Two enzymes contribute to citrate production in the mitochondrion of Toxoplasma gondii.

Citrate synthase catalyzes the first and the rate-limiting reaction of the tricarboxylic acid (TCA) cycle, producing citrate from the condensation of oxaloacetate and acetyl-coenzyme A. The parasitic protozoan Toxoplasma gondii has full TCA cycle activity, but its physiological roles remain poorly understood. In this study, we identified three proteins with predicted citrate synthase (CS) activities two of which were localized in the mitochondrion, including the 2-methylcitrate synthase (PrpC) that was thought to be involved in the 2-methylcitrate cycle, an alternative pathway for propionyl-CoA detoxification. Further analyses of the two mitochondrial enzymes showed that both had citrate synthase activity, but the catalytic efficiency of CS1 was much higher than that of PrpC. Consistently, the deletion of CS1 resulted in a significantly reduced flux of glucose-derived carbons into TCA cycle intermediates, leading to decreased parasite growth. In contrast, disruption of PrpC had little effect. On the other hand, simultaneous disruption of both CS1 and PrpC resulted in more severe metabolic changes and growth defects than a single deletion of either gene, suggesting that PrpC does contribute to citrate production under physiological conditions. Interestingly, deleting Δcs1 and Δprpc individually or in combination only mildly or negligibly affected the virulence of parasites in mice, suggesting that both enzymes are dispensable in vivo. The dispensability of CS1 and PrpC suggests that either the TCA cycle is not essential for the asexual reproduction of tachyzoites or there are other routes of citrate supply in the parasite mitochondrion.

Sedentary time associates detrimentally and physical activity beneficially with metabolic flexibility in adults with metabolic syndrome.

Metabolic flexibility (MetFlex) describes the ability to respond and adapt to changes in metabolic demand and substrate availability. The relationship between physical (in)activity and MetFlex is unclear. This study aimed to determine whether sedentary time, physical activity (PA), and cardiorespiratory fitness associate with MetFlex. Sedentary time, standing, and PA were measured with accelerometers for 4 weeks in 64 sedentary adults with metabolic syndrome [37 women, 27 men; 58.3 (SD 6.8) years]. Fitness (V̇o2max; mL·kg-1·min-1) was measured with graded maximal cycle ergometry. MetFlex was assessed with indirect calorimetry as the change in respiratory exchange ratio (ΔRER) from fasting to insulin stimulation with hyperinsulinemic-euglycemic clamp and from low-intensity to maximal exercise. Carbohydrate (CHOox) and fat oxidation (FATox) were calculated from respiratory gases. High sedentary time associated with higher fasting RER [ß = 0.35 (95% confidence interval: 0.04, 0.67)], impaired insulin-stimulated MetFlex (ΔRER) [ß=-0.41 (-0.72, -0.09)], and lower fasting FATox [ß=-0.36 (-0.67, -0.04)]. Standing associated with lower fasting RER [ß=-0.32 (-0.62, -0.02)]. Higher standing time and steps/day associated with higher fasting FATox [ß = 0.31 (0.01, 0.61), and ß = 0.26 (0.00, 0.53)]. Light-intensity and total PA associated with better insulin-stimulated MetFlex [ß = 0.33 (0.05, 0.61)], and ß = 0.33 (0.05, 0.60)]. Higher V̇o2max associated with higher CHOox during maximal exercise [ß = 0.81 (0.62, 1.00)], as well as during insulin stimulation [ß = 0.43 (0.13, 0.73)]. P values are less than 0.05 for all associations. Sedentary time and PA associate with MetFlex. Reducing sitting and increasing PA of even light intensity might aid in the prevention of metabolic diseases in risk populations through their potential effects on energy metabolism.NEW & NOTEWORTHY High accelerometer-assessed sedentary time associates with metabolic inflexibility measured during hyperinsulinemic-euglycemic clamp in adults with metabolic syndrome, and more light-intensity and total physical activity associate with more metabolic flexibility. Physical activity behaviors may thus play an important role in the regulation of fuel metabolism. This highlights the potential of reduced sedentary time and increased physical activity of any intensity to induce metabolic health benefits and help in disease prevention in risk populations.

Identifying metabotypes of insulin resistance severity in children with metabolic syndrome.

BACKGROUND: Insulin resistance is a frequent precursor of typical obesity and metabolic syndrome complications. However, accurate diagnosis remains elusive because of its pathophysiological complexity and heterogeneity. Herein, we have explored the utility of insulin secretion dynamics in response to an oral glucose tolerance test as a surrogate marker to identify distinct metabotypes of disease severity. METHODS: The study population consisted of children with obesity and insulin resistance, stratified according to the post-challenge insulin peak timing (i.e., early, middle, and late peak), from whom fasting and postprandial plasma and erythrocytes were collected for metabolomics analysis. RESULTS: Children with late insulin peak manifested worse cardiometabolic health (i.e., higher blood pressure, glycemia, and HOMA-IR scores) than early responders. These subjects also showed more pronounced changes in metabolites mirroring failures in energy homeostasis, oxidative stress, metabolism of cholesterol and phospholipids, and adherence to unhealthy dietary habits. Furthermore, delayed insulin peak was associated with impaired metabolic flexibility, as reflected in compromised capacity to regulate mitochondrial energy pathways and the antioxidant defense in response to glucose overload. CONCLUSIONS: Altogether, these findings suggest that insulin resistance could encompass several phenotypic subtypes characterized by graded disturbances in distinctive metabolic derangements occurring in childhood obesity, which serve as severity predictive markers.

Age-related flexibility of energetic metabolism in the honey bee Apis mellifera.

The mechanisms that underpin aging are still elusive. In this study, we suggest that the ability of mitochondria to oxidize different substrates, which is known as metabolic flexibility, is involved in this process. To verify our hypothesis, we used honey bees (Apis mellifera carnica) at different ages, to assess mitochondrial oxygen consumption and enzymatic activities of key enzymes of the energetic metabolism as well as ATP5A1 content (subunit of ATP synthase) and adenylic energy charge (AEC). We also measured mRNA abundance of genes involved in mitochondrial functions and the antioxidant system. Our results demonstrated that mitochondrial respiration increased with age and favored respiration through complexes I and II of the electron transport system (ETS) while glycerol-3-phosphate (G3P) oxidation was relatively decreased. In addition, glycolytic, tricarboxylic acid cycle and ETS enzymatic activities increased, which was associated with higher ATP5A1 content and AEC. Furthermore, we detected an early decrease in the mRNA abundance of subunits of NADH ubiquinone oxidoreductase subunit B2 (NDUFB2, complex I), mitochondrial cytochrome b (CYTB, complex III) of the ETS as well as superoxide dismutase 1 and a later decrease for vitellogenin, catalase and mitochondrial cytochrome c oxidase subunit 1 (COX1, complex IV). Thus, our study suggests that the energetic metabolism is optimized with aging in honey bees, mainly through quantitative and qualitative mitochondrial changes, rather than showing signs of senescence. Moreover, aging modulated metabolic flexibility, which might reflect an underpinning mechanism that explains lifespan disparities between the different castes of worker bees.

Assessing metabolic flexibility response to a multifibre diet: a randomised-controlled trial.

INTRODUCTION: Metabolic flexibility (MetF), defined as the ability to switch between fat and glucose oxidation, is increasingly recognised as a critical marker for assessing responses to dietary interventions. Previously, we showed that the consumption of multifibre bread improved insulin sensitivity and reduced low-density lipoprotein cholesterol (LDLc) levels in overweight and obese individuals. As a secondary objective, we aimed to explore whether our intervention could also improve MetF. METHODS: In this study, 39 subjects at cardiometabolic risk participated in a double-blind, randomised, crossover trial lasting 8 weeks, repeated twice. During each phase, participants consumed either 150 g of standard bread daily or bread enriched with a mixture of seven dietary fibres. MetF response was assessed using a mixed-meal tolerance test (MMTT), analysing changes in respiratory quotient (∆RQ) measured using indirect calorimetry. RESULTS: Although there were no significant differences in ∆RQ changes induced by dietary fibre between the two diets, these changes were positively correlated with postprandial triglyceride excursion (∆TG) at baseline. Subgroup analysis of baseline fasting and postprandial plasma metabolites was conducted to characterise MetF responders. These responders exhibited higher baseline fasting LDLc levels and greater post-MMTT ∆TG. CONCLUSION: In conclusion, although dietary fibres did not directly impact MetF in this study, our findings highlight potential determinants of MetF response, warranting further investigation in dedicated future interventions.

Intramammary lipopolysaccharide challenge in early- versus mid-lactation dairy cattle: Immune, production, and metabolic responses.

Study objectives were to compare the immune response, metabolism, and production following intramammary LPS (IMM LPS) administration in early and mid-lactation cows. Early (E-LPS; n = 11; 20 ± 4 DIM) and mid- (M-LPS; n = 10; 155 ± 40 DIM) lactation cows were enrolled in an experiment consisting of 2 periods (P). During P1 (5 d) cows were fed ad libitum and baseline data were collected, including liver and muscle biopsies. At the beginning of P2 (3 d) cows received 10 mL of sterile saline containing 10 µg of LPS from Escherichia coli O111:B4/mL into the left rear quarter of the mammary gland, and liver and muscle biopsies were collected at 12 h after LPS. Tissues were analyzed for metabolic flexibility, which measures substrate switching capacity from pyruvic acid to palmitic acid oxidation. Data were analyzed with the MIXED procedure in SAS 9.4. Rectal temperature was assessed hourly for the first 12 h after LPS and every 6 h thereafter for the remainder of P2. All cows developed a febrile response following LPS, but E-LPS had a more intense fever than M-LPS cows (0.7°C at 5 h after LPS). Blood samples were collected at 0, 3, 6, 9, 12, 24, 36, 48, and 72 h after LPS for analysis of systemic inflammation and metabolism parameters. Total serum Ca decreased after LPS (26% at 6 h nadir) but did not differ by lactation stage (LS). Circulating neutrophils decreased, then increased after LPS in both LS, but E-LPS had exaggerated neutrophilia (56% from 12 to 48 h) compared with M-LPS. Haptoglobin increased after LPS (15-fold) but did not differ by LS. Many circulating cytokines were increased after LPS, and IL-6, IL-10, TNF-α, MCP-1, and IP-10 were further augmented in E-LPS compared with M-LPS cows. Relative to P1, all cows had reduced milk yield (26%) and DMI (14%) on d 1 that did not differ by LS. Somatic cell score increased rapidly in response to LPS regardless of LS and gradually decreased from 18 h onwards. Milk component yields decreased after LPS. However, E-LPS had increased fat (11%) and tended to have increased lactose (8%) yield compared with M-LPS cows throughout P2. Circulating glucose was not affected by LPS. Nonesterified fatty acids (NEFA) decreased in E-LPS (29%) but not M-LPS cows. ß-Hydroxybutyrate slightly increased (14%) over time after LPS regardless of LS. Insulin increased after LPS in all cows, but E-LPS had blunted hyperinsulinemia (52%) compared with M-LPS cows. Blood urea nitrogen increased after LPS, and the relative change in BUN was elevated in E-LPS cows compared with M-LPS cows (36% and 13%, respectively, from 9 to 24 h). During P1, metabolic flexibility was increased in liver and muscle in early lactating cows compared with mid-lactation cows, but 12 h after LPS, metabolic flexibility was reduced and did not differ by LS. In conclusion, IMM LPS caused severe immune activation, and E-LPS cows had a more intense inflammatory response compared with M-LPS cows, but the effects on milk synthesis was similar between LS. Some parameters of the E-LPS metabolic profile suggest continuation of metabolic adjustments associated with early lactation to support both a robust immune system and milk synthesis.

Acute Effects of Taurine Supplementation on Maximal Fat Oxidation and FATmax in Recreational Endurance Runners: A Randomized, Placebo-Controlled, Crossover, and Triple-Blinded Study.

Taurine (TAU) has been shown to improve time to exhaustion (TTE) and fat oxidation during exercise; however, no studies have examined the effect of acute TAU supplementation on maximal fat oxidation (MFO) and related intensity to MFO (FATmax). Our study aimed to investigate the effect of acute TAU supplementation on MFO, FATmax, VO2peak, and TTE. Eleven recreationally trained male endurance runners performed three incremental running tests. The first visit included a familiarization to the test, followed by two subsequent visits in which exercise was performed 90 min after ingestion of either 6-g TAU or placebo (PLA) using a triple-blind randomized crossover design. There was no effect of TAU on MFO (p = .89, d = -0.07, TAU: 0.48 ± 0.22 g/min; PLA: 0.49 ± 0.15 g/min or FATmax (p = .26, d = -0.66; TAU: 49.17 ± 15.86 %V˙O2peak; PLA: 56.00 ± 13.27 %V˙O2peak). TTE was not significantly altered (TAU: 1,444.8 ± 88.6 s; PLA: 1,447.6 ± 87.34 s; p = .65, d = -0.04). TAU did not show any effect on V˙O2peak in comparison with PLA (TAU: 58.9 ± 8.4 ml·kg-1·min-1; PLA: 56.5 ± 5.7 ml·kg-1·min-1, p = .47, d = 0.48). However, V˙O2 TAU at most stages of exercise with large effect sizes (ηp2=.43). The acute ingestion of 6 g of TAU before exercise did not enhance MFO, FATmax, or TTE. However, it did increase the oxygen cost of running fixed intensities in recreationally trained endurance runners.

The Downregulation of the Liver Lipid Metabolism Induced by Hypothyroidism in Male Mice: Metabolic Flexibility Favors Compensatory Mechanisms in White Adipose Tissue.

In mammals, the maintenance of energy homeostasis relies on complex mechanisms requiring tight synchronization between peripheral organs and the brain. Thyroid hormones (THs), through their pleiotropic actions, play a central role in these regulations. Hypothyroidism, which is characterized by low circulating TH levels, slows down the metabolism, which leads to a reduction in energy expenditure as well as in lipid and glucose metabolism. The objective of this study was to evaluate whether the metabolic deregulations induced by hypothyroidism could be avoided through regulatory mechanisms involved in metabolic flexibility. To this end, the response to induced hypothyroidism was compared in males from two mouse strains, the wild-derived WSB/EiJ mouse strain characterized by a diet-induced obesity (DIO) resistance due to its high metabolic flexibility phenotype and C57BL/6J mice, which are prone to DIO. The results show that propylthiouracil (PTU)-induced hypothyroidism led to metabolic deregulations, particularly a reduction in hepatic lipid synthesis in both strains. Furthermore, in contrast to the C57BL/6J mice, the WSB/EiJ mice were resistant to the metabolic dysregulations induced by hypothyroidism, mainly through enhanced lipid metabolism in their adipose tissue. Indeed, WSB/EiJ mice compensated for the decrease in hepatic lipid synthesis by mobilizing lipid reserves from white adipose tissue. Gene expression analysis revealed that hypothyroidism stimulated the hypothalamic orexigenic circuit in both strains, but there was unchanged melanocortin 4 receptor (Mc4r) and leptin receptor (LepR) expression in the hypothyroid WSB/EiJ mice strain, which reflects their adaptability to maintain their body weight, in contrast to C57BL/6J mice. Thus, this study showed that WSB/EiJ male mice displayed a resistance to the metabolic dysregulations induced by hypothyroidism through compensatory mechanisms. This highlights the importance of metabolic flexibility in the ability to adapt to disturbed circulating TH levels.

Metabolic Flexibility of the Heart: The Role of Fatty Acid Metabolism in Health, Heart Failure, and Cardiometabolic Diseases.

This comprehensive review explores the critical role of fatty acid (FA) metabolism in cardiac diseases, particularly heart failure (HF), and the implications for therapeutic strategies. The heart's reliance on ATP, primarily sourced from mitochondrial oxidative metabolism, underscores the significance of metabolic flexibility, with fatty acid oxidation (FAO) being a dominant source. In HF, metabolic shifts occur with an altered FA uptake and FAO, impacting mitochondrial function and contributing to disease progression. Conditions like obesity and diabetes also lead to metabolic disturbances, resulting in cardiomyopathy marked by an over-reliance on FAO, mitochondrial dysfunction, and lipotoxicity. Therapeutic approaches targeting FA metabolism in cardiac diseases have evolved, focusing on inhibiting or stimulating FAO to optimize cardiac energetics. Strategies include using CPT1A inhibitors, using PPARα agonists, and enhancing mitochondrial biogenesis and function. However, the effectiveness varies, reflecting the complexity of metabolic remodeling in HF. Hence, treatment strategies should be individualized, considering that cardiac energy metabolism is intricate and tightly regulated. The therapeutic aim is to optimize overall metabolic function, recognizing the pivotal role of FAs and the need for further research to develop effective therapies, with promising new approaches targeting mitochondrial oxidative metabolism and FAO that improve cardiac function.

Restricted physical activity after volumetric muscle loss alters whole-body and local muscle metabolism.

Volumetric muscle loss (VML) is the traumatic loss of skeletal muscle, resulting in chronic functional deficits and pathological comorbidities, including altered whole-body metabolic rate and respiratory exchange ratio (RER), despite no change in physical activity in animal models. In other injury models, treatment with ß2 receptor agonists (e.g. formoterol) improves metabolic and skeletal muscle function. We aimed first to examine if restricting physical activity following injury affects metabolic and skeletal muscle function, and second, to enhance the metabolic and contractile function of the muscle remaining following VML injury through treatment with formoterol. Adult male C57Bl/6J mice (n = 32) underwent VML injury to the posterior hindlimb compartment and were randomly assigned to unrestricted or restricted activity and formoterol treatment or no treatment; age-matched injury naïve mice (n = 4) were controls for biochemical analyses. Longitudinal 24 h evaluations of physical activity and whole-body metabolism were conducted following VML. In vivo muscle function was assessed terminally, and muscles were biochemically evaluated for protein expression, mitochondrial enzyme activity and untargeted metabolomics. Restricting activity chronically after VML had the greatest effect on physical activity and RER, reflected in reduced lipid oxidation, although changes were attenuated by formoterol treatment. Formoterol enhanced injured muscle mass, while mitigating functional deficits. These novel findings indicate physical activity restriction may recapitulate following VML clinically, and adjunctive oxidative treatment may create a metabolically beneficial intramuscular environment while enhancing the injured muscle's mass and force-producing capacity. Further investigation is needed to evaluate adjunctive oxidative treatment with rehabilitation, which may augment the muscle's regenerative and functional capacity following VML. KEY POINTS: The natural ability of skeletal muscle to regenerate and recover function is lost following complex traumatic musculoskeletal injury, such as volumetric muscle loss (VML), and physical inactivity following VML may incur additional deleterious consequences for muscle and metabolic health. Modelling VML injury-induced physical activity restriction altered whole-body metabolism, primarily by decreasing lipid oxidation, while preserving local skeletal muscle metabolic activity. The ß2 adrenergic receptor agonist formoterol has shown promise in other severe injury models to improve regeneration, recover function and enhance metabolism. Treatment with formoterol enhanced mass of the injured muscle and whole-body metabolism while mitigating functional deficits resulting from injury. Understanding of chronic effects of the clinically available and FDA-approved pharmaceutical formoterol could be a translational option to support muscle function after VML injury.

An adipocentric perspective on the development and progression of non-alcoholic fatty liver disease.

Alongside the liver, white adipose tissue (WAT) is critical in regulating systemic energy homeostasis. Although each organ has its specialised functions, they must work coordinately to regulate whole-body metabolism. Adipose tissues and the liver are relatively resilient and can adapt to an energy surplus by facilitating triglyceride (TG) storage up to a certain threshold level without significant metabolic disturbances. However, lipid storage in WAT beyond a "personalised" adiposity threshold becomes dysfunctional, leading to metabolic inflexibility, progressive inflammation, and aberrant adipokine secretion. Moreover, the failure of adipose tissue to store and mobilise lipids results in systemic knock-on lipid overload, particularly in the liver. Factors contributing to hepatic lipid overload include lipids released from WAT, dietary fat intake, and enhanced de novo lipogenesis. In contrast, extrahepatic mechanisms counteracting toxic hepatic lipid overload entail coordinated compensation through oxidation of surplus fatty acids in brown adipose tissue and storage of fatty acids as TGs in WAT. Failure of these integrated homeostatic mechanisms leads to quantitative increases and qualitative alterations to the lipidome of the liver. Initially, hepatocytes preferentially accumulate TG species leading to a relatively "benign" non-alcoholic fatty liver. However, with time, inflammatory responses ensue, progressing into more severe conditions such as non-alcoholic steatohepatitis, cirrhosis, and hepatocellular carcinoma, in some individuals (often without an early prognostic clue). Herein, we highlight the pathogenic importance of obesity-induced "adipose tissue failure", resulting in decreased adipose tissue functionality (i.e. fat storage capacity and metabolic flexibility), in the development and progression of NAFL/NASH.

Divergence in aerobic capacity influences hepatic and systemic metabolic adaptations to bile acid sequestrant and short-term high-fat/sucrose feeding in rats.

High versus low aerobic capacity significantly impacts the risk for metabolic diseases. Rats selectively bred for high or low intrinsic aerobic capacity differently modify hepatic bile acid metabolism in response to high-fat diets (HFDs). Here we tested if a bile acid sequestrant would alter hepatic and whole body metabolism differently in rats with high and low aerobic capacity fed a 1-wk HFD. Male rats (8 mo of age) that were artificially selected to be high (HCR) and low-capacity runners (LCR) with divergent intrinsic aerobic capacities were transitioned from a low-fat diet (LFD, 10% fat) to an HFD (45% fat) with or without a bile acid sequestrant (BA-Seq, 2% cholestyramine resin) for 7 days while maintained in an indirect calorimetry system. HFD + BA-Seq increased fecal excretion of lipids and bile acids and prevented weight and fat mass gain in both strains. Interestingly, HCR rats had increased adaptability to enhance fecal bile acid and lipid loss, resulting in more significant energy loss than their LCR counterpart. In addition, BA-Seq induced a greater expression of hepatic CYP7A1 gene expression, the rate-limiting enzyme of bile acid synthesis in HCR rats both on HFD and HFD + BA-Seq diets. HCR displayed a more significant reduction of RQ in response to HFD than LCR, but HFD + BA-Seq lowered RQ in both groups compared with HFD alone, demonstrating a pronounced impact on metabolic flexibility. In conclusion, BA-Seq provides uniform metabolic benefits for metabolic flexibility and adiposity, but rats with higher aerobic capacity display adaptability for hepatic bile acid metabolism.NEW & NOTEWORTHY The administration of bile acid sequestrant (BA-Seq) has uniform metabolic benefits in terms of metabolic flexibility and adiposity in rats with high and low aerobic capacity. However, rats with higher aerobic capacity demonstrate greater adaptability in hepatic bile acid metabolism, resulting in increased fecal bile acid and lipid loss, as well as enhanced fecal energy loss.

Metabolic flexibility during a trophic transition reveals the phenotypic plasticity of greater duckweed (Spirodela polyrhiza 7498).

The greater duckweed (Spirodela polyrhiza 7498) exhibits trophic diversity (photoautotrophic, heterotrophic, photoheterotrophic, and mixotrophic growth) depending on the availability of exogenous organic carbon sources and light. Here, we show that the ability to transition between various trophic growth conditions is an advantageous trait, providing great phenotypic plasticity and metabolic flexibility in S. polyrhiza 7498. By comparing S. polyrhiza 7498 growth characteristics, metabolic acclimation, and cellular ultrastructure across these trophic modes, we show that mixotrophy decreases photosynthetic performance and relieves the CO2 limitation of photosynthesis by enhancing the CO2 supply through the active respiration pathway. Proteomic and metabolomic analyses corroborated that S. polyrhiza 7498 increases its intracellular CO2 and decreases reactive oxygen species under mixotrophic and heterotrophic conditions, which substantially suppressed the wasteful photorespiration and oxidative-damage pathways. As a consequence, mixotrophy resulted in a higher biomass yield than the sum of photoautotrophy and heterotrophy. Our work provides a basis for using trophic transitions in S. polyrhiza 7498 for the enhanced accumulation of value-added products.

Deletion of Tbc1d4/As160 abrogates cardiac glucose uptake and increases myocardial damage after ischemia/reperfusion.

BACKGROUND: Type 2 Diabetes mellitus (T2DM) is a major risk factor for cardiovascular disease and associated with poor outcome after myocardial infarction (MI). In T2DM, cardiac metabolic flexibility, i.e. the switch between carbohydrates and lipids as energy source, is disturbed. The RabGTPase-activating protein TBC1D4 represents a crucial regulator of insulin-stimulated glucose uptake in skeletal muscle by controlling glucose transporter GLUT4 translocation. A human loss-of-function mutation in TBC1D4 is associated with impaired glycemic control and elevated T2DM risk. The study's aim was to investigate TBC1D4 function in cardiac substrate metabolism and adaptation to MI. METHODS: Cardiac glucose metabolism of male Tbc1d4-deficient (D4KO) and wild type (WT) mice was characterized using in vivo [18F]-FDG PET imaging after glucose injection and ex vivo basal/insulin-stimulated [3H]-2-deoxyglucose uptake in left ventricular (LV) papillary muscle. Mice were subjected to cardiac ischemia/reperfusion (I/R). Heart structure and function were analyzed until 3 weeks post-MI using echocardiography, morphometric and ultrastructural analysis of heart sections, complemented by whole heart transcriptome and protein measurements. RESULTS: Tbc1d4-knockout abolished insulin-stimulated glucose uptake in ex vivo LV papillary muscle and in vivo cardiac glucose uptake after glucose injection, accompanied by a marked reduction of GLUT4. Basal cardiac glucose uptake and GLUT1 abundance were not changed compared to WT controls. D4KO mice showed mild impairments in glycemia but normal cardiac function. However, after I/R D4KO mice showed progressively increased LV endsystolic volume and substantially increased infarction area compared to WT controls. Cardiac transcriptome analysis revealed upregulation of the unfolded protein response via ATF4/eIF2α in D4KO mice at baseline. Transmission electron microscopy revealed largely increased extracellular matrix (ECM) area, in line with decreased cardiac expression of matrix metalloproteinases of D4KO mice. CONCLUSIONS: TBC1D4 is essential for insulin-stimulated cardiac glucose uptake and metabolic flexibility. Tbc1d4-deficiency results in elevated cardiac endoplasmic reticulum (ER)-stress response, increased deposition of ECM and aggravated cardiac damage following MI. Hence, impaired TBC1D4 signaling contributes to poor outcome after MI.

Retinoic acid regulates pyruvate dehydrogenase kinase 4 (Pdk4) to modulate fuel utilization in the adult heart: Insights from wild-type and ß-carotene 9',10' oxygenase knockout mice.

Regulation of the pyruvate dehydrogenase (PDH) complex by the pyruvate dehydrogenase kinase PDK4 enables the heart to respond to fluctuations in energy demands and substrate availability. Retinoic acid, the transcriptionally active form of vitamin A, is known to be involved in the regulation of cardiac function and growth during embryogenesis as well as under pathological conditions. Whether retinoic acid also maintains cardiac health under physiological conditions is unknown. However, vitamin A status and intake of its carotenoid precursor ß-carotene have been linked to the prevention of heart diseases. Here, we provide in vitro and in vivo evidence that retinoic acid regulates cardiac Pdk4 expression and thus PDH activity. Furthermore, we show that mice lacking ß-carotene 9',10'-oxygenase (BCO2), the only enzyme of the adult heart that cleaves ß-carotene to generate retinoids (vitamin A and its derivatives), displayed cardiac retinoic acid insufficiency and impaired metabolic flexibility linked to a compromised PDK4/PDH pathway. These findings provide novel insights into the functions of retinoic acid in regulating energy metabolism in adult tissues, especially the heart.

miR-183 and miR-96 orchestrate both glucose and fat utilization in skeletal muscle.

Our knowledge of the coordination of fuel usage in skeletal muscle is incomplete. Whether and how microRNAs are involved in the substrate selection for oxidation is largely unknown. Here we show that mice lacking miR-183 and miR-96 have enhanced muscle oxidative phenotype and altered glucose/lipid homeostasis. Moreover, loss of miR-183 and miR-96 results in a shift in substrate utilization toward fat relative to carbohydrates in mice. Mechanistically, loss of miR-183 and miR-96 suppresses glucose utilization in skeletal muscle by increasing PDHA1 phosphorylation via targeting FoxO1 and PDK4. On the other hand, loss of miR-183 and miR-96 promotes fat usage in skeletal muscle by enhancing intramuscular lipolysis via targeting FoxO1 and ATGL. Thus, our study establishes miR-183 and miR-96 as master coordinators of fuel selection and metabolic homeostasis owing to their capability of modulating both glucose utilization and fat catabolism. Lastly, we show that loss of miR-183 and miR-96 can alleviate obesity and improve glucose metabolism in high-fat diet-induced mice, suggesting that miR-183 and miR-96 may serve as therapeutic targets for metabolic diseases.

Dynamic changes in DICER levels in adipose tissue control metabolic adaptations to exercise.

DICER is a key enzyme in microRNA (miRNA) biogenesis. Here we show that aerobic exercise training up-regulates DICER in adipose tissue of mice and humans. This can be mimicked by infusion of serum from exercised mice into sedentary mice and depends on AMPK-mediated signaling in both muscle and adipocytes. Adipocyte DICER is required for whole-body metabolic adaptations to aerobic exercise training, in part, by allowing controlled substrate utilization in adipose tissue, which, in turn, supports skeletal muscle function. Exercise training increases overall miRNA expression in adipose tissue, and up-regulation of miR-203-3p limits glycolysis in adipose under conditions of metabolic stress. We propose that exercise training-induced DICER-miR-203-3p up-regulation in adipocytes is a key adaptive response that coordinates signals from working muscle to promote whole-body metabolic adaptations.

Metabolic Flexibility During Exercise in Children with Overweight/Obesity Versus Children who are Lean.

PURPOSE: This study examined metabolic flexibility with respect to fat metabolism during exercise in children who are lean (n=11; 10.9[0.9] y) and overweight/obese (OW/OB; n=9; 10.3[1.2] y). METHOD: Participants were grouped based on body mass index percentiles for age and sex. Groups were mixed in age and sex. Participants completed two 20-minute exercise bouts on a cycle ergometer, separated by a 10-minute rest. Bout 1 consisted of 10 minutes at 50% VO2peak and 10 minutes at 75% VO2peak. Bout 2 was 20 minutes at 50% VO2peak. Absolute fat oxidation rate (FOR), FOR relative to body mass, FOR relative to fat-free mass, and proportional fat use were measured at 10 minutes of bout 1 and 5, 10, 15, and 20 minutes of bout 2. RESULTS: Absolute FOR was higher in the OW/OB group (range: 117.8 [55.1]-206.2 [48.3] mg·min-1) than in the lean group (81.1 [32.2]-152.2 [38.2] mg·min-1); however, there were no significant main effects for group or significant interactions for proportional fat use, FOR relative to body mass, or FOR relative to fat-free mass. CONCLUSION: Children in this age range who are overweight/obese do not display impaired metabolic flexibility with respect to fat metabolism during exercise.

Lipid droplets, bioenergetic fluxes, and metabolic flexibility.

The capacity of cells and animals to sense and adapt to fluctuations in the availability of energetic substrates is commonly described as metabolic flexibility. This flexibility allows for example the transition from fed to fasting states and to meet the energy demands of exercise in both states. Flexibility is disrupted in pathological conditions such as the metabolic syndrome but in contrast, it is enhanced in some tumours. Lipid droplets (LDs) and mitochondria are key organelles in bioenergetics. In all eukaryotic cells, LDs store and supply essential lipids to produce signalling molecules, membrane building blocks, and the metabolic energy needed to survive during nutrient poor periods. Highly conserved, robust, and regulated mechanisms ensure these bioenergetic fluxes. Although mitochondria are recognized as the epicentre of metabolic flexibility, the contribution of LDs and LD-proteins is often neglected or considered detrimental. Here, we revisit the key roles of LDs during fasting and the intimate collaboration existing with mitochondria when cells sense and respond to fluctuations in substrate availability.

The crucial roles of mitochondria in supporting C4 photosynthesis.

C4 photosynthesis involves a series of biochemical and anatomical traits that significantly improve plant productivity under conditions that reduce the efficiency of C3 photosynthesis. We explore how evolution of the three classical biochemical types of C4 photosynthesis (NADP-ME, NAD-ME and PCK types) has affected the functions and properties of mitochondria. Mitochondria in C4 NAD-ME and PCK types play a direct role in decarboxylation of metabolites for C4 photosynthesis. Mitochondria in C4 PCK type also provide ATP for C4 metabolism, although this role for ATP provision is not seen in NAD-ME type. Such involvement has increased mitochondrial abundance/size and associated enzymatic capacity, led to changes in mitochondrial location and ultrastructure, and altered the role of mitochondria in cellular carbon metabolism in the NAD-ME and PCK types. By contrast, these changes in mitochondrial properties are absent in the C4 NADP-ME type and C3 leaves, where mitochondria play no direct role in photosynthesis. From an eco-physiological perspective, rates of leaf respiration in darkness vary considerably among C4 species but does not differ systematically among the three C4 types. This review outlines further mitochondrial research in key areas central to the engineering of the C4 pathway into C3 plants and to the understanding of variation in rates of C4 dark respiration.