Adiponectin enhances the bioenergetics of cardiac myocytes via an AMPK- and succinate dehydrogenase-dependent mechanism
Introduction
The cell's ATP production is linked to its energy requirements, which is monitored by metabolic sensors, including adenosine monophosphate-activated protein kinase (AMPK) [1]. AMPK senses the levels of AMP in the cells and its activity is accordingly adjusted, triggered by allosteric modulation and phosphorylation [2]. An increase in AMP, as a consequence of energy deprivation, results in activation of AMPK, which, thereby, enhances catabolism in muscle and reduces anabolism in the liver, in an effort to increase ATP production and reduce the synthesis and storage of metabolic substrates. It is known to exert its effects through inhibitory phosphorylation of acetyl-CoA carboxylases and glycogen synthases, thereby inhibiting lipid synthesis and glycogen storage, respectively [3,4]. Alternatively, AMPK is also regulated by hormones that maintain energy homeostasis, such as the adipocytokine Adiponectin [5].
Adiponectin was first discovered as a circulating hormone that enhances the cells' sensitivity to insulin, while a decrease in its level is associated with insulin resistance and type 2 diabetes [[6], [7], [8]]. It was soon realized though, that it also functions in maintaining systemic energy homeostasis via elevating fatty acid oxidation in skeletal muscle [9]. Moreover, transgenic mice overexpressing Adiponectin exhibit reduced body fat and longevity after a high fat diet [10]. Its effects are thought to be largely mediated by APPL1 (adaptor protein containing pleckstrin homology domain, phosphotyrosine binding (PTB) domain and leucine zipper motif) [11], as well as, AMPK [12,13].
Adiponectin has also been linked to cardioprotection, as cardiac hypertrophy and mortality increase in Adiponectin-deficient mice and are associated with reduced AMPK activity [14]. The antihypertrophic effects of this hormone on cardiac myocytes are indeed mediated by AMPK. Consistently, Adiponectin deficiency increases infarct size after ischemia-reperfusion in the mouse heart via inhibition of AMPK [15,16]. In both cases, the phenotype is rescued by supplementing the mice with exogenous Adiponectin. Some of the mechanisms underlying its function include stimulation of AMPK-mediated angiogenesis [17], APPL1-AMPK-mediated fatty acid uptake and oxidation [18], and AMPK-PGC-1α-enhanced mitochondrial biogenesis [19]. Additionally, it has been reported to reduce fibrosis by upregulating matrix metallopeptidase 9 [20], protect against inflammation by inhibiting toll-like receptor 4 signaling [21], and reduce oxidative/nitrative stress via activation of protein kinase A and inhibition of NFκB [22]. Finally, a meta-analysis study shows that high levels of Adiponectin correlate with lower risk of cardiovascular disease [23].
We have previously reported that AICAR – an AMPK activator - enhances the cell's basal and maximum oxygen consumption rates (OCR) and spare respiratory capacity (SRC), and that these effects were completely abolished by the succinate dehydrogenase (SDH) inhibitor, 3-nitroproprionic acid (3NP) [24]. Conversely, the loss of the metabolic sensor, AMPK, has been shown to be associated with lower basal, maximum, and SRC in fibroblasts from patients with X-linked adrenoleukodystrophy [25]. These results reveal a direct effect of AMPK on OCR, through regulating SDH activity. SDH, also known as complex II (cII), oxidizes succinate into fumarate while reducing FAD in the tricarboxylic acid (TCA) cycle [26]. Subsequently, electrons are transferred from FADH2, via the iron‑sulfur clusters in SDH, to CoQ/ubiquinol in the electron transport chain (ETC), but unlike the complexes I, III, and IV, CII/SDH does not pump protons across the mitochondrial inner membrane [27]. Accordingly, it does not play vital role in sustaining electron flow and ATP production. However, it is the only enzyme that is common to both the TCA cycle and the ETC and is, thus, well-positioned to boost the cell's bioenergetics through enhancing production of FADH2 and the transfer of its electrons to the ETC.
Thus, we hypothesized that Adiponectin, through AMPK, enhances mitochondrial bioenergetics via promoting the assembly of SDH. Our results confirm this hypothesis and further show that this is mediated in a Sdhaf1- and sirtuin 3 (Sirt3)-dependent manner, during normoxia and hypoxia.
Section snippets
Neonatal rat cardiac myocyte culture and treatments
Cardiac myocytes were prepared as we previously described [28]. Hearts from 1 to 2-day old Sprague-Dawley rats were isolated, chopped, and digested by collagenase to dissociate the cells. The dissociated cells were sorted by centrifugation on a Percoll gradient, followed by differential preplating for 30 min, which enriches for cardiac myocytes and depletes non-myocytes. The purified myocytes were plated in Dulbecco's modified essential medium/Ham F12 (DMEM-F12) with 10% fetal bovine serum
Adiponectin increases cellular bioenergetics via an AMPK-dependent mechanism
We have previously shown that AICAR – an AMPK activator - augments the cells‘bioenergetics via an SDH-dependent mechanism [24], which is regulated by the assembly of its holoenzyme [30]. Thus, we hypothesized that Adiponectin, a regulator of AMPK, would enhance aerobic respiration through an AMPK-SDH-dependent mechanism. To address this, we first knocked down the Prkag1 (AMPKγ1) subunit of AMPK, which binds AMP/ADP/ATP [31], using short hairpin interfering RNA targeting its mRNA (si-Prkag1)
Discussion
The circulating levels of Adiponectin is known to reciprocally correlate with caloric intake, thus, regulating energy homeostasis by increasing the appetite, boosting substrate storage, and decreasing energy expenditure [39]. Accordingly, it has been characterized as a starvation hormone. However, it should be noted that its effects are tissue-dependent. For example, in adipocytes, Adiponectin inhibits lipolysis [40,41], whereas, in skeletal muscle, it increases fatty acid oxidation, mainly
Credit author statement
Yong Heui Jeon: Investigation, Minzhen He: Investigation, Julianne Austin: Investigation, Hyewon Shin: Investigation, Jessica Pfleger: Investigation, and Maha Abdellatif: Conceptualization, Data curation, Formal analysis, Writing -review & editing, Funding acquisition.
Disclaimer
The authors declare that they have no conflicts of interest with the contents of this article.
The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
Declaration of Competing Interest
None.
Acknowlegment
We thank Dr. Junichi Sadoshima, Chairman of the Department of Cell Biology and Molecular Medicine, Rutgers University, for his support. This work was supported by National Institute of Health funding [1R01HL119726 to M.A.]
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