Differential effects of high-carbohydrate and high-fat diets on hepatic lipogenesis in rats.

PURPOSE: Hepatic fatty acid synthesis is influenced by several nutritional and hormonal factors. In this study, we have investigated the effects of distinct experimental diets enriched in carbohydrate or in fat on hepatic lipogenesis. METHODS: Male Wistar rats were divided into four groups and fed distinct experimental diets enriched in carbohydrates (70% w/w) or in fat (20 and 35% w/w). Activity and expression of the mitochondrial citrate carrier and of the cytosolic enzymes acetyl-CoA carboxylase and fatty acid synthetase were analyzed through the study with assessments at 0, 1, 2, 4, and 6 weeks. Liver lipids and plasma levels of lipids, glucose, and insulin were assayed in parallel. RESULTS: Whereas the high-carbohydrate diet moderately stimulated hepatic lipogenesis, a strong inhibition of this anabolic pathway was found in animals fed high-fat diets. This inhibition was time-dependent and concentration-dependent. Moreover, whereas the high-carbohydrate diet induced an increase in plasma triglycerides, the high-fat diets determined an accumulation of triglycerides in liver. An increase in the plasmatic levels of glucose and insulin was observed in all cases. CONCLUSIONS: The excess of sucrose in the diet is converted into fat that is distributed by bloodstream in the organism in the form of circulating triglycerides. On the other hand, a high amount of dietary fat caused a strong inhibition of lipogenesis and a concomitant increase in the level of hepatic lipids, thereby highlighting, in these conditions, the role of liver as a reservoir of exogenous fat.

The effects of queen mandibular pheromone on nurse-aged honey bee (Apis mellifera) hypopharyngeal gland size and lipid metabolism.

Queen honey bees (Apis mellifera) release Queen Mandibular Pheromone (QMP) to regulate traits in the caste of female helpers called workers. QMP signals the queen's presence and suppresses worker reproduction. In the absence of reproduction, young workers take care of the queen and her larvae (nurse tasks), while older workers forage. In nurses, QMP increases lipid stores in abdominal fat tissue (fat body) and protein content in hypopharyngeal glands (HPG). HPG are worker-specific head glands that can synthesize proteinaceous jelly used in colony nourishment. Larger HPG signifies ability to secrete proteinaceous jelly, while shrunken glands characterize foragers that do not make jelly. While it is known that QMP increases abdominal lipid stores, the mechanism is unclear: Does QMP make workers consume more pollen which provides lipids, or does QMP increase lipogenic capacity? Here, we measure abdominal lipogenic capacity as fatty acid synthase (FAS) activity while monitoring abdominal protein content and HPG size in caged workers. Cages allow us to rigorously control worker age, pheromone exposure, and diet. In our 2-factorial design, 3- vs. 8-day-old workers (age factor) were exposed to synthetic QMP or not (pheromone factor) while consuming a lipid deficient diet. We found that QMP did not influence abdominal FAS activity or protein content, but QMP still increased HPG size in the absence of dietary lipids. Our data revealed a positive correlation between abdominal protein content and HPG size. Our findings show that QMP is not a strong modulator of lipogenic capacity in caged worker bees. However, our data may reflect that QMP mobilizes abdominal protein for production of jelly, in line with previous findings on effects of honey bee Brood Pheromone. Overall, our study expands the understanding of how QMP can affect honey bee workers. Such insights are important beyond regulatory biology, as QMP is used in various aspects of beekeeping.

Her-2-directed systemic delivery of fatty acid synthase (FASN) siRNA with novel liposomal carrier systems in the breast cancer mouse model.

Despite the current advancements in the gene silencing therapy in vitro, the systemic delivery of siRNA still remains a challenging task for its transition into clinics. We have previously developed the Her2-targeted fatty acid synthase (FASN) siRNA-encapsulating immunoliposomes (ILs) with a great stability in the presence of serum. We report here the therapeutic potential of the lipid-based novel formulations in the breast cancer mouse model. The growth inhibitory and gene silencing effects of various formulations were determined by measuring the size of the tumour, cell proliferation, apoptotic index and immunoassays against Her2-overexpressed tumour xenografts in nude mice. The pegylated DSPC/Chol and DOPE/CHEMS ILs containing FASN-siRNA significantly decreased the tumour growth relative to non-targeted liposomes. They induced the 1.5-fold increase in cellular apoptosis and several fold decrease in proliferation as compared to non-targeted liposomal formulations of FASN-siRNA. Moreover, FASN-siRNA-ILs produced several fold increase in the ratios of p53/p21 and Bax/Bcl-2. The gene silencing effects of targeted FASN-liposomes were found to be significantly superior, resulting in 30-40% downregulation in FASN as compared to non-targeted similar formulations. Both types of FASN ILs provided a highly efficient approach for targeted delivery in Her-2-expressed breast cancer and thus offered a promising anticancer strategy in the clinical therapy.

Comparison of fatty acid synthase and cyclooxygenase-2 immunoexpression in embryonal, benign, and malignant odontogenic tissues.

OBJECTIVES: The aim of this study was to analyze the immunohistochemical expression of fatty acid synthase (FASN) and cyclooxygenase-2 (COX-2) in tooth germ (TG), ameloblastoma (AM), ameloblastic carcinoma (AC), ameloblastic fibroma (AF), and ameloblastic fibrosarcoma (AFS). STUDY DESIGN: Immunohistochemistry for FASN and COX-2 was performed in 10 TG, 44 AM, 10 AC, 9 AF, and 5 AFS specimens. The results were analyzed by using the immunoreactive score (IRS) and Kruskal-Wallis test followed by Dunn's post-test. RESULTS: Most TG specimens were strongly positive for FASN, whereas COX-2 was weak or negative. All AM and AC specimens expressed both proteins. In AF specimens, FASN and COX-2 were variably expressed in the epithelium and negative in the mesenchyme. In AFS specimens, FASN was strongly positive in the malignant mesenchyme and variable in the epithelium; COX-2 was focal or weak in both components. FASN expression showed significant differences in the following comparisons: TG vs AC, AM vs AC, and AF vs AFS. Differences in COX-2 were significant when comparing TG specimens with AM, AC, and AF specimens. CONCLUSIONS: The results suggest that FASN and COX-2 overexpression may have a role in the pathogenesis of AM and AC, whereas in AFS, FASN seems to be mainly involved. Further studies are necessary to clarify these mechanisms and their clinical implications.

Porphyromonas gingivalis lipopolysaccharide induces pro-inflammatory adipokine secretion and oxidative stress by regulating Toll-like receptor-mediated signaling pathways and redox enzymes in adipocytes.

Gut microbiota LPS contributes to obesity-related chronic inflammation and oxidative stress, promoting insulin resistance. Periodontal disease also represents a risk factor for type 2 diabetes and is associated with obesity. This study compared the effect of LPS from P. gingivalis periodontopathogen and E. coli enterobacteria on inflammatory adipokine secretion and redox status of 3T3-L1 adipocytes. We found that both LPS activated TLR2- and TLR4-mediated signaling pathways involving MyD88 adaptor and NFκB transcription factor, leading to an increased secretion of leptin, resistin, IL-6 and MCP-1. These effects were partly blocked by inhibitors targeting p38 MAPK, JNK and ERK. Moreover, P. gingivalis LPS reduced adiponectin secretion. Both LPS also enhanced ROS production and the expression of NOX2, NOX4 and iNOS genes. P. gingivalis LPS altered catalase gene expression. Collectively, these results showed that LPS of periodontal bacteria induced pro-inflammatory adipokine secretory profile and oxidative stress in adipocytes which may participate to obesity-related insulin resistance.

Effect of starvation on the activity of the mitochondrial tricarboxylate carrier.

The effect of starvation on the activity of the tricarboxylate carrier has been investigated in intact rat liver mitochondria and in a reconstituted system. In both experimental conditions, the rate of citrate transport, when compared to control, is greatly reduced (35-40%) in starved rats. Similar behaviour is shown by the cytosolic lipogenic enzymes. Kinetic analysis of the carrier activity in intact mitochondria and in the proteoliposomal system has showed that during starvation only the Vmax of this process decreases while there is no change in the Km. No difference in the Arrhenius plot and in the lipid composition has been detected, which indicates that the reduced transport activity in fasted animals is not due to a change in the carrier lipid microenvironment. In starved rats, a reduction of the carrier activity has occurred even after the addition of increasing cardiolipin concentrations to proteoliposomes. These findings thus suggest that starvation-induced decrease of citrate carrier activity could be due to a change of the intrinsic properties of the transport protein.

Proliferation of fibroblasts cultured from normal gingiva and hereditary gingival fibromatosis is dependent on fatty acid synthase activity.

BACKGROUND: Fatty acid synthase (FAS) is the enzyme that synthesizes palmitate from malonyl-CoA and acetyl-CoA. Recent studies have shown that FAS is overexpressed in human cancers and that its activity is necessary for cell proliferation. Hereditary gingival fibromatosis (HGF) is a genetic disease manifested as a progressive enlargement of the gingiva. The pathogenesis of this condition is not understood; however, a proliferative advantage of HGF fibroblasts in comparison with cells from normal gingiva (NG) has been described. The aim of this study was to investigate the role of FAS in NG and HGF fibroblast proliferation. METHODS: NG and HGF fibroblasts had their proliferative potential assessed by automated cell counting and immunocytochemistry against Ki-67 or proliferating cell nuclear antigen (PCNA). The production of FAS, androgen receptor (AR), and ErbB2 was analyzed by Western blot and the pattern of FAS expression studied by immunocytochemistry. FAS activity was blocked by the specific inhibitor cerulenin. RESULTS: Higher proliferation rates were found in fibroblasts isolated from HGF than from NG. HGF fibroblasts with greater proliferative potential produced more FAS and AR than the cell lines with lower growth rates, and all studied cell lines produced similar amounts of the ErbB2 protein. In addition, the FAS inhibitor cerulenin was able to significantly reduce the proliferation of both NG and HGF cells. CONCLUSIONS: These results show that FAS is expressed by gingival fibroblasts and that highly proliferative HGF cells produced more FAS and AR than the other fibroblast cell lines. Moreover, FAS inhibition significantly reduced both NG and HGF fibroblast growth, suggesting a role for the androgen-driven fatty acid biosynthesis in their proliferation.

[Effect of dehydration on lipid metabolism]. / K voprosu o vliianii degidratatsii na lipidnyi obmen.

Control of 25 sportsmen-wrestlers determined that accelerated loss in weight leads to a significant increase of ketone bodies evacuation with urine. The loss in weight for a 10-11 days period before a competition results in a less pronounced hyperketonuria. In experiment on rats it is shown that 48 and 98 hour hyperthermia and dehydration lead to the inhibition of the glucose-6-phosphate dehydrogenase activity in the liver. The mentioned permits supposing that under indicated conditions the inhibition of fat acid synthesis caused by NADPH2 and an increase of ketone bodies and cholesterol synthesis are observed.

Production of a polyhydroxyalkanoate biopolymer in insect cells with a modified eucaryotic fatty acid synthase.

A novel pathway for the synthesis of poly-3-hydroxybutyrate has been engineered by simultaneous delivery of two genes into insect cells (Spodoptera frugiperda) by use of individual baculovirus vectors. This system includes expression of a dehydrase-domain mutant rat fatty acid synthase cDNA and the phbC gene encoding polyhydroxyalkanoate synthase from Alcaligenes eutrophus. The dehydrase-deficient fatty acid synthase provides de novo synthesis of R-(-)-3-hydroxybutyryl-coenzyme A as a premature termination product rather than palmityl-coenzyme A, the normal product of wild-type rat fatty acid synthase. High levels of this mutant multifunctional protein provide a suitable precursor pool of R-(-)-3-hydroxybutyryl-coenzyme A for conversion to poly-3-hydroxybutyrate in insect cells coexpressing the phbC gene product. This strategy for redesigning a poly-3-hydroxybutyrate biosynthetic pathway suggests a new method for generating structurally diverse polyhydroxyalkanoates by metabolic engineering.

Immobilization of fatty acid synthetase from Mycobacterium smegmatis by radiation-induced polymerization.

Fatty acid synthetase of Type I from Mycobacterium smegmatis was immobilized by radiation-induced polymerization of 2-hydroxyethyl methacrylate (HEMA) in the presence of trimethylolpropane trimethacrylate (TMPTMA). The stability of immobilized synthetase toward low ionic strength increased in comparison with the free form, but the stabilities of immobilized preparations assessed by pH and temperature were identical to those of the free form. The apparent Km of immobilized enzyme for acetyl-CoA and malonyl-CoA were both 6 microM, essentially the same as those of the free form; acetyl-CoA, 5 microM and malonyl-CoA, 6 microM.

Requirement for the Candida albicans FAS2 gene for infection in a rat model of oropharyngeal candidiasis.

The virulence of Candida albicans strains deficient in fatty acid synthase activity by virtue of disruption/deletion of the FAS2 gene was examined in a rat model of oropharyngeal candidiasis. The FAS2 alleles of C. albicans CAI4 (delta ura3::imm434/delta ura3::imm434) were sequentially disrupted with a cassette that included a portion of FAS2 from which a 984 bp fragment containing the FAS condensing reaction domain was deleted and replaced with hisG-URA3-hisG sequences. Verification of fatty acid synthase inactivation was obtained from assays of enzyme activity. Strains in which a single allele was disrupted (CFD1 and CFD3) exhibited an approximately 20% reduction in activity, when compared to wild-type. In addition, fatty acid synthase activity was abolished in a FAS2 null mutant strain (CFD2), and growth of CFD2 occurred only when the growth medium was supplemented with Tween 40 and certain fatty acids. Strain CFD2 was avirulent in the rat model, indicating that fatty acid synthase activity is required for C. albicans oropharyngeal infection. Strains with a single FAS2 allele disruption colonized the oral cavity, but the number of cells recovered from infected animals was approximately fivefold less than for the parental strain. The results suggest that FAS may be exploited as a possible target for the development of new antifungal agents.

Stromal concentrations of coenzyme A and its esters are insufficient to account for rates of chloroplast fatty acid synthesis: evidence for substrate channelling within the chloroplast fatty acid synthase.

Concentrations of total CoAs in chloroplasts freshly isolated from spinach and peas were 10-20 microM, assuming a stromal volume of 66 microl per mg of chlorophyll. Acetyl-CoA and CoASH constituted at least 90% of the total CoA in freshly isolated chloroplasts. For a given chloroplast preparation, the concentration of endogenous acetyl-CoA was the same when extractions were performed using HClO4, trichloroacetic acid, propan-2-ol or chloroform/methanol, and the extracts analysed by quantitative HPLC after minimal processing. During fatty acid synthesis from acetate, concentrations of CoASH within spinach and pea chloroplasts varied from less than 0.1 to 5.0 microM. Malonyl-CoA concentrations were also very low (<0.1-3.0 microM) during fatty acid synthesis but could be calculated from radioactivity incorporated from [1-14C]acetate. Concentrations of CoASH in chloroplasts synthesizing fatty acids could be doubled in the presence of Triton X-100, suggesting that the detergent stimulates fatty acid synthesis by increasing the turnover rate of acyl-CoA. However, although taken up, exogenous CoASH (1 microM) did not stimulate fatty acid synthesis by permeabilized spinach chloroplasts. Calculated rates for acetyl-CoA synthetase, acetyl-CoA carboxylase and malonyl-CoA-acyl-carrier protein transacylase reactions at the concentrations of metabolites measured here are < 0.1-4% of the observed rates of fatty acid synthesis from acetate by isolated chloroplasts. The results suggest that CoA and its esters are probably confined within, and channelled through, the initial stages of a fatty acid synthase multienzyme complex.

Cloning of the fatty acid synthetase beta subunit from fission yeast, coexpression with the alpha subunit, and purification of the intact multifunctional enzyme complex.

We have cloned and sequenced the fission yeast (Schizosaccharomyces pombe) fas1+ gene, which encodes the fatty acid synthetase (FAS) beta subunit, by applying a PCR technique to conserved regions in the beta subunit of the alpha6beta6 types of FAS among different organisms. The deduced amino acid sequence of the Fas1 polypeptide, consisting of 2073 amino acids (Mr = 230,616), exhibits the 48.1% identity with the beta subunit from the budding yeast (Saccharomyces cerevisiae). This subunit, with five different catalytic activities, bears four distinct domains, while the alpha subunit, the sequence of which was previously reported by Saitoh et al. (S. Saitoh et al., 1996, J. Cell Biol. 134, 949-961), carries three domains. We have developed a co-expression system of the FAS alpha and beta subunits by cotransformation of two expression vectors, containing the lsd1+/fas2+ gene and the fas1+ gene, into fission yeast cells. The isolated FAS complex showed quite high specific activity, of more than 4000 mU/mg, suggesting complete purification. Its molecular weight was determined by dynamic light scattering and ultracentrifugation analysis to be 2.1-2.4 x 10(6), and one molecule of the FAS complex was found to contain approximately six FMN molecules. These results indicate that the FAS complex from S. pombe forms a heterododecameric alpha6beta6 structure. Electron micrographs of the negatively stained molecule suggest that the complex adopts a unique barrel-shaped cage architecture.

Evidence that the rate-limiting step for the biosynthesis of arachidonic acid in Mortierella alpina is at the level of the 18:3 to 20:3 elongase.

Mortierella alpina, a fungus used commercially as a source of arachidonic acid, 20:4(n-6), has been examined to see if growth on lipid-based carbon sources leads to repression of either fatty acid biosynthesis and/or fatty acid desaturation and elongation. Changes in the activities of ATP:citrate lyase, isocitrate lyase, carnitine acetyltransferase, malic enzyme, glucose-6-phosphate dehydrogenase and pyruvate kinase when the fungus was grown on fatty-acid-based (Tween) carbon sources were consistent with (i) the cells using the fatty acyl portion of the substrate as the sole carbon source, (ii) pyruvate kinase being the source of pyruvate for biosynthesis under these conditions and (iii) malic enzyme's major function being as a provider of NADPH for lipid biosynthesis. The abolition of fatty acid synthase activity when cells were grown on Tweens indicated the cessation of de novo fatty acid biosynthesis under these conditions. The fatty acyl composition of the lipid accumulated by the fungus grown on Tweens 20, 40 and 80 showed that desaturation and elongation of the substrate lipid still occurred. The absolute amount of arachidonic acid synthesized by Tween-grown cells was the same as for cells grown on glucose. The transformation of incorporated fatty acids into 20:4(n-6) was, it appeared, limited at the elongation of 18:3(n-6) to 20:3(n-6) as, in every case, 18:1, 18:2 and 18:3(n-6) increased in amount in the Tween-grown cells. These data show for the first time that fatty acid synthesis is regulated separately from fatty acid desaturation/elongation and that the latter reactions are not repressed by growth of the fungus on simple fatty acids. Furthermore, the data strongly implicate the elongation of 18:3(n-6) to 20:3(n-6) as the limiting step in arachidonic acid biosynthesis by Mort. alpina.

Saccharomyces cerevisiae expressing bacterial polyhydroxybutyrate synthase produces poly-3-hydroxybutyrate.

The polyhydroxybutyrate (PHB) synthase gene of the bacterium Alcaligenes eutrophus was used to construct a yeast plasmid which enabled expression of the functional synthase enzyme in Saccharomyces cerevisiae. Cells transformed with the synthase plasmid accumulated up to 0.5% of cell dry weight as PHB, with accumulation occurring in the stationary phase of batch growth. The identity of PHB in recombinant yeast cells was confirmed with 1H-NMR spectra of chloroform-extracted cell material. In addition, freeze-fracture electron microscopy revealed cytoplasmic granules exhibiting plastic deformations characteristic for PHB. GC results indicated a low background level of PHB in the wild-type strain, but intact polymer could not be detected by 1H-NMR. Formation of PHB in the recombinant strain implies the participation of native yeast enzymes in the synthesis of D-3-hydroxybutyryl-CoA (3-HB-CoA). Inhibition studies with cerulenin indicated that the fatty acid synthesis pathway is not involved in PHB precursor formation. Wild-type cell-free extracts showed D-3-HB-CoA dehydrogenase activity [150-200 nmol min-1 (mg protein)-1] and acetoacetyl-CoA thiolase activity [10-20 nmol min-1 (mg protein)-1], which together could synthesize monomer from acetyl-CoA. PHB accumulation was simultaneous with ethanol production, suggesting that PHB can act as an alternate electron sink in fermentative metabolism. We propose that PHB synthesis in recombinant yeast is catalysed by native cytoplasmic acetoacetyl-CoA thiolase, a native beta-oxidation protein possessing D-3-HB-CoA dehydrogenase activity and heterologous PHB synthase.

Repeat unit polysaccharides of bacteria: a model for polymerization resembling that of ribosomes and fatty acid synthetase, with a novel mechanism for determining chain length.

We report the identification and sequence from Escherichia coli and Salmonella enterica strains of the cld gene, encoding the chain-length determinant (CLD) which confers a modal distribution of chain length on the O-antigen component of lipopolysaccharide (LPS). The distribution of chain lengths in the absence of this gene fits a model in which as the chain is extended there is a constant probability of 0.165 of transfer of growing chain to LPS core, with termination of chain extension. The data for E. coli O111 fit a model in which the CLD reduces this probability for short chains and increases it to 0.4 for longer chains, leading to a reduced number of short chain molecules but an increase in numbers of longer molecules and transfer of essentially all molecules by chain length 21. We put forward a model for O-antigen polymerase which resembles the ribosome and fatty acid synthetase in having two sites, with the growing chain being transferred from a D site onto the new unit at the R site to extend the chain and then back to the D site to repeat the process. It is proposed that the CLD protein and polymerase form a complex which has two states: 'E' facilitating extension and 'T' facilitating transfer to core. The complex is postulated to enter the E state as O-antigen polymerization starts, and to shift to the T state after a predetermined time, the CLD acting as a molecular clock. The CLD is not O-antigen or species-specific but the modal value does depend on the source of the cld gene.

The mitochondrial tricarboxylate carrier: unexpected increased activity in starved silver eels.

The tricarboxylate carrier was purified to homogeneity from liver mitochondria of European eel at the silver and the yellow stage and functionally reconstituted into liposomes. Unexpectedly, the molecular activity of the tricarboxylate carrier obtained from silver eel was about twofold higher than that of the same protein from yellow eel, although eels at the silver stage stop feeding. Parallel changes were found in the activities of the lipogenic enzymes in silver eels. This suggests a functional coordination between all these proteins sequentially involved in hepatic lipogenesis. Cardiolipin added to proteoliposomes strongly stimulated the activity of the purified tricarboxylate carrier from yellow eels, whereas it slightly reduced the activity of the same protein from silver eels. The higher activity of the tricarboxylate carrier from silver eels could therefore be ascribed, at least in part, to a different composition of the lipid domain surrounding the carrier protein, possibly in response to the hormonal alterations accompanying metamorphosis from yellow to silver stage.

Purification and characterization of fatty acyl-acyl carrier protein synthetase from Vibrio harveyi.

A Vibrio harveyi enzyme which catalyzes the ATP-dependent ligation of fatty acids to acyl carrier protein (ACP) has been purified 6,000-fold to apparent homogeneity by anion-exchange, gel filtration, and ACP-Sepharose affinity chromatography. Purified acyl-ACP synthetase migrated as a single 62-kDa band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and as an 80-kDa protein by gel filtration under reducing conditions. Activity of the purified enzyme was lost within hours in the absence of glycerol and low concentrations of Triton X-100. Acyl-ACP synthetase exhibited Kms for myristic acid, ACP, and ATP of 7 microM, 18 microM, and 0.3 mM, respectively. The enzyme was specific for adenine-containing nucleotides, and AMP was the product of the reaction. No covalent acyl-enzyme intermediate was observed. Enzyme activity was stimulated up to 50% by iodoacetamide but inhibited > 80% by N-ethylmaleimide: inhibition by the latter was prevented by ATP and ACP but not myristic acid. Dithiothreitol and sulfhydryl-directed reagents also influenced enzyme size, activity, and elution pattern on anion-exchange resins. The function of acyl-ACP synthetase has not been established, but it may be related to the capacity of V. harveyi to elongate exogenous fatty acids by an ACP-dependent mechanism.