malonyl-CoA Levels Research Articles

Improved phloroglucinol production by metabolically engineered Escherichia coli

Phloroglucinol is a valuable chemical which has been successfully produced by metabolically engineered Escherichia coli. However, the low productivity remains a bottleneck for large-scale application and cost-effective production. In the present work, we cloned the key biosynthetic gene, phlD (a type III polyketide synthase), into a bacterial expression vector to produce phloroglucinol in E. coli and developed different strategies to re-engineer the recombinant strain for robust synthesis of phloroglucinol. Overexpression of E. coli marA (multiple antibiotic resistance) gene enhanced phloroglucinol resistance and elevated phloroglucinol production to 0.27 g/g dry cell weight. Augmentation of the intracellular malonyl coenzyme A (malonyl-CoA) level through coordinated expression of four acetyl-CoA carboxylase (ACCase) subunits increased phloroglucinol production to around 0.27 g/g dry cell weight. Furthermore, the coexpression of ACCase and marA caused another marked improvement in phloroglucinol production 0.45 g/g dry cell weight, that is, 3.3-fold to the original strain. Under fed-batch conditions, this finally engineered strain accumulated phloroglucinol up to 3.8 g/L in the culture 12 h after induction, corresponding to a volumetric productivity of 0.32 g/L/h. This result was the highest phloroglucinol production to date and showed promising to make the bioprocess economically feasible.

Malonyl-CoA mediates leptin hypothalamic control of feeding independent of inhibition of CPT-1a

Hypothalamic fatty acid metabolism is involved in central nervous system controls of feeding and energy balance. Malonyl-CoA, an intermediate of fatty acid biosynthesis, is emerging as a significant player in these processes. Notably, hypothalamic malonyl-CoA has been implicated in leptin's feeding effect. Leptin treatment increases malonyl-CoA level in the hypothalamic arcuate nucleus (Arc), and this increase is required for leptin-induced decrease in food intake. However, the intracellular downstream mediators of malonyl-CoA's feeding effect have not been identified. A primary biochemical action of malonyl-CoA is the inhibition of the acyltransferase activity of carnitine palmitoyltransferase-1 (CPT-1). In the hypothalamus, the predominant isoform of CPT-1 that possesses the acyltransferase activity is CPT-1 liver type (CPT-1a). To address the role of CPT-1a in malonyl-CoA's anorectic action, we used a recombinant adenovirus expressing a mutant CPT-1a that is insensitive to malonyl-CoA inhibition. We show that Arc overexpression of the mutant CPT-1a blocked the malonyl-CoA-mediated inhibition of CPT-1 activity. However, the overexpression of this mutant did not affect the anorectic actions of leptin or central cerulenin for which an increase in Arc malonyl-CoA level is also required. Thus, CPT-1a does not appear to be involved in the malonyl-CoA's anorectic actions induced by leptin. Furthermore, long-chain fatty acyl-CoAs, substrates of CPT-1a, dissociate from malonyl-CoA's actions in the Arc under different feeding states. Together, our results suggest that Arc intracellular mechanisms of malonyl-CoA's anorectic actions induced by leptin are independent of CPT-1a. The data suggest that target(s), rather than CPT-1a, mediates malonyl-CoA action on feeding.

Post-translational modifications, a key process in CD36 function: Lessons from the spontaneously hypertensive rat heart

CD36, a multifunctional protein, is involved in cardiac long chain fatty acid (LCFA) metabolism and in the etiology of heart diseases, yet the functional impact of Cd36 gene variants remains unclear. In 7-week-old spontaneously hypertensive rats (SHR), which, like humans, carry numerous mutations in Cd36, we tested the hypothesis that their restricted cardiac LCFA utilization occurs prior to hypertrophy due to defective CD36 post-translational modifications (PTM), as assessed by ex vivo perfusion of 13C-labeled substrates and biochemical techniques. Compared to their controls, SHR hearts displayed a lower (i) contribution of LCFA to β-oxidation (− 40%) and triglycerides (+ 2.8 folds), which was not explained by transcriptional changes or malonyl-CoA level, a recognized β-oxidation inhibitor, and (ii) membrane-associated CD36 protein level, but unchanged distribution. Other results demonstrate alterations in CD36 PTM in SHR hearts, specifically by N-glycosylation, and the importance of O-linked-β-N-acetylglucosamine for its membrane recruitment and role in LCFA use in the heart.

Design of small molecule inhibitors of acetyl-CoA carboxylase 1 and 2 showing reduction of hepatic malonyl-CoA levels in vivo in obese Zucker rats

Inhibition of acetyl-CoA carboxylases has the potential for modulating long chain fatty acid biosynthesis and mitochondrial fatty acid oxidation. Hybridization of weak inhibitors of ACC2 provided a novel, moderately potent but lipophilic series. Optimization led to compounds 33 and 37, which exhibit potent inhibition of human ACC2, 10-fold selectivity over inhibition of human ACC1, good physical and in vitro ADME properties and good bioavailability. X-ray crystallography has shown this series binding in the CT-domain of ACC2 and revealed two key hydrogen bonding interactions. Both 33 and 37 lower levels of hepatic malonyl-CoA in vivo in obese Zucker rats.

Cooperativity and allosteric regulation of yeast diacylglycerol acyltransferase, DGA1

The esterification of the Kennedy Pathway intermediate diacylglycerol generates the energy storage molecule, triglyceride. Alternatively, diacylglycerol can be used as a precursor for phospholipids. Therefore, diacylglycerol acyltransferase (DGAT), mainly mediated by DGA1 in S. cerevisiae, is a putative control point for the storage or structural utilization of acyl chains. Our in vitro DGAT assays using lro1are1are2 yeast microsomes indicated a distinct sigmoidal relationship between oleoyl‐CoA concentration and relative specific activity (Hill number = 3.0). This apparent cooperativity lead to our hypothesis that DGA1 is allosterically regulated. Subsequent in vitro DGAT assays included metabolites that, based on the regulation of other key enzymes in metabolism, are “metabolic indicators”. Elevated levels of ATP, NADH, acetyl‐CoA, and malonyl‐CoA are indicative of an energy‐rich cell. Elevated levels of AMP, ADP, and NAD+ are indicative of an energy‐poor cell. Initial experiments (n = 3) with estimated physiological concentrations of these metabolites found only 0.8 mM NAD+ to have an effect. The K0.5 was increased by about 20%. Such a direct connection between NAD+ levels and triglyceride synthetic enzymes has not, to our knowledge, been previously reported. Funding was provided by the Pennsylvania ‐ Delaware affiliate of the American Heart Association.

Chronic Inhibition of Pyruvate Dehydrogenase in Heart Triggers an Adaptive Metabolic Response

Diabetic cardiac dysfunction is associated with decreased rates of myocardial glucose oxidation (GO) and increased fatty acid oxidation (FAO), a fuel shift that has been shown to sensitize the heart to ischemic insult and ventricular dysfunction. We sought to evaluate the metabolic and functional consequences of chronic suppression of GO in heart as modeled by transgenic mice with cardiac-specific overexpression of pyruvate dehydrogenase kinase 4 (myosin heavy chain (MHC)-PDK4 mice), an inhibitor of pyruvate dehydrogenase. Hearts of MHC-PDK4 mice were shown to exhibit an insulin-resistant substrate utilization profile, characterized by low GO rates and high FAO flux. Surprisingly, MHC-PDK4 mice were not sensitized to cardiac ischemia-reperfusion injury despite a fuel utilization pattern that phenocopied the diabetic heart. In addition, MHC-PDK4 mice were protected against high fat diet-induced myocyte lipid accumulation, likely related to increased capacity for FAO. The high rates of mitochondrial FAO in the MHC-PDK4 heart were related to heightened activity of the AMP-activated protein kinase, reduced levels of malonyl-CoA, and increased capacity for mitochondrial uncoupled respiration. The expression of the known AMP-activated protein kinase target, peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α), a master regulator of mitochondrial function and biogenesis, was also activated in the MHC-PDK4 heart. These results demonstrate that chronic activation of PDK4 triggers transcriptional and post-transcriptional mechanisms that re-program the heart for chronic high rates of FAO without the expected deleterious functional or metabolic consequences.

Hypothalamic malonyl-CoA and CPT1c in the treatment of obesity

Metabolic integration of nutrient sensing in the central nervous system has been shown to be an important regulator of adiposity by affecting food intake and peripheral energy expenditure. Modulation of de novo fatty acid synthetic flux by cytokines and nutrient availability plays an important role in this process. Inhibition of hypothalamic fatty acid synthase by pharmacologic or genetic means leads to an increased malonyl-CoA level and suppression of food intake and adiposity. Conversely, the ectopic expression of malonyl-CoA decarboxylase in the hypothalamus is sufficient to promote feeding and adiposity. Based on these and other findings, metabolic intermediates in fatty acid biogenesis, including malonyl-CoA and long-chain acyl-CoAs, have been implicated as signaling mediators in the central control of body weight. Malonyl-CoA has been hypothesized to mediate its effects in part through an allosteric interaction with an atypical and brain-specific carnitine palmitoyltransferase-1 (CPT1c). CPT1c is expressed in neurons and binds malonyl-CoA, however, it does not perform the same biochemical function as the prototypical CPT1 enzymes. Mouse knockout models of CPT1c exhibit suppressed food intake and smaller body weight, but are highly susceptible to weight gain when fed a high-fat diet. Thus, the brain can directly sense and respond to changes in nutrient availability and composition to affect body weight and adiposity.

Abstract P4-03-02: Establishing a Relationship between Breast Cancer, Prolactin and Altered Fat Metabolism

Abstract Despite decades of research, breast cancer remains one of the most common cancers world-wide. Mammary carcinoma has been associated with a high-fat diet, and its rate in overweight post-menopausal women is up to 50% higher than in normal-weight women. The current study examined breast cancer as a metabolic disease in the context of altered fat catabolism, with a focus on examining the effects of prolactin (PRL) on the adenosine 5'-monophosphate-activated protein kinase (AMPK) energy sensing pathway that culminates with carnitine palmitoyl transferase 1 (CPT1), an enzyme that shuttles long-chain fatty acids into the mitochondrial matrix for beta oxidation. When a cell has high energy demands or is stressed, AMPK activation leads to either increased glucose uptake or the phosphorylation of acetyl-CoA carboxylase (ACC), resulting in a reduction in malonyl-CoA levels that lift an allosteric inhibition on CPT1, in turn leading to an increase in CPT1 enzyme activity. Conversely, increased malonyl-CoA levels, together with increased fatty acid synthase activity, favor lipogenesis. PRL has been shown to affect fat metabolism in adipocytes by altering malonyl-CoA levels, and we therefore examined whether CPT1 expression was altered in breast cancer cells in response to treatment with recombinant human PRL. The metabolic effects of this hormone, which is normally produced in breast tissue and has been epidemiologically linked with breast cancer, were investigated in a normal human breast epithelial cell line (184B5) and in breast cancer cells. PRL up-regulated CPT1 expression at both the mRNA and protein levels in cancer cells, but not in 184B5 cells. Of note, compared to MCF-7 or MDA-MB-231 cells, T47D cells expressed the highest levels of CPT1. The 85 kDa PRL receptor isoform was also highly expressed in this cell line compared to 184B5 or MCF-7 cells. Furthermore, PRL dose-dependently increased CPT1 enzyme activity in T47D cells, also inducing the phosphorylation of both the AMPKα catalytic subunit at threonine 172 and ACC. In T47D cells treated with AMPKα siRNA, transient knock-down of the catalytic AMPK subunit correlated with a similar knock-down in CPT1 mRNA levels. CPT1 levels in the knock-down cells could not be restored by treatment with PRL, suggesting that the PRL-mediated effect requires activation of the AMPK pathway. We also demonstrated that treatment with PRL leads to the phosphorylation of TAK1 and LKB1, two kinases that are known to activate AMPKα. It is known that PRL contributes to breast cancer by inducing cell proliferation, survival, motility, and angiogenesis. Our work proposes a novel role for this hormone in mammary carcinogenesis through its effect on fat metabolism via the induction of the AMPK pathway. Elucidating how breast cancer cells differentially produce and utilize energy compared to normal breast epithelial cells may lead to the development of new therapies for the treatment or improved management of this complex disease. (Research supported by Canadian Institutes of Health Research.) Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P4-03-02.

Increased Mitochondrial Fatty Acid Oxidation Is Sufficient to Protect Skeletal Muscle Cells from Palmitate-induced Apoptosis

The mechanisms underlying the protective effect of monounsaturated fatty acids (e.g. oleate) against the lipotoxic action of saturated fatty acids (e.g. palmitate) in skeletal muscle cells remain poorly understood. This study aimed to examine the role of mitochondrial long-chain fatty acid (LCFA) oxidation in mediating oleate's protective effect against palmitate-induced lipotoxicity. CPT1 (carnitine palmitoyltransferase 1), which is the key regulatory enzyme of mitochondrial LCFA oxidation, is inhibited by malonyl-CoA, an intermediate of lipogenesis. We showed that expression of a mutant form of CPT1 (CPT1mt), which is active but insensitive to malonyl-CoA inhibition, in C2C12 myotubes led to increased LCFA oxidation flux even in the presence of high concentrations of glucose and insulin. Furthermore, similar to preincubation with oleate, CPT1mt expression protected muscle cells from palmitate-induced apoptosis and insulin resistance by decreasing the content of deleterious palmitate derivates (i.e. diacylglycerols and ceramides). Oleate preincubation exerted its protective effect by two mechanisms: (i) in contrast to CPT1mt expression, oleate preincubation increased the channeling of palmitate toward triglycerides, as a result of enhanced diacylglycerol acyltransferase 2 expression, and (ii) oleate preincubation promoted palmitate oxidation through increasing CPT1 expression and modulating the activities of acetyl-CoA carboxylase and AMP-activated protein kinase. In conclusion, we demonstrated that targeting mitochondrial LCFA oxidation via CPT1mt expression leads to the same protective effect as oleate preincubation, providing strong evidence that redirecting palmitate metabolism toward oxidation is sufficient to protect against palmitate-induced lipotoxicity.

Whole Body Deletion of AMP-activated Protein Kinase β2 Reduces Muscle AMPK Activity and Exercise Capacity

AMP-activated protein kinase (AMPK) β subunits (β1 and β2) provide scaffolds for binding α and γ subunits and contain a carbohydrate-binding module important for regulating enzyme activity. We generated C57Bl/6 mice with germline deletion of AMPK β2 (β2 KO) and examined AMPK expression and activity, exercise capacity, metabolic control during muscle contractions, aminoimidazole carboxamide ribonucleotide (AICAR) sensitivity, and susceptibility to obesity-induced insulin resistance. We find that β2 KO mice are viable and breed normally. β2 KO mice had a reduction in skeletal muscle AMPK α1 and α2 expression despite up-regulation of the β1 isoform. Heart AMPK α2 expression was also reduced but this did not affect resting AMPK α1 or α2 activities. AMPK α1 and α2 activities were not changed in liver, fat, or hypothalamus. AICAR-stimulated glucose uptake but not fatty acid oxidation was impaired in β2 KO mice. During treadmill running β2 KO mice had reduced maximal and endurance exercise capacity, which was associated with lower muscle and heart AMPK activity and reduced levels of muscle and liver glycogen. Reductions in exercise capacity of β2 KO mice were not due to lower muscle mitochondrial content or defects in contraction-stimulated glucose uptake or fatty acid oxidation. When challenged with a high-fat diet β2 KO mice gained more weight and were more susceptible to the development of hyperinsulinemia and glucose intolerance. In summary these data show that deletion of AMPK β2 reduces AMPK activity in skeletal muscle resulting in impaired exercise capacity and the worsening of diet-induced obesity and glucose intolerance.

Coffee polyphenols suppress diet-induced body fat accumulation by downregulating SREBP-1c and related molecules in C57BL/6J mice

The prevalence of obesity is increasing globally, and obesity is a major risk factor for type 2 diabetes and cardiovascular disease. We investigated the effects of coffee polyphenols (CPP), which are abundant in coffee and consumed worldwide, on diet-induced body fat accumulation. C57BL/6J mice were fed either a control diet, a high-fat diet, or a high-fat diet supplemented with 0.5 to 1.0% CPP for 2-15 wk. Supplementation with CPP significantly reduced body weight gain, abdominal and liver fat accumulation, and infiltration of macrophages into adipose tissues. Energy expenditure evaluated by indirect calorimetry was significantly increased in CPP-fed mice. The mRNA levels of sterol regulatory element-binding protein (SREBP)-1c, acetyl-CoA carboxylase-1 and -2, stearoyl-CoA desaturase-1, and pyruvate dehydrogenase kinase-4 in the liver were significantly lower in CPP-fed mice than in high-fat control mice. Similarly, CPP suppressed the expression of these molecules in Hepa 1-6 cells, concomitant with an increase in microRNA-122. Structure-activity relationship studies of nine quinic acid derivatives isolated from CPP in Hepa 1-6 cells suggested that mono- or di-caffeoyl quinic acids (CQA) are active substances in the beneficial effects of CPP. Furthermore, CPP and 5-CQA decreased the nuclear active form of SREBP-1, acetyl-CoA carboxylase activity, and cellular malonyl-CoA levels. These findings indicate that CPP enhances energy metabolism and reduces lipogenesis by downregulating SREBP-1c and related molecules, which leads to the suppression of body fat accumulation.

AMPK activation represses the human gene promoter of the cardiac isoform of acetyl-CoA carboxylase: Role of nuclear respiratory factor-1

The cardiac-enriched isoform of acetyl-CoA carboxylase (ACCβ) produces malonyl-CoA, a potent inhibitor of carnitine palmitoyltransferase-1. AMPK inhibits ACCβ activity, lowering malonyl-CoA levels and promoting mitochondrial fatty acid β-oxidation. Previously, AMPK increased promoter binding of nuclear respiratory factor-1 (NRF-1), a pivotal transcriptional modulator controlling gene expression of mitochondrial proteins. We therefore hypothesized that NRF-1 inhibits myocardial ACCβ promoter activity via AMPK activation. A human ACCβ promoter-luciferase construct was transiently transfected into neonatal cardiomyocytes ± a NRF-1 expression construct. NRF-1 overexpression decreased ACCβ gene promoter activity by 71 ± 4.6% ( p < 0.001 vs. control). Transfections with 5′-end serial promoter deletions revealed that NRF-1-mediated repression of ACCβ was abolished with a pPIIβ-18/+65-Luc deletion construct. AMPK activation dose-dependently reduced ACCβ promoter activity, while NRF-1 addition did not further decrease it. We also investigated NRF-1 inhibition in the presence of upstream stimulatory factor 1 (USF1), a known transactivator of the human ACCβ gene promoter. Here NRF-1 blunted USF1-dependent induction of ACCβ promoter activity by 58 ± 7.5% ( p < 0.001 vs. control), reversed with a dominant negative NRF-1 construct. NRF-1 also suppressed endogenous USF1 transcriptional activity by 55 ± 6.2% ( p < 0.001 vs. control). This study demonstrates that NRF-1 is a novel transcriptional inhibitor of the human ACCβ gene promoter in the mammalian heart. Our data extends AMPK regulation of ACCβ to the transcriptional level.

Liquid-liquid extraction coupled with LC/MS/MS for monitoring of malonyl-CoA in rat brain tissue

The formation of malonyl-CoA is catalyzed by acetyl-CoA carboxylase (ACC), the rate-limiting enzyme of de novo fatty acid synthesis. Monitoring the changes of malonyl-CoA concentration in the brain in response to treatments such as pharmaceutical intervention (via ACC inhibitors) or different dietary conditions (such as varied feeding regimes) is of great interest and could help increase the understanding of how this molecule contributes to feeding behavior and overall energy balance. We have developed a sensitive analytical method for the determination of malonyl-CoA levels in rat brain tissue. The assay involved removal of tissue lipids by liquid-liquid extraction followed by LC/MS/MS analysis of the aqueous layer for malonyl-CoA. The method was sensitive enough (limit of quantitation = 50 ng/mL, or approximately 0.018 nmol/g brain tissue) to determine malonyl-CoA in individual rat brain preparations. The assay performance was sufficiently rugged to support drug discovery screening efforts and provided an additional analytical tool for monitoring brain malonyl-CoA levels.

Influence of Ghrelin and Growth Hormone Deficiency on AMP‐Activated Protein Kinase and Hypothalamic Lipid Metabolism

Current evidence demonstrates that the stomach-derived hormone ghrelin, a potent growth hormone (GH) secretagogue, promotes feeding through a mechanism involving the short-term activation of hypothalamic AMP-activated protein kinase (AMPK), which in turn results in decreased hypothalamic levels of malonyl-CoA and increased carnitine palmitoyltransferase 1 (CPT1) activity. Despite this evidence, no data have been reported about the effect of chronic, central ghrelin administration on hypothalamic fatty acid metabolism. In the present study, we examined the differences in hypothalamic fatty acid metabolism in the presence and absence of GH, by using a model for the study of GH-deficiency, namely the spontaneous dwarf rat and the effect of long-term central ghrelin treatment and starvation on hypothalamic fatty acid metabolism in this animal model. Our data showed that GH-deficiency induces reductions in both de novo lipogenesis and beta-oxidation pathways in the hypothalamus. Thus, dwarf rats display reductions in fatty acid synthase (FAS) mRNA expression both in the ventromedial nucleus of the hypothalamus (VMH) and whole hypothalamus, as well as in FAS protein and activity. CPT1 activity was also reduced. In addition, in the present study, we show that chronic ghrelin treatment does not promote AMPK-induced changes in the overall fluxes of hypothalamic fatty acid metabolism in normal rats and that this effect is independent of GH status. By contrast, we demonstrated that both chronic ghrelin and fasting decreased FAS mRNA expression in the VMH of normal rats but not dwarf rats, suggesting GH status dependency. Overall, these results suggest that ghrelin plays a dual time-dependent role in modulating hypothalamic lipid metabolism. Understanding the molecular mechanism underlying the interplay between GH and ghrelin on hypothalamic lipid metabolism will allow new strategies for the design and development of suitable drugs for the treatment of GH-deficiency, obesity and its comorbidities.

Targeting Intermediary Metabolism in the Hypothalamus as a Mechanism to Regulate Appetite

The central nervous system mediates energy balance (energy intake and energy expenditure) in the body; the hypothalamus has a key role in this process. Recent evidence has demonstrated an important role for hypothalamic malonyl CoA in mediating energy balance. Malonyl CoA is generated by the carboxylation of acetyl CoA by acetyl CoA carboxylase and is then either incorporated into long-chain fatty acids by fatty acid synthase, or converted back to acetyl-CoA by malonyl CoA decarboxylase. Increased hypothalamic malonyl CoA is an indicator of energy surplus, resulting in a decrease in food intake and an increase in energy expenditure. In contrast, a decrease in hypothalamic malonyl CoA signals an energy deficit, resulting in an increased appetite and a decrease in body energy expenditure. A number of hormonal and neural orexigenic and anorexigenic signaling pathways have now been shown to be associated with changes in malonyl CoA levels in the arcuate nucleus (ARC) of the hypothalamus. Despite compelling evidence that malonyl CoA is an important mediator in the hypothalamic ARC control of food intake and regulation of energy balance, the mechanism(s) by which this occurs has not been established. Malonyl CoA inhibits carnitine palmitoyltransferase-1 (CPT-1), and it has been proposed that the substrate of CPT-1, long-chain acyl CoA(s), may act as a mediator(s) of appetite and energy balance. However, recent evidence has challenged the role of long-chain acyl CoA(s) in this process, as well as the involvement of CPT-1 in hypothalamic malonyl CoA signaling. A better understanding of how malonyl CoA regulates energy balance should provide novel approaches to targeting intermediary metabolism in the hypothalamus as a mechanism to control appetite and body weight. Here, we review the data supporting an important role for malonyl CoA in mediating hypothalamic control of energy balance, and recent evidence suggesting that targeting malonyl CoA synthesis or degradation may be a novel approach to favorably modify appetite and weight gain.

Ghrelin effects on neuropeptides in the rat hypothalamus depend on fatty acid metabolism actions on BSX but not on gender

The orexigenic effect of ghrelin is mediated by neuropeptide Y (NPY) and agouti-related protein (AgRP) in the hypothalamic arcuate nucleus (ARC). Recent evidence also indicates that ghrelin promotes feeding through a mechanism involving activation of hypothalamic AMP-activated protein kinase (AMPK) and inactivation of acetyl-CoA carboxylase and fatty acid synthase (FAS). This results in decreased hypothalamic levels of malonyl-CoA, increased carnitine palmitoyltransferase 1 (CPT1) activity, and mitochondrial production of reactive oxygen species. We evaluated whether these molecular events are part of a unique signaling cascade or whether they represent alternative pathways mediating the orexigenic effect of ghrelin. Moreover, we examined the gender dependency of these mechanisms, because recent evidence has proposed that ghrelin orexigenic effect is reduced in female rats. We studied in both genders the effect of ghrelin on the expression of AgRP and NPY, as well as their transcription factors: cAMP response-element binding protein (CREB and its phosphorylated form, pCREB), forkhead box O1 (FoxO1 and its phosphorylated form, pFoxO1), and brain-specific homeobox transcription factor (BSX). In addition, to establish a mechanistic link between ghrelin, fatty acid metabolism, and neuropeptides, we evaluated the effect of ghrelin after blockage of hypothalamic fatty acid beta oxidation, by using the CPT1 inhibitor etomoxir. Ghrelin-induced changes in the AMPK-CPT1 pathway are associated with increased levels of AgRP and NPY mRNA expression through modulation of BSX, pCREB, and FoxO1, as well as decreased expression of endoplasmic reticulum (ER) stress markers in a gender-independent manner. In addition, blockage of hypothalamic fatty acid beta oxidation prevents the ghrelin-promoting action on AgRP and NPY mRNA expression, also in a gender-independent manner. Notably, this effect is associated with decreased BSX expression and reduced food intake. Overall, our data suggest that BSX integrates changes in neuronal metabolic status with ARC-derived neuropeptides in a gender-independent manner.

SIRT1 Takes a Backseat to AMPK in the Regulation of Insulin Sensitivity by Resveratrol

Resveratrol is a polyphenol that is enriched in the skins of red grapes. Although scientists speculated for decades about the French paradox in which red wine appeared to protect against heart disease, the mechanisms mediating these effects remained a mystery. In 2003 Sinclair and colleagues (1) identified that resveratrol activated the nicotinamide adenine dinucleotide (NAD+)-dependent histone deacetylase, SIRT1, whose yeast homolog (Sir2) was previously shown to enhance longevity in response to calorie restriction (2). Over the past several years studies in rodents have confirmed the powerful antiaging effects of resveratrol, which have been shown to range from improved exercise performance to correction of age- and obesity-induced dyslipidemia, cardiovascular dysfunction, and insulin resistance (3–5). Whereas SIRT1 has a diverse range of substrates, many of its metabolic actions are believed to be mediated by deacetylation and activation of peroxisome proliferator–activated receptor γ coactivator-α (PGC1α), which is a master regulator of mitochondrial biogenesis (4). These studies have suggested that resveratrol and SIRT1 may be the panacea for preventing the development of age-onset diseases. Resveratrol, like other polyphenols, also increases the activity of the AMP-activated protein kinase (AMPK) (3,6,7). AMPK is activated by cellular stress, including fasting and exercise, and is also regulated by circulating hormones and nutrients (8). The activation of AMPK increases fatty acid oxidation, an effect which may involve phosphorylation and inhibition of acetyl-CoA-carboxylase (ACC), reduced levels of malonyl-CoA, and increased mitochondrial fatty acid flux via carnitine palmitoyltransferase-1 (Fig. 1). At the same time, AMPK also phosphorylates …

Differential effect of prolonged food restriction and fasting on hypothalamic malonyl-CoA concentration and expression of orexigenic and anorexigenic neuropeptides genes in rats

Several lines of evidence suggest that malonyl-CoA in the hypothalamus plays an important role in monitoring and modulating body energy balance. In fasted state the level of malonyl-CoA concentration significantly decreases. Simultaneously, orexigenic neuropeptides (NPY - neuropeptide Y, AgRP - agouti-related peptide) genes are expressed at high level, whereas anorexigenic neuropeptides (CART - cocaine-and amphetamine-regulated transcript, POMC - proopiomelanocortin) genes are expressed at low level. When food intake resumes, opposite effect is observed. This study examined the effect of prolonged food restriction, common in humans trying to lose body weight on expression of orexigenic and anorexigenic neuropeptides genes and on malonyl-CoA content in rat whole hypothalamus. We observed an increase of NPY and AgRP mRNA levels in hypothalamus of rats kept on 30 days-long food restriction (50% of the amount of food consumed by controls). Simultaneously, a decrease of CART and POMC mRNA levels occurred. Refeeding caused a decrease in NPY and POMC mRNA levels without effect on AgRP and CART mRNA. Surprisingly, both prolonged food restriction and food restriction/refeeding caused the increase of malonyl-CoA level in whole hypothalamus. In contrast, fasting for 24h caused the decrease of malonyl-CoA level, which was associated with the up-regulation of NPY and AgRP genes expression and down-regulation of CART and POMC genes expression. After refeeding opposite effect was observed. These results indicate that prolonged food restriction and acute fasting, conditions in which energy expenditure exceeds intake, differentially affect malonyl-CoA concentration and similarly affect orexigenic and anorexigenic neuropeptide genes expression in whole rat hypothalamus.

κ‐Opioid receptors control the metabolic response to a high‐energy diet in mice

General opioid receptor antagonists reduce food intake and body weight in rodents, but the contributions of specific receptor subtypes are unknown. We examined whether genetic deletion of the kappa-opioid receptor (KOR) in mice alters metabolic physiology. KOR-knockout (KO) and wild-type (WT) mice were fed a high-energy diet (HED) for 16 wk. KO mice had 28% lower body weight and 45% lower fat mass when compared to WT mice fed an HED. No differences in caloric intake were found. An HED reduced energy expenditure in WT mice, but not in KO mice. KOR deficiency led to an attenuation of triglyceride synthesis in the liver. Malonyl CoA levels were also reduced in response to an HED, thereby promoting hepatic beta-oxidation. Glycemic control was also found to be improved in KO mice. These data suggest a key role for KORs in the central nervous system regulation of the metabolic adaptation to an HED, as we were unable to detect expression of KOR in liver, white adipose tissue, or skeletal muscle in WT mice. This study provides the first evidence that KORs play an essential physiological role in the control of hepatic lipid metabolism, and KOR activation is a permissive signal toward fat storage.-Czyzyk, T. A., Nogueiras, R., Lockwood, J. F., McKinzie, J. H., Coskun, T., Pintar, J. E., Hammond, C., Tschöp, M. H., Statnick, M. A. kappa-Opioid receptors control the metabolic response to a high-energy diet in mice.

Abstract 5058: Glucose OxidationStimulation Affords Dramatic Protection Against in vivo Myocardial Ischemia Reperfusion Injury

During reperfusion of the ischemic myocardium, fatty acid oxidation rates quickly recover, while glucose oxidation rates remain low, which decreases cardiac efficiency during reperfusion. Malonyl CoA decarboxylase (MCD) degrades malonyl CoA and increases fatty acid oxidation by relieving malonyl CoA-dependent inhibition of carnitine palmitoyl transferase-1. Studies have shown that MCD inhibition improves reperfusion recovery of ischemic hearts ex vivo by reducing fatty acid oxidation rates and increasing glucose oxidation rates. The objective of this study was to determine whether MCD inhibition would translate into a reduction in infarct size. Wild Type (WT, n = 7) or MCD deficient (MCD−/−, n = 7) mice had their left anterior descending coronary artery ligated for 30 min followed by 24 hr reperfusion. MCD−/− mice demonstrated a dramatic reduction in infarct size compared to WT mice (10.8±3.8 vs. 39.5±4.7 % of area at risk, P &lt; 0.05) despite similar areas at risk (53 vs. 52 % of left ventricle). Furthermore, malonyl CoA levels were higher in the area at risk of hearts from MCD−/− mice (3.9±0.7 vs. 1.9±0.5 nmol/g wet weight, P &lt; 0.05). Because the protection against ex vivo ischemia/reperfusion injury in MCD−/− mice was associated with increased myocardial glucose oxidation rates, secondary to an inhibition of fatty acid oxidation, we next determined whether direct stimulation of pyruvate dehydrogenase (PDH) and subsequent glucose oxidation would also afford protection against myocardial infarction. Infusion of the PDH activator, dichloroacetate (100 mg/kg intraperitoneal injection, followed by hourly injections of 50 mg/kg for the 24 hr duration of reperfusion), demonstrated significant protection against myocardial infarction (36.4±2.4 vs. 50.3±3.2 % of area at risk, P &lt; 0.05). We demonstrate that MCD inhibition is beneficial against ischemia/reperfusion injury, possibly due to its effects on glucose oxidation stimulation, validating the optimization of myocardial metabolism as a novel therapy for ischemic heart disease.