Glycolysis-Mediated Activation of v-ATPase by Nicotinamide Mononucleotide Ameliorates Lipid-Induced Cardiomyopathy by Repressing the CD36-TLR4 Axis.

BACKGROUND: Chronic overconsumption of lipids followed by their excessive accumulation in the heart leads to cardiomyopathy. The cause of lipid-induced cardiomyopathy involves a pivotal role for the proton-pump vacuolar-type H+-ATPase (v-ATPase), which acidifies endosomes, and for lipid-transporter CD36, which is stored in acidified endosomes. During lipid overexposure, an increased influx of lipids into cardiomyocytes is sensed by v-ATPase, which then disassembles, causing endosomal de-acidification and expulsion of stored CD36 from the endosomes toward the sarcolemma. Once at the sarcolemma, CD36 not only increases lipid uptake but also interacts with inflammatory receptor TLR4 (Toll-like receptor 4), together resulting in lipid-induced insulin resistance, inflammation, fibrosis, and cardiac dysfunction. Strategies inducing v-ATPase reassembly, that is, to achieve CD36 reinternalization, may correct these maladaptive alterations. For this, we used NAD+ (nicotinamide adenine dinucleotide)-precursor nicotinamide mononucleotide (NMN), inducing v-ATPase reassembly by stimulating glycolytic enzymes to bind to v-ATPase. METHODS: Rats/mice on cardiomyopathy-inducing high-fat diets were supplemented with NMN and for comparison with a cocktail of lysine/leucine/arginine (mTORC1 [mechanistic target of rapamycin complex 1]-mediated v-ATPase reassembly). We used the following methods: RNA sequencing, mRNA/protein expression analysis, immunofluorescence microscopy, (co)immunoprecipitation/proximity ligation assay (v-ATPase assembly), myocellular uptake of [3H]chloroquine (endosomal pH), and [14C]palmitate, targeted lipidomics, and echocardiography. To confirm the involvement of v-ATPase in the beneficial effects of both supplementations, mTORC1/v-ATPase inhibitors (rapamycin/bafilomycin A1) were administered. Additionally, 2 heart-specific v-ATPase-knockout mouse models (subunits V1G1/V0d2) were subjected to these measurements. Mechanisms were confirmed in pharmacologically/genetically manipulated cardiomyocyte models of lipid overload. RESULTS: NMN successfully preserved endosomal acidification during myocardial lipid overload by maintaining v-ATPase activity and subsequently prevented CD36-mediated lipid accumulation, CD36-TLR4 interaction toward inflammation, fibrosis, cardiac dysfunction, and whole-body insulin resistance. Lipidomics revealed C18:1-enriched diacylglycerols as lipid class prominently increased by high-fat diet and subsequently reversed/preserved by lysine/leucine/arginine/NMN treatment. Studies with mTORC1/v-ATPase inhibitors and heart-specific v-ATPase-knockout mice further confirmed the pivotal roles of v-ATPase in these beneficial actions. CONCLUSION: NMN preserves heart function during lipid overload by preventing v-ATPase disassembly.

Ketone Body Exposure of Cardiomyocytes Impairs Insulin Sensitivity and Contractile Function through Vacuolar-Type H+-ATPase Disassembly-Rescue by Specific Amino Acid Supplementation.

The heart is metabolically flexible. Under physiological conditions, it mainly uses lipids and glucose as energy substrates. In uncontrolled diabetes, the heart switches towards predominant lipid utilization, which over time is detrimental to cardiac function. Additionally, diabetes is accompanied by high plasma ketone levels and increased utilization of energy provision. The administration of exogenous ketones is currently being investigated for the treatment of cardiovascular disease. Yet, it remains unclear whether increased cardiac ketone utilization is beneficial or detrimental to cardiac functioning. The mechanism of lipid-induced cardiac dysfunction includes disassembly of the endosomal proton pump (named vacuolar-type H+-ATPase; v-ATPase) as the main early onset event, followed by endosomal de-acidification/dysfunction. The de-acidified endosomes can no longer serve as a storage compartment for lipid transporter CD36, which then translocates to the sarcolemma to induce lipid accumulation, insulin resistance, and contractile dysfunction. Lipid-induced v-ATPase disassembly is counteracted by the supply of specific amino acids. Here, we tested the effect of ketone bodies on v-ATPase assembly status and regulation of lipid uptake in rodent/human cardiomyocytes. 3-ß-hydroxybutyrate (3HB) exposure induced v-ATPase disassembly and the entire cascade of events leading to contractile dysfunction and insulin resistance, similar to conditions of lipid oversupply. Acetoacetate addition did not induce v-ATPase dysfunction. The negative effects of 3HB could be prevented by addition of specific amino acids. Hence, in sedentary/prediabetic subjects ketone bodies should be used with caution because of possible aggravation of cardiac insulin resistance and further loss of cardiac function. When these latter maladaptive conditions would occur, specific amino acids could potentially be a treatment option.

Endosomal v-ATPase as a Sensor Determining Myocardial Substrate Preference.

The heart is a metabolically flexible omnivore that can utilize a variety of substrates for energy provision. To fulfill cardiac energy requirements, the healthy adult heart mainly uses long-chain fatty acids and glucose in a balanced manner, but when exposed to physiological or pathological stimuli, it can switch its substrate preference to alternative substrates such as amino acids (AAs) and ketone bodies. Using the failing heart as an example, upon stress, the fatty acid/glucose substrate balance is upset, resulting in an over-reliance on either fatty acids or glucose. A chronic fuel shift towards a single type of substrate is linked with cardiac dysfunction. Re-balancing myocardial substrate preference is suggested as an effective strategy to rescue the failing heart. In the last decade, we revealed that vacuolar-type H+-ATPase (v-ATPase) functions as a key regulator of myocardial substrate preference and, therefore, as a novel potential treatment approach for the failing heart. Fatty acids, glucose, and AAs selectively influence the assembly state of v-ATPase resulting in modulation of its proton-pumping activity. In this review, we summarize these novel insights on v-ATPase as an integrator of nutritional information. We also describe its exploitation as a therapeutic target with focus on supplementation of AA as a nutraceutical approach to fight lipid-induced insulin resistance and contractile dysfunction of the heart.

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

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.

Putative Role of Protein Palmitoylation in Cardiac Lipid-Induced Insulin Resistance.

In the heart, inhibition of the insulin cascade following lipid overload is strongly associated with contractile dysfunction. The translocation of fatty acid transporter CD36 (SR-B2) from intracellular stores to the cell surface is a hallmark event in the lipid-overloaded heart, feeding forward to intracellular lipid accumulation. Yet, the molecular mechanisms by which intracellularly arrived lipids induce insulin resistance is ill-understood. Bioactive lipid metabolites (diacyl-glycerols, ceramides) are contributing factors but fail to correlate with the degree of cardiac insulin resistance in diabetic humans. This leaves room for other lipid-induced mechanisms involved in lipid-induced insulin resistance, including protein palmitoylation. Protein palmitoylation encompasses the reversible covalent attachment of palmitate moieties to cysteine residues and is governed by protein acyl-transferases and thioesterases. The function of palmitoylation is to provide proteins with proper spatiotemporal localization, thereby securing the correct unwinding of signaling pathways. In this review, we provide examples of palmitoylations of individual signaling proteins to discuss the emerging role of protein palmitoylation as a modulator of the insulin signaling cascade. Second, we speculate how protein hyper-palmitoylations (including that of CD36), as they occur during lipid oversupply, may lead to insulin resistance. Finally, we conclude that the protein palmitoylation machinery may offer novel targets to fight lipid-induced cardiomyopathy.

Understanding the distinct subcellular trafficking of CD36 and GLUT4 during the development of myocardial insulin resistance.

CD36 and GLUT4 are the main cardiac trans-sarcolemmal transporters for long-chain fatty acids and glucose, respectively. Together they secure the majority of cardiac energy demands. Moreover, these transporters each represent key governing kinetic steps in cardiac fatty acid and glucose fluxes, thereby offering major sites of regulation. The underlying mechanism of this regulation involves a perpetual vesicle-mediated trafficking (recycling) of both transporters between intracellular stores (endosomes) and the cell surface. In the healthy heart, CD36 and GLUT4 translocation to the cell surface is under short-term control of the same physiological stimuli, most notably increased contraction and insulin secretion. However, under chronic lipid overload, a condition that accompanies a Western lifestyle, CD36 and GLUT4 recycling are affected distinctly, with CD36 being expelled to the sarcolemma while GLUT4 is imprisoned within the endosomes. Moreover, the increased CD36 translocation towards the cell surface is a key early step, setting the heart on a route towards insulin resistance and subsequent contractile dysfunction. Therefore, the proteins making up the trafficking machinery of CD36 need to be identified with special focus to the differences with the protein composition of the GLUT4 trafficking machinery. These proteins that are uniquely dedicated to either CD36 or GLUT4 traffic may offer targets to rectify aberrant substrate uptake seen in the lipid-overloaded heart. Specifically, CD36-dedicated trafficking regulators should be inhibited, whereas such GLUT4-dedicated proteins would need to be activated. Recent advances in the identification of CD36-dedicated trafficking proteins have disclosed the involvement of vacuolar-type H+-ATPase and of specific vesicle-associated membrane proteins (VAMPs). In this review, we summarize these recent findings and sketch a roadmap of CD36 and GLUT4 trafficking compatible with experimental findings.

AMPK-dependent activation of the Cyclin Y/CDK16 complex controls autophagy.

The AMP-activated protein kinase (AMPK) is a master sensor of the cellular energy status that is crucial for the adaptive response to limited energy availability. AMPK is implicated in the regulation of many cellular processes, including autophagy. However, the precise mechanisms by which AMPK controls these processes and the identities of relevant substrates are not fully understood. Using protein microarrays, we identify Cyclin Y as an AMPK substrate that is phosphorylated at Serine 326 (S326) both in vitro and in cells. Phosphorylation of Cyclin Y at S326 promotes its interaction with the Cyclin-dependent kinase 16 (CDK16), thereby stimulating its catalytic activity. When expressed in cells, Cyclin Y/CDK16 is sufficient to promote autophagy. Moreover, Cyclin Y/CDK16 is necessary for efficient AMPK-dependent activation of autophagy. This functional interaction is mediated by AMPK phosphorylating S326 of Cyclin Y. Collectively, we define Cyclin Y/CDK16 as downstream effector of AMPK for inducing autophagy.

Augmenting Vacuolar H+-ATPase Function Prevents Cardiomyocytes from Lipid-Overload Induced Dysfunction.

The diabetic heart is characterized by a shift in substrate utilization from glucose to lipids, which may ultimately lead to contractile dysfunction. This substrate shift is facilitated by increased translocation of lipid transporter CD36 (SR-B2) from endosomes to the sarcolemma resulting in increased lipid uptake. We previously showed that endosomal retention of CD36 is dependent on the proper functioning of vacuolar H+-ATPase (v-ATPase). Excess lipids trigger CD36 translocation through inhibition of v-ATPase function. Conversely, in yeast, glucose availability is known to enhance v-ATPase function, allowing us to hypothesize that glucose availability, via v-ATPase, may internalize CD36 and restore contractile function in lipid-overloaded cardiomyocytes. Increased glucose availability was achieved through (a) high glucose (25 mM) addition to the culture medium or (b) adenoviral overexpression of protein kinase-D1 (a kinase mediating GLUT4 translocation). In HL-1 cardiomyocytes, adult rat and human cardiomyocytes cultured under high-lipid conditions, each treatment stimulated v-ATPase re-assembly, endosomal acidification, endosomal CD36 retention and prevented myocellular lipid accumulation. Additionally, these treatments preserved insulin-stimulated GLUT4 translocation and glucose uptake as well as contractile force. The present findings reveal v-ATPase functions as a key regulator of cardiomyocyte substrate preference and as a novel potential treatment approach for the diabetic heart.

AMP-Activated Protein Kinase Signalling.

AMP-activated protein kinase (AMPK) regulates energy homeostasis in eukaryotic cells and organisms [...].

Fluorescent labelling of membrane fatty acid transporter CD36 (SR-B2) in the extracellular loop.

CONTEXT: Upon palmitate oversupply, membrane fatty acid-transporter CD36 (SR-B2) permanently translocates from endosomal storage to the sarcolemma, inducing lipotoxicity. CD36 translocation results from endosomal alkalinisation elicited by palmitate-induced disattachment of the cytoplasmic V1-subcomplex from the membrane-integrated V0-subcomplex of vacuolar-type H+-ATPase. OBJECTIVE: Develop a CD36 fluorescent labeling technique as initial step towards live cell imaging. METHODS: Three human CD36 (hCD36) mutants were constructed via insertion of a tetracysteine motif at different positions within the extracellular domain. Constructs were lentivirally transduced for subsequent CD36 labeling with fluorescein-arsenical hairpin-binder (FlAsH). Cell imaging was combined with V0/V1 immunostaining and Western blotting. RESULTS: Transduction of hCD36-wildtype and mutants yielded corresponding proteins in HL-1 cardiomyocytes. Tetracysteine mutant-2 (hCD36-TC2) showed similar fatty acid uptake to wildtype. FlAsH staining revealed a speckled pattern reminiscent of endosomes. We found decreased V1 co-localization with CD36 upon high-palmitate culturing. Conversely, V0 consistently co-localized with CD36. CONCLUSION: hCD36-TC2 is a possible candidate for application of biarsenical dyes in live imaging studies pending further investigation. Our data is compatible with V0/V1 disassembly in high-palmitate-treated cells.

The endocannabinoid system: Overview of an emerging multi-faceted therapeutic target.

The endocannabinoids anandamide (AEA) and 2-arachidonoylglyerol (2-AG) are endogenous lipid mediators that exert protective roles in pathophysiological conditions, including cardiovascular diseases. In this brief review, we provide a conceptual framework linking endocannabinoid signaling to the control of the cellular and molecular hallmarks, and categorize the key components of endocannabinoid signaling that may serve as targets for novel therapeutics. The emerging picture not only reinforces endocannabinoids as potent regulators of cellular metabolism but also reveals that endocannabinoid signaling is mechanistically more complex and diverse than originally thought.

Is TAK1 a Direct Upstream Kinase of AMPK?

Alongside Liver kinase B1 (LKB1) and Ca2+/Calmodulin-dependent protein kinase kinase 2 (CaMKK2), Transforming growth factor-ß (TGF-ß)-activated kinase 1 (TAK1) has been suggested as a direct upstream kinase of AMP-activated protein kinase (AMPK). Several subsequent studies have reported on the TAK1-AMPK relationship, but the interpretation of the respective data has led to conflicting views. Therefore, to date the acceptance of TAK1 as a genuine AMPK kinase is lagging behind. This review provides with argumentation, whether or not TAK1 functions as a direct upstream kinase of AMPK. Several specific open questions that may have precluded the consensus are discussed based on available data. In brief, TAK1 can function as direct AMPK upstream kinase in specific contexts and in response to a subset of TAK1 activating stimuli. Further research is needed to define the intricate signals that are conditional for TAK1 to phosphorylate and activate AMPKα at T172.

ß1Pix exchange factor stabilizes the ubiquitin ligase Nedd4-2 and plays a critical role in ENaC regulation by AMPK in kidney epithelial cells.

Our previous work has established that the metabolic sensor AMP-activated protein kinase (AMPK) inhibits the epithelial Na+ channel (ENaC) by promoting its binding to neural precursor cell-expressed, developmentally down-regulated 4-2, E3 ubiquitin protein ligase (Nedd4-2). Here, using MS analysis and in vitro phosphorylation, we show that AMPK phosphorylates Nedd4-2 at the Ser-444 (Xenopus Nedd4-2) site critical for Nedd4-2 stability. We further demonstrate that the Pak-interacting exchange factor ß1Pix is required for AMPK-mediated inhibition of ENaC-dependent currents in both CHO and murine kidney cortical collecting duct (CCD) cells. Short hairpin RNA-mediated knockdown of ß1Pix expression in CCD cells attenuated the inhibitory effect of AMPK activators on ENaC currents. Moreover, overexpression of a ß1Pix dimerization-deficient mutant unable to bind 14-3-3 proteins (Δ602-611) increased ENaC currents in CCD cells, whereas overexpression of WT ß1Pix had the opposite effect. Using additional immunoblotting and co-immunoprecipitation experiments, we found that treatment with AMPK activators promoted the binding of ß1Pix to 14-3-3 proteins in CCD cells. However, the association between Nedd4-2 and 14-3-3 proteins was not consistently affected by AMPK activation, ß1Pix knockdown, or overexpression of WT ß1Pix or the ß1Pix-Δ602-611 mutant. Moreover, we found that ß1Pix is important for phosphorylation of the aforementioned Nedd4-2 site critical for its stability. Overall, these findings elucidate novel molecular mechanisms by which AMPK regulates ENaC. Specifically, they indicate that AMPK promotes the assembly of ß1Pix, 14-3-3 proteins, and Nedd4-2 into a complex that inhibits ENaC by enhancing Nedd4-2 binding to ENaC and its degradation.

Assessment of AMPK-Stimulated Cellular Long-Chain Fatty Acid and Glucose Uptake.

Here we describe an assay for simultaneous measurement of cellular uptake rates of long-chain fatty acids (LCFA) and glucose that can be applied to cells in suspension. The uptake assay includes the use of radiolabeled substrates at such concentrations and incubation periods that exact information is provided about unidirectional uptakes rates. Cellular uptake of both substrates is under regulation of AMPK. The underlying mechanism includes the translocation of LCFA and glucose transporters from intracellular membrane compartments to the cell surface, leading to an increase in substrate uptake. In this chapter, we explain the principles of the uptake assay before detailing the exact procedure. We also provide information of the specific LCFA and glucose transporters subject to AMPK-mediated subcellular translocation. Finally, we discuss the application of AMPK inhibitors and activators in combination with cellular substrate uptake assays.

Human embryonic stem cell-derived cardiomyocytes as an in vitro model to study cardiac insulin resistance.

Patients with type 2 diabetes (T2D) and/or insulin resistance (IR) have an increased risk for the development of heart failure (HF). Evidence indicates that this increased risk is linked to an altered cardiac substrate preference of the insulin resistant heart, which shifts from a balanced utilization of glucose and long-chain fatty acids (FAs) towards an almost complete reliance on FAs as main fuel source. This shift leads to a loss of endosomal proton pump activity and increased cardiac fat accumulation, which eventually triggers cardiac dysfunction. In this review, we describe the advantages and disadvantages of currently used in vitro models to study the underlying mechanism of IR-induced HF and provide insight into a human in vitro model: human embryonic stem cell-derived cardiomyocytes (hESC-CMs). Using functional metabolic assays we demonstrate that, similar to rodent studies, hESC-CMs subjected to 16h of high palmitate (HP) treatment develop the main features of IR, i.e., decreased insulin-stimulated glucose and FA uptake, as well as loss of endosomal acidification and insulin signaling. Taken together, these data propose that HP-treated hESC-CMs are a promising in vitro model of lipid overload-induced IR for further research into the underlying mechanism of cardiac IR and for identifying new pharmacological agents and therapeutic strategies. This article is part of a Special issue entitled Cardiac adaptations to obesity, diabetes and insulin resistance, edited by Professors Jan F.C. Glatz, Jason R.B. Dyck and Christine Des Rosiers.

Molecular mechanism of lipid-induced cardiac insulin resistance and contractile dysfunction.

Long-chain fatty acids are the main cardiac substrates from which ATP is generated continually to serve the high energy demand and sustain the normal function of the heart. Under healthy conditions, fatty acid ß-oxidation produces 50-70% of the energy demands with the remainder largely accounted for by glucose. Chronically increased dietary lipid supply often leads to excess lipid accumulation in the heart, which is linked to a variety of maladaptive phenomena, such as insulin resistance, cardiac hypertrophy and contractile dysfunction. CD36, the predominant cardiac fatty acid transporter, has a key role in setting the heart on a road to contractile dysfunction upon the onset of chronic lipid oversupply by translocating to the cell surface and opening the cellular 'doors' for fatty acids. The sequence of events after the CD36-mediated myocellular lipid accumulation is less understood, but in general it has been accepted that the excessively imported lipids cause insulin resistance, which in turn leads to contractile dysfunction. There are several gaps of knowledge in this proposed order of events which this review aims to discuss. First, the molecular mechanisms underlying lipid-induced insulin resistance are not yet completely disclosed. Specifically, several mediators have been proposed, such as diacylglycerols, ceramides, peroxisome proliferator-activated receptors (PPAR), inflammatory kinases and reactive oxygen species (ROS), but their relative contributions to the onset of insulin resistance and their putatively synergistic actions are topics of controversy. Second, there are also pieces of evidence that lipids can induce contractile dysfunction independently of insulin resistance. Perhaps, a more integrative view is needed, in which several lipid-induced pathways operate synergistically or in parallel to induce contractile dysfunction. Unraveling of these processes is expected to be important in designing effective therapeutic strategies to protect the lipid-overloaded heart.

2-Arachidonoylglycerol ameliorates inflammatory stress-induced insulin resistance in cardiomyocytes.

Several studies have linked impaired glucose uptake and insulin resistance (IR) to functional impairment of the heart. Recently, endocannabinoids have been implicated in cardiovascular disease. However, the mechanisms involving endocannabinoid signaling, glucose uptake, and IR in cardiomyocytes are understudied. Here we report that the endocannabinoid 2-arachidonoylglycerol (2-AG), via stimulation of cannabinoid type 1 (CB1) receptor and Ca2+/calmodulin-dependent protein kinase ß, activates AMP-activated kinase (AMPK), leading to increased glucose uptake. Interestingly, we have observed that the mRNA expression of CB1 and CB2 receptors was decreased in diabetic mice, indicating reduced endocannabinoid signaling in the diabetic heart. We further establish that TNFα induces IR in cardiomyocytes. Treatment with 2-AG suppresses TNFα-induced proinflammatory markers and improves IR and glucose uptake. Conversely, pharmacological inhibition or knockdown of AMPK attenuates the anti-inflammatory effect and reversal of IR elicited by 2-AG. Additionally, in human embryonic stem cell-derived cardiomyocytes challenged with TNFα or FFA, we demonstrate that 2-AG improves insulin sensitivity and glucose uptake. In conclusion, 2-AG abates inflammatory responses, increases glucose uptake, and overcomes IR in an AMPK-dependent manner in cardiomyocytes.

Palmitate-Induced Vacuolar-Type H+-ATPase Inhibition Feeds Forward Into Insulin Resistance and Contractile Dysfunction.

Dietary fat overconsumption leads to myocardial lipid accumulation through mechanisms that are incompletely resolved. Previously, we identified increased translocation of the fatty acid transporter CD36 from its endosomal storage compartment to the sarcolemma as the primary mechanism of excessive myocellular lipid import. Here, we show that increased CD36 translocation is caused by alkalinization of endosomes resulting from inhibition of proton pumping activity of vacuolar-type H+-ATPase (v-ATPase). Endosomal alkalinization was observed in hearts from rats fed a lard-based high-fat diet and in rodent and human cardiomyocytes upon palmitate overexposure, and appeared as an early lipid-induced event preceding the onset of insulin resistance. Either genetic or pharmacological inhibition of v-ATPase in cardiomyocytes exposed to low palmitate concentrations reduced insulin sensitivity and cardiomyocyte contractility, which was rescued by CD36 silencing. The mechanism of palmitate-induced v-ATPase inhibition involved its dissociation into two parts: the cytosolic V1 and the integral membrane V0 subcomplex. Interestingly, oleate also inhibits v-ATPase function, yielding triacylglycerol accumulation but not insulin resistance. In conclusion, lipid oversupply increases CD36-mediated lipid uptake that directly impairs v-ATPase function. This feeds forward to enhanced CD36 translocation and further increased lipid uptake. In the case of palmitate, its accelerated uptake ultimately precipitates into cardiac insulin resistance and contractile dysfunction.