RIP140 deficiency enhances cardiac fuel metabolism and protects mice from heart failure.

During the development of heart failure (HF), the capacity for cardiomyocyte (CM) fatty acid oxidation (FAO) and ATP production is progressively diminished, contributing to pathologic cardiac hypertrophy and contractile dysfunction. Receptor-interacting protein 140 (RIP140, encoded by Nrip1) has been shown to function as a transcriptional corepressor of oxidative metabolism. We found that mice with striated muscle deficiency of RIP140 (strNrip1-/-) exhibited increased expression of a broad array of genes involved in mitochondrial energy metabolism and contractile function in heart and skeletal muscle. strNrip1-/- mice were resistant to the development of pressure overload-induced cardiac hypertrophy, and CM-specific RIP140-deficient (csNrip1-/-) mice were protected against the development of HF caused by pressure overload combined with myocardial infarction. Genomic enhancers activated by RIP140 deficiency in CMs were enriched in binding motifs for transcriptional regulators of mitochondrial function (estrogen-related receptor) and cardiac contractile proteins (myocyte enhancer factor 2). Consistent with a role in the control of cardiac fatty acid oxidation, loss of RIP140 in heart resulted in augmented triacylglyceride turnover and fatty acid utilization. We conclude that RIP140 functions as a suppressor of a transcriptional regulatory network that controls cardiac fuel metabolism and contractile function, representing a potential therapeutic target for the treatment of HF.

Chemotherapeutic drug screening in 3D-Bioengineered human myobundles provides insight into taxane-induced myotoxicities.

Two prominent frontline breast cancer (BC) chemotherapies commonly used in combination, doxorubicin (DOX) and docetaxel (TAX), are associated with long-lasting cardiometabolic and musculoskeletal side effects. Whereas DOX has been linked to mitochondrial dysfunction, mechanisms underlying TAX-induced myotoxicities remain uncertain. Here, the metabolic and functional consequences of TAX ± DOX were investigated using a 3D-bioengineered model of adult human muscle and a drug dosing regimen designed to resemble in vivo pharmacokinetics. DOX potently reduced mitochondrial respiratory capacity, 3D-myobundle size, and contractile force, whereas TAX-induced acetylation and remodeling of the microtubule network led to perturbations in glucose uptake, mitochondrial respiratory sensitivity, and kinetics of fatigue, without compromising tetanic force generation. These findings suggest TAX-induced remodeling of the microtubule network disrupts glucose transport and respiratory control in skeletal muscle and thereby have important clinical implications related to the cardiometabolic health and quality of life of BC patients and survivors.

Myocardial Lipin 1 knockout in mice approximates cardiac effects of human LPIN1 mutations.

Lipin 1 is a bifunctional protein that is a transcriptional regulator and has phosphatidic acid (PA) phosphohydrolase activity, which dephosphorylates PA to generate diacylglycerol. Human lipin 1 mutations lead to episodic rhabdomyolysis, and some affected patients exhibit cardiac abnormalities, including exercise-induced cardiac dysfunction and cardiac triglyceride accumulation. Furthermore, lipin 1 expression is deactivated in failing heart, but the effects of lipin 1 deactivation in myocardium are incompletely understood. We generated mice with cardiac-specific lipin 1 KO (cs-Lpin1-/-) to examine the intrinsic effects of lipin 1 in the myocardium. Cs-Lpin1-/- mice had normal systolic cardiac function but mild cardiac hypertrophy. Compared with littermate control mice, PA content was higher in cs-Lpin1-/- hearts, which also had an unexpected increase in diacylglycerol and triglyceride content. Cs-Lpin1-/- mice exhibited diminished cardiac cardiolipin content and impaired mitochondrial respiration rates when provided with pyruvate or succinate as metabolic substrates. After transverse aortic constriction-induced pressure overload, loss of lipin 1 did not exacerbate cardiac hypertrophy or dysfunction. However, loss of lipin 1 dampened the cardiac ionotropic response to dobutamine and exercise endurance in association with reduced protein kinase A signaling. These data suggest that loss of lipin 1 impairs cardiac functional reserve, likely due to effects on glycerolipid homeostasis, mitochondrial function, and protein kinase A signaling.

Muscle-Liver Trafficking of BCAA-Derived Nitrogen Underlies Obesity-Related Glycine Depletion.

Glycine levels are inversely associated with branched-chain amino acids (BCAAs) and cardiometabolic disease phenotypes, but biochemical mechanisms that explain these relationships remain uncharted. Metabolites and genes related to BCAA metabolism and nitrogen handling were strongly associated with glycine in correlation analyses. Stable isotope labeling in Zucker fatty rats (ZFRs) shows that glycine acts as a carbon donor for the pyruvate-alanine cycle in a BCAA-regulated manner. Inhibition of the BCAA transaminase (BCAT) enzymes depletes plasma pools of alanine and raises glycine levels. In high-fat-fed ZFRs, dietary glycine supplementation raises urinary acyl-glycine content and lowers circulating triglycerides but also results in accumulation of long-chain acyl-coenzyme As (acyl-CoAs), lower 5' adenosine monophosphate-activated protein kinase (AMPK) phosphorylation in muscle, and no improvement in glucose tolerance. Collectively, these studies frame a mechanism for explaining obesity-related glycine depletion and also provide insight into the impact of glycine supplementation on systemic glucose, lipid, and amino acid metabolism.

Plasma MicroRNAs in Established Rheumatoid Arthritis Relate to Adiposity and Altered Plasma and Skeletal Muscle Cytokine and Metabolic Profiles.

Background: MicroRNAs have been implicated in the pathogenesis of rheumatoid arthritis (RA), obesity, and altered metabolism. Although RA is associated with both obesity and altered metabolism, expression of RA-related microRNA in the setting of these cardiometabolic comorbidities is unclear. Our objective was to determine relationships between six RA-related microRNAs and RA disease activity, inflammation, body composition, and metabolic function. Methods: Expression of plasma miR-21, miR-23b, miR-27a, miR-143, miR-146a, and miR-223 was measured in 48 persons with seropositive and/or erosive RA (mean DAS-28-ESR 3.0, SD 1.4) and 23 age-, sex-, and BMI-matched healthy controls. Disease activity in RA was assessed by DAS-28-ESR. Plasma cytokine concentrations were determined by ELISA. Body composition was assessed using CT scan to determine central and muscle adipose and thigh muscle tissue size and tissue density. Plasma and skeletal muscle acylcarnitine, amino acid, and organic acid metabolites were measured via mass-spectroscopy. Plasma lipoproteins were measured via nuclear magnetic resonance (NMR) spectroscopy. Spearman correlations were used to assess relationships for microRNA with inflammation and cardiometabolic measures. RA and control associations were compared using Fisher transformations. Results: Among RA subjects, plasma miR-143 was associated with plasma IL-6 and IL-8. No other RA microRNA was positively associated with disease activity or inflammatory markers. In RA, microRNA expression was associated with adiposity, both visceral adiposity (miR-146a, miR-21, miR-23b, and miR-27a) and thigh intra-muscular adiposity (miR-146a and miR-223). RA miR-146a was associated with greater concentrations of cardiometabolic risk markers (plasma short-chain dicarboxyl/hydroxyl acylcarnitines, triglycerides, large VLDL particles, and small HDL particles) and lower concentrations of muscle energy substrates (long-chain acylcarnitines and pyruvate). Despite RA and controls having similar microRNA levels, RA, and controls differed in magnitude and direction for several associations with cytokines and plasma and skeletal muscle metabolic intermediates. Conclusion: Most microRNAs thought to be associated with RA disease activity and inflammation were more reflective of RA adiposity and impaired metabolism. These associations show that microRNAs in RA may serve as an epigenetic link between RA inflammation and cardiometabolic comorbidities.

Systematic Dissection of the Metabolic-Apoptotic Interface in AML Reveals Heme Biosynthesis to Be a Regulator of Drug Sensitivity.

Crosstalk between metabolic and survival pathways is critical for cellular homeostasis, but the connectivity between these processes remains poorly defined. We used loss-of-function CRISPR/Cas9 knockout screening to identify metabolic genes capable of influencing cellular commitment to apoptosis, using sensitization to the BCL-2 inhibitor ABT-199 in BCL-2-dependent acute myeloid leukemia (AML) cell lines as a proxy for apoptotic disposition. This analysis revealed metabolic pathways that specifically cooperate with BCL-2 to sustain survival. In particular, our analysis singled out heme biosynthesis as an unappreciated apoptosis-modifying pathway. Although heme is broadly incorporated into the proteome, reduction of heme biosynthesis potentiates apoptosis through the loss of ETC activity, resulting in baseline depolarization of the mitochondrial membrane and an increased propensity to undergo apoptosis. Collectively, our findings chart the first apoptotic map of metabolism, motivating the design of metabolically engaged combination chemotherapies and nominating heme biosynthesis as an apoptotic modulator in AML.

Mitochondrial Diagnostics: A Multiplexed Assay Platform for Comprehensive Assessment of Mitochondrial Energy Fluxes.

Chronic metabolic diseases have been linked to molecular signatures of mitochondrial dysfunction. Nonetheless, molecular remodeling of the transcriptome, proteome, and/or metabolome does not necessarily translate to functional consequences that confer physiologic phenotypes. The work here aims to bridge the gap between molecular and functional phenomics by developing and validating a multiplexed assay platform for comprehensive assessment of mitochondrial energy transduction. The diagnostic power of the platform stems from a modified version of the creatine kinase energetic clamp technique, performed in parallel with multiplexed analyses of dehydrogenase activities and ATP synthesis rates. Together, these assays provide diagnostic coverage of the mitochondrial network at a level approaching that gained by molecular "-omics" technologies. Application of the platform to a comparison of skeletal muscle versus heart mitochondria reveals mechanistic insights into tissue-specific distinctions in energy transfer efficiency. This platform opens exciting opportunities to unravel the connection between mitochondrial bioenergetics and human disease.

Metabolic Alterations Contribute to Enhanced Inflammatory Cytokine Production in Irgm1-deficient Macrophages.

The immunity-related GTPases (IRGs) are a family of proteins that are induced by interferon (IFN)-γ and play pivotal roles in immune and inflammatory responses. IRGs ostensibly function as dynamin-like proteins that bind to intracellular membranes and promote remodeling and trafficking of those membranes. Prior studies have shown that loss of Irgm1 in mice leads to increased lethality to bacterial infections as well as enhanced inflammation to non-infectious stimuli; however, the mechanisms underlying these phenotypes are unclear. In the studies reported here, we found that uninfected Irgm1-deficient mice displayed high levels of serum cytokines typifying profound autoinflammation. Similar increases in cytokine production were also seen in cultured, IFN-γ-primed macrophages that lacked Irgm1. A series of metabolic studies indicated that the enhanced cytokine production was associated with marked metabolic changes in the Irgm1-deficient macrophages, including increased glycolysis and an accumulation of long chain acylcarnitines. Cells were exposed to the glycolytic inhibitor, 2-deoxyglucose, or fatty acid synthase inhibitors to perturb the metabolic alterations, which resulted in dampening of the excessive cytokine production. These results suggest that Irgm1 deficiency drives metabolic dysfunction in macrophages in a manner that is cell-autonomous and independent of infectious triggers. This may be a significant contributor to excessive inflammation seen in Irgm1-deficient mice in different contexts.

Metabolic Catastrophe in Mice Lacking Transferrin Receptor in Muscle.

Transferrin receptor (Tfr1) is ubiquitously expressed, but its roles in non-hematopoietic cells are incompletely understood. We used a tissue-specific conditional knockout strategy to ask whether skeletal muscle required Tfr1 for iron uptake. We found that iron assimilation via Tfr1 was critical for skeletal muscle metabolism, and that iron deficiency in muscle led to dramatic changes, not only in muscle, but also in adipose tissue and liver. Inactivation of Tfr1 incapacitated normal energy production in muscle, leading to growth arrest and a muted attempt to switch to fatty acid ß oxidation, using up fat stores. Starvation signals stimulated gluconeogenesis in the liver, but amino acid substrates became limiting and hypoglycemia ensued. Surprisingly, the liver was also iron deficient, and production of the iron regulatory hormone hepcidin was depressed. Our observations reveal a complex interaction between iron homeostasis and metabolism that has implications for metabolic and iron disorders.

Metabolite signatures of exercise training in human skeletal muscle relate to mitochondrial remodelling and cardiometabolic fitness.

AIMS/HYPOTHESIS: Targeted metabolomic and transcriptomic approaches were used to evaluate the relationship between skeletal muscle metabolite signatures, gene expression profiles and clinical outcomes in response to various exercise training interventions. We hypothesised that changes in mitochondrial metabolic intermediates would predict improvements in clinical risk factors, thereby offering novel insights into potential mechanisms. METHODS: Subjects at risk of metabolic disease were randomised to 6 months of inactivity or one of five aerobic and/or resistance training programmes (n = 112). Pre/post-intervention assessments included cardiorespiratory fitness ([Formula: see text]), serum triacylglycerols (TGs) and insulin sensitivity (SI). In this secondary analysis, muscle biopsy specimens were used for targeted mass spectrometry-based analysis of metabolic intermediates and measurement of mRNA expression of genes involved in metabolism. RESULTS: Exercise regimens with the largest energy expenditure produced robust increases in muscle concentrations of even-chain acylcarnitines (median 37-488%), which correlated positively with increased expression of genes involved in muscle uptake and oxidation of fatty acids. Along with free carnitine, the aforementioned acylcarnitine metabolites were related to improvements in [Formula: see text], TGs and SI (R = 0.20-0.31, p < 0.05). Muscle concentrations of the tricarboxylic acid cycle intermediates succinate and succinylcarnitine (R = 0.39 and 0.24, p < 0.05) emerged as the strongest correlates of SI. CONCLUSIONS/INTERPRETATION: The metabolic signatures of exercise-trained skeletal muscle reflected reprogramming of mitochondrial function and intermediary metabolism and correlated with changes in cardiometabolic fitness. Succinate metabolism and the succinate dehydrogenase complex emerged as a potential regulatory node that intersects with whole-body insulin sensitivity. This study identifies new avenues for mechanistic research aimed at understanding the health benefits of physical activity. Trial registration ClinicalTrials.gov NCT00200993 and NCT00275145 Funding This work was supported by the National Heart, Lung, and Blood Institute (National Institutes of Health), National Institute on Aging (National Institutes of Health) and National Institute of Arthritis and Musculoskeletal and Skin Diseases (National Institutes of Health).

Measurement of fatty acid oxidation rates in animal tissues and cell lines.

While much oncological research has focused on metabolic shifts in glucose and amino acid oxidation, recent evidence suggests that fatty acid oxidation (FAO) may also play an important role in the metabolic reprogramming of cancer cells. Here, we present a simple method for measuring FAO rates using radiolabeled palmitate, common laboratory reagents, and standard supplies. This protocol is broadly applicable for measuring FAO rates in cultured cancer cells as well as in both malignant and nontransformed animal tissues.

Substituting dietary monounsaturated fat for saturated fat is associated with increased daily physical activity and resting energy expenditure and with changes in mood.

BACKGROUND: The Western diet increases risk of metabolic disease. OBJECTIVE: We determined whether lowering the ratio of saturated fatty acids to monounsaturated fatty acids in the Western diet would affect physical activity and energy expenditure. DESIGN: With the use of a balanced design, 2 cohorts of 18 and 14 young adults were enrolled in separate randomized, double-masked, crossover trials that compared a 3-wk high-palmitic acid diet (HPA; similar to the Western diet fat composition) to a low-palmitic acid and high-oleic acid diet (HOA; similar to the Mediterranean diet fat composition). All foods were provided by the investigators, and the palmitic acid (PA):oleic acid (OA) ratio was manipulated by adding different oil blends to the same foods. In both cohorts, we assessed physical activity (monitored continuously by using accelerometry) and resting energy expenditure (REE). To gain insight into a possible mood disturbance that might explain changes in physical activity, the Profile of Mood States (POMS) was administered in cohort 2. RESULTS: Physical activity was higher during the HOA than during the HPA in 15 of 17 subjects in cohort 1 (P = 0.008) (mean: 12% higher; P = 0.003) and in 12 of 12 subjects in the second, confirmatory cohort (P = 0.005) (mean: 15% higher; P = 0.003). When the HOA was compared with the HPA, REE measured during the fed state was 3% higher for cohort 1 (P < 0.01), and REE was 4.5% higher in the fasted state for cohort 2 (P = 0.04). POMS testing showed that the anger-hostility score was significantly higher during the HPA (P = 0.007). CONCLUSIONS: The replacement of dietary PA with OA was associated with increased physical activity and REE and less anger. Besides presumed effects on mitochondrial function (increased REE), the dietary PA:OA ratio appears to affect behavior. The second cohort was derived from a study that was registered at clinicaltrials.gov as R01DK082803.

Peroxisome proliferator-activated receptor-gamma coactivator-1alpha overexpression increases lipid oxidation in myocytes from extremely obese individuals.

OBJECTIVE: To determine whether the obesity-related decrement in fatty acid oxidation (FAO) in primary human skeletal muscle cells (HSkMC) is linked with lower mitochondrial content and whether this deficit could be corrected via overexpression of peroxisome proliferator-activated receptor-gamma coactivator-1alpha (PGC-1alpha). RESEARCH DESIGN AND METHODS: FAO was studied in HSkMC from lean (BMI 22.4 +/- 0.9 kg/m(2); N = 12) and extremely obese (45.3 +/- 1.4 kg/m(2); N = 9) subjects. Recombinant adenovirus was used to increase HSkMC PGC-1alpha expression (3.5- and 8.0-fold), followed by assessment of mitochondrial content (mtDNA and cytochrome C oxidase IV [COXIV]), complete ((14)CO(2) production from labeled oleate), and incomplete (acid soluble metabolites [ASM]) FAO, and glycerolipid synthesis. RESULTS: Obesity was associated with a 30% decrease (P < 0.05) in complete FAO, which was accompanied by higher relative rates of incomplete FAO ([(14)C]ASM production/(14)CO(2)), increased partitioning of fatty acid toward storage, and lower (P < 0.05) mtDNA (-27%), COXIV (-35%), and mitochondrial transcription factor (mtTFA) (-43%) protein levels. PGC-1alpha overexpression increased (P < 0.05) FAO, mtDNA, COXIV, mtTFA, and fatty acid incorporation into triacylglycerol in both lean and obese groups. Perturbations in FAO, triacylglycerol synthesis, mtDNA, COXIV, and mtTFA in obese compared with lean HSkMC persisted despite PGC-1alpha overexpression. When adjusted for mtDNA and COXIV content, FAO was equivalent between lean and obese groups. CONCLUSION: Reduced mitochondrial content is related to impaired FAO in HSkMC derived from obese individuals. Increasing PGC-1alpha protein levels did not correct the obesity-related absolute reduction in FAO or mtDNA content, implicating mechanisms other than PGC-1alpha abundance.

Receptor-selective coactivators as tools to define the biology of specific receptor-coactivator pairs.

In the absence of specific high-affinity agonists and antagonists, it has been difficult to define the target genes and biological responses attributable to many of the orphan nuclear receptors (ONRs). Indeed, it appears that many members of this receptor superfamily are not regulated by classical small molecules but rather their activity is controlled by interacting cofactors. Motivated by this finding, we have developed an approach to genetically isolate specific receptor-cofactor pairs in cells, allowing us to define the biological responses attributable to each complex. This is accomplished by using combinatorial peptide phage display to engineer the receptor interacting domain of each cofactor such that it interacts selectively with one nuclear receptor. In this study, we describe the customization of PGC-1alpha and its use to study the biology of the estrogen-related receptor alpha (ERRalpha) in cultured liver cells.

Peroxisome proliferator-activated receptor-gamma co-activator 1alpha-mediated metabolic remodeling of skeletal myocytes mimics exercise training and reverses lipid-induced mitochondrial inefficiency.

Peroxisome proliferator-activated receptor-gamma co-activator 1alpha (PGC1alpha) is a promiscuous co-activator that plays a key role in regulating mitochondrial biogenesis and fuel homeostasis. Emergent evidence links decreased skeletal muscle PGC1alpha activity and coincident impairments in mitochondrial performance to the development of insulin resistance in humans. Here we used rodent models to demonstrate that muscle mitochondrial efficiency is compromised by diet-induced obesity and is subsequently rescued by exercise training. Chronic high fat feeding caused accelerated rates of incomplete fatty acid oxidation and accumulation of beta-oxidative intermediates. The capacity of muscle mitochondria to fully oxidize a heavy influx of fatty acid depended on factors such as fiber type and exercise training and was positively correlated with expression levels of PGC1alpha. Likewise, an efficient lipid-induced substrate switch in cultured myocytes depended on adenovirus-mediated increases in PGC1alpha expression. Our results supported a novel paradigm in which a high lipid supply, occurring under conditions of low PGC1alpha, provokes a disconnect between mitochondrial beta-oxidation and tricarboxylic acid cycle activity. Conversely, the metabolic remodeling that occurred in response to PGC1alpha overexpression favored a shift from incomplete to complete beta-oxidation. We proposed that PGC1alpha enables muscle mitochondria to better cope with a high lipid load, possibly reflecting a fundamental metabolic benefit of exercise training.

Hepatic expression of malonyl-CoA decarboxylase reverses muscle, liver and whole-animal insulin resistance.

Lipid infusion or ingestion of a high-fat diet results in insulin resistance, but the mechanism underlying this phenomenon remains unclear. Here we show that, in rats fed a high-fat diet, whole-animal, muscle and liver insulin resistance is ameliorated following hepatic overexpression of malonyl-coenzyme A (CoA) decarboxylase (MCD), an enzyme that affects lipid partitioning. MCD overexpression decreased circulating free fatty acid (FFA) and liver triglyceride content. In skeletal muscle, levels of triglyceride and long-chain acyl-CoA (LC-CoA)-two candidate mediators of insulin resistance-were either increased or unchanged. Metabolic profiling of 36 acylcarnitine species by tandem mass spectrometry revealed a unique decrease in the concentration of one lipid-derived metabolite, beta-OH-butyrate, in muscle of MCD-overexpressing animals. The best explanation for our findings is that hepatic expression of MCD lowered circulating FFA levels, which led to lowering of muscle beta-OH-butyrate levels and improvement of insulin sensitivity.

Effect of short-term exercise training on insulin-stimulated PI 3-kinase activity in middle-aged men.

The purpose of this study was to determine whether the improved insulin action with short-term exercise training in middle-aged individuals is associated with enhanced phosphatidylinositol (PI) 3-kinase activity in skeletal muscle. Nine men of ages 50-70 yr were studied before and after 7 consecutive days of supervised exercise (60 min/day, 70% peak O2 consumption). Insulin sensitivity was measured with a euglycemic hyperinsulinemic glucose clamp in the sedentary condition and 15-17 h after the final exercise session. Anti-phosphotyrosine-associated PI 3-kinase activity was determined from muscle samples obtained in the fasted condition and after 60 min of insulin infusion during the clamp. With exercise, the glucose infusion rate increased (P < 0.001) by 33%, indicating enhanced insulin action (mean +/- SE, 6.6 +/- 0.6 vs. 8.7 +/- 0.8 mg x kg(-1) x min(-1)). Short-term exercise training did not, however, increase insulin-stimulated (insulin stimulated/fasting) PI 3-kinase activity (1.8 +/- 0.8 vs. 1.8 +/- 0.7-fold stimulation with insulin pre- vs. posttraining, respectively). There was also no change in insulin-stimulated protein kinase B activity (1.3 +/- 0.1 vs. 1.4 +/- 0.2-fold stimulation with insulin) with training. These data suggest that insulin action is enhanced with short-term exercise training via an adaptation distal to PI 3-kinase in middle-aged, insulin-resistant individuals.