Precursor For Fatty Acid Synthesis Research Articles

Exogenous methyl jasmonate enhances lipid production in Isochrysis galbana under nitrogen deprivation and high light

Nitrogen deprivation under high light is the most employed cultivation condition for the lipid's accumulation in microalgae. However, nitrogen deprivation causes the over-reduction of the photosystem (PS) II acceptor side in the presence of high light, confirmed by an increased VJ (the relative variable fluorescence at J-step) during cultivation. This in turn results in photo-inhibition of microalgal cells and photodamage to cellular metabolism which hinders the lipid production. To explore modifications for lipid accumulation, the exogenous methyl jasmonate (MeJA) effect was investigated on antioxidant capacity and lipid accumulation in Isochrysis galbana. Treatment with MeJA under high light nitrogen deprivation, (100 μM concentration), significantly enhanced the activity of ribulose-1,5-bisphosphate carboxylase/oxygenase, and facilitated the electron transport in photosynthesis as indicated by increased ΦPSII (effective quantum yield of PSII) after MeJA treatment. The exogenous MeJA increased the antioxidant potential of related enzymes (ascorbate peroxidase and superoxide dismutase) and significantly alleviated the accumulation of reactive oxygen species (ROS) under high light nitrogen deprivation for improved cell survival and cellular metabolism in I. galbana. The improved cellular metabolism further increased the pyruvate content, which can be converted into acetyl coenzyme A (an important precursor for fatty acid synthesis). MeJA addition increased the substrate pool required for fatty acid and lipid production. Overall, exogenous MeJA enhanced lipid production in I. galbana under high light nitrogen deprivation by regulating the antioxidant capability and biosynthesis of the substrate.

High oil accumulation in tuber of yellow nutsedge compared to purple nutsedge is associated with more abundant expression of genes involved in fatty acid synthesis and triacylglycerol storage

BackgroundYellow nutsedge is a unique plant species that can accumulate up to 35% oil of tuber dry weight, perhaps the highest level observed in the tuber tissues of plant kingdom. To gain insight into the molecular mechanism that leads to high oil accumulation in yellow nutsedge, gene expression profiles of oil production pathways involved carbon metabolism, fatty acid synthesis, triacylglycerol synthesis, and triacylglycerol storage during tuber development were compared with purple nutsedge, the closest relative of yellow nutsedge that is poor in oil accumulation.ResultsCompared with purple nutsedge, high oil accumulation in yellow nutsedge was associated with significant up-regulation of specific key enzymes of plastidial RubisCO bypass as well as malate and pyruvate metabolism, almost all fatty acid synthesis enzymes, and seed-like oil-body proteins. However, overall transcripts for carbon metabolism toward carbon precursor for fatty acid synthesis were comparable and for triacylglycerol synthesis were similar in both species. Two seed-like master transcription factors ABI3 and WRI1 were found to display similar transcript patterns but were expressed at 6.5- and 14.3-fold higher levels in yellow nutsedge than in purple nutsedge, respectively. A weighted gene co-expression network analysis revealed that ABI3 was in strong transcriptional coordination with WRI1 and other key oil-related genes.ConclusionsThese results implied that pyruvate availability and fatty acid synthesis in plastid, along with triacylglycerol storage in oil bodies, rather than triacylglycerol synthesis in endoplasmic reticulum, are the major factors responsible for high oil production in tuber of yellow nutsedge, and ABI3 most likely plays a critical role in regulating oil accumulation. This study is of significance with regard to understanding the molecular mechanism controlling carbon partitioning toward oil production in oil-rich tuber and provides a valuable reference for enhancing oil accumulation in non-seed tissues of crops through genetic breeding or metabolic engineering.

Using 14C-acetate Pulse-chase Labeling to Study Fatty Acid and Glycerolipid Metabolism in Plant Leaves.

Lipids metabolism is comprised of networks of reactions occurred in different subcellular compartments. Isotopic labeling is a good way to track the transformations and movements of metabolites without perturbing overall cellular metabolism. Fatty acids, the building blocks of membrane lipids and storage triacylglycerols, are synthesized in plastids. The immediate precursor for fatty acid synthesis is acetyl-CoA. Exogenous acetate is rapidly incorporated into fatty acids in leaves and isolated plastids because it can diffuse freely through cellular membranes, enter the plastid where it is rapidly metabolized to acetyl-CoA. Therefore, isotope-labeled acetate is often used as a tracer for the investigation of fatty acid synthesis and complex lipid metabolism in plants and other organisms. The basic principle of isotope labeling and its recent technological advances have been reviewed ( Allen et al., 2015 ). The present protocol describes the use of 14C-labeled acetate to determine rates of fatty acid synthesis and degradation and to track the metabolism of glycerolipids in leaves. This method, which is often referred to as acetate pulse-chase labeling, has been widely used to probe various aspects of lipid metabolism ( Allen et al., 2015 ), including the role of autophagy in membrane lipid turnover ( Fan et al., 2019 ) and the interplay between lipid and starch metabolism pathways ( Yu et al., 2018 ).

Abstract B12: Acetate metabolism mediated by acetyl-CoA synthetase 2 in cisplatin-resistant bladder cancer

Abstract Introduction and Objective: Cisplatin is an important chemotherapeutic agent against metastatic bladder cancer, but resistance often limits its usage. With the recent recognition of lipid metabolic alterations in bladder cancers, we studied the metabolic implications of cisplatin resistance using cisplatin-sensitive (T24S) and -resistant (T24R) bladder cancer cells. Methods: To test whether there should be differences in metabolism and metabolism-associated pathways between cisplatin-sensitive and resistant bladder cancer cells, we applied the live metabolomics approach that we recently developed to isogenic bladder cancer cell lines T24S (cisplatin sensitive) and T24R (cisplatin resistant). The metabolites generated from 13C-glucose tracer were monitored with 2D 1H-13C HSQC NMR in real time. Results: Real-time live metabolomics revealed that T24R cells consume more glucose, leading to higher production of glucose-derived acetate and fatty acids. Along with the activation of general metabolic regulators, enzymes involved in acetate usage (ACSS2) and fatty acid synthesis (ACC) and a precursor for fatty acid synthesis (acetyl-CoA) were elevated in T24R cells. Consistently, metabolic analysis with 13C isotope revealed that T24R cells preferred glucose to acetate as the exogenous carbon source for the increased fatty acid synthesis, contrary to T24S cells. In addition, ACSS2, rather than the well-established ACLY, was the key enzyme that supplies acetyl-CoA in T24R cells through glucose-derived endogenous acetate. The relevance of ACSS2 in cisplatin resistance was further confirmed by the abrogation of resistance by an ACSS2 inhibitor and, finally, by the higher expression of ACSS2 in the patient tissues with cisplatin resistance. Conclusions: Our results may help improve the treatment options for chemoresistant bladder cancer patients and provide possible vulnerability targets to overcome the resistance. Citation Format: He Wen, Sujin Lee, Wei-Guo Zhu, Ok-Jun Lee, Seok Joong Yun, Jayoung Kim, Sunghyouk Park. Acetate metabolism mediated by acetyl-CoA synthetase 2 in cisplatin-resistant bladder cancer [abstract]. In: Proceedings of the AACR Special Conference on Bladder Cancer: Transforming the Field; 2019 May 18-21; Denver, CO. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(15_Suppl):Abstract nr B12.

Enhancement of docosahexaenoic acid production by overexpression of ATP-citrate lyase and acetyl-CoA carboxylase in Schizochytrium sp.

BackgroundDocosahexaenoic acid (DHA) is an important omega-3 long-chain polyunsaturated fatty acid that has a variety of physiological functions for infant development and human health. Although metabolic engineering was previously demonstrated to be a highly efficient way to rapidly increase lipid production, metabolic engineering has seldom been previously used to increase DHA accumulation in Schizochytrium spp.ResultsHere, a sensitive β-galactosidase reporter system was established to screen for strong promoters in Schizochytrium sp. Four constitutive promoters (EF-1αp, TEF-1p, ccg1p, and ubiquitinp) and one methanol-induced AOX1 promoter were characterized by the reporter system with the promoter activity ccg1p> TEF-1p > AOX1p (induced) > EF-1αp > ubiquitinp. With the strong constitutive promoter ccg1p, Schizochytrium ATP-citrate lyase (ACL) and acetyl-CoA carboxylase (ACC) were overexpressed in Schizochytrium sp. ATCC 20888. The cells were cultivated at 28 °C and 250 rpm for 120 h with glucose as the carbon source. Shake-flask fermentation results showed that the overexpression strains exhibited growth curves and biomass similar to those of the wild-type strain. The lipid contents of the wild-type strain and of the OACL, OACC, and OACL-ACC strains were 53.8, 68.8, 69.8, and 73.0%, respectively, and the lipid yields of the overexpression strains were increased by 21.9, 30.5, and 38.3%, respectively. DHA yields of the wild-type strain and of the corresponding overexpression strains were 4.3, 5.3, 6.1, and 6.4 g/L, i.e., DHA yields of the overexpression strains were increased by 23.3, 41.9, and 48.8%, respectively.ConclusionsAcetyl-CoA and malonyl-CoA are precursors for fatty acid synthesis. ACL catalyzes the conversion of citrate in the cytoplasm into acetyl-CoA, and ACC catalyzes the synthesis of malonyl-CoA from acetyl-CoA. The results demonstrate that overexpression of ACL and ACC enhances lipid accumulation and DHA production in Schizochytrium sp.

Role of Adenosine Monophosphate Deaminase during Fatty Acid Accumulation in Oleaginous Fungus Mortierella alpina.

In oleaginous micro-organisms, nitrogen limitation activates adenosine monophosphate deaminase (AMPD) and promotes lipogenesis via the inhibition of isocitrate dehydrogenase. We found that the overexpression of homologous AMPD in Mortierella alpina favored lipid synthesis over cell growth. Total fatty acid content in the recombinant strain was 15.0-34.3% higher than that in the control, even though their biomass was similar. During the early fermentation stage, the intracellular AMP level reduced by 40-60%, together with a 1.9-2.7-fold increase in citrate content compared with the control, therefore provided more precursors for fatty acid synthesis. Moreover, the decreased AMP level resulted in metabolic reprogramming, reflected by the blocked TCA cycle and reduction of amino acids, distributing more carbon to lipid synthesis pathways. By coupling the energy balance with lipogenesis, this study provides new insights into cell metabolism under nitrogen-limited conditions and targets the regulation of fatty acid accumulation in oleaginous micro-organisms.

Abstract 4355: Altered acetate metabolism in cisplatin resistant bladder cancer

Abstract Introduction and Objective: Cisplatin is an important chemotherapeutic agent against metastatic bladder cancer, but resistance often limits its usage. With the recent recognition of lipid metabolic alterations in bladder cancers, we studied the metabolic implications of cisplatin resistance using cisplatin-sensitive (T24S) and resistant (T24R) bladder cancer cells. Methods: To test whether there should be differences in metabolism and metabolism-associated pathways between cisplatin-sensitive and resistant bladder cancer cells, we applied the live metabolomics approach that we recently developed to isogenic bladder cancer cell lines T24S (cisplatin sensitive) and T24R (cisplatin resistant). The metabolites generated from 13C-glucose tracer were monitored with 2D 1H-13C HSQC NMR in real time. Results: Real-time live metabolomics revealed that T24R cells consume more glucose, leading to higher production of glucose-derived acetate and fatty acids. Along with the activation of general metabolic regulators, enzymes involved in acetate usage (ACSS2) and fatty acid synthesis (ACC) and a precursor for fatty acid synthesis (acetyl-CoA) were elevated in T24R cells. Consistently, metabolic analysis with 13C isotope revealed that T24R cells preferred glucose to acetate as the exogenous carbon source for the increased fatty acid synthesis, contrary to T24S cells. In addition, ACSS2, rather than the well-established ACLY, was the key enzyme that supplies acetyl-CoA in T24R cells through glucose-derived endogenous acetate. The relevance of ACSS2 in cisplatin resistance was further confirmed by the abrogation of resistance by an ACSS2 inhibitor and, finally, by the higher expression of ACSS2 in the patient tissues with cisplatin resistance. Conclusions: Our results may help improve the treatment options for chemoresistant bladder cancer patients and provide possible vulnerability targets to overcome the resistance. Citation Format: Jayoung Kim. Altered acetate metabolism in cisplatin resistant bladder cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4355.

Abstract 4969: Autophagy modulates lipid metabolism to support Liver Kinase B1 (LKB1) - deficient lung tumor growth

Abstract Autophagy degrades and recycles macromolecules for cells to survive starvation. In genetically engineered mouse models (GEMMs) for human non-small cell lung cancer (NSCLC), autophagy supports Kras-driven lung tumor growth with or without Trp53. Tumor suppressor liver kinase B1 (LKB1) activates 5'-adenosine monophosphate protein kinase (AMPK) to maintain energy homeostasis. LKB1 mutations are detected in 20-30% of NSCLC, causing aggressive tumor growth and resistance to chemotherapy. Identifying novel target to improve LKB1-deficient tumor treatment is urgently needed. Using GEMMs for NSCLC with oncogenic Kras and LKB1 loss (KL), we found that autophagy deficiency increased the survival of the mice bearing Atg7-/- tumors compared to mice bearing wild-type (WT) tumors. To determine the mechanism of autophagy in supporting LKB1-deficient Kras-driven lung tumor growth, tumor derived cell lines (TDCLs) were generated from the lung tumors of these mice. We found that Atg7 null TDCLs were more sensitive to glucose and glutamine deprivation-induced cell death compared to Atg7 WT TDCLs. Atg7 null TDCLs were also more sensitive to starvation-induced cell death than Atg7 WT TDCLs, which can be rescued by glucose, glutamine, pyruvate, lactate and nucleotide supplementation. Thus, autophagy is required for KL TDCLs to tolerate metabolic stress. Furthermore, we observed that palmitate supplementation successfully rescued starvation-induced Atg7 null TDCLs death, indicating that autophagy is required to maintain free fatty acid level for cells to survive starvation. We further performed metabolomics in TDCLs in normal and starvation conditions and found that level of amino acids and intermediates for glycolysis and TCA cycle metabolism were significantly lower in Atg7 null TDCLs compared to Atg7 WT TDCLs during HBSS starvation. Surprisingly, we observed a significant increase in the levels of biotin, a precursor for fatty acid synthesis, in Atg7 null TDCLs than that in WT TDCLs, indicating that accumulation of biotin in autophagy deficient cells might be due to defective fatty acid synthesis. In support of this, we found that the lipid droplets accumulation is significantly lower in Atg7 null KL TDCLs than that in Atg7 WT cells. To further evaluate the functional consequences of autophagy-mediated lipid metabolism in supporting KL tumor growth, we treated Atg7 null and WT cells with etomoxir, an irreversible inhibitor of carnitine palmitoyltransferase-1 (CPT-1) to inhibit fatty acid oxidation, in normal and starvation conditions. We found that Atg7 null TDCLs are much more sensitive to etomoxir treatment than WT cells. Taken together, autophagy plays a critical role in supporting lipid metabolism for cells to survive metabolic stress. Thus, a combination of autophagy inhibition with interruption of lipid metabolism could be a novel therapeutic strategy to treat LKB1-deficient lung tumor. Citation Format: Vrushank D. Bhatt, Zhixian Hu, Xiaoyang Su, Jessie Yanxiang Guo. Autophagy modulates lipid metabolism to support Liver Kinase B1 (LKB1) - deficient lung tumor growth [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4969.

Glucose-derived acetate and ACSS2 as key players in cisplatin resistance in bladder cancer

Cisplatin is an important chemotherapeutic agent against metastatic bladder cancer, but resistance often limits its usage. With the recent recognition of lipid metabolic alterations in bladder cancers, we studied the metabolic implications of cisplatin resistance using cisplatin-sensitive (T24S) and resistant (T24R) bladder cancer cells. Real-time live metabolomics revealed that T24R cells consume more glucose, leading to higher production of glucose-derived acetate and fatty acids. Along with the activation of general metabolic regulators, enzymes involved in acetate usage (ACSS2) and fatty acid synthesis (ACC) and a precursor for fatty acid synthesis (acetyl-CoA) were elevated in T24R cells. Consistently, metabolic analysis with 13C isotope revealed that T24R cells preferred glucose to acetate as the exogenous carbon source for the increased fatty acid synthesis, contrary to T24S cells. In addition, ACSS2, rather than the well-established ACLY, was the key enzyme that supplies acetyl-CoA in T24R cells through glucose-derived endogenous acetate. The relevance of ACSS2 in cisplatin resistance was further confirmed by the abrogation of resistance by an ACSS2 inhibitor and, finally, by the higher expression of ACSS2 in the patient tissues with cisplatin resistance. Our results may help improve the treatment options for chemoresistant bladder cancer patients and provide possible vulnerability targets to overcome the resistance.

Autophagy modulates lipid metabolism to support Liver Kinase B1 (LKB1)‐deficient lung tumor growth

Autophagy degrades and recycles macromolecules for cells to survive starvation. In genetically engineered mouse models (GEMMs) for human non‐small cell lung cancer (NSCLC), autophagy supports Kras‐driven lung tumor growth with or without Trp53. Tumor suppressor liver kinase B1 (LKB1) activates 5′‐adenosine monophosphate protein kinase (AMPK) to maintain energy homeostasis. LKB1 mutations are detected in 20–30% of NSCLC, causing aggressive tumor growth and resistance to chemotherapy. Identifying novel target to improve LKB1‐deficient tumor treatment is urgently needed. Using GEMMs for NSCLC with oncogenic Kras and LKB1 loss (KL), we found that autophagy deficiency increased the survival of the mice bearing Atg7−/− tumors compared to mice bearing wild‐type (WT) tumors. To determine the mechanism of autophagy in supporting LKB1‐deficient Kras‐driven lung tumor growth, tumor‐derived cell lines (TDCLs) were generated from the lung tumors of these mice. We found that Atg7 null TDCLs were more sensitive to glucose and glutamine deprivation‐induced cell death compared to Atg7 WT TDCLs. Atg7 null TDCLs were also more sensitive to starvation‐induced cell death than Atg7 WT TDCLs, which can be rescued by glucose, glutamine, pyruvate, lactate and nucleotide supplementation. Thus, autophagy is required for KL TDCLs to tolerate metabolic stress. Furthermore, we observed that palmitate supplementation successfully rescued starvation‐induced Atg7 null TDCLs death, indicating that autophagy is required to maintain the free fatty acid level for cells to survive starvation. We further performed metabolomics in TDCLs in normal and starvation conditions and found that level of amino acids and intermediates for glycolysis and TCA cycle metabolism were significantly lower in Atg7 null TDCLs compared to Atg7 WT TDCLs during HBSS starvation. Surprisingly, we observed a significant increase in the levels of biotin, a precursor for fatty acid synthesis, in Atg7 null TDCLs than that in WT TDCLs, indicating that accumulation of biotin in autophagy deficient cells might be due to defective fatty acid synthesis. In support of this, we found that the lipid droplets accumulation is significantly lower in Atg7 null KL TDCLs than that in Atg7 WT cells. To further evaluate the functional consequences of autophagy‐mediated lipid metabolism in supporting KL tumor growth, we treated Atg7 null and WT cells with etomoxir, an irreversible inhibitor of carnitine palmitoyltransferase‐1 (CPT‐1) to inhibit fatty acid oxidation, in normal and starvation conditions. We found that Atg7 null TDCLs are much more sensitive to etomoxir treatment than WT cells. Taken together, autophagy plays a critical role in supporting lipid metabolism for cells to survive metabolic stress. Thus, a combination of autophagy inhibition with interruption of lipid metabolism could be a novel therapeutic strategy to treat LKB1‐deficient lung tumor.Support or Funding InformationThis research has been supported by the research funds from K22 CA190521 (NIH) grant, American Cancer Society Early Investigator Pilot Award and start‐up funding from Rutgers Cancer Institute of New Jersey.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Evaluating the Metabolic Impact of Hypoxia on Pancreatic Cancer Cells.

Hypoxia is frequently observed in human cancers and induces global metabolic reprogramming that includes an increase in glucose uptake and glycolysis, alterations in NAD(P)H/NAD(P)+ and intracellular ATP levels, and increased utilization of glutamine as the major precursor for fatty acid synthesis. In this chapter, we describe in detail various physiological assays that have been adopted to study the metabolic shift propagated by exposure to hypoxic conditions in pancreatic cell culture model that includes glucose uptake, glutamine uptake, and lactate release by pancreatic cancer cell lines. We have also elaborated the assays to evaluate the ratio of NAD(P)H/NAD(P)+ and intracellular ATP estimation using the commercially available kit to assess the metabolic state of cancer cells.

High carbon (CO2) supply leads to elevated intracellular acetyl CoA levels and increased lipid accumulation in Chlorella vulgaris

Carbon dioxide, the sole carbon source in phototrophic cultivation of microalgae, is the limiting factor for photosynthesis due to its low concentration in the atmosphere (0.04% v/v). We postulate that exposure to increased CO2 concentrations can increase the steady-state specific intracellular levels of carbon metabolic intermediates, including, the specific intracellular (si) levels of the precursor for fatty acid synthesis (acetyl CoA (AcCoA)), which in turn, appears to improve lipid accumulation. The effects of higher CO2 concentrations on Chlorella vulgaris were studied. At 2.6% v/v CO2, a 6-fold increase in volumetric lipid production was achieved. A 5.4-fold increase in the specific intracellular neutral lipid level (si-NL) from 9.6 to 52.3mg triolein (TO)/g biomass was also obtained. Si-AcCoA was significantly elevated at 2.6% CO2, and showed a 41, 25 and 27% increase over the air-sparged controls in the lag, log and stationary phases, respectively. Further, we show a quantitative empirical relationship between si-AcCoA and si-NL, which provides a new outlook for the design of strategies to improve lipid accumulation. In addition, favorable biodiesel characteristics were also obtained at higher CO2. Increased si-AcCoA with increase in CO2 concentrations, a related increase in lipid levels, and a quantitative empirical relationship between them have not been reported in the literature thus far.

Acetate functions as an epigenetic metabolite to promote lipid synthesis under hypoxia.

Besides the conventional carbon sources, acetyl-CoA has recently been shown to be generated from acetate in various types of cancers, where it promotes lipid synthesis and tumour growth. The underlying mechanism, however, remains largely unknown. We find that acetate induces a hyperacetylated state of histone H3 in hypoxic cells. Acetate predominately activates lipogenic genes ACACA and FASN expression by increasing H3K9, H3K27 and H3K56 acetylation levels at their promoter regions, thus enhancing de novo lipid synthesis, which combines with its function as the metabolic precursor for fatty acid synthesis. Acetyl-CoA synthetases (ACSS1, ACSS2) are involved in this acetate-mediated epigenetic regulation. More importantly, human hepatocellular carcinoma with high ACSS1/2 expression exhibit increased histone H3 acetylation and FASN expression. Taken together, this study demonstrates that acetate, in addition to its ability to induce fatty acid synthesis as an immediate metabolic precursor, also functions as an epigenetic metabolite to promote cancer cell survival under hypoxic stress.

CARBONIC ANHYDRASE MEDIATES HEPATIC STEATOSIS AND VASCULAR STIFFNESS INDUCED BY A WESTERN DIET

The typical “Western diet” (WD) is higher in fat and high fructose corn syrup when compared to a healthful diet (CD) that is lower in fat and does not contain high fructose corn syrup. A WD leads to insulin resistance and the metabolic syndrome that is characterized by fatty steatosis in the liver, a precursor of non‐alcoholic fatty liver disease, and vascular stiffness that presages atherosclerotic vascular disease and hypertension. Bicarbonate is a necessary precursor for fatty acid synthesis and the storage of depot fat in the liver and in perivascular tissue. We hypothesized that acetazolamide (ACT), a carbonic anhydrase inhibitor that blocks bicarbonate‐dependent formation of fatty acids and triglycerides, could prevent the fatty steatosis and vascular stiffness that is induced by a WD. Laboratory mice were fed a WD (46% kcals from fat; 36% kcals from carbohydrate, sucrose [17.5%] and high fructose corn syrup [17.5%]) or CD (10% kcals from fat; 72% kcals from carbohydrate; no high fructose corn syrup) for sixteen weeks with, and without, 2mM ACT in the drinking water ad libitum. Subsequently, rodents were weighed, and fat mass, lean mass, and body water was measured with magnetic resonance imaging (MRI). Hepatic steatosis was graded on a scale of 1–9 arbitrary units (A.U.) on hematoxylin and eosin stained liver tissue. Vascular stiffness was measured by atomic force microscopy (AFM) on aorta explants. CD WD WD + ACT Total Body Weight (values in g ± SEM) 23.1 ± 0.4 31.3 ± 1.1** 20.1 ± 1.5 (N.S.) Total Fat Mass (values in g ± SEM) 4.7 ± 11.2 11.2 ± 1 ** 5.1 ± 1.2 (N.S.) Total Body Water/Total Body Weight (in % ± SEM) 65.1 ± 0.8 49.9 ± 2.0** 57.9 ± 2.6 (N.S.) Hepatic Steatosis (in arbitrary units, AU, ± SEM) 3.75 ± 1.4 7.4 ± 0.5* 5.0 ± 1.1 (N.S.) Vascular Stiffness (in kPa ± SEM) 4.72 ± 0.6 17.18 ± 3.2* 5.22 ± 1.0 (N.S.) N = 20, denotes P < 0.05, denotes P < 0.01, N.S. denotes not significantly different from control diet (CD). Compared to a CD, sixteen weeks of a WD was associated with a gain in total body weight that was completely inhibited by ACT. The increase in total adipocyte fat mass and percentage of body weight from water in WD animals were also inhibited by ACT. Notably, inhibition of carbonic anhydrase resulted in a decrease in hepatic steatosis and aortic vascular stiffness. If excess adiposity is responsible for the untoward sequelae of the metabolic syndrome, chronic inhibition of carbonic anhydrase‐dependent formation of bicarbonate and fatty acid synthesis could result in the normalization of the metabolic profile associated with an unhealthy Western dietSupport or Funding InformationSupport for this project was provided by the University of Missouri.

The role of pyruvate hub enzymes in supplying carbon precursors for fatty acid synthesis in photosynthetic microalgae.

Photosynthetic microalgae are currently the focus of basic and applied research due to an ever-growing interest in renewable energy resources. This review discusses the role of carbon-unit supply for the production of acetyl-CoA, a direct precursor of fatty acid biosynthesis and the primary building block of the growing acyl chains for the purpose of triacylglycerol (TAG) production in photosynthetic microalgae under stressful conditions. It underscores the importance of intraplastidic acetyl-CoA generation for storage lipid accumulation. The main focus is placed on two enzymatic steps linking the central carbon metabolism and fatty acid synthesis, namely the reactions catalyzed by the plastidic isoform of pyruvate kinase and the chloroplastic pyruvate dehydrogenase complex. Alternative routes for plastidic acetyl-CoA synthesis are also reviewed. A separate section is devoted to recent advances in functional genomics studies related to fatty acid and TAG biosynthesis.

Evidence for Proteomic and Metabolic Adaptations Associated with Alterations of Seed Yield and Quality in Sulfur-limited Brassica napus L

In Brassica napus, seed yield and quality are related to sulfate availability, but the seed metabolic changes in response to sulfate limitation remain largely unknown. To address this question, proteomics and biochemical studies were carried out on mature seeds obtained from plants grown under low sulfate applied at the bolting (LS32), early flowering (LS53), or start of pod filling (LS70) stage. The protein quality of all low-sulfate seeds was reduced and associated with a reduction of S-rich seed storage protein accumulation (as Cruciferin Cru4) and an increase of S-poor seed storage protein (as Cruciferin BnC1). This compensation allowed the protein content to be maintained in LS70 and LS53 seeds but was not sufficient to maintain the protein content in LS32 seeds. The lipid content and quality of LS53 and LS32 seeds were also affected, and these effects were primarily associated with a reduction of C18-derivative accumulation. Proteomics changes related to lipid storage, carbohydrate metabolism, and energy (reduction of caleosins, phosphoglycerate kinase, malate synthase, ATP-synthase β-subunit, and thiazole biosynthetic enzyme THI1 and accumulation of β-glucosidase and citrate synthase) provide insights into processes that may contribute to decreased oil content and altered lipid composition (in favor of long-chain fatty acids in LS53 and LS32 seeds). These data indicate that metabolic changes associated with S limitation responses affect seed storage protein composition and lipid quality. Proteins involved in plant stress response, such as dehydroascorbate reductase and Cu/Zn-superoxide dismutase, were also accumulated in LS53 and LS32 seeds, and this might be a consequence of reduced glutathione content under low S availability. LS32 treatment also resulted in (i) reduced germination vigor, as evidenced by lower germination indexes, (ii) reduced seed germination capacity, related to a lower seed viability, and (iii) a strong decrease of glyoxysomal malate synthase, which is essential for the use of fatty acids during seedling establishment.

Milk fatty acids profile and arterial blood milk fat precursors concentration of dairy goats fed increasing doses of soybean oil

The provision of fatty acid sources in ruminant diets allows the manipulation of fatty acid profiles in milk and meat, permitting to increase the fatty acids of interest, such as conjugated linoleic acid. The objectives of this experiment were to evaluate the inclusion of increasing doses of soybean oil (30, 60 or 90g/d) on intake, total tract digestibility of nutrients, milk production and composition, arterial and milk fatty acid profiles, and arterial concentrations of glucose, acetate, and β-hydroxybutyrate in dairy goats. Four multiparous Saanen goats with mean initial DIM 32±6 and mean initial BW 63±7kg were assigned in a 4×4 Latin square. Does were housed in tie stalls and fed a 40% of corn silage and 60% concentrate diet. Experimental treatments consisted of: (1) basal diet without soybean oil infusion (control); (2) basal diet plus an oral infusion of 30mL soybean oil; (3) basal diet plus an oral infusion of 60mL soybean oil; and (4) basal diet plus an oral infusion of 90mL soybean oil. The DM and OM intakes decreased linearly (P<0.05) with increasing soybean oil supply. Dry matter digestibility was negatively influenced by oil (P<0.01); however, the OM digestibility was not affected by soybean oil doses. Milk production tended to decrease (P=0.07) by soybean oil supply, although when this was corrected to 3.5% fat, it decreased linearly (P<0.01) as a result of the decreasing (P<0.05) milk fat content. Vaccenic acid concentration in arterial blood increased (P<0.01) 127% with 90g/d of soybean oil addition. Appearance of C18:2 t10, c12 in milk caused a reduction (P<0.01) in milk fat and reached a 31% reduction when 90g/d of soybean oil was offered compared to the control diet, while the opposite occurred with C18:2 c9, t11, which decreased (P<0.05) with increasing doses of soybean oil. Increasing soybean oil had no effect on arterial blood concentrations of glucose, acetate and β-hydroxybutyrate. Therefore, the decrease in milk fat concentration was due to the inhibition of mammary synthesis of fatty acids, but not by a limitation of its precursors for fatty acid synthesis.

Abstract 1001: Reductive tricarboxylic acid metabolism of glutamine mediates lipogenesis under hypoxia

Abstract Acetyl coenzyme A (AcCoA) is the central biosynthetic precursor for fatty acid synthesis and protein acetylation. In the conventional view of mammalian cell metabolism, AcCoA is primarily generated from glucose-derived pyruvate through the citrate shuttle and adenosine triphosphate citrate lyase (ACL) in the cytosol. However, proliferating cells that exhibit aerobic glycolysis and those exposed to hypoxia convert glucose to lactate at near stoichiometric levels, directing glucose carbon away from the tricarboxylic acid cycle (TCA) and fatty acid synthesis. Although glutamine is consumed at levels exceeding that required for nitrogen biosynthesis, the regulation and utilization of glutamine metabolism in hypoxic cells is not well understood. Here we show that human cells employ reductive metabolism of alpha-ketoglutarate (aKG) to synthesize AcCoA for lipid synthesis. This isocitrate dehydrogenase 1 (IDH1) dependent pathway is active in most cell lines under normal culture conditions, but cells grown under hypoxia rely almost exclusively on the reductive carboxylation of glutamine-derived aKG for de novo lipogenesis. Furthermore, renal cell lines deficient in the von Hippel-Lindau (VHL) tumor suppressor protein preferentially utilize reductive glutamine metabolism for lipid biosynthesis even at normal oxygen levels. These results identify a critical role for oxygen in regulating carbon utilization in order to produce AcCoA and support lipid synthesis in mammalian cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1001. doi:1538-7445.AM2012-1001

Malonyl-CoA Synthetase, Encoded by ACYL ACTIVATING ENZYME13, Is Essential for Growth and Development of Arabidopsis

Malonyl-CoA is the precursor for fatty acid synthesis and elongation. It is also one of the building blocks for the biosynthesis of some phytoalexins, flavonoids, and many malonylated compounds. In plants as well as in animals, malonyl-CoA is almost exclusively derived from acetyl-CoA by acetyl-CoA carboxylase (EC 6.4.1.2). However, previous studies have suggested that malonyl-CoA may also be made directly from malonic acid by malonyl-CoA synthetase (EC 6.2.1.14). Here, we report the cloning of a eukaryotic malonyl-CoA synthetase gene, Acyl Activating Enzyme13 (AAE13; At3g16170), from Arabidopsis thaliana. Recombinant AAE13 protein showed high activity against malonic acid (K(m) = 529.4 ± 98.5 μM; V(m) = 24.0 ± 2.7 μmol/mg/min) but little or no activity against other dicarboxylic or fatty acids tested. Exogenous malonic acid was toxic to Arabidopsis seedlings and caused accumulation of malonic and succinic acids in the seedlings. aae13 null mutants also grew poorly and accumulated malonic and succinic acids. These defects were complemented by an AAE13 transgene or by a bacterial malonyl-CoA synthetase gene under control of the AAE13 promoter. Our results demonstrate that the malonyl-CoA synthetase encoded by AAE13 is essential for healthy growth and development, probably because it is required for the detoxification of malonate.

Opening a New Path to Lipoic Acid

Lipoic acid (Fig. 1) is a cofactor that is critical to the catalytic function of several key enzymes in intermediary metabolism that are found in all domains of life. There are two pathways to lipoyl-modified proteins. The de novo pathway from octanoylacyl carrier protein (ACP) involves the consecutive action of LipB and LipA (Fig. 2A), and the LplA-dependent salvage pathway activates and transfers environmental lipoic acid to the lipoyl protein domains (Fig. 2B). In this issue, Hermes and Cronan (6) identify gain-of-function mutations in LplA that allow cells to bypass the requirement for LipB in de novo lipoic acid formation (Fig. 2C). Normally, lipB null strains require either a lipoate or an octanoate supplement for growth, but these lplA mutants have increased affinities for octanoic acid, permitting them to utilize the low concentrations of intracellular octanoate, thus substituting for the LipB reaction. The characterization of these suppressor strains provides new information on the biochemistry of lipoate metabolism and reveals the existence of intracellular pools of medium-chain fatty acids that previously were not thought to exist. Functions of lipoic acid. Lipoic acid is essential for the functions of several enzymes in oxidative metabolism (2). A good example is pyruvate dehydrogenase (PDH), where covalently bound lipoate shuttles reaction intermediates between the active sites of a multisubunit complex (11). Lipoic acid is bound to the dihydrolipoyl transacetylase (E2) subunit in its oxidized state as a five-membered, disulfide-linked ring (Fig. 1). The E1 subunit of PDH decarboxylates pyruvate, forming 2-hydroxyethyl-2-thiamine pyrophosphate, and the lipoic acid disulfide is reduced when E2 accepts an acetaldehyde moiety from the thiamine adduct. Acetyl-coenzyme A is then released via a thioester exchange reaction with coenzyme A. The disulfide ring of lipoate is reformed by the FAD-dependent dihydrolipoyl dehydrogenase component (E3). Other important bacterial lipoyl enzymes include the glycine cleavage system, -ketoglutarate dehydrogenase, and the branched-chain ketoacid dehydrogenase that is involved in supplying branched-chain precursors for fatty acid synthesis. In Escherichia coli, strains defective in lipoic acid synthesis are unable to grow aerobically on glucose minimal medium unless it is supplemented with acetate and succinate to bypass the requirement for PDH and -ketoglutarate dehydrogenase (5, 13). Bypassing LipB. The de novo biosynthetic pathway in E. coli requires lipA and lipB (Fig. 2A). Genetic studies identified the product of the lipA gene as responsible for inserting the two sulfur atoms into the octanoate precursor (13). LipA is an S-adenosylmethionine-dependent enzyme that inserts sulfur at