Enzymes Of Fatty Acid Synthesis Research Articles

Artemisia capillarisInhibits Lipid Accumulation in 3T3-L1 Adipocytes and Obesity in C57BL/6J Mice Fed a High Fat Diet

The purpose of the current study was to determine the effect of the Artemisia capillaris ethyl acetate (ACE) fraction on diet-induced obesity and to elucidate the underlying mechanism. The ACE fraction treatment decreased the leptin level and fat accumulation in cultured 3T3-L1 adipocytes through the free fatty acids released in the medium. The ACE fraction significantly suppressed the expression of peroxisome proliferator-activated receptor-gamma in cultured 3T3-L1 adipocytes. To determine the effect of the ACE fraction on C57BL/6J male mice, the mice were separated into six groups: normal control (N), N plus 0.1 g/kg body weight ACE (NB), high fat control group (HF), HF plus 0.05 g/kg of body weight ACE (HFA), HF plus 0.1 g/kg of body weight ACE (HFB), and HF plus 0.03 g/kg of body weight rosiglitazone (RG) groups. We speculate that the HFB group exhibits a lipid-lowering effect via increased mitochondrial beta-oxidation, of which the rate-limiting enzyme is carnitine palmitoyl transferase I, the activity of which was significantly increased. Also, the activity of fatty acid synthase, a key enzyme of fatty acid synthesis, was markedly suppressed (19%) in the HFB group, as compared to the HF group, and glycerol-3-phosphate dehydrogenase activity, which is very useful in studying adipogenic differentiation in vitro, was markedly suppressed (30%) in the HFB group compared with the HF group. Furthermore, the HFB group showed lowered hepatic lipid droplet accumulation and adipose tissue weight and size. We suggest that 0.1 g of the ACE fraction/kg of body weight may exert an anti-obesity effect in C57BL/6J mice by enhancing lipid metabolism.

Functions of the Clostridium acetobutylicium FabF and FabZ proteins in unsaturated fatty acid biosynthesis

BackgroundThe original anaerobic unsaturated fatty acid biosynthesis pathway proposed by Goldfine and Bloch was based on in vivo labeling studies in Clostridium butyricum ATCC 6015 (now C. beijerinckii) but to date no dedicated unsaturated fatty acid biosynthetic enzyme has been identified in Clostridia. C. acetobutylicium synthesizes the same species of unsaturated fatty acids as E. coli, but lacks all of the known unsaturated fatty acid synthetic genes identified in E. coli and other bacteria. A possible explanation was that two enzymes of saturated fatty acid synthesis of C. acetobutylicium, FabZ and FabF might also function in the unsaturated arm of the pathway (a FabZ homologue is known to be an unsaturated fatty acid synthetic enzyme in enterococci).ResultsWe report that the FabF homologue located within the fatty acid biosynthetic gene cluster of C. acetobutylicium functions in synthesis of both unsaturated fatty acids and saturated fatty acids. Expression of this protein in E. coli functionally replaced both the FabB and FabF proteins of the host in vivo and replaced E. coli FabB in a defined in vitro fatty acid synthesis system. In contrast the single C. acetobutylicium FabZ homologue, although able to functionally replace E. coli FabZ in vivo and in vitro, was unable to replace FabA, the key dehydratase-isomerase of E. coli unsaturated fatty acid biosynthesis in vivo and lacked isomerase activity in vitro.ConclusionThus, C. acetobutylicium introduces the double of unsaturated fatty acids by use of a novel and unknown enzyme.

Acetyl-CoA carboxylase-α inhibitor TOFA induces human cancer cell apoptosis

Acetyl-CoA carboxylase-α (ACCA) is a rate-limiting enzyme in long chain fatty acid synthesis, playing a critical role in cellular energy storage and lipid synthesis. ACCA is upregulated in multiple types of human cancers and small interfering RNA-mediated ACCA silencing in human breast and prostate cancer cells results in oxidative stress and apoptosis. This study reports for the first time that TOFA (5-tetradecyloxy-2-furoic acid), an allosteric inhibitor of ACCA, is cytotoxic to lung cancer cells NCI-H460 and colon carcinoma cells HCT-8 and HCT-15, with an IC50 at approximately 5.0, 5.0, and 4.5μg/ml, respectively. TOFA at 1.0–20.0μg/ml effectively blocked fatty acid synthesis and induced cell death in a dose-dependent manner. The cell death was characterized with PARP cleavage, DNA fragmentation, and annexin-V staining, all of which are the features of the apoptosis. Supplementing simultaneously the cells with palmitic acids (100μM), the end-products of the fatty acid synthesis pathway, prevented the apoptosis induced by TOFA. Taken together, these data suggest that TOFA is a potent cytotoxic agent to lung and colon cancer cells, inducing apoptosis through disturbing their fatty acid synthesis.

Nutritional and hormonal regulation of fatty acid synthase.

Fasting causes a decrease in the rate of synthesis of fatty acid synthase, the central enzyme in fatty acid synthesis, while refeeding carbohydrate increases synthesis. Insulin also increases the synthesis of fatty acid synthase, while glucagon causes a decline. The mechanism was shown to be transcriptional activation-mediated through a 2.1-kb stretch of the 5'-flanking sequence of the fatty acid synthase gene promoter that contains an insulin response element. These effects were confirmed by in vivo experiments with transgenic mice.

Gene expression in primate liver during viral hemorrhagic fever

BackgroundRhesus macaques infected with lymphocytic choriomeningitis virus (LCMV) provide a model for human Lassa fever. Disease begins with flu-like symptoms and progresses rapidly with fatal consequences. Previously, we profiled the blood transcriptome of LCMV-infected monkeys (M. Djavani et al J. Virol. 2007) showing distinct pre-viremic and viremic stages that discriminated virulent from benign infections. In the present study, changes in liver gene expression from macaques infected with virulent LCMV-WE were compared to gene expression in uninfected monkeys as well as to monkeys that were infected but not diseased.ResultsBased on a functional pathway analysis of differentially expressed genes, virulent LCMV-WE had a broader effect on liver cell function than did infection with non-virulent LCMV-Armstrong. During the first few days after infection, LCMV altered expression of genes associated with energy production, including fatty acid and glucose metabolism. The transcriptome profile resembled that of an organism in starvation: mRNA for acetyl-CoA carboxylase, a key enzyme of fatty acid synthesis was reduced while genes for enzymes in gluconeogenesis were up-regulated. Expression was also altered for genes associated with complement and coagulation cascades, and with signaling pathways involving STAT1 and TGF-β.ConclusionMost of the 4500 differentially expressed transcripts represented a general response to both virulent and mild infections. However, approximately 250 of these transcripts had significantly different expression in virulent infections as compared to mild infections, with approximately 30 of these being differentially regulated during the pre-viremic stage of infection. The genes that are expressed early and differently in mild and virulent disease are potential biomarkers for prognosis and triage of acute viral disease.

MTOR modulates cellular fat metabolism by regulating stearoyl-CoA desaturase 1 transcription.

Abstract Abstract #6030 mTOR (mammalian target of rapamycin) signaling is a central regulator of cell growth and proliferation. Rapamycin and its analogues have been shown to inhibit breast cancer cell growth in preclinical models, and are actively being pursued in clinical trials. We performed a high-throughput microarray polysome analysis to identify novel genes whose expression was regulated by rapamycin at the level of transcription or translation. In three breast cancer cell lines representing different breast cancer subtypes, we found a significant decline in polysome-associated mRNA for stearoyl-CoA desaturase 1 (SCD1), the rate-limiting enzyme in monounsaturated fatty acid synthesis. Insulin and insulin-like growth factor, both activators of mTOR, increase SCD1 expression. Rapamycin and PI3K inhibitors decrease SCD1 protein, as does mTOR siRNA. Quantitative RT-PCR of total, polysomal and monosomal RNA, northern analysis and stability studies demonstrate that rapamycin regulates SCD1 at the level of transcription. Furthermore, rapamycin inhibits the SCD1 promoter activation by mevinolin, an inhibitor of cholesterol biosynthesis. We demonstrate that mTOR regulates SCD1 expression. This establishes a novel link between cell signaling and cancer cell fat metabolism. In doing so, we also present SCD1 as a potential marker of response to treatment by mTOR inhibitors. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 6030.

Type II fatty acid synthesis is essential only for malaria parasite late liver stage development

Intracellular malaria parasites require lipids for growth and replication. They possess a prokaryotic type II fatty acid synthesis (FAS II) pathway that localizes to the apicoplast plastid organelle and is assumed to be necessary for pathogenic blood stage replication. However, the importance of FAS II throughout the complex parasite life cycle remains unknown. We show in a rodent malaria model that FAS II enzymes localize to the sporozoite and liver stage apicoplast. Targeted deletion of FabB/F, a critical enzyme in fatty acid synthesis, did not affect parasite blood stage replication, mosquito stage development and initial infection in the liver. This was confirmed by knockout of FabZ, another critical FAS II enzyme. However, FAS II-deficient Plasmodium yoelii liver stages failed to form exo-erythrocytic merozoites, the invasive stage that first initiates blood stage infection. Furthermore, deletion of FabI in the human malaria parasite Plasmodium falciparum did not show a reduction in asexual blood stage replication in vitro. Malaria parasites therefore depend on the intrinsic FAS II pathway only at one specific life cycle transition point, from liver to blood.

Functional expression of Francisella tularensis FabH and FabI, potential antibacterial targets

Francisella tularensis is an extremely infectious airborne pathogen that has long been considered as a potential biological weapon. Enzymes of fatty acid synthesis (FAS) pathway are attractive targets for the development of new antibacterial agents because of differences between the biosynthesis pathways of bacteria and mammals. We report here the first expression of three functional enzymes in F. tularensis FAS-II pathway: FabH (3-oxoacyl-acyl carrier protein synthase III) which initiates elongation in FAS-II; FabD (Malonyl-CoA-acyl carrier protein transacylase) which catalyzes the transfer of a malonyl moiety from malonyl-CoA to ACP generating malonyl-ACP, and FabI (enoyl-ACP reductase) which catalyzes the reduction of enoyl-acyl-ACP derivatives. The genes encoding the FabD, FabH, and FabI were custom synthesized and cloned in pET15b expression vector. Each recombinant His-tagged fusion protein was overexpressed by IPTG induction, and then purified by affinity chromatography on a Ni-NTA column. The purified FabH and FabI have been used as targets for new drug development. Screening of a class of indole-2-carboxylic acid compounds has led to the discovery of several new compounds with promising activity against F. tularensis FabH or FabI enzymes. For example, indole derivative WIUAKP-001 inhibited 80% the FabH enzyme at 40 μM with IC 50 value of 2 μM whereas WIUAKP-031 inhibited 98% the FabI enzyme at 37.5 μM with IC 50 value of 6 μM. These compounds hold great promise for future development of new indole derivatives as inhibitors of type II FAS enzymes, and as potential new treatment for tularemia.

AMP-activated Protein Kinase Contributes to UV- and H2O2-induced Apoptosis in Human Skin Keratinocytes

AMP-activated protein kinase or AMPK is an evolutionarily conserved sensor of cellular energy status, activated by a variety of cellular stresses that deplete ATP. However, the possible involvement of AMPK in UV- and H(2)O(2)-induced oxidative stresses that lead to skin aging or skin cancer has not been fully studied. We demonstrated for the first time that UV and H(2)O(2) induce AMPK activation (Thr(172) phosphorylation) in cultured human skin keratinocytes. UV and H(2)O(2) also phosphorylate LKB1, an upstream signal of AMPK, in an epidermal growth factor receptor-dependent manner. Using compound C, a specific inhibitor of AMPK and AMPK-specific small interfering RNA knockdown as well as AMPK activator, we found that AMPK serves as a positive regulator for p38 and p53 (Ser(15)) phosphorylation induced by UV radiation and H(2)O(2) treatment. We also observed that AMPK serves as a negative feedback signal against UV-induced mTOR (mammalian target of rapamycin) activation in a TSC2-dependent manner. Inhibiting mTOR and positively regulating p53 and p38 might contribute to the pro-apoptotic effect of AMPK on UV- or H(2)O(2)-treated cells. Furthermore, activation of AMPK also phosphorylates acetyl-CoA carboxylase or ACC, the pivotal enzyme of fatty acid synthesis, and PFK2, the key protein of glycolysis in UV-radiated cells. Collectively, we conclude that AMPK contributes to UV- and H(2)O(2)-induced apoptosis via multiple mechanisms in human skin keratinocytes and AMPK plays important roles in UV-induced signal transduction ultimately leading to skin photoaging and even skin cancer.

High content secondary screening assay to model NRTI-induced mitochondrial toxicity

Several recent reports suggest that stearoyl-CoA desaturase 1 (SCD1), the rate-limiting enzyme in monounsaturated fatty acid synthesis, plays an important role in regulating lipid homeostasis and lipid oxidation in metabolically active tissues. As several manifestations of type 2 diabetes and related metabolic disorders are associated with alterations in intracellular lipid partitioning, pharmacological manipulation of SCD1 activity might be of benefit in the treatment of these disease states. In an effort to identify small molecule inhibitors of SCD1, we have developed a mass spectrometry based high-throughput screening (HTS) assay using deuterium labeled stearoyl-CoA substrate and induced rat liver microsomes. The methodology developed allows the use of a nonradioactive substrate which avoids interference by the endogenous SCD1 substrate and/or product that exist in the non-purified enzyme source. Throughput of the assay was up to twenty 384-well assay plates per day. The assay was linear with protein concentration and time, and was saturable for stearoyl-CoA substrate (Km = 10.5 μM). The assay was highly reproducible with an average Z′ value = 0.6. Conjugated linoleic acid and sterculic acid, known inhibitors of SCD1, exhibited IC50 values of 0.88 and 0.12 μM, respectively. High-throughput mass spectrometry screening of over 1.7 million compounds in compressed format demonstrated that the enzyme target is druggable. A total of 2515 hits were identified (0.1% hit rate), and 346 were confirmed active (>40% inhibition of total SCD activity at 20 μM – 14% conformation rate). Of the confirmed hits 172 had IC50 values of <10 μM, including 111 <1 μM and 48 <100 nM. A large number of potent drug-like (MW < 450) hits representing six different chemical series were identified. The application of mass spectrometry to high-throughput screening permitted the development of a high-quality screening protocol for an otherwise intractable target, SCD1. Further medicinal chemistry and characterization of SCD inhibitors should lead to the development of reagents to treat metabolic disorders.

Central Resistin Regulates Hypothalamic and Peripheral Lipid Metabolism in a Nutritional-Dependent Fashion

Evidence suggests that the adipocyte-derived hormone resistin (RSTN) directly regulates both feeding and peripheral metabolism through, so far, undefined hypothalamic-mediated mechanisms. Here, we demonstrate that the anorectic effect of RSTN is associated with inappropriately decreased mRNA expression of orexigenic (agouti-related protein and neuropeptide Y) and increased mRNA expression of anorexigenic (cocaine and amphetamine-regulated transcript) neuropeptides in the arcuate nucleus of the hypothalamus. Of interest, RSTN also exerts a profound nutrition-dependent inhibitory effect on hypothalamic fatty acid metabolism, as indicated by increased phosphorylation levels of both AMP-activated protein kinase and its downstream target acetyl-coenzyme A carboxylase, associated with decreased expression of fatty acid synthase in the ventromedial nucleus of the hypothalamus. In addition, we also demonstrate that chronic central RSTN infusion results in decreased body weight and major changes in peripheral expression of lipogenic enzymes, in a tissue-specific and nutrition-dependent manner. Thus, in the fed state central RSTN is associated with induced expression of fatty acid synthesis enzymes and proinflammatory cytokines in liver, whereas its administration in the fasted state does so in white adipose tissue. Overall, our results indicate that RSTN controls feeding and peripheral lipid metabolism and suggest that hepatic RSTN-induced insulin resistance may be mediated by central activation of de novo lipogenesis in liver.

Development of a high-throughput screening assay for stearoyl-CoA desaturase using rat liver microsomes, deuterium labeled stearoyl-CoA and mass spectrometry

Several recent reports suggest that stearoyl-CoA desaturase 1 (SCD1), the rate-limiting enzyme in monounsaturated fatty acid synthesis, plays an important role in regulating lipid homeostasis and lipid oxidation in metabolically active tissues. As several manifestations of type 2 diabetes and related metabolic disorders are associated with alterations in intracellular lipid partitioning, pharmacological manipulation of SCD1 activity might be of benefit in the treatment of these disease states. In an effort to identify small molecule inhibitors of SCD1, we have developed a mass spectrometry based high-throughput screening (HTS) assay using deuterium labeled stearoyl-CoA substrate and induced rat liver microsomes. The methodology developed allows the use of a nonradioactive substrate which avoids interference by the endogenous SCD1 substrate and/or product that exist in the non-purified enzyme source. Throughput of the assay was up to twenty 384-well assay plates per day. The assay was linear with protein concentration and time, and was saturable for stearoyl-CoA substrate ( K m = 10.5 μM). The assay was highly reproducible with an average Z′ value = 0.6. Conjugated linoleic acid and sterculic acid, known inhibitors of SCD1, exhibited IC 50 values of 0.88 and 0.12 μM, respectively. High-throughput mass spectrometry screening of over 1.7 million compounds in compressed format demonstrated that the enzyme target is druggable. A total of 2515 hits were identified (0.1% hit rate), and 346 were confirmed active (>40% inhibition of total SCD activity at 20 μM – 14% conformation rate). Of the confirmed hits 172 had IC 50 values of <10 μM, including 111 <1 μM and 48 <100 nM. A large number of potent drug-like (MW < 450) hits representing six different chemical series were identified. The application of mass spectrometry to high-throughput screening permitted the development of a high-quality screening protocol for an otherwise intractable target, SCD1. Further medicinal chemistry and characterization of SCD inhibitors should lead to the development of reagents to treat metabolic disorders.

Fatty Acid Synthase Expression in Barrett's Esophagus and Its Detection in Endoscopic Surveillance

Background and Aims: Fatty acid synthase (FAS) is a key enzyme of fatty acid synthesis from carbohydrate. FAS also expresses in regenerating epithelium, intestinal metaplastic epithelium, and dysplastic lesions of the stomach, suggesting that FAS expression may be indicative of a pre-cancerous stage. We found FAS expression in all the cases with Barrett's cancer and in some cases with Barrett's esophagus (BE) by immunohistochemical and RT-PCR methods. Therefore, FAS expression may play an important role in BE and its carcinogenesis. In this study, the significance of FAS expression in BE and the endoscopic method for detecting FAS-positive BE were investigated. Patients and Methods: 180 patients (mean age 65.0+/−12.3 years, M:F = 124:56) histologically proven BE were enrolled. Prior to endoscopic examination, they were assessed for GERD and administration of PPIs and NSAIDs using a structured questionnaire. H. pylori infection and endoscopic findings of reflux esophagitis, hiatal hernia, and gastric mucosal atrophy were also investigated. Using immunohistochemistry, mucin-phenotyping of BE; expression of FAS, COX-2, and Cdx2; cellular proliferation and apoptotic activity were evaluated in biopsy samples. Factors influencing FAS expression were determined by logistic multivariate analysis. Fifty consecutive patients with BE were surveyed by crystal violet chromoendoscopy and the relationship between the pit pattern and FAS expression was also evaluated by RT-PCR methods and immunohistochemistry. The pit pattern was classified as previously proposed by us (Dig Liv Dis 2006). Results: FAS expression was observed in 52.6% of patients with BE. BE with FAS expression showed an expression of COX-2 protein, an accelerated cellular proliferation and in inhibited apoptosis. Presence of reflux esophagitis (OR 4.28, p = 0.045), intestinal mucin-phenotype (OR 6.78, p = 0.008), Cdx2 protein (OR 13.29, p = 0.038), COX-2 protein (OR 1.76, p = 0.021), elevated PCNA index (OR 1.09, p = 0.009), and lower apoptosis index (OR 0.75, p = 0.039) were confirmed to be positive independent factors for the expression of FAS. FAS-positive BE significantly showed an open-type pit pattern (p < 0.001). Discussion and Conclusion: FAS is strongly expressed in intestinal mucin-phenotype BE with accelerated cell proliferation and inhibited apoptosis, suggesting that FAS expression is a marker for malignant potential of BE. Moreover, BE with FAS expression could be easily detected by crystal violet chromoendoscopy and this may play an important role in endoscopic Barrett's surveillance. However, the role of FAS for the carcinogenesis of BE should be more clearly elucidated in the future study.

Contribution of de novo fatty acid synthesis to hepatic steatosis and insulin resistance: lessons from genetically engineered mice

Nonalcoholic fatty liver disease (NAFLD) is associated with obesity, insulin resistance, and type 2 diabetes. NAFLD represents a large spectrum of diseases ranging from (i) fatty liver (hepatic steatosis); (ii) steatosis with inflammation and necrosis; and (iii) cirrhosis. Although the molecular mechanism leading to the development of hepatic steatosis in the pathogenesis of NAFLD is complex, recent animal models have shown that modulating important enzymes in fatty acid synthesis in liver may be key for the treatment of NAFLD. This review discusses recent advances in the field.

UV and H2O2 induce AMPK activation in cultured skin keratinocytes

AMP‐activated protein kinase or AMPK is an evolutionarily conserved sensor of cellular energy status, activated by a variety of cellular stresses that deplete ATP as well as other stresses. However, the possible involvement of AMPK in UV‐ and H2O2 ‐induced skin aging is not fully studied. We show for the first time that UV and H2O2 induce AMPK activation in cultured human skin keratinocytes (HaCaT cells). UV and H2O2 also induce LKB1, the possible upstream signal of AMPK, in an EGFR dependent manner. Using Compound C, a specific inhibitor of AMPK and AMPK specific siRNA knockdown as well as AMPK activator AICAR, we found that AMPK serves as a positive regulator for p38 and p53 (ser 15) activation induced by UV radiation or H2O2 treatment. We also observed that AMPK activation pathway serves a negative feedback signal pathway against UV‐induced mTOR activation in a TSC2 dependent manner. Inhibiting mTOR and activating p53 and p38 may mediate AMPK's pro‐apoptotic effect in UV or H2O2 treated cells. Furthermore, activation AMPK also phosphorylates acetyl‐CoA carboxylase or ACC, the pivotal enzyme of fatty acid synthesis, and PFK2, the key protein of glycolysis in UV‐radiated cells which may explain reduced adipogenesis in UV or H2O2 treated 3T3‐L1 adipocyte and skin keratinocytes. Collectively, our results demonstrate that UV and H2O2 induce EGFR/LKB1/AMPK signal pathway activation in cultured skin keratinocytes, which plays important role on UV or H2O2‐induced signal transduction and skin aging.

Low hepatic stearoyl-CoA desaturase 1 activity is associated with fatty liver and insulin resistance in obese humans

Stearoyl-CoA desaturase 1 (SCD1) is the rate-limiting enzyme in monounsaturated fatty acid synthesis. It is imperative for the assembly of VLDL particles, which transport triacylglycerol (TG) from liver to adipose tissue and other sites. We aimed to determine the role of hepatic SCD1 activity in human glucose and lipid metabolism. We studied 54 people participating in a lifestyle intervention programme with diet modification and increased physical activity. Insulin sensitivity was determined during a euglycaemic-hyperinsulinaemic clamp and estimated from an OGTT. Liver fat was quantified by (1)H-magnetic resonance spectroscopy at baseline and after 9 months of intervention. The pattern of fatty acids in serum VLDL-TGs was determined by ultracentrifugation followed by thin layer and gas chromatography, with the 18:1 n-9: 18:0 ratio providing an index of hepatic SCD1 activity. The hepatic SCD1 activity index correlated negatively with liver fat (r= -0.29, p=0.04) and positively with insulin sensitivity, both OGTT-derived (r=0.42, p=0.003) and clamp-derived (r=0.27, p=0.07). These correlations depended on overall adiposity. They were absent in leaner participants (n=27, liver fat: p=0.34, insulin sensitivity [OGTT]: p=0.75, insulin sensitivity [clamp]: p=0.24), but were strong in obese individuals (n=27, p=0.004, p=0.0002 and p=0.006, respectively). Furthermore, during intervention a high SCD1 activity index at baseline predicted a decrease in liver fat only in obese participants (r= -0.46, p=0.02). Our data suggest that high hepatic SCD1 activity may regulate fat accumulation in the liver and possibly protects from insulin resistance in obesity.

The Synthetic Triterpenoid CDDO-Im Inhibits Fatty Acid Synthase Expression and Has Antiproliferative and Proapoptotic Effects in Human Liposarcoma Cells

Liposarcomas constitute a rare group of tumors of mesenchymal origin that are often poorly responsive to therapy. This study characterizes a novel human liposarcoma cell line (LiSa-2) and defines the mechanism of its response to a synthetic triterpenoid. Fatty acid synthase (FAS) is a key enzyme of de-novo fatty acid synthesis and is highly expressed in both human liposarcoma tissue specimens and LiSa-2 cells. Treatment of the LiSa-2 cell line with the synthetic triterpenoid 2-cyano-3,12-dioxooleana-1,9-dien-28-oic imidazolide (CDDO-Im) markedly inhibited FAS mRNA expression, FAS protein production and FAS gene promoter activity. As expected, fatty acid synthesis was down regulated, but there was no effect on cellular fatty acid uptake or glycerol-3-phosphate synthesis suggesting a selective inhibition of endogenous fatty acid synthesis. Importantly, CDDO-Im produced a dose-dependent apoptotic effect in the LiSa-2 cell line, and simultaneous treatment with CDDO-Im and the fatty acid synthase inhibitor Cerulenin produced a synergistic cytotoxic effect. Thus, CDDO-Im and Cerulenin act at different loci to inhibit long chain fatty acid synthesis in liposarcoma cells. This study's demonstration of CDDO-Im inhibition of FAS and Spot 14 (S14) expression is the first report of triterpenoid compounds affecting the fatty acid synthesis pathway. The observed dependence of liposarcomas on lipogenesis to support their growth and survival provides a novel approach to the treatment of liposarcomas with agents that target fatty acid production.

Nuclear transcription factors and lipid homeostasis in liver

Nuclear transcription factors and lipid homeostasis in liver

In Vivo Functional Analyses of the Type II Acyl Carrier Proteins of Fatty Acid Biosynthesis

Acyl carrier protein (ACP) is a key component of the fatty acid synthesis pathways of both type I and type II synthesis systems. A large number of structure-function studies of various type II ACPs have been reported, but all are in vitro studies that assayed function or interaction of mutant ACPs with various enzymes of fatty acid synthesis or transfer. Hence in these studies functional properties of various mutant ACPs were assayed with only a subset of the many ACP-interacting proteins, which may not give an accurate overall view of the function of these proteins in vivo. This is especially so because Escherichia coli ACP has been reported to interact with several proteins that have no known roles in lipid metabolism. We therefore tested a large number of mutant derivatives of E. coli ACP carrying single amino acid substitutions for their abilities to restore growth to an E. coli strain carrying a temperature-sensitive mutation in acpP, the gene that encodes ACP. Many of these mutant proteins had previously been tested in vitro thus providing data for comparison with our results. We found that several mutant ACPs containing substitutions of ACP residues reported previously to be required for ACP function in vitro support normal growth of the acpP mutant strain. However, several mutant proteins reported to be severely defective in vitro failed to support growth of the acpP strain in vivo (or supported only weak growth). A collection of ACPs from diverse bacteria and from three eukaryotic organelles was also tested. All of the bacterial ACPs tested restored growth to the E. coli acpP mutant strain except those from two related bacteria, Enterococcus faecalis and Lactococcus lactis. Only one of the three eukaryotic organellar ACPs allowed growth. Strikingly the ACP is that of the apicoplast of Plasmodium falciparum (the protozoan that causes malaria). The fact that an ACP from a such diverse organism can replace AcpP function in E. coli suggests that some of the protein-protein interactions detected for AcpP may be not be essential for growth of E. coli.

Competition between 24:5n-3 and ALA for Δ6 desaturase may limit the accumulation of DHA in HepG2 cell membranes

The use of Delta 6 desaturase (D6D) twice in the conversion of alpha-linolenic acid (ALA; 18:3n-3) to docosahexaenoic acid (DHA; 22:6n-3) suggests that this enzyme may play a key regulatory role in the synthesis and accumulation of DHA from ALA. We examined this using an in vitro model of fatty acid metabolism to measure the accumulation of the long-chain metabolites of ALA in HepG2 cell phospholipids. The accumulation of ALA, eicosapentaenoic acid (20:5n-3), docosapentaenoic acid (22:5n-3), and 24:5n-3 in cell phospholipids was linearly related to the concentration of supplemented ALA over the range tested (1.8-72 microM). The accumulation of the post-D6D products of 22:5n-3, 24:6n-3 and DHA, in cell phospholipids was saturated at concentrations of >18 microM ALA. Supplementation of HepG2 cells with preformed DHA revealed that, although the accumulation of DHA in cell phospholipids approached saturation, the level of DHA in cell phospholipids was significantly greater compared with the accumulation of DHA from ALA, indicating that the accumulation of DHA from ALA was not limited by incorporation. The parallel pattern of accumulation of 24:6n-3 and DHA in response to increasing concentrations of ALA suggests that the competition between 24:5n-3 and ALA for D6D may contribute to the limited accumulation of DHA in cell membranes.