Ketone Body Oxidation Research Articles

The microenvironment of secondary lymphedema. The key to finding effective treatments?

Lymphedema is characterized by the swelling of extremities due to the accumulation of interstitial fluids. It is a painful and devastating disease that increases the risk of infections and destroys patients' quality of life. Secondary lymphedema is caused by damage to the lymphatic system due to infections, obesity, surgery, and cancer treatments. This damage fails to be repaired and leads to fluid accumulation, tissue remodeling, inflammation, and ultimately fibrosis. The lymphedema microenvironment is altered by stress, immune dysfunction, and changes in metabolism. Stress in the microenvironment includes increased hypoxia and oxidative stress but how this contributes to lymphedema progression is unclear. The immune system plays a critical role in lymphedema through T cell helper type 2 (Th2) immune responses and the infiltration of macrophages into lymphedematous tissue. The inflammatory cytokines released by immune cells lead to tissue remodeling and fibrosis. There are also changes in metabolism in the lymphedema microenvironment with altered lipid oxidation, ketone body oxidation, and glycolysis. How these changes affect lymphedema and treatment interventions has been the focus of clinical trials. Lymphedema is also associated with cancer and obesity through damage to the lymphatic system. This review will illustrate microenvironmental changes in lymphedema and how this relates to cancer and obesity. In addition, we will discuss new therapeutic strategies to treat lymphedema. Finally, we will address the prospects of lymphedema research in the context of the microenvironment.

A murine model of acute and prolonged abdominal sepsis, supported by intensive care, reveals time-dependent metabolic alterations in the heart

BackgroundSepsis-induced cardiomyopathy (SICM) often occurs in the acute phase of sepsis and is associated with increased mortality due to cardiac dysfunction. The pathogenesis remains poorly understood, and no specific treatments are available. Although SICM is considered reversible, emerging evidence suggests potential long-term sequelae. We hypothesized that metabolic and inflammatory cardiac changes, previously observed in acute sepsis as potential drivers of SICM, partially persist in prolonged sepsis.MethodsIn 24-week-old C57BL/6J mice, sepsis was induced by cecal ligation and puncture, followed by intravenous fluid resuscitation, subcutaneous analgesics and antibiotics, and, in the prolonged phase, by parenteral nutrition. Mice were killed after 5 days of sepsis (prolonged sepsis, n = 15). For comparison, we included acutely septic mice killed at 30 h (acute sepsis, n = 15) and healthy controls animals (HC, n = 15). Cardiac tissue was collected for assessment of inflammatory and metabolic markers through gene expression, metabolomic analysis and histological assessment.ResultsIn prolonged sepsis, cardiac expression of IL-1β and IL-6 and macrophage infiltration remained upregulated (p ≤ 0.05). In contrast, tissue levels of Krebs cycle intermediates and adenosine phosphates were normal, whereas NADPH levels were low in prolonged sepsis (p ≤ 0.05). Gene expression of fatty acid transporters and of the glucose transporter Slc2a1 was upregulated in prolonged sepsis (p ≤ 0.01). Lipid staining and glycogen content were elevated in prolonged sepsis together with increased gene expression of enzymes responsible for lipogenesis and glycogen synthesis (p ≤ 0.05). Intermediate glycolytic metabolites (hexose-phosphates, GADP, DHAP) were elevated (p ≤ 0.05), but gene expression of several enzymes for glycolysis and mitochondrial oxidation of pyruvate, fatty-acyl-CoA and ketone bodies to acetyl-CoA were suppressed in prolonged sepsis (p ≤ 0.05). Key metabolic transcription factors PPARα and PGC-1α were downregulated in acute, but upregulated in prolonged, sepsis (p ≤ 0.05 for both). Ketone body concentrations were normal but ketolytic enzymes remained suppressed (p ≤ 0.05). Amino acid metabolism showed mild, mixed changes.ConclusionsOur results suggest myocardial lipid and glycogen accumulation and suppressed mitochondrial oxidation, with a functionally intact Krebs cycle, in the prolonged phase of sepsis, together with ongoing myocardial inflammation. Whether these alterations have functional consequences and predispose to long-term sequelae of SICM needs further research.

Dietary Restriction Enhances CD8+ T Cell Ketolysis to Limit Exhaustion and Boost Anti-Tumor Immunity.

Reducing calorie intake without malnutrition limits tumor progression but the underlying mechanisms are poorly understood. Here we show that dietary restriction (DR) suppresses tumor growth by enhancing CD8+ T cell-mediated anti-tumor immunity. DR reshapes CD8+ T cell differentiation within the tumor microenvironment (TME), promoting the development of effector T cell subsets while limiting the accumulation of exhausted T (Tex) cells, and synergizes with anti-PD1 immunotherapy to restrict tumor growth. Mechanistically, DR enhances CD8+ T cell metabolic fitness through increased ketone body oxidation (ketolysis), which boosts mitochondrial membrane potential and fuels tricarboxylic acid (TCA) cycle-dependent pathways essential for T cell function. T cells deficient for ketolysis exhibit reduced mitochondrial function, increased exhaustion, and fail to control tumor growth under DR conditions. Our findings reveal a critical role for the immune system in mediating the anti-tumor effects of DR, highlighting nutritional modulation of CD8+ T cell fate in the TME as a critical determinant of anti-tumor immunity.

The impact of exogenous ketone body suppletion on energy and pH balance in skeletal muscle during exercise in chronic heart failure patients: a prospective randomized double-blind cross-over study

Abstract Background Heart Failure (HF) is characterized by impediments in exercise capacity. Disruptions in the energy metabolism of skeletal muscle contribute to this condition. Pre-clinical and clinical evidence showed that reductions in carbohydrate and FA metabolism in HF may be partially overcome by a compensatory increase in ketone body oxidation and suggest that suppletion of ketone bodies (KB) may improve energy metabolism in HF. Purpose We tested the effect of exogenous KB suppletion on energy and pH balance during cycling exercise using 31P Magnetic Resonance (MR) Spectroscopy of the exercising quadriceps muscle in patients with HF. Methods In a randomized, double-blind, placebo-controlled cross-over study, 14 stable HF patients with reduced ejection fraction and diminished exercise capacity underwent maximal exercise testing in a MR scanner fitted with a cycling ergometer after KB or placebo treatment. Phosphocreatine (PCr) and inorganic phosphate (Pi) resonance amplitudes and frequencies were measured continuously in the quadriceps muscle during rest, exercise and recovery using in-vivo 31P MR spectroscopy (Figure 1). Results Patients had a median age of 66 [53 – 75] years, were 14% female, had a median LVEF of 37% [27 – 40] and circulating NT-pro BNP levels of 411 [182 – 946] pg/mL. Peak oxygen uptake (VO2) was 18.2 [15.2 – 20.3] ml/kg/min, which is 68% [62 – 78%] of predicted. After treatment, exercise performance in terms of cycling duration and workload were comparable between treatment arms (Figure 2). Quadriceps acidification during exercise was larger after KB suppletion compared to placebo (ΔpH: -0.2 [-0.3 - -0.1] vs -0.1 [-0.3 - -0.1] respectively; p=0.041) while quadriceps energy depletion during exercise was not altered by treatment (ΔPi/PCr 0.77 [0.55 – 2.51] vs 0.66 [0.46 – 0.81] respectively; p=0.087). Post-exercise mitochondrial oxidative function was similar between treatment arms (KB: PCr resynthesis rate 48 sec [25 – 59] versus placebo: 42 sec [36 – 63]; p=0.767). Conclusions Ketone body suppletion increased acidification and did not improve energy balance in skeletal muscle during exercise or mitochondrial function during recovery in patients with chronic HF. These results question the value of ketone bodies as treatment to boost muscular energy metabolism during exercise in HF.31P MRS Spectrum during exercise in HFExercise and energy parameters

Ketone body oxidation and susceptibility to ethyl acetoacetate in a novel hemolytic multidrug-resistant strain Leptospira interrogans KeTo originated from sewage water

Terrestrial and aquatic environments contaminated with animal urine may contribute to the transmission of Leptospira, a causative agent of leptospirosis in humans and wild/domesticated animals. Although enormous amounts of work have been done decoding the ecophysiology, the factors governing the cell growth and virulence in Leptospires derived from environmental samples still remain elusive. Here, we show oxidation of a wide array of organic acids including acetoacetate by a new strain of Leptospira interrogans designated as KeTo, isolated from a sewage sample originating from a wildlife enclosure located at Mangalore, India. We further demonstrate the susceptibility of KeTo to ethyl ester of acetoacetate (ethyl acetoacetate, EA). A 4.7 Mbp genome of KeTo shared the highest relatedness to pathogenic L. interrogans RGAT (99.3%), followed by L. kirschneri 3522CT (91.3%) and other related species of Leptospira (80.8‒74.3%), and harbored genes encoding acetoacetyl-CoA synthetase and acetoacetate decarboxylase respectively involved in the acetoacetate utilization and acetone formation. In line with this, strain KeTo oxidized acetoacetate when supplied as a sole carbon. Aqueous EA suppressed biofilm formation (p < 0.0001) of KeTo in basal Ellinghausen–McCullough–Johnson–Harris (EMJH) medium. Similarly, significant inhibition in the growth/biofilm of Leptospira was recorded in semisolid EMJH with/without blood supplementation when exposed to volatile EA. The extent of ketone body oxidation and susceptibility to EA was found to vary with strain as evident through the analysis of L. interrogans serogroup Australis sv. Australis strain Ballico and L. interrogans serogroup Icterohaemorrhagiae sv. Lai Like strain AF61. In conclusion, our study demonstrated the ketone body metabolic ability and susceptibility to an esterified derivative of a major ketone body in the tested strains of L. interrogans. Molecular aspects governing EA-driven growth inhibition warrant further investigations to develop optimal therapeutics for leptospirosis.

Potential therapeutic effect of a ketogenic diet for the treatment of lymphoedema: Results of an exploratory study.

Lymphoedema is a chronic and progressive disease characterised by excessive accumulation of lymph in the interstitial compartment, leading to tissue swelling and fibroadipose deposition. Lymphangiogenesis is partly regulated by ketone body oxidation, and a ketogenic diet (KD) has shown therapeutic efficacy in a preclinical mouse tail lymphoedema model. Therefore, we aimed to investigate the potential therapeutic effect of a KD in patients with secondary lymphoedema. Nine patients with unilateral stage 2 lymphoedema secondary to lymphadenectomy were included in this quasi-experimental exploratory study consisting of a short run-in phase to gradually induce ketosis, followed by a classic KD (CKD) and modified Atkins diet (MAD) phase during which patients consumed a CKD and MAD, respectively. Lymphatic function and oedema volume, the primary outcomes, were assessed at baseline and at the end of both the CKD and MAD phase. Secondary outcomes included health-related and lymphedema-specific quality of life (QoL). Seven out of nine patients completed the study protocol. Lymphatic function was improved upon consumption of both a CKD (dermal backflow score [mean ± SD]: 7.29 ± 2.98 vs. 10.86 ± 2.19 at baseline; p = 0.03) and MAD (6.71 ± 2.06; p = 0.02), whereas oedema volume did not decrease during the course of the study (excess limb volume [mean ± SD]: 20.13 ± 10.25% at end of CKD and 24.07 ± 17.77% at end of MAD vs. 20.79 ± 12.96% at baseline; p > 0.99 and p > 0.30, respectively). No changes were observed in health-related, nor lymphoedema-specific QoL at the end of CKD and MAD. The consumption of a KD improved lymphatic function and was associated with a clinically meaningful reduction in oedema volume in some patients (3/7 at end of CKD, 2/7 at end of MAD) with unilateral stage 2 secondary lymphoedema. These results highlight the potential of a KD to improve lymphatic function in patients with lymphoedema. However, further studies are required to substantiate our findings.

DETERMINATION OF PRODUCTIVITY OF COWS IN KETOSIS USING PROBIOTICS

High-yielding cows are prone to negative energy balance due to high lactation and insufficient dry matter intake after calving. This leads to the mobilization of fat and protein in the body to meet the nutrient needs of the lactating cow. High levels of nonesterified fatty acids are maintained by fat and protein mobilization, leading to ketosis due to incomplete oxidation of ketone bodies. The purpose of the research was to investigate the effect of probiotics on the productivity of cows and the quality of milk in ketosis. Research was conducted in the period from October to November 2021 on Holstein cows in the limited liability company "Lan" Agricultural Company of the North-Eastern region of Ukraine. It was found that in cows in the post-calving group, seven days after calving, the level of ketone bodies in the blood of the cows ranged from 1.0 to 2.1 mmol/l, which is higher than in the control by 71.42–185.71%. Two weeks after probiotic administration, ketone levels were between 0.5 and 1.2 mmol/L, 16.65–100% higher than controls. On the 28th day of the study, the content of ketone bodies in cows was within the physiological norm. In cows suffering from ketosis, productivity was reduced on the seventh day of research by 22.42–336.4%, on the 14th day by 11.6–29.35%. During the study of productivity, its restoration to the indicators of healthy animals on the 28th day after calving was established at the level of 28-29 kg/day. At the same time, the content of microorganisms and somatic cells had a direct correlation with productivity, and on the 28th day, the milk of all experimental cows was suitable for consumption and corresponded to the "extra" variety. In the first week after calving, the ratio of milk fat and protein in the milk of cows with ketosis was slightly imbalanced, which is a sign of ketosis. On the seventh day of the study, the milk fat level exceeded the protein content in cow 6905 by 32%, in cow 6852 by 39.25%, in cow 6916 by 30.34%, in cow 7642 by 41.17%, in cow 6187 – by 33.62%, in cow 2563 – by 36.40%, in cow 3891 – by 41.63%. At the end of the study on the 28th day, the fat/protein ratio approached the reference level of 1:1 in all experimental animals.

The role of lymphatic endothelial cell metabolism in lymphangiogenesis and disease.

Lymphatic endothelial cells (LECs) line lymphatic vessels, which play an important role in the transport of lymph fluid throughout the human body. An organized lymphatic network develops via a process termed "lymphangiogenesis." During development, LECs respond to growth factor signaling to initiate the formation of a primary lymphatic vascular network. These LECs display a unique metabolic profile, preferring to undergo glycolysis even in the presence of oxygen. In addition to their reliance on glycolysis, LECs utilize other metabolic pathways such as fatty acid β-oxidation, ketone body oxidation, mitochondrial respiration, and lipid droplet autophagy to support lymphangiogenesis. This review summarizes the current understanding of metabolic regulation of lymphangiogenesis. Moreover, it highlights how LEC metabolism is implicated in various pathological conditions.

Unraveling the Mechanism Behind the Ketogenic Diet-Mediated Reversal of Heart Failure in Mice

It has become clear that heart failure involves a host of metabolic alterations, and nutritional or pharmacologic modulation of cardiac metabolism can improve heart failure. We previously studied the role of the mitochondrial pyruvate carrier (MPC) in heart failure, and observed that pyruvate transport into the mitochondria of cardiac myocytes was critical for maintenance of normal cardiac size and function. However, we were also able to prevent or reverse heart failure in cardiac-specific MPC2−/− (cs-MPC2−/−) mice by feeding a low carbohydrate, high fat “ketogenic” diet. Intriguingly, while ketosis was associated with this reversal in heart failure, it was observed that cardiac ketone body oxidation enzymes were downregulated in these hearts, and direct administration of ketone bodies without altering dietary fat did not improve heart failure. The objective of this current study was to define whether ketone body oxidation was necessary for improving heart failure with a ketogenic diet. Wildtype mice were subjected to combined transverse aortic constriction and apical myocardial infarction (TAC-MI) to induce heart failure, were imaged by echocardiography two weeks later and randomized to either low fat control or ketogenic diet for an additional two weeks before repeat echocardiography and euthanasia. Cardiac size and function was also assessed in cs-MPC2−/− mice, mice with cardiac deletion of betahydroxybutyrate dehydrogenase 1 (cs-BDH1−/−, the first enzyme in ketone body oxidation), and cs-MPC2/BDH1−/− double KO mice. Mice were aged to 16 weeks, when MPC−/− hearts have developed dilated cardiomyopathy, and then fed either low fat control or ketogenic diet for 3 weeks before echocardiography and euthanasia. Of the WT mice subjected to TAC-MI, being fed a LF control diet led to further cardiac remodeling and worsened contractile function. However, ketogenic diet feeding completely prevented the progression of cardiac remodeling. cs-BDH1−/− hearts maintained normal size and function, suggesting that lack of ketone oxidation has no overt effect on cardiac function or remodeling. However, as previously reported, cs-MPC2−/− hearts developed dilated cardiomyopathy, which was not significantly altered by combined deletion of BDH1. Switching cs-MPC2−/− or cs-MPC2/BDH1−/− mice to a ketogenic diet was able to significantly reverse the heart failure, suggesting that enhanced ketone oxidation is not the mechanism for improved heart failure. Gene expression from these hearts suggests that ketogenic diet suppresses ketolytic gene expression and enhances expression of fat oxidation genes. Altogether, these findings suggest that improving heart failure with a ketogenic diet is due to stimulation of cardiac fat oxidation and not ketone body metabolism. Project was funded by NIH R00 HL136658 and SLU institutional funds. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Podophyllotoxin via SIRT1/PPAR /NF-κB axis induced cardiac injury in rats based on the toxicological evidence chain (TEC) concept

BackgroundThe study of cardiotoxicity of drugs has become an important part of clinical safety evaluation of drugs. It is commonly known that podophyllotoxin (PPT) and its many derivatives and congeners are broad-spectrum pharmacologically active substances. Clinical cardiotoxicity of PPT and its derivatives has been raised, basic research on the mechanism of cardiotoxicity remains insufficient. PurposeIn present study, our group's innovative concept of toxicological evidence chain (TEC) was applied to reveal the cardiac toxicity mechanism of PPT by targeted metabolomics, TMT-based quantitative proteomics and western blot. MethodsThe injury phenotype evidence (IPE) acquired from the toxicity manifestations, such as weight and behavior observation of Sprague-Dawley rat. The damage to rat hearts were assessed through histopathological examination and myocardial enzymes levels, which were defined as Adverse Outcomes Evidence (AOE). The damage to rat hearts was assessed through histopathological examination and myocardial enzyme levels, which were defined as evidence of adverse outcomes.Overall measurements of targeted metabolomics based on energy metabolism and TMT-based quantitative proteomics were obtained after exposure to PPT to acquire the Toxic Event Evidence (TEE). The mechanism of cardiac toxicity was speculated based on the integrated analysis of targeted metabolomics and TMT-based quantitative proteomics, which was verified by western blot. ResultsThe results indicated that exposure to PPT could result in significant elevation of myocardial enzymes and pathological alterations in rat hearts. In addition, we found that PPT caused disorders in cardiac energy metabolism, characterized by a decrease in energy metabolism fuels. TMT-based quantitative proteomics revealed that the PPAR (Peroxisome proliferators-activated receptor) signaling pathway needs further study. It is worth noting that PPT may suppress the expression of SIRT1, subsequently inhibiting AMPK, decreasing the expression of PGC-1α, PPARα and PPARγ. This results in disorders of glucose oxidation, glycolysis and ketone body metabolism. Additionally, the increase in the expression of p-IKK and p-IκBα, leads to the nuclear translocation of NF-κB p65 from the cytosol, thus triggering inflammation. ConclusionThis study comprehensively evaluated cardiac toxicity of PPT and initially revealed the mechanism of cardiotoxicity,suggesting that PPT induced disorders of energy metabolism and inflammation via SIRT1/PPAR/NF-κB axis, potentially contributing to cardiac injury.

OXCT1 regulates hippocampal neurogenesis and alleviates cognitive impairment via the Akt/GSK-3β/β-catenin pathway after subarachnoid hemorrhage

BackgroundSubarachnoid hemorrhage (SAH) is a life-threatening neurological disease that usually has a poor prognosis. Neurogenesis is a potential therapeutic target for brain injury. Ketone metabolism also plays neuroprotective roles in many neurological disorders. OXCT1 (3-Oxoacid CoA-Transferase 1) is the rate-limiting enzyme of ketone body oxidation. In this study, we explored whether increasing ketone oxidation by upregulating OXCT1 in neurons could promote neurogenesis after SAH, and evaluated the potential mechanism involved in this process. MethodsThe β-hydroxybutyrate content was measured using an enzymatic colorimetric assay. Adeno-associated virus targeting neurons was injected to overexpress OXCT1, and the expression and localization of proteins were evaluated by western blotting and immunofluorescence staining. Adult hippocampal neurogenesis was evaluated by dual staining with doublecortin and 5-Ethynyl-2′-Deoxyuridine. LY294002 was intracerebroventricularly administered to inhibit Akt activity. The Morris water maze and Y-maze tests were employed to assess cognitive function after SAH. ResultsThe results showed that OXCT1 expression and hippocampal neurogenesis significantly decreased in the early stage of SAH. Overexpression of OXCT1 successfully increased hippocampal neurogenesis via activation of Akt/GSK-3β/β-catenin signaling and improved cognitive function, both of which were reversed by administration of LY294002. ConclusionsOXCT1 regulated hippocampal ketone body metabolism and increased neurogenesis through mechanisms mediated by the Akt/GSK-3β/β-catenin pathway, improving cognitive impairment after SAH.

OXCT1 functions as a succinyltransferase, contributing to hepatocellular carcinoma via succinylating LACTB

Metabolic reprogramming is an important feature of cancers that has been closely linked to post-translational protein modification (PTM). Lysine succinylation is a recently identified PTM involved in regulating protein functions, whereas its regulatory mechanism and possible roles in tumor progression remain unclear. Here, we show that OXCT1, an enzyme catalyzing ketone body oxidation, functions as a lysine succinyltransferase to contribute to tumor progression. Mechanistically, we find that OXCT1 functions as a succinyltransferase, with residue G424 essential for this activity. We also identified serine beta-lactamase-like protein (LACTB) as a main target of OXCT1-mediated succinylation. Extensive succinylation of LACTB K284 inhibits its proteolytic activity, resulting in increased mitochondrial membrane potential and respiration, ultimately leading to hepatocellular carcinoma (HCC) progression. In summary, this study establishes lysine succinyltransferase function of OXCT1 and highlights a link between HCC prognosis and LACTB K284 succinylation, suggesting a potentially valuable biomarker and therapeutic target for further development.

Abstract 17387: The Ketone Body Beta-Hydroxybutyrate Attenuates Cardiomyocyte Hypertrophy Independent of BDH1-Mediated Oxidation

Previously, we and others identified evidence for increased myocardial ketone body oxidation in failing mouse and human hearts and demonstrated that increased levels of beta-hydroxybutyrate (ßOHB), the most abundant ketone body, reduced pathologic cardiac remodeling in preclinical models of heart failure. To better understand how ßOHB prevents pathologic cardiac hypertrophy, we sought to determine whether: 1) ßOHB has direct effects on cardiomyocyte (CM) hypertrophy; and 2) if ßOHB oxidation is required for this effect. CMs were isolated from hearts of adult male C57BL/6 cardiac-specific beta-hydroxybutyrate dehydrogenase 1 knockout (csBDH1KO) mice and wildtype (WT) littermate controls. csBDH1KO CMs are unable to oxidize ßOHB given that BDH1 catalyzes the first step of R-ßOHB oxidation. CMs were cultured in media containing 5.5mM glucose ± 3mM R-ßOHB in the presence or absence of the hypertrophic agonist phenylephrine (PE, 100μM). CM width and length were quantified to assess hypertrophic growth. Stimulation with PE increased CM width:length ratio compared to glucose control by 31% and 33% in WT and BDH1KO CMs, respectively. Addition of R-ßOHB in the absence of PE had no effect on CM morphology, whereas R-ßOHB limited PE-induced increases in width:length ratio to 2% and 4% in WT and BDH1KO CMs, respectively (p&lt;0.01 vs PE without R-ßOHB). In addition, PE-induced hypertrophy was prevented in WT CMs treated with S-ßOHB (this stereoisomer cannot be oxidized). To confirm that the cultured CMs utilized ßOHB, WT and BDH1KO CMs were cultured in media containing [U- 13 C] R-ßOHB. Stable isotope tracing showed significant fractional enrichment of citrate by R-ßOHB in WT CMs with limited enrichment in BDH1KO CMs (17.8% vs 4.5%, p=0.02). These findings support a role for ßOHB-mediated attenuation of cardiac hypertrophy through direct action on CMs via a mechanism independent of BDH1-mediated oxidation. Taken together with our previous results, these findings strongly suggest that ketone bodies improve heart failure by providing both oxidation-dependent (fuel source) and oxidation-independent as yet undefined cardioprotective mechanisms.

Beta-hydroxybutyrate regulation of metabolic stress responses to Staphylococcus aureusinfection in macrophages

Abstract Macrophages orchestrate the initiation, intensity, and duration of the inflammatory response induced during Staphylococcus aureusinfection. The pleiotropic role of macrophages is dictated by the capacity to adapt to different metabolic cues. During acute inflammation, macrophages are highly glycolytic; during resolution, these cells shift to oxidative metabolism. While glycolysis drives the expression of pro-inflammatory genes, oxidative phosphorylation leads to fatty acid oxidation and synthesis of ketone bodies, such as beta-hydroxybutyrate (BHB). Therefore, identifying the events that drive the shift from glycolytic to oxidative phosphorylation could provide clues for inflammation and homeostasis. If exogenous BHB influences macrophage responses to S. aureusinfection is not well understood. We hypothesize that BHB is a bridge between glycolysis and oxidative phosphorylation during S. aureusinfection. BHB increases mitochondrial respiration in S. aureus-infected immortalized bone-marrow-derived macrophages (iBMDM). During infection, treatment with BHB increased PDHA1(Pyruvate Dehydrogenase E1 Subunit Alpha 1) but decreased HK1 (Hexokinase 1) expression in iBMDMs. Furthermore, BHB decreased macrophage phagocytic capacity in S. aureusinfected peritoneal macrophages, indicating a decrease in glycolytic activity. Cumulatively, these findings suggest that BHB may cause a shift toward aerobic respiration and increase the anti-inflammatory capacity of macrophages. Therefore, exogenous BHB or ketogenic diets could be detrimental during S. aureusinfection by decreasing both antimicrobial effector functions and inflammatory responses. NIH R01 HL124159-09 The SyBBURE Searle Undergraduate Research Program

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 which 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 to 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. Further, 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.

Ketone Bodies and Cardiovascular Disease: An Alternate Fuel Source to the Rescue.

The increased metabolic activity of the heart as a pump involves a high demand of mitochondrial adenosine triphosphate (ATP) production for its mechanical and electrical activities accomplished mainly via oxidative phosphorylation, supplying up to 95% of the necessary ATP production, with the rest attained by substrate-level phosphorylation in glycolysis. In the normal human heart, fatty acids provide the principal fuel (40-70%) for ATP generation, followed mainly by glucose (20-30%), and to a lesser degree (<5%) by other substrates (lactate, ketones, pyruvate and amino acids). Although ketones contribute 4-15% under normal situations, the rate of glucose use is drastically diminished in the hypertrophied and failing heart which switches to ketone bodies as an alternate fuel which are oxidized in lieu of glucose, and if adequately abundant, they reduce myocardial fat delivery and usage. Increasing cardiac ketone body oxidation appears beneficial in the context of heart failure (HF) and other pathological cardiovascular (CV) conditions. Also, an enhanced expression of genes crucial for ketone break down facilitates fat or ketone usage which averts or slows down HF, potentially by avoiding the use of glucose-derived carbon needed for anabolic processes. These issues of ketone body utilization in HF and other CV diseases are herein reviewed and pictorially illustrated.

700 EPICARDIAL ADIPOSE TISSUE AND ATRIAL FIBRILLATION, A REVIEW

Abstract Aims Atrial fibrillation (AF) is the most common cardiac arrhythmia closely associated with stroke, heart failure, and decreased quality of life. Excess adiposity has long been associated with increased cardiovascular disease and risk factor of AF. Methods Epicardial Adipose Tissue (EAT) is the fat depot located between the myocardium and the epicardium supplied by branches of the coronary arteries. By contrast, pericardial adipose tissue (PAT) is located externally and is supplied by non-​coronary arteries. EAT fat thickness was first visualized by and measured with Transthoracic Two-dimensional (2D) Echocardiography because is visible as an echo free space between the outer wall of the myocardium and the visceral layer of the pericardium. Moreover, computed tomography (CT) has been used to measure EAT through accurate volumetric measurement. CMR imaging is instead considered the ‘gold standard’ modality for imaging adipose tissue.EAT function and morphology in normal conditions is protective but it function changes under pathological conditions.Lots of studies, infact, indicated higher pericardial fat volume was associated with a nearly 40% higher odds of prevalent AF, and the association remained significant even after adjustment for other AF risk factors including age, sex, myocardial infarction, heart failure, BMI, and other regional fat deposits. Infact, EAT volume is related to impaired diastolic function, elevated left ventricular mass, and enlarged left atrium. Moreover, a higher degree of EAT accumulation is often observed in patients with systematic inflammatory disease and oxidative stress, such as rheumatoid arthritis and psoriasis, compared with those without systematic inflammatory disease even after adjusting other risk factors. Free fatty acids can be transported from EAT to the myocardium and lead to electromechanical changes in atrial tissue: they separate cardiomyocytes and this situation result in conduction slowing, loss of side-​to-​side cell connections and myocardial disorganization that leads to conduction delay and re-​entry. Results EAT is a modifiable cardiovascular risk factor and a potential novel therapeutic target owing to its responsiveness to drugs with pleiotropic effects such as GLP1R agonists (visceral fat reduction is one of the non-​glycaemic effects of the GLP1R agonists), SGLT2 inhibitors (reduction of intramyocardial lipid content by increasing EAT lipolysis and ketone body oxidation) and statins (reduction of EAT thickness through modulation of peroxisome proliferator-​activated receptors (PPARs)). Furthemore, suppression of AF after injecting botulinum toxin into EAT in patients with paroxysmal AF, both during postoperative cardiac surgery and catheter ablation of epicardial fat pad can also result in EAT reduction. Conclusion EAT is a measurable and modifiable cardiovascular risk factor that adds qualitative value to the stratification of cardiovascular risk. In the context of atrial fibrillation, EAT represents a new pathogenic substrate through the regional secretion of factors that induce fibrosis and neurohormonal disarray of the atrial myocytes. Genetic studies are also optional to uncover pathophysiological mechanism of EAT-AF interaction and clarify causal relationship. Finally, more efforts should be made to realize application of EAT quantification in risk evaluation and management for AF.

The Effects of Exercise Training on Glucose Homeostasis and Muscle Metabolism in Type 1 Diabetic Female Mice.

Although exercise training is an important recommendation for the management of type 1 diabetes (T1D), most of the available research studies predominantly focus on male subjects. Given the importance of sex as a biological variable, additional studies are required to improve the knowledge gap regarding sex differences in T1D research. Therefore, the purpose of this study was to examine the role of exercise training in mediating changes in glucose homeostasis and skeletal muscle metabolism in T1D female mice. Female mice were injected with streptozotocin (STZ) to induce T1D. Two weeks after STZ injection, control (CON) and STZ mice were exercise trained on a treadmill for 4 weeks. Aerobic exercise training failed to improve glucose tolerance, prevent the decrease in body weight and adipose tissue mass, or attenuate muscle atrophy in T1D female mice. However, insulin sensitivity was improved in T1D female mice after exercise training. Aerobic exercise training maintained skeletal muscle triglyceride content but did not prevent depletion of skeletal muscle or liver glycogen in T1D mice. Gene expression analysis suggested that T1D resulted in decreased glucose transport, decreased ketone body oxidation, and increased fatty acid metabolism in the skeletal muscle, which was not altered by exercise training. These data demonstrate that 4 weeks of aerobic exercise training of a moderate intensity is insufficient to counteract the negative effects of T1D in female mice, but does lead to an improvement in insulin sensitivity.

Proteomic and phosphoproteomic profiling in heart failure with preserved ejection fraction (HFpEF)

Abstract Background The lack of therapies for HFpEF patients is a major unmet need, thus identifying cardiac specific pathways in HFpEF is a priority. Purpose Identify molecular features of protein and phosphoprotein in a murine model of HFpEF Methods Studies followed the principles of laboratory animal care (NIH Pub no. 85–23 revised 1985). HFpEF (N=4) was induced by NaCl drinking water, unilateral nephrectomy, and chronic aldosterone infusion (SAUNA) or saline (Sham; N=4) for 4 wks. Mice were euthanized and LV tissue was collected. Samples were homogenized, proteins extracted and subjected to digestion followed by phosphopeptide enrichment for proteomics and phosphoproteomics profiling. Results HFpEF mice had moderate hypertension (137.8±7.0 vs. Sham; 115.4±6.0mmHg; P&amp;lt;0.05), lung congestion (4.5±0.1 vs. 4.0±0.1; P&amp;lt;0.01), and LVH (3.7±0.1 vs. 3.3±0.1mg/g; P&amp;lt;0.05). ECHO showed normal LVEF with evidence of diastolic dysfunction in HFpEF mice. Mitral E velocity was reduced (1347±154 vs. 1971±284mm/s; P&amp;lt;0.05) and IVRT increased (24.3±2.6 vs. 14.4±1.6ms; P&amp;lt;0.05) vs. Sham. Protein and protein phosphosites from the LV were quantified by mass spectrometry. Analysis of the proteomics datasets revealed marked changes in sarcomere proteins, such as skeletal alpha (α)-actin (ACTA1; P=0.000039), beta (β)-myosin heavy chain (MYH7; P=0.006963) and myosin heavy chain 9 (MYH9; P=0.000408); the mitochondria-related proteins mitofusin 1 (MFN1; P=0.001059), mitochondrial dynamin like GTPase (aka optic atrophy protein 1, OPA1; P=0.046441) and transcription factor A mitochondrial (TFAM; P=0.005837); and the NAD-dependent protein deacetylase sirtuin-3 (SIRT3; P=0.000914). There was also a reduction in proteins involved in the oxidation of free fatty acid, pyruvate, and ketone bodies in the LV from HFpEF vs. Sham. Phosphoproteomics analysis also showed aberrant protein phosphorylation patterns linked to disparate subcellular compartments, ranging from sarcomere proteins (LIM domain-binding protein 3, LDB3; myozenin 2, Myoz2; titin, TTN), to nuclear-localized proteins (BAG family molecular chaperone regulator 3, BAG3; high mobility group protein HMG-I/HMG-Y, HMGA1) with known links to contractile dysfunction, LVH and/or cardiomyopathy. Additional GSEA analysis revealed the most relevant and enriched biological annotations in LV from HFpEF, to be processes involving immune system modulation and muscle contraction. Downregulated pathways were mainly related to multitude of GO terms associated with mitochondrial metabolism. Conclusion(s) This study presents the systematic, quantitative proteomics and phosphoproteomic analysis of the LV from the SAUNA HFpEF mice. We observed profound changes in proteins related to mitochondrial metabolism and function and heart contractile dysfunction in HFpEF which may be mediated by Sirtuin 3. Additional studies are warranted to investigate the specific role of Sirtuin 3 in mitochondrial metabolism and heart contractile dysfunction in HFpEF. Funding Acknowledgement Type of funding sources: Public grant(s) – EU funding. Main funding source(s): National Institute of Heath (NIH/ NHLBI)

Metabolic signatures of pregnancy-induced cardiac growth.

The goal of this study was to develop an atlas of the metabolic, transcriptional, and proteomic changes that occur with pregnancy in the maternal heart. Timed pregnancy studies in FVB/NJ mice revealed a significant increase in heart size by day 8 of pregnancy (midpregnancy; MP), which was sustained throughout the rest of the term compared with nonpregnant control mice. Cardiac hypertrophy and myocyte cross-sectional area were highest 7 days after birth (postbirth; PB) and were associated with significant increases in end-diastolic and end-systolic left ventricular volumes and higher cardiac output. Metabolomics analyses revealed that by day 16 of pregnancy (late pregnancy; LP) metabolites associated with nitric oxide production as well as acylcholines, sphingomyelins, and fatty acid species were elevated, which coincided with a lower activation state of phosphofructokinase and higher levels of pyruvate dehydrogenase kinase 4 (Pdk4) and β-hydroxybutyrate dehydrogenase 1 (Bdh1). In the postpartum period, urea cycle metabolites, polyamines, and phospholipid levels were markedly elevated in the maternal heart. Cardiac transcriptomics in LP revealed significant increases in not only Pdk4 and Bdh1 but also genes that regulate glutamate and ketone body oxidation, which were preceded in MP by higher expression of transcripts controlling cell proliferation and angiogenesis. Proteomics analysis of the maternal heart in LP and PB revealed significant reductions in several contractile filament and mitochondrial subunit complex proteins. Collectively, these findings describe the coordinated molecular changes that occur in the maternal heart during and after pregnancy.NEW & NOTEWORTHY Little is known of the underlying molecular and cellular mechanisms that contribute to pregnancy-induced cardiac growth. Several lines of evidence suggest that changes in cardiac metabolism may contribute. Here, we provide a comprehensive metabolic atlas of the metabolomic, proteomic, and transcriptomic changes occurring in the maternal heart. We show that pregnancy-induced cardiac growth is associated with changes in glycerophospholipid, nucleotide, and amino acid metabolism, with reductions in cardiac glucose catabolism. Collectively, these results suggest that substantial metabolic changes occur in the maternal heart during and after pregnancy.