FoxO1 negatively regulates leptin-induced POMC transcription through its direct interaction with STAT3.

FoxO1, which is up-regulated during early stages of diet-induced leptin resistance, directly interacts with STAT3 and prevents STAT3 from binding to specificity protein 1 (SP1)-pro-opiomelanocortin (POMC) promoter complex, and thereby inhibits STAT3-mediated regulation of POMC transcription. FoxO1 also binds directly to the POMC promoter and negatively regulates its transcription. The present study aims to understand the relative contribution of the two interactions in regulating POMC expression. We studied the structural requirement of FoxO1 for its interaction with STAT3 and POMC promoter, and tested the inhibitory action of FoxO1 mutants by using biochemical assays, molecular biology and computer modelling. FoxO1 mutant with deletion of residues Ala137-Leu160 failed to bind to STAT3 or inhibit STAT3-mediated POMC activation, although its binding to the POMC promoter was unaffected. Further analysis mapped Gly140-Leu160 to be critical for STAT3 binding. The identified region Gly140-Leu160 was conserved among mammalian FoxO1 proteins, and showed a high degree of sequence identity with FoxO3, but not FoxO4. Consistently, FoxO3 could interact with STAT3 and inhibit POMC promoter activity, whereas FoxO4 could not bind to STAT3 or affect POMC promoter activity. We further identified that five residues (Gln145, Arg147, Lys148, Arg153 and Arg154) in FoxO1 were necessary in FoxO1-STAT3 interaction, and mutation of these residues abolished its interaction with STAT3 and inhibition of POMC promoter activity. Finally, a FoxO1-STAT3 interaction interface model generated by computational docking simulations confirmed that the identified residues of FoxO1 were in close proximity to STAT3. These results show that FoxO1 inhibits STAT3-mediated leptin signalling through direct interaction with STAT3.

Robust hyperpolarized (13)C metabolic imaging with selective non-excitation of pyruvate (SNEP).

In vivo metabolic imaging using hyperpolarized [1-(13)C]pyruvate provides localized biochemical information and is particularly useful in detecting early disease changes, as well as monitoring disease progression and treatment response. However, a major limitation of hyperpolarized magnetization is its unrecoverable decay, due not only to T1 relaxation but also to radio-frequency (RF) excitation. RF excitation schemes used in metabolic imaging must therefore be able to utilize available hyperpolarized magnetization efficiently and robustly for the optimal detection of substrate and metabolite activities. In this work, a novel RF excitation scheme called selective non-excitation of pyruvate (SNEP) is presented. This excitation scheme involves the use of a spectral selective RF pulse to specifically exclude the excitation of [1-(13)C]pyruvate, while uniformly exciting the key metabolites of interest (namely [1-(13)C]lactate and [1-(13)C]alanine) and [1-(13)C]pyruvate-hydrate. By eliminating the loss of hyperpolarized [1-(13)C]pyruvate magnetization due to RF excitation, the signal from downstream metabolite pools is increased together with enhanced dynamic range. Simulation results, together with phantom measurements and in vivo experiments, demonstrated the improvement in signal-to-noise ratio (SNR) and the extension of the lifetime of the [1-(13)C]lactate and [1-(13)C]alanine pools when compared with conventional non-spectral selective (NS) excitation. SNEP has also been shown to perform comparably well with multi-band (MB) excitation, yet SNEP possesses distinct advantages, including ease of implementation, less stringent demands on gradient performance, increased robustness to frequency drifts and B0 inhomogeneity as well as easier quantification involving the use of [1-(13)C]pyruvate-hydrate as a proxy for the actual [1-(13)C] pyruvate signal. SNEP is therefore a promising alternative for robust hyperpolarized [1-(13)C]pyruvate metabolic imaging with high fidelity.

In vivo hyperpolarized carbon-13 magnetic resonance spectroscopy reveals increased pyruvate carboxylase flux in an insulin-resistant mouse model.

UNLABELLED: The pathogenesis of type 2 diabetes is characterized by impaired insulin action and increased hepatic glucose production (HGP). Despite the importance of hepatic metabolic aberrations in diabetes development, there is currently no molecular probe that allows measurement of hepatic gluconeogenic pathways in vivo and in a noninvasive manner. In this study, we used hyperpolarized carbon 13 ((13)C)-labeled pyruvate magnetic resonance spectroscopy (MRS) to determine changes in hepatic gluconeogenesis in a high-fat diet (HFD)-induced mouse model of type 2 diabetes. Compared with mice on chow diet, HFD-fed mice displayed higher levels of oxaloacetate, aspartate, and malate, along with increased (13)C label exchange rates between hyperpolarized [1-(13) C]pyruvate and its downstream metabolites, [1-(13)C]malate and [1-(13)C]aspartate. Biochemical assays using liver extract revealed up-regulated malate dehydrogenase activity, but not aspartate transaminase activity, in HFD-fed mice. Moreover, the (13) C label exchange rate between [1-(13)C]pyruvate and [1-(13)C]aspartate (k(pyr->asp)) exhibited apparent correlation with gluconeogenic pyruvate carboxylase (PC) activity in hepatocytes. Finally, up-regulated HGP by glucagon stimulation was detected by an increase in aspartate signal and k(pyr->asp), whereas HFD mice treated with metformin for 2 weeks displayed lower production of aspartate and malate, as well as reduced k(pyr->asp) and (13)C-label exchange rate between pyruvate and malate, consistent with down-regulated gluconeogenesis. CONCLUSION: Taken together, we demonstrate that increased PC flux is an important pathway responsible for increased HGP in diabetes development, and that pharmacologically induced metabolic changes specific to the liver can be detected in vivo with a hyperpolarized (13)C-biomolecular probe. Hyperpolarized (13)C MRS and the determination of metabolite exchange rates may allow longitudinal monitoring of liver function in disease development.

Seipin differentially regulates lipogenesis and adipogenesis through a conserved core sequence and an evolutionarily acquired C-terminus.

Homozygous mutations in BSCL2 (Berardinelli-Seip congenital lipodystrophy)/seipin cause CGL2 (congenital generalized lipodystrophy type 2). Recent data suggest that seipin regulates LD (lipid droplet) dynamics and adipocyte differentiation, but whether these roles are mechanistically linked remains unclear. To understand how seipin regulates these processes, we investigated the evolutionary changes of seipin orthologues, and studied individual domains in regulating lipid accumulation in non-adipocytes and adipocytes. Mammalian seipins comprise at least two distinct functional domains, a conserved core sequence and an evolutionarily acquired C-terminus. Despite its requirement for adipocyte formation, seipin overexpression inhibited oleate-induced LD formation and accumulation in nonadipocytes, which was mediated by the core sequence. In contrast, seipin overexpression did not inhibit LD accumulation during adipocyte differentiation or the adipogenic process in 3T3-L1 cells. However, adipogenesis and LD accumulation were impaired in 3T3-L1 cells expressing a seipin mutant lacking the C-terminus. Furthermore, expression of the same mutant without the C-terminus failed to rescue the adipogenic defects in seipin-knockdown cells, demonstrating the importance of the C-terminus for seipin's function in adipocyte development. We propose that seipin is involved in lipid homoeostasis by restricting lipogenesis and LD accumulation in non-adipocytes, while promoting adipogenesis to accommodate excess energy storage.

Regulation of adipogenesis by cytoskeleton remodelling is facilitated by acetyltransferase MEC-17-dependent acetylation of α-tubulin.

Cytoskeleton remodelling is a prerequisite step for the morphological transition from preadipocytes to mature adipocytes. Although microtubules play a pivotal role in organizing cellular structure, regulation of microtubule dynamics during adipogenesis remains unclear. In the present paper we show that acetylation of α-tubulin is up-regulated during adipogenesis, and adipocyte development is dependent on α-tubulin acetylation, as expression of an acetylation-resistant α-tubulin mutant significantly inhibits adipogenesis. Moreover, acetylation of α-tubulin is under the control of the acetyltransferase MEC-17 and deacetylases SIRT2 (Sirtuin 2) and HDAC6 (histone deacetylase 6). Adipocyte development is inhibited in MEC-17-knockdown cells, but enhanced in MEC-17-overexpressing cells. Finally, we show that katanin, a microtubule-severing protein with enhanced activity on acetylated α-tubulin, is actively involved in adipogenesis. We propose that co-ordinated up-regulation of α-tubulin acetylation initiates cytoskeleton remodelling by promoting α-tubulin severing by katanin which, in turn, allows expansion of lipid droplets and accelerates the morphological transition toward mature adipocytes.

Role of pyruvate dehydrogenase inhibition in the development of hypertrophy in the hyperthyroid rat heart: a combined magnetic resonance imaging and hyperpolarized magnetic resonance spectroscopy study.

BACKGROUND: Hyperthyroidism increases heart rate, contractility, cardiac output, and metabolic rate. It is also accompanied by alterations in the regulation of cardiac substrate use. Specifically, hyperthyroidism increases the ex vivo activity of pyruvate dehydrogenase kinase, thereby inhibiting glucose oxidation via pyruvate dehydrogenase. Cardiac hypertrophy is another effect of hyperthyroidism, with an increase in the abundance of mitochondria. Although the hypertrophy is initially beneficial, it can eventually lead to heart failure. The aim of this study was to use hyperpolarized magnetic resonance spectroscopy to investigate the rate and regulation of in vivo pyruvate dehydrogenase flux in the hyperthyroid heart and to establish whether modulation of flux through pyruvate dehydrogenase would alter cardiac hypertrophy. METHODS AND RESULTS: Hyperthyroidism was induced in 18 male Wistar rats with 7 daily intraperitoneal injections of freshly prepared triiodothyronine (0.2 mg x kg(-1) x d(-1)). In vivo pyruvate dehydrogenase flux, assessed with hyperpolarized magnetic resonance spectroscopy, was reduced by 59% in hyperthyroid animals (0.0022 ± 0.0002 versus 0.0055 ± 0.0005 second(-1); P=0.0003), and this reduction was completely reversed by both short- and long-term delivery of dichloroacetic acid, a pyruvate dehydrogenase kinase inhibitor. Hyperpolarized [2-(13)C]pyruvate was also used to evaluate Krebs cycle metabolism and demonstrated a unique marker of anaplerosis, the level of which was significantly increased in the hyperthyroid heart. Cine magnetic resonance imaging showed that long-term dichloroacetic acid treatment significantly reduced the hypertrophy observed in hyperthyroid animals (100 ± 20 versus 200 ± 30 mg; P=0.04) despite no change in the increase observed in cardiac output. CONCLUSIONS: This work has demonstrated that inhibition of glucose oxidation in the hyperthyroid heart in vivo is mediated by pyruvate dehydrogenase kinase. Relieving this inhibition can increase the metabolic flexibility of the hyperthyroid heart and reduce the level of hypertrophy that develops while maintaining the increased cardiac output required to meet the higher systemic metabolic demand.

Determining the in vivo regulation of cardiac pyruvate dehydrogenase based on label flux from hyperpolarised [1-13C]pyruvate.

Pyruvate dehydrogenase (PDH) is a key regulator of cardiac substrate selection and is regulated by both pyruvate dehydrogenase kinase (PDK)-mediated phosphorylation and feedback inhibition. The extent to which chronic upregulation of PDK protein levels, acutely increased PDK activity and acute feedback inhibition limit PDH flux remains unclear because existing in vitro assessment methods inherently disrupt the regulation of the enzyme complex. We have demonstrated previously that hyperpolarised (13)C-labelled metabolic tracers coupled with MRS can monitor flux through PDH in vivo. The aim of this study was to determine the relative contributions of acute and chronic changes in PDK and PDH activities to in vivo myocardial PDH flux. We examined both fed and fasted rats with either hyperpolarised [1-(13)C]pyruvate alone or hyperpolarised [1-(13)C]pyruvate co-infused with malate [to modulate mitochondrial nicotinamide adenine dinucleotide (NADH/NAD(+)) and acetyl-coenzyme A (acetyl-CoA)/CoA ratios, which alter both PDH activity and flux]. To confirm the metabolic fate of infused malate, we performed in vitro (1)H NMR spectroscopy on cardiac tissue extracts. We observed that, in fed rats, where PDH activity was high, the presence of malate increased PDH flux by 27%, whereas, in the fasted state, malate infusion had no effect on PDH flux. These observations suggest that pyruvate oxidation is limited by feedback inhibition from acetyl-CoA only when PDH activity is high. Therefore, in the case of PDH, and potentially other enzymes, hyperpolarised (13)C MRI can be used to assess noninvasively enzymatic regulation.

Validation of the in vivo assessment of pyruvate dehydrogenase activity using hyperpolarised 13C MRS.

Many diseases of the heart are characterised by changes in substrate utilisation, which is regulated in part by the activity of the enzyme pyruvate dehydrogenase (PDH). Consequently, there is much interest in the in vivo evaluation of PDH activity in a range of physiological and pathological states to obtain information on the metabolic mechanisms of cardiac diseases. Hyperpolarised [1-(13)C]pyruvate, detected using MRS, is a novel technique for the noninvasive evaluation of PDH flux. PDH flux has been assumed to directly reflect in vivo PDH activity, although to date this assumption remains unproven. Control animals and animals undergoing interventions known to modulate PDH activity, namely high fat feeding and dichloroacetate infusion, were used to investigate the relationship between in vivo hyperpolarised MRS measurements of PDH flux and ex vivo measurements of PDH enzyme activity (PDH(a)). Further, the plasma concentrations of pyruvate and other important metabolites were evaluated following pyruvate infusion to assess the metabolic consequences of pyruvate infusion during hyperpolarised MRS experiments. Hyperpolarised MRS measurements of PDH flux correlated significantly with ex vivo measurements of PDH(a), confirming that PDH activity influences directly the in vivo flux of hyperpolarised pyruvate through cardiac PDH. The maximum plasma concentration of pyruvate reached during hyperpolarised MRS experiments was approximately 250 µM, equivalent to physiological pyruvate concentrations reached during exercise or with dietary interventions. The concentrations of other metabolites, including lactate, glucose and ß-hydroxybutyrate, did not vary during the 60 s following pyruvate infusion. Hence, during the 60-s data acquisition period, metabolism was minimally affected by pyruvate infusion.

In vivo assessment of pyruvate dehydrogenase flux in the heart using hyperpolarized carbon-13 magnetic resonance.

The advent of hyperpolarized (13)C magnetic resonance (MR) has provided new potential for the real-time visualization of in vivo metabolic processes. The aim of this work was to use hyperpolarized [1-(13)C]pyruvate as a metabolic tracer to assess noninvasively the flux through the mitochondrial enzyme complex pyruvate dehydrogenase (PDH) in the rat heart, by measuring the production of bicarbonate (H(13)CO(3)(-)), a byproduct of the PDH-catalyzed conversion of [1-(13)C]pyruvate to acetyl-CoA. By noninvasively observing a 74% decrease in H(13)CO(3)(-) production in fasted rats compared with fed controls, we have demonstrated that hyperpolarized (13)C MR is sensitive to physiological perturbations in PDH flux. Further, we evaluated the ability of the hyperpolarized (13)C MR technique to monitor disease progression by examining PDH flux before and 5 days after streptozotocin induction of type 1 diabetes. We detected decreased H(13)CO(3)(-) production with the onset of diabetes that correlated with disease severity. These observations were supported by in vitro investigations of PDH activity as reported in the literature and provided evidence that flux through the PDH enzyme complex can be monitored noninvasively, in vivo, by using hyperpolarized (13)C MR.

Impaired insulin secretion and glucose intolerance in synaptotagmin-7 null mutant mice.

Vertebrates express at least 15 different synaptotagmins with the same domain structure but diverse localizations and tissue distributions. Synaptotagmin-1,-2, and -9 act as calcium sensors for the fast phrase of neurotransmitter release, and synaptotagmin-12 acts as a calcium-independent modulator of release. The exact functions of the remaining 11 synaptotagmins, however, have not been established. By analogy to the role of synaptotagmin-1, -2, and -9 in neurotransmission, these other synaptotagmins may serve as Ca(2+) transducers regulating other Ca(2+)-dependent membrane processes, such as insulin secretion in pancreatic beta-cells. Of these other synaptotagmins, synaptotagmin-7 is one of the most abundant and is present in pancreatic beta-cells. To determine whether synaptotagmin-7 regulates Ca(2+)-dependent insulin secretion, we analyzed synaptotagmin-7 null mutant mice for glucose tolerance and insulin release. Here, we show that synaptotagmin-7 is required for the maintenance of systemic glucose tolerance and glucose-stimulated insulin secretion. Mutant mice have normal insulin sensitivity, insulin production, islet architecture and ultrastructural organization, and metabolic and calcium responses but exhibit impaired glucose-induced insulin secretion, indicating a calcium-sensing defect during insulin-containing secretory granule exocytosis. Taken together, our findings show that synaptotagmin-7 functions as a positive regulator of insulin secretion and may serve as a calcium sensor controlling insulin secretion in pancreatic beta cells.

Real-time assessment of Krebs cycle metabolism using hyperpolarized 13C magnetic resonance spectroscopy.

The Krebs cycle plays a fundamental role in cardiac energy production and is often implicated in the energetic imbalance characteristic of heart disease. In this study, we measured Krebs cycle flux in real time in perfused rat hearts using hyperpolarized magnetic resonance spectroscopy (MRS). [2-(13)C]Pyruvate was hyperpolarized and infused into isolated perfused hearts in both healthy and postischemic metabolic states. We followed the enzymatic conversion of pyruvate to lactate, acetylcarnitine, citrate, and glutamate with 1 s temporal resolution. The appearance of (13)C-labeled glutamate was delayed compared with that of other metabolites, indicating that Krebs cycle flux can be measured directly. The production of (13)C-labeled citrate and glutamate was decreased postischemia, as opposed to lactate, which was significantly elevated. These results showed that the control and fluxes of the Krebs cycle in heart disease can be studied using hyperpolarized [2-(13)C]pyruvate.

Nanotheranostic agents for neurodegenerative diseases.

Neurodegenerative diseases (NDDs), including Alzheimer's disease (AD) and Parkinson's disease (PD), affect the ageing population worldwide and while severely impairing the quality of life of millions, they also cause a massive economic burden to countries with progressively ageing populations. Parallel with the search for biomarkers for early detection and prediction, the pursuit for therapeutic approaches has become growingly intensive in recent years. Various prospective therapeutic approaches have been explored with an emphasis on early prevention and protection, including, but not limited to, gene therapy, stem cell therapy, immunotherapy and radiotherapy. Many pharmacological interventions have proved to be promising novel avenues, but successful applications are often hampered by the poor delivery of the therapeutics across the blood-brain-barrier (BBB). To overcome this challenge, nanoparticle (NP)-mediated drug delivery has been considered as a promising option, as NP-based drug delivery systems can be functionalized to target specific cell surface receptors and to achieve controlled and long-term release of therapeutics to the target tissue. The usefulness of NPs for loading and delivering of drugs has been extensively studied in the context of NDDs, and their biological efficacy has been demonstrated in numerous preclinical animal models. Efforts have also been made towards the development of NPs which can be used for targeting the BBB and various cell types in the brain. The main focus of this review is to briefly discuss the advantages of functionalized NPs as promising theranostic agents for the diagnosis and therapy of NDDs. We also summarize the results of diverse studies that specifically investigated the usage of different NPs for the treatment of NDDs, with a specific emphasis on AD and PD, and the associated pathophysiological changes. Finally, we offer perspectives on the existing challenges of using NPs as theranostic agents and possible futuristic approaches to improve them.

Synaptotagmin-7 is a principal Ca2+ sensor for Ca2+ -induced glucagon exocytosis in pancreas.

Hormones such as glucagon are secreted by Ca(2+)-induced exocytosis of large dense-core vesicles, but the mechanisms involved have only been partially elucidated. Studies of pancreatic beta-cells secreting insulin revealed that synaptotagmin-7 alone is not sufficient to mediate Ca(2+)-dependent insulin granule exocytosis, and studies of chromaffin cells secreting neuropeptides and catecholamines showed that synaptotagmin-1 and -7 collaborate as Ca(2+) sensors for exocytosis, and that both are equally involved. As no other peptide secretion was analysed, it remains unclear whether synaptotagmins generally act as Ca(2+) sensors in large dense-core vesicle exocytosis in endocrine cells, and if so, whether synaptotagmin-7 always functions with a partner in that role. In particular, far less is known about the mechanisms underlying Ca(2+)-triggered glucagon release from alpha-cells than insulin secretion from beta-cells, even though insulin and glucagon together regulate blood glucose levels. To address these issues, we analysed the role of synaptotagmins in Ca(2+)-triggered glucagon exocytosis. Surprisingly, we find that deletion of a single synaptotagmin isoform, synaptotagmin-7, nearly abolished Ca(2+)-triggered glucagon secretion. Moreover, single-cell capacitance measurements confirmed that pancreatic alpha-cells lacking synaptotagmin-7 exhibited little Ca(2+)-induced exocytosis, whereas all other physiological and morphological parameters of the alpha-cells were normal. Our data thus identify synaptotagmin-7 as a principal Ca(2+) sensor for glucagon secretion, and support the notion that synaptotagmins perform a universal but selective function as individually acting Ca(2+) sensors in neurotransmitter, neuropeptide, and hormone secretion.

The effect of hyperpolarized tracer concentration on myocardial uptake and metabolism.

Hyperpolarized (13)C-labeled substrates directly provide a source of magnetic resonance (MR) signal to observe the substrates' real-time uptake and enzymatic conversion. The aim of this study was to optimize the concentration of hyperpolarized [1-(13)C]pyruvate infused as a metabolic tracer, by observing the mitochondrial conversion of pyruvate to H(13)CO(3)(-) in heart tissue. Hyperpolarized pyruvate was infused into rats at concentrations between 20 mM and 80 mM and the relationships between [1-(13)C]lactate, [1-(13)C]alanine, and H(13)CO(3)(-) production and the infused pyruvate concentration were investigated. H(13)CO(3)(-) production reached saturation above 40 mM infused pyruvate concentration, indicating that hyperpolarized MR experiments performed at this concentration maximize the H(13)CO(3)(-) signal with minimal alterations to in vivo substrate composition. Additionally, the linear dependence of alanine production on pyruvate concentration confirmed that hyperpolarized MR methods in the heart reveal enzyme activity, rather than cellular uptake. H(13)CO(3)(-) production demonstrated evidence of sigmoidal enzyme kinetics, a reflection of the allosteric nature of the pyruvate dehydrogenase (PDH) enzyme complex. This protocol could be useful to optimize the infused concentration of other hyperpolarized metabolites in different organs, to ensure adequate MR signal with minimum metabolic perturbation.

A new hyperpolarized 13C ketone body probe reveals an increase in acetoacetate utilization in the diabetic rat heart.

Emerging studies have recently shown the potential importance of ketone bodies in cardio-metabolic health. However, techniques to determine myocardial ketone body utilization in vivo are lacking. In this work, we developed a novel method to assess myocardial ketone body utilization in vivo using hyperpolarized [3-13C]acetoacetate and investigated the alterations in myocardial ketone body metabolism in diabetic rats. Within a minute upon injection of [3-13C]acetoacetate, the production of [5-13C]glutamate and [1-13C] acetylcarnitine can be observed real time in vivo. In diabetic rats, the production of [5-13C]glutamate was elevated compared to controls, while [1-13C]acetylcarnitine was not different. This suggests an increase in ketone body utilization in the diabetic heart, with the produced acetyl-CoA channelled into the tricarboxylic acid cycle. This observation was corroborated by an increase activity of succinyl-CoA:3-ketoacid-CoA transferase (SCOT) activity, the rate-limiting enzyme of ketone body utilization, in the diabetic heart. The increased ketone body oxidation in the diabetic hearts correlated with cardiac hypertrophy and dysfunction, suggesting a potential coupling between ketone body metabolism and cardiac function. Hyperpolarized [3-13C]acetoacetate is a new probe with potential for non-invasive and real time monitoring of myocardial ketone body oxidation in vivo, which offers a powerful tool to follow disease progression or therapeutic interventions.

Loss of BCAA Catabolism during Carcinogenesis Enhances mTORC1 Activity and Promotes Tumor Development and Progression.

Tumors display profound changes in cellular metabolism, yet how these changes aid the development and growth of tumors is not fully understood. Here we use a multi-omic approach to examine liver carcinogenesis and regeneration, and find that progressive loss of branched-chain amino acid (BCAA) catabolism promotes tumor development and growth. In human hepatocellular carcinomas and animal models of liver cancer, suppression of BCAA catabolic enzyme expression led to BCAA accumulation in tumors, though this was not observed in regenerating liver tissues. The degree of enzyme suppression strongly correlated with tumor aggressiveness, and was an independent predictor of clinical outcome. Moreover, modulating BCAA accumulation regulated cancer cell proliferation in vitro, and tumor burden and overall survival in vivo. Dietary BCAA intake in humans also correlated with cancer mortality risk. In summary, loss of BCAA catabolism in tumors confers functional advantages, which could be exploited by therapeutic interventions in certain cancers.

Abnormal cardiac morphology, function and energy metabolism in the dystrophic mdx mouse: an MRI and MRS study.

Patients with muscular dystrophy have abnormal cardiac function and decreased high-energy phosphate metabolism. Here, we have determined whether the 8 month old mdx mouse, an animal model of muscular dystrophy, also has abnormal cardiac function and energetics. In vivo cardiac MRI revealed 33% and 104% larger right ventricular end-diastolic and end-systolic volumes, respectively, and 17% lower right ventricular ejection fractions in mdx mice compared with controls. Evidence of left ventricular diastolic dysfunction included 18% lower peak filling rates in mdx mouse hearts. Abnormal cardiac function was accompanied by necrosis and lower citrate synthase activity in the mdx mouse heart, suggesting decreased mitochondrial content. Decreased mitochondrial numbers were associated with 38% lower phosphocreatine concentration, 22% lower total creatine, 36% higher cytosolic free ADP concentration and 1.3 kJ/mol lower free-energy available from ATP hydrolysis in whole isolated, perfused mdx mouse hearts than in controls. Transsarcolemmal creatine uptake was 12% lower in mdx mouse hearts. We conclude that the absence of dystrophin in adult mdx mouse heart, as in the heart of human patient, is associated with right ventricular dilatation, left ventricular diastolic dysfunction and abnormal energy metabolism.

Insulin resistance, abnormal energy metabolism and increased ischemic damage in the chronically infarcted rat heart.

OBJECTIVE: Many patients with heart failure have whole-body insulin resistance and reduced cardiac fluorodeoxyglucose uptake, but whether these metabolic changes have detrimental effects on the heart is unknown. Here, we tested whether there is a link between insulin resistance and ischemic damage in the chronically infarcted Wistar rat heart, postulating that the heart would have decreased insulin sensitivity, with lower GLUT4 glucose transporter protein levels due to high circulating free fatty acid (FFA) concentrations. A decreased capacity for glucose uptake would lower glycolytic adenosine triphosphate (ATP) production and thereby increase ischemic injury in the infarcted heart. METHODS AND RESULTS: In vivo left ventricular ejection fractions, measured using echocardiography, were 40% lower in rats 10 weeks after coronary artery ligation than in sham-operated control rats. Insulin-stimulated D[2-3H]glucose uptake was 42% lower in isolated, perfused, infarcted hearts. Myocardial GLUT4 glucose transporter protein levels were 28% lower in the infarcted hearts and correlated negatively with ejection fractions and with fasting plasma FFA concentrations. Compared with controls, chronically infarcted hearts had 46% lower total glucose uptake and three-fold faster ATP hydrolysis rates, measured using phosphorus-31 nuclear magnetic resonance spectroscopy, during 32-min ischemia at 0.4 ml/min/gww. During reperfusion, recovery of left ventricular developed pressure in infarcted hearts was 42% lower than in control hearts. CONCLUSIONS: Glucose uptake, in response to insulin or ischemia, was lower in the chronically infarcted rat heart and associated with increased circulating FFA concentrations and decreased GLUT4 levels. Thus, infarcted hearts had greater ATP depletion, and consequently incurred greater damage, during ischemia.

Downregulating serine hydroxymethyltransferase 2 (SHMT2) suppresses tumorigenesis in human hepatocellular carcinoma.

Serine-glycine biosynthetic pathway diverts the glycolytic intermediate 3-phosphoglycerate to synthesize serine and glycine, of which the latter was found to correlate with cancer cell proliferation. Increased de novo biosynthesis of glycine by serine hydroxymethyltransferase 2 (SHMT2) is the central mechanism to fuel one-carbon pools supporting tumorigenesis. However, the therapeutic potential in targeting SHMT2 in hepatocellular carcinoma (HCC) is unknown. In this study we showed that SHMT2 inhibition significantly suppressed liver tumorigenesis. In vitro, SHMT2-knockdown was found to reduce cell growth and tumorigenicity in Huh-7 and HepG2 liver cancer cells. Moreover SHMT2-knockdown Huh-7 cells failed to form tumor xenograft after subcutaneous inoculation into nude mice. Similarly, inducible SHMT2 inhibition, via doxycycline-added drinking water, was found to reduce tumor incidence and tumor growth in a human tumor xenograft mouse model. SHMT2-knockdown increased the susceptibility of Huh-7 cells to doxorubicin suggesting its potential in combination chemotherapy. Through isotopomer tracing of [2-13C] glycine metabolism, we demonstrated that SHMT2 activity is associated with cancer phenotype. However, overexpression of SHMT2 was insufficient to transform immortalized hepatic cells to malignancy, suggesting that SHMT2 is one of the building blocks in liver cancer metabolism but does not initiate malignant transformation. Moreover, our results suggest that glycine, but not 5,10-methylenetetrahydrofolate, from the SHMT2-mediated enzymatic reaction is instrumental in tumorigenesis. Indeed, we found that SHMT2-knockdown cells exhibited increased glycine uptake. Taken together, our data suggest that SHMT2 may be a potential target in the treatment of human HCC.