/MS analysis and co-immunoprecipitation analysis (Co-IP) unveiled Csk as a direct downstream regulator of Adipsin. Csk knockdown increased Src phosphorylation (Tyr416) and VE-cadherin phosphorylation (Tyr685 and Tyr731), while abolishing Adipsin-induced inhibition of VE-cadherin internalization. Furthermore, Csk knockdown counteracted Adipsin-induced protective effects on endothelial hyperpermeability in vitro and endothelial barrier integrity of coronary microvessels in vivo. CONCLUSIONS: Together, these findings favor the vital role of Adipsin in the regulation of CMECs adherens junctions integrity, revealing its promises as a treatment target against diabetic coronary microvascular dysfunction. Graphical abstract depicting the mechanisms of action behind Adipsin-induced regulation of diabetic coronary microvascular dysfunction.

Complement factor D derived from epicardial adipose tissue participates in cardiomyocyte apoptosis after myocardial infarction by mediating PARP-1 activity.

BACKGROUND: Acute myocardial infarction (MI) is considered to be the main cause of congestive heart failure. The aim of this study was to provide an in-depth analysis of athophysiological processes and provide key targets for intervention in the occurrence of acute MI. METHODS: A rat model of MI was established by ligation of left anterior descending branch. Heart tissue, epicardial adipose tissue (EAT) and subcutaneous adipose tissue (SAT) were collected. H9c2 cells were used to explore the mechanism of complement factor D (CFD) regulating cardiomyocyte apoptosis. RESULTS: Myocardial apoptosis were observed in MI rat, and more EAT was found in the MI group in vivo. The conditioned medium prepared by EAT (EAT-CM) significantly reduced the activity of H9c2 cells. The content of CFD in EAT was significantly increased, and CFD promoted cardiomyocyte apoptosis in vitro and CFD-IN1 (a selective inhibitor of CFD) could revised this effect. CFD induced poly ADP-ribosepolymerase-1 (PARP-1) overactivation. Furthermore, the addition of pan-caspase inhibitor Z-VAD in the SAT-CM + CFD group couldn't affect H9c2 cell apoptosis. CFD induced cell apoptosis via PARP-1 activation and PARP-1 inhibitor 3-Aminobenzamide could revise this effect. The injection of CFD-IN1 in MI rat model confirmed that inhibition of CFD activity alleviated cardiomyocytes apoptosis. CONCLUSION: Our findings indicate that EAT mediating cardiomyocyte apoptosis after MI through secretion of CFD and activation of PARP-1 activity.

Suprachoroidal delivery enables targeting, localization and durability of small molecule suspensions.

Drug delivery to the suprachoroidal space (SCS®) has become a clinical reality after the 2021 FDA approval of CLS-TA, a triamcinolone acetonide injectable suspension for suprachoroidal use (XIPERE®), administered via a microneedle-based device, the SCS Microinjector®. Suprachoroidal (SC) delivery facilitates targeting, compartmentalization, and durability of small molecule suspensions, thereby potentially addressing some of the efficacy, safety, and treatment burden limitations of current retinal therapies. Herein, the design features of the SCS Microinjector are reviewed, along with the biomechanics of SC drug delivery. Also presented are preclinical evaluations of SC small molecule suspensions from 4 different therapeutic classes (plasma kallikrein inhibitor, receptor tyrosine kinase inhibitor, corticosteroid, complement factor D inhibitor), highlighting their potential for durability, targeted compartmentalization, and acceptable safety profiles following microinjector-based SC delivery. The clinical evaluations of the safety, tolerability and efficacy of SC delivered triamcinolone further supports potential of SC small molecule suspensions as a clinically viable strategy for the treatment of chorioretinal diseases. Also highlighted are current limitations, key pharmacological considerations, and future opportunities to optimize the SC microinjector platform for safe, effective, and potentially long-acting drug delivery for the treatment of chorioretinal disorders.

Exercise Promotes Bone Marrow Microenvironment by Inhibiting Adipsin in Diet-Induced Male Obese Mice.

Obesity is a growing global epidemic linked to many diseases, including diabetes, cardiovascular diseases, and musculoskeletal disorders. Exercise can improve bone density and decrease excess bone marrow adipose tissue (BMAT) in obese individuals. However, the mechanism of exercise regulating bone marrow microenvironment remains unclear. This study examines how exercise induces bone marrow remodeling in diet-induced obesity. We employed unbiased RNA-Seq to investigate the effect of exercise on the bone marrow of diet-induced obese male mice. Bone mesenchymal stem cells (BMSCs) were isolated to explore the regulatory effects of exercise in vitro. Our data demonstrated that exercise could slow down the progression of obesity and improve trabecular bone density. RNA-seq data revealed that exercise inhibited secreted phosphoprotein 1 (Spp1), which was shown to mediate bone resorption through mechanosensing mechanisms. Interactome analysis of Spp1 using the HINT database showed that Spp1 interacted with the adipokine adipsin. Moreover, exercise decreased BMAT, which induced osteoclast differentiation and promoted bone loss. Our study reveals that exercise improves the bone marrow microenvironment by at least partially inhibiting the adipsin-Spp1 signaling pathway so as to inhibit the alternative complement system from activating osteoclasts in diet-induced obese mice.

Adipsin preserves beta cells in diabetic mice and associates with protection from type 2 diabetes in humans.

Type 2 diabetes is characterized by insulin resistance and a gradual loss of pancreatic beta cell mass and function1,2. Currently, there are no therapies proven to prevent beta cell loss and some, namely insulin secretagogues, have been linked to accelerated beta cell failure, thereby limiting their use in type 2 diabetes3,4. The adipokine adipsin/complement factor D controls the alternative complement pathway and generation of complement component C3a, which acts to augment beta cell insulin secretion5. In contrast to other insulin secretagogues, we show that chronic replenishment of adipsin in diabetic db/db mice ameliorates hyperglycemia and increases insulin levels while preserving beta cells by blocking dedifferentiation and death. Mechanistically, we find that adipsin/C3a decreases the phosphatase Dusp26; forced expression of Dusp26 in beta cells decreases expression of core beta cell identity genes and sensitizes to cell death. In contrast, pharmacological inhibition of DUSP26 improves hyperglycemia in diabetic mice and protects human islet cells from cell death. Pertaining to human health, we show that higher concentrations of circulating adipsin are associated with a significantly lower risk of developing future diabetes among middle-aged adults after adjusting for body mass index (BMI). Collectively, these data suggest that adipsin/C3a and DUSP26-directed therapies may represent a novel approach to achieve beta cell health to treat and prevent type 2 diabetes.

Total Synthesis of A54145 Factor D.

An efficient total synthesis of A54145 factor D (A5D), a member of the A54145 family of cyclic lipodepsipeptide antibiotics, is reported. The peptide was constructed by attaching the peptide to the 2'-chlorotrityl polystyrene resin via Sar5 and developing conditions that avoided diketopiperazine formation upon subsequent elaboration using 9-fluorenylmethoxycarbonyl solid-phase peptide synthesis. This route allowed for facile formation of the crucial depsi bond. A branched acyclic precursor was cyclized off-resin and then globally deprotected to obtain A5D. Consistent with recent studies by others, we found that the MeOAsp residue has the 2S,3R configuration. We also established that the configuration of the stereocenter in the anteiso-undecanoyl lipid tail does not affect biological activity.

Senescent dermal fibroblasts negatively influence fibroblast extracellular matrix-related gene expression partly via secretion of complement factor D.

Aging is associated with a decrease of extracellular matrix and an increase of senescent cells in the dermal layer. Here, to examine whether and how senescent cells are involved in aging-related deterioration of the dermal layer, we cocultured dermal young fibroblasts (low-passage number) with senescent cells (high-passage number) in Transwells, in which the two cell types are separated by a semipermeable membrane. Young fibroblasts in coculture showed decreased collagen type I alpha 1 chain and elastin gene expression, and increased matrix metalloproteinase 1 (MMP1) gene expression. To identify causative factors, we compared gene expression of young and senescent cells and selected candidate secretory factors whose expression was increased by ≥2.5 in senescent fibroblasts. Then, we used siRNAs to knock down each of the 11 candidate genes in senescent fibroblasts in the coculture system. Knockdown of complement factor D (CFD) in senescent fibroblasts significantly reduced the increase of MMP1 in the cocultured young fibroblasts. In monocultures, treatment of young fibroblasts with CFD resulted in increased MMP1 gene expression, while knockdown of CFD in senescent fibroblasts decreased MMP1 gene expression. In addition, production of CFD was increased in culture medium of untreated senescent fibroblasts. Furthermore, CFD gene and protein expression were increased in the dermal layer of skin specimens from aged subjects (>70 years old), compared to young subjects (<20 years old). Overall, these results suggest that senescent cells negatively influence matrix production and promote degradation of nearby fibroblasts in the dermal layer, in part through secretion of CFD.

Eosinophil granule cationic proteins regulate complement. I. Activity on the alternative pathway.

Eosinophil granules contain several cationic proteins that mediate tissue damage in allergic disease. The present study examined the capacity and mechanisms by which these cationic proteins regulate activity of the alternative pathway of C. Eosinophil peroxidase and eosinophil cationic protein inhibited formation of cell-bound alternative pathway C3 convertase, causing 50% inhibition of lysis at about 0.19 and 0.75 microgram/10(7) cellular intermediates, respectively. Major basic protein inhibited alternative pathway C3 activity by only 19% at 1.5 micrograms/10(7) cellular intermediates. Eosinophil-derived neurotoxin had no activity on the alternative pathway. The eosinophil granule proteins were examined for the mechanism by which they inhibited alternative pathway activity. Eosinophil peroxidase and major basic protein inhibited fluid phase factor B consumption in a reaction mixture that also contained factors D and C3b, eosinophil-derived neurotoxin had no activity on factor B consumption, and eosinophil cationic protein consumed factor B in the absence of C3b and factor D. Both eosinophil cationic protein and eosinophil peroxidase enhanced the decay of preformed alternative pathway convertase. Lysis of EAC4b,3b cellular intermediates formed to contain a low surface amount of C3b was more inhibited than was lysis of cells formed with a standard amount of C3b on the surface. This suggests that these eosinophil proteins acted predominantly on C3b to regulate alternative pathway activity. We also found that none of the eosinophil granule cationic proteins had any effect on later events after the formation of the C3 convertase. We conclude that although eosinophil-derived neurotoxin (isoelectric pH value (pI) = 8.9) does not regulate alternative pathway activity, the more highly charged eosinophil granule cationic proteins--major basic protein (pI = 10.9), eosinophil cationic protein (pI = 10.8), and eosinophil peroxidase (pI = 10.8)--do share the capacity to regulate C activity and may exert this activity in vivo.

A novel cleavage product of human complement component C3 with structural and functional properties of cobra venom factor.

The generation of two cleavage products of human C3, termed C3o and C3p, by incubation with a C3-cleaving protease isolated from cobra venom (Naja naja siamensis) is described. The venom protease removes the C3p fragment (Mr approximately 33,000) from the C3dg region of the C3 alpha-chain. The major cleavage fragment C3o (Mr approximately 140,000) contains the unaltered beta-chain of C3 and two alpha-chain-derived polypeptides of Mr approximately 29,000 and Mr approximately 38,000, respectively. Amino-terminal amino acids sequence analysis of C3p and the three chains of C3o allowed the identification of the exact location of the two alpha-chain-derived fragments of C3o and the three cleavage sites of the venom protease. The chain structure of C3o resembles those of C3c and cobra venom factor. In contrast to C3c but like cobra venom factor (and C3b), C3o was found to support the activation of the serine protease Factor B by cleavage in the presence of Factor D and Mg2+ into Bb and Ba, generating an enzymatically active complex that is able to cleave a fluorogenic peptide substrate for C3 convertases. Since the only stretch of amino acid residues of C3o not present in C3c is the carboxyl terminus of the Mr approximately 29,000 chain of C3o, it is suggested that this region is important for the interaction with Factor B and convertase formation.

A reexamination of the role of magnesium in the human alternative pathway of complement.

The formation of the alternative-pathway C3 convertase has been previously suggested to have an absolute requirement for Mg2+, especially at the level of complex formation between C3b and factor B (B). In the course of defining spectral probes that could be used to monitor the C3b-B interaction (e.g. 1-anilino-8-naphthalene sulfonic acid fluorescence and near-u.v. circular dichroism) we observed that the signal change reporting on this binding was not completely reversed upon addition of excess ethylene-diaminetetraacetic acid (EDTA). Using sucrose gradient ultracentrifugation, we have directly demonstrated a Mg2+-independent C3b-B complex in the fluid phase. B thus bound was not only susceptible to specific proteolytic activation by factor D, but the resulting C3bBb enzyme was able to convert native C3 to C3b. Interestingly, we were unable to detect Mg2+-independent specific binding of 125I-B to C3b which was particle-bound. Using a sensitive hemolytic assay, however, we estimated that the functional activity of B with surface-bound C3b is 80-fold greater in the presence of physiological Mg2+ (0.5 mM) than in 2 mM EDTA. In contrast, the fluid-phase association is estimated to differ less than three-fold under the same conditions. These data demonstrate that the requirement for Mg2+ in the formation of the fluid-phase alternative-pathway C3 convertase is not absolute. Furthermore, they suggest a difference in the stable functional properties of fluid-phase and surface-bound C3b.

Activation of factor B of the complement system by kallikrein and its light chain.

The cleavage of factor B, a protein of the alternative pathway of complement, by kallikrein was studied. Like factor D, kallikrein can cleave B to generate the alternative pathway C3 convertase C3bBb. When this convertase was formed on erythrocytes previously coated with C3b, lysis was observed indicating that a functionally active C3 convertase was formed. B was also cleaved by kallikrein in the presence of fluid phase C3b, and this resulted in B fragments comparable in size to those generated in the presence of D. The capacity of kallikrein to cleave B is localised in the light chain of the kallikrein molecule, which is the same chain of kallikrein that is responsible for its other enzymatic activities. Since on a molar basis D is much more active then kallikrein in cleaving B, a physiological role for B activation by kallikrein is only likely under certain conditions, and still has to be established.

The activation of the alternative pathway C3 convertase by human plasma kallikrein.

Human plasma kallikrein can replace factor D for the activation of the alternative pathway C3 convertase of human complement. The factor B cleavage patterns by factor D and kallikrein are indistinguishable. The ability of kallikrein to cleave factor B is influenced by the magnesium ion concentration and the C3b concentration. Factor D is about ten-fold more effective on a molar basis, for the alternative pathway C3 convertase activation than is kallikrein. The physiological role of the action of kallikrein on the alternative pathway C3 convertase is discussed.

Properdin factor D: effects on thrombin-induced platelet aggregation.

Factor D, when preincubated with platelet suspensions, at concentrations as low as 1.2 micrograms/ml, inhibited thrombin-induced platelet aggregation. No inhibition of collagen or arachidonic acid-induced platelet aggregation was found. Inhibition occurred, but to a lesser extent, when thrombin and factor D were added to platelets at the same time. No inhibition occurred when factor D was added after thrombin. Thrombin was able to overcome inhibition by factor D by increasing its concentration. Diisopropyl-phosphorofluoridate-inactivated factor D also inhibited thrombin-induced platelet aggregation so that enzymatic activity of factor D was not required for inhibition. Factor D absorbed with hirudin coupled to Sepharose 6B showed no decrease in inhibitory capacity. 125I-Factor D bound to platelets in a manner suggesting an equilibrium reaction similar to thrombin. At low factor D input, binding was linear, whereas at higher input, binding began to approach saturation. Binding of 125I-labeled thrombin to platelets was inhibited by factor D. Analysis of these data show that factor D does not alter the total number of thrombin molecules which bind to the platelet surface at saturation. However, the dissociation constant for thrombin is altered from 2.78 to 6.90 nM in the presence of factor D (20 micrograms/ml). Factor D is thus a competitive inhibitor of thrombin binding, although the affinity of factor D for the platelet thrombin receptor is much less than that of thrombin. These phenomena occur at physiologic concentrations of factor D. Therefore, factor D may function in vivo as an inhibitor of platelet aggregation.

The alternative pathway C3/C5 convertase: chemical basis of factor B activation.

The structural basis of activation of the alternative pathway C3 convertase was explored. For this purpose a modified isolation procedure of the activating enzyme, Factor D, was elaborated. The procedure affords a 70,000-fold purification of the enzyme with a 20% yield. A simple assay was designed for the quantitation of both Factor D and Factor B activity. On the basis of activity measurements and amino acid analysis, Factor D concentration in plasma was estimated to be 1 microgram/ml. Highly purified Factor D was used to activate Factor B in the presence of C3b and Mg++. The resulting fragments, Ba and Bb, were characterized with respect to their circular dichroism spectra, amino acid compositions, reactive sulfhydryl groups, and partial amino- and carboxy-terminal sequences. The results indicate that the Ba fragment constitutes the amino-terminal region and the Bb fragment the carboxy-terminal region of Factor B. The bond in Factor B that is cleaved by Factor D is proposed to be an arginyl-lysine bond.