Inhibition Of Fatty Acid Synthesis Research Articles

Abstract 2323: Involvement of glutamine in tolerance of human pancreatic cancer cell to inhibition of fatty acid synthesis

Abstract Pancreatic cancer (PC) is the fifth leading cause of cancer-related mortality in Japan. Although the standard chemotherapy regimens, FOLFIRINOX (fluorouracil, leucovorin, irinotecan and oxaliplatin), gemcitabine and nab-paclitaxel, are widely used to treat patients with advanced pancreatic cancer, their antitumor effects (progression and survival rates) are less potent against this cancer type than other solid tumors. Previously, we found that inhibition of acetyl-CoA carboxylase (ACC), a major rate-controlling enzyme of fatty acid synthetic pathway, suppressed the proliferation of PC cells. However, in this study, we found the tolerance to the inhibition in a part of PC cells. Therefore, we investigated the mechanism of the tolerance to ACC inhibition in PC cells. The human PC cell lines, AsPC-1, BxPC-3 and PANC-1 were obtained from the American Type Culture Collection. Live and apoptotic cell numbers were determined using the MUSE Annexin V and Dead Cell Kit according to the manufacturer's instructions. PANC-1 cells showed the tolerance to inhibition of fatty acid synthesis by ACC inhibitor, 5-(tetradecyloxy)-2-furoic acid (TOFA), whereas TOFA induced apoptosis in AsPC-1 and BxPC-3 cells. In addition, PANC-1 did not show autophagy even under the condition of fatty acid starvation. Next, we investigated whether PANC-1 has the tolerance to nutrient starvation in nutrient-depletion medium, Earle's Balanced Salt Solution (EBSS) and serum-free medium respectively. PANC-1 showed the survival even in such mediums, whereas AsPC-1 and BxPC-3 almost dead. This result suggests that the tolerance of fatty acid starvation of PANC-1 are related to that of nutrient starvation. Interestingly, proliferation of PANC-1 cultured in glutamine-depletion medium dramatically was suppressed, even though low glucose medium did not affect. BPTES, a selective inhibitor of glutaminase, also suppressed the proliferation and induced apoptosis in PANC-1, whereas there are little effects of 2-dexy-glucose, inhibitor of glycolysis, on them. These results suggest that the TCA cycle plays an important role on the tolerance to inhibition of fatty acid synthesis and nutrient starvation of PANC-1, and can be novel therapeutic target of PC cells which have the tolerance to nutrient starvation like PANC-1. Citation Format: Koji Nishi, Mina Suzuki, Noriko Yamamoto, Yumiko Iwase, Nagahiko Yumita. Involvement of glutamine in tolerance of human pancreatic cancer cell to inhibition of fatty acid synthesis [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 2323.

Abstract 2395: Acid-induced autophagic protein products are stored as adiposomes in breast cancer cells

Abstract Malignant tumors exhibit altered metabolism and consume higher levels of glucose compared to surrounding normal tissue, resulting in an acidic extracellular microenvironment. Adaptation to acidic conditions is a pre-requisite for tumor cells to survive and to out-compete the stroma into which they invade. Our previous studies demonstrated that acid adaptation is associated with survival mechanisms like chronic activation of autophagy and redistribution of the lysosomal proteins to the plasma membrane. When grown under acidic pH, breast cancer cells accumulate lipids as revealed by staining with Nile Red and perilipin 2, a protein that coats lipid droplets (adiposomes). Adiposomes are dynamic organelles that store neutral lipids surrounded by a shell of proteins and phospholipid monolayer. The lipids stored in adiposomes are produced de novo, as acid-induced lipogenic phenotype is maintained, even if cells are grown with de-lipidated serum. Fatty acid synthesis inhibition was selectively toxic under acidic conditions as compared to neutral pH and attenuated acid-induced adiposome accumulation. Using 13C isotopomer analysis, we observed a major shift in glucose metabolism from Embden Meyerhof to the Pentose Phosphate Pathway, resulting in increased production of NADPH, necessary for de novo lipid synthesis. To identify the carbon source of the lipid precursors in adiposomes, we employed 13C tracer analysis using [UL] 13C Glucose, 13C3 Leucine (labeled to steady-state) 13C2 Acetate and [UL] 13C glutamine as sources of lipids in the media. 13C label incorporation was determined by LC-MS/MS from adiposomes isolated from cells grown in media containing delipidated serum at pH 6.5. Label incorporation to glycerol backbone and acyl chains of lipids was observed when 13C glucose was used as the source. We could detect (M+1 ~6% of total) labeled 13C in triglycerides from adiposomes isolated from 13C3 leucine labeled MCF7, T47D and ZR75.1 cells. These data indicate that ketogenic amino acids arising from the autophagic breakdown of proteins are the major source of carbons in adiposomal lipids. Additionally, adiposome accumulation was significantly inhibited when cells were treated with autophagy inhibitors indicating that acid induced adiposomogenesis depends on autophagy. Further, we investigated the role of various acid-sensing GPCRs such as OGR1 and TDAG8 in transducing the acid signal. CRISPR/Cas9 mediated depletion of these receptors demonstrated that only OGR1 depletion abrogated acid induced adiposome accumulation and were defective in autophagy. In addition, acid-induced adiposomogenesis required PI3K and pAkt signaling. Hence, accumulation of adiposomes is a highly regulated metabolic process related to storing autophagic products, and appears to be important in cell survival under acidosis. This increased dependence on lipid metabolism reveals novel therapeutic vulnerabilities. Citation Format: Smitha R. Pillai, Jonathan W. Wojtkowiak, Jonathan Nguyen, Mehdi Damaghi, Marilyn M. Bui, Timothy Garrett, Robert J. Gillies. Acid-induced autophagic protein products are stored as adiposomes in breast cancer cells [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 2395.

De novo lipogenesis represents a therapeutic target in mutant Kras non-small cell lung cancer.

Oncogenic Kras mutations are one of the most common alterations in non-small cell lung cancer and are associated with poor response to treatment and reduced survival. Driver oncogenes, such as Kras are now appreciated for their ability to promote tumor growth via up-regulation of anabolic pathways. Therefore, we wanted to identify metabolic vulnerabilities in Kras-mutant lung cancer. Using the Kras LSL-G12D lung cancer model, we show that mutant Kras drives a lipogenic gene-expression program. Stable-isotope analysis reveals that mutant Kras promotes de novo fatty acid synthesis in vitro and in vivo. The importance of fatty acid synthesis in Kras-induced tumorigenesis was evident by decreased tumor formation in Kras LSL-G12D mice after treatment with a fatty acid synthesis inhibitor. Importantly, with gain and loss of function models of mutant Kras, we demonstrate that mutant Kras potentiates the growth inhibitory effects of several fatty acid synthesis inhibitors. These studies highlight the potential to target mutant Kras tumors by taking advantage of the lipogenic phenotype induced by mutant Kras.-Singh, A., Ruiz, C., Bhalla, K., Haley, J. A., Li, Q. K., Acquaah-Mensah, G., Montal, E., Sudini, K. R., Skoulidis, F., Wistuba, I. I., Papadimitrakopoulou, V., Heymach, J. V., Boros, L. G., Gabrielson, E., Carretero, J., Wong, K.-k., Haley, J. D., Biswal, S., Girnun, G. D. De novo lipogenesis represents a therapeutic target in mutant Kras non-small cell lung cancer.

Global metabolite profiling analysis of lipotoxicity in HER2/neu-positive breast cancer cells.

Recent work has shown that HER2/neu-positive breast cancer cells rely on a unique Warburg-like metabolism for survival and aggressive behavior. These cells are dependent on fatty acid (FA) synthesis, show markedly increased levels of stored fats and disruption of the synthetic process results in apoptosis. In this study, we used global metabolite profiling and a multi-omics network analysis approach to model the metabolic changes in this physiology under palmitate-supplemented growth conditions to gain insights into the molecular mechanism and its relevance to disease prevention and treatment. Computational analyses were used to define pathway enrichment based on the dataset of significantly altered metabolites and to integrate metabolomics and transcriptomics data in a multi-omics network analysis. Network-predicted changes and functional relationships were tested with cell assays in vitro. Palmitate-supplemented growth conditions induce distinct metabolic alterations. Growth of HER2-normal MCF7 cells is unaffected under these conditions whereas HER2/neu-positive cells display unchanged neutral lipid content, AMPK activation, inhibition of fatty acid synthesis and significantly altered glutamine, glucose and serine/glycine metabolism. The predominant upregulated lipid species is the novel bioactive lipid N-palmitoylglycine, which is non-toxic to these cells. Limiting the availability of glutamine significantly ameliorates the lipotoxic effects of palmitate, reduces CHOP and XBP1(s) induction and restores the expression levels of HER2 and HER3. The study shows that HER2/neu-positive breast cancer cells change their metabolic phenotype in the presence of palmitate. Palmitate induces AMPK activation and inhibition of fatty acid synthesis that feeds back into glycolysis as well as anaplerotic glutamine metabolism.

Staphylococcus aureus Utilizes Host-Derived Lipoprotein Particles as Sources of Fatty Acids.

Methicillin-resistant Staphylococcus aureus (MRSA) is a threat to global health. Consequently, much effort has focused on the development of new antimicrobials that target novel aspects of S. aureus physiology. Fatty acids are required to maintain cell viability, and bacteria synthesize fatty acids using the type II fatty acid synthesis (FASII) pathway. FASII is significantly different from human fatty acid synthesis, underscoring the therapeutic potential of inhibiting this pathway. However, many Gram-positive pathogens incorporate exogenous fatty acids, bypassing FASII inhibition and leaving the clinical potential of FASII inhibitors uncertain. Importantly, the source(s) of fatty acids available to pathogens within the host environment remains unclear. Fatty acids are transported throughout the body by lipoprotein particles in the form of triglycerides and esterified cholesterol. Thus, lipoproteins, such as low-density lipoprotein (LDL), represent a potentially rich source of exogenous fatty acids for S. aureus during infection. We sought to test the ability of LDLs to serve as a fatty acid source for S. aureus and show that cells cultured in the presence of human LDLs demonstrate increased tolerance to the FASII inhibitor triclosan. Using mass spectrometry, we observed that host-derived fatty acids present in the LDLs are incorporated into the staphylococcal membrane and that tolerance to triclosan is facilitated by the fatty acid kinase A, FakA, and Geh, a triacylglycerol lipase. Finally, we demonstrate that human LDLs support the growth of S. aureus fatty acid auxotrophs. Together, these results suggest that human lipoprotein particles are a viable source of exogenous fatty acids for S. aureus during infection.IMPORTANCE Inhibition of bacterial fatty acid synthesis is a promising approach to combating infections caused by S. aureus and other human pathogens. However, S. aureus incorporates exogenous fatty acids into its phospholipid bilayer. Therefore, the clinical utility of targeting bacterial fatty acid synthesis is debated. Moreover, the fatty acid reservoir(s) exploited by S. aureus is not well understood. Human low-density lipoprotein particles represent a particularly abundant in vivo source of fatty acids and are present in tissues that S. aureus colonizes. Herein, we establish that S. aureus is capable of utilizing the fatty acids present in low-density lipoproteins to bypass both chemical and genetic inhibition of fatty acid synthesis. These findings imply that S. aureus targets LDLs as a source of fatty acids during pathogenesis.

Early Inhibition of Fatty Acid Synthesis Reduces Generation of Memory Precursor Effector T cells in Chronic Infection

Abstract Understanding the mechanisms of CD4 memory T cell (Tmem) differentiation in malaria is critical for vaccine development. However, the metabolic regulation of CD4 Tmem differentiation is not clear, particularly in persistent infections. In this study, we investigated the role of fatty acid synthesis (FAS) in Tmem development in Plasmodium chabaudi chronic mouse malaria infection. We show that T cell-specific deletion, and early pharmaceutical inhibition of acetyl coA carboxylase 1, the rate limiting step of FAS, inhibit generation of early memory precursor effector T cells (MPEC). To compare the role of FAS during early differentiation or survival of Tmem in chronic infection, a specific inhibitor of acetyl coA carboxylase 1, 5-(Tetradecyloxy)-2-furoic acid, was administered at different times postinfection. Strikingly, the number of Tmem was only reduced when FAS was inhibited during T cell priming, but not during the Tmem survival phase. FAS inhibition during priming increased effector T cells (Teff) proliferation, and strongly decreased peak parasitemia, which is consistent with improved Teff function. Conversely, MPEC were decreased, in a T cell-intrinsic manner, upon early FAS inhibition in chronic, but not acute, infection. Early cure of infection also increased mitochondrial volume in Tmem compared to Teff, supporting previous reports in acute infection. We demonstrate that the MPEC-specific effect was due to the higher fatty acid content and synthesis in MPEC compared to terminally differentiated Teff. In conclusion, FAS in CD4 T cells regulates the early divergence of Tmem from Teff in chronic infection.

Prostate cancer chemoprevention by sulforaphane in a preclinical mouse model is associated with inhibition of fatty acid metabolism.

Increased de novo synthesis of fatty acids is a rather unique and targetable mechanism of human prostate cancer. We have shown previously that oral administration of sulforaphane (SFN) significantly inhibits the incidence and/or burden of prostatic intraepithelial neoplasia and well-differentiated adenocarcinoma in TRansgenic Adenocarcinoma of Mouse Prostate (TRAMP) mice. The present study used cellular models of prostate cancer and archived plasma/adenocarcinoma tissues and sections from the TRAMP study to demonstrate inhibition of fatty acid synthesis by SFN treatment in vitro and in vivo. Treatment of androgen-responsive (LNCaP) and castration-resistant (22Rv1) human prostate cancer cells with SFN (5 and 10 μM) resulted in downregulation of protein and mRNA levels of acetyl-CoA carboxylase 1 (ACC1) and fatty acid synthase (FASN), but not ATP citrate lyase. Protein and mRNA levels of carnitine palmitoyltransferase 1A (CPT1A), which facilitates fatty acid uptake by mitochondria for β-oxidation, were also decreased following SFN treatment in both cell lines. Immunohistochemistry revealed a significant decrease in expression of FASN and ACC1 proteins in prostate adenocarcinoma sections of SFN-treated TRAMP mice when compared with controls. SFN administration to TRAMP mice resulted in a significant decrease in plasma and/or prostate adenocarcinoma levels of total free fatty acids, total phospholipids, acetyl-CoA and ATP. Consistent with these results, number of neutral lipid droplets was lower in the prostate adenocarcinoma sections of SFN-treated TRAMP mice than in control tumors. Collectively, these observations indicate that prostate cancer chemoprevention by SFN in TRAMP mice is associated with inhibition of fatty acid metabolism.

Metabonomics Indicates Inhibition of Fatty Acid Synthesis, β-Oxidation, and Tricarboxylic Acid Cycle in Triclocarban-Induced Cardiac Metabolic Alterations in Male Mice

Triclocarban (TCC) has been identified as a new environmental pollutant that is potentially hazardous to human health; however, the effects of short-term TCC exposure on cardiac function are not known. The aim of this study was to use metabonomics and molecular biology techniques to systematically elucidate the molecular mechanisms of TCC-induced effects on cardiac function in mice. Our results show that TCC inhibited the uptake, synthesis, and oxidation of fatty acids, suppressed the tricarboxylic acid (TCA) cycle, and increased aerobic glycolysis levels in heart tissue after short-term TCC exposure. TCC also inhibited the nuclear peroxisome proliferator-activated receptor α (PPARα), confirming its inhibitory effects on fatty acid uptake and oxidation. Histopathology and other analyses further confirm that TCC altered mouse cardiac physiology and pathology, ultimately affecting normal cardiac metabolic function. We elucidate the molecular mechanisms of TCC-induced harmful effects on mouse cardiac metabolism and function from a new perspective, using metabonomics and bioinformatics analysis data.

Betaine Supplementation Enhances Lipid Metabolism and Improves Insulin Resistance in Mice Fed a High-Fat Diet.

Obesity is a major driver of metabolic diseases such as nonalcoholic fatty liver disease, certain cancers, and insulin resistance. However, there are no effective drugs to treat obesity. Betaine is a nontoxic, chemically stable and naturally occurring molecule. This study shows that dietary betaine supplementation significantly inhibits the white fat production in a high-fat diet (HFD)-induced obese mice. This might be due to betaine preventing the formation of new white fat (WAT), and guiding the original WAT to burn through stimulated mitochondrial biogenesis and promoting browning of WAT. Furthermore, dietary betaine supplementation decreases intramyocellular lipid accumulation in HFD-induced obese mice. Further analysis shows that betaine supplementation reduced intramyocellular lipid accumulation might be associated with increasing polyunsaturated fatty acids (PUFA), fatty acid oxidation, and the inhibition of fatty acid synthesis in muscle. Notably, by performing insulin-tolerance tests (ITTs) and glucose-tolerance tests (GTTs), dietary betaine supplementation could be observed for improvement of obesity and non-obesity induced insulin resistance. Together, these findings could suggest that inhibiting WAT production, intramyocellular lipid accumulation and inflammation, betaine supplementation limits HFD-induced obesity and improves insulin resistance.

Early Inhibition of Fatty Acid Synthesis Reduces Generation of Memory Precursor Effector T Cells in Chronic Infection.

Understanding the mechanisms of CD4 memory T cell (Tmem) differentiation in malaria is critical for vaccine development. However, the metabolic regulation of CD4 Tmem differentiation is not clear, particularly in persistent infections. In this study, we investigated the role of fatty acid synthesis (FAS) in Tmem development in Plasmodium chabaudi chronic mouse malaria infection. We show that T cell-specific deletion and early pharmaceutical inhibition of acetyl CoA carboxylase 1, the rate limiting step of FAS, inhibit generation of early memory precursor effector T cells (MPEC). To compare the role of FAS during early differentiation or survival of Tmem in chronic infection, a specific inhibitor of acetyl CoA carboxylase 1, 5-(tetradecyloxy)-2-furoic acid, was administered at different times postinfection. Strikingly, the number of Tmem was only reduced when FAS was inhibited during T cell priming and not during the Tmem survival phase. FAS inhibition during priming increased effector T cell (Teff) proliferation and strongly decreased peak parasitemia, which is consistent with improved Teff function. Conversely, MPEC were decreased, in a T cell-intrinsic manner, upon early FAS inhibition in chronic, but not acute, infection. Early cure of infection also increased mitochondrial volume in Tmem compared with Teff, supporting previous reports in acute infection. We demonstrate that the MPEC-specific effect was due to the higher fatty acid content and synthesis in MPEC compared with terminally differentiated Teff. In conclusion, FAS in CD4 T cells regulates the early divergence of Tmem from Teff in chronic infection.

Using [2H]water to quantify the contribution of de novo palmitate synthesis in plasma: enabling back-to-back studies.

An increased contribution of de novo lipogenesis (DNL) may play a role in cases of dyslipidemia and adipose accretion; this suggests that inhibition of fatty acid synthesis may affect clinical phenotypes. Since it is not clear whether modulation of one step in the lipogenic pathway is more important than another, the use of tracer methods can provide a deeper level of insight regarding the control of metabolic activity. Although [2H]water is generally considered a reliable tracer for quantifying DNL in vivo (it yields a homogenous and quantifiable precursor labeling), the relatively long half-life of body water is thought to limit the ability of performing repeat studies in the same subjects; this can create a bottleneck in the development and evaluation of novel therapeutics for inhibiting DNL. Herein, we demonstrate the ability to perform back-to-back studies of DNL using [2H]water. However, this work uncovered special circumstances that affect the data interpretation, i.e., it is possible to obtain seemingly negative values for DNL. Using a rodent model, we have identified a physiological mechanism that explains the data. We show that one can use [2H]water to test inhibitors of DNL by performing back-to-back studies in higher species [i.e., treat nonhuman primates with platensimycin, an inhibitor of fatty acid synthase]; studies also demonstrate the unsuitability of [13C]acetate.

Abstract B142: The anticancer agent Dp44mT upregulates the AMPK-dependent energy homeostasis pathway in cancer cells

Abstract Background: Iron plays a crucial role in cellular energy transducing pathways and tumor cell proliferation. Therefore, metals (such as iron) could play an important role in the regulation of the AMPK-dependent pathway regulating cellular energy homeostasis. This investigation examined the effect of the iron and copper chelator and potent anticancer agent, di-2-pyridylketone 4,4-dimethyl-3-thiosemicarbazone (Dp44mT), on AMPK-mediated energy homeostasis. Methods and Results: These studies demonstrated that Dp44mT, which forms intracellular redox-active complexes with iron and copper, significantly activated AMPK (i.e., p-AMPK/AMPK ratio) in 5 different tumor cell-types. Furthermore, examination of the Dp44mT-metal complexes demonstrated that the effect of Dp44mT on AMPK was due to a dual mechanism: (1) its ability to chelate metal ion; and (2) the generation of reactive oxygen species (ROS). The activation of the AMPK pathway by Dp44mT was mediated by the upstream kinase, liver kinase B1 (LKB1), which is a known tumor suppressor. Moreover, using AMPKα1-selective silencing, we demonstrated that Dp44mT activated AMPK, resulting in inhibition of acetyl CoA carboxylase 1 (ACC1) and raptor, and activation of Unc-51 like kinase (ULK1). Conclusions: These effects are vital for inhibition of fatty acid synthesis, suppression of protein synthesis, and autophagic activation, respectively. Together, this AMPK-mediated repair response aims to rescue the loss of metal ions via chelation and the induction of cytotoxic damage mediated by redox cycling of the Dp44mT-metal ion complex. In conclusion, this study demonstrates for the first time that chelators target the AMPK-dependent energy homeostasis pathway. Citation Format: Sukriti Krishan, Sumit Sahni, Des R. Richardson. The anticancer agent Dp44mT upregulates the AMPK-dependent energy homeostasis pathway in cancer cells [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr B142.

Metabolomics reveals the mechanism of (−)-hydroxycitric acid promotion of protein synthesis and inhibition of fatty acid synthesis in broiler chickens

(−)-Hydroxycitric acid (HCA), a major component of Garcinia cambogia extracts, has been shown to suppress BW gain and fat accumulation in animals and humans. However, the mechanism remains unknown. In this study, gas chromatography-mass spectrometry was used to analyse serum metabolites, and principal component analysis and partial least-squares-discriminant analysis models were generated to analyse serum metabolite changes in broiler chickens after the administration of (−)-HCA at 0, 1000, 2000 and 3000 mg/kg diets for 28 days. Metabolites showing significant changes were screened by ‘variable importance in the projection’ plots. The results showed that 20 metabolites in the 1000 mg/kg (−)-HCA treatment group and 16 metabolites in 3000 mg/kg (−)-HCA treatment group were significantly altered. Metabolites pathway enrichment analysis indicated that these metabolites were mainly associated with metabolism of amino acids, protein synthesis, citric acid cycle, and uric acid and fatty acid synthesis. The data indicated that (−)-HCA promoted protein synthesis by regulating the metabolic directions of amino acids. At the same time, (−)-HCA treatment inhibited fatty acid synthesis by promoting the citric acid cycle, resulting in reduced cytosolic acetyl-CoA content in broiler chickens. The present study identified global changes in metabolites and analysed the main canonical metabolic pathways in broiler chickens supplemented with (−)-HCA. These results will deepen our understanding of the mechanism of (−)-HCA’s effects in animals.

Berberine inhibits glucose oxidation and insulin secretion in rat islets.

Glucose promotes insulin secretion primarily via its metabolism and the production of metabolic coupling factors in beta-cells. The activation of AMP-activated protein kinase (AMPK), an energy sensor, results in a decrease in insulin secretion from beta-cells, but its mechanism remains largely unknown. Berberine, an oral anti-diabetic drug, has been shown to activate AMPK in multiple peripheral tissues. Here, we examined the effects of berberine and AMPK activation on insulin secretion and glucose oxidation in rat islets. Our results showed that berberine inhibited glucose-stimulated insulin secretion from rat islets with AMPK activation. When glucose concentration was elevated to 25 mmol/L, the inhibitory action of berberine on insulin secretion disappeared. Furthermore, berberine significantly decreased oxygen consumption rate (OCR) and ATP production induced by high glucose in rat islets. Although adenovirus-mediated overexpression of constituent-activated AMPK markedly decreased GSIS and OCR in rat islets, the inhibition of AMPK by compound C did not reverse berberine-suppressed OCR. In addition, berberine attenuated glucose-stimulated expression of fatty acid synthase. These results indicate that berberine-mediated deceleration of glucose oxidation is tightly link to the decreased insulin secretion in islets independent of AMPK activation and inhibition of fatty acid synthesis may also contribute to the effect of berberine on insulin secretion.

Multiple Roles of Sms2 in White and Brown Adipose Tissues from Dietinduced Obese Mice

Background/Objectives: Adipose tissue (AT) has an important role in energy homeostasis. The dysfunction of AT or the hyper-accumulation of neutral lipids leads to various metabolic diseases. Recent studies indicate that sphingo lipid metabolism associates with the development of metabolic diseases. Sphingomyelin, a major sphingolipid in mammals, requires sphingomyelin synthase (SMS) for biosynthesis. Previously, we reported that Sms2 deficiency inhibited diet-induced obesity, fatty liver, and insulin resistance in mice. However, the contribution of Sms2 to obesity and insulin resistance in AT is largely unknown. In this study, we investigated whether Sms2 deficiency in ATs affects obesity and insulin resistance.Subjects/Methods: Wild-type and Sms2 knockout (KO) mice were fed a high-fat for 12 weeks. Body and AT weights, and the food intake, were recorded. The AT status and macrophage infiltration were evaluated by histological analysis. The expression levels of genes and proteins involved in adipogenesis, inflammation, energy expenditure, and fatty acid metabolism were examined.Results: In white adipose tissue (WAT) from Sms2 KO mice, the number of small adipocytes increased but the adipocyte size decreased. In epididymal WAT, Sms2 deficiency inhibited inflammation and macrophage infiltration. Moreover, adipogenesis was moderately suppressed. In subcutaneous WAT from Sms2 KO mice, the expression of genes involved in energy expenditure and browning (Ucp1, Cidea, Tbx1) was elevated. In brown adipose tissue (BAT) from Sms2 KO mice, the lipid droplet surface area was lower than that of WT mice and the expression of genes involved in fatty acid synthesis (Fasn, Scd1) decreased.Conclusion: These results demonstrate that Sms2 deficiency leads to moderate adipogenesis and inflammatory suppression in epididymal WAT, increased energy expenditure by the browning of subcutaneous WAT, and suppression of fatty acid synthesis in BAT, suggesting that these synergetic effects in ATs from Sms2 KO mice contribute to the suppression of diet-induced obesity and insulin resistance.

Trans10, cis12 conjugated linoleic acid increases triacylglycerol accumulation in goat mammary epithelial cells invitro.

Trans10, cis12 conjugated linoleic acid (t10c12-CLA) is well-established in decreasing milk fat content and causing milk fat depression (MFD) in dairy cattle and goats. However, the detailed mechanisms of its effect are not completely understood. Therefore, we used goat mammary epithelial cells (GMECs) to further study the molecular mechanisms whereby t10c12-CLA regulates milk fat synthesis. The optimal concentration of t10c12-CLA (100μmol/L) for cell culture was determined through a cell vitality and morphology assay, and evaluation of abundance of apoptosis-related proteins. Oil red O stain indicated that t10c12-CLA increased concentration of cytoplasmic lipid droplets. Furthermore, t10c12-CLA increased the intracellular triacylglycerol (TG) content (P<0.05). Among 16 genes related to lipid metabolism that were measured by quantitative real-time PCR, t10c12-CLA down-regulated (P<0.05) genes involved in de novo fatty acid synthesis including FASN, ACACA and SCD1, and also down-regulated the protein expression of FASN and SCD1 but up-regulated (P<0.05) the expression of CD36 and ADRP. Overall, the data indicate that a side effect of de novo fatty acid synthesis inhibition by t10c12-CLA is the up-regulation of fatty acid uptake and accumulation of lipid droplets in GMECs. The biologic reason for such an effect merits further study.

Effect of dietary betaine on growth performance, antioxidant capacity and lipid metabolism in blunt snout bream fed a high-fat diet.

An 8-week feeding experiment was conducted to determine the effect of dietary betaine levels on the growth performance, antioxidant capacity, and lipid metabolism in high-fat diet-fed blunt snout bream (Megalobrama amblycephala) with initial body weight 4.3±0.1g [mean±SEM]. Five practical diets were formulated to contain normal-fat diet (NFD), high-fat diet (HFD), and high-fat diet with betaine addition (HFB) at difference levels (0.6, 1.2, 1.8%), respectively. The results showed that the highest final body weight (FBW), weight gain ratio (WGR), specific growth rate (SGR), condition factor (CF), and feed intake (FI) (P<0.05) were obtained in fish fed 1.2% betaine supplementation, whereas feed conversion ratio (FCR) was significantly lower in the same group compared to others. Hepatosomatic index (HSI) and abdominal fat rate (AFR) were significantly high in fat group compared to the lowest in NDF and 1.2% betaine supplementation, while VSI and survival rate (SR) were not affected by dietary betaine supplementation. Significantly higher (P<0.05), plasma total cholesterol (TC), triglycerides (TG), low-density lipoprotein (LDL), aspartate transaminase (AST), alanine transaminase (ALT), cortisol, and lower high-density lipoprotein (HDL) content were observed in HFD but were improved when supplemented with 1.2% betaine. In addition, increase in superoxide dismutase (SOD), catalase (CAT), and reduced glutathione (GSH) in 1.2% betaine inclusion could reverse the increasing malondialdehyde (MDA) level induced by HFD. Based on the second-order polynomial analysis, the optimum growth of blunt snout bream was observed in fish fed HFD supplemented with 1.2% betaine. HFD upregulated fatty acid synthase messenger RNA (mRNA) expression and downregulated carnitine palmitoyltransferase 1, peroxisome proliferator-activated receptor α, and microsomal triglyceride transfer protein mRNA expression; nevertheless, 1.2% betaine supplementation significantly reversed these HFD-induced effects, implying suppression of fatty acid synthesis, β-oxidation, and lipid transport. This present study indicated that inclusion of betaine (1.2%) can significantly improve growth performance and antioxidant defenses, as well as reduce fatty acid synthesis and enhance mitochondrial β-oxidation and lipid transportation in high-fat diet-fed blunt snout bream, thus effectively alleviating fat accumulation in the liver by changing lipid metabolism.

Andrographolide Suppresses MV4-11 Cell Proliferation through the Inhibition of FLT3 Signaling, Fatty Acid Synthesis and Cellular Iron Uptake.

Background: Andrographolide (ADR), the main active component of Andrographis paniculata, displays anticancer activity in various cancer cell lines, among which leukemia cell lines exhibit the highest sensitivity to ADR. In particular, ADR was also reported to have reduced drug resistance in multidrug resistant cell lines. However, the mechanism of action (MOA) of ADR’s anticancer and anti-drug-resistance activities remain elusive. Methods: In this study, we used the MV4-11 cell line, a FLT3 positive acute myeloid leukemia (AML) cell line that displays multidrug resistance, as our experimental system. We first evaluated the effect of ADR on MV4-11 cell proliferation. Then, a quantitative proteomics approach was applied to identify differentially expressed proteins in ADR-treated MV4-11 cells. Finally, cellular processes and signal pathways affected by ADR in MV4-11 cell were predicted with proteomic analysis and validated with in vitro assays. Results: ADR inhibits MV4-11 cell proliferation in a dose- and time-dependent manner. With a proteomic approach, we discovered that ADR inhibited fatty acid synthesis, cellular iron uptake and FLT3 signaling pathway in MV4-11 cells. Conclusions: ADR inhibits MV4-11 cell proliferation through inhibition of fatty acid synthesis, iron uptake and protein synthesis. Furthermore, ADR reduces drug resistance by blocking FLT3 signaling.

Artesunate Activates the Intrinsic Apoptosis of HCT116 Cells through the Suppression of Fatty Acid Synthesis and the NF-κB Pathway.

The artemisinin compounds, which are well-known for their potent therapeutic antimalarial activity, possess in vivo and in vitro antitumor effects. Although the anticancer effect of artemisinin compounds has been extensively reported, the precise mechanisms underlying its cytotoxicity remain under intensive study. In the present study, a high-throughput quantitative proteomics approach was applied to identify differentially expressed proteins of HCT116 colorectal cancer cell line with artesunate (ART) treatment. Through Ingenuity Pathway Analysis, we discovered that the top-ranked ART-regulated biological pathways are abrogation of fatty acid biosynthetic pathway and mitochondrial dysfunction. Subsequent assays showed that ART inhibits HCT116 cell proliferation through suppressing the fatty acid biosynthetic pathway and activating the mitochondrial apoptosis pathway. In addition, ART also regulates several proteins that are involved in NF-κB pathway, and our subsequent assays showed that ART suppresses the NF-κB pathway. These proteomic findings will contribute to improving our understanding of the underlying molecular mechanisms of ART for its therapeutic cytotoxic effect towards cancer cells.

Epe1 contributes to activation of AMPK by promoting phosphorylation of AMPK alpha subunit, Ssp2

AMP-activated protein kinase (AMPK) is a pivotal cellular energy sensor. It is activated by stresses that cause depletion of energy and initiates adaptive responses by regulating metabolism balance. AMPK forms αβγ heterotrimer. In fission yeast, activation of AMPK mainly depends on the phosphorylation of AMPKα subunit Ssp2 at Thr189 by upstream kinase Ssp1. However, not much is known about the regulation of this process. In this study, we identified Epe1 as a novel positive regulator of AMPK. Epe1, a jmjC-domain-containing protein, is best-known as a negative regulator of heterochromatin spreading. Although the novel role of Epe1 in regulation of AMPK relies on predicted iron- and 2-oxyglutarate-binding residues inside jmjC domain, it seems to be irrelevant to inhibition of heterochromatin spreading. Epe1 is associated with Ssp2 directly and promotes phosphorylation of Ssp2 upon various environmental stresses, including low-glucose, high-sodium, high-pH and oxidative conditions. Similar to Epe1, Jmj1 and Msc1 also contribute to phosphorylation of Ssp2. Deletion of epe1+ impairs downstream events following phosphorylation of Ssp2, including nuclear translocation of Ssp2, sexual differentiation and inhibition of fatty acid synthesis. Our study reveals a novel way in which a jmjC-domain-containing protein regulates adaptive response by directly binding to a principal sensor.