Metabolic programs drive function of therapeutic NK cells in hypoxic tumor environments.

Limited oxygen (hypoxia) in solid tumors poses a challenge to successful immunotherapy with natural killer (NK) cells. NK cells have impaired cytotoxicity when cultured in hypoxia (1% oxygen) but not physiologic (>5%) or atmospheric oxygen (20%). We found that changes to cytotoxicity were regulated at the transcriptional level and accompanied by metabolic dysregulation. Dosing with interleukin-15 (IL-15) enhanced NK cell cytotoxicity in hypoxia, but preactivation with feeder cells bearing IL-21 and 4-1BBL was even better. Preactivation resulted in less perturbed metabolism in hypoxia; greater resistance to oxidative stress; and no hypoxia-induced loss of transcription factors (T-bet and Eomes), activating receptors, adhesion molecules (CD2), and cytotoxic proteins (TRAIL and FasL). There remained a deficit in CD122/IL-2Rß when exposed to hypoxia, which affected IL-15 signaling. However, tri-specific killer engager molecules that deliver IL-15 in the context of anti-CD16/FcγRIII were able to bypass this deficit, enhancing cytotoxicity of both fresh and preactivated NK cells in hypoxia.

Ketolysis drives CD8+ T cell effector function through effects on histone acetylation.

Environmental nutrient availability influences T cell metabolism, impacting T cell function and shaping immune outcomes. Here, we identified ketone bodies (KBs)-including ß-hydroxybutyrate (ßOHB) and acetoacetate (AcAc)-as essential fuels supporting CD8+ T cell metabolism and effector function. ßOHB directly increased CD8+ T effector (Teff) cell cytokine production and cytolytic activity, and KB oxidation (ketolysis) was required for Teff cell responses to bacterial infection and tumor challenge. CD8+ Teff cells preferentially used KBs over glucose to fuel the tricarboxylic acid (TCA) cycle in vitro and in vivo. KBs directly boosted the respiratory capacity and TCA cycle-dependent metabolic pathways that fuel CD8+ T cell function. Mechanistically, ßOHB was a major substrate for acetyl-CoA production in CD8+ T cells and regulated effector responses through effects on histone acetylation. Together, our results identify cell-intrinsic ketolysis as a metabolic and epigenetic driver of optimal CD8+ T cell effector responses.

Metabolic and Signaling Roles of Ketone Bodies in Health and Disease.

Ketone bodies play significant roles in organismal energy homeostasis, serving as oxidative fuels, modulators of redox potential, lipogenic precursors, and signals, primarily during states of low carbohydrate availability. Efforts to enhance wellness and ameliorate disease via nutritional, chronobiological, and pharmacological interventions have markedly intensified interest in ketone body metabolism. The two ketone body redox partners, acetoacetate and D-ß-hydroxybutyrate, serve distinct metabolic and signaling roles in biological systems. We discuss the pleiotropic roles played by both of these ketones in health and disease. While enthusiasm is warranted, prudent procession through therapeutic applications of ketogenic and ketone therapies is also advised, as a range of metabolic and signaling consequences continue to emerge. Organ-specific and cell-type-specific effects of ketone bodies are important to consider as prospective therapeutic and wellness applications increase.

Nonalcoholic Steatohepatitis: A Review.

Importance: Nonalcoholic steatohepatitis (NASH) is the inflammatory subtype of nonalcoholic fatty liver disease (NAFLD) and is associated with disease progression, development of cirrhosis, and need for liver transplant. Despite its importance, NASH is underrecognized in clinical practice. Observations: NASH affects an estimated 3% to 6% of the US population and the prevalence is increasing. NASH is strongly associated with obesity, dyslipidemia, type 2 diabetes, and metabolic syndrome. Although a number of noninvasive tests and scoring systems exist to characterize NAFLD and NASH, liver biopsy is the only accepted method for diagnosis of NASH. Currently, no NASH-specific therapies are approved by the US Food and Drug Administration. Lifestyle modification is the mainstay of treatment, including dietary changes and exercise, with the primary goal being weight loss. Substantial improvement in histologic outcomes, including fibrosis, is directly correlated with increasing weight loss. In some cases, bariatric surgery may be indicated to achieve and maintain the necessary degree of weight loss required for therapeutic effect. An estimated 20% of patients with NASH will develop cirrhosis, and NASH is predicted to become the leading indication for liver transplants in the US. The mortality rate among patients with NASH is substantially higher than the general population or patients without this inflammatory subtype of NAFLD, with annual all-cause mortality rate of 25.56 per 1000 person-years and a liver-specific mortality rate of 11.77 per 1000 person-years. Conclusions and Relevance: Nonalcoholic steatohepatitis affects 3% to 6% of the US population, is more prevalent in patients with metabolic disease and obesity, progresses to cirrhosis in approximately 20% of cases, and is associated with increased rates of liver-specific and overall mortality. Early identification and targeted treatment of patients with nonalcoholic steatohepatitis are needed to improve patient outcomes, including directing patients toward intensive lifestyle modification to promote weight loss and referral for bariatric surgery as indicated for management of obesity and metabolic disease.

Hepatocyte-Macrophage Acetoacetate Shuttle Protects against Tissue Fibrosis.

Metabolic plasticity has been linked to polarized macrophage function, but mechanisms connecting specific fuels to tissue macrophage function remain unresolved. Here we apply a stable isotope tracing, mass spectrometry-based untargeted metabolomics approach to reveal the metabolome penetrated by hepatocyte-derived glucose and ketone bodies. In both classically and alternatively polarized macrophages, [13C]acetoacetate (AcAc) labeled ∼200 chemical features, but its reduced form D-[13C]ß-hydroxybutyrate (D-ßOHB) labeled almost none. [13C]glucose labeled ∼500 features, and while unlabeled AcAc competed with only ∼15% of them, the vast majority required the mitochondrial enzyme succinyl-coenzyme A-oxoacid transferase (SCOT). AcAc carbon labeled metabolites within the cytoplasmic glycosaminoglycan pathway, which regulates tissue fibrogenesis. Accordingly, livers of mice lacking SCOT in macrophages were predisposed to accelerated fibrogenesis. Exogenous AcAc, but not D-ßOHB, ameliorated diet-induced hepatic fibrosis. These data support a hepatocyte-macrophage ketone shuttle that segregates AcAc from D-ßOHB, coordinating the fibrogenic response to hepatic injury via mitochondrial metabolism in tissue macrophages.

Transport-exclusion pharmacology to localize lactate dehydrogenase activity within cells.

BACKGROUND: Recent in vitro and in vivo work has shown that lactate provides an important source of carbon for metabolic reactions in cancer cell mitochondria. An interesting question is whether lactate is oxidized by lactate dehydrogenase (LDH) in the cytosol and/or in mitochondria. Since metabolic processes in the cytosol and mitochondria are affected by redox balance, the location of LDH may have important regulatory implications in cancer metabolism. METHODS: Within most mammalian cells, metabolic processes are physically separated by membrane-bound compartments. Our general understanding of this spatial organization and its role in cellular function, however, suffers from the limited number of techniques to localize enzymatic activities within a cell. Here, we describe an approach to assess metabolic compartmentalization by monitoring the activity of pharmacological inhibitors that cannot be transported into specific cellular compartments. RESULTS: Oxamate, which chemically resembles pyruvate, is transported into mitochondria and inhibits LDH activity in purified mitochondria. GSK-2837808A, in contrast, is a competitive inhibitor of NAD, which cannot cross the inner mitochondrial membrane. GSK-2837808A did not inhibit the LDH activity of intact mitochondria, but GSK-2837808A did inhibit LDH activity after the inner mitochondrial membrane was disrupted. CONCLUSIONS: Our results are consistent with some mitochondrial LDH that is accessible to oxamate, but inaccessible to GSK-2837808A until mitochondria are homogenized. This strategy of using inhibitors with selective access to subcellular compartments, which we refer to as transport-exclusion pharmacology, is broadly applicable to localize other metabolic reactions within cells.

Multi-dimensional Roles of Ketone Bodies in Fuel Metabolism, Signaling, and Therapeutics.

Ketone body metabolism is a central node in physiological homeostasis. In this review, we discuss how ketones serve discrete fine-tuning metabolic roles that optimize organ and organism performance in varying nutrient states and protect from inflammation and injury in multiple organ systems. Traditionally viewed as metabolic substrates enlisted only in carbohydrate restriction, observations underscore the importance of ketone bodies as vital metabolic and signaling mediators when carbohydrates are abundant. Complementing a repertoire of known therapeutic options for diseases of the nervous system, prospective roles for ketone bodies in cancer have arisen, as have intriguing protective roles in heart and liver, opening therapeutic options in obesity-related and cardiovascular disease. Controversies in ketone metabolism and signaling are discussed to reconcile classical dogma with contemporary observations.

Lactate metabolism is associated with mammalian mitochondria.

It is well established that lactate secreted by fermenting cells can be oxidized or used as a gluconeogenic substrate by other cells and tissues. It is generally assumed, however, that within the fermenting cell itself, lactate is produced to replenish NAD+ and then is secreted. Here we explore the possibility that cytosolic lactate is metabolized by the mitochondria of fermenting mammalian cells. We found that fermenting HeLa and H460 cells utilize exogenous lactate carbon to synthesize a large percentage of their lipids. Using high-resolution mass spectrometry, we found that both 13C and 2-2H labels from enriched lactate enter the mitochondria. The lactate dehydrogenase (LDH) inhibitor oxamate decreased respiration of isolated mitochondria incubated in lactate, but not of isolated mitochondria incubated in pyruvate. Additionally, transmission electron microscopy (TEM) showed that LDHB localizes to the mitochondria. Taken together, our results demonstrate a link between lactate metabolism and the mitochondria of fermenting mammalian cells.

Comprehensive and Quantitative Analysis of Polyphosphoinositide Species by Shotgun Lipidomics Revealed Their Alterations in db/db Mouse Brain.

Polyphosphoinositides (PPI) play crucial roles in cellular signaling and functions. However, comprehensively determining the changed levels of these species during different cellular processes has faced difficulties. Herein, we applied a novel methylation pattern recognition and simulation approach, and we exploited newly derived fragmentation patterns of methylated PPI species for comprehensive analysis of PPI species including phosphate position(s) and fatty acyl chains capable of circumpassing previous limitations. The developed method was applied for quantitative analysis of PPI species present in diabetic mouse cortex and liver, and it allowed us to unravel the marked reduction of PPI levels in brain cortices of db/db mice for the first time. Taken together, we developed a powerful and high-throughput method for comprehensive analysis of PPI species, which should greatly contribute to the elucidation of PPI biology under different disease states.

The ketone metabolite ß-hydroxybutyrate blocks NLRP3 inflammasome-mediated inflammatory disease.

The ketone bodies ß-hydroxybutyrate (BHB) and acetoacetate (AcAc) support mammalian survival during states of energy deficit by serving as alternative sources of ATP. BHB levels are elevated by starvation, caloric restriction, high-intensity exercise, or the low-carbohydrate ketogenic diet. Prolonged fasting reduces inflammation; however, the impact that ketones and other alternative metabolic fuels produced during energy deficits have on the innate immune response is unknown. We report that BHB, but neither AcAc nor the structurally related short-chain fatty acids butyrate and acetate, suppresses activation of the NLRP3 inflammasome in response to urate crystals, ATP and lipotoxic fatty acids. BHB did not inhibit caspase-1 activation in response to pathogens that activate the NLR family, CARD domain containing 4 (NLRC4) or absent in melanoma 2 (AIM2) inflammasome and did not affect non-canonical caspase-11, inflammasome activation. Mechanistically, BHB inhibits the NLRP3 inflammasome by preventing K(+) efflux and reducing ASC oligomerization and speck formation. The inhibitory effects of BHB on NLRP3 are not dependent on chirality or starvation-regulated mechanisms like AMP-activated protein kinase (AMPK), reactive oxygen species (ROS), autophagy or glycolytic inhibition. BHB blocks the NLRP3 inflammasome without undergoing oxidation in the TCA cycle, and independently of uncoupling protein-2 (UCP2), sirtuin-2 (SIRT2), the G protein-coupled receptor GPR109A or hydrocaboxylic acid receptor 2 (HCAR2). BHB reduces NLRP3 inflammasome-mediated interleukin (IL)-1ß and IL-18 production in human monocytes. In vivo, BHB or a ketogenic diet attenuates caspase-1 activation and IL-1ß secretion in mouse models of NLRP3-mediated diseases such as Muckle-Wells syndrome, familial cold autoinflammatory syndrome and urate crystal-induced peritonitis. Our findings suggest that the anti-inflammatory effects of caloric restriction or ketogenic diets may be linked to BHB-mediated inhibition of the NLRP3 inflammasome.

Cardiomyocyte-specific deficiency of ketone body metabolism promotes accelerated pathological remodeling.

OBJECTIVE: Exploitation of protective metabolic pathways within injured myocardium still remains an unclarified therapeutic target in heart disease. Moreover, while the roles of altered fatty acid and glucose metabolism in the failing heart have been explored, the influence of highly dynamic and nutritionally modifiable ketone body metabolism in the regulation of myocardial substrate utilization, mitochondrial bioenergetics, reactive oxygen species (ROS) generation, and hemodynamic response to injury remains undefined. METHODS: Here we use mice that lack the enzyme required for terminal oxidation of ketone bodies, succinyl-CoA:3-oxoacid CoA transferase (SCOT) to determine the role of ketone body oxidation in the myocardial injury response. Tracer delivery in ex vivo perfused hearts coupled to NMR spectroscopy, in vivo high-resolution echocardiographic quantification of cardiac hemodynamics in nutritionally and surgically modified mice, and cellular and molecular measurements of energetic and oxidative stress responses are performed. RESULTS: While germline SCOT-knockout (KO) mice die in the early postnatal period, adult mice with cardiomyocyte-specific loss of SCOT (SCOT-Heart-KO) remarkably exhibit no overt metabolic abnormalities, and no differences in left ventricular mass or impairments of systolic function during periods of ketosis, including fasting and adherence to a ketogenic diet. Myocardial fatty acid oxidation is increased when ketones are delivered but cannot be oxidized. To determine the role of ketone body oxidation in the remodeling ventricle, we induced pressure overload injury by performing transverse aortic constriction (TAC) surgery in SCOT-Heart-KO and αMHC-Cre control mice. While TAC increased left ventricular mass equally in both groups, at four weeks post-TAC, myocardial ROS abundance was increased in myocardium of SCOT-Heart-KO mice, and mitochondria and myofilaments were ultrastructurally disordered. Eight weeks post-TAC, left ventricular volume was markedly increased and ejection fraction was decreased in SCOT-Heart-KO mice, while these parameters remained normal in hearts of control animals. CONCLUSIONS: These studies demonstrate the ability of myocardial ketone metabolism to coordinate the myocardial response to pressure overload, and suggest that the oxidation of ketone bodies may be an important contributor to free radical homeostasis and hemodynamic preservation in the injured heart.

Ketogenesis prevents diet-induced fatty liver injury and hyperglycemia.

Nonalcoholic fatty liver disease (NAFLD) spectrum disorders affect approximately 1 billion individuals worldwide. However, the drivers of progressive steatohepatitis remain incompletely defined. Ketogenesis can dispose of much of the fat that enters the liver, and dysfunction in this pathway could promote the development of NAFLD. Here, we evaluated mice lacking mitochondrial 3-hydroxymethylglutaryl CoA synthase (HMGCS2) to determine the role of ketogenesis in preventing diet-induced steatohepatitis. Antisense oligonucleotide-induced loss of HMGCS2 in chow-fed adult mice caused mild hyperglycemia, increased hepatic gluconeogenesis from pyruvate, and augmented production of hundreds of hepatic metabolites, a suite of which indicated activation of the de novo lipogenesis pathway. High-fat diet feeding of mice with insufficient ketogenesis resulted in extensive hepatocyte injury and inflammation, decreased glycemia, deranged hepatic TCA cycle intermediate concentrations, and impaired hepatic gluconeogenesis due to sequestration of free coenzyme A (CoASH). Supplementation of the CoASH precursors pantothenic acid and cysteine normalized TCA intermediates and gluconeogenesis in the livers of ketogenesis-insufficient animals. Together, these findings indicate that ketogenesis is a critical regulator of hepatic acyl-CoA metabolism, glucose metabolism, and TCA cycle function in the absorptive state and suggest that ketogenesis may modulate fatty liver disease.

Role of choline deficiency in the Fatty liver phenotype of mice fed a low protein, very low carbohydrate ketogenic diet.

Though widely employed for clinical intervention in obesity, metabolic syndrome, seizure disorders and other neurodegenerative diseases, the mechanisms through which low carbohydrate ketogenic diets exert their ameliorative effects still remain to be elucidated. Rodent models have been used to identify the metabolic and physiologic alterations provoked by ketogenic diets. A commonly used rodent ketogenic diet (Bio-Serv F3666) that is very high in fat (~94% kcal), very low in carbohydrate (~1% kcal), low in protein (~5% kcal), and choline restricted (~300 mg/kg) provokes robust ketosis and weight loss in mice, but through unknown mechanisms, also causes significant hepatic steatosis, inflammation, and cellular injury. To understand the independent and synergistic roles of protein restriction and choline deficiency on the pleiotropic effects of rodent ketogenic diets, we studied four custom diets that differ only in protein (5% kcal vs. 10% kcal) and choline contents (300 mg/kg vs. 5 g/kg). C57BL/6J mice maintained on the two 5% kcal protein diets induced the most significant ketoses, which was only partially diminished by choline replacement. Choline restriction in the setting of 10% kcal protein also caused moderate ketosis and hepatic fat accumulation, which were again attenuated when choline was replete. Key effects of the 5% kcal protein diet - weight loss, hepatic fat accumulation, and mitochondrial ultrastructural disarray and bioenergetic dysfunction - were mitigated by choline repletion. These studies indicate that synergistic effects of protein restriction and choline deficiency influence integrated metabolism and hepatic pathology in mice when nutritional fat content is very high, and support the consideration of dietary choline content in ketogenic diet studies in rodents to limit hepatic mitochondrial dysfunction and fat accumulation.

Hepatic steatosis, inflammation, and ER stress in mice maintained long term on a very low-carbohydrate ketogenic diet.

Low-carbohydrate diets are used to manage obesity, seizure disorders, and malignancies of the central nervous system. These diets create a distinctive, but incompletely defined, cellular, molecular, and integrated metabolic state. Here, we determine the systemic and hepatic effects of long-term administration of a very low-carbohydrate, low-protein, and high-fat ketogenic diet, serially comparing these effects to a high-simple-carbohydrate, high-fat Western diet and a low-fat, polysaccharide-rich control chow diet in C57BL/6J mice. Longitudinal measurement of body composition, serum metabolites, and intrahepatic fat content, using in vivo magnetic resonance spectroscopy, reveals that mice fed the ketogenic diet over 12 wk remain lean, euglycemic, and hypoinsulinemic but accumulate hepatic lipid in a temporal pattern very distinct from animals fed the Western diet. Ketogenic diet-fed mice ultimately develop systemic glucose intolerance, hepatic endoplasmic reticulum stress, steatosis, cellular injury, and macrophage accumulation, but surprisingly insulin-induced hepatic Akt phosphorylation and whole-body insulin responsiveness are not impaired. Moreover, whereas hepatic Pparg mRNA abundance is augmented by both high-fat diets, each diet confers splice variant specificity. The distinctive nutrient milieu created by long-term administration of this low-carbohydrate, low-protein ketogenic diet in mice evokes unique signatures of nonalcoholic fatty liver disease and whole-body glucose homeostasis.

Akt2 deficiency promotes cardiac induction of Rab4a and myocardial ß-adrenergic hypersensitivity.

Patients with diabetes mellitus can develop cardiac dysfunction in the absence of underlying coronary artery disease or hypertension; a condition defined as diabetic cardiomyopathy. Mice lacking the intracellular protein kinase Akt2 develop a syndrome that is similar to diabetes mellitus type 2. Expression profiling of akt2(-/-) myocardium revealed that Rab4a, a GTPase involved in glucose transporter 4 translocation and ß-adrenergic receptor (ßAR) recycling to the plasma membrane, was significantly induced. We therefore hypothesized that Akt2 deficiency increases myocardial ß-adrenergic sensitivity. Confirmatory analysis revealed up-regulation of Rab4a mRNA and protein in akt2(-/-) myocardium. In cultured cardiomyocyte experiments, Rab4a was induced by pharmacological inhibition of Akt as well as by specific knockdown of Akt2 with siRNA. Isolated akt2(-/-) hearts were hypersensitive to isoproterenol (ISO) but exhibited normal sensitivity to forskolin. Prolonged ISO treatment led to increased cardiac hypertrophy in akt2(-/-) mice compared to wild type mice. In addition, spontaneous hypertrophy was noted in aged akt2(-/-) hearts that was inhibited by treatment with the ßAR blocker propranolol. In agreement with previous results demonstrating increased fatty acid oxidation rates in akt2(-/-) myocardium, we found increased peroxisome proliferator-activated receptor α (PPARα) activity in the hearts of these animals. Interestingly, increased myocardial Rab4a expression was present in mice with cardiac-specific overexpression of PPARα and was also observed upon stimulation of PPARα activity in cultured cardiomyocytes. Accordingly, propranolol attenuated the development of cardiac hypertrophy in the PPARα transgenic mice as well. Our results indicate that reduced Akt2 leads to up-regulation of Rab4a expression in cardiomyocytes in a cell-autonomous fashion that may involve activation of PPARα. This maladaptive response is associated with hypersensitivity of akt2(-/-) myocardium to ß-adrenergic stimulation.

Postnatal lymphatic partitioning from the blood vasculature in the small intestine requires fasting-induced adipose factor.

Lymphatic vessels develop from specialized venous endothelial cells. Using knockout mice, we found that fasting-induced adipose factor (Fiaf) is required for functional partitioning of postnatal intestinal lymphatic and blood vessels. In wild-type animals, levels of intestinal Fiaf expression rise during the first postnatal day and peak at day 2, which coincides with the onset of the lymphatico-venous partitioning abnormality in Fiaf-/- mutants on a mixed 129/SvJ:C57BL/6 genetic background. Fiaf deficiency is not associated with disruption of the blood vasculature or with lymphatic endothelial recruitment of smooth muscle cells. We identified Prox1, a critical regulator of lymphangiogenesis, as a downstream target for Fiaf signaling in the intestinal lymphatic endothelium. This organ-specific lymphovascular abnormality can be rescued by allowing embryonic Fiaf-/- intestinal isografts to develop in Fiaf+/+ recipients.

Microbial regulation of intestinal radiosensitivity.

We describe a method for treating germ-free (GF) mice with gamma-irradiation and transplanting them with normal or genetically manipulated bone marrow while maintaining their GF status. This approach revealed that GF mice are markedly resistant to lethal radiation enteritis. Furthermore, administering lethal doses of total body irradiation to GF mice produces markedly fewer apoptotic endothelial cells and lymphocytes in the mesenchymal cores of their small intestinal villi, compared with conventionally raised animals that have acquired a microbiota from birth. Analysis of GF and conventionally raised Rag1-/- mice disclosed that mature lymphocytes are not required for the development of lethal radiation enteritis or the microbiota-associated enhancement of endothelial radiosensitivity. Studies of gnotobiotic knockout mice that lack fasting-induced adipose factor (Fiaf), a fibrinogen/angiopoietin-like protein normally secreted from the small intestinal villus epithelium and suppressed by the microbiota, showed that Fiaf deficiency results in loss of resistance of villus endothelial and lymphocyte populations to radiation-induced apoptosis. Together, these findings provide insights about the cellular and molecular targets involved in microbial regulation of intestinal radiosensitivity.