Genetic analysis of acyl-CoA carboxylases involved in lipid accumulation in Rhodococcus jostii RHA1.

In actinomycetes, the acyl-CoA carboxylases, including the so-called acetyl-CoA carboxylases (ACCs), are biotin-dependent enzymes that exhibit broad substrate specificity and diverse domain and subunit arrangements. Bioinformatic analyses of the Rhodococcus jostii RHA1 genome found that this microorganism contains a vast arrange of putative acyl-CoA carboxylases domains and subunits. From the thirteen putative carboxyltransferase domains, only the carboxyltransferase subunit RO01202 and the carboxyltransferase domain present in the multidomain protein RO04222 are highly similar to well-known essential ACC subunits from other actinobacteria. Mutant strains in each of these genes showed that none of these enzymes is essential for R. jostii growth in rich or in minimal media with high nitrogen concentration, presumably because of their partial overlapping activities. A mutant strain in the ro04222 gene showed a decrease in triacylglycerol and mycolic acids accumulation in rich and minimal medium, highlighting the relevance of this multidomain ACC in the biosynthesis of these lipids. On the other hand, RO01202, a carboxyltransferase domain of a putative ACC complex, whose biotin carboxylase and biotin carboxyl carrier protein domain were not yet identified, was found to be essential for R. jostii growth only in minimal medium with low nitrogen concentration. The results of this study have identified a new component of the TAG-accumulating machinery in the oleaginous R. jostii RHA1. While non-essential for growth and TAG biosynthesis in RHA1, the activity of RO04222 significantly contributes to lipogenesis during single-cell oil production. Furthermore, this study highlights the high functional diversity of ACCs in actinobacteria, particularly regarding their essentiality under different environmental conditions. KEY POINTS: • R. jostii possess a remarkable heterogeneity in their acyl-carboxylase complexes. • RO04222 is a multidomain acetyl-CoA carboxylase involved in lipid accumulation. • RO01202 is an essential carboxyltransferase only at low nitrogen conditions.

Identification of acetyl-CoA carboxylase alpha as a prognostic and targeted candidate for hepatocellular carcinoma.

PURPOSE: The de novo lipogenesis has been a longstanding observation in hepatocellular carcinoma (HCC). However, the prognostic value and carcinogenic roles of the enzyme Acetyl-CoA carboxylase alpha (ACACA) in HCC remains unknown. METHODS: The proteins with remarkable prognostic significance were screened out from The Cancer Proteome Atlas Portal (TCPA) database. Furthermore, the expression characteristics and prognostic value of ACACA were evaluated in multiple databases and the local HCC cohort. The loss-of-function assays were performed to uncover the potential roles of ACACA in steering malignant behaviors of HCC cells. The underlying mechanisms were conjectured by bioinformatics and validated in HCC cell lines. RESULTS: ACACA was identified as a crucial factor of HCC prognosis. Bioinformatics analyses showed that HCC patients with higher expression of ACACA protein or mRNA levels had poor prognosis. Knockdown of ACACA remarkably crippled the proliferation, colony formation, migration, invasion, epithelial-mesenchymal transition (EMT) process of HCC cells and induced the cell cycle arrest. Mechanistically, ACACA might facilitate the malignant phenotypes of HCC through aberrant activation of Wnt/ß-catenin signaling pathway. In addition, ACACA expression was associated with the dilute infiltration of immune cells including plasmacytoid DC (pDC) and cytotoxic cells by utilization of relevant database analysis. CONCLUSION: ACACA could be a potential biomarker and molecular target for HCC.

Deficiency of Acetyl-CoA Carboxylase Impairs Digestion, Lipid Synthesis, and Reproduction in the Kissing Bug Rhodnius prolixus.

Rhodnius prolixus is a hematophagous insect, vector of Chagas disease. After feeding, as blood is slowly digested, amino acids are used as substrates to fuel lipid synthesis, and adult females accumulate lipids in the fat body and produce eggs. In order to evaluate the importance of de novo fatty acid synthesis for this insect metabolism, we generated acetyl-CoA carboxylase (ACC) deficient insects. The knockdown (AccKD) females had delayed blood digestion and a shorter lifespan. Their fat bodies showed reduced de novo lipogenesis activity, did not accumulate triacylglycerol during the days after blood meal, and had smaller lipid droplets. At 10 days after feeding, there was a general decrease in the amounts of neutral lipids and phospholipids in the fat body. In the hemolymph, no difference was observed in lipid composition at 5 days after blood meal, but at day ten, there was an increase in hydrocarbon content and a decrease in phospholipids. Total protein concentration and amino acid composition were not affected. The AccKD females laid 60% fewer eggs than the control ones, and only 7% hatched (89% for control), although their total protein and triacylglycerol contents were not different. Scanning electron microscopy of the egg surface showed that chorion (eggshell) from the eggs laid by the AccKD insects had an altered ultrastructural pattern when compared to control ones. These results show that ACC has a central role in R. prolixus nutrient homeostasis, and its appropriate activity is important to digestion, lipid synthesis and storage, and reproductive success.

Blood meal drives de novo lipogenesis in the fat body of Rhodnius prolixus.

In insects, lipids are stored in the fat body mainly as triacylglycerol. Lipids can be directly provided by digestion and incorporated from the hemolymph, or synthesized de novo from other substrates such as carbohydrates and amino acids. The first step in de novo lipid synthesis is catalyzed by acetyl-CoA carboxylase (ACC), which carboxylates acetyl-CoA to form malonyl-CoA. Rhodnius prolixus is a hematophagous insect vector of Chagas disease and feeds exclusively on large and infrequent blood meals. Adult females slowly digest the blood and concomitantly accumulate lipids in the fat body. In this study, we investigated the regulation of R. prolixus ACC (RhoprACC) expression and de novo lipogenesis activity in adult females at different nutritional and metabolic conditions. A phylogenetic analysis showed that insects, similar to other arthropods and unlike vertebrate animals, have only one ACC gene. In females on the fourth day after a blood meal, RhoprACC transcript levels were similar in the anterior and posterior midgut, fat body and ovary and higher in the flight muscles. In the fat body, gene expression was higher in fasted females and decreased after a blood meal. In the posterior midgut it increased after feeding, and no variation was observed in the flight muscle. RhoprACC protein content analysis of the fat body revealed a profile similar to the gene expression, with higher protein contents before feeding and in the first two days after a blood meal. Radiolabeled acetate was used to follow de novo lipid synthesis in the fat body and it was incorporated mainly into triacylglycerol, diacylglycerol and phospholipids. This lipogenic activity was inhibited by soraphen A, an ACC inhibitor, and it varied according to the insect metabolic status. De novo lipogenesis was very low in starved females and increased during the initial days after a blood meal. The flight muscles had a very low capacity to synthesize lipids when compared to the fat body. Radiolabeled leucine was also used as a substrate for de novo lipogenesis and the same lipid classes were formed. In conclusion, our results indicate that the blood meal induces the utilization of diet-derived amino acids by de novo lipogenesis in the fat body, and that the control of this activity does not occur at the RhoprACC gene or protein expression level.

Revisiting the coupling of fatty acid to phospholipid synthesis in bacteria with FapR regulation.

A key aspect in membrane biogenesis is the coordination of fatty acid to phospholipid synthesis rates. In most bacteria, PlsX is the first enzyme of the phosphatidic acid synthesis pathway, the common precursor of all phospholipids. Previously, we proposed that PlsX is a key regulatory point that synchronizes the fatty acid synthase II with phospholipid synthesis in Bacillus subtilis. However, understanding the basis of such coordination mechanism remained a challenge in Gram-positive bacteria. Here, we show that the inhibition of fatty acid and phospholipid synthesis caused by PlsX depletion leads to the accumulation of long-chain acyl-ACPs, the end products of the fatty acid synthase II. Hydrolysis of the acyl-ACP pool by heterologous expression of a cytosolic thioesterase relieves the inhibition of fatty acid synthesis, indicating that acyl-ACPs are feedback inhibitors of this metabolic route. Unexpectedly, inactivation of PlsX triggers a large increase of malonyl-CoA leading to induction of the fap regulon. This finding discards the hypothesis, proposed for B. subtilis and extended to other Gram-positive bacteria, that acyl-ACPs are feedback inhibitors of the acetyl-CoA carboxylase. Finally, we propose that the continuous production of malonyl-CoA during phospholipid synthesis inhibition provides an additional mechanism for fine-tuning the coupling between phospholipid and fatty acid production in bacteria with FapR regulation.

Fatty acid biosynthesis is enhanced in Escherichia coli strains with deletion in genes encoding the PII signaling proteins.

The committed and rate-limiting step in fatty acid biosynthesis is catalyzed by acetyl-CoA carboxylase (ACC). In previous studies we showed that ACC activity is inhibited through interactions with the PII signaling proteins in vitro. Here we provide in vivo support for that model; we noted that PII proteins are able to reduce malonyl-CoA levels in vivo in Escherichia coli. Furthermore, we show that fatty acid biosynthesis is strongly enhanced in E. coli strains carrying deletions in PII coding genes. Given that PII proteins act as conserved negative regulators of ACC in Bacteria, our findings may be explored to engineer other prokaryotes to improve fatty acid yields, thereby turning microbial biofuel production economically competitive in the future.

Adipose tissue is less responsive to food restriction anti-inflammatory effects than liver, muscle, and brain in mice

High caloric intake promotes chronic inflammation, insulin resistance, and chronic diseases such as type-2 diabetes, which may be prevented by food restriction (FR). The effect of FR on expression of pro-inflammatory and anti-inflammatory genes in adipose tissue, liver, muscle, and brain was compared. Male Swiss mice were submitted to FR (FR group) or had free access to food (control group) during 56 days. The liver, gastrocnemius muscle, brain, and epididymal white adipose tissue (WAT) were collected for analysis of gene expressions. FR attenuated inflammation in the liver, brain, and gastrocnemius muscle but did not markedly change inflammatory gene expression in epididymal WAT. We concluded that adipose tissue was less responsive to FR in terms of gene expression of pro-inflammatory and anti-inflammatory genes.

Intermittent food restriction in female rats induces SREBP high expression in hypothalamus and immediately postfasting hyperphagia.

OBJECTIVE: We investigated the effect of intermittent food restriction (IFR) cycles on hypothalamic expression of lipogenic proteins and induction of overeating. METHODS: Female Wistar rats were distributed in three groups: free access to feed (control, C), 2 d feed restriction at 50% of C intake followed by 3 d (restricted 3, R3) or 5 d (restricted 5, R5) ad libitum feeding. After 6 wk, the rats were submitted to euthanasia and collected the hypothalamus and blood. The deposits of retroperitoneal, mesenteric, and gonadal fat were weighed. The expression of the mRNA for sterol regulatory element binding protein (SREBP) 1c and 2 and acetyl-CoA carboxylase in the hypothalamus were determined by real-time polymerase chain reaction, and glucose and triacylglycerol were evaluated by a commercial kit. Body mass and food intake were measured daily. RESULTS: IFR promoted increased expression of SREBP-2 in both treated groups and, in R5, increased expression of SREBP-1c. The serum triacylglycerol, mesenteric deposit, and total fat content were higher in R3. Neither of the treatment intervals altered the expression of the mRNA of acetyl-CoA carboxylase enzyme but induced hyperglycemia and higher food intake immediately after food restriction. CONCLUSION: IFR affected the expression of SREBP-1c in R5 and SREBP-2 in the hypothalamus and caused overeating immediately after fasting in both groups. We suggest that hypothalamic and peripheral alterations, coupled with compulsive eating behavior in the ad libitum period, indicate risks for diabetes mellitus and recovery of body mass after interruption of IFR.

Maternal conjugated linoleic acid modulates TAG metabolism in adult rat offspring.

Conjugated linoleic acid (CLA) might regulate the lipid depots in liver and adipose tissue. As there is an association between maternal nutrition, fat depots and risk of offspring chronic disease, the aim was to investigate the effect of maternal CLA consumption on TAG regulation and some inflammatory parameters in adult male rat offspring receiving or not receiving CLA. Female Wistar rats were fed control (C) or CLA-supplemented (1 %, w/w) diets during 4 weeks before and throughout pregnancy and lactation. After weaning, male offspring of CLA rats were fed C or CLA diets (CLA/C and CLA/CLA groups, respectively), whereas C male rat offspring were fed a C diet (C/C group) for 9 weeks. Serum TAG levels were increased in the CLA/CLA and CLA/C groups, associated with a reduction of lipoprotein lipase activity and weights of adipose tissue. The liver TAG levels were decreased in the CLA/CLA group, related to a significant reduction of fatty acid synthase (FAS), acetyl-CoA carboxylase (ACC) and glucose-6-phosphate dehydrogenase enzyme activities, as well as to the mRNA levels of FAS, ACC, stearoyl-CoA desaturase-1 and sterol regulatory element-binding protein-1c. Even though normal TAG levels were found in the liver of CLA/C rats, a reduction of lipogenesis was also observed. Thus, these results demonstrated a programming effect of CLA on the lipid metabolic pathways leading to a preventive effect on the TAG accretion in adipose tissue and the liver of male rat offspring. This knowledge could be important to develop some dietary strategies leading to a reduced incidence of obesity and fatty acid liver disease in humans.

Perinatal maternal high-fat diet induces early obesity and sex-specific alterations of the endocannabinoid system in white and brown adipose tissue of weanling rat offspring.

Perinatal maternal high-fat (HF) diet programmes offspring obesity. Obesity is associated with overactivation of the endocannabinoid system (ECS) in adult subjects, but the role of the ECS in the developmental origins of obesity is mostly unknown. The ECS consists of endocannabinoids, cannabinoid receptors (cannabinoid type-1 receptor (CB1) and cannabinoid type-2 receptor (CB2)) and metabolising enzymes. We hypothesised that perinatal maternal HF diet would alter the ECS in a sex-dependent manner in white and brown adipose tissue of rat offspring at weaning in parallel to obesity development. Female rats received standard diet (9 % energy content from fat) or HF diet (29 % energy content from fat) before mating, during pregnancy and lactation. At weaning, male and female offspring were killed for tissue harvest. Maternal HF diet induced early obesity, white adipocyte hypertrophy and increased lipid accumulation in brown adipose tissue associated with sex-specific changes of the ECS's components in weanling rats. In male pups, maternal HF diet decreased CB1 and CB2 protein in subcutaneous adipose tissue. In female pups, maternal HF diet increased visceral and decreased subcutaneous CB1. In brown adipose tissue, maternal HF diet increased CB1 regardless of pup sex. In addition, maternal HF diet differentially changed oestrogen receptor across the adipose depots in male and female pups. The ECS and oestrogen signalling play an important role in lipogenesis, adipogenesis and thermogenesis, and we observed early changes in their targets in adipose depots of the offspring. The present findings provide insights into the involvement of the ECS in the developmental origins of metabolic disease induced by inadequate maternal nutrition in early life.

Reciprocal regulation of acetyl-CoA carboxylase 1 and senescence in human fibroblasts involves oxidant mediated p38 MAPK activation.

We sought to explore the fate of the fatty acid synthesis pathway in human fibroblasts exposed to DNA damaging agents capable of inducing senescence, a state of irreversible growth arrest. Induction of premature senescence by doxorubicin or hydrogen peroxide led to a decrease in protein and mRNA levels of acetyl-CoA carboxylase 1 (ACC1), the enzyme that catalyzes the rate-limiting step in fatty-acid biosynthesis. ACC1 decay accompanied the activation of the DNA damage response (DDR), and resulted in decreased lipid synthesis. A reduction in protein and mRNA levels of ACC1 and in lipid synthesis was also observed in human primary fibroblasts that underwent replicative senescence. We also explored the consequences of inhibiting fatty acid synthesis in proliferating non-transformed cells. Using shRNA technology, we knocked down ACC1 in human fibroblasts. Interestingly, this metabolic perturbation was sufficient to arrest proliferation and trigger the appearance of several markers of the DDR and increase senescence associated ß-galactosidase activity. Reactive oxygen species and p38 mitogen activated protein kinase phosphorylation participated in the induction of senescence. Similar results were obtained upon silencing of fatty acid synthase (FAS) expression. Together our results point towards a tight coordination of fatty acid synthesis and cell proliferation in human fibroblasts.

The effects of trans-fatty acids on TAG regulation in mice depend on dietary unsaturated fatty acids.

The aim of this study was to investigate the effects of trans-fatty acids (TFA) on liver and serum TAG regulation in mice fed diets containing different proportions of n-3, n-6 and n-9 unsaturated fatty acids (UFA) from olive (O), maize (C) or rapeseed (R) oils partially substituted or not with TFA (Ot, Ct and Rt, respectively). Male CF1 mice were fed (30 d) one of these diets. The effects of the partial substitution (1 %, w/w) of different UFA with TFA on the activity and expression of hepatic enzymes involved in lipogenesis and fatty acids oxidation were evaluated, as well as their transcription factor expressions. Some of the mechanisms involved in the serum TAG regulation, hepatic VLDL rich in TAG (VLDL-TAG) secretion rate and lipoprotein lipase (LPL) activity were assessed. In liver, TFA induced an increase in TAG content in the Ot and Rt groups, and this effect was associated with an imbalance between lipogenesis and ß-oxidation. In the Ot group, exacerbated lipogenesis may be one of the mechanisms responsible for the liver steatosis induced by TFA, whereas in Rt it has been related to a decreased ß-oxidation, compared with their respective controls. The enhanced hepatic VLDL-TAG secretion in the Ot and Rt groups was compensated with a differential removal of TAG by LPL enzyme in extrahepatic tissues, leading to unchanged serum TAG levels. In brief, the effects of low levels of TFA on liver and serum TAG regulation in mice depend on the dietary proportions of n-3, n-6 and n-9 UFA.

Genistein stimulates fatty acid oxidation in a leptin receptor-independent manner through the JAK2-mediated phosphorylation and activation of AMPK in skeletal muscle.

Obesity is a public health problem that contributes to the development of insulin resistance, which is associated with an excessive accumulation of lipids in skeletal muscle tissue. There is evidence that soy protein can decrease the ectopic accumulation of lipids and improves insulin sensitivity; however, it is unknown whether soy isoflavones, particularly genistein, can stimulate fatty acid oxidation in the skeletal muscle. Thus, we studied the mechanism by which genistein stimulates fatty acid oxidation in the skeletal muscle. We showed that genistein induced the expression of genes of fatty acid oxidation in the skeletal muscle of Zucker fa/fa rats and in leptin receptor (ObR)-silenced C2C12 myotubes through AMPK phosphorylation. Furthermore, the genistein-mediated AMPK phosphorylation occurred via JAK2, which was possibly activated through a mechanism that involved cAMP. Additionally, the genistein-mediated induction of fatty acid oxidation genes involved PGC1α and PPARδ. As a result, we observed that genistein increased fatty acid oxidation in both the control and silenced C2C12 myotubes, as well as a decrease in the RER in mice, suggesting that genistein can be used in strategies to decrease lipid accumulation in the skeletal muscle.

Elevated tissue omega-3 fatty acid status prevents age-related glucose intolerance in fat-1 transgenic mice.

The objective of this study was to investigate the impact of elevated tissue omega-3 (n-3) polyunsaturated fatty acids (PUFA) status on age-related glucose intolerance utilizing the fat-1 transgenic mouse model, which can endogenously synthesize n-3 PUFA from omega-6 (n-6) PUFA. Fat-1 and wild-type mice, maintained on the same dietary regime of a 10% corn oil diet, were tested at two different ages (2 months old and 8 months old) for various glucose homeostasis parameters and related gene expression. The older wild-type mice exhibited significantly increased levels of blood insulin, fasting blood glucose, liver triglycerides, and glucose intolerance, compared to the younger mice, indicating an age-related impairment of glucose homeostasis. In contrast, these age-related changes in glucose metabolism were largely prevented in the older fat-1 mice. Compared to the older wild-type mice, the older fat-1 mice also displayed a lower capacity for gluconeogenesis, as measured by pyruvate tolerance testing (PTT) and hepatic gene expression of phosphoenolpyruvate carboxykinase (PEPCK) and glucose 6 phosphatase (G6Pase). Furthermore, the older fat-1 mice showed a significant decrease in body weight, epididymal fat mass, inflammatory activity (NFκ-B and p-IκB expression), and hepatic lipogenesis (acetyl-CoA carboxylase (ACC) and fatty acid synthase (FAS) expression), as well as increased peroxisomal activity (70-kDa peroxisomal membrane protein (PMP70) and acyl-CoA oxidase1 (ACOX1) expression). Altogether, the older fat-1 mice exhibit improved glucose homeostasis in comparison to the older wild-type mice. These findings support the beneficial effects of elevated tissue n-3 fatty acid status in the prevention and treatment of age-related chronic metabolic diseases.

Effects on Bacillus subtilis of conditional expression of the accBC operon encoding subunits of acetyl coenzyme A carboxylase, the first enzyme of fatty acid synthesis.

A Bacillus subtilis strain was constructed in which the operon accBC, encoding the biotin carboxyl carrier protein (BCCP) and biotin carboxylase (BC) subunits of acetyl-CoA carboxylase (ACC), was placed under the control of the IPTG-inducible promoter spac. A reduction in the levels of BCCP resulted in a decrease of de novo fatty acid synthesis and in the total content of membrane fatty acids. This strain was dependent upon the presence of IPTG for a normal growth phenotype. Growth was specifically restored by supplying exogenous long branched-chain fatty acids in the medium, indicating that the inducer-dependent phenotype was specifically related to a conditional block in fatty acid biosynthesis. The strain showed a strong decrease in sporulation frequency when it was induced to sporulate in an IPTG-free medium. Germination and outgrowth were both delayed in spores of the mutant obtained in the absence of IPTG.