Inhibition Of Fatty Acid Oxidation Research Articles

High-Fat Diet-Induced Obesity Alters Dendritic Cell Homeostasis by Enhancing Mitochondrial Fatty Acid Oxidation.

Obesity is associated with increased cancer risk and weak responses to vaccination and sepsis treatment. Although dendritic cells (DCs) are fundamental for the initiation and maintenance of competent immune responses against pathogens and tumors, how obesity alters the normal physiology of these myeloid cells remains largely unexplored. In this study, we report that obesity caused by prolonged high-fat diet feeding disrupts the metabolic and functional status of mouse splenic DCs (SpDCs). High-fat diet-induced obesity drastically altered the global transcriptional profile of SpDCs, causing severe changes in the expression of gene programs implicated in lipid metabolism and mitochondrial function. SpDCs isolated from obese mice demonstrated enhanced mitochondrial respiration provoked by increased fatty acid oxidation (FAO), which drove the intracellular accumulation of reactive oxygen species that impaired Ag presentation to T cells. Accordingly, treatment with the FAO inhibitor etomoxir, or antioxidants such as vitamin E or N-acetyl-l-cysteine, restored the Ag-presenting capacity of SpDCs isolated from obese mice. Our findings reveal a major detrimental effect of obesity in DC physiology and suggest that controlling mitochondrial FAO or reactive oxygen species overproduction may help improve DC function in obese individuals.

Contribution of fatty acid oxidation to the pathogenesis of pulmonary hypertension.

Dysregulated metabolism characterizes both animal and human forms of pulmonary hypertension (PH). Enzymes involved in fatty acid metabolism have previously not been assessed in human pulmonary arteries affected by pulmonary arterial hypertension (PAH), and how inhibition of fatty acid oxidation (FAO) may attenuate PH remains unclear. Fatty acid metabolism gene transcription was quantified in laser-dissected pulmonary arteries from 10 explanted lungs with advanced PAH (5 idiopathic, 5 associated with systemic sclerosis), and 5 donors without lung diseases. Effects of oxfenicine, a FAO inhibitor, on female Sugen 5416-chronic hypoxia (SuHx) rats were studied in vivo using right heart catheterization, and ex vivo using perfused lungs and pulmonary artery ring segments. The impact of pharmacologic (oxfenicine) and genetic (carnitine palmitoyltransferase 1a heterozygosity) FAO suppression was additionally probed in mouse models of Schistosoma and hypoxia-induced PH. Potential mechanisms underlying FAO-induced PH pathogenesis were examined by quantifying ATP and mitochondrial mass in oxfenicine-treated SuHx pulmonary arterial cells, and by assessing pulmonary arterial macrophage infiltration with immunohistochemistry. We found upregulated pulmonary arterial transcription of 26 and 13 FAO genes in idiopathic and systemic sclerosis-associated PAH, respectively. In addition to promoting de-remodeling of pulmonary arteries in SuHx rats, oxfenicine attenuated endothelin-1-induced vasoconstriction. FAO inhibition also conferred modest benefit in the two mouse models of PH. Oxfenicine increased mitochondrial mass in cultured rat pulmonary arterial cells, and decreased the density of perivascular macrophage infiltration in pulmonary arteries of treated SuHx rats. In summary, FAO inhibition attenuated experimental PH, and may be beneficial in human PAH.

228-LB: Mitochondrial Architecture Controls Fatty Acid Utilization by Regulating CPT1 Sensitivity to Malonyl-CoA

Mitochondrial network fragmentation has been associated with metabolic dysregulation, although the underlying mechanisms remain unknown. Herein, using multiple cellular models where the physiological relevance of fatty acid is well-established, we show that mitochondrial fragmentation is highly associated with increased fatty acid oxidation (FAO) rates. Through confocal imaging and stable isotope tracing analysis, forced mitochondrial elongation using Mfn2 over-expression or Drp1 depletion significantly decreased FAO rates, while Mfn2 knockout leads to enhanced FAO rates (P-value =0.0003) , suggesting that mitochondrial morphology regulates fatty acid utilization. To determine the mechanism by which mitochondrial architecture controls fatty acid utilization we studied the effects of mitochondrial fragmentation and elongation on CPT1 activity. Remarkably, we find that CPT1 sensitivity to its main regulator, malonyl-CoA is strongly affected by molecular interventions that inhibit mitochondrial fusion and fission. Notably, the reduction in hepatic FAO rates upon mitochondrial elongation is attributed to a 14 fold increase in CPT1 sensitivity to malonyl-CoA. Consistent with this mechanism we show that the inhibition of FAO imposed by mitochondrial elongation can be overcome using CPT1 pharmacological activator that is malonyl-CoA-independent, but not by the malonyl-CoA-sensitive activator. Furthermore, we tested the functional implications of this mechanism in hepatocytes and beta cells. In hepatocytes we show that forced elongation by the inhibition of fission reduced gluconeogenesis by 30-70%. Whereas in islets, forced mitochondrial fragmentation through molecular inhibition of fusion, resulted in a 2.5 fold increase in insulin secretion at basal glucose concentrations, a phenomenon associated with prediabetes and obesity. Taken together, our study provides a biochemical and mechanistic explanation by which changes to mitochondrial architecture affects fuel utilization and lipid metabolism. Disclosure J. Ngo: None. D. Choi: None. L. Stiles: None. A. S. Divakaruni: None. M. Liesa: None. N. N. Danial: None. O. Shirihai: None. Funding American Diabetes Association (1-19-IBS-049)

Acbp is essential for decidualization during early pregnancy in mice.

Decidualization of uterine stromal cells plays an important role in the establishment of normal pregnancy. Previous studies have demonstrated that Acyl-CoA binding protein (Acbp) is critical to cellular proliferation, differentiation, mitochondrial functions, and autophagy. The characterization and physiological function of Acbp during decidualization remain largely unknown. In the present study, we conducted the expression profile of Acbp in the endometrium of early pregnant mice. With the occurrence of decidualization, the expression of Acbp gradually increased. Similarly, Acbp expression was also strongly expressed in decidualized cells following artificial decidualization, both in vivo and in vitro. We applied the mice pseudopregnancy model to reveal that the expression of Acbp in the endometrium of early pregnant mice was not induced by embryonic signaling. Moreover, P4 significantly upregulated the expression of Acbp, whereas E2 appeared to have no regulating effect on Acbp expression in uterine stromal cells. Concurrently, we found that interfering with Acbp attenuated decidualization, and that might due to mitochondrial dysfunctions and the inhibition of fatty acid oxidation. The level of autophagy was increased after knocking down Acbp. During induced decidualization, the expression of ACBP was decreased with the treatment of rapamycin (an autophagy inducer), while increased with the addition of Chloroquine (an autophagy inhibitor). Our work suggests that Acbp plays an essential role in the proliferation and differentiation of stromal cells during decidualization through regulating mitochondrial functions, fatty acid oxidation, and autophagy.

Trained immunity of alveolar macrophages requires metabolic rewiring and type 1 interferon signaling

Environmental microbial triggers shape the development and functionality of the immune system. Alveolar macrophages (AMs), tissue-resident macrophages of the lungs, are in constant and direct contact with inhaled particles and microbes. Such exposures likely impact AM reactivity to subsequent challenges by immunological imprinting mechanisms referred to as trained immunity. Here, we investigated whether a ubiquitous microbial compound has the potential to induce AM training in vivo. We discovered that intranasal exposure to ambient amounts of lipopolysaccharide (LPS) induced a pronounced AM memory response, characterized by enhanced reactivity upon pneumococcal challenge. Exploring the mechanistic basis of AM training, we identified a critical role of type 1 interferon signaling and found that inhibition of fatty acid oxidation and glutaminolysis significantly attenuated the training effect. Notably, adoptive transfer of trained AMs resulted in increased bacterial loads and tissue damage upon subsequent pneumococcal infection. In contrast, intranasal pre-exposure to LPS promoted bacterial clearance, highlighting the complexity of stimulus-induced immune responses, which likely involve multiple cell types and may depend on the local immunological and metabolic environment. Collectively, our findings demonstrate the profound impact of ambient microbial exposure on pulmonary immune memory and reveal tissue-specific features of trained immunity.

Fatty acid oxidation protects cancer cells from apoptosis by increasing mitochondrial membrane lipids.

Overcoming resistance to chemotherapies remains a major unmet need for cancers, such as triple-negative breast cancer (TNBC). Therefore, mechanistic studies to provide insight for drug development are urgently needed to overcome TNBC therapy resistance. Recently, an important role of fatty acid β-oxidation (FAO) in chemoresistance has been shown. But how FAO might mitigate tumor cell apoptosis by chemotherapy is unclear. Here, we show that elevated FAO activates STAT3 by acetylation via elevated acetyl-coenzyme A (CoA). Acetylated STAT3 upregulates expression of long-chain acyl-CoA synthetase 4 (ACSL4), resulting in increased phospholipid synthesis. Elevating phospholipids in mitochondrial membranes leads to heightened mitochondrial integrity, which in turn overcomes chemotherapy-induced tumor cell apoptosis. Conversely, in both cultured tumor cells and xenograft tumors, enhanced cancer cell apoptosis by inhibiting ASCL4 or specifically targeting acetylated-STAT3 is associated with a reduction in phospholipids within mitochondrial membranes. This study demonstrates a critical mechanism underlying tumor cell chemoresistance.

Supramolecular nanotherapeutics enable metabolic reprogramming of tumor-associated macrophages to inhibit tumor growth.

Tumor-associated macrophages (TAMs) exist in multiple phenotypes across the spectrum, defined by an M1 antitumorigenic phenotype and an M2 pro-tumorigenic phenotype on two ends of the spectrum. A largely immunosuppressive tumor-microenvironment aids the polarization of the infiltrating macrophages to a pro-tumorigenic M2 phenotype that promotes tumor progression and metastasis. Recent developments in macrophage immunotherapy have focused on strategies to re-educate TAMs from an M2 to M1 phenotype. Recent findings in the realm of immuno-metabolism have indicated that distinct metabolic signatures accompany macrophages based on their polarization states (M1-Glycolysis and M2-TCA cycle). These metabolites are important drivers of cellular signaling responsible for acquiring these polarization states, with evidence showing that metabolism is essential to facilitate the energy requirements of immune cells and regulate immune cell response. We hypothesized that TAMs could be reprogrammed metabolically by co-delivery of drugs using a supramolecular nanoparticle system that could effectively rewire macrophage metabolism by simultaneous inhibition of the TCA cycle and upregulation of the glycolytic metabolic pathway. TLR7/8 agonist and Fatty Acid Oxidation (FAO) inhibitor loaded metabolic supramolecular nanoparticles (MSNPs) were synthesized. In vitro assays showed macrophages treated with MSNPs were reprogrammed from an M2 phenotype to an M1 phenotype while significantly upregulating phagocytosis. When injected in 4T1 tumor-bearing mice, MSNPs treatment reduced tumor growth progression more than other treatments. Hence, the delivery of TLR7/8 agonist combined with an FAO inhibitor can enhance antitumor efficacy through metabolic reprogramming of tumor-associated macrophages.

BSCL2/Seipin deficiency in hearts causes cardiac energy deficit and dysfunction via inducing excessive lipid catabolism.

BackgroundHeart failure (HF) is one of the leading causes of death worldwide and is associated with cardiac metabolic perturbations. Human Type 2 Berardinelli‐Seip Congenital Lipodystrophy (BSCL2) disease is caused by mutations in the BSCL2 gene. Global lipodystrophic Bscl2−/− mice exhibit hypertrophic cardiomyopathy with reduced cardiac steatosis. Whether BSCL2 plays a direct role in regulating cardiac substrate metabolism and/or contractile function remains unknown.MethodsWe generated mice with cardiomyocyte‐specific deletion of Bscl2 (Bscl2cKO ) and studied their cardiac substrate utilisation, bioenergetics, lipidomics and contractile function under baseline or after either a treatment regimen using fatty acid oxidation (FAO) inhibitor trimetazidine (TMZ) or a prevention regimen with high‐fat diet (HFD) feeding. Mice with partial ATGL deletion and cardiac‐specific deletion of Bscl2 were also generated followed by cardiac phenotyping.ResultsDifferent from hypertrophic cardiomyopathy in Bscl2−/− mice, mice with cardiac‐specific deletion of Bscl2 developed systolic dysfunction with dilation. Myocardial BSCL2 deletion led to elevated ATGL expression and FAO along with reduced cardiac lipid contents. Cardiac dysfunction in Bscl2cKO mice was independent of mitochondrial dysfunction and oxidative stress, but associated with decreased metabolic reserve and ATP levels. Importantly, cardiac dysfunction in Bscl2cKO mice could be partially reversed by FAO inhibitor TMZ, or prevented by genetic abolishment of one ATGL allele or HFD feeding. Lipidomic analysis further identified markedly reduced glycerolipids, glycerophospholipids, NEFA and acylcarnitines in Bscl2cKO hearts, which were partially normalised by TMZ or HFD.ConclusionsWe identified a new form of cardiac dysfunction with excessive lipid utilisation which ultimately causes cardiac substrate depletion and bioenergetics failure. Our findings also uncover a crucial role of BSCL2 in controlling cardiac lipid catabolism and contractile function and provide novel insights into metabolically treating energy‐starved HF using FAO inhibitor or HFD.

Gut microbiota production of trimethyl-5-aminovaleric acid reduces fatty acid oxidation and accelerates cardiac hypertrophy

Numerous studies found intestinal microbiota alterations which are thought to affect the development of various diseases through the production of gut-derived metabolites. However, the specific metabolites and their pathophysiological contribution to cardiac hypertrophy or heart failure progression still remain unclear. N,N,N-trimethyl-5-aminovaleric acid (TMAVA), derived from trimethyllysine through the gut microbiota, was elevated with gradually increased risk of cardiac mortality and transplantation in a prospective heart failure cohort (n = 1647). TMAVA treatment aggravated cardiac hypertrophy and dysfunction in high-fat diet-fed mice. Decreased fatty acid oxidation (FAO) is a hallmark of metabolic reprogramming in the diseased heart and contributes to impaired myocardial energetics and contractile dysfunction. Proteomics uncovered that TMAVA disturbed cardiac energy metabolism, leading to inhibition of FAO and myocardial lipid accumulation. TMAVA treatment altered mitochondrial ultrastructure, respiration and FAO and inhibited carnitine metabolism. Mice with γ-butyrobetaine hydroxylase (BBOX) deficiency displayed a similar cardiac hypertrophy phenotype, indicating that TMAVA functions through BBOX. Finally, exogenous carnitine supplementation reversed TMAVA induced cardiac hypertrophy. These data suggest that the gut microbiota-derived TMAVA is a key determinant for the development of cardiac hypertrophy through inhibition of carnitine synthesis and subsequent FAO.

Fatty acid oxidation fuels glioblastoma radioresistance with CD47-mediated immune evasion

Glioblastoma multiforme (GBM) remains the top challenge to radiotherapy with only 25% one-year survival after diagnosis. Here, we reveal that co-enhancement of mitochondrial fatty acid oxidation (FAO) enzymes (CPT1A, CPT2 and ACAD9) and immune checkpoint CD47 is dominant in recurrent GBM patients with poor prognosis. A glycolysis-to-FAO metabolic rewiring is associated with CD47 anti-phagocytosis in radioresistant GBM cells and regrown GBM after radiation in syngeneic mice. Inhibition of FAO by CPT1 inhibitor etomoxir or CRISPR-generated CPT1A−/−, CPT2−/−, ACAD9−/− cells demonstrate that FAO-derived acetyl-CoA upregulates CD47 transcription via NF-κB/RelA acetylation. Blocking FAO impairs tumor growth and reduces CD47 anti-phagocytosis. Etomoxir combined with anti-CD47 antibody synergizes radiation control of regrown tumors with boosted macrophage phagocytosis. These results demonstrate that enhanced fat acid metabolism promotes aggressive growth of GBM with CD47-mediated immune evasion. The FAO-CD47 axis may be targeted to improve GBM control by eliminating the radioresistant phagocytosis-proofing tumor cells in GBM radioimmunotherapy.

Short-term intermittent cigarette smoke exposure enhances alveolar type 2 cell stemness via fatty acid oxidation

BackgroundCigarette smoke (CS) is associated with chronic obstructive pulmonary disease (COPD) and cancer. However, the underlying pathological mechanisms are not well understood. We recently reported that mice exposed to long-term intermittent CS for 3 months developed more severe emphysema and higher incidence of adenocarcinoma than mice exposed to long-term continuous CS for 3 months and long-term continuous CS exposure activated alveolar stem cell proliferation. However, the influence of variations in the CS exposure pattern in alveolar stem cell in unknown. Here, we exposed mice to 3 weeks of continuous or intermittent CS to identify whether different CS exposure patterns would result in differential effects on stem cells and the mechanisms underlying these potential differences.MethodsFemale mice expressing GFP in alveolar type 2 (AT2) cells, which are stem cells of the alveolar compartment, were exposed to mainstream CS via nasal inhalation. AT2 cells were collected based on their GFP expression by flow cytometry and co-cultured with fibroblasts in stem cell 3D organoid/colony-forming assays. We compared gene expression profiles of continuous and intermittent CS-exposed AT2 cells using microarray analysis and performed a functional assessment of a differentially expressed gene to confirm its involvement in the process using activator and inhibitor studies.ResultsAT2 cells sorted from intermittent CS-exposed mice formed significantly more colonies compared to those from continuous CS-exposed mice, and both CS-exposed groups formed significantly more colonies when compared to air-exposed cells. Comparative microarray analysis revealed the upregulation of genes related to fatty acid oxidation (FAO) pathways in AT2 cells from intermittent CS-exposed mice. Treatment of intermittent CS-exposed mice with etomoxir, an inhibitor of the FAO regulator Cpt1a, for 5 weeks resulted in a significant suppression of the efficiency of AT2 cell colony formation. In vitro treatment of naïve AT2 cells with a FAO activator and inhibitor further confirmed the relationship between FAO and AT2 stem cell function.ConclusionsAlveolar stem cell function was more strongly activated by intermittent CS exposure than by continuous CS exposure. We provide evidence that AT2 stem cells respond to intermittent CS exposure by activating stem cell proliferation via the activation of FAO.

Norisoboldine induces the development of Treg cells by promoting fatty acid oxidation-mediated H3K27 acetylation of Foxp3.

Norisoboldine (NOR), an alkaloid isolated from Radix Lindera, was previously reported to promote the differentiation of regulatory T cells (Treg cells), an important subtype of lymphocytes capable of controlling autoimmune diseases. The present study was performed to explore the mechanism of NOR in the view of cellular metabolism. A global metabolomic analysis indicated that NOR preferentially altered the fatty acid oxidation (FAO) pathway and elevated the content of related metabolites during Treg cell differentiation. The detection of oxygen consumption rate (OCR) and mRNA expression of FAO-related enzymes demonstrated that NOR promoted FAO in the early stage of Treg cell differentiation. Consistently, pharmacological or genetic inhibition of FAO markedly diminished the induction of NOR on Treg cell differentiation. Furthermore, NOR was shown to elevate the level of acetyl-CoA derived from FAO and acetylation of lysine 27 on histone 3 (H3K27) at the Foxp3 promoter and CNS2 regions. A knockdown of CPT1, the rate-limiting enzyme of FAO, weakened the promotion of NOR on the development, acetyl-CoA level, and acetylation of H3K27 of Treg cells in vitro and in the mice with collagen-induced arthritis, and attenuated the anti-arthritic effect of NOR. These findings demonstrate that NOR induces the development of Treg cells through promoting FAO, therefore, facilitating gene transcription of Foxp3 via acetyl-CoA-mediated H3K27 acetylation modification, and FAO might serve as a novel target to induce Treg cell development.

S-15176 Difumarate Salt Can Impair Mitochondrial Function through Inhibition of the Respiratory Complex III and Permeabilization of the Inner Mitochondrial Membrane.

Simple SummaryThe metabolic agent S-15176 difumarate salt belongs to a potent class of drugs called partial fatty acid oxidation inhibitors. The possible therapeutic effects of this agent have been widely investigated in models of oxidative damage to different tissues, but the underlying mechanisms are currently unclear and require further research. In the present study, we aimed to investigate in more detail the effect of S-15176 on the mitochondria, power stations inside the cell, and pivotal regulators of many cellular processes. We found that S-15176 affected the key indicators of the function of rat liver and thymocyte mitochondria at the concentrations reached in target tissues. S-15176 disturbed the catalytic function of enzyme complex III of the mitochondrial electron transfer system and induced nonspecific membrane permeability, which was related to the dissipation of the mitochondrial membrane potential and a decline in ATP production. These findings lead us to predict that S-15176 can induce mitochondrial dysfunction in mammal tissues and organs that are most vulnerable to chemical toxicity or exposed to higher concentrations of the drug.S-15176 difumarate salt, a derivative of the anti-ischemic metabolic drug trimetazidine, has been intensively studied for its impact on cellular metabolism in animal models of ischemia-reperfusion injury of the liver, heart, spinal cord, and other organs. Despite evidence of some reduction in oxidative damage to cells, the results of therapy with S-15176 have been mostly disappointing, possibly because of the lack of data on its underlying mechanisms. Here, we aimed to investigate in more detail the role of complexes I-IV of the electron transport chain and membrane permeability transition in mitochondrial toxicity associated with S-15176. Using rat thymocyte and liver mitochondria, we demonstrated that: (1) acute exposure to S-15176 (10 to 50 μM) dose-dependently decreased the mitochondrial membrane potential; (2) S-15176 suppressed the ADP-stimulated (State 3) and uncoupled (State 3UDNP) respiration of mitochondria energized with succinate or malate/glutamate, but not ascorbate/TMPD, and increased the resting respiration (State 4) when using all the substrate combinations; (3) S-15176 directly inhibited the activity of the respiratory complex III; (4) low doses of S-15176 diminished the rate of H2O2 production by mitochondria; (5) at concentrations of above 30 μM, S-15176 reduced calcium retention capacity and contributed to mitochondrial membrane permeabilization. Taken together, these findings suggest that S-15176 at tissue concentrations reached in animals can impair mitochondrial function through suppression of the cytochrome bc1 complex and an increase in the nonspecific membrane permeability.

Fatty acids secreted from head and neck cancer induce M2-like Macrophages.

Tumor-infiltrating monocytes can mature into Macrophages that support tumor survival or that display antitumor properties. To explore mechanisms steering Macrophage maturation, we assessed the effects of supernatants from squamous cell carcinoma cell lines (FaDu and SCC) on monocyte-derived Macrophage maturation. Purified monocytes were incubated in medium or medium supplemented with supernatants from FaDu and SCC9 or the leukemia monocytic cell line, THP-1. Macrophages were examined for markers of maturation (CD14, CD68), activation (HLA-DR, CD86, IL15R), scavenger receptor (CD36), toll-like receptor (TLR4), M2 marker (CD206), immune checkpoint (PD-L1), and intracellular chemokine expression (IP-10). Compared to other conditions, cells incubated with FaDu or SCC9 supernatants displayed enhanced survival, down-regulation of cell surface HLA-DR, CD86, IL-15R, CD36, and intracellular IP-10 expression, and increased cell surface PD-L1, CD14, and CD206 expression. Despite expressing TLR4 and CD14, Macrophages matured in tumor supernatants failed to respond to stimulation with the canonical TLR4 agonist, LPS. These changes were accompanied by a decrease in intracellular phospho-p38 expression in tumor supernatant conditioned Macrophages. Depletion of fatty acids from tumor supernatants or treatment of cell cultures with an inhibitor of fatty acid oxidation, Etomoxir, reversed a number of these phenotypic changes induced by tumor supernatants. Additionally, Macrophages incubated with either palmitic acid or oleic acid developed similar phenotypes as cells incubated in tumor supernatants. Together, these data suggest that fatty acids derived from tumor cells can mediate the maturation of Macrophages into a cell type with limited pro-inflammatory characteristics.

Abstract P5-05-06: Metformin concentration is a deciding factor of its pro- or anti-tumor function in triple negative breast cancer

Abstract We have previously reported that triple negative (TN) breast cancer (BC), an aggressive subtype of BC, has high dependency for mitochondrial fatty acid oxidation (FAO). We have also reported that FAO activates c-Src, one of the frequently upregulated oncopathways in TNBC. Our mechanism-based mathematical model of cancer metabolism by coupling the master gene regulators (AMPK, HIF-1) suggested that the combination of mitochondrial electron transport chain (ETC) and FAO repressions acquire the highest efficacy in suppressing the bioenergetics (i.e., cellular ATP production) of the TNBC cells. Biguanides such as metformin are one of the most widely administered anti-diabetic agents. Phenformin, a biguanide derivative similar to metformin, has greater potency than metformin. Both these drugs have been widely studied for their antitumor activities but showed only limited clinical advantages in BC. Biguanides can suppress oxidative phosphorylation (OXPHOS) by inhibiting the complex-I activity of mitochondrial ETC. However, biguanides also activate the energy sensor AMPK and phosphorylate its downstream protein ACC. Since ACC inhibits the transport of fatty acids to mitochondria, its phosphorylation can lead to FAO activation. Thus, biguanides can play opposing roles in mitochondrial metabolism (suppression of ETC and activation of FAO). Here we have analyzed the significance of these opposing roles of biguanides in TNBC metabolism and oncopathway activation. Molecular biology and RNASeq analyses suggest that while higher concentrations of biguanides suppress ETC activity and Src pathway despite the activation of AMPK, lower concentrations of biguanides activate FAO-driven ETC and Src pathway in TNBC cells. To understand the translational significance of this observation, we have performed preclinical studies using TNBC organoids and patient-derived xenografts (PDX) models. Combination therapy with metformin and FAO inhibitor, etomoxir significantly improved the antitumor activity compared to the single drug therapy. For a better clinically relevant approach, we did preclinical studies using the combination of metformin and Src inhibitor, dasatinib in TNBC organoids and PDX models. As expected, the combination of metformin and dasatinib synergistically inhibited tumor growth and enhanced the overall survival of the mice. Our observations suggested that the tumor bioavailability of biguanides is critical for its anti-tumor activity, especially in FAO-dependent tumors like TNBC. Using metabolomic, genomic and preclinical experiments, we are currently evaluating the translational potential of this findings in FAO-dependent TNBCs. Citation Format: Jun Hyoung Park, Dongya Jia, Sukjin Yang, Kwang Hwa Jung, Abha Tiwari, Debasmita Dutta, Meron Ghidey, Nagireddy Putluri, Cristian Coarfa, Chad Creighton, José N. Onuchic, Benny A Kaipparettu. Metformin concentration is a deciding factor of its pro- or anti-tumor function in triple negative breast cancer [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P5-05-06.

Cholangiocarcinoma progression depends on the uptake and metabolization of extracellular lipids.

Cholangiocarcinoma (CCA) includes a heterogeneous group of biliary cancers with a dismal prognosis. We investigated if lipid metabolism is disrupted in CCA and its role in tumor proliferation. The in vitro and in vivo tumorigenic capacity of five human CCA cell lines was analyzed. Proteome, lipid content, and metabolic fluxes were evaluated in CCA cells and compared with normal human cholangiocytes (NHC). The Akt1/NOTCH1 intracellular cytoplasmic domain (Nicd1)-driven CCA mouse model was also evaluated. The proteome of CCA cells was enriched in pathways involved in lipid and lipoprotein metabolism. The EGI1 CCA cell line presented the highest tumorigenic capacity. Metabolic studies in high (EGI1) versus low (HUCCT1) proliferative CCA cells in vitro showed that both EGI1 and HUCCT1 incorporated more fatty acids (FA) than NHC, leading to increased triglyceride storage, also observed in Akt1/Nicd1-driven CCA mouse model. The highly proliferative EGI1 CCA cells showed greater uptake of very-low-density and HDLs than NHC and HUCCT1 CCA cells and increased cholesteryl ester content. The FA oxidation (FAO) and related proteome enrichment were specifically up-regulated in EGI1, and consequently, pharmacological blockade of FAO induced more pronounced inhibition of their tumorigenic capacity compared with HUCCT1. The expression of acyl-CoA dehydrogenase ACADM, the first enzyme involved in FAO, was increased in human CCA tissues and correlated with the proliferation marker PCNA. Highly proliferative human CCA cells rely on lipid and lipoprotein uptake to fuel FA catabolism, suggesting that inhibition of FAO and/or lipid uptake could represent a therapeutic strategy for this CCA subclass.

Anticancer activity and metabolic profile alterations by ortho-topolin riboside in in vitro and in vivo models of non-small cell lung cancer.

Lung cancer has the highest incidence and mortality rates among all types of cancer worldwide, and 80%-85% of patients with lung cancer are diagnosed with non-small cell lung cancer (NSCLC), which has 5-year survival rate of only 5% at advanced stages. Development of new therapeutic agents and strategies is required to enhance the treatment efficiency in patients with NSCLC. Metabolic alterations and anticancer effects of plant hormones and their derivatives have not been investigated in NSCLC in vitro and in vivo. The present study investigated the cytotoxic effects of 11 plant hormones and their derivatives against NSCLC cell lines; ortho-topolin riboside (oTR) showed the highest cytotoxicity among all tested compounds against NSCLC cells. Alteration of metabolites and lipids was investigated using gas chromatography-mass spectrometry and nano electrospray ionization-mass spectrometry in oTR-treated NSCLC cells and a xenograft mouse model. oTR reduced amino acid and pyrimidine synthesis in NSCLC cells and xenograft tumors. Moreover, oTR reduced glycolytic function and decreased mitochondrial respiration function by inhibiting glutamine and fatty acid oxidation. Increased levels of phosphatidylcholine, phosphatidylethanolamine, and phosphatidylserine species suggested that oTR might act as a fatty acid oxidation inhibitor. In addition, the increased level of phosphatidylserine species implied that phosphatidylserine-mediated apoptosis occurred in oTR-treated NSCLC cells and xenograft tumor. The antiproliferative and apoptotic effects of oTR were mediated by the reduced p-ERK and p-AKT levels and increased cleaved Caspase-3levels, respectively. This is the first study to investigate the metabolic alterations and anticancer activity of oTR in in vitro and in vivo models of NSCLC. Our results provide basis for the development of oTR-based therapeutic agent for patients with NSCLC.

A Planar Culture Model of Human Absorptive Enterocytes Reveals Metformin Increases Fatty Acid Oxidation and Export.

Background & AimsFatty acid oxidation by absorptive enterocytes has been linked to the pathophysiology of type 2 diabetes, obesity, and dyslipidemia. Caco-2 and organoids have been used to study dietary lipid-handling processes including fatty acid oxidation, but are limited in physiological relevance or preclude simultaneous apical and basal access. Here, we developed a high-throughput planar human absorptive enterocyte monolayer system for investigating lipid handling, and then evaluated the role of fatty acid oxidation in fatty acid export, using etomoxir, C75, and the antidiabetic drug metformin.MethodsSingle-cell RNA-sequencing, transcriptomics, and lineage trajectory was performed on primary human jejunum. In vivo absorptive enterocyte maturational states informed conditions used to differentiate human intestinal stem cells (ISCs) that mimic in vivo absorptive enterocyte maturation. The system was scaled for high-throughput drug screening. Fatty acid oxidation was modulated pharmacologically and BODIPY (Thermo Fisher Scientific, Waltham, MA) (B)–labeled fatty acids were used to evaluate fatty acid handling via fluorescence and thin-layer chromatography.ResultsSingle-cell RNA-sequencing shows increasing expression of lipid-handling genes as absorptive enterocytes mature. Culture conditions promote ISC differentiation into confluent absorptive enterocyte monolayers. Fatty acid-handling gene expression mimics in vivo maturational states. The fatty acid oxidation inhibitor etomoxir decreased apical-to-basolateral export of medium-chain B-C12 and long-chain B-C16 fatty acids, whereas the CPT1 agonist C75 and the antidiabetic drug metformin increased apical-to-basolateral export. Short-chain B-C5 was unaffected by fatty acid oxidation inhibition and diffused through absorptive enterocytes.ConclusionsPrimary human ISCs in culture undergo programmed maturation. Absorptive enterocyte monolayers show in vivo maturational states and lipid-handling gene expression profiles. Absorptive enterocytes create strong epithelial barriers in 96-Transwell format. Fatty acid export is proportional to fatty acid oxidation. Metformin enhances fatty acid oxidation and increases basolateral fatty acid export, supporting an intestine-specific role.

Twist1 downregulation of PGC-1α decreases fatty acid oxidation in tubular epithelial cells, leading to kidney fibrosis.

Rationale: A deficiency of fatty acid oxidation (FAO) is the metabolic hallmark in proximal tubular cells (PTCs) in renal fibrosis owing to utilization of fatty acids by PTCs as the main energy source. Lipid accumulation may promote lipotoxicity-induced pathological injury in renal tissue. However, the molecular mechanism underlying lipotoxicity and renal tubulointerstitial fibrosis (TIF) remains unclear. Twist1 has been identified to play an essential role in fatty acid metabolism. We hypothesized that Twist1 may regulate FAO in PTCs and consequently facilitate lipotoxicity-induced TIF.Methods: We used hypoxia-induced Twist1 overexpression to incite defective mitochondrial FAO in PTCs, and used renal ischemia-reperfusion or unilateral ureteral obstruction to induce renal injury in mice. We used knockout cells, mice of Twist1, and Harmine to determine the role of Twist1 in FAO and TIF.Results: Overexpression of Twist1 downregulates the transcription of PGC-1α and further inhibits the expression of FAO-associated genes, such as PPARα, CPT1 and ACOX1. Consequently, reduced FAO and increased intracellular lipid droplet accumulation in a human PTC line (HK-2), leads to mitochondrial dysfunction, and production of increased profibrogenic factors. Twist1 knockout mice with renal injury had increased expression of PGC-1α, which restored FAO and obstructed progression of TIF. Strikingly, pharmacological inhibition of Twist1 by using Harmine reduced lipid accumulation and restored FAO in vitro and in vivo.Conclusion: Our findings suggest that Twist1-mediated inhibition of FAO in PTCs results in TIF and suggest that Twist1-targeted inhibition could provide a potential strategy for the treatment of renal fibrosis.

Abstract 12092: IMB-1018792, an Investigational Agent Designed to Augment Cardiac Glucose Utlization and Energetics Reduces Cardiac Remodelling and Preserves Cardiac Function in a Model of Pressure Overload-Induced Heart Failure

Introduction: Energetic impairments play a key role in heart failure (HF). Contributors include reduced pyruvate dehydrogenase activity partially uncoupling glycolysis from glucose oxidation, increased dependence on fatty acid (FA) metabolism yielding less ATP than glucose per O 2 molecule consumed, a lower phosphate-to-O 2 ratio, mitochondrial uncoupling and futile cycling of FA intermediates. IMB-1018972 (IMB-101) is a novel investigational agent designed to increase cardiac glucose uptake to promote glucose oxidation (Randle effect) through partial inhibition of FA oxidation enhancing cardiac energetics and mechanical efficiency. Hypotheses: Administration of IMB-101 will: 1) increase cardiac glucose uptake and 2) attenuate LV hypertrophic remodelling and progression of HF resulting from chronic pressure overload. Methods: 1) Cardiac glucose metabolism was evaluated using dynamic 18 F-FDG PET/CT imaging in vivo in fasted normal rats following a subcutaneous dose of IMB-101 or saline. Each animal had 2 randomized sequenced scan sessions (n=16). 2) Male C57BL/6 mice aged 12 weeks underwent transverse aortic constriction (TAC) or sham surgery (n=15) and were treated with IMB-101 (10 mg/kg/day, n=15) or saline (n=15) from day 1 (D1) for 6 weeks by osmotic minipump. Cardiac function was assessed by conscious echocardiography (D1, week 3 and 6). Cardiac fibrosis was measured by Masson’s Trichrome staining. Results: IMB-101 robustly increased in vivo cardiac 18 F-FDG uptake vs saline in rats. In mice subjected to TAC, compared to saline controls, IMB-101 treatment: preserved LV systolic function (fractional shortening 46%±3% vs 34%±3%, at week 6, p<0.05); improved LV relaxation with less prolonged isovolumic relaxation time (32±1 ms vs 36±1 ms, p<0.05); reduced cardiac hypertrophy (LV mass 156±10 mg vs 195±12 mg, p<0.05; and heart:body weight ratio 7.4±0.3 mg/g vs. 9.1±0.5 mg/g, p<0.05); and attenuated cardiac fibrosis (6.6±0.6% vs 10.7±1%, p<0.01). Conclusions: IMB-101 increases cardiac glucose uptake and protects against LV dysfunction, hypertrophic remodelling and interstitial fibrosis after chronic pressure overload.