Incorporation Of 14C-acetate Research Articles

Toll-like receptor stimulants in processed meats promote lipid accumulation in macrophages and atherosclerosis in Apoe−/− mice

Dietary intake of processed meat is a risk factor for cardiovascular disease. However, the effects of processed meats on lipid metabolism in macrophages, a key regulator of cardiovascular risk, have remained largely unexplored.Extracts of processed meats, but not their fresh non-processed equivalents, were found to promote a significant increase in macrophage lipid accumulation in vitro. Calibrated receptor-dependent reporter assays revealed that pro-inflammatory stimulants of Toll-like receptor (TLR)-2 and TLR4 were low or undetectable in fresh meats, but rose dramatically following chopping and storage at 4 °C. Lipid accumulation in response to processed meats correlated well with TLR-stimulant content, was significantly reduced in TLR4-deficient macrophages, and was absent in response to meats stored frozen to prevent bacterial growth. TLR-stimulation significantly increased the incorporation of 14C-acetate into cellular lipids, and induced lipid accumulation in the absence of exogenous lipoproteins, suggesting a key role for de novo lipid synthesis in this process. Aortic atherosclerosis was also significantly accelerated in Apoe−/− mice receiving a diet supplemented with TLR-stimulants at concentrations relevant to those measured in processed meats, compared to normal chow.The findings reveal novel mechanisms which may be of relevance to the observed connections between processed meat consumption, inflammatory markers and cardiovascular risk.

A Systematic Review of the Randomized Controlled Trials of Gemcabene and Its Therapeutic Uses.

Statins are the most widely used drugs for reducing lipid and cholesterol levels in the blood. However, statins have shown some adverse effects and less acceptance among patients; hence, new drugs have been promoted in the market. Furthermore, gemcabenes were discovered in 1995 and are now in phase II and III clinical trials. Gemcabene acts by inhibiting incorporation of 14C-acetate into hepatocytes and stops the mechanism of fatty acids and cholesterol synthesis. In this review the six randomized controlled trials (RCTs) were scrutinized from the two databases by using keywords "Gemcabene" AND "Randomized Controlled Trial." The trials were mainly on animal models, and two studies were found to be associated with human subjects. The study concluded that gemcabene was effective as an anti-inflammatory agent and reduced lipid levels and the progression of fibrosis. Hence, further controlled trials are needed to determine its efficacy and safety in human subjects, along with the identification of adverse effects.

Acetyl-CoA carboxylase inhibition alters tubulin acetylation and aggregation in thrombin-stimulated platelets

Abstract Introduction Acetyl-CoA carboxylase (ACC), the first enzyme regulating lipid synthesis, promotes thrombus formation by increasing platelet phospholipid content. Inhibition of its activity decreases lipogenesis and increases the content in acetyl-CoA which can serve as a substrate for protein acetylation. This posttranslational modification plays a key role in the regulation of platelet aggregation, via tubulin acetylation. Purpose To demonstrate that ACC inhibition may affect platelet functions via an alteration of lipid content and/or tubulin acetylation. Methods Platelets were treated 2 hours with CP640.186, a pharmacological ACC inhibitor, prior to thrombin stimulation. Platelet functions were assessed by aggregometry and flow cytometry. Lipogenesis was measured via 14C-acetate incorporation into lipids. Lipidomics analysis was carried out on the commercial Lipidyzer platform. Protein phosphorylation and acetylation were evaluated by western blot. Results Treatment with CP640.186 drastically decreased platelet lipogenesis. However, the quantitative lipidomics analyses showed that preincubation with the compound did not affect global platelet lipid content. Interestingly, this short-term ACC inhibition was sufficient to increase tubulin acetylation level, at basal state and after thrombin stimulation. It was associated with an impaired platelet aggregation, in response to low thrombin concentration, while granules secretion was not affected. Mechanistically, we highlighted a decrease in Rac1 activity, associated with a reduced phosphorylation of its downstream effector PAK2. Surprisingly, actin cytoskeleton was not impacted but we evidenced a significant decrease in ROS production which could result from a decreased NOX2 activity. Conclusion Pharmacological ACC inhibition decreases platelet aggregation upon thrombin stimulation. The mechanism depends on increased tubulin acetylation, with subsequent alteration of the Rac1/PAK2/NOX2 signaling pathway Funding Acknowledgement Type of funding sources: Other. Main funding source(s): Fonds pour la formation à la Recherche dans l'Industrie et l'Agriculture (FRIA)

Acetyl-CoA carboxylase inhibition alters tubulin acetylation and aggregation in thrombin-stimulated platelets

Acetyl-CoA carboxylase (ACC), the first enzyme regulating lipid synthesis, promotes thrombus formation by increasing platelet phospholipid content. Inhibition of its activity decreases lipogenesis and increases the content in acetyl-CoA which can serve as a substrate for protein acetylation. This posttranslational modification plays a key role in the regulation of platelet aggregation, via tubulin acetylation. To demonstrate that ACC inhibition may affect platelet functions via an alteration of lipid content and/or tubulin acetylation. Platelets were treated 2 hours with CP640.186, a pharmacological ACC inhibitor, prior to thrombin stimulation. Platelet functions were assessed by aggregometry and flow cytometry. Lipogenesis was measured via 14 C-acetate incorporation into lipids. Lipidomics analysis was carried out on the commercial Lipidyzer platform. Protein phosphorylation and acetylation were evaluated by western blot. Treatment with CP640.186 drastically decreased platelet lipogenesis. However, the quantitative lipidomics analyses showed that preincubation with the compound did not affect global platelet lipid content. Interestingly, this short-term ACC inhibition was sufficient to increase tubulin acetylation level, at basal state and after thrombin stimulation. It was associated with an impaired platelet aggregation, in response to low thrombin concentration, while granules secretion was not affected. Mechanistically, we highlighted a decrease in Rac1 activity, associated with a reduced phosphorylation of its downstream effector PAK2. Surprisingly, actin cytoskeleton was not impacted but we evidenced a significant decrease in ROS production, which could result from a decreased NOX2 activity. Pharmacological ACC inhibition decreases platelet aggregation upon thrombin stimulation. The mechanism depends on increased tubulin acetylation, with subsequent alteration of the Rac1/PAK2/NOX2 signaling pathway.

Pharmacological ACC inhibition decreases thrombin-induced platelet aggregation by a mechanism independent of lipid content

Acetyl-CoA carboxylase (ACC) is the first committed enzyme regulating lipids synthesis. We have recently demonstrated that the overexpression of a constitutively active form of ACC in platelets leads to an increase in the phospholipid content which stimulates thrombus formation. Based on these data, we believe that ACC inhibition might be a counter-regulatory mechanism limiting endogenous lipogenesis and platelet reactivity in certain pathological circumstances. Our study aims to assess the impact of pharmacological ACC1 inhibition on platelet functions, de novo lipogenesis and lipid content. Platelets were treated with the long-chain fatty acid (FA) analog TOFA (30 μM) or the active site-directed CP640.186 (CP, 60 μM), two ACC inhibitors, for 2H before thrombin stimulation. Lipogenesis was measured via 14C-acetate incorporation into FA. Platelet functions were assessed by aggregometry. Lipidomics analysis was carried out on the commercial Lipidyzer platform. Both ACC inhibitors decreased lipogenesis and thrombin-induced platelet aggregation. TOFA, but not CP, drastically decreased mitochondrial oxygen consumption rate, an effect associated with an increased reactive oxygen species production and AMPK activation. Surprisingly, the quantitative lipidomics analyses showed that a preincubation with CP did not affect global platelet lipid content. However, the short-term ACC inhibition (and potential acetyl-CoA accumulation) was sufficient to modify the level of acetylated proteins such as tubulin, a key player in the regulation of platelet shape change and aggregation. Given the effect of TOFA on mitochondrial respiration, oxidative stress and AMPK activation, CP seems more appropriate to investigate the impact of ACC inhibition in platelets. A short-term acute treatment with CP inhibits thrombin-induced aggregation by a mechanism which doesn’t depend on lipid content but might involve changes in protein acetylation.

Can enzymatic stoichiometry be used to determine growth-limiting nutrients for microorganisms? - A critical assessment in two subtropical soils

The measurement of potential enzymatic activities has been proposed as an efficient method to infer nutrient limitations for microorganisms in environmental samples. To validate this use, confirmation with direct methods of microbial growth responses to resource additions are required. We experimentally manipulated nutrient-poor soils from the afromontane subtropics with relatively low (grassland soils, ca. 4% soil carbon (C)) or high organic matter content (forest soils, ca. 13% soil C) with nutrient additions (plant material added at 8 mg C g−1 soil combined with mineral N and/or P to reach C:N:P mass-ratios of 10:1:1) in a multifactorial design for one month in order to shift the microbial community towards C-, N- or P-limitation. We then measured the responses of the most commonly measured indicator enzymes used to infer growth limiting nutrients, using ß-1,4-glucosidase, ß-1,4-N-acetylglucosaminidase and leucine aminopeptidase, and acid phosphatase as indicators for C-, N- and P-acquiring enzymatic activities, respectively. In the same soil samples, we also determined the responses in bacterial (3H-leucine incorporation) and fungal growth rates (14C-acetate incorporation into ergosterol) to nutrient supplements, and also verified these with biomass responses (microbial PLFA and ergosterol concentrations) to the factorial nutrient loading amendments. Ratios of C-, N-, and P-acquiring enzymes indicated that the grassland soils were primarily P-limited, and secondarily co-limited by C and N, while the forest soils were co-limited by C and P. However, short-term responses in growth rates and respiration to nutrient additions, along with long-term growth rate, respiration and biomass responses to nutrient loading treatments all indicated that bacterial growth, fungal growth and respiration were primarily limited by C in both grassland and forest soils. We conclude that enzymatic ratios do not capture the growth-limiting factors for bacterial growth, fungal growth, or respiration in soil. Furthermore, the addition of C-rich plant material could shift the fungal community into N-limitation, while bacteria were shifted into co-limitation by both C and N, revealing that bacteria and fungi can be limited by different nutrients within the same soil environment.

“Trans-nonachlor increases extracellular free fatty acid accumulation and de novo lipogenesis to produce hepatic steatosis in McArdle-RH7777 cells”

Recent studies suggest there may be an environmental exposure component to the development and progression of non-alcoholic fatty liver disease (NAFLD) involving the organochlorine (OC) pesticides or their metabolites. However, the roles of OC compounds in the development of NAFLD has not been fully elucidated. Therefore, the current study was designed to determine if exposure to trans-nonachlor, a prevalent OC compound, could promote hepatocyte lipid accumulation and determine potential pro-steatotic mechanisms. McArdle-RH7777 (McA) hepatoma cells were incubated with trans-nonachlor for 24 h then neutral lipid accumulation was determined by Oil Red O staining. Exposure to trans-nonachlor produced a concentration dependent increase in neutral lipid accumulation. Trans-nonachlor also increased extracellular free fatty acid-induced neutral lipid accumulation which appears to be due at least in part to increased free fatty acid accumulation as evident by increased accumulation of Bodipy labeled dodecanoic acid. Additionally, 14C-acetate incorporation into total cellular lipids was increased by trans-nonachlor implicating increased de novo lipogenesis (DNL) as a potential mediator of trans-nonachlor-induced neutral lipid accumulation. Taken together, the present data indicate exposure to trans-nonachlor has a direct, pro-steatotic effect on hepatocytes to increase lipid accumulation through the combinatorial actions of extracellular free fatty acid accumulation and increased DNL.

Development of a Self-Assembled Nanoparticle Formulation of Orlistat, Nano-ORL, with Increased Cytotoxicity against Human Tumor Cell Lines.

Fatty acid synthase (FASN), the enzyme that catalyzes de novo synthesis of fatty acids, is expressed in many cancer types. Its potential as a therapeutic target is well recognized, but inhibitors of FASN have not yet been approved for cancer therapy. Orlistat (ORL), an FDA-approved lipase inhibitor, is also an effective inhibitor of FASN. However, ORL is extremely hydrophobic and has low systemic uptake after oral administration. Thus, new strategies are required to formulate ORL for cancer treatment as a FASN inhibitor. Here, we report the development of a nanoparticle (NP) formulation of ORL using amphiphilic bioconjugates that are derived from hyaluronic acid (HA), termed Nano-ORL. The NPs were loaded with up to 20 wt % weight of ORL at greater than 95% efficiency. The direct inhibition of the human recombinant thioesterase domain of FASN by ORL extracted from Nano-ORL was similar to that of stock ORL. Nano-ORL demonstrated a similar ability to inhibit cellular FASN activity when compared to free ORL, as demonstrated by analysis of (14)C-acetate incorporation into lipids. Nano-ORL treatment also disrupted mitochondrial function similarly to ORL by reducing adenosine triphosphate turnover in MDA-MB-231 and LNCaP cells. Nano-ORL demonstrated increased potency compared to ORL toward prostate and breast cancer cells. Nano-ORL decreased viability of human prostate and breast cancer cell lines to 55 and 57%, respectively, while free ORL decreased viability to 71 and 79% in the same cell lines. Moreover, Nano-ORL retained cytotoxic activity after a 24 h preincubation in aqueous conditions. Preincubation of ORL dramatically reduced the efficacy of ORL as indicated by high cell viability (>85%) in both breast and prostate cell lines. These data demonstrate that NP formulation of ORL using HA-derived polymers retains similar levels of FASN, lipid synthesis, and ATP turnover inhibition while significantly improving the cytotoxic activity against cancer cell lines.

Inhibitory effect of imperatorin on insulin-like growth factor-1-induced sebum production in human sebocytes cultured in vitro.

Acne is a common skin disease that originates in the sebaceous gland. The pathogenesis of acne is very complex, involving the increase of sebum production and perifollicular inflammation. In this study, we screened the anti-lipogenic material and demonstrated its effect using cultured human sebocytes. Normal human sebocytes were cultured by explanting the sebaceous glands. To evaluate the anti-lipogenic effect, sebocytes were treated with test materials and (14)C-acetate incorporation assay was performed. To screen the anti-lipogenic materials, we tested the effect of many herbal plant extracts. We found that Angelica dahurica extract inhibited the insulin-like growth factor-1 (IGF-1)-induced sebum production in terms of squalene synthesis in sebocytes. Furthermore, imperatorin isolated from A. dahurica showed remarkable inhibitory effect on squalene production as well as squalene synthase promoter activity. To investigate the putative action mechanism, we tested the effect of imperatorin on intracellular signaling. The results showed that imperatorin inhibited IGF-1-induced phosphorylation of Akt. In addition, imperatorin significantly down-regulated PPAR-γ and SREBP-1, the important transcription factors for lipid synthesis. These results suggest that imperatorin has a potential for reducing sebum production in sebocytes, and can be applicable for acne treatment.

Fungal contributions to carbon flow and nutrient cycling during decomposition of standing Typha domingensis leaves in a subtropical freshwater marsh

Summary Despite the well‐known occurrence of ‘standing‐dead’ emergent plant litter in freshwater marshes, the role of fungi in its decomposition is poorly known. Here, we quantified the growth and biomass dynamics of fungi associated with standing‐dead Typha domingensis leaves, estimated the contribution of fungi to carbon flow during decomposition and assessed their contribution to nutrient (nitrogen and phosphorus) cycling. In a subtropical freshwater marsh, standing leaves of T. domingensis were sampled in August while living (green) and then monthly during leaf senescence and standing‐dead decomposition for 1 year. Leaf samples were analysed for mass loss, fungal biomass (ergosterol), rates of fungal production (14C‐acetate incorporation) and microbial respiration (CO2 evolution), and for litter chitin (glucosamine), carbon, N and P concentrations. Losses in T. domingensis leaf carbon (37%) occurred during senescence and standing decomposition. During this time, increases in ergosterol and chitin concentrations were observed in the standing litter, indicating the rapid colonisation of decaying Typha leaves by fungi. Estimated fungal biomass (from ergosterol) reached a maximum of 37 mg C g−1 detrital C. Over the entire study period, estimated cumulative fungal production in standing Typha litter was 39 mg C g−1 initial detrital C, indicating that 11% of leaf C was converted to fungal C. The corresponding estimate of cumulative microbial respiration was 136 mg C g−1 initial detrital C, indicating that 37% of Typha leaf litter C was mineralised by microorganisms (bacteria and fungi) during decomposition. Fungi also immobilised up to c.27% and c.55% of the total detrital N and P, respectively. Fungi play an important role in the cycling of C and nutrients in freshwater marshes, and this should be integrated into current models that describe major biogeochemical pathways.

Abstract 801: Sensitivity of cell lines to Fatty Acid Synthase inhibitors depends on the lipid content in the cellular environment

Abstract Fatty Acid Synthase (FASN) catalyzes the final step in palmitate (PA) synthesis, using acetyl-CoA, malonyl-CoA and NADPH. Most normal tissues express low levels of FASN and rely on uptake of fatty acids (FA) from the diet. It has been proposed that FASN overexpressing tumors including prostate and breast tumors depend on de novo FA synthesis, which is advantageous to tumors by providing lipids for membrane synthesis and increased growth factor receptor expression/signaling in lipid rafts. Overexpression of FASN leads to a higher amount of saturated lipids in membranes which can lead to resistance to cytotoxic chemotherapy. Lastly, NADPH consumption during PA synthesis keeps the redox balance in check. All of the above imply that FASN represents a potential therapeutic target for the treatment of multiple cancer types. At this AACR we report two novel chemical series (posters Connolly et al., Lu et al.). JNJ-53793220 and JNJ-54302833 potently and selectively (< 100nM) inhibit the FASN enzyme and proliferative activity in cells in lipid reduced medium (LRM). Using these, we investigated the underlying hypothesis that tumor cells do not utilize circulating FA and are dependent on de novo synthesis of FA. In a lipid reduced environment many cell lines, particularly of prostate, breast, ovarian or heme origin, proved to be sensitive to JNJ-53793220. However co-administration of PA dose-dependently reversed the anti-proliferative effects. Also androgen driven proliferation of LNCaP cells was potently blocked by JNJ-53793220 (EC50 30 nM), and decreased PSA levels. Both effects were partially rescued by the addition of PA. While the rescue of tumor cells by PA confirmed the on-target activity of the compounds, it also suggested that cancer cells are capable of using external FA. To extend these findings, we screened more than 400 cell lines in lipid containing medium (LCM) for their sensitivity to JNJ-53793220. In LCM sensitivity to FASN inhibition was lower than in LRM conditions. In most, but not all, cases the addition of PA reverted the antiproliferative effects of JNJ-53793220, although target engagement was not reduced in LCM conditions. The EC50 of 14C-acetate incorporation in lipids of ∼30 nM corresponded well with enzymatic and anti-proliferative effects in LRM (27 and 13 nM respectively). Furthermore, growth of pre-established LNCaP xenografts in vivo was not blocked significantly by JNJ-53793220, even though malonyl-CoA levels were increased as expected upon FASN inhibition in the tumor. While circulating lipids in vivo are likely culprits for the lack of efficacy, other factors may play a role as well. In a 3D culture model (poster Vidic et al.) the growth of LNCaP and PC346c spheroids was blocked by JNJ-54302833 (1µM), but growth of PC346c spheroids co-cultured with cancer associated fibroblasts was not inhibited. Taken together our data suggest that the outcome of FASN inhibition is influenced by the tumor environment. Citation Format: Karine A. Smans, Sabine De Breucker, Norbert Esser, Erwin Fraiponts, Ron Gilissen, Ralph Graeser, Boudewijn Janssen, Lieven Meerpoel, Danielle Peeters, Geert Van Hecke, Luc Van Nuffel, Yolanda Chong, Peter Vermeulen, Gilles Bignan, James Bischoff, Peter Connolly, Bruce Grasberger, Tianbao Lu, Donald Ludovici, Carsten Schubert, Michael Parker, Christophe Meyer, Suzana Vidic. Sensitivity of cell lines to Fatty Acid Synthase inhibitors depends on the lipid content in the cellular environment. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 801. doi:10.1158/1538-7445.AM2014-801

Activating transcription factor 6 is necessary and sufficient for alcoholic fatty liver disease in zebrafish.

Fatty liver disease (FLD) is characterized by lipid accumulation in hepatocytes and is accompanied by secretory pathway dysfunction, resulting in induction of the unfolded protein response (UPR). Activating transcription factor 6 (ATF6), one of three main UPR sensors, functions to both promote FLD during acute stress and reduce FLD during chronic stress. There is little mechanistic understanding of how ATF6, or any other UPR factor, regulates hepatic lipid metabolism to cause disease. We addressed this using zebrafish genetics and biochemical analyses and demonstrate that Atf6 is necessary and sufficient for FLD. atf6 transcription is significantly upregulated in the liver of zebrafish with alcoholic FLD and morpholino-mediated atf6 depletion significantly reduced steatosis incidence caused by alcohol. Moreover, overexpression of active, nuclear Atf6 (nAtf6) in hepatocytes caused FLD in the absence of stress. mRNA-Seq and qPCR analyses of livers from five day old nAtf6 transgenic larvae revealed upregulation of genes promoting glyceroneogenesis and fatty acid elongation, including fatty acid synthase (fasn), and nAtf6 overexpression in both zebrafish larvae and human hepatoma cells increased the incorporation of 14C-acetate into lipids. Srebp transcription factors are key regulators of lipogenic enzymes, but reducing Srebp activation by scap morpholino injection neither prevented FLD in nAtf6 transgenics nor synergized with atf6 knockdown to reduce alcohol-induced FLD. In contrast, fasn morpholino injection reduced FLD in nAtf6 transgenic larvae and synergistically interacted with atf6 to reduce alcoholic FLD. Thus, our data demonstrate that Atf6 is required for alcoholic FLD and epistatically interacts with fasn to cause this disease, suggesting triglyceride biogenesis as the mechanism of UPR induced FLD.

Effect of liraglutide on adipose insulin resistance and hepatic de-novo lipogenesis in non-alcoholic steatohepatitis: substudy of a phase 2, randomised placebo-controlled trial

BackgroundAdipose tissue insulin resistance and lipotoxicity are key pathognomonic features in non-alcoholic steatohepatitis (NASH). Liraglutide is a once daily, glucagon-like peptide 1 (GLP-1) analogue that significantly improves glycaemic control, weight, and hepatic steatosis. The aim of this phase 2 study was to determine the effect of liraglutide on insulin sensitivity (hepatic, muscle, adipose), hepatic lipogenesis, and markers of adipose inflammation. Methods14 patients with biopsy-proven NASH were randomly assigned to either 1·8 mg liraglutide or placebo (once a day subcutaneously) for 12 weeks as part of the metabolic substudy of the double-blind, randomised placebo-controlled LEAN trial. At baseline and 12 weeks, patients underwent paired two-step hyperinsulinaemic euglycaemic clamps incorporating stable isotopes with concomitant adipose tissue microdialysis. Serum adipocytokines were measured with Fluorokine MAP multiplex kits (R&D Systems, UK). In-vitro isotope experiments were performed with Huh-7 and primary human hepatocytes. The LEAN trial is registered with ClinicalTrials.gov, number NCT01237119. FindingsLiraglutide significantly decreased weight, waist circumference, HbA1c, fasting glucose, LDL, and liver enzymes compared with placebo. Liraglutide significantly increased the suppression of hepatic glucose production with low-dose insulin. Liraglutide significantly decreased circulating non-esterified fatty acid (NEFA) in the fasting, low-dose, and high-dose insulin states. Liraglutide significantly reduced the insulin concentration required to half-maximally suppress circulating NEFA and significantly decreased adipose tissue lipolysis, as shown by a reduction in interstitial fluid glycerol concentrations. Furthermore, liraglutide significantly improved serum markers of adipose inflammation—namely, leptin, adiponectin, and chemokine ligand 2. In addition, liraglutide significantly decreased de-novo lipogenesis in vivo, as measured by incorporation of deuterated 2H20 into palmitate, compared with placebo. In support of our clinical observations, in-vitro experiments using both the Huh-7 cell line and primary cultures of human hepatocytes showed decreased de-novo lipogenesis, measured by incorporation of 14C-acetate into cellular lipid after treatment with GLP-1 (exendin-4). InterpretationLiraglutide significantly reduces metabolic dysfunction, hepatic lipogenesis, hepatic and adipose insulin resistance, and adipose inflammation in patients with NASH. GLP-1 analogue therapy might be a novel treatment for patients with this condition. FundingWellcome Trust, National Institute of Health Research, Novo Nordisk.

Biosynthesis of lipophilic compounds in tomato fruit

Metabolic engineering approaches to increasing lycopene contents in tomato fruit have always led to moderate increases of this metabolite. This reflects, at least in part, the lack of knowledge about the mechanisms underlying the regulation of carotenoid biosynthesis and accumulation as well as the rest of metabolic processes operating in the chromoplasts. A study performed with chromoplasts to know the origin of the precursors for carotenoids biosynthesis using a variety of 14C-labelled precursors showed that the most important incorporation was found in lipids. This study aims to understand the biochemical and metabolic processes operating during tomato fruit ripening. Labelling studies showed an efficient incorporation of 14Cacetate not only into fatty acids and sterols (as expected) but also into lycopene, suggesting that the cytosolic mevalonate pathway was also contributing to carotenoid biosynthesis during fruit ripening. This observation has been confirmed using 14C-mevalonate, which served as a precursor for the synthesis of sterols and carotenoids.Keywords: Carotenoid, sterol, labelling assay, pericarp, chromoplasts.

Long-chain fatty acids differentially alter lipogenesis in bovine and caprine mammary slices

Indirect comparisons from studies in vivo have suggested that caprine mammary tissue is less sensitive than bovine mammary tissue to the anti-lipogenic effect of long-chain fatty acids (LCFA), including specific rumen biohydrogenation (RBH) intermediates of polyunsaturated fatty acids (PUFA). Our objective was to investigate the effects on lipogenesis of 18-carbon LCFA differing in the degree of unsaturation and/or double bond conformation using cultured slices of bovine and caprine mammary tissues. Mammary tissues were collected from five multiparous Holstein × Normande cows and six multiparous Alpine goats in mid lactation. The expression of genes involved in milk component synthesis was measured in tissues collected at slaughter and after slice preparation: FASN, SCD1, CD36, SREBF1 and PPARG1 mRNA levels were higher in bovine than caprine samples, whereas the opposite was observed for CSN2 mRNA levels. Bovine and caprine mammary slices were incubated for 20 h in a medium with BSA (control), cis-9-18 : 1, 18 : 2n-6, 18 : 3n-3, cis-9, trans-11-CLA, or trans-10, cis-12-CLA (the latter at 3 increasing concentrations: C1 (0.11 mm), C2 (0.16 mm), C3 (0.37 mm)). Lipogenesis was estimated by measuring the incorporation of 14C-acetate into total lipid. Significant differences of individual LCFA (P < 0.05) were observed between species: bovine tissue showed a decrease in total lipogenesis with 18 : 2n-6, 18 : 3n-3, trans-10,cis-12-CLA (C2 and C3) while caprine tissue showed an increase after treatment with 18 : 3n-3, cis-9, trans-11-CLA or trans-10, cis-12-CLA (C3). These results were not related to the mRNA abundance of our set of genes in the mammary slices after incubation. In conclusion, this study demonstrates that caprine mammary slices reacted differently from bovine mammary slices to the anti-lipogenic activity of specific LCFA and suggests that regulation of lipogenesis via other genes and/or at protein level and enzyme activity may be involved.

Molecular Structures of Citrus Flavonoids Determine Their Effects on Lipid Metabolism in HepG2 Cells by Primarily Suppressing ApoB Secretion

This study investigated the underlying mechanisms of action for blood lipid lowering effects of citrus flavonoids and their methoxylated analogues (n = 19; dose range: 0-100 μM) in HepG2 cells. Cholesterol (CH) and triglyceride (TG) syntheses were assessed by measuring the incorporation of (14)C-acetate and (14)C-glycerol, respectively, whereas apoB secretion was determined by ELISA. Results show that two polymethoxylated citrus flavonoids (PMFs), tangeretin and nobiletin, potently inhibited apoB secretion (IC(50) = 13 and 29 μM, respectively) and modestly inhibited CH synthesis (IC(50) = 49 and 68 μM) and TG synthesis (IC(50) = 14 and 73 μM), without effecting LDL-receptor activity. Other PMFs (e.g., sinensetin) and non-PMFs (e.g., hesperetin and naringenin) had only weak effects on CH and TG syntheses and apoB secretion (IC(50) > 100 μM). The structure-activity analysis indicated that a fully methoxylated A-ring of the flavonoid structure was associated with a potent inhibitory activity on hepatic apoB secretion. In conclusion, this study using HepG2 cells indicates that citrus flavonoids with a fully methoxylated A-ring may lower blood CH and TG concentrations primarily by suppressing hepatic apoB secretion as a main underlying mode of action.

Activation of the nuclear receptor PPARα regulates lipid metabolism in foetal liver from diabetic rats: implications in diabetes‐induced foetal overgrowth

peroxisome proliferator-activated receptor α (PPARα) is a crucial regulator of liver lipid metabolism. As maternal diabetes impairs foetal lipid metabolism and growth, we aimed to determine whether PPARα activation regulates lipid metabolism in the foetal liver from diabetic rats as well as foetal weight and foetal liver weight. diabetes was induced by neonatal streptozotocin administration (90 mg/kg). For ex vivo studies, livers from 21-day-old foetuses from control and diabetic rats were explanted and incubated in the presence of PPARα agonists (clofibrate and leukotriene B(4) ) for further evaluation of lipid levels (by thin layer chromatography and densitometry), de novo lipid synthesis (by (14) C-acetate incorporation) and lipid peroxidation (by thiobarbituric reactive substances evaluation). For in vivo studies, foetuses were injected through the uterine wall with leukotriene B(4) on days 19, 20 and 21 of gestation. On day 21 of gestation, foetal liver concentrations of lipids and lipoperoxides were evaluated. foetuses from diabetic rats showed increased body weight and liver weight, as well as accumulation of triglycerides and cholesteryl esters, increased de novo lipid synthesis and lipid peroxidation in the liver when compared to controls. Ex vivo studies showed that PPARα ligands reduced both the concentrations and synthesis of the lipid species studied and lipid peroxidation in the foetal liver from diabetic rats. In vivo experiments showed that leukotriene B(4) reduced the concentrations of triglycerides, cholesteryl esters and phospholipids, as well as lipid peroxidation, foetal weight and foetal liver weight in diabetic rats. PPARα activation regulates the impaired foetal liver lipid metabolism, prevents hepatomegaly and reduces foetal overgrowth induced by maternal diabetes.

Abstract 68: Nampt regulates fatty acid synthesis, ER homeostasis, and survival in prostate cancer

Abstract Nicotinamide adenine dinucleotide (NAD) is crucial for the survival of living organisms. This coenzyme functions in both redox metabolism and as a substrate for several proteins, notably Sirtuin deacetylases and poly-(ADP ribose) polymerases, which control stress response and apoptosis in cancer. The rate limiting step of NAD biosynthesis is catalyzed by Nicotinamide phosphoribosyl transferase (Nampt). Recent evidence suggests Nampt may play a role in cancer by regulating survival. The Nampt inhibitor FK866 is in phase II clinical trials against a variety of solid malignancies, however no studies have focused on the role of NAD in prostate cancer (PCa). Because tumor cells have a high requirement for NAD and PCa has increased fatty acid synthesis, we hypothesized that Nampt is important for PCa cell survival and metabolism. Our studies demonstrate that Nampt activity is required for the survival of prostate tumor cells (PC-3, LNCaP, and DU145), but not normal prostate epithelial cells, as determined by treatment with FK866. Exogenous NAD rescued cell death, indicating FK866 is a specific inhibitor of Nampt. To test whether PCa cells can rely on de novo NAD synthesis, we treated cell lines with FK866 and added back nicotinic acid, the substrate for the de novo pathway. Only DU145 cell survival was rescued by nicotinic acid demonstrating that NAD biosynthesis pathways are heterogeneous in PCa. Furthermore, expression of Nicotinic acid phosphoribosyl transferase (Naprt1), the rate-limiting step of the de novo pathway, correlated with the rescue by nicotinic acid in DU145 cells. Additionally, Nampt inhibition activated endoplasmic reticulum (ER) stress and autophagy in a pro-survival manner in all cell lines. Interestingly, FK866 also dramatically reduced fatty acid synthesis in PC-3 and DU145 cells, independent of fatty acid synthase levels. However, LNCaP cells showed no decrease in 14C-acetate incorporation. Robust phosphorylation of ACC occured in DU145 and PC-3 cells upon treatment with FK866, and this effect was rescued by adding back NAD. This correlated with reduced ATP levels and activation (phosphorylation) of AMPK at T172. LNCaP cells had a less dramatic depletion of ATP and no changes in AMPK or ACC phosphorylation, indicating a potential mechanism of fatty acid synthesis shutdown. However, lipid synthesis (as measured by incorporation of 14C-choline) was reduced in all 3 cell lines, albeit to a lesser extent in LNCaP cells. Overall, these findings demonstrate a novel role for Nampt in PCa, and highlight the contribution of NAD to ER homeostasis, fatty acid synthesis, and survival. Targeting NAD biosynthesis could be a novel approach to disrupting PCa cell survival and lipid synthesis. 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 68.

Overexpression of fatty acid synthase is associated with palmitoylation of Wnt1 and cytoplasmic stabilization of β-catenin in prostate cancer

Fatty acid synthase (FASN), a key metabolic enzyme for liponeogenesis highly expressed in several human cancers, displays oncogenic properties such as resistance to apoptosis and induction of proliferation when overexpressed. To date, no mechanism has been identified to explain the oncogenicity of FASN in prostate cancer. We generated immortalized prostate epithelial cells (iPrECs) overexpressing FASN, and found that 14C-acetate incorporation into palmitate synthesized de novo by FASN was significantly elevated in immunoprecipitated Wnt-1 when compared to isogenic cells not overexpressing FASN. Overexpression of FASN caused membranous and cytoplasmic β-catenin protein accumulation and activation, whereas FASN knockdown by short-hairpin RNA resulted in a reduction in the extent of β-catenin activation. Orthotopic transplantation of iPrECs overexpressing FASN in nude mice resulted in invasive tumors that overexpressed β-catenin. A strong significant association between FASN and cytoplasmic (stabilized) β-catenin immunostaining was found in 862 cases of human prostate cancer after computerized subtraction of the membranous β-catenin signal (P<0.001, Spearman's ρ=0.33). We propose that cytoplasmic stabilization of β-catenin through palmitoylation of Wnt-1 and subsequent activation of the pathway is a potential mechanism of FASN oncogenicity in prostate cancer.

The Role of Acetyl-Coenzyme A Synthetase in Arabidopsis

The acs1 knockout mutant that has a disruption in the plastidic acetyl-coenzyme A (CoA) synthetase (ACS; At5g36880) gene was used to explore the role of this protein and plastidic acetate metabolism in Arabidopsis (Arabidopsis thaliana). Disruption of the ACS gene decreased ACS activity by 90% and largely blocked the incorporation of exogenous (14)C-acetate and (14)C-ethanol into fatty acids. Whereas the disruption had no significant effect on the synthesis of bulk seed triacylglycerols, the acs1 plants were smaller and flowered later. This suggests that the pyruvate dehydrogenase bypass provided by the aerobic fermentation pathway that converts pyruvate to acetate and probably on to fatty acids is important to the plants during normal growth. The role of ACS in destroying fermentative intermediates is supported by the increased sensitivity of the acs1 mutant to exogenous acetate, ethanol, and acetaldehyde compared to wild-type plants. Whereas these observations suggest that flux through the aerobic fermentation pathway is important, the reason for this flux is unclear. Interestingly, acetate is able to support high rates of plant growth on medium and this growth is blocked in the acs1 mutant.