An APPL1-AMPK signaling axis mediates beneficial metabolic effects of adiponectin in the heart

Abstract

Adiponectin promotes cardioprotection by various mechanisms, and this study used primary cardiomyocytes and the isolated working perfused heart to investigate cardiometabolic effects. We show in adult cardiomyocytes that adiponectin increased CD36 translocation and fatty acid uptake as well as insulin-stimulated glucose transport and Akt phosphorylation. Coimmunoprecipitation showed that adiponectin enhanced association of AdipoR1 with APPL1, subsequent binding of APPL1 with AMPKα2, which led to phosphorylation and inhibition of ACC and increased fatty acid oxidation. Using siRNA to effectively knockdown APPL1 in neonatal cardiomyocytes, we demonstrated an essential role for APPL1 in mediating increased fatty acid uptake and oxidation by adiponectin. Importantly, enhanced fatty acid oxidation in conjunction with AMPK and ACC phosphorylation was also observed in the isolated working heart. Despite increasing fatty acid oxidation and myocardial oxygen consumption, adiponectin increased hydraulic work and maintained cardiac efficiency. In summary, the present study documents several beneficial metabolic effects mediated by adiponectin in the heart and provides novel insight into the mechanisms behind these effects, in particular the importance of APPL1.

Fig. 1.

Adiponectin (Ad) increases fatty acid (FA) uptake and cell surface CD36 and sensitizes insulin's actions in adult cardiomyocytes. Isolated rat cardiomyocytes were left unstimulated (0 min, Control) or stimulated with 10 μg Ad or with 100 nM insulin for the durations indicated in the figures. Representative confocal images and quantification of FA uptake (A) or cell surface CD36 (>100 cells per condition; B). C: quantification of 2-deoxyglucose uptake in cardiomyocytes stimulated with 10 nM insulin for 5 min with or without pretreatment with 10 μg Ad for 15 min. D: representative immunoblot showing that Ad sensitizes insulin stimulation of Akt phosphorylation (Thr³⁰⁸). Data are means ± SE of ≥4 independent experiments. *P < 0.05 vs. Control (0 min Ad treatment); †P < 0.05 vs. insulin alone.

Fig. 2.

Functional participation of APPL1 in Ad's physiological actions in isolated rat cardiomyocytes. Representative immunoblots (IB) showing interaction between APPL1 and adiponectin receptor 1 (AdipoR1) or AdipoR2 (A) and AMPKα1 or AMPKα2 catalytic subunits (C). B: representative confocal images (optical single slice) showing individual and merged staining of cell surface APPL1 (green) or sarcolemma marker dystrophin (red). Enlarged segment is an optical single slice, magnification ×60. D: immunoblot showing knockdown of endogenous APPL1 (si-APPL1) compared with nontransfected (Control) neonatal rat cardiomyocytes. Palmitate uptake (E), palmitate oxidation (F), and CD36 translocation (G) in isolated neonatal rat cardiomyocytes not transfected (Control) or transfected with an unrelated siRNA (SCR) or siRNA against APPL1 (si-APPL1) and were treated with or without 10 μg Ad for 1 h (E and F) or 30 min (G). H: representative immunoblot showing AMPK phosphorylation (Thr¹⁷²) in isolated neonatal rat cardiomyocytes transfected with SCR or si-APPL1 and subsequently treated with or without 10 μg Ad for 10 min. Data are means ± SE of ≥4 independent experiments and are expressed relative to Control (0 min). *P < 0.05 vs. Control (0 min).

Fig. 3.

Ad activates LKB1-AMPK signaling to increase FA metabolism in adult primary rat cardiomyocytes. A: representative immunoblot and quantification of LKB1 in cytosolic and nuclear fractions in cells untreated or treated with 10 μg Ad for 5 min. B: representative immunoblot showing interaction between LKB1 and APPL1. C: representative immunoblot and quantification of AMPK phosphorylation (Thr¹⁷²). D: quantification of palmitate uptake in unstimulated (Control) or stimulated with 10 μg Ad for indicated times with or without 30-min pretreatment with 10 μM compound C. Data are means ± SE of ≥4 independent experiments and are expressed relative to Control (0 min or without adiponectin). *P < 0.05 vs. Control.

Fig. 4.

Ad increases acetyl-CoA carboxylase (ACC) phosphorylation and FA oxidation and decreases ACC activity and intracellular lipid content. A: representative immunoblot and quantification of ACC phosphorylation (Ser⁷⁹). B: quantification of ACC activity in adult cardiomyocytes. ACC activity determined by incorporation of [¹⁴C]sodium bicarbonate into malonyl-CoA in adult cardiomyocytes stimulated with or without 10 μg Ad for 1 or 2 h or with 1 mM 5-aminoimidazole-4-carboxamide-1-β-d-ribofuranoside (AICAR) for 2 h. C: quantification of palmitate oxidation in cardiomyocytes untreated (0 h) or treated with 10 μg Ad for 2 or 6 h or with 1 mM AICAR for 2 h. Representative confocal images (D) and Quantification of intracellular lipid content (E) assayed using Oil red O staining in cardiomyocytes treated with 10 μg Ad or without (CON) for 20 h. Data are means ± SE of ≥4 independent experiments and are expressed relative to time-matched control (0 min). *P < 0.05 vs. Control.

Fig. 5.

Effects of Ad on substrate metabolism, signaling molecules, and cardiac performance in perfused working mouse hearts. Representative immunoblot and quantification of phospho-AMPK^Thr172 (A), phospho-ACC^Ser79 (B), and total AMPKα1, AMPKα2, ACC, LKB1, and APPL1 (C) in the same hearts in which metabolism was measured. Quantification of Ad's effects on palmitate oxidation (D), glucose oxidation (E), glycolysis (F), oxygen consumption (MV̇o₂; G), hydraulic work (H), and cardiac efficiency (I) in perfused hearts. All parameters were measured in hearts (n = 5) isolated from 6-wk-old mice perfused with 4 μg/ml Ad or without (CON) for 60 min. Data are means ± SE from, and are expressed relative to, hearts perfused without Ad for 60 min (CON). *P < 0.05 vs. CON.