PFKFB3-dependent glucose metabolism regulates 3T3-L1 adipocyte development.

Proliferation and differentiation of preadipocytes, and other cell types, is accompanied by an increase in glucose uptake. Previous work showed that a pulse of high glucose was required during the first 3 days of differentiation in vitro, but was not required after that. The specific glucose metabolism pathways required for adipocyte differentiation are unknown. Herein, we used 3T3-L1 adipocytes as a model system to study glucose metabolism and expansion of the adipocyte metabolome during the first 3 days of differentiation. Our primary outcome measures were GLUT4 and adiponectin, key proteins associated with healthy adipocytes. Using complete media with 0 or 5 mM glucose, we distinguished between developmental features that were dependent on the differentiation cocktail of dexamethasone, insulin, and isobutylmethylxanthine alone or the cocktail plus glucose. Cocktail alone was sufficient to activate the capacity for 2-deoxglucose uptake and glycolysis, but was unable to support the expression of GLUT4 and adiponectin in mature adipocytes. In contrast, 5 mM glucose in the media promoted a transient increase in glucose uptake and glycolysis as well as a significant expansion of the adipocyte metabolome and proteome. Using genetic and pharmacologic approaches, we found that the positive effects of 5 mM glucose on adipocyte differentiation were specifically due to increased expression of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 3 (PFKFB3), a key regulator of glycolysis and the ancillary glucose metabolic pathways. Our data reveal a critical role for PFKFB3 activity in regulating the cellular metabolic remodeling required for adipocyte differentiation and maturation.

A biallelic pathogenic variant in the OGDH gene results in a neurological disorder with features of a mitochondrial disease.

2-Oxoglutarate dehydrogenase (OGDH) is a rate-limiting enzyme in the mitochondrial TCA cycle, encoded by the OGDH gene. α-Ketoglutarate dehydrogenase (OGDH) deficiency was previously reported in association with developmental delay, hypotonia, and movement disorders and metabolic decompensation, with no genetic data provided. Using whole exome sequencing, we identified two individuals carrying a homozygous missense variant c.959A>G (p.N320S) in the OGDH gene. These individuals presented with global developmental delay, elevated lactate, ataxia and seizure. Fibroblast analysis and modeling of the mutation in Drosophila were used to evaluate pathogenicity of the variant. Skin fibroblasts from subject # 2 showed a decrease in both OGDH protein and enzyme activity. Transfection of human OGDH cDNA in HEK293 cells carrying p.N320S also produced significantly lower protein levels compared to those with wild-type cDNA. Loss of Drosophila Ogdh (dOgdh) caused early developmental lethality, rescued by expressing wild-type dOgdh (dOgdhWT ) or human OGDH (OGDHWT ) cDNA. In contrast, expression to the mutant OGDH (OGDHN320S ) or dOgdh carrying homologous mutations to human OGDH p.N320S variant (dOgdhN324S ) failed to rescue lethality of dOgdh null mutants. Knockdown of dOgdh in the nervous system resulted in locomotion defects which were rescued by dOgdhWT expression but not by dOgdhN324S expression. Collectively, the results indicate that c.959A>G variant in OGDH leads to an amino acid change (p.N320S) causing a severe loss of OGDH protein function. Our study establishes in the first time a genetic link between an OGDH gene mutation and OGDH deficiency.

Enhancing cardiac glycolysis causes an increase in PDK4 content in response to short-term high-fat diet.

The healthy heart has a dynamic capacity to respond and adapt to changes in nutrient availability. Metabolic inflexibility, such as occurs with diabetes, increases cardiac reliance on fatty acids to meet energetic demands, and this results in deleterious effects, including mitochondrial dysfunction, that contribute to pathophysiology. Enhancing glucose usage may mitigate metabolic inflexibility and be advantageous under such conditions. Here, we sought to identify how mitochondrial function and cardiac metabolism are affected in a transgenic mouse model of enhanced cardiac glycolysis (GlycoHi) basally and following a short-term (7-day) high-fat diet (HFD). GlycoHi mice constitutively express an active form of phosphofructokinase-2, resulting in elevated levels of the PFK-1 allosteric activator fructose 2,6-bisphosphate. We report that basally GlycoHi mitochondria exhibit augmented pyruvate-supported respiration relative to fatty acids. Nevertheless, both WT and GlycoHi mitochondria had a similar shift toward increased rates of fatty acid-supported respiration following HFD. Metabolic profiling by GC-MS revealed distinct features based on both genotype and diet, with a unique increase in branched-chain amino acids in the GlycoHi HFD group. Targeted quantitative proteomics analysis also supported both genotype- and diet-dependent changes in protein expression and uncovered an enhanced expression of pyruvate dehydrogenase kinase 4 (PDK4) in the GlycoHi HFD group. These results support a newly identified mechanism whereby the levels of fructose 2,6-bisphosphate promote mitochondrial PDK4 levels and identify a secondary adaptive response that prevents excessive mitochondrial pyruvate oxidation when glycolysis is sustained after a high-fat dietary challenge.

Loss of mitochondrial protease ClpP protects mice from diet-induced obesity and insulin resistance.

Caseinolytic peptidase P (ClpP) is a mammalian quality control protease that is proposed to play an important role in the initiation of the mitochondrial unfolded protein response (UPRmt), a retrograde signaling response that helps to maintain mitochondrial protein homeostasis. Mitochondrial dysfunction is associated with the development of metabolic disorders, and to understand the effect of a defective UPRmt on metabolism, ClpP knockout (ClpP-/-) mice were analyzed. ClpP-/- mice fed ad libitum have reduced adiposity and paradoxically improved insulin sensitivity. Absence of ClpP increased whole-body energy expenditure and markers of mitochondrial biogenesis are selectively up-regulated in the white adipose tissue (WAT) of ClpP-/- mice. When challenged with a metabolic stress such as high-fat diet, despite similar caloric intake, ClpP-/- mice are protected from diet-induced obesity, glucose intolerance, insulin resistance, and hepatic steatosis. Our results show that absence of ClpP triggers compensatory responses in mice and suggest that ClpP might be dispensable for mammalian UPRmt initiation. Thus, we made an unexpected finding that deficiency of ClpP in mice is metabolically beneficial.

GC-MS metabolic profiling reveals fructose-2,6-bisphosphate regulates branched chain amino acid metabolism in the heart during fasting.

INTRODUCTION: As an insulin sensitive tissue, the heart decreases glucose usage during fasting. This response is mediated, in part, by decreasing phosphofructokinase-2 (PFK-2) activity and levels of its product fructose-2,6-bisphosphate. However, the importance of fructose-2,6-bisphosphate in the fasting response on other metabolic pathways has not been evaluated. OBJECTIVES: The goal of this study is to determine how sustaining cardiac fructose-2,6-bisphosphate levels during fasting affects the metabolomic profile. METHODS: Control and transgenic mice expressing a constitutively active form of PFK-2 (GlycoHi) were subjected to either 12-h fasting or regular feeding. Animals (n = 4 per group) were used for whole-heart extraction, followed by gas chromatography-mass spectrometry metabolic profiling and multivariate data analysis. RESULTS: Principal component analysis displayed differences between Control and GlycoHi groups under both fasting and fed conditions while a clear response to fasting was observed only for Control animals. However, pathway analysis revealed that these smaller changes in the GlycoHi group were significantly associated with branched-chain amino acid (BCAA) metabolism (~ 40% increase in all BCAAs). Correlation network analysis demonstrated clear differences in response to fasting between Control and GlycoHi groups amongst most parameters. Notably, fasting caused an increase in network density in the Control group from 0.12 to 0.14 while the GlycoHi group responded oppositely (0.17-0.15). CONCLUSIONS: Elevated cardiac PFK-2 activity during fasting selectively increases BCAAs levels and decreases global changes in metabolism.

Decreased Mitochondrial Pyruvate Transport Activity in the Diabetic Heart: ROLE OF MITOCHONDRIAL PYRUVATE CARRIER 2 (MPC2) ACETYLATION.

Alterations in mitochondrial function contribute to diabetic cardiomyopathy. We have previously shown that heart mitochondrial proteins are hyperacetylated in OVE26 mice, a transgenic model of type 1 diabetes. However, the universality of this modification and its functional consequences are not well established. In this study, we demonstrate that Akita type 1 diabetic mice exhibit hyperacetylation. Functionally, isolated Akita heart mitochondria have significantly impaired maximal (state 3) respiration with physiological pyruvate (0.1 mm) but not with 1.0 mm pyruvate. In contrast, pyruvate dehydrogenase activity is significantly decreased regardless of the pyruvate concentration. We found that there is a 70% decrease in the rate of pyruvate transport in Akita heart mitochondria but no decrease in the mitochondrial pyruvate carriers 1 and 2 (MPC1 and MPC2). The potential role of hyperacetylation in mediating this impaired pyruvate uptake was examined. The treatment of control mitochondria with the acetylating agent acetic anhydride inhibits pyruvate uptake and pyruvate-supported respiration in a similar manner to the pyruvate transport inhibitor α-cyano-4-hydroxycinnamate. A mass spectrometry selective reactive monitoring assay was developed and used to determine that acetylation of lysines 19 and 26 of MPC2 is enhanced in Akita heart mitochondria. Expression of a double acetylation mimic of MPC2 (K19Q/K26Q) in H9c2 cells was sufficient to decrease the maximal cellular oxygen consumption rate. This study supports the conclusion that deficient pyruvate transport activity, mediated in part by acetylation of MPC2, is a contributor to metabolic inflexibility in the diabetic heart.

Prognostic Significance of QT Interval Dispersion in the Response to Intravenous Immunoglobulin Therapy in Kawasaki Disease.

BACKGROUND: Kawasaki disease (KD) is classified as a systemic vasculitis syndrome and QT interval dispersion (QTD) has been associated with cardiac involvement and disease activity in patients with cardiovasculitis. We examined whether baseline QTD could predict a response to intravenous immunoglobulin (IVIG) in KD.Methods and Results:QTD was recorded in 86 patients with KD before IVIG, who were separated into IVIG responders (R group; n=62) and nonresponders (N group; n=24). The association between baseline QTD and response to IVIG was investigated, and the predictive response value was compared with conventional risk scores from Gunma and Kurume universities. Baseline-corrected QTDs with Bazett's (QTbcD) and Fridericia's (QTfcD) formulae were significantly increased in the N group (R group vs. N group: 31.6 [28.3, 44.0] ms vs. 66.6 [50.5, 76.3] ms and 27.4 [25.2, 39.1] ms vs. 55.2 [42.4, 66.3] ms, respectively, both P<0.001). Multiple logistic regression analysis revealed QTfcD as an independent predictor of a response to IVIG after adjustment for conventional scores (odds ratio: 1.133, 95% confidence interval: 1.061-1.210, P<0.001). Moreover, QTfcD provided incremental predictive value for IVIG nonresponders over Gunma score (increment in global χ2=25.46, P<0.001). CONCLUSIONS: QTD was significantly associated with a response to IVIG in KD patients and may represent a useful identifier of IVIG nonresponders with high risk of coronary aneurysm.

Decreased Aortic Elasticity in Children With Marfan Syndrome or Loeys-Dietz Syndrome.

BACKGROUND: The characteristics of aortic elasticity are unclear in children with connective tissue disorders (CTDs) such as Marfan syndrome (MFS) and Loeys-Dietz syndrome (LDS), especially in those with a non-dilated aortic root (AoR). This study evaluated the aortic elasticity properties of pediatric MFS and LDS patients with either dilated or non-dilated AoR.Methods and Results:The 31 children with MFS or LDS were classified into dilated (Z score of AoR diameter ≥2.5; n=17) or non-dilated (Z score of AoR diameter <2.5; n=14) AoR groups and compared with controls. Using transthoracic echocardiography, we analyzed the aortic elasticity parameters of distensibility, strain, and stiffness index at the levels of the AoR, sinotubular junction, ascending aorta, and descending aorta. Aortic distensibility and strain were significantly lower in both test groups than in controls at the AoR level. The Z score of AoR diameter significantly correlated with aortic distensibility (R=-0.63, P<0.001), strain (R=-0.54, P=0.002), and stiffness index (R=0.52, P=0.002) in the patients' groups. Multivariate analysis revealed that aortic distensibility and the type of CTD were independently associated with AoR dilatation. CONCLUSIONS: Aortic elasticity at the level of the AoR may be decreased in children with MFS or LDS even before AoR dilatation progresses. Less aortic distensibility and CTD type are considered important parameters in estimating AoR dilatation in these patients. (Circ J 2016; 80: 2369-2375).

Successful treatment of arrhythmia-induced cardiomyopathy in an infant with tuberous sclerosis complex.

BACKGROUND: Tuberous sclerosis complex (TSC) is an autosomal-dominant tumor suppressor gene syndrome that is characterized by the development of distinctive benign tumors and malformations in multiple organ systems (N Eng J Med 355:1345-1356, 2006). Cardiac rhabdomyomas are intracavitary or intramural tumors observed in 50-70 % of infants with TSC but only cause serious clinical problems in a very small fraction of these patients (N Eng J Med 355:1345-1356, 2006; Pediatrics 118:1146-1151, 2006; Eur J Pediatr 153:155-7, 1994); most individuals have no clinical symptoms and their tumors spontaneously regress. However, despite being clinically silent, these lesions can provoke arrhythmias and heart failure (Pediatrics 118:1146-1151, 2006; Eur J Pediatr 153:155-7, 1994). CASE PRESENTATION: We here report the clinical findings of an infant suffering from TSC complicated with dilated cardiomyopathy (DCM) after the regression of cardiac rhabdomyomas. Although his tumors improved spontaneously, tachycardia and irregular heart rate due to frequent premature ventricular and supraventricular contractions persisted from the newborn period and were refractory to several medications. His cardiomyopathy was suspected to have been induced by the tachycardia or arrhythmia. We found carvedilol therapy to be safe and highly effective in treating the cardiomyopathy. To our knowledge, this is the first case report of TSC with DCM after regression of cardiac tumors and its successful treatment. CONCLUSION: The patient's clinical course suggests that careful life-long disease management is important, even in TSC patients without apparent symptoms.

Left ventricular non-compaction revealed by aortic regurgitation due to Kawasaki disease in a boy with LDB3 mutation.

Kawasaki disease (KD) is an acute febrile illness of childhood characterized by systemic vasculitis, especially coronary arteritis. Aortic valve regurgitation (AVR) is a relatively common complication. There have been no reports to date of heart failure and left ventricular non-compaction (LVNC) after acute KD, although the precise etiology of this condition remains unclear. A 6-month-old boy with KD was admitted to hospital. Despite high-dose i.v. gammaglobulin for dilation of the coronary artery, moderate AVR appeared, and thereafter he developed heart failure. A rough, dense LV myocardium indicated LVNC. On genetic testing a heterogenous 163G > A substitution changing a valine to isoleucine in LIM domain binding protein 3 (LDB3) was identified. Additional cardiac stress, such as that caused by AVR and/or KD might have triggered cardiac failure in the form of LVNC due to LDB3 mutation.

Selective inhibition of deactivated mitochondrial complex I by biguanides.

Biguanides are widely used antihyperglycemic agents for diabetes mellitus and prediabetes treatment. Complex I is the rate-limiting step of the mitochondrial electron transport chain (ETC), a major source of mitochondrial free radical production, and a known target of biguanides. Complex I has two reversible conformational states, active and de-active. The deactivated state is promoted in the absence of substrates but is rapidly and fully reversed to the active state in the presence of NADH. The objective of this study was to determine the relative sensitivity of active/de-active complex I to biguanide-mediated inhibition and resulting superoxide radical (O2(•⁻)) production. Using isolated rat heart mitochondria, we show that deactivation of complex I sensitizes it to metformin and phenformin (4- and 3-fold, respectively), but not to other known complex I inhibitors, such as rotenone. Mitochondrial O2(•⁻) production by deactivated complex I was measured fluorescently by NADH-dependent 2-hydroxyethidium formation at alkaline pH to impede reactivation. Superoxide production was 260.4% higher than in active complex I at pH 9.4. However, phenformin treatment of de-active complex I decreased O2(•⁻) production by 14.9%, while rotenone increased production by 42.9%. Mitochondria isolated from rat hearts subjected to cardiac ischemia, a condition known to induce complex I deactivation, were sensitized to phenformin-mediated complex I inhibition. This supports the idea that the effects of biguanides are likely to be influenced by the complex I state in vivo. These results demonstrate that the complex I active and de-active states are a determinant in biguanide-mediated inhibition.

Reversible cerebral vasoconstriction syndrome and posterior reversible encephalopathy syndrome in a boy with Loeys-Dietz syndrome.

Loeys-Dietz syndrome (LDS) is an autosomal dominant connective tissue disorder, caused by heterozygous mutations in TGFBR1 or TGFBR2 and characterized by vascular complications (cerebral, thoracic, and abdominal arterial aneurysms and/or dissections) and skeletal manifestations. We here report the first patient with LDS presenting with reversible cerebral vasoconstriction syndrome (RCVS), a clinico-radiological condition characterized by recurrent thunderclap headaches, with or without neurological symptoms, and reversible vasoconstriction of cerebral arteries. The patient was a 9-year-old boy with a heterozygous TGFBR2 mutation, manifesting camptodactyly, talipes equinovarus, and lamboid craniosynostosis. He complained of severe recurrent headaches 2 months after total aortic replacement for aortic root dilatation and a massive Stanford type B aortic dissection. A thoracic CT scan revealed a left subclavian artery dissection. Brain MRI and MRA detected bilateral internal carotid artery constriction along with a cortical subarachnoid hemorrhage without intracranial aneurysms. Subsequently, he developed visual disturbance and a generalized seizure associated with multiple legions of cortical and subcortical increased signals including the left posterior lobe, consistent with posterior reversible encephalopathy syndrome (PRES), a condition characterized by headaches, visual disorders, seizures, altered mentation, consciousness disturbances, focal neurological signs, and vasogenic edema predominantly in the white matter of the posterior lobe. Vasoconstriction of the internal carotid artery was undetectable 2 months later, and he was diagnosed as having RCVS. Endothelial dysfunction, associated with impaired TGF-ß signaling, might have been attributable to the development of RCVS and PRES.

Metabolic inflexibility and protein lysine acetylation in heart mitochondria of a chronic model of type 1 diabetes.

Diabetic cardiomyopathy refers to the changes in contractility that occur to the diabetic heart that can arise in the absence of vascular disease. Mitochondrial bioenergetic deficits and increased free radical production are pathological hallmarks of diabetic cardiomyopathy, but the mechanisms and causal relationships between mitochondrial deficits and the progression of disease are not understood. We evaluated cardiac mitochondrial function in a rodent model of chronic Type 1 diabetes (OVE26 mice) before the onset of contractility deficits. We found that the most pronounced change in OVE26 heart mitochondria is severe metabolic inflexibility. This inflexibility is characterized by large deficits in mitochondrial respiration measured in the presence of non-fatty acid substrates. Metabolic inflexibility occurred concomitantly with decreased activities of PDH (pyruvate dehydrogenase) and complex II. Hyper-acetylation of protein lysine was also observed. Treatment of control heart mitochondria with acetic anhydride (Ac2O), an acetylating agent, preferentially inhibited respiration by non-fatty acid substrates and increased superoxide production. We have concluded that metabolic inflexibility, induced by discrete enzymatic and molecular changes, including hyper-acetylation of protein lysine residues, precedes mitochondrial defects in a chronic rodent model of Type 1 diabetes.

Loss of Cardiac PFKFB2 Drives Metabolic, Functional, and Electrophysiological Remodeling in the Heart.

BACKGROUND: Phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFK-2) is a critical glycolytic regulator responsible for upregulation of glycolysis in response to insulin and adrenergic signaling. PFKFB2, the cardiac isoform of PFK-2, is degraded in the heart in the absence of insulin signaling, contributing to diabetes-induced cardiac metabolic inflexibility. However, previous studies have not examined how the loss of PFKFB2 affects global cardiac metabolism and function. METHODS AND RESULTS: To address this, we have generated a mouse model with a cardiomyocyte-specific knockout of PFKFB2 (cKO). Using 9-month-old cKO and control mice, we characterized the impacts of PFKFB2 on cardiac metabolism, function, and electrophysiology. cKO mice have a shortened life span of 9 months. Metabolically, cKO mice are characterized by increased glycolytic enzyme abundance and pyruvate dehydrogenase activity, as well as decreased mitochondrial abundance and beta oxidation, suggesting a shift toward glucose metabolism. This was supported by a decrease in the ratio of palmitoyl carnitine to pyruvate-dependent mitochondrial respiration in cKO relative to control animals. Metabolomic, proteomic, and Western blot data support the activation of ancillary glucose metabolism, including pentose phosphate and hexosamine biosynthesis pathways. Physiologically, cKO animals exhibited impaired systolic function and left ventricular dilation, represented by reduced fractional shortening and increased left ventricular internal diameter, respectively. This was accompanied by electrophysiological alterations including increased QT interval and other metrics of delayed ventricular conduction. CONCLUSIONS: Loss of PFKFB2 results in metabolic remodeling marked by cardiac ancillary pathway activation. This could delineate an underpinning of pathologic changes to mechanical and electrical function in the heart.

The loss of cardiac SIRT3 decreases metabolic flexibility and proteostasis in an age-dependent manner.

SIRT3 is a longevity factor that acts as the primary deacetylase in mitochondria. Although ubiquitously expressed, previous global SIRT3 knockout studies have shown primarily a cardiac-specific phenotype. Here, we sought to determine how specifically knocking out SIRT3 in cardiomyocytes (SIRTcKO mice) temporally affects cardiac function and metabolism. Mice displayed an age-dependent increase in cardiac pathology, with 10-month-old mice exhibiting significant loss of systolic function, hypertrophy, and fibrosis. While mitochondrial function was maintained at 10 months, proteomics and metabolic phenotyping indicated SIRT3 hearts had increased reliance on glucose as an energy substrate. Additionally, there was a significant increase in branched-chain amino acids in SIRT3cKO hearts without concurrent increases in mTOR activity. Heavy water labeling experiments demonstrated that, by 3 months of age, there was an increase in protein synthesis that promoted hypertrophic growth with a potential loss of proteostasis in SIRT3cKO hearts. Cumulatively, these data show that the cardiomyocyte-specific loss of SIRT3 results in severe pathology with an accelerated aging phenotype.

Loss of cardiac PFKFB2 drives Metabolic, Functional, and Electrophysiological Remodeling in the Heart.

Background: Phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFK-2) is a critical glycolytic regulator responsible for upregulation of glycolysis in response to insulin and adrenergic signaling. PFKFB2, the cardiac isoform of PFK-2, is degraded in the heart in the absence of insulin signaling, contributing to diabetes-induced cardiac metabolic inflexibility. However, previous studies have not examined how the loss of PFKFB2 affects global cardiac metabolism and function. Methods: To address this, we have generated a mouse model with a cardiomyocyte-specific knockout of PFKFB2 (cKO). Using 9-month-old cKO and control (CON) mice, we characterized impacts of PFKFB2 on cardiac metabolism, function, and electrophysiology. Results: cKO mice have a shortened lifespan of 9 months. Metabolically, cKO mice are characterized by increased glycolytic enzyme abundance and pyruvate dehydrogenase (PDH) activity, as well as decreased mitochondrial abundance and beta oxidation, suggesting a shift toward glucose metabolism. This was supported by a decrease in the ratio of palmitoyl carnitine to pyruvate-dependent mitochondrial respiration in cKO relative to CON animals. Metabolomic, proteomic, and western blot data support the activation of ancillary glucose metabolism, including pentose phosphate and hexosamine biosynthesis pathways. Physiologically, cKO animals exhibited impaired systolic function and left ventricular (LV) dilation, represented by reduced fractional shortening and increased LV internal diameter, respectively. This was accompanied by electrophysiological alterations including increased QT interval and other metrics of delayed ventricular conduction. Conclusions: Loss of PFKFB2 results in metabolic remodeling marked by cardiac ancillary pathway activation. This could delineate an underpinning of pathologic changes to mechanical and electrical function in the heart. Clinical Perspective: What is New?: We have generated a novel cardiomyocyte-specific knockout model of PFKFB2, the cardiac isoform of the primary glycolytic regulator Phosphofructokinase-2 (cKO).The cKO model demonstrates that loss of cardiac PFKFB2 drives metabolic reprogramming and shunting of glucose metabolites to ancillary metabolic pathways.The loss of cardiac PFKFB2 promotes electrophysiological and functional remodeling in the cKO heart.What are the Clinical Implications?: PFKFB2 is degraded in the absence of insulin signaling, making its loss particularly relevant to diabetes and the pathophysiology of diabetic cardiomyopathy.Changes which we observe in the cKO model are consistent with those often observed in diabetes and heart failure of other etiologies.Defining PFKFB2 loss as a driver of cardiac pathogenesis identifies it as a target for future investigation and potential therapeutic intervention.

Increased cardiac PFK-2 protects against high-fat diet-induced cardiomyopathy and mediates beneficial systemic metabolic effects.

A healthy heart adapts to changes in nutrient availability and energy demands. In metabolic diseases like type 2 diabetes (T2D), increased reliance on fatty acids for energy production contributes to mitochondrial dysfunction and cardiomyopathy. A principal regulator of cardiac metabolism is 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFK-2), which is a central driver of glycolysis. We hypothesized that increasing PFK-2 activity could mitigate cardiac dysfunction induced by high-fat diet (HFD). Wild type (WT) and cardiac-specific transgenic mice expressing PFK-2 (GlycoHi) were fed a low fat or HFD for 16 weeks to induce metabolic dysfunction. Metabolic phenotypes were determined by measuring mitochondrial bioenergetics and performing targeted quantitative proteomic and metabolomic analysis. Increasing cardiac PFK-2 had beneficial effects on cardiac and mitochondrial function. Unexpectedly, GlycoHi mice also exhibited sex-dependent systemic protection from HFD, including increased glucose homeostasis. These findings support improving glycolysis via PFK-2 activity can mitigate mitochondrial and functional changes that occur with metabolic syndrome.

Identification of mitochondrial electron transport chain-mediated NADH radical formation by EPR spin-trapping techniques.

The mitochondrial electron transport chain (ETC) is a major source of free radical production. However, due to the highly reactive nature of radical species and their short lifetimes, accurate detection and identification of these molecules in biological systems is challenging. The aim of this investigation was to determine the free radical species produced from the mitochondrial ETC by utilizing EPR spin-trapping techniques and the recently commercialized spin-trap, 5-(2,2-dimethyl-1,3-propoxycyclophosphoryl)-5-methyl-1-pyrroline N-oxide (CYPMPO). We demonstrate that this spin-trap has the preferential quality of having minimal mitochondrial toxicity at concentrations required for radical detection. In rat heart mitochondria and submitochondrial particles supplied with NADH, the major species detected under physiological pH was a carbon-centered radical adduct, indicated by markedly large hyperfine coupling constant with hydrogen (a(H) > 2.0 mT). In the presence of the ETC inhibitors, the carbon-centered radical formation was increased and exhibited NADH concentration dependency. The same carbon-centered radical could also be produced with the NAD biosynthesis precursor, nicotinamide mononucleotide, in the presence of a catalytic amount of NADH. The results support the conclusion that the observed species is a complex I derived NADH radical. The formation of the NADH radical could be blocked by hydroxyl radical scavengers but not SOD. In vitro experiments confirmed that an NADH-radical is readily formed by hydroxyl radical but not superoxide anion, further implicating hydroxyl radical as an upstream mediator of NADH radical production. These findings demonstrate the identification of a novel mitochondrial radical species with potential physiological significance and highlight the diverse mechanisms and sites of production within the ETC.

Maintenance of cardiac energy metabolism by histone deacetylase 3 in mice.

Histone deacetylase (HDAC) inhibitors show remarkable therapeutic potential for a variety of disorders, including cancer, neurological disease, and cardiac hypertrophy. However, the specific HDAC isoforms that mediate their actions are unclear, as are the physiological and pathological functions of individual HDACs in vivo. To explore the role of Hdac3 in the heart, we generated mice with a conditional Hdac3 null allele. Although global deletion of Hdac3 resulted in lethality by E9.5, mice with a cardiac-specific deletion of Hdac3 survived until 3-4 months of age. At this time, they showed massive cardiac hypertrophy and upregulation of genes associated with fatty acid uptake, fatty acid oxidation, and electron transport/oxidative phosphorylation accompanied by fatty acid-induced myocardial lipid accumulation and elevated triglyceride levels. These abnormalities in cardiac metabolism can be attributed to excessive activity of the nuclear receptor PPARalpha. The phenotype associated with cardiac-specific Hdac3 gene deletion differs from that of all other Hdac gene mutations. These findings reveal a unique role for Hdac3 in maintenance of cardiac function and regulation of myocardial energy metabolism.

Inhibition of mitochondrial respiration by phosphoenolpyruvate.

The cytosolic factors that influence mitochondrial oxidative phosphorylation rates are relatively unknown. In this report, we examine the effects of phosphoenolpyruvate (PEP), a glycolytic intermediate, on mitochondrial function. It is reported here that in rat heart mitochondria, PEP delays the onset of state 3 respiration in mitochondria supplied with either NADH-linked substrates or succinate. However, the maximal rate of state 3 respiration is only inhibited when oxidative phosphorylation is supported by NADH-linked substrates. The capacity of PEP to delay and/or inhibit state 3 respiration is dependent upon the presence or absence of ATP. Inhibition of state 3 is exacerbated in uncoupled mitochondria, with a 40% decrease in respiration seen with 0.1mM PEP. In contrast, ATP added exogenously or produced by oxidative phosphorylation completely prevents PEP-mediated inhibition. Mechanistically, the results support the conclusion that the main effects of PEP are to impede ADP uptake and inhibit NADH oxidation. By altering the NADH/NAD(+) status of mitochondria, it is demonstrated that PEP enhances succinate dehydrogenase activity and increase free radical production. The results of this study indicate PEP may be an important modulator of mitochondrial function under conditions of decreased ATP.