Fig 2: Overexpression of Adipsin inhibits mitochondrial translocation of Irak2. a Western blotting results and quantitative analysis of Irak2 expression in cytoplasm and mitochondria of cardiomyocytes in the CHD + NTg, CHD + Adipsin-Tg, NTg + HFD, and Adipsin-Tg + HFD groups (n = 6). b Electron microscopy images of Irak2 stained with immunogold and quantitative analysis of Irak2-positive dots (red arrow) in the myocardial mitochondria of NTg or Adipsin-Tg mice fed with CHD or HFD (n = 5). Scale bar = 200 nm. c Western blotting results and quantitative analysis of Phb and Opa1 in myocardial mitochondria of NTg or Adipsin-Tg mice fed with CHD or HFD (n = 6). Statistical significance was determined using one-way ANOVA. All data are represented with mean ± SD. †P < 0.05 vs. CHD + NTg; ‡P < 0.05 vs. CHD + Adipsin-Tg; ¶P < 0.05 vs. HFD + NTg. Mito mitochondria, Cyto cytosol, CHD chow diet, HFD high-fat diet, Adipsin-Tg Adipsin tissue-specific transgenic mice, NTg nontransgenic mice, Irak2 interleukin-1 receptor-associated kinase-like 2, VDAC1 voltage-dependent anion channel 1, Phb prohibitin, Opa1 optic atrophy protein 1

Fig 3: Adipsin improves cardiomyocyte FAO through the Irak2 signaling pathway. a Transmission electron microscopic images and quantitative analyses of mouse hearts depicting larger lipid droplets accumulation in various treatment groups (n = 6). Lipid droplets were marked by asterisks. Scale bar = 1 μm. b TG and MDA content in myocardial tissues (n = 8). c FAO evaluating with [3H]-oleic acid uptake (n = 6). d Oxidation pressure test of palmitate in cardiomyocytes from various treatment groups, and evaluation of FAO using quantitative analysis of maximum ΔOCR. Statistical significance was determined using one-way ANOVA. All data are represented with mean ± SD. †P < 0.05 vs. CHD + WT; ‡P < 0.05 vs. HFD + NTg + Ad-shControl. CHD chow diet, HFD high-fat diet, Adipsin-Tg Adipsin tissue-specific transgenic mice, NTg nontransgenic mice, TG triglyceride, MDA malonaldehyde, FAO fatty acid β-oxidation, BSA bovine serum albumin, ETO etomoxir, PA palmitate, OCR oxygen consumption rate, OM oligomycin, FCCP fluoro-carbonyl cyanide phenylhydrazone, Rot/AA rotenone and antimycin A, △OCR oxygen consumption rate[PA-(PA+ETO)]—oxygen consumption rate[BSA-(BSA+ETO)], Irak2 interleukin-1 receptor-associated kinase-like 2

Fig 4: Adipsin and Irak2 bind directly in cardiomyocytes. a Schematic representation of the experimental protocol. The cardiomyocytes lysis prey protein was obtained by immobilizing GST-Adipsin with equilibrated glutathione agarose. Then, the eluted protein complexes were employed for mass spectrometry analysis and subsequent immunoblotting. b Proteins bound and pulled down by purified GST-Adipsin in cardiomyocytes. c, d Co-IP results indicating the interaction between Adipsin and Irak2 following the infection with Ad-Adipsin. e Co-IP results indicating the interaction between Adipsin and Irak2 with or without PA challenge following the infection with Ad-Adipsin (n = 5). f Colocalization images and analysis of Adipsin (green) and Irak2 (red) proteins in cardiomyocytes. Scale bar = 50 μm. Statistical significance was determined using a two-tailed Student’s t test. All data are represented with mean ± SD. *P < 0.05 vs. Control. LC–MS/MS liquid chromatography-tandem mass spectrometry, Co-IP Co-immunoprecipitation, DAPI 4′,6-diamidino-2-phenylindole, Irak2 interleukin-1 receptor-associated kinase-like 2, PA palmitate, GST glutathione-S-transferase, IB immunoblotting

Fig 5: Adipsin improves cardiac function, reduces mitochondrial cristae damage, and improves mitochondrial function through Irak2 signaling pathway. a Echocardiographic results and quantitative analysis of echocardiographic data including LVEF, LVFS, LVIDd and LVIDs (n = 8). b Pulse-wave Doppler results and quantitative analysis of the E/A ratio (n = 8). c Transmission electron microscopic images of myocardium from different groups (n = 8). Scale bar = 500 nm. d OCR in cardiomyocytes from various treatment groups, and quantitative statistical analysis of OCR including basal respiration, ATP production, maximal respiration and spare respiration capacity. Statistical significance was determined using one-way ANOVA. All data are represented with mean ± SD. †P < 0.05 vs. CHD + WT; ‡P < 0.05 vs. HFD + NTg + Ad-shControl; *P < 0.05 vs. Con; §P < 0.05 vs. PA + Ad-Control + siControl. CHD chow diet, HFD high-fat diet, Adipsin-Tg Adipsin tissue-specific transgenic mice, NTg nontransgenic mice, LVEF left ventricular ejection fraction, LVFS left ventricular fractional shortening, LVIDd end-diastolic left ventricular internal diameters, LVIDs end-systolic left ventricular internal diameters, E/A the ratio of mitral peak velocity of early filling (E) to mitral peak velocity of late filling (A), OCR oxygen consumption rate, OM oligomycin, FCCP fluoro-carbonyl cyanide phenylhydrazone, Rot/AA rotenone and antimycin A, PA palmitate, Irak2 interleukin-1 receptor-associated kinase-like 2, siControl control small interfering RNA