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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.

Metformin is a widely prescribed medication whose mechanism of action is not completely defined and whose role in gestational diabetes management remains controversial. In addition to increasing risks of fetal growth abnormalities and preeclampsia, gestational diabetes is associated with abnormalities in placental development including impairments in trophoblast differentiation. Given that metformin impacts cellular differentiation events in other systems, we assessed metformin’s impact on trophoblast metabolism and differentiation. Using established cell culture models of trophoblast differentiation, oxygen consumption rates and relative metabolite abundance were determined following 200 μM (therapeutic range) and 2000 μM (supra-therapeutic range) metformin treatment using Seahorse and mass-spectrometry approaches. While no differences in oxygen consumption rates or relative metabolite abundance were detected between vehicle and 200 μM metformin treated cells, 2000 μM metformin impaired oxidative metabolism and increased abundance of lactate and TCA cycle intermediates, α-ketoglutarate, succinate, and malate. Examining differentiation, treatment with 2000 μM, but not 200 μM metformin, impaired HCG production and expression of multiple trophoblast differentiation markers. Overall, this work suggests that supra-therapeutic concentrations of metformin impairs trophoblast metabolism and differentiation whereas metformin concentrations in the therapeutic range do not strongly impact these processes.

Cytotrophoblasts fuse to form and renew syncytiotrophoblasts necessary to maintain placental health throughout gestation. During cytotrophoblast to syncytiotrophoblast differentiation, cells undergo regulated metabolic and transcriptional reprogramming. Mitochondria play a critical role in differentiation events in cellular systems, thus we hypothesized that mitochondrial metabolism played a central role in trophoblast differentiation. In this work, we employed static and stable isotope tracing untargeted metabolomics methods along with gene expression and histone acetylation studies in an established cell culture model of trophoblast differentiation. Trophoblast differentiation was associated with increased abundance of the TCA cycle intermediates citrate and α-ketoglutarate. Citrate was preferentially exported from mitochondria in the undifferentiated state but was retained to a larger extent within mitochondria upon differentiation. Correspondingly, differentiation was associated with decreased expression of the mitochondrial citrate transporter (CIC). CRISPR/Cas9 disruption of the mitochondrial citrate carrier showed that CIC is required for biochemical differentiation of trophoblasts. Loss of CIC resulted in broad alterations in gene expression and histone acetylation. These gene expression changes were partially rescued through acetate supplementation. Taken together, these results highlight a central role for mitochondrial citrate metabolism in orchestrating histone acetylation and gene expression during trophoblast differentiation.

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-β-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.

Ketogenic diets are emerging as protective interventions in preclinical and clinical models of somatosensory nervous system disorders. Additionally, dysregulation of succinyl-CoA 3-oxoacid CoA-transferase 1 (SCOT, encoded byOxct1), the fate-committing enzyme in mitochondrial ketolysis, has recently been described in Friedreich’s ataxia and amyotrophic lateral sclerosis. However, the contribution of ketone metabolism in the normal development and function of the somatosensory nervous system remains poorly characterized. We generated sensory neuron-specific, Advillin-Cre knockout of SCOT (Adv-KO-SCOT) mice and characterized the structure and function of their somatosensory system. We used histological techniques to assess sensory neuronal populations, myelination, and skin and spinal dorsal horn innervation. We also examined cutaneous and proprioceptive sensory behaviors with the von Frey test, radiant heat assay, rotarod, and grid-walk tests. Adv-KO-SCOT mice exhibited myelination deficits, altered morphology of putative Aδ soma from the dorsal root ganglion, reduced cutaneous innervation, and abnormal innervation of the spinal dorsal horn compared to wildtype mice. Synapsin 1-Cre-driven knockout ofOxct1confirmed deficits in epidermal innervation following a loss of ketone oxidation. Loss of peripheral axonal ketolysis was further associated with proprioceptive deficits, yet Adv-KO-SCOT mice did not exhibit drastically altered cutaneous mechanical and thermal thresholds. Knockout ofOxct1in peripheral sensory neurons resulted in histological abnormalities and severe proprioceptive deficits in mice. We conclude that ketone metabolism is essential for the development of the somatosensory nervous system. These findings also suggest that decreased ketone oxidation in the somatosensory nervous system may explain the neurological symptoms of Friedreich’s ataxia.

Environmental nutrient availability influences T cell metabolism, impacting T cell function and shaping immune outcomes. However, the metabolic pathways critical for optimal T cell responses remain poorly understood. Here, we identify ketone bodies (KBs) – including β-hydroxybutyrate (βOHB) and acetoacetate (AcAc) – as essential fuels supporting CD8+ T cell metabolism and effector function. Ketolysis is an intrinsic feature of highly functional CD8+ T effector (Teff) cells and βOHB directly increases CD8+ Teff cell IFN-γ production and cytolytic activity. Using metabolic tracers, we establish that CD8+ Teff cells preferentially use KBs over glucose to fuel the tricarboxylic acid (TCA) cycle in vitro and in vivo. KBs directly boost the respiratory capacity of CD8+ T cells and TCA cycle-dependent metabolic pathways that fuel T cell growth. Mechanistically, we find that βOHB is a major substrate for acetyl-CoA production in CD8+ T cells and regulates 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.One Sentence summaryKetone bodies promote CD8+ T cell metabolism and effector function through regulation of epigenetic programming

Exercise confers metabolic benefits to both normal weight and overweight populations. Responses of the metabolome to acute aerobic exercise may predict cardiovascular fitness and longer-term outcomes, including the development of diabetes and its complications. We applied an LC-MS based untargeted metabolomics platform to evaluate the effect of acute exercise on the serum metabolome of overweight trained (OWT, n = 11, HOMA-IR 1.6 ± 0.1) and normal weight trained (NWT, n = 14, HOMA-IR, 0.9 ± 0.2, p < 0.01) runners. Trained individuals were studied to limit the effects of a deconditioned acute stress response. While NWT and OWT metabolic profiles shared increased circulating acylcarnitines and free fatty acids (FFAs) with exercise, intermediates of adenine metabolism, inosine and hypoxanthine, were strongly correlated with body fat percentage (R = 0.67) and VO2max (R = 0.70) . Untargeted metabolomics-guided follow-up quantitative lipidomic analysis revealed that, as expected, baseline levels of fatty acid esters of hydroxy fatty acids (FAHFAs) were generally diminished in the OWT group. FAHFAs negatively correlated with visceral fat mass and HOMA-IR. Strikingly, a 4-fold decrease in FAHFAs was provoked by acute aerobic running in NWT, an effect that negatively correlated with circulating IL-6 (R ≤ -0.7) . These effects of exercise were not observed in the OWT group. Machine learning models based on a profile including FAHFAs, FFAs, and adenine intermediates predicted 53% of the variation in cardiovascular fitness. Taken together, these findings in relatively insulin resistant overweight human participants and healthy controls have implications for metabolic crosstalk between adipose tissue and skeletal muscle and support the notion that FAHFAs could modulate the inflammatory response, fuel utilization, and insulin resistance. Disclosure A.B.Nelson: None. L.S.Chow: Other Relationship; Dexcom, Inc. C.C.Hughey: None. C.Myers: None. X.Han: None. P.A.Crawford: Advisory Panel; Abbott Diabetes, Johnson & Johnson Global Services. P.Puchalska: None. Funding NIH (DK091538, AG069781, DK098203, TR000114, UL1TR002494)

Interest in intermittent fasting, ketogenic diets, and exogenous ketone therapies for prediabetes, diabetes, and nonalcoholic fatty liver disease (NAFLD) is increasing, with numerous studies showing connectivity between ketone body metabolism and NAFLD progression. Previous studies from our group showed that exogenously administered ketone bodies have divergent effects on liver health: acetoacetate (AcAc) ameliorated, while its reduced redox partner D-B-hydroxybutyrate (D-BOHB) exacerbated lobular fibrosis. Here, we study the role of hepatic redox dependent interconversion of AcAc and D-BOHB in the setting of obesity and insulin resistance. Mice deficient in hepatocyte D-BOHB dehydrogenase (BDH1) and their littermate controls were maintained on high fat western diet (WD) for 18 weeks. Post WD challenge, these mice showed similar body weight, glucose, insulin, and circulating lipids in comparison to littermate controls. While livers of hepatocyte-specific BDH1-deficient mice lost the ability to generate D-BOHB, these mice remained capable to interconvert ketone bodies in extrahepatic tissues, leading to substantial circulating D-BOHB. Hepatocyte-specific BDH1 deficient mice maintained on WD displayed diminished glucose production and hepatic oxidative fluxes. Surprisingly, BDH1 deficient mice maintained normal static energy charge and NAD+/NADH ratios. Although insulin signaling in the liver and muscle was comparable between genotypes, knockout mice exhibited improved insulin sensitivity after acute insulin challenge. In addition, livers of knockout mice showed diminished liver fibrosis and oxidative stress. Taken together, these data support the notion for salutary roles of local AcAc, but detrimental roles of local D-BOHB on liver health and suggest that hepatic BDH1-dependent ketone body interconversion is a significant contributor to liver physiology in the setting of obese insulin resistance. Disclosure D.B.Stagg: None. A.R.Seay: None. A.B.Nelson: None. D.A.D’avignon: None. C.C.Hughey: None. P.Puchalska: None. P.A.Crawford: Advisory Panel; Abbott Diabetes, Johnson & Johnson Global Services. Funding NIH DK091538, and NIH AG069781

In addition to fatty acids, glucose and lactate are important myocardial substrates under physiologic and stress conditions. They are metabolized to pyruvate, which enters mitochondria via the mitochondrial pyruvate carrier (MPC) for citric acid cycle metabolism. In the present study, we show that MPC-mediated mitochondrial pyruvate utilization is essential for the partitioning of glucose-derived cytosolic metabolic intermediates, which modulate myocardial stress adaptation. Mice with cardiomyocyte-restricted deletion of subunit 1 of MPC (cMPC1−/−) developed age-dependent pathologic cardiac hypertrophy, transitioning to a dilated cardiomyopathy and premature death. Hypertrophied hearts accumulated lactate, pyruvate and glycogen, and displayed increased protein O-linked N-acetylglucosamine, which was prevented by increasing availability of non-glucose substrates in vivo by a ketogenic diet (KD) or a high-fat diet, which reversed the structural, metabolic and functional remodelling of non-stressed cMPC1−/− hearts. Although concurrent short-term KDs did not rescue cMPC1−/− hearts from rapid decompensation and early mortality after pressure overload, 3 weeks of a KD before transverse aortic constriction was sufficient to rescue this phenotype. Together, our results highlight the centrality of pyruvate metabolism to myocardial metabolism and function. Metabolic substrate partitioning underlies myocardial homeostatic capacity and adaptation to stress. Zhang et al. show that decreasing mitochondrial pyruvate carrier expression redirects glycolytic intermediates and leads to heart failure, which is prevented or reversed with a high-fat or ketogenic diet.

Objective Throughout the last decade, interest has intensified in intermittent fasting, ketogenic diets, and exogenous ketone therapies as prospective health-promoting, therapeutic, and performance-enhancing agents. However, the regulatory roles of ketogenesis and ketone metabolism on liver homeostasis remain unclear. Therefore, we sought to develop a better understanding of the metabolic consequences of hepatic ketone body metabolism by focusing on the redox-dependent interconversion of acetoacetate (AcAc) and D-β-hydroxybutyrate (D-βOHB). Methods Using targeted and isotope tracing high-resolution liquid chromatography-mass spectrometry, dual stable isotope tracer nuclear magnetic resonance spectroscopy-based metabolic flux modeling, and complementary physiological approaches in novel cell type-specific knockout mice, we quantified the roles of hepatocyte D-β-hydroxybutyrate dehydrogenase (BDH1), a mitochondrial enzyme required for NAD+/NADH-dependent oxidation/reduction of ketone bodies. Results Exogenously administered AcAc is reduced to D-βOHB, which increases hepatic NAD+/NADH ratio and reflects hepatic BDH1 activity. Livers of hepatocyte-specific BDH1-deficient mice did not produce D-βOHB, but owing to extrahepatic BDH1, these mice nonetheless remained capable of AcAc/D-βOHB interconversion. Compared to littermate controls, hepatocyte-specific BDH1 deficient mice exhibited diminished liver tricarboxylic acid (TCA) cycle flux and impaired gluconeogenesis, but normal hepatic energy charge overall. Glycemic recovery after acute insulin challenge was impaired in knockout mice, but they were not more susceptible to starvation-induced hypoglycemia. Conclusions Ketone bodies influence liver homeostasis. While liver BDH1 is not required for whole body equilibration of AcAc and D-βOHB, loss of the ability to interconvert these ketone bodies in hepatocytes results in impaired TCA cycle flux and glucose production. Therefore, through oxidation/reduction of ketone bodies, BDH1 is a significant contributor to hepatic mitochondrial redox, liver physiology, and organism-wide ketone body homeostasis., Graphical abstract Image 1, Highlights • Exogenously administered acetoacetate is reduced to D-β-hydroxybutyrate, increasing hepatic NAD+/NADH ratio. • Liver BDH1 is not required for whole body equilibration of acetoacetate and D-β-hydroxybutyrate. • Hepatocyte-specific loss of BDH1 reduces hepatic TCA cycle flux, and TCA-cycle sourced gluconeogenesis. • Hepatocyte-specific loss of BDH1 impairs glycemic recovery without provoking starvation-induced hypoglycemia.

The prevalence of non-alcoholic fatty liver disease (NAFLD) is increasing globally. NAFLD includes non-alcoholic fatty liver (NAFL) and non-alcoholic steatohepatitis (NASH). NASH is the pathological form of the disease characterized by liver steatosis, inflammation, cell injury, and fibrosis. A fundamental contributor to NASH is the imbalance between lipid accretion and disposal. The accumulation of liver lipids precipitates lipotoxicity and the inflammatory contributions to disease progression. This review defines the role of dysregulated of lipid disposal in NAFLD pathophysiology. The characteristic changes in mitochondrial oxidative metabolism pathways and the factors promoting these changes across the spectrum of NAFLD severity are detailed. This includes pathway-specific and integrative perturbations in mitochondrial β-oxidation, citric acid cycle flux, oxidative phosphorylation, and ketogenesis. Moreover, well-recognized and emerging mechanisms through which dysregulated mitochondrial oxidative metabolism mediates inflammation, fibrosis, and disease progression are highlighted.

Summary We apply stable isotope tracing, mass-spectrometry-based untargeted metabolomics, to reveal the biochemical space labeled by 13C-substrates in bone-marrow-derived macrophages. At the pathway level, classically (lipopolysaccharide [LPS]-polarized, M1) and alternatively (interleukin [IL]-4-polarized, M2) polarized macrophages were 13C-labeled with surprising concordance. Total pools of uridine diphosphate N-acetylglucosamine (UDP-GlcNAc), an intermediate in the hexosamine biosynthetic pathway, were equally abundant in LPS- and IL-4-polarized macrophages. Informatic scrutiny of 13C-isotopologues revealed that LPS-polarized macrophages leverage the pentose phosphate pathway to generate UDP-GlcNAc, whereas IL-4-polarized macrophages rely on intact glucose and mitochondrial metabolism of glucose carbon. Labeling from [13C]glucose is competed by unlabeled fatty acids and acetoacetate, underscoring the broad roles for substrate metabolism beyond energy conversion. Finally, the LPS-polarized macrophage metabolite itaconate is imported into IL-4-polarized macrophages, in which it reprograms [13C]glucose metabolism. Thus, use of fully unsupervised isotope tracing metabolomics in macrophages reveals polarization-state-specific metabolic pathway connectivity, substrate competition, and metabolite allocation among cellular compartments., Graphical Abstract, Highlights • Classically and alternatively polarized macrophages leverage hexosamine biosynthesis • Multiple carbon sources support hexosamine and itaconate biosynthesis • Intracellular metabolic compartmentation varies with macrophage polarization • Itaconate is imported into alternatively polarized macrophages, Components of the Immune System; Immunology; Metabolomics

The pathogenesis of human T2D is linked to the progression of nonalcoholic fatty liver disease and steatohepatitis (NAFLD/NASH). In the normal fed state, impairments of hepatocyte ketogenesis and Kupffer cell mitochondrial metabolism of ketone bodies conspire to drive hepatic injury, and lobular fibrosis - key features increasing insulin resistance and T2D. Preliminary data suggest that a local circuit of intrahepatic ketone metabolism may protect the liver from lobular injury, which involves distribution of ketone body carbon into numerous metabolic pathways. Nonetheless, the metabolic products of local hepatic ketone body metabolism that confer protection to the liver, and which are therefore linked to insulin resistance and T2D, remain unknown. Here, we use stable isotope tracing untargeted metabolomics (ITUM) to reveal the chemical space penetrated in vivo by D-[13C]β-hydroxybutyrate (D-βOHB) and its oxidized redox partner [13C]acetoacetate (AcAc). A kinetic study revealed rapid clearance (∼30 min) of both ketones from the bloodstream when introduced as intraperitoneal boluses into wild type mice. Many 13C-labeled metabolites were tissue-specific, particularly distinctly acetylated amino acids. Labeling of TCA cycle intermediates by 13C-labeled ketone bodies also varied by tissue, and by the specific labeled ketone body delivered. Finally, recurrent administration of unlabeled AcAc protected mice from high fat, fibrogenic diet-induced hepatic injury and fibrosis, while unlabeled D-βOHB exacerbated injury and fibrosis. In conclusion, the two ketone bodies AcAc and D-βOHB, strikingly exhibit distinct metabolic and phenotypic effects in the context of NAFLD/NASH, and insulin resistance. Disclosure P. Puchalska: None. J.E. Lengfeld: None. D.B. Stagg: None. P.A. Crawford: Consultant; Spouse/Partner; Medtronic.

The progression of nonalcoholic fatty liver disease (NAFLD) increases the risks of cirrhosis and cardiovascular disease. Marked alteration of both cytosolic and mitochondrial metabolism, and in combination with insulin resistance, increases hepatic glucose production. Utilization of stable isotope tracers to study liver metabolism offers deep insight into rearrangements of metabolic pathways and substrate-product relationships under the conditions leading to fatty liver and induced by diseases, drugs, toxins, or genetic manipulations. Isotope tracing untargeted metabolomics (ITUM) recently emerged as a powerful platform in which the label can be tracked in an untargeted fashion, revealing the penetration of substrates into metabolic pathways, even at low abundance. Here, we describe a protocol that can be utilized to study the changes in utilization of any labeled substrate toward a wide range of metabolites either in isolated liver cells or whole liver tissue under conditions mimicking various stages of fatty liver disease. Furthermore, a routine protocol for extraction, separation, and mass spectrometric detection of isotopically labeled metabolites in an untargeted or targeted fashion. An informatic approach to analyze stable isotope untargeted metabolomic datasets is also described.

The progression of nonalcoholic fatty liver disease (NAFLD) increases the risks of cirrhosis and cardiovascular disease. Marked alteration of both cytosolic and mitochondrial metabolism, and in combination with insulin resistance, increases hepatic glucose production. Utilization of stable isotope tracers to study liver metabolism offers deep insight into rearrangements of metabolic pathways and substrate-product relationships under the conditions leading to fatty liver and induced by diseases, drugs, toxins, or genetic manipulations. Isotope tracing untargeted metabolomics (ITUM) recently emerged as a powerful platform in which the label can be tracked in an untargeted fashion, revealing the penetration of substrates into metabolic pathways, even at low abundance. Here, we describe a protocol that can be utilized to study the changes in utilization of any labeled substrate toward a wide range of metabolites either in isolated liver cells or whole liver tissue under conditions mimicking various stages of fatty liver disease. Furthermore, a routine protocol for extraction, separation, and mass spectrometric detection of isotopically labeled metabolites in an untargeted or targeted fashion. An informatic approach to analyze stable isotope untargeted metabolomic datasets is also described.

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 (AcAc) and D-β-hydroxybutyrate (D-βOHB) serve distinct metabolic and signaling roles in biological systems. A highly efficient, specific, and reliable approach to simultaneously quantify AcAc and D-βOHB in biological specimens is lacking, due to challenges of separating the structural isomers and enantiomers of βOHB, and to the chemical instability of AcAc. Here we present a single UPLC-MS/MS method that simultaneously quantifies both AcAc and βOHB using independent stable isotope internal standards for both ketones. This method incorporates one sample preparation step requiring only 7 min of analysis per sample. The output is linear over three orders of magnitude, shows very low limits of detection and quantification, is highly specific, and shows favorable recovery yields from mammalian serum and tissue samples. Tandem MS discriminates D-βOHB from structural isomers 2- or 4-hydroxybutyrate as well as 3-hydroxyisobutyrate (3-HIB). Finally, a simple derivatization distinguishes D- and L-enantiomers of βOHB, 3-HIB, and 2-OHB, using the same rapid chromatographic platform. Together, this simple, efficient, reproducible, scalable, and all-encompassing method will support basic and clinical research laboratories interrogating ketone metabolism and redox biochemistry.

Peptides with antioxidant and ACE-inhibitory capacities have been obtained from a plum processing by-product. The aim of this work was to fractionate most active plum hydrolysates, to identify peptides in these fractions, and to evaluate their resistance to gastrointestinal enzymes. Highest antioxidant capacity was observed in the whole Alcalase hydrolysate while peptides yielding the highest ability to inhibit ACE were concentrated in the fraction, Novozymes Spain S.A.

Summary 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]β-hydroxybutyrate (D-βOHB) 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-βOHB, ameliorated diet-induced hepatic fibrosis. These data support a hepatocyte-macrophage ketone shuttle that segregates AcAc from D-βOHB, coordinating the fibrogenic response to hepatic injury via mitochondrial metabolism in tissue macrophages.

While several molecular targets are under consideration, mechanistic underpinnings of the transition from uncomplicated nonalcoholic fatty liver disease (NAFLD) to nonalcoholic steatohepatitis (NASH) remain unresolved. Here we apply multiscale chemical profiling technologies to mouse models of deranged hepatic ketogenesis to uncover potential NAFLD driver signatures. Use of stable-isotope tracers, quantitatively tracked by nuclear magnetic resonance (NMR) spectroscopy, supported previous observations that livers of wild-type mice maintained long term on a high-fat diet (HFD) exhibit a marked increase in hepatic energy charge. Fed-state ketogenesis rates increased nearly 3-fold in livers of HFD-fed mice, a greater proportionate increase than that observed for tricarboxylic acid (TCA) cycle flux, but both of these contributors to overall hepatic energy homeostasis fueled markedly increased hepatic glucose production (HGP). Thus, to selectively determine the role of the ketogenic conduit on HGP and oxidative hepatic fluxes, we studied a ketogenesis-insufficient mouse model generated by knockdown of the mitochondrial isoform of 3-hydroxymethylglutaryl-CoA synthase (HMGCS2). In response to ketogenic insufficiency, TCA cycle flux in the fed state doubled and HGP increased more than 60%, sourced by a 3-fold increase in glycogenolysis. Finally, high-resolution untargeted metabolomics and shotgun lipidomics performed using ketogenesis-insufficient livers in the fed state revealed accumulation of bis(monoacylglycero)phosphates, which also accumulated in livers of other models commonly used to study NAFLD. In summary, natural and interventional variations in ketogenesis in the fed state strongly influence hepatic energy homeostasis, glucose metabolism, and the lipidome. Importantly, HGP remains tightly linked to overall hepatic energy charge, which includes both terminal fat oxidation through the TCA cycle and partial oxidation via ketogenesis.

Background Saliva and muscle derived mosquito allergens have been purified and characterized. However, the complete set of allergens remains to be elucidated. In this study, we identified and characterized IgE-binding proteins from the mosquito species A. aegypti. Methods Serum was obtained from 15 allergic individuals with asthma and/or rhinitis and sensitized to mosquito. IgE-binding was determined by ELISA. Total proteins from freeze dried bodies of A. aegypti were extracted and IgE reactive proteins were identified by 2D-gel electrophoresis, followed by Western blot with pooled or individual sera. IgE-reactive spots were further characterized by mass spectrometry. Results Twenty five IgE-reactive spots were identified, corresponding to 10 different proteins, some of which appeared as different variants or isoforms. Heat shock cognate 70 (HSC-70) and Tropomyosin showed IgE reactivity with 60% of the sera, Lysosomal aspartic protease and “AAEL006070-PA” (Uniprot: Q177P3) with 40% and the other proteins with less than 33.3% of the sera. Different variants, or isoforms of Tropomyosin, Arginine or Creatine kinase, Glyceraldehyde-3-phosphate dehydrogenase (GPDH), Calcium-binding protein and Phosphoglycerate mutase were also identified. The mixture of three allergens (Aed a 6, Aed a 8 and Aed a 10) seems to identify more than 80% of A. aegypti sensitized individuals, indicating that these allergens should be considered when designing of improved mosquito allergy diagnostic tools. Conclusions The newly identified allergens may play a role in the pathophysiology of mosquito allergy in the Tropics, and some of them might be important arthropod-related proteins involved in cross-reactivity between A. aegypti and other allergenic arthropods. This article is protected by copyright. All rights reserved.

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, recent 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.

This work evaluates, the presence of native antihypertensive peptides in five soybean-based infant formulas (SBIFs). SBIFs peptide extracts (

Soybean peptide VLIVP presents a very high antihypertensive activity (IC50 value 1.69 μM), even higher than extensively studied IPP and VPP peptides from milk. Nevertheless, no much attention has been paid to this peptide and there is no method enabling its determination in soybeans. The aim of this work was the development of an analytical methodology for this purpose. A methodology consisting of the extraction of soybean proteins, their digestion with Protease P enzyme, their chromatographic separation using capillary-HPLC, and IT-MS detection was optimized. Protein extraction was performed by the use of high intensity focused ultrasounds to obtain a reduced extraction time. Optimization of chromatographic and mass spectrometry parameters enabled the separation of VLIVP peptide within just 7 min and its sensitive detection. The analytical characteristics of the capillary-HPLC–IT-MS method were evaluated through the study of linearity, LOD, LOQ, study of the presence of matrix interferences, precision, and recovery. The method enabled to detect as low as 3.6 ng of peptide and to determine as low as 12 ng of peptide in 1 g of soybean (as dry basis). Finally, the developed method was applied to the determination of the antihypertensive peptide VLIVP in different soybean varieties. The results showed the highest yield of VLIVP peptide in variety Mazowiecka II from Poland.

A capillary electrophoresis method with UV detection was developed for the first time for the determination of the pyridine betaine trigonelline (N-methylnicotinic acid) in seeds and vegetable oils. Analytical characteristics of the method showed its good performance in terms of linearity (r > 0.999), precision (relative standard deviations < 5%), and limits of detection (up to 0.9 μM or 1 ng/g for oils). The developed method was applied to the analysis of soy and sunflower seeds, three varieties of olives, and sunflower, soy, and extra virgin olive oils. Trigonelline was determined in soy and sunflower seeds and their respective oils, whereas it was not detected in olives or olive oils. Different mixtures of extra virgin olive oil with seed oils were analyzed, detecting up to 10% of soy oil in olive oil. As a consequence, trigonelline is proposed in this work as a novel marker for the detection of adulterations of olive oils with other vegetable oils such as soy and sunflower oils.

A new chiral stationary phase based on continuous bed (CB) technology using L-prolinamide as a chiral selector was prepared. Its ability for enantioseparation of amino acids and alpha-hydroxy acids by ligand-exchange CEC was compared with that of a CB containing L-4-hydroxyproline as chiral selector. Preparation was done by a one-step in situ copolyermerization procedure using methacrylamide as monomer, vinylsulfonic acid as charge providing agent and piperazine diacrylamide as crosslinker. L-Prolinamide showed marked enantioselective properties for the separation of amino acids and alpha-hydroxy acids. Furthermore, we show the possibility of simultaneous enantioseparation of amino acids and alpha-hydroxy acids using the CB with L-4-hydroxyproline as chiral selector.

Hypertension is one of the main causes of cardiovascular diseases. Synthetic drugs inhibiting ACE activity present high effectiveness in the treatment of hypertension but cause undesirable side effects. Unlike these synthetic drugs, antihypertensive peptides do not show any adverse effect. These peptides are naturally present in some foods and since hypertension is closely related to modern diet habits, the interest for this kind of foods is increasing. Different methods for the purification, isolation, and characterization of antihypertensive peptides in foods have been developed. Nevertheless, there is no revision work summarizing and comparing these strategies. In this review, in vivo and in vitro pathways to obtain antihypertensive peptides have been summarized. The ACE mechanism and the methodologies developed to assay the ACE inhibitory activity have also been described. Moreover, a comprehensive overview on the isolation, purification, and identification techniques focusing on the discovery of new antihypertensive peptides with high activity has been included. Finally, it is worthy to highlight that the quantitation of antihypertensive peptides in foods is a new trend since genotype and processing conditions could affect their presence. Analytical methodologies using mass spectrometry constitute an interesting option for this purpose.

Soybean-based infant formulas (SBIFs) based on soybean protein isolate (90% of proteins) are an interesting alternative to cow's milk infant formulas. Different works have demonstrated the presence of bioactive peptides in different soybean-based foodstuffs. The aim of this work was the evaluation, for the first time, of antioxidant peptides in five different commercially available SBIFs. Ultrafiltration through 10 kDa molecular weight cut-off filters was the most suitable extraction method. Despite peptide concentrations ranging between 1.19 and 2.27 mg/mL, similar antioxidant capacities were detected in all SBIF extracts. Extracts were further fractionated according to their molecular weight by ultrafiltration, and fractions from 5 to 10 kDa, 3 to 5 kDa, and below 3 kDa were obtained. The most active fraction was further fractionated by off-gel isoelectrofocusing and reversed-phase chromatography. Antioxidant fractions were also submitted to simulated gastrointestinal digestion (GI) with pepsin and pancreatin to evaluate their antioxidant capacity after digestion. Peptides were identified by HPLC-ESI-Q-ToF-MS/MS. At least 120 peptides were identified in every antioxidant fraction, with 42 peptides common to all SBIFs.

The simultaneous quantification of three highly antihypertensive peptides (LRP, LSP, and LQP) in six maize crops using novel HPLC-ESI-Q-ToF methodology is presented. The method included the extraction of α-zein proteins from maize, their purification by acetone precipitation, digestion with thermolysin and HPLC separation in a fused-core column. Several MS parameters were optimized to increase sensitivity and reduce spontaneous fragmentation of peptide ions into the ESI source. The ions with m/z 193.1315, 385.2558 (for LRP), 316.1867 (for LSP), and 357.2132 (for LQP) were monitored in the optimization and characterization of the method. In order to improve the repeatability, sensitivity, and the stability of peptides in the sample, the removal of urea was required. The use of two solid-phase extraction methods to remove urea from digested extract was evaluated. For the first time filter-aided sample preparation approach for the study of bioactive peptides in foodstuffs has been proposed. The optimized HPLC-ESI-Q-ToF method was characterized by the evaluation of linearity, LOD, LOQ, precision, and recovery. A study on the existence of matrix interferences was also performed. The developed method was applied to the quantification of LRP, LQP, and LSP peptides in maize lines using the standard addition method. The results showed the highest yield of LSP peptide in EZ11A line and LRP and LQP peptides in A632 line.

Despite less explored than foods from animal origin, plant derived foods also contain biologically active proteins and peptides. Bioactive peptides can be present as an independent entity in the food or, more frequently, can be in a latent state as part of the sequence of a protein. Release from that protein requires protein hydrolysis by enzymatic digestion, fermentation or autolysis. Different methodologies have been used to test proteins and peptides bioactivities. Fractionation, separation, and identification techniques have also been employed for the isolation and identification of bioactive proteins or peptides. In this work, proteins and peptides from plant derived foods exerting antihypertensive, antioxidant, hypocholesterolemic, antithrombotic, and immunostimulating capacities or ability to reduce food intake have been reviewed.

The aim of this work was to estimate the content of three highly antihypertensive peptides (LQP, LSP, and LRP) in different maize crops. For that purpose, a method consisting of the extraction of the protein containing these peptides (α-zeins), releasing of peptides by thermolysin digestion, and separation and detection of peptides was designed. The rapid and efficient ultrasound assisted extraction of α-zeins proteins from whole maize kernels was achieved using 70% of ethanol followed by precipitation with acetone. A 10 mM Tris–HCl (pH 8.0) buffer containing 8 M urea enabled to dissolve the precipitated α-zeins. This buffer was diluted to reach a 6 M urea concentration before digestion to keep active the enzyme. Other digestion parameters that were optimized were: enzyme to substrate ratio (5:100 was selected), digestion temperature (50 °C) and digestion time (6 h). The RP-HPLC separation in a fused-core column was also optimized allowing the separation of the three peptides extracted from maize kernels in 6 min. The presence of the three antihypertensive peptides in the digested extract was confirmed using HPLC–Q-TOF-MS analysis and by comparison with peptide standards. Clear differences were observed in the content of the three antihypertensive peptides and, thus, in the antihypertensive activity of the analyzed crops. The content of LRP peptide was very low regardless of the maize variety while the content of LQP and LSP significantly varied among studied maize lines.