Inhibitor Of Carnitine Palmitoyltransferase Research Articles

Suppression of fatty acid β-oxidation and energy deficiency as a cause of inhibitory effect of E. coli lipopolysaccharide on osmotic water transport in the frog urinary bladder

Previously we showed that arginine-vasotocin (AVT)-stimulated osmotic water permeability (OWP) of the frog urinary bladder was decreased if the mucosal side of the bladder has been naturally colonized by Gram-negative bacteria, or if bacterial lipopolysaccharide (LPS) was introduced into the lumen of the isolated bladder (J. Exp. Zool., 2013, 319, 487–494). Taking into account that in different tissues and cell types, challenge with LPS causes significant metabolic shift and energy deficiency, we hypothesized that an LPS-induced decrease of AVT-stimulated OWP could depend on the reduction of fatty acid oxidation (FAO), which is important for generation of ATP in epithelia. Using an isolated frog Rana temporaria urinary bladder we showed that the AVT-induced increase of OWP did not depend on the external glucose, but was inhibited by oligomycin, an ATP-synthase inhibitor, and by etomoxir, an inhibitor of carnitine palmitoyltransferase-1. In primary cultured epithelial cells isolated from the bladder mucosa, LPS E. coli (25 μg/ml, 21 h), as well as etomoxir (100 μM), decreased FAO accompanied by triacylglycerol accumulation. Both drugs impaired mitochondrial functions demonstrated by decreased ATP production and a reduced maximal oxygen consumption rate (OCR) and OCR directed at ATP synthesis. Additionally, we found that LPS decreased the expression of peroxisome proliferator-activated receptor alpha, a key player in the regulation of FAO. These data indicate that the impairment of AVT-induced water transport in osmoregulatory epithelium caused by LPS depends at least partly on defects in FAO and FAO-dependent energy production.

Различие влияния липополисахарида E. coli , олеиновой кислоты и этомоксира на экспрессию белка ADRP в эпителиальных клетках

Intracellularly neutral lipids are deposited in lipid bodies (LB) – special organelles composed of triglyceride or cholesterol esters core surrounded by a phospholipid monolayer. LBs play a central role in cellular lipid metabolism. The biogenesis of LB and consumption of neutral lipids are controlled by proteins of the PAT family, which are expressed on the surface of LB and regulate lipids storage and degradation providing the interaction of LB with mitochondria and access of lipases to their substrates. The ADRP (adipose differentiation-related protein), a member of the PAT family, is a quantitatively major LB surface protein expressed in many cell types. The goal of the present work was to study the involvement of ADRP in the formation of LB and intracellular TAG accumulation under the action of different stimuli –lipopolysaccharide (LPS), exogenous oleic acid and etomoxir, an inhibitor of carnitine-palmitoyltransferase 1. The experiments were performed on epithelial cells isolated from the mucosal surface of the frog urinary bladder. Incubation of the cells for 21 hours with E. coli LPS (25 μg / ml), oleic acid (50 μM) and etomoxir (100 μM) led to a significant increase in the number and size of LBs and an increase in intracellular TAG accumulation. Despite the fact that all the substances used effectively stimulated LB growth, only incubation with LPS was accompanied by a sharp increase in the expression of ADRP protein, whereas in the presence of etomoxir or oleic acid the expression of this protein did not change. The obtained results indicate that the molecular mechanisms underlying the increase in LB formation and TAG accumulation under the action of different stimuli in the same cell type can be differed in relation to the involvement of LB surface proteins.

AMPK activating and anti adipogenic potential of Hibiscus rosa sinensis flower in 3T3-L1 cells

Ethnopharmacological relevanceThe flowers of Hibiscus rosa sinensis has array of pharmacological actions. They are used in preparation of herbal decoction and teas, which have been used traditionally to reduce body weight and for its effect on metabolic syndrome. Aim of the studyTo investigate the anti adipogenic efficacy of major fraction from ethyl acetate extract of the Hibiscus rosa sinensis flower at 25 and 50 µg/mL (HRF 25 and 50 µg/mL) in 3T3-L1 cells and delineate its possible mechanism of action. Materials and methodsPre adipocyte 3T3-L1 cells were differentiated in the presence and absence of HRF 25 and 50 µg/mL, their lipid accumulation was measured qualitatively by Oil red O staining and quantitatively by triglyceride estimation. Effect on adipolysis was determined, adipogenic and its regulatory gene and protein expression were studied and effect of HRF 25 and 50 µg/mL on AMPK was confirmed in the presence of dorsomorphin. ResultsTreatment with HRF 25 and 50 µg/mL activated AMP-activated protein kinase (AMPK) and was found to alleviate triglyceride accumulation significantly (p < 0.001) by 1.6 and 2.3 times respectively in pre adipocytes during differentiation. HRF 25 and 50 µg/mL also nonsignificantly reduced lipolysis which releases free fatty acids, a major contributing factor for insulin resistance. Activation of AMPK by phosphorylation has led to reduced gene and protein expression of adipogenic factors Peroxisome proliferator- activated receptor gamma (PPAR-γ), CCAT/enhancer binding protein alpha (C/EBPα), Sterol regulatory element- binding protein-1c (SREBP-1c) and their targets Fatty acid binding protein 4 (FABP4), Fatty acid synthase (FAS), Perilipin and enhanced Adiponectin expression. Treatment with HRF 25 and 50 µg/mL also resulted in inactivation of Acetyl-CoA carboxylase (ACC) by enhancing ACC phosphorylation, which reduced the levels of malonyl-CoA an allosteric inhibitor of carnitine palmitoyl transferase 1 (CPT1). Enhanced CPT1 levels causes induction of fatty acid β- oxidation. Effects of HRF were nullified in the presence of AMPK antagonist dorsomorphin. ConclusionIn summary, HRF treatments reduced adipogenesis, enhanced factors regulating fatty acid oxidation and this is mediated by AMPK activation. The results conclusively showed anti-obesity potential of HRF and it might be helpful in treatment of associated complications.

Noninvasive evaluation of fat-carbohydrate metabolic switching in heart and contracting skeletal muscle.

The ability of heart and skeletal muscle (SM) to switch between fat and carbohydrate oxidation is of high interest in the study of metabolic diseases and exercise physiology. Positron emission tomography (PET) imaging with the glucose analog 2-[18F]fluoro-2-deoxy-glucose (18F-FDG) provides a noninvasive means to quantitate glucose metabolic rates. However, evaluation of fatty acid oxidation (FAO) rates by PET has been limited by the lack of a suitable FAO probe. We have developed a metabolically trapped oleate analog, ( Z)-18-[18F]fluoro-4-thia-octadec-9-enoate (18F-FTO), and investigated the feasibility of using 18F-FTO and 18F-FDG to measure FAO and glucose uptake, respectively, in heart and SM of rats in vivo. To enhance the metabolic rates in SM, the vastus lateralis (VL) muscle was electrically stimulated in fasted rats for 30 min before and 30 min following radiotracer injection. The responses of radiotracer uptake patterns to pharmacological inhibition of FAO were assessed by pretreatment of the rats with the carnitine palmitoyl-transferase-1 (CPT-1) inhibitor sodium 2-[5-(4-chlorophenyl)-pentyl]oxirane-2-carboxylate (POCA). Small-animal PET images and biodistribution data with 18F-FTO and 18F-FDG demonstrated profound metabolic switching for energy provision in the myocardium from exogenous fatty acids to glucose in control and CPT-1-inhibited rats, respectively. Uptake of both radiotracers was low in unstimulated SM. In stimulated VL muscle, 18F-FTO and 18F-FDG uptakes were increased 4.4- and 28-fold, respectively, and CPT-1 inhibition only affected 18F-FTO uptake (66% decrease). 18F-FTO is a FAO-dependent PET probe that may allow assessment of energy substrate metabolic switching in conjunction with 18F-FDG and other metabolic probes.

Abstract LB-249: HDAC11 regulates lysine acetylation of enolase 1

Abstract Therapeutic molecules targeting the activity of histone deacetylases (HDACs) are currently under investigation for the treatment of several malignancies. There are currently eighteen human HDACs and while histone deacetylation is associated with transcriptional repression, acetylated lysine targets are functionally diverse and include cytoplasmic, nuclear, and mitochondrial proteins. Here, we used a new class of novel small molecule inhibitors that are highly selective for HDAC11 to identify its role in the regulation of non-histone proteins. Stable isotope labeling with amino acids in cell culture (SILAC) followed by mass spectrometry in the presence of HDAC11 selective inhibitors identified proteins with acetylation and/or expression changes after treatment and were compared to known HDAC substrates to establish a unique set of putative HDAC11 target proteins. Metabolic processes were highly enriched in this data set. Specifically, acetylated enolase 1 (ENO1, 2-phospho-D-glycerate hydrolase) which catalyzes the conversion of 2-phosphoglycerate to phosphoenolpyruvate (PEP) in the glycolytic pathway was highly altered after HDAC11 inhibition. Using acetylated lysine specific immunoprecipitation, we validated the hyperacetylated state of ENO1 upon HDAC11 inhibition. Functional assays confirmed that the HDAC11 inhibition lowered ENO1-mediated PEP production, and reduced proliferation and viability of hematopoietic and solid tumor cells. Similar observations were obtained in HDAC11 knock down cell lines confirming that HDAC11 is a required molecule in the regulation of ENO1-mediated metabolic regulation. The proteomics data also mapped three distinct target lysine residues of HDAC11 in ENO1 and each of these residues were substituted to either an acetylated or an un-acetylated lysine mimic to test their function in ENO1 activity and stability. We confirmed that K335 is the major target site of HDAC11 and its substitution to the acetylated mimic (glutamine) causes loss of enolase activity. Concomitantly, using proton nuclear magnetic resonance spectroscopy we identified some glycolytic intermediates upstream of ENO1 to be increased and downstream intermediates quantitatively reduced after HDAC11 inhibition suggesting that glycolysis is functionally suppressed. Glycolytic pathway disruption was associated with a compensatory increase in oxygen consumption and ATP production through oxidative phosphorylation in these oncogene transformed tumor cells, but not in their non-transformed counterparts. Suppression of fatty acid oxidation by inhibiting carnitine palmitoyltransferase 1 (CPT-1) or blocking glutamine utilization by inhibiting glutaminase (GLS1) in combination with HDAC11 inhibition resulted in a cooperative reduction in cellular ATP levels further supporting a direct role of HDAC11 in regulating glycolysis in tumor cells. For the first time, this study mechanistically and functionally defines a cytoplasmic non-histone protein regulated by HDAC11. Citation Format: Vasundhara Sharma, Agni Christodoulidou, Lanzhu Yue, Aileen Y. Alontaga, William E. Goodheart, Rebecca Hesterberg, Xiaozhang Zheng, Matthew W. Martin, Jennifer Y. Lee, Pearlie K. Burnette, Kenneth L. Wright. HDAC11 regulates lysine acetylation of enolase 1 [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 LB-249.

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.

Etomoxir Inhibits Macrophage Polarization by Disrupting CoA Homeostasis

Long-chain fatty acid (LCFA) oxidation has been shown to play an important role in interleukin-4 (IL-4)-mediated macrophage polarization (M(IL-4)). However, many of these conclusions are based on the inhibition of carnitine palmitoyltransferase-1 with high concentrations of etomoxir that far exceed what is required to inhibit enzyme activity (EC90< 3μM). We employ genetic and pharmacologic models to demonstrate that LCFA oxidation is largely dispensable for IL-4-driven polarization. Unexpectedly, high concentrations of etomoxir retained the ability to disrupt M(IL-4) polarization in the absence of Cpt1a or Cpt2 expression. Although excess etomoxir inhibits the adenine nucleotide translocase, oxidative phosphorylation is surprisingly dispensable for M(IL-4). Instead, the block in polarization was traced to depletion of intracellular free coenzymeA (CoA), likely resulting from conversion of the pro-drug etomoxir into active etomoxiryl CoA. These studies help explain the effect(s) of excess etomoxir on immune cells and reveal an unappreciated role for CoA metabolism in macrophage polarization.

\u03b2-Oxidation in ghrelin-producing cells is important for ghrelin acyl-modification

Ghrelin is a unique fatty acid-modified peptide hormone produced in the stomach and has important roles in energy homeostasis and gastrointestinal motility. However, the medium-chain fatty acid source for ghrelin acyl-modification is not known. We found that a fat-free diet and the removal of intestinal microbiota did not decrease acyl-ghrelin production in the stomach or plasma acyl-ghrelin levels in mice. RT-PCR analysis showed that genes involving fatty acid synthesis, metabolism, and transport were expressed in pancreas-derived ghrelinoma (PG-1) cells. Treatment with an irreversible inhibitor of carnitine palmitoyltransferase-1 (CPT-1) strongly decreased acylated ghrelin levels but did not affect ghrelin or ghrelin o-acyl transferase (GOAT) mRNA levels in PG-1 cells. Our results suggest that the medium-chain fatty acid used for the acyl-modification of ghrelin is produced in ghrelin-producing cells themselves by β-oxidation of long-chain fatty acids provided from the circulation.

Protective role of the ELOVL2/docosahexaenoic acid axis in glucolipotoxicity-induced apoptosis in rodent beta cells and human islets.

Dietary n-3 polyunsaturated fatty acids, especially docosahexaenoic acid (DHA), are known to influence glucose homeostasis. We recently showed that Elovl2 expression in beta cells, which regulates synthesis of endogenous DHA, was associated with glucose tolerance and played a key role in insulin secretion. The present study aimed to examine the role of the very long chain fatty acid elongase 2 (ELOVL2)/DHA axis on the adverse effects of palmitate with high glucose, a condition defined as glucolipotoxicity, on beta cells. We detected ELOVL2 in INS-1 beta cells and mouse and human islets using quantitative PCR and western blotting. Downregulation and adenoviral overexpression of Elovl2 was carried out in beta cells. Ceramide and diacylglycerol levels were determined by radio-enzymatic assay and lipidomics. Apoptosis was quantified using caspase-3 assays and poly (ADP-ribose) polymerase cleavage. Palmitate oxidation and esterification were determined by [U-14C]palmitate labelling. We found that glucolipotoxicity decreased ELOVL2 content in rodent and human beta cells. Downregulation of ELOVL2 drastically potentiated beta cell apoptosis induced by glucolipotoxicity, whereas adenoviral Elovl2 overexpression and supplementation with DHA partially inhibited glucolipotoxicity-induced cell death in rodent and human beta cells. Inhibition of beta cell apoptosis by the ELOVL2/DHA axis was associated with a decrease in ceramide accumulation. However, the ELOVL2/DHA axis was unable to directly alter ceramide synthesis or metabolism. By contrast, DHA increased palmitate oxidation but did not affect its esterification. Pharmacological inhibition of AMP-activated protein kinase and etomoxir, an inhibitor of carnitine palmitoyltransferase 1 (CPT1), the rate-limiting enzyme in fatty acid β-oxidation, attenuated the protective effect of the ELOVL2/DHA axis during glucolipotoxicity. Downregulation of CPT1 also counteracted the anti-apoptotic action of the ELOVL2/DHA axis. By contrast, a mutated active form of Cpt1 inhibited glucolipotoxicity-induced beta cell apoptosis when ELOVL2 was downregulated. Our results identify ELOVL2 as a critical pro-survival enzyme for preventing beta cell death and dysfunction induced by glucolipotoxicity, notably by favouring palmitate oxidation in mitochondria through a CPT1-dependent mechanism.

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.

Dronedarone-Induced Cardiac Mitochondrial Dysfunction and Its Mitigation by Epoxyeicosatrienoic Acids.

Dronedarone and amiodarone are structurally similar antiarrhythmic drugs. Dronedarone worsens cardiac adverse effects with unknown causes while amiodarone has no cardiac adversity. Dronedarone induces preclinical mitochondrial toxicity in rat liver and exhibits clinical hepatotoxicity. Here, we further investigated the relative potential of the antiarrhythmic drugs in causing mitochondrial injury in cardiomyocytes. Differentiated rat H9c2 cardiomyocytes were treated with dronedarone, amiodarone, and their respective metabolites namely N-desbutyldronedarone (NDBD) and N-desethylamiodarone (NDEA). Intracellular ATP content, mitochondrial membrane potential (Δψm), and inhibition of carnitine palmitoyltransferase I (CPT1) activity and arachidonic acid (AA) metabolism were measured in H9c2 cells. Inhibition of electron transport chain (ETC) activities and uncoupling of ETC were further studied in isolated rat heart mitochondria. Dronedarone, amiodarone, NDBD and NDEA decreased intracellular ATP content significantly (IC50 = 0.49, 1.84, 1.07, and 0.63 µM, respectively) and dissipated Δψm potently (IC50 = 0.5, 2.94, 12.8, and 7.38 µM, respectively). Dronedarone, NDBD, and NDEA weakly inhibited CPT1 activity while amiodarone (IC50 > 100 µM) yielded negligible inhibition. Only dronedarone inhibited AA metabolism to its regioisomeric epoxyeicosatrienoic acids (EETs) consistently and potently. NADH-supplemented ETC activity was inhibited by dronedarone, amiodarone, NDBD and NDEA (IC50 = 3.07, 5.24, 11.94, and 16.16 µM, respectively). Cytotoxicity, ATP decrease and Δψm disruption were ameliorated via exogenous pre-treatment of H9c2 cells with 11, 12-EET and 14, 15-EET. Our study confirmed that dronedarone causes mitochondrial injury in cardiomyocytes by perturbing Δψm, inhibiting mitochondrial complex I, uncoupling ETC and dysregulating AA-EET metabolism. We postulate that cardiac mitochondrial injury is one potential contributing factor to dronedarone-induced cardiac failure exacerbation.

Abstract 3538: Enhanced dependence on lipid metabolism is a cellular adaptation to acidic microenvironment

Abstract Malignant tumors exhibit altered metabolism and consume higher levels of glucose compared to surrounding normal tissue, resulting in highly acidic microenvironment. Adaptation to acidic conditions is a pre-requisite for tumor cells to survive and thrive and to out-compete the stroma into which they invade. Acid adaptation is associated with chronic activation of autophagy as well as redistribution of the lysosomal proteins to the plasma membrane. These processes are major survival mechanisms adopted by tumor cells under acidic conditions. We have also observed that under acidic conditions, there is a rapid, reversible and robust increase in the accumulation of cytoplasmic lipid droplets (adiposomes) in a panel of breast cancer cells. Adiposomes are dynamic organelles that store neutral lipids surrounded by a shell of proteins and a phospholipid monolayer. Breast cancer cells when grown in acidic media accumulated adiposomes as revealed by nile red and perilipin-2 staining followed by confocal microscopy. The acid-induced lipogenic phenotype persists even when the cells are grown in de-lipidated serum, indicating that the source of lipids is de-novo and endogenous. When cells were treated with inhibitors of fatty acid synthesis such as TOFA, an inhibitor of Acetyl CoA Carboxylase or FAS inhibitor C75, adiposome formation at low pH was attenuated. Inhibition of either lipid anabolic or catabolic pathways was specifically cytotoxic in acid-adapted cells, but not in control cells where the FAS inhibitor, C75, is selectively toxic under acidic conditions. Additionally, when treated with etomoxir, an inhibitor of carnitine palmitoyltransferase 1, the rate limiting step in βeta-oxidation, acid adapted cells showed increased sensitivity. To investigate the role of de novo fatty acid synthesis further, we employed high resolution NMR spectroscopy to measure 13C enriched lactate isotopomers following metabolism of D-[1,2-13C] glucose. These analyses showed that glucose flux through the pentose phosphate pathway (PPP) was significantly (&amp;gt;2.5 fold) higher in low pH exposed cells, compared to controls, representing a major shift in glucose metabolism from Embden Meyerhof to PPP, which results in increased production of NADPH, necessary for de novo lipid synthesis. Additional metabolic profiling using the Seahorse XF revealed that cells at low pH had higher rates of oxygen consumption (OCR) and that this was reversible. Further, we investigated the role of various acid sensing G-protein coupled receptors such as OGR1, TDAG8 and GPR4 in transducing the acid signal that results in the accumulation of lipid droplets. CRISPR/Cas9 mediated depletion of these receptors indicates that they might play a major role in inducing adiposome accumulation under acidic conditions. Taken together, increased dependence on lipid metabolism by cancer cells under acidic conditions reveals novel therapeutic vulnerabilities. Citation Format: Smitha Pillai, Jonathan W. Wojtkowiak, Mehdi Damaghi, Robert Gatenby, Robert Gillies. Enhanced dependence on lipid metabolism is a cellular adaptation to acidic microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3538. doi:10.1158/1538-7445.AM2017-3538

Neuronal nitric oxide synthase modulation of intracellular Ca2+ handling overrides fatty acid potentiation of cardiac inotropy in hypertensive rats.

Cardiac neuronal nitric oxide synthase (nNOS) is an important molecule that regulates intracellular Ca2+ homeostasis and contractility of healthy and diseased hearts. Here, we examined the effects of nNOS on fatty acid (FA) regulation of left ventricular (LV) myocyte contraction in sham and angiotensin II (Ang II)-induced hypertensive (HTN) rats. Our results showed that palmitic acid (PA, 100μM) increased the amplitudes of sarcomere shortening and intracellular ATP in sham but not in HTN despite oxygen consumption rate (OCR) was increased by PA in both groups. Carnitine palmitoyltransferase-1 inhibitor, etomoxir (ETO), reduced OCR and ATP with PA in sham and HTN but prevented PA potentiation of sarcomere shortening onlyin sham. PA increased nNOS-derived NO only in HTN. Inhibition of nNOS with S-methyl-L-thiocitrulline (SMTC) prevented PA-induced OCR and restored PA potentiation of myocyte contraction in HTN. Mechanistically, PA increased intracellular Ca2+ transient ([Ca2+]i) without changing Ca2+ influx via L-type Ca2+ channel (I-LTCC) and reduced myofilament Ca2+ sensitivity in sham. nNOS inhibition increased [Ca2+]i, I-LTCC and reduced myofilament Ca2+ sensitivity prior to PA supplementation; as such, normalized PA increment of [Ca2+]i. In HTN, PA reduced I-LTCC without affecting [Ca2+]i or myofilament Ca2+ sensitivity. However, PA increased I-LTCC, [Ca2+]i and reduced myofilament Ca2+ sensitivity following nNOS inhibition. Myocardial FA oxidation (18F-fluoro-6-thia-heptadecanoic acid, 18F-FTHA) was comparable between groups, but nNOS inhibition increased it only in HTN. Collectively, PA increases myocyte contraction through stimulating [Ca2+]i and mitochondrial activity in healthy hearts. PA-dependent cardiac inotropy was limited by nNOS in HTN, predominantly due to its modulatory effect on [Ca2+]i handling.

Pharmacological inhibition of carnitine palmitoyltransferase 1 restores mitochondrial oxidative phosphorylation in human trifunctional protein deficient fibroblasts

BackgroundMitochondrial Trifunctional Protein deficiency (TFPD) is a severe genetic disease characterized by altered energy metabolism and accumulation of long-chain (LC) acylcarnitines in blood and tissues. This accumulation could impair the mitochondrial oxidative phosphorylation (OxPhos), contributing to the non-optimal outcome despite conventional diet therapy with medium-chain triglycerides (MCT). MethodAcylcarnitine and OxPhos parameters were measured in TFPD-fibroblasts obtained from 8 children and cultured in medium mimicking fasting (LCFA) or conventional treatment (MCT), with or without Etomoxir (ETX) an inhibitor of carnitine palmitoyltransferase 1 (CPT1) activity, and were compared to results obtained with fibroblasts from 5 healthy-control children. The effects of various acylcarnitines were also tested on control fibroblasts. ResultsIn the LCFA-condition, TFPD-fibroblasts demonstrated a large accumulation of LC-acylcarnitines associated with decreased O2-consumption (63±3% of control, P<0.001) and ATP production (67±5%, P<0.001) without modification of coupling efficiency. A dose-dependent decrease in O2-consumption was reproduced in control fibroblasts by addition of increasing dose of LC-acylcarnitines, while it was almost preserved with MC-acylcarnitines. The MCT-condition reduced LC-acylcarnitine accumulation and partially improved O2-consumption (80±3%, P<0.01) in TFPD-fibroblasts. The addition of ETX in both LCFA- and MCT-conditions normalized acylcarnitine profiles and restored O2-consumption and ATP production at the same levels than control. ConclusionAccumulation of LC-acylcarnitines plays a major role in the pathophysiology of TFPD, reducing OxPhos capacities. These deleterious effects could be partially prevented by MCT-therapy and totally corrected by ETX. Inhibition of CPT1 may be view as a new therapeutic target for patients with a severe form of TFPD.

Nutrient deprivation in neuroblastoma cells alters 4-hydroxynonenal-induced stress response.

4-hydroxy-2-nonenal (HNE), a toxic lipid peroxidation product, is associated with oxidative damage in cells and involved in various diseases including the initiation and progression of cancer. Cancer cells have a high, adaptable metabolism with a shift from oxidative phosphorylation to glycolysis and rely on high levels of glucose and glutamine as essential nutrients for cell growth. Here we investigated whether the toxic effects of HNE on the mitochondrial membrane potential (MMP) of cancer cells depends on their metabolic state by deprivation of glucose and/or glutamine. The addition of 16 μM HNE to N18TG2 neuroblastoma cells incubated in glucose medium led to a severe reduction of MMP, which was similar to the MMP of cells fed with both glucose and glutamine. In contrast, HNE addition to cells starved in glutamine medium increased their MMP slightly for a prolonged time period and this was accompanied by increased cellular survival. We found that ß-oxidation of HNE did not cause the increased MMP, since the aldehyde dehydrogenase was distinctly more active in cells with glucose medium. However, after blocking fatty acid ß-oxidation in cells starved in glutamine medium with etomoxir, which inhibits carnitine palmitoyltransferase 1, HNE addition induced a strong reduction of MMP similar to cells in glucose medium. Surprisingly, the effect of more toxic 4-oxo-2-nonenal was less pronounced. Our results suggest that in contrast to cells fed with glucose, glutamine-fed cancer cells are capable of ß-oxidizing fatty acids to maintain their MMP to combat the toxic effects of HNE.

The Fatty Acid Synthase Inhibitor Platensimycin Improves Insulin Resistance without Inducing Liver Steatosis in Mice and Monkeys.

ObjectivesPlatensimycin (PTM) is a natural antibiotic produced by Streptomyces platensis that selectively inhibits bacterial and mammalian fatty acid synthase (FAS) without affecting synthesis of other lipids. Recently, we reported that oral administration of PTM in mouse models (db/db and db/+) with high de novo lipogenesis (DNL) tone inhibited DNL and enhanced glucose oxidation, which in turn led to net reduction of liver triglycerides (TG), reduced ambient glucose, and improved insulin sensitivity. The present study was conducted to explore translatability and the therapeutic potential of FAS inhibition for the treatment of diabetes in humans.MethodsWe tested PTM in animal models with different DNL tones, i.e. intrinsic synthesis rates, which vary among species and are regulated by nutritional and disease states, and confirmed glucose-lowering efficacy of PTM in lean NHPs with quantitation of liver lipid by MRS imaging. To understand the direct effect of PTM on liver metabolism, we performed ex vivo liver perfusion study to compare FAS inhibitor and carnitine palmitoyltransferase 1 (CPT1) inhibitor.ResultsThe efficacy of PTM is generally reproduced in preclinical models with DNL tones comparable to humans, including lean and established diet-induced obese (eDIO) mice as well as non-human primates (NHPs). Similar effects of PTM on DNL reduction were observed in lean and type 2 diabetic rhesus and lean cynomolgus monkeys after acute and chronic treatment of PTM. Mechanistically, PTM lowers plasma glucose in part by enhancing hepatic glucose uptake and glycolysis. Teglicar, a CPT1 inhibitor, has similar effects on glucose uptake and glycolysis. In sharp contrast, Teglicar but not PTM significantly increased hepatic TG production, thus caused liver steatosis in eDIO mice.ConclusionsThese findings demonstrate unique properties of PTM and provide proof-of-concept of FAS inhibition having potential utility for the treatment of diabetes and related metabolic disorders.

Inhibition of β-oxidation is not a valid therapeutic tool for reducing oxidative stress in conditions of neurodegeneration

According to recent reports, systemic treatment of rats with methylpalmoxirate (carnitine palmitoyltransferase-1 inhibitor) decreased peroxidation of polyunsaturated fatty acids in brain tissue. This was taken as evidence of mitochondrial β-oxidation in brain, thereby contradicting long-standing paradigms of cerebral metabolism, which claim that β-oxidation of activated fatty acids has minor importance for brain energy homeostasis. We addressed this controversy. Our experiments are the first direct experimental analysis of this question. We fueled isolated brain mitochondria or rat brain astrocytes with octanoic acid, but octanoic acid does not enhance formation of reactive oxygen species, neither in isolated brain mitochondria nor in astrocytes, even at limited hydrogen delivery to mitochondria. Thus, octanoic acid or l-octanoylcarnitine does not stimulate H2O2 release from brain mitochondria fueled with malate, in contrast to liver mitochondria (2.25-fold rise). This does obviously not support the possible occurrence of β-oxidation of the fatty acid octanoate in the brain. We conclude that a proposed inhibition of β-oxidation does not seem to be a helpful strategy for therapies aiming at lowering oxidative stress in cerebral tissue. This question is important, since oxidative stress is the cause of neurodegeneration in numerous neurodegenerative or inflammatory disease situations.

L-carnitine ameliorates the liver inflammatory response by regulating carnitine palmitoyltransferase I-dependent PPARγ signaling.

The liver is crucial for systemic inflammation in cancer cachexia. Previous studies have shown that L-carnitine, as the key regulator of lipid metabolism, exerts an anti-inflammatory effect in several diseases, and ameliorates the symptoms of cachexia by regulating the expression and activity of carnitine palmitoyltransferase (CPT) in the liver. However, the effect of L-carnitine on the liver inflammatory response in cancer cachexia remains to be elucidated. The aim of the present study was to examine the role of the CPT I-dependent peroxisome proliferator-activated receptor (PPAR)γ signaling pathway in the ameliorative effect of L-carnitine on the liver inflammatory response. This was investigated in a colon-26 tumor-bearing mouse model with cancer cachexia. Liver sections were immunohistochemically analyzed, and mRNA and protein levels of representative molecules of the CPT-associated PPARγ signaling pathway were assessed using PCR and western blot analysis, respectively. The results showed that oral administration of L-carnitine in these mice improved hepatocyte necrosis, liver cell cord derangement and hydropic or fatty degeneration of the liver cells in the liver tissues, decreased serum levels of malondialdehyde, increased serum levels of superoxide dismutase and glutathione peroxidase, and elevated the expression levels of PPARα and PPARγ at the mRNA and protein levels. These changes induced by L-carnitine were reversed by treatment with etomoxir, an inhibitor of CPT I. The inhibitory effect of L-carnitine on the increased expression level of nuclear factor (NF)-κB p65 in the peripheral blood mononuclear cells was markedly weakened by GW9662, a selective inhibitor of PPAR-γ. GW9662 also eliminated the inhibitory effect of L-carnitine on the expression of cyclooxygenase-2 (Cox-2) in the liver, and on the serum expression levels of pro-inflammatory prostaglandin E2, C-reactive protein, tumor necrosis factor-α and interleukin-6 in the cancer cachexia model mice. This reversing effect of GW9662 on L-carnitine was restored by pyrrolidine dithiocarbamate, a specific inhibitor of NF-κB signaling. Taken together, these results demonstrated that L-carnitine ameliorated liver inflammation and serum pro-inflammatory markers in cancer cachexia through regulating CPT I-dependent PPARγ signaling, including the downstream molecules of NF-κB p65 and Cox-2.

Acetyl CoA Carboxylase 2 Is Dispensable for CD8+ T Cell Responses.

Differentiation of T cells is closely associated with dynamic changes in nutrient and energy metabolism. However, the extent to which specific metabolic pathways and molecular components are determinative of CD8+ T cell fate remains unclear. It has been previously established in various tissues that acetyl CoA carboxylase 2 (ACC2) regulates fatty acid oxidation (FAO) by inhibiting carnitine palmitoyltransferase 1 (CPT1), a rate-limiting enzyme of FAO in mitochondria. Here, we explore the cell-intrinsic role of ACC2 in T cell immunity in response to infections. We report here that ACC2 deficiency results in a marginal increase of cellular FAO in CD8+ T cells, but does not appear to influence antigen-specific effector and memory CD8+ T cell responses during infection with listeria or lymphocytic choriomeningitis virus. These results suggest that ACC2 is dispensable for CD8+ T cell responses.

Abstract 1483: Lipid oxidation via CPT1 as a target for prostate cancer imaging and therapy

Abstract Introduction: Prostate cancer (PCa) is the most common cancer in males and is currently treated medically with androgen deprivation therapy. However, resistance to androgen receptor (AR) targeted therapy develops in almost all patients over time. Simultaneously targeting multiples pathways may prevent the development of this resistance. Recent studies suggest PCa relies on lipid fuel over glycolysis. Blocking the ability of PCa to use lipids inhibits its growth and leads to apoptosis but enhances glucose uptake initially, potentially enhancing glucose-based FDG-PET (18F-dexyglucose-Positron Emission Tomography) imaging of PCa tumors. Experimental approach: To address the role of lipid metabolism in PCa we have used etomoxir, a clinically utilized drug that blocks lipid oxidation by inhibiting carnitine palmitoyltransferase (CPT1) in the mitochondria. Enzalutamide is a clinically available anti-androgen, which was used by itself or in combination with etomoxir. The gene/drug interaction was studied in LNCaP cells with decreased expression of CPT1A (via shRNA). PET-FDG of mouse xenografts were used to evaluated the glycolytic switch induced by systemic etomoxir treatment in 24 hours and the therapeutic effect over time. Results: Treatment with etomoxir alone significantly decreased cell viability and AR content, including ARv7 variant. Combinatorial treatment with enzalutamide synergistically enhanced this effect on viability. CPT1A Knockdown clones were also more sensitive to enzalutamide (2 fold, p&amp;lt;0.001) compared to control clones, and this effect was associated with reduced AR expression. Systemic treatment with etomoxir alone in nude mice resulted in decreased xenograft growth over 21 days, underscoring the therapeutic potential of blocking lipid catabolism to decrease PCa tumor growth. Interestingly, FDG uptake by VCaP xenografts was increased with systemic etomoxir in 24 hours (∼1.5 fold, P&amp;lt;0.05), enhancing the visualization of the tumors in the PET scans. FDG uptake in the mouse non-cancerous prostate tissue was negligible with etomoxir. Conclusions: Safely blocking lipid oxidation in PCa results in decreased viability with temporarily increased glucose uptake, due to a flare of glucose uptake to compensate for the fat oxidation blockade. Validation in human PCa patients with localized disease is needed to confirm these preclinical studies. Additionally, the decreased viability over time suggests that lipid metabolism is needed to maintain AR expression and combination of fat burning inhibitors and enzalutamide may offer novel approach to anti-AR resistance in PCa. Citation Format: Isabel R. Schlaepfer, Maren Salzmann-Sullivan, Lih-Jen Su, L.Michael Glode, Thomas Flaig. Lipid oxidation via CPT1 as a target for prostate cancer imaging and therapy. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1483. doi:10.1158/1538-7445.AM2015-1483