Specific amino acid supplementation rescues the heart from lipid overload-induced insulin resistance and contractile dysfunction by targeting the endosomal mTOR–v-ATPase axis

Abstract

Objective The diabetic heart is characterized by extensive lipid accumulation which often leads to cardiac contractile dysfunction. The underlying mechanism involves a pivotal role for vacuolar-type H + -ATPase (v-ATPase, functioning as endosomal/lysosomal proton pump). Specifically, lipid oversupply to the heart causes disassembly of v-ATPase and endosomal deacidification. Endosomes are storage compartments for lipid transporter CD36. However, upon endosomal deacidification, CD36 is expelled to translocate to the sarcolemma, thereby inducing myocardial lipid accumulation, insulin resistance, and contractile dysfunction. Hence, the v-ATPase assembly may be a suitable target for ameliorating diabetic cardiomyopathy. Another function of v-ATPase involves the binding of anabolic master-regulator mTORC1 to endosomes, a prerequisite for the activation of mTORC1 by amino acids (AAs). We examined whether the relationship between v-ATPase and mTORC1 also operates reciprocally; specifically, whether AA induces v-ATPase reassembly in a mTORC1-dependent manner to prevent excess lipids from entering and damaging the heart. Methods Lipid overexposed rodent/human cardiomyocytes and high-fat diet-fed rats were treated with a specific cocktail of AAs (lysine/leucine/arginine). Then, v-ATPase assembly status/activity, cell surface CD36 content, myocellular lipid uptake/accumulation, insulin sensitivity, and contractile function were measured. To elucidate underlying mechanisms, specific gene knockdown was employed, followed by subcellular fractionation, and coimmunoprecipitation. Results In lipid-overexposed cardiomyocytes, lysine/leucine/arginine reinternalized CD36 to the endosomes, prevented/reversed lipid accumulation, preserved/restored insulin sensitivity, and contractile function. These beneficial AA actions required the mTORC1–v-ATPase axis, adaptor protein Ragulator, and endosomal/lysosomal AA transporter SLC38A9, indicating an endosome-centric inside-out AA sensing mechanism. In high-fat diet-fed rats, lysine/leucine/arginine had similar beneficial actions at the myocellular level as in vitro in lipid-overexposed cardiomyocytes and partially reversed cardiac hypertrophy. Conclusion Specific AAs acting through v-ATPase reassembly reduce cardiac lipid uptake raising the possibility for treatment in situations of lipid overload and associated insulin resistance.

Effects of Lipid Overload on Heart in Metabolic Diseases

“…It has also been shown that supplementing or promoting non-lipid myocardial metabolic substrates can improve the state of high-fat induced cardiac energy metabolism imbalance. In vitro and in vivo studies have demonstrated that specific amino acid supplementation (lysine, leucine, arginine) reinternalized CD36 to the endosomes by regulating the mTORC1-v-ATPase axis, reduces myocardial lipid uptake and reverses/prevents lipid accumulation 39 .…”

Effects of Individual Amino Acids on PPARα Transactivation, mTORC1 Activation, ApoA-I Transcription and pro-ApoA-I Secretion

“…Georgila et al showed that amino acid deprivation inhibited mTORC1 activity and thereby reduced ApoA-I protein levels, but not ApoA-I mRNA expression in HepG2 cells [ 18 ]. Moreover, a recent study by Wang et al showed that a cocktail of leucine, arginine, and lysine increased mTORC1 activation in HL-1 cells [ 29 ]. Therefore, we also evaluated the effects of individual amino acids on mTORC1 activity.…”

“…Interestingly, specific mTORC1 activation by using the activator MHY1485 did not further increase ApoA-I mRNA expression or pro-ApoA-I levels. It is important to note that the previously mentioned studies used amino acid deprived cells that were only treated for 1–6 h [ 18 , 29 ]. Our cells were, however, treated for 48 h and were probably not enough deprived of amino acids to reduce the activity of the mTORC1 pathway due to the amino acids already present in the culture medium ( Supplementary Table S2 ).…”

CD36 (SR-B2) as master regulator of cellular fatty acid homeostasis

“…Recently, it was observed that specific amino acids also stimulate v-ATPase (re-)assembly to retain CD36 in the endosomes and reduce myocardial fatty acid uptake [ 29 ▪ ]. These novel findings combined indicate the application of specific amino acid supplementation for tuning of the contributions of fatty acids and of glucose to their current cellular demand so as to avoid both fatty acid overload (lipotoxicity) and glucose overload (glucotoxicity).…”

“…The recent disclosure of v-ATPase as regulator of CD36 recycling makes this endosomal enzyme an interesting candidate for modulation of sarcolemmal CD36 content. Indeed, as outlined above, specific amino acids increase myocardial v-ATPase activity thereby reducing sarcolemmal CD36, and ultimately preventing or restoring lipid overexposure-induced contractile dysfunction [ 29 ▪ ]. However, whether these amino acids act specifically on the heart or would also affect v-ATPase in other tissues has not yet been addressed.…”