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BackgroundAlthough metabolic reprogramming is critical in the pathogenesis of heart failure, studies to date have focused principally on fatty acid and glucose metabolism. Contribution of amino acid metabolic regulation in the disease remains understudied.Methods and resultsTranscriptomic and metabolomic analyses were performed in mouse failing heart induced by pressure overload. Suppression of branched-chain amino acid (BCAA) catabolic gene expression along with concomitant tissue accumulation of branched-chain α-keto acids was identified as a significant signature of metabolic reprogramming in mouse failing hearts and validated to be shared in human cardiomyopathy hearts. Molecular and genetic evidence identified the transcription factor Krüppel-like factor 15 as a key upstream regulator of the BCAA catabolic regulation in the heart. Studies using a genetic mouse model revealed that BCAA catabolic defect promoted heart failure associated with induced oxidative stress and metabolic disturbance in response to mechanical overload. Mechanistically, elevated branched-chain α-keto acids directly suppressed respiration and induced superoxide production in isolated mitochondria. Finally, pharmacological enhancement of branched-chain α-keto acid dehydrogenase activity significantly blunted cardiac dysfunction after pressure overload.ConclusionsBCAA catabolic defect is a metabolic hallmark of failing heart resulting from Krüppel-like factor 15-mediated transcriptional reprogramming. BCAA catabolic defect imposes a previously unappreciated significant contribution to heart failure.

Classic physiology studies dating to the 1930s demonstrate that moderate or transient glucocorticoid (GC) exposure improves muscle performance. The ergogenic properties of GCs are further evidenced by their surreptitious use as doping agents by endurance athletes and poorly understood efficacy in Duchenne muscular dystrophy (DMD), a genetic muscle-wasting disease. A defined molecular basis underlying these performance-enhancing properties of GCs in skeletal muscle remains obscure. Here, we demonstrate that ergogenic effects of GCs are mediated by direct induction of the metabolic transcription factor KLF15, defining a downstream pathway distinct from that resulting in GC-related muscle atrophy. Furthermore, we establish that KLF15 deficiency exacerbates dystrophic severity and muscle GC-KLF15 signaling mediates salutary therapeutic effects in the mdx mouse model of DMD. Thus, although glucocorticoid receptor (GR)-mediated transactivation is often associated with muscle atrophy and other adverse effects of pharmacologic GC administration, our data define a distinct GR-induced gene regulatory pathway that contributes to therapeutic effects of GCs in DMD through proergogenic metabolic programming.

Skeletal muscle is a major determinant of systemic metabolic homeostasis that plays a critical role in glucose metabolism and insulin sensitivity. By contrast, despite being a major user of fatty acids, and evidence that muscular disorders can lead to abnormal lipid deposition (e.g., nonalcoholic fatty liver disease in myopathies), our understanding of skeletal muscle regulation of systemic lipid homeostasis is not well understood. Here we show that skeletal muscle Kruppel-like factor 15 (KLF15) coordinates pathways central to systemic lipid homeostasis under basal conditions and in response to nutrient overload. Mice with skeletal muscle-specific KLF15 deletion demonstrated (a) reduced expression of key targets involved in lipid uptake, mitochondrial transport, and utilization, (b) elevated circulating lipids, (c) insulin resistance/glucose intolerance, and (d) increased lipid deposition in white adipose tissue and liver. Strikingly, a diet rich in short-chain fatty acids bypassed these defects in lipid flux and ameliorated aspects of metabolic dysregulation. Together, these findings establish skeletal muscle control of lipid flux as critical to systemic lipid homeostasis and metabolic health., Introduction Beyond the structural and locomotive functions classically ascribed to skeletal muscle, this tissue represents the largest metabolic organ by mass and is a major site for the metabolism of [...]

Abdominal aortic aneurysm (AAA) is a major cause of morbidity and mortality; however, the mechanisms that are involved in disease initiation and progression are incompletely understood. Extracellular matrix proteins play an integral role in modulating vascular homeostasis in health and disease. Here, we determined that the expression of the matricellular protein CCN3 is strongly reduced in rodent AAA models, including angiotensin Il-induced AAA and elastase perfusion-stimulated AAA. CCN3 levels were also reduced in human AAA biopsies compared with those in controls. In murine models of induced AAA, germline deletion of Ccn3 resulted in severe phenotypes characterized by elastin fragmentation, vessel dilation, vascular inflammation, dissection, heightened ROS generation, and smooth muscle cell loss. Conversely, overexpression of CCN3 mitigated both elastase- and angiotensin Il-induced AAA formation in mice. BM transplantation experiments suggested that the AAA phenotype of CCN3-deficient mice is intrinsic to the vasculature, as AAA was not exacerbated in WT animals that received CCN3-deficient BM and WT BM did not reduce AAA severity in CCN3-deficient mice. Genetic and pharmacological approaches implicated the ERK1/2 pathway as a critical regulator of CCN3-dependent AAA development. Together, these results demonstrate that CCN3 is a nodal regulator in AAA biology and identify CCN3 as a potential therapeutic target for vascular disease., Introduction Disruption of aortic homeostasis arising from genetic defects or exposure to environmental risk factors leads to localized abnormal widening of the aorta, a degenerative disease state termed aortic aneurysm [...]

Mitochondrial homeostasis is critical for tissue health, and mitochondrial dysfunction contributes to numerous diseases, including heart failure. Here, we have shown that the transcription factor Kruppel-like factor 4 (KLF4) governs mitochondrial biogenesis, metabolic function, dynamics, and autophagic clearance. Adult mice with cardiac-specific Klf4 deficiency developed cardiac dysfunction with aging or in response to pressure overload that was characterized by reduced myocardial ATP levels, elevated ROS, and marked alterations in mitochondrial shape, size, ultrastructure, and alignment. Evaluation of mitochondria isolated from KLF4-deficient hearts revealed a reduced respiration rate that is likely due to defects in electron transport chain complex I. Further, cardiac- specific, embryonic Klf4 deletion resulted in postnatal premature mortality, impaired mitochondrial biogenesis, and altered mitochondrial maturation. We determined that KLF4 binds to, cooperates with, and is requisite for optimal function of the estrogen-related receptor/PPARγ coactivator 1 (ERR/PGC-1) transcriptional regulatory module on metabolic and mitochondrial targets. Finally, we found that KLF4 regulates autophagy flux through transcriptional regulation of a broad array of autophagy genes in cardiomyocytes. Collectively, these findings identify KLF4 as a nodal transcriptional regulator of mitochondrial homeostasis., Introduction The heart has an unrelenting need for energy throughout life to sustain contractile function. Despite its high-energy demand, the heart contains relatively low ATP reserves, and thus, a continual [...]

Activation of cells intrinsic to the vessel wall is central to the initiation and progression of vascular inflammation. As the dominant cellular constituent of the vessel wall, vascular smooth muscle [...]

Vascular calcification is a multifaceted process involving gain of calcification inducers and loss of calcification inhibitors. One such inhibitor is inorganic pyrophosphate ([PP.sub.i]), and regulated generation and homeostasis of extracellular [PP.sub.i] is a critical determinant of soft-tissue mineralization. We recently described an autocrine mechanism of extracellular [PP.sub.i] generation in cultured rat aortic vascular smooth muscle cells (VSMC) that involves both ATP release coupled to the ectophosphodiesterase/pyrophosphatase ENPP1 and efflux of intracellular [PP.sub.i] mediated or regulated by the plasma membrane protein ANK. We now report that increased cAMP signaling and elevated extracellular inorganic phosphate ([P.sub.i]) act synergistically to induce calcification of these VSMC that is correlated with progressive reduction in ability to accumulate extracellular [PP.sub.i]. Attenuated [PP.sub.i] accumulation was mediated in part by cAMP-dependent decrease in ANK expression coordinated with cAMP-dependent increase in expression of TNAP, the tissue nonselective alkaline phosphatase that degrades [PP.sub.i]. Stimulation of cAMP signaling did not alter ATP release or ENPP1 expression, and the cAMP-induced changes in ANK and TNAP expression were not sufficient to induce calcification. Elevated extracellular [P.sub.i] alone elicited only minor calcification and no significant changes in ANK, TNAP, or ENPP1. In contrast, combined with a cAMP stimulus, elevated [P.sub.i] induced decreases in the ATP release pathway(s) that supports ENPP1 activity: this resulted in markedly reduced rates of [PP.sub.i] accumulation that facilitated robust calcification. Calcified VSMC were characterized by maintained expression of multiple SMC differentiation marker proteins including smooth muscle (SM) [alpha]-actin, SM22[alpha], and calponin. Notably, addition of exogenous ATP (or [PP.sub.i] per se) rescued cAMP + phosphate-treated VSMC cultures from progression to the calcified state. These observations support a model in which extracellular [PP.sub.i] generation mediated by both ANK- and ATP release-dependent mechanisms serves as a critical regulator of VSMC calcification. ectonucleotide pyrophosphatase/phosphodiesterase-1; ANK; adenosine 3',5'-cyclic monophosphate; ATP release doi:10.1152/ajpcell.00419.2009.

Extracellular inorganic pyrophosphate ([PP.sub.i]) is a potent suppressor of physiological calcification in bone and pathological calcification in blood vessels. Ectonucleotide pyrophosphatase/phosphodiesterases (eNPPs) generate [PP.sub.i] via the hydrolysis of ATP released into extracellular compartments by poorly understood mechanisms. Here we report that cultured vascular smooth muscle cells (VSMC) from rat aorta generate extracellular [PP.sub.i] via an autocrine mechanism that involves ATP release tightly coupled to eNPP activity. The nucleotide analog [beta], [gamma]-methylene ATP (MeATP or AMPPCP) was used to selectively suppress ATP metabolism by eNPPs but not the CD39-type ecto-ATPases. In the absence of MeATP, VSMC generated extracellular [PP.sub.i] to accumulate [greater than or equal to] 600 nM within 2 h while steadily maintaining extracellular ATP at 1 nM. Conversely, the presence of MeATP completely suppressed [PP.sub.i] accumulation while increasing ATP accumulation. Probenecid, which inhibits [PP.sub.i] efflux dependent on ANK, a putative [PP.sub.i] transporter or transport regulator, reduced extracellular [PP.sub.i] accumulation by approximately twofold. This indicates that autocrine ATP release coupled to eNPP activity comprises [greater than or equal to] 50% of the extracellular PP,-generating capacity of VSMC. The accumulation of extracellular [PP.sub.i] and ATP was markedly attenuated by reduced temperature but was insensitive to brefeldin A, which suppresses constitutive exocytosis of Golgi-derived secretory vesicles. The magnitude of extracellular [PP.sub.i] accumulation in VSMC cultures increased with time postplating, suggesting that ATP release coupled to [PP.sub.i] generation is upregulated as cultured VSMC undergo contact-inhibition of proliferation or deposit extracellular matrix. adenosine 5'-triphosphate release; vascular calcification; ectonucleotide pyrophosphatase; ectoadenosine 5'-triphosphatase

The role of anaerobic glycolysis and oxidative substrate selection on contractile function and mechanical efficiency during moderate severity myocardial ischemia is unclear. We hypothesize that 1) preventing anaerobic glycolysis worsens contractile function and mechanical efficiency and 2) increasing glycolysis and glucose oxidation while inhibiting free fatty acid oxidation improves contractile function during ischemia. Experiments were performed in anesthetized pigs, with regional ischemia induced by a 60% decrease in left anterior descending coronary artery blood flow for 40 min. Three groups were studied: 1) no treatment, 2) inhibition of glycolysis with iodoacetate (IAA), or 3) hyperinsulinemia and hyperglycemia (HI + HG). Glucose and free fatty acid oxidation were measured using radioisotopes and anaerobic glycolysis from net lactate efflux and myocardial lactate content. Regional contractile power was assessed from left ventricular pressure and segment length in the anterior wall. We found that preventing anaerobic glycolysis with IAA during ischemia in the absence of alterations in free fatty acid and glucose oxidation did not adversely affect contractile function or mechanical efficiency during myocardial ischemia, suggesting that anaerobic glycolysis is not essential for maintaining residual contractile function. Increasing glycolysis and glucose oxidation with HI + HG inhibited free fatty acid oxidation and improved contractile function and mechanical efficiency. In conclusion, these results show a dissociation between myocardial function and anaerobic glycolysis during moderate severity ischemia in vivo, suggesting that metabolic therapies should not be aimed at inhibiting anaerobic glycolysis per se, but rather activating insulin signaling and/or enhancing carbohydrate oxidation and/or decreasing fatty acid oxidation. angina; fatty acids; glucose; insulin

Adipose tissue stores energy in the form of triglycerides. The ability to regulate triglyceride synthesis and breakdown based on nutrient status (e.g., fed versus fasted) is critical for physiological homeostasis and dysregulation of this process can contribute to metabolic disease. Whereas much is known about hormonal control of this cycle, transcriptional regulation is not well understood. Here, we show that the transcription factor Kruppel-like factor 15 (KLF15) is critical for the control of adipocyte lipid turnover. Mice lacking Klf15 in adipose tissue (AK15KO) display decreased adiposity and are protected from diet-induced obesity. Mechanistic studies suggest that adipose KLF15 regulates key genes of triglyceride synthesis and inhibits lipolytic action, thereby promoting lipid storage in an insulin-dependent manner. Finally, AK15KO mice demonstrate accelerated lipolysis and altered systemic energetics (e.g., locomotion, ketogenesis) during fasting conditions. Our study identifies adipose KLF15 as an essential regulator of adipocyte lipid metabolism and systemic energy balance. [ABSTRACT FROM AUTHOR]

The metabolic myocardium is an omnivore and utilizes various carbon substrates to meet its energetic demand. While the adult heart preferentially consumes fatty acids (FAs) over carbohydrates, myocardial fuel plasticity is essential for organismal survival. This metabolic plasticity governing fuel utilization is under robust transcriptional control and studies over the past decade have illuminated members of the nuclear receptor family of factors (e.g., PPARα) as important regulators of myocardial lipid metabolism. However, given the complexity of myocardial metabolism in health and disease, it is likely that other molecular pathways are likely operative and elucidation of such pathways may provide the foundation for novel therapeutic approaches. We previously demonstrated that Kruppel-like factor 15 (KLF15) is an independent regulator of cardiac lipid metabolism thus raising the possibility that KLF15 and PPARα operate in a coordinated fashion to regulate myocardial gene expression requisite for lipid oxidation. In the current study, we show that KLF15 binds to, cooperates with, and is required for the induction of canonical PPARα-mediated gene expression and lipid oxidation in cardiomyocytes. As such, this study establishes a molecular module involving KLF15 and PPARα and provides fundamental insights into the molecular regulation of cardiac lipid metabolism. [ABSTRACT FROM AUTHOR]

The role of anaerobic glycolysis and oxidative substrate selection on contractile function and mechanical efficiency during moderate severity myocardial ischemia is unclear. We hypothesize that 1) preventing anaerobic glycolysis worsens contractile function and mechanical efficiency and 2) increasing glycolysis and glucose oxidation while inhibiting free fatty acid oxidation improves contractile function during ischemia. Experiments were performed in anesthetized pigs, with regional ischemia induced by a 60% decrease in left anterior descending coronary artery blood flow for 40 mm. Three groups were studied: 1) no treatment, 2) inhibition of glycolysis with iodoacetate (IAA), or 3) hyperinsulinemia and hyperglycemia (HI + HG). Glucose and free fatty acid oxidation were measured using radioisotopes and anaerobic glycolysis from net lactate efflux and myocardial lactate content. Regional contractile power was assessed from left ventricular pressure and segment length in the anterior wall. We found that preventing anaerobic glycolysis with IAA during ischemia in the absence of alterations in free fatty acid and glucose oxidation did not adversely affect contractile function or mechanical efficiency during myocardial ischemia, suggesting that anaerobic glycolysis is not essential for maintaining residual contractile function. Increasing glycolysis and glucose oxidation with HI + HG inhibited free fatty acid oxidation and improved contractile function and mechanical efficiency. In conclusion, these results show a dissociation between myocardial function and anaerobic glycolysis during moderate severity ischemia in vivo, suggesting that metabolic therapies should not be aimed at inhibiting anaerobic glycolysis per se, but rather activating insulin signaling and/or enhancing carbohydrate oxidation and/or decreasing fatty acid oxidation. [ABSTRACT FROM AUTHOR]

Extracellular Pi and PPi are critical regulators of tissue calcification with PPi acting to inhibit mineralization. The balance between extracellular Pi and PPi concentration modulates hydroxyapatite crystal formation in soft tissue, and disruption of this balance results in aberrant mineralization. The objective of this study was to compare the expression and function of NPP1/PC-1, a PPi producing ecto-ATPase, versus NTPD1/CD39, a Pi producing ecto-ATPase, in primary rat aortic smooth muscle cells (SMC) and the A7r5 rat aortic SMC line. An online luciferase assay directly measured extracellular ATP hydrolysis in the presence/absence of bg-methylene ATP as a selective NPP1 inhibitor, while an indirect luciferase-based protocol measured extracellular PPi levels. NPP1 and NTPD1 mRNA transcripts (assayed by RT-PCR) were present in both primary SMC and A7r5 cells. NPP1 and NTPD1 contributed equally in metabolizing extracellular [ATP] less than 1 uM, while NPTD1 was the predominant ecto-ATPase at [ATP] greater than 10 uM. Hydrolysis of submicromolar ATP was correlated with PPi accumulation. These finding indicate that both NPP1 and NTPD1 play important ATP scavenging roles in vascular smooth muscle cells, but the respective Kd of these enzymes dictates the relative contributions these enzymes have in metabolizing extracellular ATP. [ABSTRACT FROM AUTHOR]