malonyl-CoA Concentration Research Articles

S-1-Propenylcysteine Enhances Endurance Capacity of Mice by Stimulating Fatty Acid Metabolism via Muscle Isoform of Carnitine Acyltransferase-1

BackgroundEndurance is an important capacity to sustain healthy lifestyles. Aged garlic extract (AGE) has been reported to exert an endurance-enhancing effect in clinical and animal studies, although little is known about its active ingredients and mechanism of action. ObjectivesThis study investigated the potential effect of S-1-propenylcysteine (S1PC), a characteristic sulfur amino acid in AGE, on the swimming endurance of mice, and examined its mechanism of action by a metabolomics-based approach. MethodsMale Institute of Cancer Research (ICR) mice (6 wk old) were orally administered either water (control) or S1PC (6.5 mg/kg/d) for 2 wk. The swimming duration to exhaustion was measured at 24 h after the final administration. Nontargeted metabolomic analysis was conducted on the plasma samples obtained from mice after 40-min submaximal swimming bouts. Subsequently, the enzyme activity of carnitine acyltransferase-1 (CPT-1) and the content of malonyl-coenzyme A (CoA), acetyl-CoA, and adenosine triphosphate (ATP) were quantified in heart, skeletal muscles, and liver of mice. ResultsThe duration time of swimming was substantially increased in the S1PC-treated mice as compared with the control group. Metabolomic analysis revealed significant alterations in the plasma concentration of the metabolites involved in fatty acid metabolism, in particular medium- or long-chain acylcarnitines in the mice treated with S1PC. Moreover, the administration of S1PC significantly enhanced the CPT-1 activity with the concomitant decrease in the malonyl-CoA content in the heart and skeletal muscles. These effects of S1PC were accompanied by the elevation of the acetyl-CoA and ATP levels to enhance the energy production in those tissues. ConclusionsS1PC is a key constituent responsible for the endurance-enhancing effect of AGE. This study suggests that S1PC helps provide energy during endurance exercise by increasing fatty acid metabolism via CPT-1 activation in the heart and skeletal muscles.

Reciprocal regulation of cardiac β-oxidation and pyruvate dehydrogenase by insulin

The heart alters the rate and relative oxidation of fatty acids and glucose based on availability and energetic demand. Insulin plays a crucial role in this process diminishing fatty acid and increasing glucose oxidation when glucose availability increases. Loss of insulin sensitivity and metabolic flexibility can result in cardiovascular disease. It is therefore important to identify mechanisms by which insulin regulates substrate utilization in the heart. Mitochondrial pyruvate dehydrogenase (PDH) is the key regulatory site for the oxidation of glucose for ATP production. Nevertheless, the impact of insulin on PDH activity has not been fully delineated, particularly in the heart. We sought invivo evidence that insulin stimulates cardiac PDH and that this process is driven by the inhibition of fatty acid oxidation. Mice injected with insulin exhibited dephosphorylation and activation of cardiac PDH. This was accompanied by an increase in the content of malonyl-CoA, an inhibitor of carnitine palmitoyltransferase 1 (CPT1), and, thus, mitochondrial import of fatty acids. Administration of the CPT1 inhibitor oxfenicine was sufficient to activate PDH. Malonyl-CoA is produced by acetyl-CoA carboxylase (ACC). Pharmacologic inhibition or knockout of cardiac ACC diminished insulin-dependent production of malonyl-CoA and activation of PDH. Finally, circulating insulin and cardiac glucose utilization exhibit daily rhythms reflective of nutritional status. We demonstrate that time-of-day-dependent changes in PDH activity are mediated, in part, by ACC-dependent production of malonyl-CoA. Thus, by inhibiting fatty acid oxidation, insulin reciprocally activates PDH. These studies identify potential molecular targets to promote cardiac glucose oxidation and treat heart disease.

Structural and mechanistic insights into Quinolone Synthase to address its functional promiscuity

Quinolone synthase from Aegle marmelos (AmQNS) is a type III polyketide synthase that yields therapeutically effective quinolone and acridone compounds. Addressing the structural and molecular underpinnings of AmQNS and its substrate interaction in terms of its high selectivity and specificity can aid in the development of numerous novel compounds. This paper presents a high-resolution AmQNS crystal structure and explains its mechanistic role in synthetic selectivity. Additionally, we provide a model framework to comprehend structural constraints on ketide insertion and postulate that AmQNS’s steric and electrostatic selectivity plays a role in its ability to bind to various core substrates, resulting in its synthetic diversity. AmQNS prefers quinolone synthesis and can accommodate large substrates because of its wide active site entrance. However, our research suggests that acridone is exclusively synthesized in the presence of high malonyl-CoA concentrations. Potential implications of functionally relevant residue mutations were also investigated, which will assist in harnessing the benefits of mutations for targeted polyketide production. The pharmaceutical industry stands to gain from these findings as they expand the pool of potential drug candidates, and these methodologies can also be applied to additional promising enzymes.

Phosphorus-induced greater enhancement in carbon supply and storage for oil synthesis during the crucial period made cottonseed kernel oil yield have a higher increment than protein

Cottonseed has a high utilization value due to its luxuriant oil and protein, but low phosphorus (P) in cropland reduces its yield and quality. A limited understanding of the physiological mechanism underlying these results restricted the exploration of P efficient management in cotton cultivation. A 3-year experiment was performed with Lu 54 (low-P sensitive) and Yuzaomian 9110 (low-P tolerant) under 0 (deficient-P), 100 (critical-P), and 200 (excessive-P) kg P2O5 ha−1 in a field having 16.9 mg kg−1 available P to explore the key pathway for P to regulate cottonseed oil and protein formation. P application markedly increased cottonseed oil and protein yields, with the enhanced acetyl-CoA and oxaloacetate contents during 20–26 days post anthesis being a vital reason. Notably, during the crucial period, decreased phosphoenolpyruvate carboxylase activity weakened the carbon allocation to protein, making malonyl-CoA content increase greater than free amino acid; Meanwhile, P application accelerated the carbon storage in oil but retarded that in protein. Consequently, cottonseed oil yield increased more than protein. Oil and protein synthesis in Lu 54 was more susceptible to P, resulting in greater increments in oil and protein yields than Yuzaomian 9110. Based on acetyl-CoA and oxaloacetate contents (the key substrates), the critical P content in the subtending leaf to cotton boll needed by oil and protein synthesis in Lu 54 (0.35%) was higher than Yuzaomian 9110 (0.31%). This study provided a new perception of the regulation of P on cottonseed oil and protein formation, contributing to the efficient P management in cotton cultivation.

Malonyl-CoA Accumulation as a Compensatory Cytoprotective Mechanism in Cardiac Cells in Response to 7-Ketocholesterol-Induced Growth Retardation

The major oxidized product of cholesterol, 7-Ketocholesterol (7KCh), causes cellular oxidative damage. In the present study, we investigated the physiological responses of cardiomyocytes to 7KCh. A 7KCh treatment inhibited the growth of cardiac cells and their mitochondrial oxygen consumption. It was accompanied by a compensatory increase in mitochondrial mass and adaptive metabolic remodeling. The application of [U-13C] glucose labeling revealed an increased production of malonyl-CoA but a decreased formation of hydroxymethylglutaryl-coenzyme A (HMG-CoA) in the 7KCh-treated cells. The flux of the tricarboxylic acid (TCA) cycle decreased, while that of anaplerotic reaction increased, suggesting a net conversion of pyruvate to malonyl-CoA. The accumulation of malonyl-CoA inhibited the carnitine palmitoyltransferase-1 (CPT-1) activity, probably accounting for the 7-KCh-induced suppression of β-oxidation. We further examined the physiological roles of malonyl-CoA accumulation. Treatment with the inhibitor of malonyl-CoA decarboxylase, which increased the intracellular malonyl-CoA level, mitigated the growth inhibitory effect of 7KCh, whereas the treatment with the inhibitor of acetyl-CoA carboxylase, which reduced malonyl-CoA content, aggravated such a growth inhibitory effect. Knockout of malonyl-CoA decarboxylase gene (Mlycd-/-) alleviated the growth inhibitory effect of 7KCh. It was accompanied by improvement of the mitochondrial functions. These findings suggest that the formation of malonyl-CoA may represent a compensatory cytoprotective mechanism to sustain the growth of 7KCh-treated cells.

Engineering a Novel Metabolic Pathway for Improving Cellular Malonyl-CoA Levels in Escherichia coli.

Cellular pool of malonyl-CoA in Escherichia coli is small, which impedes its utility for overproduction of natural products such as phenylpropanoids, polyketides, and flavonoids. In this study, we report the use of a new metabolic pathway to increase the malonyl-CoA concentration as a limiting metabolite in E. coli. For this purpose, the malonate/sodium symporter from Malonomonas rubra, and malonyl-CoA synthetase (MCS) from Bradyrhizobium japonicum were co-expressed in E. coli. This new pathway allows the cell to actively import malonate from the culture medium and to convert malonate and CoA to malonyl-CoA via an ATP-dependent ligation reaction. HPLC analysis confirmed elevated levels of malonyl-CoA and (2S)-naringenin as a malonyl-CoA-dependent metabolite, in E. coli. A 6.8-fold and more than 3.5-fold increase in (2S)-naringenin production were achieved in the engineered host in comparison with non-engineered E. coli and previously reported passive transport MatBMatC pathway, respectively. This observation suggests that using active transporters of malonate not only improves malonyl-CoA-dependent production but also makes it possible to harness low concentrations of malonate in culture media.

Effects of the potassium application rate on lipid synthesis and eating quality of two rice cultivars

Lipid content has an important effect on rice eating quality, but the effects of fertilizer application rate on the lipid synthesis and eating quality of rice are not well understood. Potassium (K) has a strong influence on rice quality and the requirement for K fertilizer in rice is greater than for nitrogen (N) and phosphorus (P) fertilizers. To investigate the effects of K fertilizer on the lipid synthesis and eating quality of rice, we used Nanjing 9108 (NJ9108, japonica) and IR72 (indica) rice as experimental materials and four K levels: K0 (0 kg ha–1), K1 (90 kg ha–1), K2 (135 kg ha–1) and K3 (180 kg ha–1). The results showed that the lipid content, free fatty acid (FFA) content, unsaturated fatty acid (UFA) content, malonyl-CoA (MCA) content, phosphatidic acid (PA) content, lipid synthesis-related enzyme activities and eating quality first increased and then decreased with increasing K in both cultivars. The maximum values were obtained under K2. However, the saturated fatty acid (SFA) content showed the opposite trend. No significant differences were found in pyruvate (PYR) content among the K treatments. The protein and oxaloacetic acid (OAA) contents and phosphoenolpyruvate carboxylase (PEPCase) activity of NJ9108 first decreased and then increased with increasing K, and the minimum values were obtained under K2; while IR72 showed the opposite trend and the maximum values were obtained under K1. Overall, increasing K optimized the fatty acid components and increased the lipid content and eating quality of rice by enhancing lipid synthesis-related enzyme activities and regulating substrate competition for lipid and protein synthesis. The optimal K application rate for lipid synthesis, eating quality and grain yield was 135 kg ha–1 for both cultivars.

122-LB: Effect of Dapagliflozin on Mitochondrial Metabolism and Cardiac Function in the Failing Heart

Several studies highlight the potential for SGLT2 inhibitors (i) to exert their cardioprotective effects by shifting myocardial fuel utilization towards ketone oxidation. However, direct in vivo measurements of cardiac mitochondrial substrate oxidation in awake rodents during heart failure are lacking, and whether SGLT2i-mediated increases in ketone availability and oxidation are able to improve cardiac efficiency remains unclear. Here, we examined the effect of acute SGLT2 inhibition with dapagliflozin (dapa) on cardiac function and mitochondrial metabolism in awake male Sprague Dawley rats 2 weeks after induction of heart failure by permanent ligation of the left anterior descending coronary artery. Echocardiography and in vivo metabolic flux studies were performed 4-6 h after oral dapa (1.5 mg/kg) or vehicle treatment in sham-surgerized rats and rats with myocardial infarction (MI) . Acute dapa treatment caused a 15% decrease in fasting plasma glucose concentrations and 50% increase in fasting plasma NEFAs, whole-body βOHB turnover and plasma βOHB levels in sham and MI rats (all P<0.05 vs. VEH) . Dapa treatment decreased left ventricular (LV) [14C]deoxyglucose uptake and relative rates of pyruvate oxidation to total mitochondrial oxidation (VPDH/VCS) by 40-60%. Dapa treatment also caused a 60% increase in relative rates of LV ketone oxidation to total mitochondrial oxidation (VβOHB/VCS) in sham and MI rats (all P<0.05 vs. VEH) , independently of changes in cardiac metabolic gene expression. Interestingly, the dramatic shift in substrate utilization caused by acute dapa treatment did not significantly alter cardiac output or LV ejection fraction. Acute dapa treatment also did not significantly alter myocardial acetyl-CoA content, malonyl-CoA content, ATP/ADP ratios or ATP/AMP ratios in the LV myocardium remote from the infarct. Conclusion: Taken together, these results demonstrate that single-dose dapa treatment, which acutely decreased LV myocardial pyruvate oxidation and increased LV ketone oxidation, was not sufficient to alter cardiac output or LV ejection fraction. Disclosure L. Goedeke: Other Relationship; The Liver Company, Inc. . G. I. Shulman: Advisory Panel; 89bio, Inc., AstraZeneca, Equator Therapeutics, Inc., Janssen Research & Development, LLC, Merck & Co., Inc., Consultant; DiCerna Pharmaceuticals, Inc., Novo Nordisk, Other Relationship; Generian Pharmaceuticals, iMetabolic Biopharma Corporation, Maze Therapeutics, The Liver Company, Stock/Shareholder; Levels Health, Inc. . Y. Ma: None. J. Zhang: n/a. N. Guerrera: None. X. Wu: None. D. Zhang: None. M. Kahn: None. X. Zhang: None. L. H. Young: None. Funding NIH (P30 DK045735 and K99 HL150234) , AstraZeneca (NCR-19-20028)

Effects of Phosphorus Application Rate on Lipid Synthesis and Eating Quality of Two Rice Grains

Lipids are an important nutritional component of rice, and lipid content has an important effect on rice eating quality. However, the effects of the fertilizer application rate on lipid synthesis and eating quality of rice are poorly understood. To investigate the effects of phosphorus (P) fertilizer on lipid synthesis and eating quality of rice, we used Nanjing (NJ) 9108 (japonica) and IR72 (indica) rice as experimental materials, and four P levels, P0 (0 kg ha−1), P1 (45 kg ha−1), P2 (67.5 kg ha−1) and P3 (135 kg ha−1). The results showed that the lipid, free fatty acid (FFA) content, unsaturated fatty acid (UFA) content, malonyl-CoA (MCA) content, phosphatidic acid (PA) content, lipid synthesis-related enzyme activities and eating quality of both cultivars increased with increasing P. However, the saturated fatty acid (SFA) content showed the opposite trend. No significant differences were found in pyruvate (PYR) content between P treatments. Protein and oxaloacetic acid (OAA) contents and phosphoenolpyruvate carboxylase (PEPCase) activity first increased and then decreased with increasing P, which indicated that high P levels could stimulate lipid synthesis more than protein synthesis. Overall, increasing P optimized fatty acid components and increased the lipid content and eating quality of rice by enhancing lipid synthesis-related enzyme activities and regulating substrate competition for lipid and protein synthesis. The optimal P application rate for lipid synthesis and eating quality of both cultivars was 135 kg ha−1.

Improvement of Rimocidin Biosynthesis by Increasing Supply of Precursor Malonyl-CoA via Over-expression of Acetyl-CoA Carboxylase in Streptomyces rimosus M527.

Precursor engineering is an effective strategy for the overproduction of secondary metabolites. The polyene macrolide rimocidin, which is produced by Streptomyces rimosus M527, exhibits a potent activity against a broad range of phytopathogenic fungi. It has been predicted that malonyl-CoA is used as extender units for rimocidin biosynthesis. Based on a systematic analysis of three sets of time-series transcriptome microarray data of S. rimosus M527 fermented in different conditions, the differentially expressed accsr gene that encodes acetyl-CoA carboxylase (ACC) was found. To understand how the formation of rimocidin is being influenced by the expression of the accsr gene and by the concentration of malonyl-CoA, the accsr gene was cloned and over-expressed in the wild-type strain S. rimosus M527 in this study. The recombinant strain S. rimosus M527-ACC harboring the over-expressed accsr gene exhibited better performances based on the enzymatic activity of ACC, intracellular malonyl-CoA concentrations, and rimocidin production compared to S. rimosus M527 throughout the fermentation process. The enzymatic activity of ACC and intracellular concentration of malonyl-CoA of S. rimosus M527-ACC were 1.0- and 1.5-fold higher than those of S. rimosus M527, respectively. Finally, the yield of rimocidin produced by S. rimosus M527-ACC reached 320.7mg/L, which was 34.0% higher than that of S. rimosus M527. These results confirmed that malonyl-CoA is an important precursor for rimocidin biosynthesis and suggested that an adequate supply of malonyl-CoA caused by accsr gene over-expression led to the improvement in rimocidin production.

Selective Acetyl-CoA Carboxylase 1 Inhibitor Improves Hepatic Steatosis and Hepatic Fibrosis in a Preclinical Nonalcoholic Steatohepatitis Model.

Acetyl-CoA carboxylase (ACC) 1 and ACC2 are essential rate-limiting enzymes that synthesize malonyl-CoA (M-CoA) from acetyl-CoA. ACC1 is predominantly expressed in lipogenic tissues and regulates the de novo lipogenesis flux. It is upregulated in the liver of patients with nonalcoholic fatty liver disease (NAFLD), which ultimately leads to the formation of fatty liver. Therefore, selective ACC1 inhibitors may prevent the pathophysiology of NAFLD and nonalcoholic steatohepatitis (NASH) by reducing hepatic fat, inflammation, and fibrosis. Many studies have suggested ACC1/2 dual inhibitors for treating NAFLD/NASH; however, reports on selective ACC1 inhibitors are lacking. In this study, we investigated the effects of compound-1, a selective ACC1 inhibitor for treating NAFLD/NASH, using preclinical in vitro and in vivo models. Compound-1 reduced M-CoA content and inhibited the incorporation of [14C] acetate into fatty acids in HepG2 cells. Additionally, it reduced hepatic M-CoA content and inhibited de novo lipogenesis in C57BL/6J mice after a single dose. Furthermore, compound-1 treatment of 8 weeks in Western diet-fed melanocortin 4 receptor knockout mice-NAFLD/NASH mouse model-improved liver hypertrophy and reduced hepatic triglyceride content. The reduction of hepatic M-CoA by the selective ACC1 inhibitor was highly correlated with the reduction in hepatic steatosis and fibrosis. These findings support further investigations of the use of this ACC1 inhibitor as a new treatment of NFLD/NASH. SIGNIFICANCE STATEMENT: This is the first study to demonstrate that a novel selective inhibitor of acetyl-CoA carboxylase (ACC) 1 has anti-nonalcoholic fatty liver disease (NAFLD) and anti-nonalcoholic steatohepatitis (NASH) effects in preclinical models. Treatment with this compound significantly improved hepatic steatosis and fibrosis in a mouse model. These findings support the use of this ACC1 inhibitor as a new treatment for NAFLD/NASH.

Design and synthesis of a monocyclic derivative as a selective ACC1 inhibitor by chemical modification of biphenyl ACC1/2 dual inhibitors

A structure–activity relationship (SAR) study towards novel ACC1-selective inhibitors was carried out by modifying the molecular length of the linker in biaryl derivative 1 g, an ACC1/2 dual inhibitor. Ultimately, this leads us to discover novel phenoxybenzyloxy derivative 1i as a potent ACC1-selective inhibitor. Further chemical modification of this scaffold to improve cellular potency as well as physicochemical and pharmacokinetic (PK) properties produced N-2-(pyridin-2-ylethyl)acetamide derivative 1n, which showed highly potent ACC1-selective inhibition as well as sufficient PK profile for further in vivo evaluations. Oral administration of 1n significantly reduced the concentration of malonyl-CoA in HCT-116 xenograft tumors at doses of 100 mg/kg. Accordingly, our novel series of potent ACC1-selective inhibitors represents a set of useful orally-available research tools, as well as potential therapeutic agents for cancer and fatty acid-related diseases.

A novel long noncoding RNA, ENSGALG00000021686, regulates the intracellular transport of fatty acids by targeting the FABP3 gene in chicken

Fatty acids (FAs) are essential for the vital movement of humans and animals. Their metabolism is, in part, regulated by FABP3. In our previous study, a novel lncRNA (ENSGALG00000021686, L21686) was identified, and FABP3 was predicted as its target gene. Here, using chicken myocytes, lymphocytes, and different tissues, L21686 target on the FABP3 gene, FABP3 mRNA expression, and their effect on FA metabolism are explored. The results show that the highest expression of L21686 is in muscle tissue, a significant energy-consuming tissue. L21686 expression is consistent with FABP3 mRNA expression. We also show that under the different treatments, the levels of FABP3 mRNA and protein in myocytes and lymphocytes change in tandem with L21686 expression. Moreover, the dual-luciferase reporter assay provided direct evidence that L21686 targets the FABP3 gene. Finally, it was found that the content of free FAs increases along with the up-regulation of L21686 and the FABP3 gene. Malonyl CoA content does not change under the different treatments, suggesting that L21686 regulates the intake of extracellular FAs in chicken. Further, the changes in lipoprotein lipase (LPL), sterol-regulatory element binding protein 1 (SREBP-1), fatty acid synthase (FASN), and acetyl-CoA carboxylase (ACC) mRNA levels support this view. In summary, our data show that the new lncRNA (L21686) regulates the intake of extracellular FAs in chicken cells in vitro by targeting the expression of the FABP3 gene. Our findings will help to establish the groundwork and provide a new clue for deciphering the regulation of FAs metabolism in chicken.

A Synthetic Malonyl-CoA Metabolic Oscillator in Komagataella phaffii.

Malonyl-CoA is a key metabolic molecule that participates in a diverse range of physiological responses and can act as a building block for a variety of value-added pharmaceuticals and chemicals. The cytosolic malonyl-CoA concentration is usually very low, and thus dynamic metabolic control of malonyl-CoA variation will aid its stable formation and efficient consumption. We developed a synthetic malonyl-CoA metabolic oscillator in yeast. A synthetic regulatory protein, Prm1-FapR, was constructed by fusing a yeast transcriptional activator, Prm1, with a bacterial malonyl-CoA-sensitive transcription repressor, FapR. Two oppositely regulated biosensors were then engineered. A total of 18 hybrid promoter variants were designed, each carrying the operator sequence (fapO) of FapR and the core promoter of PAOX1 (cPAOX1), which is naturally regulated by Prm1. The promoter activities of these variants, regulated by Prm1-FapR, were tested. Through this process, a sensor for Prm1-FapR/(-52)fapO-PAOX1 carrying an activation/deactivation regulation module was built. Meanwhile, 24 promoter variants of PGAP with fapO inserted were designed and tested using the fusion regulator, giving a sensor for Prm1-FapR/PGAP-(+22) fapO that contained a repression/derepression regulation module. Both sensors were subsequently integrated into a single cell, which exhibited correct metabolic switching of eGFP and mCherry reporters following manipulation of cytosolic malonyl-CoA levels. The Prm1-FapR/(-52)fapO-PAOX1 and the Prm1-FapR/PGAP-(+22)fapO were also used to control the malonyl-CoA source and sink pathways, respectively, for the synthesis of 6-methylsalicylic acid. This finally led to an oscillatory metabolic mode of cytosolic malonyl-CoA. Such a metabolator is useful in exploring potential industrial and biomedical applications not limited by natural cellular behavior.

The identification and pharmacological evaluation of potent, selective and orally available ACC1 inhibitor.

In our effort to explore the potential of ACC1-selective inhibitor as in vivo probe molecule, a series of 1,3-benzoxazole derivatives was synthesized. Previously, we reported a series of novel bicyclic and monocyclic ACC1-selective inhibitors. Among them, compound 1a exhibited highly potent cellular activity (acetate uptake IC50 = 0.76 nM) as well as promising in vivo PD efficacy. However, compound 1a caused severe body weight reduction in repeated dose administration in the mouse model. Since 1a showed potent inhibitory activity against mouse ACC1 as well as strong inhibition of mouse ACC2, we further examined a series of 1a analogues in order to reduce undesirable body weight change. The replacement of acetamide moiety with ureido moiety dramatically improved selectivity of mouse ACC1 against ACC2. In addition, analogue 1b displayed favorable bioavailability in mouse cassette dosing PK study, hence in vivo PD studies were also carried out. Oral administration of 1b significantly reduced the concentration of malonyl-CoA in HCT-116 xenograft tumors at doses of more than 30 mg/kg. Furthermore, compound 1b showed significant antitumor efficacy in 786-O xenograft mice at an oral dose of 30 mg/kg (T/C = 0.5%). Accordingly, our novel potent ACC1-selective inhibitor represents a set of useful orally-available research tools, as well as potential therapeutic agents particularly in terms of new cancer therapies.

Deficiency in the short-chain acyl-CoA dehydrogenase protects mice against diet-induced obesity and insulin resistance.

Acyl-CoA dehydrogenases (CADs) participate in mitochondrial fatty acid oxidation; abnormal fatty acid oxidation is associated with obesity and related metabolic disorders. We decipher the impact of short-chain CAD (SCAD) on adiposity and insulin resistance. BALB/cBy strain mice derived from BALB/c strain are deficient in SCAD activity because of a spontaneous deletion in the acyl-CoA dehydrogenases (Acads) gene. Adiposity, lipogenesis, and insulin sensitivity were compared in BALB/c and BALB/cBy mice subjected to high-fat diets (HFDs). A whole hepatic transcriptome profiling experiment with microarrays was performed to evaluate the mechanisms by which SCAD deficiency protects against insulin resistance. Acads-deficient mice were significantly resistant to HFD-induced obesity and insulin resistance as compared with control mice. Reduced obesity results from decreased triglyceride content due to activation of AMPK in liver that would reduce hepatic content of malonyl-CoA, resulting in decreased hepatic de novo lipogenesis. Improved insulin sensitivity was associated with reduced diacylglycerol content commensurate with reduced PKC-ε activity and increased protein kinase B (AKT) activation in liver and skeletal muscle. Additionally, Acads-deficient mice displayed significantly higher expression of the endoplasmic chaperone 78-kDa glucose-regulated protein, which was further associated with the AKT activation in the primary hepatocytes. Modulation of SCAD expression may therefore be a novel therapeutic approach to manage and prevent obesity and related metabolic diseases, such as diabetes.-Chen, Y., Chen, J., Zhang, C., Yang, S., Zhang, X., Liu, Y., Su, Z. Deficiency in the short-chain acyl-CoA dehydrogenase protects mice against diet-induced obesity and insulin resistance.

Design and synthesis of a novel 1H-pyrrolo[3,2-b]pyridine-3-carboxamide derivative as an orally available ACC1 inhibitor.

We initiated our structure-activity relationship (SAR) studies for novel ACC1 inhibitors from 1a as a lead compound. Our initial SAR studies of 1H-Pyrrolo[3,2-b]pyridine-3-carboxamide scaffold revealed the participation of HBD and HBA for ACC1 inhibitory potency and identified 1-methyl-1H-pyrrolo[3,2-b]pyridine-3-carboxamide derivative 1c as a potent ACC1 inhibitor. Although compound 1c had physicochemical and pharmacokinetic (PK) issues, we investigated the 1H-pyrrolo[3,2-b]pyridine core scaffold to address these issues. Accordingly, this led us to discover a novel 1-isopropyl-1H-pyrrolo[3,2-b]pyridine-3-carboxamide derivative 1k as a promising ACC1 inhibitor, which showed potent ACC1 inhibition as well as sufficient cellular potency. Since compound 1k displayed favorable bioavailability in mouse cassette dosing PK study, we conducted in vivo Pharmacodynamics (PD) studies of this compound. Oral administration of 1k significantly reduced the concentration of malonyl-CoA in HCT-116 xenograft tumors at a dose of 100 mg/kg. Accordingly, our novel series of potent ACC1 inhibitors represent useful orally-available research tools, as well as potential therapeutic agents for cancer and fatty acid related diseases.

Magnesium starvation improves production of malonyl-CoA-derived metabolites in Escherichia coli

Starvation of essential nutrients, such as nitrogen, sulfur, magnesium, and phosphorus, leads cells into stationary phase and potentially enhances target metabolite production because cells do not consume carbon for the biomass synthesis. The overall metabolic behavior changes depend on the type of nutrient starvation in Escherichia coli. In the present study, we determined the optimum nutrient starvation type for producing malonyl-CoA-derived metabolites such as 3-hydroxypropionic acid (3HP) and naringenin in E. coli. For 3HP production, high production titer (2.3 or 2.0 mM) and high specific production rate (0.14 or 0.28 mmol gCDW−1 h−1) was observed under sulfur or magnesium starvation, whereas almost no 3HP production was detected under nitrogen or phosphorus starvation. Metabolic profiling analysis revealed that the intracellular malonyl-CoA concentration was significantly increased under the 3HP producing conditions. This accumulation should contribute to the 3HP production because malonyl-CoA is a precursor of 3HP. Strong positive correlation (r = 0.95) between intracellular concentrations of ATP and malonyl-CoA indicates that the ATP level is important for malonyl-CoA synthesis due to the ATP requirement by acetyl-CoA carboxylase. For naringenin production, magnesium starvation led to the highest production titer (144 ± 15 μM) and specific productivity (127 ± 21 μmol gCDW−1). These results demonstrated that magnesium starvation is a useful approach to improve the metabolic state of strains engineered for the production of malonyl-CoA derivatives.

Discovery of Novel Selective Acetyl-CoA Carboxylase (ACC) 1 Inhibitors.

We initiated our structure-activity relationship (SAR) studies for selective ACC1 inhibitors from 1a as a lead compound. SAR studies of bicyclic scaffolds revealed many potent and selective ACC1 inhibitors represented by 1f; however most of them had physicochemical issues, particularly low aqueous solubility and potent CYP inhibition. To address these two issues and improve the druglikeness of this chemical series, we converted the bicyclic scaffold into a monocyclic framework. Ultimately, this lead us to discover a novel monocyclic derivative 1q as a selective ACC1 inhibitor, which showed highly potent and selective ACC1 inhibition as well as acceptable solubility and CYP inhibition profiles. Since compound 1q displayed favorable bioavailability in mouse cassette dosing testing, we conducted in vivo PD studies of this compound. Oral administration of 1q significantly reduced the concentration of malonyl-CoA in HCT-116 xenograft tumors at doses of more than 30 mg/kg. Accordingly, our novel series of selective ACC1 inhibitors represents a set of useful orally available research tools, as well as potential therapeutic agents for cancer and fatty acid related diseases.

Biosensor-aided high-throughput screening of hyper-producing cells for malonyl-CoA-derived products

BackgroundMalonyl-coenzyme A (CoA) is an important biosynthetic precursor in vivo. Although Escherichia coli is a useful organism for biosynthetic applications, its malonyl-CoA level is too low.ResultsTo identify strains with the best potential for enhanced malonyl-CoA production, we developed a whole-cell biosensor for rapidly reporting intracellular malonyl-CoA concentrations. The biosensor was successfully applied as a high-throughput screening tool for identifying targets at a genome-wide scale that could be critical for improving the malonyl-CoA biosynthesis in vivo. The mutant strains selected synthesized significantly higher titers of the type III polyketide triacetic acid lactone (TAL), phloroglucinol, and free fatty acids compared to the wild-type strain, using malonyl-CoA as a precursor.ConclusionThese results validated this novel whole-cell biosensor as a rapid and sensitive malonyl-CoA high-throughput screening tool. Further analysis of the mutant strains showed that the iron ion concentration is closely related to the intracellular malonyl-CoA biosynthesis.