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.

Ketones and the Heart: Metabolic Principles and Therapeutic Implications.

The ketone bodies beta-hydroxybutyrate and acetoacetate are hepatically produced metabolites catabolized in extrahepatic organs. Ketone bodies are a critical cardiac fuel and have diverse roles in the regulation of cellular processes such as metabolism, inflammation, and cellular crosstalk in multiple organs that mediate disease. This review focuses on the role of cardiac ketone metabolism in health and disease with an emphasis on the therapeutic potential of ketosis as a treatment for heart failure (HF). Cardiac metabolic reprogramming, characterized by diminished mitochondrial oxidative metabolism, contributes to cardiac dysfunction and pathologic remodeling during the development of HF. Growing evidence supports an adaptive role for ketone metabolism in HF to promote normal cardiac function and attenuate disease progression. Enhanced cardiac ketone utilization during HF is mediated by increased availability due to systemic ketosis and a cardiac autonomous upregulation of ketolytic enzymes. Therapeutic strategies designed to restore high-capacity fuel metabolism in the heart show promise to address fuel metabolic deficits that underpin the progression of HF. However, the mechanisms involved in the beneficial effects of ketone bodies in HF have yet to be defined and represent important future lines of inquiry. In addition to use as an energy substrate for cardiac mitochondrial oxidation, ketone bodies modulate myocardial utilization of glucose and fatty acids, two vital energy substrates that regulate cardiac function and hypertrophy. The salutary effects of ketone bodies during HF may also include extra-cardiac roles in modulating immune responses, reducing fibrosis, and promoting angiogenesis and vasodilation. Additional pleotropic signaling properties of beta-hydroxybutyrate and AcAc are discussed including epigenetic regulation and protection against oxidative stress. Evidence for the benefit and feasibility of therapeutic ketosis is examined in preclinical and clinical studies. Finally, ongoing clinical trials are reviewed for perspective on translation of ketone therapeutics for the treatment of HF.

Ketone ester administration improves glycemia in obese mice.

During periods of prolonged fasting/starvation, the liver generates ketones [i.e., ß-hydroxybutyrate (ßOHB)] that primarily serve as alternative substrates for ATP production. Previous studies have demonstrated that elevations in skeletal muscle ketone oxidation contribute to obesity-related hyperglycemia, whereas inhibition of succinyl CoA:3-ketoacid CoA transferase (SCOT), the rate-limiting enzyme of ketone oxidation, can alleviate obesity-related hyperglycemia. As circulating ketone levels are a key determinant of ketone oxidation rates, we tested the hypothesis that increases in circulating ketone levels would worsen glucose homeostasis secondary to increases in muscle ketone oxidation. Accordingly, male C57BL/6J mice were subjected to high-fat diet-induced obesity, whereas their lean counterparts received a standard chow diet. Lean and obese mice were orally administered either a ketone ester (KE) or placebo, followed by a glucose tolerance test. In tandem, we conducted isolated islet perifusion experiments to quantify insulin secretion in response to ketones. We observed that exogenous KE administration robustly increases circulating ßOHB levels, which was associated with an improvement in glucose tolerance only in obese mice. These observations were independent of muscle ketone oxidation, as they were replicated in mice with a skeletal muscle-specific SCOT deficiency. Furthermore, the R-isomer of ßOHB produced greater increases in perifusion insulin levels versus the S-isomer in isolated islets from obese mice. Taken together, acute elevations in circulating ketones promote glucose-lowering in obesity. Given that only the R-isomer of ßOHB is oxidized, further studies are warranted to delineate the precise role of ß-cell ketone oxidation in regulating insulin secretion.NEW & NOTEWORTHY It has been demonstrated that increased skeletal muscle ketone metabolism contributes to obesity-related hyperglycemia. Since increases in ketone supply are key determinants of organ ketone oxidation rates, we determined whether acute elevations in circulating ketones following administration of an oral ketone ester may worsen glucose homeostasis in lean or obese mice. Our work demonstrates the opposite, as acute elevations in circulating ketones improved glucose tolerance in obese mice.

Loss of hepatic phosphoenolpyruvate carboxykinase 1 dysregulates metabolic responses to acute exercise but enhances adaptations to exercise training in mice.

Acute exercise increases liver gluconeogenesis to supply glucose to working muscles. Concurrently, elevated liver lipid breakdown fuels the high energetic cost of gluconeogenesis. This functional coupling between liver gluconeogenesis and lipid oxidation has been proposed to underlie the ability of regular exercise to enhance liver mitochondrial oxidative metabolism and decrease liver steatosis in individuals with nonalcoholic fatty liver disease. Herein we tested whether repeated bouts of increased hepatic gluconeogenesis are necessary for exercise training to lower liver lipids. Experiments used diet-induced obese mice lacking hepatic phosphoenolpyruvate carboxykinase 1 (KO) to inhibit gluconeogenesis and wild-type (WT) littermates. 2H/13C metabolic flux analysis quantified glucose and mitochondrial oxidative fluxes in untrained mice at rest and during acute exercise. Circulating and tissue metabolite levels were determined during sedentary conditions, acute exercise, and refeeding postexercise. Mice also underwent 6 wk of treadmill running protocols to define hepatic and extrahepatic adaptations to exercise training. Untrained KO mice were unable to maintain euglycemia during acute exercise resulting from an inability to increase gluconeogenesis. Liver triacylglycerides were elevated after acute exercise and circulating ß-hydroxybutyrate was higher during postexercise refeeding in untrained KO mice. In contrast, exercise training prevented liver triacylglyceride accumulation in KO mice. This was accompanied by pronounced increases in indices of skeletal muscle mitochondrial oxidative metabolism in KO mice. Together, these results show that hepatic gluconeogenesis is dispensable for exercise training to reduce liver lipids. This may be due to responses in ketone body metabolism and/or metabolic adaptations in skeletal muscle to exercise.NEW & NOTEWORTHY Exercise training reduces hepatic steatosis partly through enhanced hepatic terminal oxidation. During acute exercise, hepatic gluconeogenesis is elevated to match the heightened rate of muscle glucose uptake and maintain glucose homeostasis. It has been postulated that the hepatic energetic stress induced by elevating gluconeogenesis during acute exercise is a key stimulus underlying the beneficial metabolic responses to exercise training. This study shows that hepatic gluconeogenesis is not necessary for exercise training to lower liver lipids.

Defective muscle ketone body oxidation disrupts BCAA catabolism by altering mitochondrial branched-chain aminotransferase.

Ketone bodies are an endogenous fuel source generated primarily by the liver to provide alternative energy for extrahepatic tissues during prolonged fasting and exercise. Skeletal muscle is an important site of ketone body oxidation that occurs through a series of reactions requiring the enzyme succinyl-CoA:3-ketoacid-CoA transferase (SCOT/Oxct1). We have previously shown that deleting SCOT in the skeletal muscle protects against obesity-induced insulin resistance by increasing pyruvate dehydrogenase (PDH) activity, the rate-limiting enzyme of glucose oxidation. However, it remains unclear whether inhibiting muscle ketone body oxidation causes hypoglycemia and affects fuel metabolism in the absence of obesity. Here, we show that lean mice lacking skeletal muscle SCOT (SCOTSkM-/-) exhibited no overt phenotypic differences in glucose and fat metabolism from their human α-skeletal actin-Cre (HSACre) littermates. Of interest, we found that plasma and muscle branched-chain amino acid (BCAA) levels are elevated in SCOTSkM-/- lean mice compared with their HSACre littermates. Interestingly, this alteration in BCAA catabolism was only seen in SCOTSkM-/- mice under low-fat feeding and associated with decreased expression of mitochondrial branched-chain aminotransferases (BCATm/Bcat2), the first enzyme in BCAA catabolic pathway. Loss- and gain-of-function studies in C2C12 myotubes demonstrated that suppressing SCOT markedly diminished BCATm expression, whereas overexpressing SCOT resulted in an opposite effect without influencing BCAA oxidation enzymes. Furthermore, SCOT overexpression in C2C12 myotubes significantly increased luciferase activity driven by a Bcat2 promoter construct. Together, our findings indicate that SCOT regulates the expression of the Bcat2 gene, which, through the abundance of its product BCATm, may influence circulating BCAA concentrations.NEW & NOTEWORTHY Most studies investigated ketone body metabolism under pathological conditions, whereas the role of ketone body metabolism in regulating normal physiology has been relatively understudied. To address this gap, we used lean mice lacking muscle ketone body oxidation enzyme SCOT. Our work demonstrates that deleting muscle SCOT has no impact on glucose and fat metabolism in lean mice, but it disrupts muscle BCAA catabolism and causes an accumulation of BCAAs by altering BCATm.

Artifactual FA dimers mimic FAHFA signals in untargeted metabolomics pipelines.

FA esters of hydroxy FAs (FAHFAs) are lipokines with extensive structural and regional isomeric diversity that impact multiple physiological functions, including insulin sensitivity and glucose homeostasis. Because of their low molar abundance, FAHFAs are typically quantified using highly sensitive LC-MS/MS methods. Numerous relevant MS databases house in silico-spectra that allow identification and speciation of FAHFAs. These provisional chemical feature assignments provide a useful starting point but could lead to misidentification. To address this possibility, we analyzed human serum with a commonly applied high-resolution LC-MS untargeted metabolomics platform. We found that many chemical features are putatively assigned to the FAHFA lipid class based on exact mass and fragmentation patterns matching spectral databases. Careful validation using authentic standards revealed that many investigated signals provisionally assigned as FAHFAs are in fact FA dimers formed in the LC-MS pipeline. These isobaric FA dimers differ structurally only by the presence of an olefinic bond. Furthermore, stable isotope-labeled oleic acid spiked into human serum at subphysiological concentrations showed concentration-dependent formation of a diverse repertoire of FA dimers that analytically mimicked FAHFAs. Conversely, validated FAHFA species did not form spontaneously in the LC-MS pipeline. Together, these findings underscore that FAHFAs are endogenous lipid species.  However, nonbiological FA dimers forming in the setting of high concentrations of FFAs can be misidentified as FAHFAs. Based on these results, we assembled a FA dimer database to identify nonbiological FA dimers in untargeted metabolomics datasets.

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.

Ketone bodies as epigenetic modifiers.

PURPOSE OF REVIEW: Ketone body metabolism is a dynamic and integrated metabolic node in human physiology, whose roles include but extend beyond alternative fuel provision during carbohydrate restriction. Here we discuss the most recent observations suggesting that ketosis coordinates cellular function via epigenomic regulation. RECENT FINDINGS: Ketosis has been linked to covalent modifications, including lysine acetylation, methylation, and hydroxybutyrylation, to key histones that serve as dynamic regulators of chromatin architecture and gene transcription. Although it remains to be fully established whether these changes to the epigenome are attributable to ketone bodies themselves or other aspects of ketotic states, the regulated genes mediate classical responses to carbohydrate restriction. SUMMARY: Direct regulation of gene expression may occur in-vivo via through ketone body-mediated histone modifications during adherence to low-carbohydrate diets, fasting ketosis, exogenous ketone body therapy, and diabetic ketoacidosis. Additional convergent functional genomics, metabolomics, and proteomics studies are required in both animal models and in humans to identify the molecular mechanisms through which ketosis regulates nuclear signaling events in a myriad of conditions relevant to disease, and the contexts in which the benefits of ketosis might outweigh the risks.

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.

Intra- and inter-subject variability for increases in serum ketone bodies in patients with type 2 diabetes treated with the sodium glucose co-transporter 2 inhibitor canagliflozin.

Sodium glucose co-transporter 2 (SGLT2) inhibitors have been associated with increased serum ketone body levels in patients with type 2 diabetes mellitus (T2DM). In the present analysis we evaluated serum ketone body levels and variability in 1278 Japanese patients with T2DM treated with canagliflozin 100 or 200 mg. Similar mean increases in ketone body concentrations of ~2-fold were seen with both canagliflozin doses. The median (interquartile range) percent change from baseline was 62% (0;180) for acetoacetate and 78% (2;236) for ß-hydroxybutyrate. Approximately two-thirds of the variability in each ketone measure was attributed to intra-subject variability. Intra-subject variability was higher for serum ketones than other metabolites. Patients in the lowest response tertile exhibited no increase in ketones. Those in the highest response tertile tended to be male and have higher fasting plasma glucose levels, lower insulin levels, and longer T2DM duration at baseline. Moreover, changes in serum ketones were not fully explained by changes in plasma fatty acids, suggesting downstream effects of SGLT2 inhibition on hepatic metabolism that favour ketogenesis. In summary, increases in serum ketone bodies with canagliflozin were greater and more variable than changes in other metabolic measures in Japanese patients with T2DM.

The Failing Heart Relies on Ketone Bodies as a Fuel.

BACKGROUND: Significant evidence indicates that the failing heart is energy starved. During the development of heart failure, the capacity of the heart to utilize fatty acids, the chief fuel, is diminished. Identification of alternate pathways for myocardial fuel oxidation could unveil novel strategies to treat heart failure. METHODS AND RESULTS: Quantitative mitochondrial proteomics was used to identify energy metabolic derangements that occur during the development of cardiac hypertrophy and heart failure in well-defined mouse models. As expected, the amounts of proteins involved in fatty acid utilization were downregulated in myocardial samples from the failing heart. Conversely, expression of ß-hydroxybutyrate dehydrogenase 1, a key enzyme in the ketone oxidation pathway, was increased in the heart failure samples. Studies of relative oxidation in an isolated heart preparation using ex vivo nuclear magnetic resonance combined with targeted quantitative myocardial metabolomic profiling using mass spectrometry revealed that the hypertrophied and failing heart shifts to oxidizing ketone bodies as a fuel source in the context of reduced capacity to oxidize fatty acids. Distinct myocardial metabolomic signatures of ketone oxidation were identified. CONCLUSIONS: These results indicate that the hypertrophied and failing heart shifts to ketone bodies as a significant fuel source for oxidative ATP production. Specific metabolite biosignatures of in vivo cardiac ketone utilization were identified. Future studies aimed at determining whether this fuel shift is adaptive or maladaptive could unveil new therapeutic strategies for heart failure.

Circulating acylcarnitine profile in human heart failure: a surrogate of fatty acid metabolic dysregulation in mitochondria and beyond.

Heart failure (HF) is associated with metabolic perturbations, particularly of fatty acids (FAs), which remain to be better understood in humans. This study aimed at testing the hypothesis that HF patients with reduced ejection fraction display systemic perturbations in levels of energy-related metabolites, especially those reflecting dysregulation of FA metabolism, namely, acylcarnitines (ACs). Circulating metabolites were assessed using mass spectrometry (MS)-based methods in two cohorts. The main cohort consisted of 72 control subjects and 68 HF patients exhibiting depressed left ventricular ejection fraction (25.9 ± 6.9%) and mostly of ischemic etiology with ≥2 comorbidities. HF patients displayed marginal changes in plasma levels of tricarboxylic acid cycle-related metabolites or indexes of mitochondrial or cytosolic redox status. They had, however, 22-79% higher circulating ACs, irrespective of chain length (P < 0.0001, adjusted for sex, age, renal function, and insulin resistance, determined by shotgun MS/MS), which reflects defective mitochondrial ß-oxidation, and were significantly associated with levels of NH2-terminal pro-B-type natriuretic peptide levels, a disease severity marker. Subsequent extended liquid chromatography-tandem MS analysis of 53 plasma ACs in a subset group from the primary cohort confirmed and further substantiated with a comprehensive lipidomic analysis in a validation cohort revealed in HF patients a more complex circulating AC profile. The latter included dicarboxylic-ACs and dihydroxy-ACs as well as very long chain (VLC) ACs or sphingolipids with VLCFAs (>20 carbons), which are proxies of dysregulated FA metabolism in peroxisomes. Our study identified alterations in circulating ACs in HF patients that are independent of biological traits and associated with disease severity markers. These alterations reflect dysfunctional FA metabolism in mitochondria but also beyond, namely, in peroxisomes, suggesting a novel mechanism contributing to global lipid perturbations in human HF.NEW & NOTEWORTHY Mass spectrometry-based profiling of circulating energy metabolites, including acylcarnitines, in two cohorts of heart failure versus control subjects revealed multiple alterations in fatty acid metabolism in peroxisomes in addition to mitochondria, thereby highlighting a novel mechanism contributing to global lipid perturbations in heart failure.Listen to this article's corresponding podcast at http://ajpheart.podbean.com/e/acylcarnitines-in-human-heart-failure/.

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.

Impact of peripheral ketolytic deficiency on hepatic ketogenesis and gluconeogenesis during the transition to birth.

Preservation of bioenergetic homeostasis during the transition from the carbohydrate-laden fetal diet to the high fat, low carbohydrate neonatal diet requires inductions of hepatic fatty acid oxidation, gluconeogenesis, and ketogenesis. Mice with loss-of-function mutation in the extrahepatic mitochondrial enzyme CoA transferase (succinyl-CoA:3-oxoacid CoA transferase, SCOT, encoded by nuclear Oxct1) cannot terminally oxidize ketone bodies and develop lethal hyperketonemic hypoglycemia within 48 h of birth. Here we use this model to demonstrate that loss of ketone body oxidation, an exclusively extrahepatic process, disrupts hepatic intermediary metabolic homeostasis after high fat mother's milk is ingested. Livers of SCOT-knock-out (SCOT-KO) neonates induce the expression of the genes encoding peroxisome proliferator-activated receptor γ co-activator-1a (PGC-1α), phosphoenolpyruvate carboxykinase (PEPCK), pyruvate carboxylase, and glucose-6-phosphatase, and the neonate's pools of gluconeogenic alanine and lactate are each diminished by 50%. NMR-based quantitative fate mapping of (13)C-labeled substrates revealed that livers of SCOT-KO newborn mice synthesize glucose from exogenously administered pyruvate. However, the contribution of exogenous pyruvate to the tricarboxylic acid cycle as acetyl-CoA is increased in SCOT-KO livers and is associated with diminished terminal oxidation of fatty acids. After mother's milk provokes hyperketonemia, livers of SCOT-KO mice diminish de novo hepatic ß-hydroxybutyrate synthesis by 90%. Disruption of ß-hydroxybutyrate production increases hepatic NAD(+)/NADH ratios 3-fold, oxidizing redox potential in liver but not skeletal muscle. Together, these results indicate that peripheral ketone body oxidation prevents hypoglycemia and supports hepatic metabolic homeostasis, which is critical for the maintenance of glycemia during the adaptation to birth.

Impairments of hepatic gluconeogenesis and ketogenesis in PPARα-deficient neonatal mice.

Peroxisome proliferator activated receptor-α (PPARα) is a master transcriptional regulator of hepatic metabolism and mediates the adaptive response to fasting. Here, we demonstrate the roles for PPARα in hepatic metabolic adaptations to birth. Like fasting, nutrient supply is abruptly altered at birth when a transplacental source of carbohydrates is replaced by a high-fat, low-carbohydrate milk diet. PPARα-knockout (KO) neonatal mice exhibit relative hypoglycemia due to impaired conversion of glycerol to glucose. Although hepatic expression of fatty acyl-CoA dehydrogenases is imparied in PPARα neonates, these animals exhibit normal blood acylcarnitine profiles. Furthermore, quantitative metabolic fate mapping of the medium-chain fatty acid [(13)C]octanoate in neonatal mouse livers revealed normal contribution of this fatty acid to the hepatic TCA cycle. Interestingly, octanoate-derived carbon labeled glucose uniquely in livers of PPARα-KO neonates. Relative hypoketonemia in newborn PPARα-KO animals could be mechanistically linked to a 50% decrease in de novo hepatic ketogenesis from labeled octanoate. Decreased ketogenesis was associated with diminished mRNA and protein abundance of the fate-committing ketogenic enzyme mitochondrial 3-hydroxymethylglutaryl-CoA synthase (HMGCS2) and decreased protein abundance of the ketogenic enzyme ß-hydroxybutyrate dehydrogenase 1 (BDH1). Finally, hepatic triglyceride and free fatty acid concentrations were increased 6.9- and 2.7-fold, respectively, in suckling PPARα-KO neonates. Together, these findings indicate a primary defect of gluconeogenesis from glycerol and an important role for PPARα-dependent ketogenesis in the disposal of hepatic fatty acids during the neonatal period.

X13CMS: global tracking of isotopic labels in untargeted metabolomics.

Studies of isotopically labeled compounds have been fundamental to understanding metabolic pathways and fluxes. They have traditionally, however, been used in conjunction with targeted analyses that identify and quantify a limited number of labeled downstream metabolites. Here we describe an alternative workflow that leverages recent advances in untargeted metabolomic technologies to track the fates of isotopically labeled metabolites in a global, unbiased manner. This untargeted approach can be applied to discover novel biochemical pathways and characterize changes in the fates of labeled metabolites as a function of altered biological conditions such as disease. To facilitate the data analysis, we introduce X(13)CMS, an extension of the widely used mass spectrometry-based metabolomic software package XCMS. X(13)CMS uses the XCMS platform to detect metabolite peaks and perform retention-time alignment in liquid chromatography/mass spectrometry (LC/MS) data. With the use of the XCMS output, the program then identifies isotopologue groups that correspond to isotopically labeled compounds. The retrieval of these groups is done without any a priori knowledge besides the following input parameters: (i) the mass difference between the unlabeled and labeled isotopes, (ii) the mass accuracy of the instrument used in the analysis, and (iii) the estimated retention-time reproducibility of the chromatographic method. Despite its name, X(13)CMS can be used to track any isotopic label. Additionally, it detects differential labeling patterns in biological samples collected from parallel control and experimental conditions. We validated the ability of X(13)CMS to accurately retrieve labeled metabolites from complex biological matrices both with targeted LC/MS/MS analysis of a subset of the hits identified by the program and with labeled standards spiked into cell extracts. We demonstrate the full functionality of X(13)CMS with an analysis of cultured rat astrocytes treated with uniformly labeled (U-)(13)C-glucose during lipopolysaccharide (LPS) challenge. Our results show that out of 223 isotopologue groups enriched from U-(13)C-glucose, 95 have statistically significant differential labeling patterns in astrocytes challenged with LPS compared to unchallenged control cells. Only two of these groups overlap with the 32 differentially regulated peaks identified by XCMS, indicating that X(13)CMS uncovers different and complementary information from untargeted metabolomic studies. Like XCMS, X(13)CMS is implemented in R. It is available from our laboratory website at http://pattilab.wustl.edu/x13cms.php .

Gender-based heterogeneity of FAHFAs in trained runners.

Fatty acid esters of hydroxy fatty acid (FAHFA) are anti-diabetic and anti-inflammatory lipokines. Recently FAHFAs were also found to predict cardiorespiratory fitness in a cross-sectional study of recreationally trained runners. Here we report the influences of body composition and gender on static FAHFA abundances in circulation. We compared the association between circulating FAHFA concentrations and body composition, determined by dual x-ray absorptiometry, in female recreational runners who were lean (BMI < 25 kg/m2, n = 6), to those who were overweight (BMI ≥ 25 kg/m2, n = 7). To characterize the effect of gender we also compared circulating FAHFAs in lean male recreational runners (n = 8) to recreationally trained lean female (n = 6) runner group. Circulating FAHFAs were increased in females in a manner that was modulated by specific adipose depot sizes, blood glucose, and lean body mass. As expected, circulating FAHFAs were diminished in the overweight group, but strikingly, within the lean cohort, increases in circulating FAHFAs were promoted by increased fat mass, relative to lean mass, while the overweight group showed a significantly attenuated relationship. These studies suggest multimodal regulation of circulating FAHFAs and raise hypotheses to test endogenous FAHFA dynamic sources and sinks in health and disease, which will be essential for therapeutic target development. Baseline circulating FAHFA concentrations could signal sub-clinical metabolic dysfunction in metabolically healthy obesity.

Aerobic capacity and exercise mediate protection against hepatic steatosis via enhanced bile acid metabolism.

High cardiorespiratory fitness and exercise show evidence of altering bile acid (BA) metabolism and are known to protect or treat diet-induced hepatic steatosis, respectively. Here, we tested the hypothesis that high intrinsic aerobic capacity and exercise both increase hepatic BA synthesis measured by the incorporation of 2 H 2 O. We also leveraged mice with inducible liver-specific deletion of Cyp7a1 (LCyp7a1KO), which encodes the rate-limiting enzyme for BA synthesis, to test if exercise-induced BA synthesis is critical for exercise to reduce hepatic steatosis. The synthesis of hepatic BA, cholesterol, and de novo lipogenesis was measured in rats bred for either high (HCR) vs. low (LCR) aerobic capacity consuming acute and chronic high-fat diets. HCR rats had increased synthesis of cholesterol and certain BA species in the liver compared to LCR rats. We also found that chronic exercise with voluntary wheel running (VWR) (4 weeks) increased newly synthesized BAs of specific species in male C57BL/6J mice compared to sedentary mice. Loss of Cyp7a1 resulted in fewer new BAs and increased liver triglycerides compared to controls after a 10-week high-fat diet. Additionally, exercise via VWR for 4 weeks effectively reduced hepatic triglycerides in the high-fat diet-fed control male and female mice as expected; however, exercise in LCyp7a1KO mice did not lower liver triglycerides in either sex. These results show that aerobic capacity and exercise increase hepatic BA metabolism, which may be critical for combatting hepatic steatosis. Highlights: Rats with intrinsic high aerobic capacity have more significant reductions in de novo lipogenesis and increased cholesterol and bile acid synthesis on a high-fat diet compared to rats with low aerobic capacity. Chronic exercise increases hepatic bile acid synthesis in mice. Loss of Cyp7a1 blunts the capacity for exercise to increase bile acid synthesis and treat hepatic steatosis in male and female mice fed a high-fat diet.

Ketogenesis supports hepatic polyunsaturated fatty acid homeostasis via fatty acid elongation.

Therapeutic interventions targeting hepatic lipid metabolism in metabolic dysfunction-associated steatotic liver disease (MASLD) and steatohepatitis (MASH) remain elusive. Using mass spectrometry-based stable isotope tracing and shotgun lipidomics, we established a novel link between ketogenesis and MASLD pathophysiology. Our findings show that mouse liver and primary hepatocytes consume ketone bodies to support fatty acid (FA) biosynthesis via both de novo lipogenesis (DNL) and FA elongation. Analysis of 13 C-labeled FAs in hepatocytes lacking mitochondrial D-ß-hydroxybutyrate dehydrogenase (BDH1) revealed a partial reliance on mitochondrial conversion of D-ßOHB to acetoacetate (AcAc) for cytoplasmic DNL contribution, whereas FA elongation from ketone bodies was fully dependent on cytosolic acetoacetyl-CoA synthetase (AACS). Ketone bodies were essential for polyunsaturated FA (PUFA) homeostasis in hepatocytes, as loss of AACS diminished both free and esterified PUFAs. Ketogenic insufficiency depleted liver PUFAs and increased triacylglycerols, mimicking human MASLD, suggesting that ketogenesis supports PUFA homeostasis, and may mitigate MASLD-MASH progression in humans.