Enzymes Of Fatty Acid Synthesis Research Articles

Pseudomonas aeruginosa aerobic fatty acid desaturase DesB is important for virulence factor production

Unsaturated fatty acids (UFAs) play a pivotal role in maintaining a functional cellular membrane in response to changes in environmental factors. Unlike in other gram-negative bacteria, in Pseudomonas aeruginosa, UFA synthesis is governed by 2 pathways: (1) the anaerobic FabAB-mediated pathway and (2) the aerobic inducible DesA/DesB desaturase pathway. Although fatty acids are functional constituents of several known virulence factors, the roles of Pseudomonas aeruginosa fatty acid synthesis enzymes in virulence factor production and pathogenesis have not yet been examined. Previous studies have shown that the mycobacterial DesA1 and DesA3 proteins are required for full virulence. Therefore, we assessed the effect, if any, of mutations affecting the various UFA synthesis enzymes on virulence factor production. Testing of individual mutations or combinations of mutations revealed that desB mutants were severely deficient in the production of proteolytic enzymes, pyocyanin, and rhamnolipid. In addition, the desB mutants showed impaired swarming and twitching motilities and reduced virulence in the Caenorhabditis elegans infection model. Taken together, these results demonstrate that DesB is not only a fatty acid desaturase but also a factor required for full virulence in Pseudomonas aeruginosa. DesB may thus constitute a novel drug target.

Rapamycin Regulates Stearoyl CoA Desaturase 1 Expression in Breast Cancer

Mammalian target of rapamycin (mTOR) signaling is a central regulator of protein translation, cell growth, and metabolism. Alterations of the mTOR signaling pathway are common in cancer, making mTOR a promising therapeutic target. In clinical trials, rapamycin analogs have shown modest response rates for most cancer types, including breast cancer. Therefore, there is an urgent need to better understand the mechanism of action of rapamycin to improve patient selection and to monitor pathway inhibition. To identify novel pharmacodynamic markers of rapamycin activity, we carried out transcriptional profiling of total and polysome-associated RNA in three breast cancer cell lines representing different subtypes. In all three cell lines, we found that rapamycin significantly decreased polysome-associated mRNA for stearoyl-CoA desaturase 1 (SCD1), the rate-limiting enzyme in monounsaturated fatty acid synthesis. Activators of mTOR increased SCD1 protein expression, whereas rapamycin, LY294002, and BEZ235 decreased SCD1 protein expression. Rapamycin decreased total SCD1 RNA expression without inducing a significant decline in its relative polysomal recruitment (polysome/total ratio). Rapamycin did not alter SCD1 mRNA stability. Instead, rapamycin inhibited SCD1 promoter activity and decreased expression of mature transcription factor sterol regulatory element binding protein 1 (SREBP1). Eukaryotic initiation factor 4E (eIF4E) small interfering RNA (siRNA) decreased both SCD1 and SREBP1 expression, suggesting that SCD1 may be regulated through the mTOR/eIF4E-binding protein 1 axis. Furthermore, SCD1 siRNA knockdown inhibited breast cancer cell growth, whereas overexpression increased growth. Taken together these findings show that rapamycin decreases SCD1 expression, establishing an important link between cell signaling and cancer cell fatty acid synthesis and growth.

TRB3: an oxidant stress-induced pseudokinase with a potential to negatively modulate MCP-1 cytokine in diabetic nephropathy

TRB3: an oxidant stress-induced pseudokinase with a potential to negatively modulate MCP-1 cytokine in diabetic nephropathy

Down-Regulation of Adipogenesis and Hyperglycemia in Diet-Induced Obesity Mouse Model by Aloe QDM

Obesity-induced disorders contribute to the development of metabolic diseases such as insulin resistance, fatty liver diseases, and type 2 diabetes (T2D). In this study, we evaluated the hypoglycemic and hypolipidemic effects of aloe formula in high fat diet (HFD)-fed C57BL/6N mice. Male mice fed HFD for 28 weeks received a supplement of aloe formula, PAG, ALS, Aloe QDM, and an Aloe QDM complex for a further 8 weeks and were then compared with regular diet fed mice. After the experimental period, the blood glucose levels of the Aloe QDM complex-and PGZ-supplemented mice were significantly lower than those of the HFD-fed mice. Aloe formula, especially the Aloe QDM complex, and the PGZ treatment group profoundly affected the IPGTT and HOMA-IR. Immunochemistry was done for the morphological observation and the resulting sizes of adipocytes around the epididymis were significantly decreased when comparing the aloe formula-treated and HFD-fed groups. Further, aloe formula decreased mRNA expression of fatty acid synthesis enzymes and led to reduced hepatic steatosis in both liver and WAT. These results suggest that supplementation of Aloe QDM complex in the HFD-fed mice improved insulin resistance by lowering blood glucose levels and reducing adipocytes. Our data suggest that dietary aloe formula reduces obesity-induced glucose tolerance by suppressing fatty acid synthesis in the WAT and liver, both of which are important peripheral tissues affecting insulin resistance. The Aloe QDM complex could be used as a nutritional intervention against T2D.

Induced polymerization of mammalian acetyl-CoA carboxylase by MIG12 provides a tertiary level of regulation of fatty acid synthesis

Acetyl-CoA carboxylase (ACC), the first committed enzyme in fatty acid (FA) synthesis, is regulated by phosphorylation/dephosphorylation, transcription, and an unusual mechanism of protein polymerization. Polymerization of ACC increases enzymatic activity and is induced in vitro by supraphysiological concentrations of citrate (> 5 mM). Here, we show that MIG12, a 22 kDa cytosolic protein of previously unknown function, binds to ACC and lowers the threshold for citrate activation into the physiological range (< 1 mM). In vitro, recombinant MIG12 induced polymerization of ACC (as determined by nondenaturing gels, FPLC, and electron microscopy) and increased ACC activity by > 50-fold in the presence of 1 mM citrate. In vivo, overexpression of MIG12 in liver induced ACC polymerization, increased FA synthesis, and produced triglyceride accumulation and fatty liver. Thus, in addition to its regulation by phosphorylation and transcription, ACC is regulated at a tertiary level by MIG12, which facilitates ACC polymerization and enhances enzymatic activity.

Abstract 950: Energy deprivation by Inositol Hexaphosphate inhibits prostate tumor growth and progression in TRAMP mice

Abstract Prostate cancer (PCa) is the second leading cause of cancer-associated deaths in elderly males in Western world, suggesting that newer approaches are needed to prevent/control PCa. Herein, for the first time, by employing anatomical and dynamic contrast-enhanced (DCE) magnetic resonance imaging (MRI), we evaluated non-invasively, the in vivo chemopreventive efficacy of inositol hexaphosphate (IP6), a major constituent of high fiber diets, against prostate tumor growth and progression in transgenic adenocarcinoma of the mouse prostate (TRAMP) model. The study was conducted on a longitudinal basis to simultaneously assess both prostate sizes and changes in tumor vascularization (perfusion and permeability) due to IP6 feeding. Male TRAMP mice, beginning 4 weeks of age, were fed with 1, 2 or 4% (w/v) IP6 in drinking water or only drinking water till 28 weeks of age and monitored using MRI over the course of treatment. Longitudinal assessment of volumes of prostate and seminal vesicles showed profound effect of 2% and 4% IP6 doses on selectively reducing prostate volume without noticeable changes in seminal vesicles. Further, gadolinium-based DCE-MRI showed decreased tumor perfusion and permeability, indicative of anti-angiogenic effects of IP6 treatment. Histopathological analysis at the end of the study corroborated the MRI results, and showed that IP6-fed mice had less severe prostatic lesions compared to positive controls. Specifically, the mice in IP6-fed groups showed higher incidence of prostatic intraepithelial neoplasia (PIN) at the expense of a strong decrease in adenocarcinoma incidence. Importantly, the mice in IP6-fed groups also had prostatic regions with typical normal histology, which was totally absent in positive controls. IHC and WB results indicated anti-proliferative, pro-apoptotic, and anti-angiogenic effect of IP6 feeding, as evidenced by decreased number of PCNA-, PECAM-1/CD-31-, VEGF- and iNOS- positive cells, together with increased number of TUNEL positive cells. As potential mechanisms of IP6 efficacy, decrease in the expression of glucose transporter (GLUT) proteins together with an increase in phosphorylated levels of AMP-activated kinase (AMPKTh172) and acetyl-CoA carboxylase (ACCSer79) were observed in the prostatic tissue of mice from IP6 fed-groups. Since phosphorylation results in decreased activity of ACC which is an essential enzyme for fatty acid synthesis, our findings suggest that IP6 is interfering with the metabolic events occurring in TRAMP prostate tumor tissue. Together, these findings show that oral IP6 feeding blocks PCa growth and progression in TRAMP mouse model via modulation of metabolic events involved in tumor sustenance and thereby results in energy deprivation within the tumor, suggesting its practical and translational potential in suppressing growth and progression of PCa in humans (NCI RO1 grant CA116636). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 950.

Abstract 3790: Molecular mechanism of cell death by silibinin in colorectal cancer: Apoptosis versus autophagy

Abstract Recently, considerable research efforts have been directed toward the identification of naturally occurring dietary/non-dietary agents for both prevention/intervention of colorectal cancer (CRC). One such agent is silibinin, a flavonolignan isolated from the seeds of milk thistle (Silybum marianum), which has been used clinically and as dietary supplement against liver toxicity for over three decades. Several recent studies conducted by us and others have shown that silibinin effectively inhibits the growth of various cancers of epithelial origin. Regarding CRC, silibinin has been shown to inhibit CRC growth and progression in various pre-clinical models; however, the mechanistic basis for its efficacy against CRC remains largely unknown. Here, we demonstrate for the first time that silibinin rapidly induces oxidative stress in human CRC SW480 cells due to ROS generation with a concomitant dissipation of mitochondrial potential, cytochrome c release and apoptotic death. ROS generation preceded the changes in DiOC6 retention indicating that silibinin triggers oxidative stress prior to mitochondrial disruption and apoptosis. Longer-time exposure of cells with silibinin caused an intensified cytoplasmic vacuolization followed by sequestration of the organelles, which inhibited the release of cytochrome c. Interestingly, this decrease in apoptotic response co-related with increased autophagic events as evidenced by tracking the dynamics of LC3-II within the cells. Further mechanistic studies revealed that silibinin strongly inhibits the activation of PI3K-Akt-mTOR pathway. Co-incident with this effect, energy restriction was induced by silibinin where an enhanced AMP/ATP ratio caused an increase in phospho AMPKTh172 resulting in increased phosphorylation and thereby decreased activity of acetyl-CoA carboxylase, an essential enzyme for fatty acid synthesis. Down regulation of mTOR kinase activity by silibinin also resulted in suppression of protein translation initiation complex leading to suppression of Cap-dependent translation. To further confirm that silibinin causes starvation-induced autophagic death, additional studies were performed to assess the metabolic profile of the cells employing investigative metabolomics study utilizing quantitative high-resolution proton nuclear magnetic resonance spectroscopy (1H-NMRS), which showed that silibinin significantly decreases glucose uptake in the cells eventually leading to cell death. Our completed studies show dual efficacy of silibinin where first it initiates an apoptotic response triggered by ROS generation and then imposes energy restriction within the cell activating autophagic machinery to induce autophagic cell death. Together, these might be major molecular mechanisms of silibinin in its chemopreventive efficacy against colon cancer. This work was supported by NCI RO1 grant CA112304. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3790.

LXR-SREBP-1c-Phospholipid Transfer Protein Axis Controls Very Low Density Lipoprotein (VLDL) Particle Size

Liver X receptors (LXRs) activate triglyceride synthesis in liver directly and indirectly by inducing sterol regulatory element-binding protein-1c (SREBP-1c). When administered to wild-type mice, the LXR activator T0901317 produces a mild and transient hypertriglyceridemia. Here, we show that T0901317 produces massive hypertriglyceridemia when given to mice lacking low density lipoprotein (LDL) receptors (Ldlr(-/-) mice). Triglycerides ranged from 4000 to 6000 mg/dl, and the plasma turned milky. The median diameter of VLDL particles, measured by electron microscopy, increased from 43 to 112 nm, 87% exceeding 80 nm, the size of chylomicrons. Hypertriglyceridemia was prevented in Ldlr(-/-) recipient mice that lacked SREBP-1c (Ldlr(-/-);Srebp-1c(-/-) double knock-out mice). In Ldlr(-/-) mice, T0901317 increased mRNAs not only for enzymes of fatty acid and triglyceride synthesis, but also for phospholipid transfer protein (PLTP), which transfers phospholipids into nascent VLDL, allowing particle expansion. The PLTP increase was blunted in Ldlr(-/-);Srebp-1c(-/-) animals. When Ldlr(-/-);Srebp-1c(-/-) mice received an adenovirus encoding Pltp, the hypertriglyceridemic response to T0901317 was partially restored and the VLDL size increased. We conclude that LXR agonists activate triglyceride synthesis and Pltp transcription by activating Srebp-1c. In concert with the increase in TG synthesis, the increased PLTP permits triglyceride incorporation into abnormally large VLDL, which are removed from plasma by LDL receptors. In the absence of LDL receptors, the large VLDLs accumulate and produce massive hypertriglyceridemia.

Expression of Vibrio harveyi Acyl-ACP Synthetase Allows Efficient Entry of Exogenous Fatty Acids into the Escherichia coli Fatty Acid and Lipid A Synthetic Pathways

Although the Escherichia coli fatty acid synthesis (FAS) pathway is the best studied type II fatty acid synthesis system, a major experimental limitation has been the inability to feed intermediates into the pathway in vivo because exogenously supplied free fatty acids are not efficiently converted to the acyl-acyl carrier protein (ACP) thioesters required by the pathway. We report that expression of Vibrio harveyi acyl-ACP synthetase (AasS), a soluble cytosolic enzyme that ligates free fatty acids to ACP to form acyl-ACPs, allows exogenous fatty acids to enter the E. coli fatty acid synthesis pathway. The free fatty acids are incorporated intact and can be elongated or directly incorporated into complex lipids by acyltransferases specific for acyl-ACPs. Moreover, expression of AasS strains and supplementation with the appropriate fatty acid restored growth to E. coli mutant strains that lack essential fatty acid synthesis enzymes. Thus, this strategy provides a new tool for circumventing the loss of enzymes essential for FAS function.

β‐Adrenergic‐AMPK Pathway Phosphorylates Acetyl‐CoA Carboxylase in a High‐epinephrine Rat Model, SPORTS

We established a new animal model called SPORTS (Spontaneously-Running Tokushima-Shikoku) rats, which show high-epinephrine (Epi) levels. Recent reports show that Epi activates adenosine monophosphate (AMP)-activated protein kinase (AMPK) in adipocytes. Acetyl-CoA carboxylase (ACC) is the rate-limiting enzyme in fatty acid synthesis, and the enzymatic activity is suppressed when its Ser-79 is phosphorylated by AMPK. The aim of this study was to investigate the in vivo effect of Epi on ACC and abdominal visceral fat accumulation. We divided both 6-week male control and SPORTS rats into two groups, which were fed either normal diet or high fat and sucrose (HFS) diet for 16 weeks. At the end of diet treatment, retroperitoneal fat was collected for western blotting and histological analysis. Food intake was not different among the groups, but SPORTS rats showed significantly lower weight gain than control rats in both diet groups. After 10 weeks of diet treatment, glucose tolerance tests (GTTs) revealed that SPORTS rats had increased insulin sensitivity. Furthermore, SPORTS rats had lower quantities of both abdominal fat and plasma triglyceride (TG). In abdominal fat, elevated ACC Ser-79 phosphorylation was observed in SPORTS rats and suppressed by an antagonist of beta-adrenergic receptor (AR), propranolol, or an inhibitor of AMPK, Compound C. From these results, high level of Epi induced ACC phosphorylation mediated through beta-AR and AMPK signaling pathways in abdominal visceral fat of SPORTS rats, which may contribute to reduce abdominal visceral fat accumulation and increase insulin sensitivity. Our results suggest that beta-AR-regulated ACC activity would be a target for treating lifestyle-related diseases, such as obesity.

Inverse relation between FASN expression in human adipose tissue and the insulin resistance level

BackgroundAdipose tissue is a key regulator of energy balance playing an active role in lipid storage and may be a dynamic buffer to control fatty acid flux. Just like PPARγ, fatty acid synthesis enzymes such as FASN have been implicated in almost all aspects of human metabolic alterations such as obesity, insulin resistance or dyslipemia. The aim of this work is to investigate how FASN and PPARγ expression in human adipose tissue is related to carbohydrate metabolism dysfunction and obesity.MethodsThe study included eighty-seven patients which were classified according to their BMI and to their glycaemia levels in order to study FASN and PPARγ gene expression levels, anthropometric and biochemical variables.ResultsThe main result of this work is the close relation between FASN expression level and the factors that lead to hyperglycemic state (increased values of glucose levels, HOMA-IR, HbA1c, BMI and triglycerides). The correlation of the enzyme with these parameters is inversely proportional. On the other hand, PPARγ is not related to carbohydrate metabolism.ConclusionsWe can demonstrate that FASN expression is a good candidate to study the pathophysiology of type II diabetes and obesity in humans.

Inhibitory effects on bacterial growth and b‐ketoacyl‐ACP reductase by different species of maple leaf extracts and tannic acid

It is important to develop new antibiotics aimed at novel targets. The investigation found that the leaf extracts from five maples (Acer platanoides, Acer campestre, Acer rubrum, Acer saccharum and Acer truncatum Bunge collected in Denmark, Canada and China) and their component tannic acid displayed antibacterial ability against 24 standard bacteria strains with the minimum inhibitory concentration of 0.3-8.0 mg/mL. Unlike the standard antibiotic levofloxacin (LFX), these samples inhibited Gram-positive bacteria more effectively than they inhibited Gram-negative bacteria. These samples effectively inhibited two antidrug bacterial strains. The results show that these samples inhibit bacteria by a different mechanism from LFX. These samples potently inhibited b-ketoacyl-ACP reductase (FabG), which is an important enzyme in bacterial fatty acid synthesis. Tannic acid showed the strongest inhibition on FabG with a half inhibition concentration of 0.78 microM (0.81 microg/mL). Furthermore, tannic acid and two maple leaf extracts showed time-dependent irreversible inhibition of FabG. These three samples also exhibited better inhibition on bacteria. It is suggested that FabG is the antibacteria target of maple leaf extracts and tannic acid, and both reversible and irreversible inhibitions of FabG are important for the antibacterial effect.

SREBP pathway genes as candidate markers in country ham production

Country hams are dry-cured products from the Southeastern region of the USA. This high value product requires quality fresh meat to avoid later processing problems. The marker SREBF1 is a transcription factor involved in the regulation of fatty acid synthesis and anti-oxidative enzyme transcription. The SREBF1 gene and its regulators, SCAP and MBTPS1, were investigated for associations with several meat quality traits in country hams. After single nucleotide polymorphism (SNP) identification, PCR-RFLP tests were designed for one polymorphism in each of the three investigated genes. Meat quality and physical traits were collected on 299 fresh hams. Significant associations were found with ham yield (MBTPS1, P<0.05 and SREBF1×MBTPS1, P<0.05) and ham circumference (MBTPS1, P<0.05 and SREBF1×MBTPS1, P<0.01), Hunter A colour score on fresh meat (SREBF1, P<0.05), Hunter B colour score on cured meat (MBTPS1, P<0.05 and SREBF1×MBTPS1, P<0.01), moisture (SCAP×MBTPS1, P<0.05) and salt percentage (SREBF1×SCAP, P<0.05). Our findings provide initial evidence that SNPs in SREBF1, SCAP and MBTPS1 are associated with some country ham quality traits. Breeders could use these gene tests to improve their animals, which would in turn improve country ham processing and other desired production goals.

Sodium lauryl sulphate alters the mRNA expression of lipid‐metabolizing enzymes and PPAR signalling in normal human skin in vivo

Detergents irritate skin and affect skin barrier homeostasis. In this study, healthy skin was exposed to 1% sodium lauryl sulphate (SLS) in water for 24 h. Biopsies were taken 6 h to 8 days post exposure. Lipid patterns were stained in situ and real-time polymerase chain reaction (PCR) was used to examine mRNA expression of enzymes synthesizing barrier lipids, peroxisome proliferator-activated receptors (PPAR) and lipoxygenases. The lipid pattern was disorganized from 6 h to 3 days after SLS exposure. Concomitant changes in mRNA expression included: (i) reduction, followed by induction, of ceramide-generating beta-glucocerebrosidase, (ii) increase on day 1 of two other enzymes for ceramide biosynthesis and (iii) persistent reduction of acetyl-CoA carboxylase-B, a key enzyme in fatty acid synthesis. Surprisingly, the rate-limiting enzyme in cholesterol synthesis, HMG-CoA reductase, was unaltered. Among putative regulators of barrier lipids synthesis, PPARalpha and PPARgamma exhibited reduced mRNA expression, while PPARbeta/delta and LXRbeta were unaltered. Epidermal lipoxygenase-3, which may generate PPARalpha agonists, exhibited reduced expression. In conclusion, SLS induces reorganization of lipids in the stratum corneum, which play a role in detergents' destruction of the barrier. The changes in mRNA expression of enzymes involved in synthesizing barrier lipids are probably important for the restoration of the barrier.

Isolation, structure and biological activities of platensimycin B4 from Streptomyces platensis

Platensimycin and platencin are inhibitors of FabF and FabF/H bacterial fatty acid synthesis enzymes, respectively. Discovery of natural congeners provides one of the ways to understand the relationship of chemical structure and biological function. Efforts to discover the natural analogs of platensimycin by chemical screening led to the isolation of platensimycin B(4), a glucoside congener of platensimycin. This analog showed significantly attenuated activity and critically defined the limited binding space around the aromatic ring and established the importance of the free phenolic and carboxyl group for the activity.

Decrease in FASN Expression in Adipose Tissue of Hypertensive Individuals

Fatty acid (FA) synthesis enzymes (FA synthase (FASN) and acetyl-CoA carboxylase (ACC)) are related to metabolic alterations such as obesity, insulin resistance, or dyslipidemia. Due to the fact that there is no literature that relates FASN and ACC expression with hypertension, we investigate how FASN and ACC expression in adipose tissue is related to hypertension. This study included 87 patients, undergoing laparoscopic surgery procedures after an overnight fast. These patients were classified according to hypertension levels into two groups, normotensive and hypertensive, with a wide range of body mass index (BMI) in order to measure gene expression levels of FASN and ACC and anthropometric and biochemical variables. The main result of this work is a significant decrease of FASN expression in adipose tissue of hypertensive vs. normotensive patients, and that FASN may predict systolic blood pressure (SBP) values by multiple regression analysis and there was also an inverse correlation between FASN expression and SBP. In conclusion, to our knowledge it has been proven, for the first time, that there is a decrease of FASN expression in hypertensive individuals. The clinical significance of this work represents an exciting challenge because it would need to be clarified whether the reduced values of FASN expression may be associated with hypertension or whether this is an adaptive mechanism to the presence of hypertension. Once this aspect is clarified, it could be a new target for the treatment of hypertension.

Arachidonic acid induces acetyl-CoA carboxylase 1 expression via activation of CREB1

Acetyl-CoA carboxylase (ACC; EC 6.4.1.2) is the major enzyme of fatty acid synthesis and oxidation in response to dietary changes. In animals, there are two major isoforms of ACCs, ACC1 and ACC2, which are encoded by different genes and display distinct tissue and cellular distribution. We examined the effect of high concentration of arachidonic acid (AA) on the expression of ACC1 mRNA in HepG2 hepatoma cells cultured in the absence of insulin. After 12h of treatment, AA was found to significantly up-regulate ACC1 mRNA level as well as that of cAMP regulatory element binding protein 1 (CREB1), implying the possible interactions between ACC1 and CREB1. In support of the hypothesis, several potential CREB1 binding sites were identified within the PII promoter of ACC1. Further experiments demonstrated that transient over-expression of CREB1 in HepG2 cells activates ACC1 PII promoter and induces the production of triacylglycerol in response to AA, indicating that the effect of AA on ACC1 is possibly regulated via CREB1.

Use of metabolic control analysis to give quantitative information on control of lipid biosynthesis in the important oil crop, Elaeis guineensis (oilpalm)

* Oil crops are a very important commodity. Although many genes and enzymes involved in lipid accumulation have been identified, much less is known of regulation of the overall process. To address the latter we have applied metabolic control analysis to lipid synthesis in the important crop, oilpalm (Elaeis guineensis). * Top-down metabolic control analysis (TDCA) was applied to callus cultures capable of accumulating appreciable triacylglycerol. The biosynthetic pathway was divided into two blocks, connected by the intermediate acyl-CoAs. Block A comprised enzymes for fatty acid synthesis and Block B comprised enzymes of lipid assembly. * Double manipulation TDCA used diflufenican and bromooctanoate to inhibit Block A and Block B, respectively, giving Block flux control coefficients of 0.61 and 0.39. Monte Carlo simulations provided extra information from previously-reported single manipulation TDCA data, giving Block flux control coefficients of 0.65 and 0.35 for A and B. * These experiments are the first time that double manipulation TDCA has been applied to lipid biosynthesis in any organism. The data show that approaching two-thirds of the total control of carbon flux to lipids in oilpalm cultures lies with the fatty acid synthesis block of reactions. This quantitative information will assist future, informed, genetic manipulation of oilpalm.

Role of Nrf2 in prevention of high-fat diet-induced obesity by synthetic triterpenoid CDDO-Imidazolide

The synthetic oleanolic triterpenoid 1-[2-cyano-3,12-dioxooleana-1,9(11)-dien-28-oyl]imidazole (CDDO-Imidazolide or CDDO-Im) is an extremely potent activator of Nrf2 signaling. In cells undergoing adipogenesis, CDDO-Im prevents lipid accumulation in an Nrf2-dependent manner. However, in vivo evidence for effects of CDDO-Im on obesity is lacking. The goals of these studies were to determine if CDDO-Im can prevent high-fat diet-induced obesogenesis in the mouse, and to elucidate the molecular target of drug action. Wild-type and Nrf2-disrupted C57BL/6J female mice were dosed 3 times per week with 30 μmol/kg CDDO-Im or vehicle by oral gavage, during 95 days of access to a control diet or a high-fat diet. Body weights, organ weights, hepatic fat accumulation and gene expression were measured. Treatment with CDDO-Im effectively prevented high-fat diet-induced increases in body weight, adipose mass, and hepatic lipid accumulation in wild-type mice but not in Nrf2-disrupted mice. Wild-type mice on a high-fat diet and treated with CDDO-Im exhibited higher oxygen consumption and energy expenditure than vehicle-treated mice, while food intake was lower in CDDO-Im-treated than vehicle-treated mice. Levels of gene transcripts for fatty acid synthesis enzymes were downregulated after CDDO-Im treatment in the liver of wild-type mice. This inhibitory effect of CDDO-Im on lipogenic gene expression was significantly reduced in Nrf2-disrupted mice. The results indicate that CDDO-Im is an exceedingly potent agent for preventing obesity, and identify the Nrf2 pathway as a novel target for management of obesogenesis.

Metabolic Adaptations During Lactogenesis

1. Mammary-tissue biopsies were obtained from multiparous cows at 30 and 7 days pre partum and 7 and 40 days postpartum. Investigations of the effect of lactogenesis on fatty acid and lactose synthesis involved measurements of biosynthetic capacity (tissue-slice incubations in vitro) and activities ofrelevant enzymes. 2. Fatty acid synthesis from acetate increased over 20-fold from 30 days pre partum to 40 days post partum. Changes in the lipogenic capacity of mammary-tissue slices more closely paralleled increases in the activities of acetyl-CoA carboxylase (EC 6.4.1.2) and acetyl-CoA synthetase (EC 6.2.1.1) than of other enzymes involved in acetate incorporation into fatty acids or in NADPH generation. 3. Lactose biosynthesis by mammary-tissue slices, lactose synthetase activity (EC 2.4.1.22) and α-lactalbumin concentration were all negligible at 30 days pre partum but increased 2.5-4-fold between 7 days pre partum and 40 days post partum. Phosphoglucomutase (EC 2.7.5.1), UDP-glucose pyrophosphorylase (EC 2.7.7.9) and UDPglucose 4-epimerase (EC 5.1.3.2) had substantial activities at 30 days pre partum and increased less dramatically during lactogenesis. 4. Results are consistent with acetyl-CoA carboxylase and perhaps acetyl-CoA synthetase representing the regulatory enzyme(s) in fatty acid synthesis, with lactose synthetase (α-lactalbumin) serving a similar function in lactose biosynthesis.