Enhanced supply of acetyl-CoA by exogenous pantothenate kinase promotes synthesis of poly(3-hydroxybutyrate).

BACKGROUND: Coenzyme A (CoA) is a carrier of acyl groups. This cofactor is synthesized from pantothenic acid in five steps. The phosphorylation of pantothenate is catalyzed by pantothenate kinase (CoaA), which is a key step in the CoA biosynthetic pathway. To determine whether the enhancement of the CoA biosynthetic pathway is effective for producing useful substances, the effect of elevated acetyl-CoA levels resulting from the introduction of the exogenous coaA gene on poly(3-hydroxybutyrate) [P(3HB)] synthesis was determined in Escherichia coli, which express the genes necessary for cyanobacterial polyhydroxyalkanoate synthesis (phaABEC). RESULTS: E. coli containing the coaA gene in addition to the pha genes accumulated more P(3HB) compared with the transformant containing the pha genes alone. P(3HB) production was enhanced by precursor addition, with P(3HB) content increasing from 18.4% (w/w) to 29.0% in the presence of 0.5 mM pantothenate and 16.3%-28.2% by adding 0.5 mM ß-alanine. Strains expressing the exogenous coaA in the presence of precursors contained acetyl-CoA in excess of 1 nmol/mg of dry cell wt, which promoted the reaction toward P(3HB) formation. The amount of acetate exported into the medium was three times lower in the cells carrying exogenous coaA and pha genes than in the cells carrying pha genes alone. This was attributed to significantly enlarging the intracellular pool size of CoA, which is the recipient of acetic acid and is advantageous for microbial production of value-added materials. CONCLUSIONS: Enhancing the CoA biosynthetic pathway with exogenous CoaA was effective at increasing P(3HB) production. Supplementing the medium with pantothenate facilitated the accumulation of P(3HB). ß-Alanine was able to replace the efficacy of adding pantothenate.

Complete genome sequence of Pseudoalteromonas sp. PS1M3, a psychrotrophic bacterium isolated from deep-sea sediment off the Boso Peninsula, Japan Trench.

Herein, we report the complete genome sequence of Pseudoalteromonas sp. PS1M3 (= NCBI 87791), which is a psychrotrophic bacterium that inhabits in seabed off the Boso Peninsula, Japan Trench. Analysis of the genomic sequence revealed that PS1M3 possesses 2 circular chromosomal DNAs and 2 circular plasmid DNAs. The genome of PS1M3 had a total size of 4,351,630 bp, an average GC content of 39.9%, and contained a total of 3811 predicted protein coding sequences, 28 rRNAs, and 100 tRNAs. The Kyoto Encyclopedia of Genes and Genomes (KEGG) was utilized to annotate the genes and KofamKOALA within KEGG assigned a gene cluster involved in glycogen biosynthesis and metabolic pathways with regard to heavy metal resistance (copper; cop and mercury; mer), indicating that PS1M3 can potentially use a stored glycogen as an energy source under oligotrophic environment and cope with multi-heavy metal contamination. To assess available genome relatedness indices, whole-genome average nucleotide identity analysis was examined using the complete genome sequences of Pseudoalteromonas spp., showing that 67.29-97.40% sequence similarity with PS1M3. This study may be useful in understanding the roles of a psychrotrophic Pseudoalteromonas in cold deep-sea sediment adaptation mechanisms.

Complete Genome Sequence of a Psychrophilic Bacterium, Pseudoalteromonas sp. Strain APM04, Isolated from the Seafloor of the South Mariana Trough, Pacific Ocean.

The complete genome sequence of Pseudoalteromonas sp. strain APM04, which is a psychrophilic bacterium that inhabits the seabed of the South Mariana Trough, Pacific Ocean, was determined to characterize the genetic features associated with evolution in extremophilic and oligotrophic deep seawater.

Fatty Acid Production by Enhanced Malonyl-CoA Supply in Escherichia coli.

The expression of exogenous genes encoding acetyl-CoA carboxylase (Acc) and pantothenate kinase (CoaA) in Escherichia coli enable highly effective fatty acid production. Acc-only strains grown at 37 °C or 23 °C produced an approximately twofold increase in fatty acid content, and additional expression of CoaA achieved a further twofold accumulation. In the presence of pantothenate, which is the starting material for the CoA biosynthetic pathway, the size of the intracellular CoA pool at 23 °C was comparable to that at 30 °C during cultivation, and more than 500 mg/L of culture containing cellular fatty acids was produced, even at 23 °C. However, the highest yield of cellular fatty acids (1100 mg/L of culture) was produced in cells possessing the gene encoding type I bacterial fatty acid synthase (FasA) along with the acc and coaA, when the transformant was cultivated at 30 °C in M9 minimal salt medium without pantothenate or IPTG. This E. coli transformant contained 141 mg/L of oleic acid attributed to FasA under noninducible conditions. The increased fatty acid content was brought about by a greatly improved specific productivity of 289 mg/g of dry cell weight. Thus, the effectiveness of the foreign acc and coaA in fatty acid production was unambiguously confirmed at culture temperatures of 23 °C to 37 °C. Cofactor engineering in E. coli using the exogenous coaA and acc genes resulted in fatty acid production over 1 g/L of culture and could effectively function at 23 °C.

Sensing of nutrients by CPT1C controls SAC1 activity to regulate AMPA receptor trafficking.

Carnitine palmitoyltransferase 1C (CPT1C) is a sensor of malonyl-CoA and is located in the ER of neurons. AMPA receptors (AMPARs) mediate fast excitatory neurotransmission in the brain and play a key role in synaptic plasticity. In the present study, we demonstrate across different metabolic stress conditions that modulate malonyl-CoA levels in cortical neurons that CPT1C regulates the trafficking of the major AMPAR subunit, GluA1, through the phosphatidyl-inositol-4-phosphate (PI(4)P) phosphatase SAC1. In normal conditions, CPT1C down-regulates SAC1 catalytic activity, allowing efficient GluA1 trafficking to the plasma membrane. However, under low malonyl-CoA levels, such as during glucose depletion, CPT1C-dependent inhibition of SAC1 is released, facilitating SAC1's translocation to ER-TGN contact sites to decrease TGN PI(4)P pools and trigger GluA1 retention at the TGN. Results reveal that GluA1 trafficking is regulated by CPT1C sensing of malonyl-CoA and provide the first report of a SAC1 inhibitor. Moreover, they shed light on how nutrients can affect synaptic function and cognition.

Enhancement of fatty acid biosynthesis by exogenous acetyl-CoA carboxylase and pantothenate kinase in Escherichia coli.

OBJECTIVES: To establish a technique for efficient fatty acid production through enhancement of coenzyme A (CoA) biosynthesis and malonyl-CoA supply by introducing exogenous pantothenate kinase (coaA) and acetyl-CoA carboxylase (acc) in Escherichia coli. RESULTS: The expression of acc, obtained from Corynebacterium glutamicum, accumulated 2.2-fold more fatty acids in E. coli. The addition of coaA from Pseudomonas putaida or fatty acid synthase (fasA) from C. glutamicum resulted in a 3.1- and 3.6-fold increase in fatty acid synthesis in E. coli cells, which expressed acc and coaA, or acc and fasA, respectively. The transformants, simultaneously possessing all three genes, produced 5.6-fold more fatty acids. The strain possessing acc, coaA, and fasA stored 691 mg/L of fatty acids, primarily as phospholipids, inside the inner membrane after 72-h cultivation. In addition, 19% of the total CoA pool was occupied by malonyl-CoA. CONCLUSIONS: Increased malonyl-CoA significantly contributed to fatty acid production, and the effect was boosted by the expanded total CoA pool. Manipulation of the intracellular CoA species is effective for fatty acid production in E. coli.

Coenzyme A and Its Thioester Pools in Obese Zucker and Zucker Diabetic Fatty Rats.

Feeding behavior is closely related to hypothalamic malonyl-CoA level in the brain and diet-induced obesity affects total CoA pools in liver. Herein, we performed a comprehensive analysis of the CoA pools formed in thirteen tissues of Zucker and Zucker diabetic fatty (ZDF) rats. Hypothalamic malonyl-CoA levels in obese rats remained low and were almost the same as those of lean rats, despite obese rats having much higher content of leptin, insulin, and glucose in their sera. Regardless of the fa-genotypes, larger total CoA pools were formed in the livers of ZDF rats and the size of hepatic total CoA pools in Zucker rats showed almost one tenth of the size of ZDF rats. The decreased total CoA pool sizes in Zucker rats was observed in the brown adipose tissues, while ZDF-fatty rats possessed 6% of total CoA pool in the lean rats in response to fa deficiency. This substantially lower CoA content in the obese rats would be disadvantageous to non-shivering thermogenesis. Thus, comparing the intracellular CoA behaviors between Zucker and ZDF rats, as well as the lean and fatty rats of each strain would help to elucidate features of obesity and type 2 diabetes in combination with result (s) of differential gene expression analysis and/or comparative genomics.

Decreased hepatic contents of coenzyme A molecular species in mice after subchronic mild social defeat stress.

Social stress may precipitate psychiatric disorders such as depression, which is related to the occurrence of the metabolic syndrome, including obesity and type 2 diabetes. We have evaluated the effects of social stress on central and peripheral metabolism using a model of depression in mice. In the present study, we focused on coenzyme A (CoA) molecular species [i.e. non-esterified CoA (CoASH), acetyl-CoA and malonyl-CoA] which play important roles in numerous metabolic pathways, and we analyzed changes in expression of these molecules in the hypothalamus and liver of adult male mice (C57BL/6J) subjected to 10 days of subchronic mild social defeat stress (sCSDS) with ICR mice as aggressors. Mice (n = 12) exposed to showed hyperphagia- and polydipsia-like symptoms and increased body weight gain compared with control mice which were not affected by exposure to ICR mice (n = 12). To elucidate the underlying metabolic features in the sCSDS model, acetyl-CoA, malonyl-CoA and CoASH tissue levels were analyzed using the acyl-CoA cycling method. The levels of hypothalamic malonyl-CoA, which decreases feeding behavior, were not influenced by sCSDS. However, sCSDS reduced levels of acetyl-CoA, malonyl-CoA and total CoA (sum of the three CoA molecular species) in the liver. Hence, hyperphagia-like symptoms in sCSDS mice evidently occurred independently of hypothalamic malonyl-CoA, but might consequently lead to down-regulation of hepatic CoA via altered expression of nudix hydrolase 7. Future studies should investigate the molecular mechanism(s) underlying the down-regulation of liver CoA pools in sCSDS mice.

Efficient butanol recovery from acetone-butanol-ethanol fermentation cultures grown on sweet sorghum juice by pervaporation using silicalite-1 membrane.

We investigated butanol recovery by pervaporation separation, using a silicalite-1 membrane, from batch cultures of butanol-producing Clostridium beijerinckii SBP2 grown on sweet sorghum juice as a fermentation medium. The pervaporation system yielded 73% (w/v) butanol from intact feed cultures containing 1% (w/v) butanol, and had a butanol permeation flux of 11 g m(-2) h(-1). Upon neutralization and activated charcoal treatment of the feed cultures, butanol yield and total flux increased to 82% (w/v) and 40 g m(-2) h(-1), respectively. This system is applicable to refining processes for practical biobutanol production from a promising energy crop, sweet sorghum.

Genetic engineering and metabolite profiling for overproduction of polyhydroxybutyrate in cyanobacteria.

Genetic engineering and metabolite profiling for the overproduction of polyhydroxybutyrate (PHB), which is a carbon material in biodegradable plastics, were examined in the unicellular cyanobacterium Synechocystis sp. PCC 6803. Transconjugants harboring cyanobacterial expression vectors that carried the pha genes for PHB biosynthesis were constructed. The overproduction of PHB by the engineering cells was confirmed through microscopic observations using Nile red, transmission electron microscopy (TEM), or nuclear magnetic resonance (NMR). We successfully recovered PHB from transconjugants prepared from nitrogen-depleted medium without sugar supplementation in which PHB reached approximately 7% (w/w) of the dry cell weight, showing a value of 12-fold higher productivity in the transconjugant than that in the control strain. We also measured the intracellular levels of acetyl-CoA, acetoacetyl-CoA, and 3-hydroxybutyryl-CoA (3HB-CoA), which are intermediate products for PHB. The results obtained indicated that these products were absent or at markedly low levels when cells were subjected to the steady-state growth phase of cultivation under nitrogen depletion for the overproduction of bioplastics. Based on these results, efficient factors were discussed for the overproduction of PHB in recombinant cyanobacteria.

Effects of chronic mild food restriction on behavior and the hypothalamic malonyl-CoA signaling pathway.

Depression induces anorexia, leading to suppressed feeding behaviors and energy intake. Previously, we revealed that chronic social defeat induced a mild suppression of feeding in rats with elevated levels of hypothalamic malonyl-CoA which regulates feeding. Therefore, we attempted to elucidate the effects of chronic mild food restriction on behavior and on hypothalamic malonyl-CoA. The chronic mild food restricted rats were fed a restricted diet approximately 80% to 90% amount of diet compared to the control for 5 weeks. Ratios of restriction were adjusted with feed consumption in the chronic social defeat stressed rats. Chronic mild food restricted rats exhibited a suppression of body weight gain similar to that of the chronic social defeat stressed rats. Also these rats showed increased time spent in the center area of an open field (OF), prolonged immobility time in forced swim, increased phosphorylation of hypothalamic adenosine monophosphate-activated protein kinase (AMPK) and acetyl-CoA carboxylase and a decreased concentration of hypothalamic malonyl-CoA. Weight of the adrenal glands, locomotion in an OF, mitogen-activated protein kinase cascade and calcium/calmodulin-dependent protein kinases II in the hippocampus were not affected by chronic mild food restriction. Our findings suggest that chronic mild food restriction activates AMPK following a decreased hypothalamic malonyl-CoA.

Effects of chronic social defeat stress on peripheral leptin and its hypothalamic actions.

BACKGROUND: Suppression of body weight and symptom of anorexia are major symptoms of depression. Recently, we reported that chronic social defeat stress (CSDS) induced suppression of body weight gain and anorexic feeding behavior in rats. These abnormalities were the result of disrupted malonyl-coenzyme A (CoA) signaling pathway in the hypothalamus. However, the condition of peripheral leptin and its hypothalamic downstream signal molecules which regulate hypothalamic malonyl-CoA level in the CSDS-exposed rats (CSDS rats) is still unknown. RESULTS: CSDS rats showed suppressed body weight gain and food intake. The weight of the CSDS rats' epididymal white adipose tissues was decreased when compared to the control rats. The plasma cholesterol concentration was decreased significantly in the CSDS rats compared to the control rats (P < 0.05). The plasma glucose concentration was slightly decreased in the CSDS rats compared to the control rats (P < 0.1). The expression of leptin mRNA in epididymal white adipose tissues and the plasma leptin concentration were decreased in CSDS rats. Furthermore, the phosphorylation of the hypothalamic downstream signals of leptin, including extracellular signal-regulated kinase 1/2 (ERK1/2) and signal transducer and activator of transcription 3 (STAT3), was decreased in CSDS rats. CONCLUSIONS: Our results indicated that decreased peripheral leptin expression in CSDS rats could down-regulate the hypothalamic downstream signaling pathways of leptin while suppressed food intake. These data indicate that CSDS induces the down-regulation of hypothalamic AMPK following the elevation of hypothalamic malonyl-CoA levels and is independent of peripheral leptin and glucose.

Long-term increased carnitine palmitoyltransferase 1A expression in ventromedial hypotalamus causes hyperphagia and alters the hypothalamic lipidomic profile.

Lipid metabolism in the ventromedial hypothalamus (VMH) has emerged as a crucial pathway in the regulation of feeding and energy homeostasis. Carnitine palmitoyltransferase (CPT) 1A is the rate-limiting enzyme in mitochondrial fatty acid ß-oxidation and it has been proposed as a crucial mediator of fasting and ghrelin orexigenic signalling. However, the relationship between changes in CPT1A activity and the intracellular downstream effectors in the VMH that contribute to appetite modulation is not fully understood. To this end, we examined the effect of long-term expression of a permanently activated CPT1A isoform by using an adeno-associated viral vector injected into the VMH of rats. Peripherally, this procedure provoked hyperghrelinemia and hyperphagia, which led to overweight, hyperglycemia and insulin resistance. In the mediobasal hypothalamus (MBH), long-term CPT1AM expression in the VMH did not modify acyl-CoA or malonyl-CoA levels. However, it altered the MBH lipidomic profile since ceramides and sphingolipids increased and phospholipids decreased. Furthermore, we detected increased vesicular γ-aminobutyric acid transporter (VGAT) and reduced vesicular glutamate transporter 2 (VGLUT2) expressions, both transporters involved in this orexigenic signal. Taken together, these observations indicate that CPT1A contributes to the regulation of feeding by modulating the expression of neurotransmitter transporters and lipid components that influence the orexigenic pathways in VMH.

Role of prokaryotic type I and III pantothenate kinases in the coenzyme A biosynthetic pathway of Bacillus subtilis.

Pantothenate kinases (CoaAs) catalyze the phosphorylation of pantothenate in the first step of the coenzyme A (CoA) biosynthetic pathway. These bacterial enzymes have been categorized into 3 types, the prokaryotic type I, II, and III CoaAs. Bacteria typically carry a single CoaA gene on their genome, but Bacillus subtilis possesses 2 proteins homologous to type I and III CoaAs, known as BsCoaA and BsCoaX, respectively. Both recombinant proteins exhibited the expected kinase activity and the characteristic properties of type I and III CoaAs, i.e., regulation by CoASH and acyl-CoAs in BsCoaA and the requirement of a monovalent cation in BsCoaX. Both gene disruptants appeared to grow in a manner similar to the wild-type strain. With the BsCoaX disruptant, the BsCoaA had the ability to completely fill the intracellular CoA pool, whereas the BsCoaA disruptant did not. These findings clearly indicate that these 2 CoaAs are employed together in the CoA biosynthetic pathway in B. subtilis and that the contribution of the type I CoaA (BsCoaA) to the formation of the intracellular CoA pool is larger than that of the type III CoaA (BsCoaX).

Antimicrobial activity of pantothenol against staphylococci possessing a prokaryotic type II pantothenate kinase.

Pantothenol is a provitamin of pantothenic acid (vitamin B5) that is widely used in healthcare and cosmetic products. This analog of pantothenate has been shown to markedly inhibit the phosphorylation activity of the prokaryotic type II pantothenate kinase of Staphylococcus aureus, which catalyzes the first step of the coenzyme A biosynthetic pathway. Since type II enzymes are found exclusively in staphylococci, pantothenol suppresses the growth of S. aureus, S. epidermidis, and S. saprophyticus, which inhabit the skin of humans. Therefore, the addition of this provitamin to ointment and skincare products may be highly effective in preventing infections by opportunistic pathogens.

The importance of tissue environment surrounding the tumor on the development of cancer cachexia.

The relationship between host factors and cancer cachexia was investigated. A single cell clone (clone 5 tumor) established from colon 26 adenocarcinoma by limiting dilution cell cloning methods was employed to eliminate the inoculation site-dependent differences in the composition of cell clones. Clone 5 tumor did not provoke manifestations of cancer cachexia when inoculated in subcutaneous tissue. However, when inoculated in the gastrocnemius muscle, the peritoneal cavity or the thoracic cavity of CD2F1 male mice, typical manifestations of cancer cachexia were observed in all groups of mice with intergroup variations. The blood levels of various cytokines, chemokines and hormones were increased but with wide intergroup variations. Analyses by stepwise multiple regression models revealed that serum interleukin-10 was the most significant factor associated with manifestations of cancer cachexia, suggesting the possible involvement of mechanisms similar to cancer patients suffering cancer cachexia. White blood cells, especially neutrophils, seemed to have some roles on the induction of cancer cachexia, because massive infiltrations and an increase in peripheral blood were observed in cachectic mice bearing clone 5 tumors. The amount of malonyl-CoA in liver correlated with manifestations of cancer cachexia, however the mRNA levels of spermidine/spermine N-1 acetyl transferase (SSAT) (of which overexpression has been shown to provoke manifestations similar to cancer cachexia) were not necessarily associated with cancer cachexia. These data suggest that the induction of cancer cachexia depends on the environment in which the tumor grows and that the infiltration of host immune cells into the tumor and the resultant increase in inflammation result in the production of cachectic factors, such as cytokines, leading to SSAT activation. Further, multiple factors likely mediate the mechanisms of cancer cachexia. Finally, this animal model was suitable for the investigation of the mechanisms involved in cachexia of cancer patients.

Marked phenotypic differences of endurance performance and exercise-induced oxygen consumption between AMPK and LKB1 deficiency in mouse skeletal muscle: changes occurring in the diaphragm.

LKB1 phosphorylates members of the AMP-activated protein kinase (AMPK) family. LKB1 and AMPK in the skeletal muscle are believed to regulate not only fuel oxidation during exercise but also exercise capacity. LKB1 was also required to prevent diaphragm fatigue, which was shown to affect exercise performance. Using mice expressing dominant negative (DN) mutants of LKB1 and AMPK, specifically in the skeletal muscle but not in the heart, we investigated the roles of LKB1 and AMPK activity in exercise performance and the effects of these kinases on the characteristics of respiratory and locomotive muscles. In the diaphragm and gastrocnemius, both AMPK-DN and LKB1-DN mice showed complete loss of AMPKα2 activity, and LKB1-DN mice showed a reduction in LKB1 activity. Exercise capacity was significantly reduced in LKB1-DN mice, with a marked reduction in oxygen consumption and carbon dioxide production during exercise. The diaphragm from LKB1-DN mice showed an increase in myosin heavy chain IIB and glycolytic enzyme expression. Normal respiratory chain function and CPT I activity were shown in the isolated mitochondria from LKB1-DN locomotive muscle, and the expression of genes related to fiber type, mitochondria function, glucose and lipid metabolism, and capillarization in locomotive muscle was not different between LKB1-DN and AMPK-DN mice. We concluded that LKB1 in the skeletal muscle contributes significantly to exercise capacity and oxygen uptake during exercise. LKB1 mediated the change of fiber-type distribution in the diaphragm independently of AMPK and might be responsible for the phenotypes we observed.

Identification of pantoate kinase and phosphopantothenate synthetase from Methanospirillum hungatei.

Pantothenate synthetase (PanC) and pantothenate kinase which function in the canonical coenzyme A (CoA) biosynthetic pathway cannot be found in most archaea. COG1829 and COG1701 intrinsic to archaea were proposed as the candidate proteins for producing 4'-phosphopantothenate instead, and the COG1701 protein from Methanosarcina mazei was assigned as PanC. Meanwhile, the Thermococcus kodakarensis COG1829 and COG1701 proteins were biochemically identified as novel enzymes, i.e., pantoate kinase (PoK) and phosphopantothenate synthetase (PPS). In this study, the functions of Mhun_0831 (COG1829) and Mhun_0832 (COG1701) from Methanospirillum hungatei were identified, and the recombinant enzymes were partially characterized. Plasmids simultaneously possessing the two genes encoding Mhun_0831 and Mhun_0832 complemented the poor growth of the temperature-sensitive Escherichia coli pantothenate kinase mutant ts9. The recombinant Mhun_0831 and Mhun_0832 expressed in E. coli cells exhibited PoK and PPS activities, respectively, being in accord with the functions of T. kodakarensis proteins. The PoK activity was most active at pH 8.5 and 40°C, and accepted ATP and UTP as a phosphate donor. Although CoA did not affect the PoK activity, the end product considerably accelerated the PPS activity. The homologs of both proteins are widely conserved in most archaeal genomes. Taken together, our findings indicate that archaea can synthesize CoA through the unique pathway involving PoK and PPS, in addition to the canonical one that the order Thermoplasmatales employs.

Effect of diet composition on coenzyme A and its thioester pools in various rat tissues.

Three coenzyme A (CoA) molecular species, i.e., acetyl-CoA, malonyl-CoA, and nonesterified CoA (CoASH), in 13 types of fasted rat tissue were analyzed. A relatively larger pool size of total CoA, consisting of acetyl-CoA, malonyl-CoA, and CoASH, was observed in the medulla oblongata, liver, heart, and brown adipose tissue. Focusing on changes in the CoA pool size in response to the nutrient composition of the diet given, total CoA pools in rats continuously fed a high-fat diet for 4 weeks were significantly higher in the hypothalamus, cerebellum, and kidney, and significantly lower in the liver and skeletal muscle than those of rats fed a high-carbohydrate or high-protein diet. In particular, reductions in the liver were remarkable and were caused by decreased CoASH levels. Consequently, the total CoA pool size was reduced by approximately one-fifth of the hepatic contents of rats fed the other diets. In the hypothalamus, which monitors energy balance, all three CoA molecular species measured were at higher levels when rats were fed the high-fat diet. Thus, it was of interest that feeding rats a high-fat diet affected the behaviors of CoA pools in the hypothalamus, liver, and skeletal muscle, suggesting a significant relationship between CoA pools, especially malonyl-CoA and/or CoASH pools, and lipid metabolism in vivo.