Sodium-glucose co-transporter 2 Inhibitors Act Independently of SGLT2 to Confer Benefit for Heart Failure with Reduced Ejection Fraction in Mice.

Sodium-glucose co-transporter 2 inhibitors (SGLT2i) are novel, potent heart failure medications with an unknown mechanism of action. We sought to determine if the beneficial actions of SGLT2i in heart failure were on- or off-target, and related to metabolic reprogramming, including increased lipolysis and ketogenesis. The phenotype of mice treated with empagliflozin and genetically engineered mice constitutively lacking SGLT2 mirrored metabolic changes seen in human clinical trials (including reduced blood glucose, increased ketogenesis, and profound glucosuria). In a mouse heart failure model, SGLT2i treatment, but not generalized SGLT2 knockout, resulted in improved systolic function and reduced pathologic cardiac remodeling. SGLT2i treatment of the SGLT2 knockout mice sustained the cardiac benefits, demonstrating an off-target role for these drugs. This benefit is independent of metabolic changes, including ketosis. The mechanism of action and target of SGLT2i in HF remain elusive.

HIF2α-dependent inhibition of mitochondrial clustering of glutaminase suppresses clear cell renal cell carcinoma.

Clear cell renal cell carcinomas (ccRCC) are largely driven by HIF2α and are avid consumers of glutamine. However, inhibitors of glutaminase1 (GLS1), the first step in glutaminolysis, have not shown benefit in phase III trials, and HIF2α inhibition, recently FDA-approved for treatment of ccRCC, shows great but incomplete benefits, underscoring the need to better understand the roles of glutamine and HIF2α in ccRCC. Here, we report that glutamine deprivation rapidly redistributes GLS1 into isolated clusters within mitochondria across diverse cell types, excluding ccRCC. GLS1 clustering is rapid (1-3 hours) and reversible, is specifically driven by the level of intracellular glutamate, and is mediated by mitochondrial fission. Clustered GLS1 has markedly enhanced glutaminase activity and promotes cell death under glutamine-deprived conditions. We further show that HIF2α prevents GLS1 clustering, independently of its transcriptional activity, thereby protecting ccRCC cells from cell death induced by glutamine deprivation. Reversing this protection, by genetic expression of GLS1 mutants that constitutively cluster, enhances ccRCC cell death in culture and suppresses ccRCC growth in vivo . These finding provide multiple insights into cellular glutamine handling, including a novel metabolic pathway by which HIF2α promotes ccRCC, and reveals a potential therapeutic avenue to synergize with HIF2α inhibition in the treatment of ccRCC.

Off-target depletion of plasma tryptophan by allosteric inhibitors of BCKDK.

The activation of branched chain amino acid (BCAA) catabolism has garnered interest as a potential therapeutic approach to improve insulin sensitivity, enhance recovery from heart failure, and blunt tumor growth. Evidence for this interest relies in part on BT2, a small molecule that promotes BCAA oxidation and is protective in mouse models of these pathologies. BT2 and other analogs allosterically inhibit branched chain ketoacid dehydrogenase kinase (BCKDK) to promote BCAA oxidation, which is presumed to underlie the salutary effects of BT2. Potential "off-target" effects of BT2 have not been considered, however. We therefore tested for metabolic off-target effects of BT2 in Bckdk-/- animals. As expected, BT2 failed to activate BCAA oxidation in these animals. Surprisingly, however, BT2 strongly reduced plasma tryptophan levels and promoted catabolism of tryptophan to kynurenine in both control and Bckdk-/- mice. Mechanistic studies revealed that none of the principal tryptophan catabolic or kynurenine-producing/consuming enzymes (TDO, IDO1, IDO2, or KATs) were required for BT2-mediated lowering of plasma tryptophan. Instead, using equilibrium dialysis assays and mice lacking albumin, we show that BT2 avidly binds plasma albumin and displaces tryptophan, releasing it for catabolism. These data confirm that BT2 activates BCAA oxidation via inhibition of BCKDK but also reveal a robust off-target effect on tryptophan metabolism via displacement from serum albumin. The data highlight a potential confounding effect for pharmaceutical compounds that compete for binding with albumin-bound tryptophan.

Acetate controls endothelial-to-mesenchymal transition.

Endothelial-to-mesenchymal transition (EndMT), a process initiated by activation of endothelial TGF-ß signaling, underlies numerous chronic vascular diseases and fibrotic states. Once induced, EndMT leads to a further increase in TGF-ß signaling, thus establishing a positive-feedback loop with EndMT leading to more EndMT. Although EndMT is understood at the cellular level, the molecular basis of TGF-ß-driven EndMT induction and persistence remains largely unknown. Here, we show that metabolic modulation of the endothelium, triggered by atypical production of acetate from glucose, underlies TGF-ß-driven EndMT. Induction of EndMT suppresses the expression of the enzyme PDK4, which leads to an increase in ACSS2-dependent Ac-CoA synthesis from pyruvate-derived acetate. This increased Ac-CoA production results in acetylation of the TGF-ß receptor ALK5 and SMADs 2 and 4 leading to activation and long-term stabilization of TGF-ß signaling. Our results establish the metabolic basis of EndMT persistence and unveil novel targets, such as ACSS2, for the potential treatment of chronic vascular diseases.

The mitochondrial Ca2+ channel MCU is critical for tumor growth by supporting cell cycle progression and proliferation.

Introduction: The mitochondrial uniporter (MCU) Ca2+ ion channel represents the primary means for Ca2+ uptake by mitochondria. Mitochondrial matrix Ca2+ plays critical roles in mitochondrial bioenergetics by impinging upon respiration, energy production and flux of biochemical intermediates through the TCA cycle. Inhibition of MCU in oncogenic cell lines results in an energetic crisis and reduced cell proliferation unless media is supplemented with nucleosides, pyruvate or α-KG. Nevertheless, the roles of MCU-mediated Ca2+ influx in cancer cells remain unclear, in part because of a lack of genetic models. Methods: MCU was genetically deleted in transformed murine fibroblasts for study in vitro and in vivo. Tumor formation and growth were studied in murine xenograft models. Proliferation, cell invasion, spheroid formation and cell cycle progression were measured in vitro. The effects of MCU deletion on survival and cell-death were determined by probing for live/death markers. Mitochondrial bioenergetics were studied by measuring mitochondrial matrix Ca2+ concentration, membrane potential, global dehydrogenase activity, respiration, ROS production and inactivating-phosphorylation of pyruvate dehydrogenase. The effects of MCU rescue on metabolism were examined by tracing of glucose and glutamine utilization for fueling of mitochondrial respiration. Results: Transformation of primary fibroblasts in vitro was associated with increased MCU expression, enhanced MCU-mediated Ca2+ uptake, altered mitochondrial matrix Ca2+ concentration responses to agonist stimulation, suppression of inactivating-phosphorylation of pyruvate dehydrogenase and a modest increase of mitochondrial respiration. Genetic MCU deletion inhibited growth of HEK293T cells and transformed fibroblasts in mouse xenograft models, associated with reduced proliferation and delayed cell-cycle progression. MCU deletion inhibited cancer stem cell-like spheroid formation and cell invasion in vitro, both predictors of metastatic potential. Surprisingly, mitochondrial matrix [Ca2+], membrane potential, global dehydrogenase activity, respiration and ROS production were unaffected. In contrast, MCU deletion elevated glycolysis and glutaminolysis, strongly sensitized cell proliferation to glucose and glutamine limitation, and altered agonist-induced cytoplasmic Ca2+ signals. Conclusion: Our results reveal a dependence of tumorigenesis on MCU, mediated by a reliance on MCU for cell metabolism and Ca2+ dynamics necessary for cell-cycle progression and cell proliferation.

The mitochondrial Ca 2+ channel MCU is critical for tumor growth by supporting cell cycle progression and proliferation.

The mitochondrial uniporter (MCU) Ca 2+ ion channel represents the primary means for Ca 2+ uptake into mitochondria. Here we employed in vitro and in vivo models with MCU genetically eliminated to understand how MCU contributes to tumor formation and progression. Transformation of primary fibroblasts in vitro was associated with increased MCU expression, enhanced mitochondrial Ca 2+ uptake, suppression of inactivating-phosphorylation of pyruvate dehydrogenase, a modest increase of basal mitochondrial respiration and a significant increase of acute Ca 2+ -dependent stimulation of mitochondrial respiration. Inhibition of mitochondrial Ca 2+ uptake by genetic deletion of MCU markedly inhibited growth of HEK293T cells and of transformed fibroblasts in mouse xenograft models. Reduced tumor growth was primarily a result of substantially reduced proliferation and fewer mitotic cells in vivo , and slower cell proliferation in vitro associated with delayed progression through S-phase of the cell cycle. MCU deletion inhibited cancer stem cell-like spheroid formation and cell invasion in vitro , both predictors of metastatic potential. Surprisingly, mitochondrial matrix Ca 2+ concentration, membrane potential, global dehydrogenase activity, respiration and ROS production were unchanged by genetic deletion of MCU in transformed cells. In contrast, MCU deletion elevated glycolysis and glutaminolysis, strongly sensitized cell proliferation to glucose and glutamine limitation, and altered agonist-induced cytoplasmic Ca 2+ signals. Our results reveal a dependence of tumorigenesis on MCU, mediated by a reliance on mitochondrial Ca 2+ uptake for cell metabolism and Ca 2+ dynamics necessary for cell-cycle progression and cell proliferation.

Regulation of maternal-fetal metabolic communication.

Pregnancy may be the most nutritionally sensitive stage in the life cycle, and improved metabolic health during gestation and early postnatal life can reduce the risk of chronic disease in adulthood. Successful pregnancy requires coordinated metabolic, hormonal, and immunological communication. In this review, maternal-fetal metabolic communication is defined as the bidirectional communication of nutritional status and metabolic demand by various modes including circulating metabolites, endocrine molecules, and other secreted factors. Emphasis is placed on metabolites as a means of maternal-fetal communication by synthesizing findings from studies in humans, non-human primates, domestic animals, rabbits, and rodents. In this review, fetal, placental, and maternal metabolic adaptations are discussed in turn. (1) Fetal macronutrient needs are summarized in terms of the physiological adaptations in place to ensure their proper allocation. (2) Placental metabolite transport and maternal physiological adaptations during gestation, including changes in energy budget, are also discussed. (3) Maternal nutrient limitation and metabolic disorders of pregnancy serve as case studies of the dynamic nature of maternal-fetal metabolic communication. The review concludes with a summary of recent research efforts to identify metabolites, endocrine molecules, and other secreted factors that mediate this communication, with particular emphasis on serum/plasma metabolomics in humans, non-human primates, and rodents. A better understanding of maternal-fetal metabolic communication in health and disease may reveal novel biomarkers and therapeutic targets for metabolic disorders of pregnancy.

Maternal Lipid Metabolism Directs Fetal Liver Programming following Nutrient Stress.

The extreme metabolic demands of pregnancy require coordinated metabolic adaptations between mother and fetus to balance fetal growth and maternal health with nutrient availability. To determine maternal and fetal contributions to metabolic flexibility during gestation, pregnant mice with genetic impairments in mitochondrial carbohydrate and/or lipid metabolism were subjected to nutrient deprivation. The maternal fasting response initiates a fetal liver transcriptional program marked by upregulation of lipid- and peroxisome proliferator-activated receptor alpha (Pparα)-regulated genes. Impaired maternal lipid metabolism alters circulating lipid metabolite concentrations and enhances the fetal response to fasting, which is largely dependent on fetal Pparα. Maternal fasting also improves metabolic deficits in fetal carbohydrate metabolism by increasing the availability of alternative substrates. Impairment of both carbohydrate and lipid metabolism in pregnant dams further exacerbates the fetal liver transcriptional response to nutrient deprivation. Together, these data demonstrate a regulatory role for mitochondrial macronutrient metabolism in mediating maternal-fetal metabolic communication, particularly when nutrients are limited.

The ADP/ATP translocase drives mitophagy independent of nucleotide exchange.

Mitochondrial homeostasis depends on mitophagy, the programmed degradation of mitochondria. Only a few proteins are known to participate in mitophagy. Here we develop a multidimensional CRISPR-Cas9 genetic screen, using multiple mitophagy reporter systems and pro-mitophagy triggers, and identify numerous components of parkin-dependent mitophagy1. Unexpectedly, we find that the adenine nucleotide translocator (ANT) complex is required for mitophagy in several cell types. Whereas pharmacological inhibition of ANT-mediated ADP/ATP exchange promotes mitophagy, genetic ablation of ANT paradoxically suppresses mitophagy. Notably, ANT promotes mitophagy independently of its nucleotide translocase catalytic activity. Instead, the ANT complex is required for inhibition of the presequence translocase TIM23, which leads to stabilization of PINK1, in response to bioenergetic collapse. ANT modulates TIM23 indirectly via interaction with TIM44, which regulates peptide import through TIM232. Mice that lack ANT1 show blunted mitophagy and consequent profound accumulation of aberrant mitochondria. Disease-causing human mutations in ANT1 abrogate binding to TIM44 and TIM23 and inhibit mitophagy. Together, our findings show that ANT is an essential and fundamental mediator of mitophagy in health and disease.

Loss of ACOT7 potentiates seizures and metabolic dysfunction.

Neurons uniquely antagonize fatty acid utilization by hydrolyzing the activated form of fatty acids, long chain acyl-CoAs, via the enzyme acyl-CoA thioesterase 7, Acot7. The loss of Acot7 results in increased fatty acid utilization in neurons and exaggerated stimulus-evoked behavior such as an increased startle response. To understand the contribution of Acot7 to seizure susceptibility, we generated Acot7 knockout (KO) mice and assayed their response to kainate-induced seizures. Acot7 KO mice exhibited potentiated behavioral and molecular indices of seizure severity following kainic acid administration, suggesting that fatty acid metabolism in neurons can be a critical regulator of neuronal activity. These data are consistent with the presentation of seizures in a human with genomic deletion of ACOT7 demonstrating the conservation of function across species. To further understand the metabolic complications arising from a deletion in Acot7, we subjected Acot7 KO mice to a high-fat diet. While the loss of Acot7 did not result in metabolic complications following a normal chow diet, a high-fat diet induced greater body weight gain, adiposity, and glucose intolerance in Acot7 KO mice. These data demonstrate that Acot7, a fatty acid metabolic enzyme highly enriched in neurons, regulates both brain-specific metabolic processes related to seizure susceptibility and the whole body response to dietary lipid.

Metabolic perturbations after pediatric TBI: It's not just about glucose.

Improved patient survival following pediatric traumatic brain injury (TBI) has uncovered a currently limited understanding of both the adaptive and maladaptive metabolic perturbations that occur during the acute and long-term phases of recovery. While much is known about the redundancy of metabolic pathways that provide adequate energy and substrates for normal brain growth and development, the field is only beginning to characterize perturbations in these metabolic pathways after pediatric TBI. To date, the majority of studies have focused on dysregulated oxidative glucose metabolism after injury; however, the immature brain is well-equipped to use alternative substrates to fuel energy production, growth, and development. A comprehensive understanding of metabolic changes associated with pediatric TBI cannot be limited to investigations of glucose metabolism alone. All energy substrates used by the brain should be considered in developing nutritional and pharmacological interventions for pediatric head trauma. This review summarizes post-injury changes in brain metabolism of glucose, lipids, ketone bodies, and amino acids with discussion of the therapeutic potential of altering substrate utilization to improve pediatric TBI outcomes.

Role of the malonyl-CoA synthetase ACSF3 in mitochondrial metabolism.

Malonyl-CoA is a central metabolite in fatty acid biochemistry. It is the rate-determining intermediate in fatty acid synthesis but is also an allosteric inhibitor of the rate-setting step in mitochondrial long-chain fatty acid oxidation. While these canonical cytoplasmic roles of malonyl-CoA have been well described, malonyl-CoA can also be generated within the mitochondrial matrix by an alternative pathway: the ATP-dependent ligation of malonate to Coenzyme A by the malonyl-CoA synthetase ACSF3. Malonate, a competitive inhibitor of succinate dehydrogenase of the TCA cycle, is a potent inhibitor of mitochondrial respiration. A major role for ACSF3 is to provide a metabolic pathway for the clearance of malonate by the generation of malonyl-CoA, which can then be decarboxylated to acetyl-CoA by malonyl-CoA decarboxylase. Additionally, ACSF3-derived malonyl-CoA can be used to malonylate lysine residues on proteins within the matrix of mitochondria, possibly adding another regulatory layer to post-translational control of mitochondrial metabolism. The discovery of ACSF3-mediated generation of malonyl-CoA defines a new mitochondrial metabolic pathway and raises new questions about how the metabolic fates of this multifunctional metabolite intersect with mitochondrial metabolism.

Etomoxir Inhibits Macrophage Polarization by Disrupting CoA Homeostasis.

Long-chain fatty acid (LCFA) oxidation has been shown to play an important role in interleukin-4 (IL-4)-mediated macrophage polarization (M(IL-4)). However, many of these conclusions are based on the inhibition of carnitine palmitoyltransferase-1 with high concentrations of etomoxir that far exceed what is required to inhibit enzyme activity (EC90 < 3 µM). We employ genetic and pharmacologic models to demonstrate that LCFA oxidation is largely dispensable for IL-4-driven polarization. Unexpectedly, high concentrations of etomoxir retained the ability to disrupt M(IL-4) polarization in the absence of Cpt1a or Cpt2 expression. Although excess etomoxir inhibits the adenine nucleotide translocase, oxidative phosphorylation is surprisingly dispensable for M(IL-4). Instead, the block in polarization was traced to depletion of intracellular free coenzyme A (CoA), likely resulting from conversion of the pro-drug etomoxir into active etomoxiryl CoA. These studies help explain the effect(s) of excess etomoxir on immune cells and reveal an unappreciated role for CoA metabolism in macrophage polarization.

Fatty acid oxidation by the osteoblast is required for normal bone acquisition in a sex- and diet-dependent manner.

Postnatal bone formation is influenced by nutritional status and compromised by disturbances in metabolism. The oxidation of dietary lipids represents a critical source of ATP for many cells but has been poorly studied in the skeleton, where the prevailing view is that glucose is the primary energy source. Here, we examined fatty acid uptake by bone and probed the requirement for fatty acid catabolism during bone formation by specifically disrupting the expression of carnitine palmitoyltransferase 2 (Cpt2), an obligate enzyme in fatty acid oxidation, in osteoblasts and osteocytes. Radiotracer studies demonstrated that the skeleton accumulates a significant fraction of postprandial fatty acids, which was equal to or in excess of that acquired by skeletal muscle or adipose tissue. Female, but not male, Cpt2 mutant mice exhibited significant impairments in postnatal bone acquisition, potentially due to an inability of osteoblasts to modify fuel selection. Intriguingly, suppression of fatty acid utilization by osteoblasts and osteocytes also resulted in the development of dyslipidemia and diet-dependent modifications in body composition. Taken together, these studies demonstrate a requirement for fatty acid oxidation during bone accrual and suggest a role for the skeleton in lipid homeostasis.

Developmental regulation and localization of carnitine palmitoyltransferases (CPTs) in rat brain.

While the brain's high energy demands are largely met by glucose, brain is also equipped with the ability to oxidize fatty acids for energy and metabolism. The brain expresses the carnitine palmitoyltransferases (CPTs) that mediate carnitine-dependent entry of long-chain acyl-CoAs into the mitochondrial matrix for ß-oxidation - CPT1a and CPT2 located on the outer and inner mitochondrial membranes, respectively. Their developmental profile, regional distribution and activity as well as cell type expression remain unknown. We determined that brain CPT1a RNA and total protein expression were unchanged throughout post-natal development (PND0, PND7, PND14, PND21 and PND50); however, CPT2 RNA peaked at PND 21 and remained unchanged through PND50 in all regions studied (cortex, hippocampus, midbrain, and cerebellum). Both long-chain acyl CoA dehydrogenase and medium acyl-CoA dehydrogenase showed a similar developmental profile to CPT2. Acylcarnitines, generated as a result of CPT1a activity, significantly increased with age and peaked at PND21 in all brain regions, concurrent with the increased expression of enzymes involved in mitochondrial ß-oxidation. The CPT system is highly enriched in vivo in hippocampus and cerebellum, relative to cortex and midbrain, and is exclusively present in astrocytes and neural progenitor cells, while absent in neurons, microglia, and oligodendrocytes. Using radiolabeled oleate, we demonstrate regional differences in brain fatty acid oxidation that may be blocked by the irreversible CPT1a inhibitor etomoxir. This study contributes to the field of knowledge in brain cell-specific metabolic pathways, which are important for understanding normal brain development and aging, as well as pathophysiology of neurological diseases. Read the Editorial Comment for this article on page 347.

The Mammalian Malonyl-CoA Synthetase ACSF3 Is Required for Mitochondrial Protein Malonylation and Metabolic Efficiency.

Malonyl-coenzyme A (malonyl-CoA) is a central metabolite in mammalian fatty acid biochemistry generated and utilized in the cytoplasm; however, little is known about noncanonical organelle-specific malonyl-CoA metabolism. Intramitochondrial malonyl-CoA is generated by a malonyl-CoA synthetase, ACSF3, which produces malonyl-CoA from malonate, an endogenous competitive inhibitor of succinate dehydrogenase. To determine the metabolic requirement for mitochondrial malonyl-CoA, ACSF3 knockout (KO) cells were generated by CRISPR/Cas-mediated genome editing. ACSF3 KO cells exhibited elevated malonate and impaired mitochondrial metabolism. Unbiased and targeted metabolomics analysis of KO and control cells in the presence or absence of exogenous malonate revealed metabolic changes dependent on either malonate or malonyl-CoA. While ACSF3 was required for the metabolism and therefore detoxification of malonate, ACSF3-derived malonyl-CoA was specifically required for lysine malonylation of mitochondrial proteins. Together, these data describe an essential role for ACSF3 in dictating the metabolic fate of mitochondrial malonate and malonyl-CoA in mammalian metabolism.

Requirement for the Mitochondrial Pyruvate Carrier in Mammalian Development Revealed by a Hypomorphic Allelic Series.

Glucose and oxygen are two of the most important molecules transferred from mother to fetus during eutherian pregnancy, and the metabolic fates of these nutrients converge at the transport and metabolism of pyruvate in mitochondria. Pyruvate enters the mitochondrial matrix through the mitochondrial pyruvate carrier (MPC), a complex in the inner mitochondrial membrane that consists of two essential components, MPC1 and MPC2. Here, we define the requirement for mitochondrial pyruvate metabolism during development with a progressive allelic series of Mpc1 deficiency in mouse. Mpc1 deletion was homozygous lethal in midgestation, but Mpc1 hypomorphs and tissue-specific deletion of Mpc1 presented as early perinatal lethality. The allelic series demonstrated that graded suppression of MPC resulted in dose-dependent metabolic and transcriptional changes. Steady-state metabolomics analysis of brain and liver from Mpc1 hypomorphic embryos identified compensatory changes in amino acid and lipid metabolism. Flux assays in Mpc1-deficient embryonic fibroblasts also reflected these changes, including a dramatic increase in mitochondrial alanine utilization. The mitochondrial alanine transaminase GPT2 was found to be necessary and sufficient for increased alanine flux upon MPC inhibition. These data show that impaired mitochondrial pyruvate transport results in biosynthetic deficiencies that can be mitigated in part by alternative anaplerotic substrates in utero.

Loss of CTRP5 improves insulin action and hepatic steatosis.

The gene that encodes C1q/TNF-related protein 5 (CTRP5), a secreted protein of the C1q family, is mutated in individuals with late-onset retinal degeneration. CTRP5 is widely expressed outside the eye and also circulates in plasma. Its physiological role in peripheral tissues, however, has yet to be elucidated. Here, we show that Ctrp5 expression is modulated by fasting and refeeding, and by different diets, in mice. Adipose expression of CTRP5 was markedly upregulated in obese and diabetic humans and in genetic and dietary models of obesity in rodents. Furthermore, human CTRP5 expression in the subcutaneous fat depot positively correlated with BMI. A genetic loss-of-function mouse model was used to address the metabolic function of CTRP5 in vivo. On a standard chow diet, CTRP5-deficient mice had reduced fasting insulin but were otherwise comparable with wild-type littermate controls in body weight and adiposity. However, when fed a high-fat diet, CTRP5-deficient animals had attenuated hepatic steatosis and improved insulin action. Loss of CTRP5 also improved the capacity of chow-fed aged mice to respond to subsequent high-fat feeding, as evidenced by decreased insulin resistance. In cultured adipocytes and myotubes, recombinant CTRP5 treatment attenuated insulin-stimulated Akt phosphorylation. Our results provide the first genetic and physiological evidence for CTRP5 as a negative regulator of glucose metabolism and insulin sensitivity. Inhibition of CTRP5 action may result in the alleviation of insulin resistance associated with obesity and diabetes.

A multi-omic analysis of human naïve CD4+ T cells.

BACKGROUND: Cellular function and diversity are orchestrated by complex interactions of fundamental biomolecules including DNA, RNA and proteins. Technological advances in genomics, epigenomics, transcriptomics and proteomics have enabled massively parallel and unbiased measurements. Such high-throughput technologies have been extensively used to carry out broad, unbiased studies, particularly in the context of human diseases. Nevertheless, a unified analysis of the genome, epigenome, transcriptome and proteome of a single human cell type to obtain a coherent view of the complex interplay between various biomolecules has not yet been undertaken. Here, we report the first multi-omic analysis of human primary naïve CD4+ T cells isolated from a single individual. RESULTS: Integrating multi-omics datasets allowed us to investigate genome-wide methylation and its effect on mRNA/protein expression patterns, extent of RNA editing under normal physiological conditions and allele specific expression in naïve CD4+ T cells. In addition, we carried out a multi-omic comparative analysis of naïve with primary resting memory CD4+ T cells to identify molecular changes underlying T cell differentiation. This analysis provided mechanistic insights into how several molecules involved in T cell receptor signaling are regulated at the DNA, RNA and protein levels. Phosphoproteomics revealed downstream signaling events that regulate these two cellular states. Availability of multi-omics data from an identical genetic background also allowed us to employ novel proteogenomics approaches to identify individual-specific variants and putative novel protein coding regions in the human genome. CONCLUSIONS: We utilized multiple high-throughput technologies to derive a comprehensive profile of two primary human cell types, naïve CD4+ T cells and memory CD4+ T cells, from a single donor. Through vertical as well as horizontal integration of whole genome sequencing, methylation arrays, RNA-Seq, miRNA-Seq, proteomics, and phosphoproteomics, we derived an integrated and comparative map of these two closely related immune cells and identified potential molecular effectors of immune cell differentiation following antigen encounter.