acetyl-CoA For Fatty Acid Synthesis Research Articles

Brown fat ATP-citrate lyase links carbohydrate availability to thermogenesis and guards against metabolic stress.

Brown adipose tissue (BAT) engages futile fatty acid synthesis-oxidation cycling, the purpose of which has remained elusive. Here, we show that ATP-citrate lyase (ACLY), which generates acetyl-CoA for fatty acid synthesis, promotes thermogenesis by mitigating metabolic stress. Without ACLY, BAT overloads the tricarboxylic acid cycle, activates the integrated stress response (ISR) and suppresses thermogenesis. ACLY's role in preventing BAT stress becomes critical when mice are weaned onto a carbohydrate-plentiful diet, while removing dietary carbohydrates prevents stress induction in ACLY-deficient BAT. ACLY loss also upregulates fatty acid synthase (Fasn); yet while ISR activation is not caused by impaired fatty acid synthesis per se, deleting Fasn and Acly unlocks an alternative metabolic programme that overcomes tricarboxylic acid cycle overload, prevents ISR activation and rescues thermogenesis. Overall, we uncover a previously unappreciated role for ACLY in mitigating mitochondrial stress that links dietary carbohydrates to uncoupling protein 1-dependent thermogenesis and provides fundamental insight into the fatty acid synthesis-oxidation paradox in BAT.

Abstract 1784: Glucose metabolism and de novo palmitate synthesis under normoxia and hypoxia in breast cancer cells that preferentially metastasize to lung and liver

Abstract Cancer cells are exposed to variable oxygen and nutrient availability during the metastatic process which can influence metabolic adaptations of cancer cells to metastasize to distant sites. Thus, we investigated the metabolic adaptations of breast cancer cells that preferentially metastasize to lung (metM-WntLung cells; MLg) and liver (metM-WntLiver cells; MLr) in normoxia and hypoxia and at different glucose concentrations. In normoxia, 14C-glucose uptake is similar, but mRNA abundance of hexokinase, the initial rate-limiting step in glycolysis, was 22% higher in MLg cells. This is consistent with higher 13C6-glucose flux into glycolytic metabolites pyruvate (M+3) and lactate (M+3) in MLg compared to MLr. Also, de novo palmitate synthesis from 13C6-glucose was 7.6% higher in MLg and was accompanied by higher mRNA expression of ATP-citrate lyase (ACLY), which converts citrate to acetyl-CoA for fatty acid synthesis. These suggest greater glucose metabolism in MLg compared to MLr in normoxia, consistent with reduced viability of MLr by 39% in high glucose (25 mM) concentrations. Further, mRNA abundance of CPT1A, a rate-limiting step in fatty acid oxidation, is 23% higher in MLg. Moreover, the fatty acid oxidation inhibition (etomoxir) reduced viability of MLg by 26.7% compared to MLr, suggesting that fatty acid synthesis and oxidation are critical for MLg. Protein expression of HIF1α, a transcription factor that induces glycolysis during hypoxia, is 1.5-fold higher in MLr than MLg in normoxia, consistent with higher MLr mRNA abundance of pyruvate dehydrogenase kinase (PDK1), a target of HIF1α that inhibits pyruvate dehydrogenase (PDH) activity. Surprisingly, mRNA abundance of pyruvate carboxylase (PC), the enzyme that converts pyruvate to oxaloacetate, is also higher in MLr (39%) than MLg, thus supporting a potential alternative mechanism for glucose metabolites to enter the TCA cycle in normoxia in MLr. In hypoxia, viability of MLr, relative to MLg, increased by 11% at high glucose concentration. This was accompanied by higher hexokinase (33%) and PDK1 (83%) mRNA abundance suggesting MLr’s shift towards a glycolytic phenotype. However, de novo palmitate synthesis from 13C6-glucose is higher in MLg, suggesting that palmitate synthesis is maintained in MLg even in hypoxia. Altogether, results demonstrate that glucose utilization shifts in breast cancer cells that preferentially metastasize to lung vs liver, but de novo fatty acid synthesis is higher in MLg in different oxygen conditions. Thus, targeting breast cancer cells’ adaptation to glucose and de novo fatty acid synthesis may be a potential strategy to prevent breast cancer progression to distant organs. Citation Format: Marjorie Anne A. Layosa, Morgan Conrad, Stephen Hursting, Dorothy Teegarden. Glucose metabolism and de novo palmitate synthesis under normoxia and hypoxia in breast cancer cells that preferentially metastasize to lung and liver [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1784.

13C-metabolic flux analysis of lipid accumulation in the green microalgae Tetradesmus obliquus under nitrogen deficiency stress

Metabolic fluxes (MF) serve as the functional phenotypes of biochemical processes and are crucial to describe the distribution of precursors within metabolic networks. There is a lack of experimental observations for carbon flux towards lipids, which is important for biodiesel generation. Here, the accumulation of lipid, and MF in Tetradesmus obliquus under nitrogen deficiency stress (NF) using a 13C isotope tracer at different time intervals was investigated. The 13C based MF showed enhanced de novo synthesis of G3P and PEP, indicating increased carbon flux from CO2 into lipid synthesis. An increase in palmitic acid (3500 μmol/mg), linoleic acid (2100 μmol/mg), and oleic acid (2000 μmol/mg) was observed. The accumulation of C16:0 under NF was mainly related to de novo synthesis while C18:3 was accumulated through a non de novo pathway. Under NF stress, T. obliquus had higher flux in PPP and glycolysis pathway, together, it might provide more NADPH and substrate acetyl-CoA for fatty acid synthesis.

Androgen Receptor Signaling and Metabolic and Cellular Plasticity During Progression to Castration Resistant Prostate Cancer.

Metabolic reprogramming is associated with re/activation and antagonism of androgen receptor (AR) signaling that drives prostate cancer (PCa) progression to castration resistance, respectively. In particular, AR signaling influences the fates of citrate that uniquely characterizes normal and malignant prostatic metabolism (i.e., mitochondrial export and extracellular secretion in normal prostate, mitochondrial retention and oxidation to support oxidative phenotype of primary PCa, and extra-mitochondrial interconversion into acetyl-CoA for fatty acid synthesis and epigenetics in the advanced PCa). The emergence of castration-resistant PCa (CRPC) involves reactivation of AR signaling, which is then further targeted by androgen synthesis inhibitors (abiraterone) and AR-ligand inhibitors (enzalutamide, apalutamide, and daroglutamide). However, based on AR dependency, two distinct metabolic and cellular adaptations contribute to development of resistance to these agents and progression to aggressive and lethal disease, with the tumor ultimately becoming highly glycolytic and with imaging by a tracer of tumor energetics, 18F-fluorodoxyglucose (18F-FDG). Another major resistance mechanism involves a lineage alteration into AR-indifferent carcinoma such a neuroendocrine which is diagnostically characterized by robust 18F-FDG uptake and loss of AR signaling. PCa is also characterized by metabolic alterations such as fatty acid and polyamine metabolism depending on AR signaling. In some cases, AR targeting induces rather than suppresses these alterations in cellular metabolism and energetics, which can be explored as therapeutic targets in lethal CRPC.

Insights into oleaginous phenotype of the yeast Papiliotrema laurentii

Oleaginous yeasts have stood out due to their ability to accumulate oil, which can be used for fatty acid-derived biofuel production. Papiliotrema laurentii UFV-1 is capable of starting the lipid accumulation in the late exponential growth phase and achieves maximum lipid content at 48 h of growth; it is, therefore, interesting to study how its oleaginous phenotype is regulated. Herein, we provide for the first time insights into the regulation of this phenotype in P. laurentii UFV-1. We sequenced and assembled its genome, performed comparative genomic analyses and investigated its phylogenetic relationships with other yeasts. Gene expression and metabolomic analyses were carried out on the P. laurentii UFV-1 cultivated under a nitrogen-limiting condition. Our results indicated that the lipogenesis of P. laurentii might have taken place during evolution after the divergence of genera in the phylum Basidiomycota. Metabolomic data indicated the redirection of the carbon flux towards fatty acid synthesis in response to the nitrogen limitation. Furthermore, purine seems to be catabolized to recycle nitrogen and leucine catabolization may provide acetyl-CoA for fatty acid synthesis. Analyses of the expression of genes encoding certain enzymes involved with the oleaginous phenotype indicated that the NADP+-dependent malic enzyme seems to play an important role in the supply of NADPH for fatty acid synthesis. There was a surprising decrease in the expression of the ACC1 gene, which encodes acetyl-CoA carboxylase, during lipid accumulation. Taken together, our results provided a basis for understanding lipid accumulation in P. laurentii under nitrogen limiting conditions.

13C-metabolic flux analysis of lipid accumulation in the oleaginous fungus Mucor circinelloides

The oleaginous fungus Mucor circinelloides is of industrial interest because it can produce high levels of polyunsaturated fatty acid γ-linolenic acid. M. circinelloides CBS 277.49 is able to accumulate less than 15% of cell dry weight as lipids, while M. circinelloides WJ11 can accumulate lipid up to 36%. In order to better understand the mechanisms behind the differential lipid accumulation in these two strains, tracer experiments with 13C-glucose were performed with the growth of M. circinelloides and subsequent gas chromatography–mass spectrometric detection of 13C-patterns in proteinogenic amino acids was carried out to identify the metabolic network topology and estimate intracellular fluxes. Our results showed that the high oleaginous strain WJ11 had higher flux of pentose phosphate pathway and malic enzyme, lower flux in tricarboxylic acid cycle, higher flux in glyoxylate cycle and ATP: citrate lyase, together, it might provide more NADPH and substrate acetyl-CoA for fatty acid synthesis.

Reciprocal Regulation of Protein Synthesis and Carbon Metabolism for Thylakoid Membrane Biogenesis

Author SummaryMetabolic control of gene expression coordinates the levels of specific gene products to meet cellular demand for their activities. This control can be exerted by metabolites acting as regulatory signals on a class of metabolic enzymes with dual functions as regulators of gene expression. However, little is known about how metabolic signals affect the balance between enzymatic and regulatory roles of these proteins. Here, we report an example of a protein with dual functions in gene expression and carbon metabolism. The chloroplast pyruvate dehydrogenase complex is well-known to produce activated di-carbon precursors for fatty acid, which is required for lipid synthesis. Our results show that a subunit of this enzyme forms ribonucleoprotein particles and influences chloroplast mRNA translation. Conversely, RNA binding affects pyruvate dehydrogenase (metabolic) activity. These findings offer insight into how intracellular metabolic signaling and gene expression are reciprocally regulated during membrane biogenesis. In addition, our results suggest that these dual roles of the protein might exist in evolutionary distant organisms ranging from cyanobacteria to humans.

PGC1α Promotes Tumor Growth by Inducing Gene Expression Programs Supporting Lipogenesis

Despite the role of aerobic glycolysis in cancer, recent studies highlight the importance of the mitochondria and biosynthetic pathways as well. PPARγ coactivator 1α (PGC1α) is a key transcriptional regulator of several metabolic pathways including oxidative metabolism and lipogenesis. Initial studies suggested that PGC1α expression is reduced in tumors compared with adjacent normal tissue. Paradoxically, other studies show that PGC1α is associated with cancer cell proliferation. Therefore, the role of PGC1α in cancer and especially carcinogenesis is unclear. Using Pgc1α(-/-) and Pgc1α(+/+) mice, we show that loss of PGC1α protects mice from azoxymethane-induced colon carcinogenesis. Similarly, diethylnitrosamine-induced liver carcinogenesis is reduced in Pgc1α(-/-) mice as compared with Pgc1α(+/+) mice. Xenograft studies using gain and loss of PGC1α expression showed that PGC1α also promotes tumor growth. Interestingly, while PGC1α induced oxidative phosphorylation and tricarboxylic acid cycle gene expression, we also observed an increase in the expression of two genes required for de novo fatty acid synthesis, ACC and FASN. In addition, SLC25A1 and ACLY, which are required for the conversion of glucose into acetyl-CoA for fatty acid synthesis, were also increased by PGC1α, thus linking the oxidative and lipogenic functions of PGC1α. Indeed, using stable (13)C isotope tracer analysis, we show that PGC1α increased de novo lipogenesis. Importantly, inhibition of fatty acid synthesis blunted these progrowth effects of PGC1α. In conclusion, these studies show for the first time that loss of PGC1α protects against carcinogenesis and that PGC1α coordinately regulates mitochondrial and fatty acid metabolism to promote tumor growth.

Nuclear transcription factors and lipid homeostasis in liver

Nuclear transcription factors and lipid homeostasis in liver

Simulation of the Metabolism of Absorbed Energy-Yielding Nutrients in Young Sheep: Efficiency of Utilization of Acetate

A previously described mathematical model that simulates the metabolism of absorbed nutrients was used to examine factors influencing the efficiency of utilization of acetate in a sheep weighing 25 kg. A significant component of the model was a function representing substrate cycles that degraded ATP when its concentration reached twice initial levels. Acetate utilization was simulated when up to 2 g mol/d were added to four diets (representing forages and concentrates) that supplied varying proportions of absorbed volatile fatty acids, glucose and protein. The basal diets provided either 4.375 (near energy balance) or 8.75 MJ/d absorbed energy. The predicted net efficiency of utilization (kf) of the energy in added acetate was high (0.58–0.70) for all combinations of absorbed nutrients at the low level of energy absorption, whereas at the higher level, it ranged from 0.16 (forage) to 0.49 (high protein concentrate). Low kf values were always associated with a considerable flux of ATP through the degradation pathway. The increase in ATP concentration that caused this pathway to operate was linked to an inhibition in the utilization of acetyl-CoA for fatty acid synthesis and an increase in its oxidation. The simulated addition of exogenous NADPH or its precursors (particularly glucose and propionate) to these diets repartitioned acetyl-CoA flux towards fatty acid synthesis instead of oxidation, decreased the flux of ATP through the degradation pathway and increased the kf of added acetate. Although a negative relationship was predicted between kf of added acetate and NADPH production for diets with increasing protein content, kf still depended on ATP flux through the degradation pathway. Addition of glucose to the high protein diets decreased this flux by decreasing acetyl-CoA oxidation and increasing fatty acid synthesis. The predictions suggest that the efficiency of acetate utilization in ruminants may be influenced by NADPH availability when a considerable amount of absorbed energy is derived from acetate. Increased absorption of glucose or propionate enhanced NADPH production, but the major effect on acetate utilization of increasing dietary protein was not through NADPH production.

The activities of 2-oxoglutarate dehydrogenase and pyruvate dehydrogenase in hearts and mammary glands from ruminants and non-ruminants

1. The activities of 2-oxoglutarate dehydrogenase (EC 1.2.4.2) were measured in hearts and mammary glands of rats, mice, rabbits, guinea pigs, cows, sheep, goats and in the flight muscles of several Hymenoptera. 2. The activity of 2-oxoglutarate dehydrogenase was similar to the maximum flux through the tricarboxylic acid cycle in vivo. Therefore measuring the activity of this enzyme may provide a simple method for estimating the maximum flux through the cycle for comparative investigations. 3. The activities of pyruvate dehydrogenase (EC 1.2.4.1) in mammalian hearts were similar to those of 2-oxoglutarate dehydrogenase, suggesting that in these tissues the tricarboxylic acid cycle can be supplied (under some conditions) by acetyl-CoA derived from pyruvate alone. 4. In the lactating mammary glands of the rat and mouse, the activities of pyruvate dehydrogenase exceeded those of 2-oxoglutarate dehydrogenase, reflecting a flux of pyruvate to acetyl-CoA for fatty acid synthesis in addition to that of oxidation via the tricarboxylic acid cycle. In ruminant mammary glands the activities of pyruvate dehydrogenase were similar to those of 2-oxoglutarate dehydrogenase, reflecting the absence of a significant flux of pyruvate to fatty acids in these tissues.

Inhibition of lipogenesis by halothane in isolated rat liver cells

1. Halothane at clinically effective concentrations [2.5 and 4% (v/v) of the gas phase of the incubation flask] was found to inhibit significantly lipogenesis from endogenous substrates, e.g., glycogen, or from added lactate plus pyruvate. This was accompanied by a decrease in the ratio of the free [NAD+]/[NADH] of the mitochondrion and the cytoplasm, as shown by the [3-hydroxybutyrate]/[acetoacetate] ratio and the [lactate]/[pyruvate] ratio. 2. Acetoacetate or pyruvate decreased the inhibitory effect of halothane and restored lipogenesis to control rates. They were reduced rapidly by 3-hydroxybutyrate dehydrogenase or lactate dehydrogenase respectively, with the concomitant oxidation of NADH and the generation of NAD+. 3. These results suggest that the mechanism by which halothane inhibits lipogenesis from glycogen or lactate is by inhibition of the oxidation of NADH; this results in inhibition of flux of carbon through pyruvate dehydrogenase and a shortage of acetyl-CoA for fatty acid synthesis. Thus when NADH acceptors are added in the presence of halothane, the concentration of mitochondrial NAD+ is raised so that the flux of carbon through pyruvate dehydrogenase increases and lipogenesis is restored.

Regulation of ketogenesis and clinical aspects of the ketotic state

Recent studies of the regulation of ketogenesis are reviewed. Under circumstances of relative or absolute insulin deficiency there is a mobilization of free fatty acids from adipose tissue to the liver. While an increased delivery of fatty acids to this organ is important in providing substrate for ketone body formation, it is emphasized that enhanced uptake of fatty acids by the liver is not sufficient in itself to initiate maximal ketogenesis. It appears likely that a major determinant of the rate of ketogenesis is competition for the fatty acid substrate between the β-oxidative and triglyceride synthesizing pathways. While it is widely held that the rate of triglyceride synthesis is primary and that only those fatty acids not utilized for esterification become available for oxidation, evidence for the reverse sequence is presented. It is considered likely that fatty acids are utilized for triglyceride synthesis only insofar as they escape uptake and oxidation in the mitochondria. Regardless of the mechanism, fatty acid oxidation is increased in the ketotic state with the consequence that acetyl-CoA production is accelerated. Since the utilization of acetyl-CoA for fatty acid synthesis and, to a much lesser extent, its oxidation in the Krebs cycle is impaired, the synthesis of acetoacetate and β-hydroxybutyrate is stimulated to a remarkable degree. The hepatic overproduction of ketones appears to be coupled to a limited capacity for their utilization by peripheral tissues, the combined effect of which accounts for the life-threatening acidosis seen in diabetic coma. From a clinical standpoint, newer studies relating to the treatment of diabetic ketoacidosis have been covered, with particular attention paid to the problems of late cerebral edema, paradoxical acidification of the cerebrospinal fluid during treatment, shifts of the oxygen dissociation curve due to 2,3-diphosphoglycerate depletion and initial hypokalemia. Recommendations for therapy designed to minmize complications are presented.

Citrate and the conversion of carbohydrate into fat. Citrate cleavage in obesity and lactation

1. The specific activity of ATP citrate lyase is two to four times as great in livers of mice with hereditary obesity as in livers of their non-obese siblings. The enzyme activity in both types of mice can be reduced by starvation, and can be increased by refeeding starved animals. The specific activity of acetyl-CoA synthetase is approximately the same in both types of mice. 2. ATP citrate lyase of mammary gland of the rat undergoes large increases in activity after the onset of lactation. It declines rapidly upon weaning. 3. The changes in activity of ATP citrate lyase in obesity and lactation are consistent with the hypothesis that the enzyme supplies extramitochondrial acetyl-CoA for fatty acid synthesis.

The fate of acetyl groups derived from glucose in the isolated perfused goat udder.

1. In the isolated perfused goat mammary gland, both glucose and acetate contribute 20-30% of carbon to citrate via acetyl-CoA. 2. Carbon dioxide does not contribute significantly to the formation of acetyl-CoA. 3. ATP citrate lyase, which, in rats, yields acetyl-CoA for fatty acid synthesis, is absent from or very low in high-speed supernatant fraction of ruminant tissue. 4. It is reasonable to infer that intramitochondrial acetyl-CoA, to which both glucose and acetate contribute, is separated from extramitochondrial acetyl-CoA to which glucose does not contribute and which is used for fatty acid synthesis.