Fatty Acid Synthase Expression Research Articles

Anti-adipogenic effect of heat-treated probiotic Lactiplantibacillus plantarum WB2719 and Lacticaseibacillus rhamnosus WB2804

Lactiplantibacillus plantarum WB2719 and Lacticaseibacillus rhamnosus WB2804 were isolated from fermented foods. These strains were demonstrated probiotic traits and anti-adipogenic effects. These strains exhibited tolerance to artificial gastric conditions and adhesion activities to intestine cells. Furthermore, heat-treated L. plantarum WB2719 and L. rhamnosus WB2804 inhibited preadipocyte differentiation by modulating peroxisome proliferator-activated receptor-γ (PPAR-γ), CCAAT/enhancer binding proteins α (C/EBP-α), and fatty acid synthase (FAS) expression. In particular, heat-treated L. plantarum WB2719 decreased mitosis and exerted anti-obesity effects by inducing apoptosis. Therefore, this study proposes the potential commercialization of heat-treated L. plantarum WB2719 and L. rhamnosus WB2804 as novel food-derived anti-obesity materials in the expanding health-functional food sector.

Glucocorticoid Resistance Increases PPARγ Activity and Drives Hepatic Lipid Accumulation via Increased Eicosanoid and Monoacylglyceride Synthesis

A diet high in fat induces adiposity and obesity, and chronic feeding can lead to multiple metabolic complications, including type II diabetes, non-alcoholic fatty liver disease, and cardiovascular disease. The glucocorticoid receptor (GR) regulates genes that control metabolism. The GR has two isoforms (GRα and GRβ) that arise from the GR gene ( NR3C1) due to the alternative splicing of exon 9. The GRα isoform is the classical receptor that binds to glucocorticoids. However, the GRβ isoform has a truncated C-terminus. It cannot bind glucocorticoid steroids, and as a result, this isoform induces a glucocorticoid-resistant state when its expression is increased. We hypothesized that increasing levels of GRβ from long-term excess glucocorticoid exposure or a high-fat diet induces adiposity by affecting lipid metabolism and lipogenesis, causing fat to accumulate in the adipose and liver tissues. We first overexpressed GRβ (GRβ-Ad) or control (Vec-Ad) using adenovirus delivery and fed the mice a normal chow diet. The GRβ-Ad mice had significantly higher lipid accumulation within five days. Western blotting analysis revealed a significant increase (p-value <0.05) in the GRβ-Ad livers for fatty acid synthase (FAS) expression, and there was a remodeling of the hepatic lipid content as measured by mass spectrometry lipidomic analysis. The data indicate an increased level of monoacylglyceride lipid species and some species of long-chain triacylglycerides and diacylglycerides. mRNA analysis revealed a significant increase (p-value <0.05) in Carbohydrate-responsive element-binding protein (ChREBP), PPARγ, and Cyp2j6, indicating an increase in de novo lipogenesis and eicosanoid synthesis. We conclude that glucocorticoid resistance due to increased levels of GRβ drives de novo lipogenesis and increases eicosanoid synthesis, possibly via synergism with PPARγ. A better understanding of the GR isoforms and glucocorticoid resistance could benefit people with obesity and other metabolic disorders to improve obesity-associated comorbidities. R01DK121797 (T.D.H.J.), R01DA058933 (T.D.H.J.). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Exosomal NAT10 from esophageal squamous cell carcinoma cells modulates macrophage lipid metabolism and polarization through ac4C modification of FASN

N-acetyltransferase 10 (NAT10) is acknowledged as a tumor promoter in various cancers due to its role as a regulator of acetylation modification. Tumor-associated macrophages (TAMs) play a pivotal role in the tumor microenvironment (TME). However, the intercellular communication between esophageal squamous cell carcinoma (ESCC) cells and TAMs involving NAT10 remains poorly understood. This study aimed to elucidate the regulatory mechanism of NAT10 in modulating macrophage lipid metabolism and polarization. Experimental evidence was derived from in vitro and in vivo analyses. We explored the association between upregulated NAT10 in ESCC tissues, macrophage polarization, and the therapeutic efficacy of PD-1. Furthermore, we investigated the impact of methyltransferase 3 (METTL3)-induced m6A modification on the increased expression of NAT10 in ESCC cells. Additionally, we examined the role of exosomal NAT10 in stabilizing the expression of fatty acid synthase (FASN) and promoting macrophage M2 polarization through mediating the ac4C modification of FASN. Results indicated that NAT10, packaged by exosomes derived from ESCC cells, promotes macrophage M2 polarization by facilitating lipid metabolism. In vivo animal studies demonstrated that targeting NAT10 could enhance the therapeutic effect of PD-1 on ESCC by mediating macrophage reprogramming. Our findings offer novel insights into improving ESCC treatment through NAT10 targeting.

Overexpression of Fatty Acid Synthase Upregulates Glutamine-Fructose-6-Phosphate Transaminase 1 and O-Linked N-Acetylglucosamine Transferase to Increase O-GlcNAc Protein Glycosylation and Promote Colorectal Cancer Growth.

Fatty acid synthesis has been extensively investigated as a therapeutic target in cancers, including colorectal cancer (CRC). Fatty acid synthase (FASN), a key enzyme of de novo lipid synthesis, is significantly upregulated in CRC, and therapeutic approaches of targeting this enzyme are currently being tested in multiple clinical trials. However, the mechanisms behind the pro-oncogenic action of FASN are still not completely understood. Here, for the first time, we show that overexpression of FASN increases the expression of glutamine-fructose-6-phosphate transaminase 1 (GFPT1) and O-linked N-acetylglucosamine transferase (OGT), enzymes involved in hexosamine metabolism, and the level of O-GlcNAcylation in vitro and in vivo. Consistently, expression of FASN significantly correlates with expression of GFPT1 and OGT in human CRC tissues. shRNA-mediated downregulation of GFPT1 and OGT inhibits cellular proliferation and the level of protein O-GlcNAcylation in vitro, and knockdown of GFPT1 leads to a significant decrease in tumor growth and metastasis in vivo. Pharmacological inhibition of GFPT1 and OGT leads to significant inhibition of cellular proliferation and colony formation in CRC cells. In summary, our results show that overexpression of FASN increases the expression of GFPT1 and OGT as well as the level of protein O-GlcNAcylation to promote progression of CRC; targeting the hexosamine biosynthesis pathway could be a therapeutic approach for this disease.

Complexes of Soluble Dietary Fiber and Polyphenols from Lotus Root Regulate High-Fat Diet-Induced Hyperlipidemia in Mice.

In this paper, complexes of soluble dietary fiber (SDF) and polyphenols (PPs) isolated from lotus roots were prepared (SDF-PPs), as well as physical mixtures (SDF&PPs), which were given to high-fat-diet (HFD)-fed mice. The results demonstrated that SDF-PPs improve lipid levels and reverse liver injury in hyperlipidemic mice. Western blotting and real-time quantitative Polymerase Chain Reaction (RT-qPCR) results showed that SDF-PPs regulated liver lipids by increasing the phosphorylation of Adenine monophosphate activated protein kinase (AMPK), up-regulating the expression of Carnitine palmitoyltransferase1 (CPT1), and down-regulating the expression of Fatty acid synthase (FAS) and 3-hydroxy-3-methyl glutaryl coenzyme A (HMG-CoA), as well as the transcription factor sterol-regulatory element binding protein (SPEBP-1) and its downstream liposynthesis genes. Additionally, the intervention of SDF-PPs could modulate the composition of intestinal gut microbes, inducing an increase in Lachnospiraceae and a decrease in Desulfovibrionaceae and Prevotellaceae in high-fat-diet-fed mice. Thus, the research provides a theoretical basis for the application of lotus root active ingredients in functional foods and ingredients.

A tipping point in dihydroxyacetone exposure: mitochondrial stress and metabolic reprogramming alter survival in rat cardiomyocytes H9c2 cells

Exogenous exposures to the triose sugar dihydroxyacetone (DHA) occur from sunless tanning products and electronic cigarette aerosol. Once inhaled or absorbed, DHA enters cells, is converted to dihydroxyacetone phosphate (DHAP), and incorporated into several metabolic pathways. Cytotoxic effects of DHA vary across the cell types depending on the metabolic needs of the cells, and differences in the generation of reactive oxygen species (ROS), cell cycle arrest, and mitochondrial dysfunction have been reported. We have shown that cytotoxic doses of DHA induced metabolic imbalances in glycolysis and oxidative phosphorylation in liver and kidney cell models. Here, we examine the dose-dependent effects of DHA on the rat cardiomyocyte cell line, H9c2. Cells begin to experience cytotoxic effects at low millimolar doses, but an increase in cell survival was observed at 2 mM DHA. We confirmed that 2 mM DHA increased cell survival compared to the low cytotoxic 1 mM dose and investigated the metabolic differences between these two low DHA doses. Exposure to 1 mM DHA showed changes in the cell's fuel utilization, mitochondrial reactive oxygen species (ROS), and transient changes in the glycolysis and mitochondrial energetics, which normalized 24 h after exposure. The 2 mM dose induced robust changes in mitochondrial flux through acetyl CoA and elevated expression of fatty acid synthase. Distinct from the 1 mM dose, the 2 mM exposure increased mitochondrial ROS and NAD(P)H levels, and sustained changes in LDHA/LDHB and acetyl CoA-associated enzymes were observed. Although the cells were exposed to low cytotoxic (1 mM) and non-cytotoxic (2 mM) acute doses of DHA, significant changes in mitochondrial metabolic pathways occurred. Further, the proliferation increase at the acute 2 mM DHA dose suggests a metabolic adaption occurred with sustained consequences in survival and proliferation. With increased exogenous exposure to DHA through e-cigarette aerosol, this work suggests cell metabolic changes induced by acute or potentially chronic exposures could impact cell function and survival.

Proanthocyanidins and Phenolic Compounds from the Twigs of Salix chaenomeloides and Their Anti-Lipogenic Effects on 3T3-L1 Preadipocytes.

The present study investigated potential bioactive natural products from the EtOH extract of Salix chaenomeloides twigs using column chromatography, leading to the isolation of six compounds (1-6), which were characterized as two proanthocyanidins, procyanidin B2 (1) and procyanidin B1 (2), and four phenolic compounds, 4-hydroxybenzoic acid β-D-glucosyl ester (3), di-O-methylcrenatin (4), p-coumaric acid glucoside (5), and syringin (6) by the comparison of their NMR spectra with the reported data and high-resolution (HR)-electrospray ionization mass spectroscopy (ESI-MS) analysis. We investigated the potential of six compounds (1-6) to inhibit adipogenesis in 3T3-L1 preadipocytes, which showed that the compounds (1-6) significantly reduced lipid accumulation in 3T3-L1 adipocytes without affecting cell proliferation. Notably, compound 1 demonstrated a remarkable 60% and 90% reduction in lipid levels with 50 and 100 µM treatments, respectively. Oil Red O staining results indicated that compound 1 significantly inhibits the formation of lipid droplets, comparable to the effect of T863, an inhibitor of triglyceride used as a positive control, in adipocytes. Compound 1 had no effect on the regulators PPARγ, C/EBPα, and SREBF1 of adipocyte differentiation in 3T3-L1 preadipocytes, but compound 1 activated the fatty acid oxidation regulator, PPARα, compared to the lipogenic-induced control. It also suppressed fatty acid synthesis by downregulating the expression of fatty acid synthase (FAS). Finally, compound 1 induced the mRNA and protein levels of CPT1A, an initial marker of mitochondrial fatty acid oxidation in 3T3-L1. This finding substantiates the anti-lipogenic and lipolytic effects of procyanidin B2 (1) in 3T3-L1 preadipocytes, emphasizing its pivotal role in modulating obesity-related markers.

Lipid droplet accumulation in Wdr45-deficient cells caused by impairment of chaperone-mediated autophagic degradation of Fasn

Backgroundβ-Propeller protein-associated neurodegeneration (BPAN) is a genetic neurodegenerative disease caused by mutations in WDR45. The impairment of autophagy caused by WDR45 deficiency contributes to the pathogenesis of BPAN; however, the pathomechanism of this disease is largely unknown. Lipid dyshomeostasis is involved in neurogenerative diseases, but whether lipid metabolism is affected by Wdr45 deficiency and whether lipid dyshomeostasis contributes to the progression of BPAN are unclear.MethodsWe generated Wdr45 knockout SN4741 cell lines using CRISPR‒Cas9-mediated genome editing, then lipid droplets (LDs) were stained using BODIPY 493/503. Chaperone-mediated autophagy was determined by RT-qPCR and western blotting. The expression of fatty acid synthase (Fasn) was detected by western blot in the presence or absence of the lysosomal inhibitor NH4Cl and the CMA activator AR7. The interaction between Fasn and HSC70 was analyzed using coimmunoprecipitation (Co-IP) assay. Cell viability was measured by a CCK-8 kit after treatment with the Fasn inhibitor C75 or the CMA activator AR7.ResultsDeletion of Wdr45 impaired chaperone-mediated autophagy (CMA), thus leading to lipid droplet (LD) accumulation. Moreover, Fasn can be degraded via CMA, and that defective CMA leads to elevated Fasn, which promotes LD formation. LD accumulation is toxic to cells; however, cell viability was not rescued by Fasn inhibition or CMA activation. Inhibition of Fasn with a low concentration of C75 did not affect cell viability but decreases LD density.ConclusionsThese results suggested that Fasn is essential for cell survival but that excessive Fasn leads to LD accumulation in Wdr45 knockout cells.

Abstract 2784: Fatty acid synthase enhances stem-like properties of normal intestinal and colorectal cancer cells via an increase in notum expression and secretion

Abstract Introduction: Overexpression of lipogenic enzymes has been associated with a poor clinical outcome in colorectal cancer (CRC). Fatty acid synthase (FASN) synthesizes palmitate which can be utilized for post-translational modifications of various proteins. Notum, a palmitoleoyl-protein, prevents the downstream activation of Wnt signaling by de-palmitoylating Wnt ligands. Notum is also a CRC stem cell marker, and its expression correlates with poor prognosis in CRC. We found that upregulation of FASN is associated with an increase in Notum expression in mouse adenoma tissues. Therefore, the purpose of this study is to elucidate the mechanisms and contribution of FASN/Notum axis in CRC. Methods: Adenoma and normal intestinal organoids, established from ApcMin/VillinCre-ERT2 and Apc/VillinCre-ERT2 mouse models with hetero- and homozygous deletion of FASN. 4-hydroxytamoxifen (4-OHT) and TVB-3664 (FASN inhibitor) treatments were used to assess the effect of FASN deletion/inhibition on organoid growth and viability. LIVE/DEAD™ Viability/Cytotoxicity and Cell Titer-Glo® 3D Cell Viability Assays were used for quantitative analysis of growth. Human NOTUM/Protein notum homolog ELISA Kit was used to analyze Notum secretion. CRC cells, NTC and FASN shRNA, and control and FASN overexpression, were used for Notum analysis. Results: The TCGA data analysis shows that Notum is significantly upregulated in CRC and its expression correlates with expression of FASN in tumor tissue. Utilizing qRT-PCR and Western blot analyses, we show that downregulation of FASN leads to a decrease in expressions of active β-catenin, Notum, and other stem cell markers in transgenic mouse models. Moreover, 4-OHT-mediated inhibition of FASN results in decrease viability and size in ApcMin/FASN/VillinCre-ERT2 and Apc/FASN/VillinCre-ERT2 organoids. Consistently, shRNA-mediated deletion of FASN decreases expression of Notum in CRC cells. In contrast, overexpression of FASN increases the expression levels of active and total β-catenin, Notum, and other stem cell markers in SW480 cells. We show that normal epithelial cells exposed to CRC cell medium exhibit stem-like phenotype which is reduced when cells are exposed to medium collected from Notum shRNA knockdown cells. Moreover, the addition of recombinant Notum restores this phenotype. Conclusion: In summary, downregulation of FASN leads to a decrease in expression of Notum and is associated with morphological changes and significant decrease in viability in organoid models. Conversely, FASN overexpression upregulates Notum expression suggesting a potential cross talk between de novo lipid synthesis and Notum. Delineating the role of FASN regulation of stem-like properties via upregulation of β-catenin signaling and expression of Notum and other stem cell markers will provide the rationale for targeting the FASN/Notum axis in CRC. Citation Format: Courtney Olivia Kelson, Josiane Weber Tessmann, Daheng He, Chi Wang, Yekaterina Zaytseva. Fatty acid synthase enhances stem-like properties of normal intestinal and colorectal cancer cells via an increase in notum expression and secretion [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2784.

Abstract 1796: Role of FASN in pancreatic neuroendocrine cancer cells survival

Abstract Introduction. Pancreatic neuroendocrine tumors (pNETs) are among the most frequently occurring neuroendocrine neoplasms. Up-regulation of fatty acid synthase (FASN) has been reported in many cancers, but the role of lipid metabolism in pNETs remains unclear. The purpose of this study was to determine whether FASN inhibition can be used as a target for pNET treatment. Methods. Immunohistochemistry and western blot analysis were used to determine protein levels of FASN in pNET tissues and cell lines. Expression of FASN was evaluated in pancreatic neuroendocrine primary and metastatic tumors and scored by a pathologist. Neutral lipid droplet levels in pNET cell lines were determined with BODIPY 493/503 staining. Clonogenic and SRB assays were used to evaluate cytotoxic effects of FASN inhibition by TVB-3664 in QGP-1 and BON pancreatic NET cell lines. Western blot analysis was used to determine levels of cleaved PARP and cyclin D1 after FASN inhibition. FASN knockdown was used to determine the effects of FASN downregulation on proliferation and lipid synthesis. Results. FASN expression was determined at the maximum scores of 6 and 5 in 98% of patient samples. BON, QGP-1 and NT-3 cell lines were stained with BODIPY 493/503; the highest levels of neutral lipid droplets were observed in the BON cell line. BON and QGP-1 cells were treated with TVB-3664 in either complete media with fatty acids (FA+) or complete media prepared with fatty acid depleted FBS (FA-). Treatment with TVB-3664 resulted in significant inhibition of BON and QGP-1 cell proliferation and cyclin D1 expression in both FA+ and FA- conditions. Depletion of extracellular FA enhanced the effect of TVB-3664 therapy on NET cells, especially in the BON cell line. FASN inhibition with TVB-3634 suppressed pNET cell proliferation and colony formation. Conclusions. Our study identified high protein levels of FASN in pNET patient samples and cell lines. Moreover, we demonstrated variability in the response of pNET cells to FASN inhibition. QGP cell viability was significantly decreased with FASN inhibition even in the presence of extracellular lipids. FASN inhibition in BON cells had only a minimal effect on cell viability and required depletion of extracellular lipids to achieve a decrease in cell viability and cyclin D1 expression. These data demonstrate that de novo lipid synthesis is involved in the pathogenesis and progression of pNETs. Importantly, our findings show that some pNETs are highly sensitive to FASN inhibition, suggesting that FASN inhibition may be considered as a treatment option in selected pNETs. Citation Format: Benjamin M. Evers, Zeta Chow, Yekaterina Zaytseva, Eun Y. Lee, Courtney M. Townsend, Tianyan Gao, B. Mark Evers, Piotr G. Rychahou. Role of FASN in pancreatic neuroendocrine cancer cells survival [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1796.

Abstract 4760: Inhibition of FASN postpones development of resistance to BRAF inhibitors in colorectal cancer

Abstract Introduction: Mutations in proto-oncogene BRAF occur in about 10-15% of CRC patients and BRAFV600E is the most common. For patients who undergo BRAF-targeted therapy, resistance develops 4-6 months after treatment initiation and results in more aggressive disease. We found that development of resistance to BRAF inhibitors (BRAFi) is associated with an increase in lipid metabolism and expression of fatty acid synthase (FASN). FASN is a key enzyme of lipid synthesis overexpressed in CRC. Therefore, our hypothesis is that inhibition lipid metabolism via FASN will postpone development of resistance to BRAFi. Methods: We established CRC cells resistant to PLX8394, a novel BRAFi. To evaluate differences in parental and resistance cells, CellTiterGlo 2.0, PrestoBlue, CytoSelectTM Invasion Assay, Triglyceride Assay, Seahorse XF, and confocal microscopy were used. Combination of PLX8394 and TVB3664 or C75 (FASN inhibitors) was tested on cell viability, colony formation, and synergy studies in parental and BRAFi resistant cells. Results: The development of resistance to BRAFi promotes cellular proliferation and increases cyclin D and survivin expression in vitro and in vivo. BRAFi resistance is also associated with an increase in invasive properties and loss of E-cadherin expression. Metabolic changes include an increase in lipid metabolism, oxidative phosphorylation, and triglycerides storage. RNAseq and western blot analysis show significant upregulation of FASN in BRAFi resistant cells. Using cell viability and soft agar colony formation assays, we show that combined PLX8394 and TVB3664 treatment leads to a significantly higher decrease in cell viability and colony formation as compared to each drug alone in parental cells but not in BRAFi resistant cells. The calculation of a Bliss synergy score confirms that combination treatment with C75 and PLX8394 has synergetic effect in BRAFV600E cells. To further confirm that FASN contributes to resistance to BRAFi, we show that HT29 FASN shRNA cells are more susceptible to PLX8394 treatment as compared to control cells. Importantly, our data show that the long-term treatment with PLX8394 in combination with TVB3664 postpones development of resistance to BRAFi as compared to cells treated with PLX8394 alone. Conclusion: Our study demonstrates that resistance to BRAFi is associated with a significant increase in proliferation, metastasis, and lipid metabolism. We demonstrate that combination of FASN inhibitors and BRAFi postpones development of resistance in BRAFV600E cells. However, FASN inhibition does not sensitize cells to BRAFi in already resistance cells, suggesting that this approach cannot be used to overcome acquired resistance to BRAFi. In summary our data suggest that an addition of TVB3664 at the beginning of BRAF treatment regimen could be an efficacious treatment strategy for BRAFV600E CRC patients. Citation Format: Mariah Geisen, Josiane weber-Tessmann, Courtney Kelson, Daheng He, Chi Wang, Abu Saleh Mosa Faisal, Jill Kolesar, Yekaterina Zaytseva. Inhibition of FASN postpones development of resistance to BRAF inhibitors in colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4760.

Abstract 7645: Differentially expressed genes as prognostic markers in racial disparities of prostate cancer

Abstract Racial disparities in prostate cancer are prevalent, with African American (AA) men experiencing 60% greater incidence and 2 to 3 times higher mortality than Caucasian American (CA) men. Consequently, there is a pressing need for improved means of interrogating race/ethnicity-based prostate cancer biology in order to identify biomarkers to help stratify men at high-risk for prostate cancer-specific progression and death and offer treatment recommendation that would mostly benefit AA men undergoing early aggressive therapies. Current predictive models rely on clinical variables but do not account for these disparities. The overall goal is to construct a race-specific prostate cancer prognostic prediction model for risk assessment. Here we performed comprehensive bioinformatics analysis using the data from 17 pairs of gene expression array from AA and CA cohorts and identified 63 differentially expressed genes (DEGs) by race. Among them, chemokine receptor 4 (CXCR4), viz. p-Akt (Ser473), fatty acid synthase (FASN), interleukin-6 (IL-6) and matrix metallopeptidase 9 (MMP-9) gene expressions were significantly altered in AA groups. Immunohistochemistry was performed on 163 prostate cancer patient specimens to validate DEG expression and immunoreactive scores (IRS) were calculated. CXCR4 exhibited significantly higher expression in AA tumors, with > 85% of AA prostate tumors showing weak to strong CXCR4 expression, compared to < 5% in CA specimens. While immunostaining for IL-6, FASN, MMP9 and p-Akt did not provide a significant distinction between CA and AA cases. IRS analysis confirmed higher expression of CXCR4 in AA cancers, compared to CA prostate cancer (p < 0.001). MMP9 and IL6 expression were moderately higher in AA cancer compared to CA prostate cancer (p < 0.05). In contrast, FASN expression was significantly higher in CA cancer, compared to AA prostate cancer (p < 0.001). We further investigated the impact of these genes on biochemical recurrence-free survival (BCRFS) using Kaplan-Meier analysis to examine the differences between low-risk versus high-risk groups. BCRFS analysis demonstrated that CXCR4 is a determinant factor of recurrence risk in AA prostate cancer patients. The activation of the PI3K-Akt signaling pathway, particularly in AA patients, suggests its role in prostate cancer aggressiveness. Taken together, this study demonstrates the importance of differential expression of biomarkers which constitute the bulk tumors in driving cancer. The distinct gene expression patterns, especially the upregulation of CXCR4, in AA prostate cancer highlight its potential as a prognostic biomarker and a target for personalized treatment strategies. These findings encourage further study into the role of population-specific difference in adding CXCR4 in risk model construction, especially in machine learning models. Citation Format: Amel A. Ahmed, Shiv Verma, Ibrahim M. Atawia, Daniel L. Shen, Gregory T. MacLennan, Pingfu Fu, Sanjay Gupta. Differentially expressed genes as prognostic markers in racial disparities of prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7645.

Abstract 4642: Fatty acid synthase as a novel prognostic biomarker and a potential therapeutic target in diffuse malignant peritoneal mesothelioma

Abstract Diffuse malignant peritoneal mesothelioma (DMPM) is a rare and locally aggressive tumor with a dismal prognosis. The availability of new therapies and biomarkers for patients’ selection is still an urgent need. Altered lipid metabolism, which has been reported in several tumors, has not yet been studied in DMPM. By comparatively analyzing gene expression profiles of tumors form relapsed (n=18) and non-relapsed (n=18) DMPM patients, we initially found that fatty acid synthase (FASN) was up-modulated in relapsed tumors, with a significant linear correlation between FASN expression and shorter patient relapse-free survival (RFS), suggesting a potential role of FASN in DMPM progression. The prognostic significance of FASN was further assessed in a cohort of 80 DMPM patients who underwent cytoreductive surgery combined with hyperthermic intraperitoneal chemotherapy. Our results indicated that high FASN staining was significantly associated with reduced RFS (hazard ration (HR): 1.930; p=0.014) and Overall Survival (OS) (HR: 1.854; p=0.032) as assessed by immunohistochemistry in tissues microarray samples. The prognostic value of FASN, along with Ki67 staining and patient age, was also maintained in multivariate analysis in terms of both RFS (HR: 2.240; p=0.007) and OS (HR: 2.246; p=0.014). To validate FASN as a therapeutic target, we investigated the cytotoxic effects of non-selective (orlistat) and selective (cerulenin and C75) FASN inhibitors on patient-derived DMPM cell lines, both as single agents and in combination with selinexor -a selective inhibitor of the exportin chromosome maintenance protein. FASN pharmacological inhibition in DMPM cells resulted in a significant reduction of cell proliferation, especially upon C75 exposure. In combination experiments, we observed a synergistic activity of FASN inhibitors and selinexor, as determined by Chou and Talalay method. Through western blotting and flow cytometry analysis, we gained further insight into the mechanisms by which FASN inhibitors and selinexor synergize to reduce DMPM cell proliferation. Compared to single agents, the combined treatments resulted in increased apoptosis response and cell cycle impairment, as indicated by caspase-dependent apoptosis activation and cell accumulation in the G2-phase. Overall, our results support the relevance of FASN as a prognostic biomarker for DMPM patients and underscore its potential as a novel therapeutic target. Citation Format: Valentina Doldi, Silvia Martini, Chiara Maura Ciniselli, Alessia Beretta, Marcello Deraco, Shigeki Kusamura, Dario Baratti, Paolo Gandellini, Paolo Verderio, Nadia Zaffaroni. Fatty acid synthase as a novel prognostic biomarker and a potential therapeutic target in diffuse malignant peritoneal mesothelioma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4642.

Improving the Effects of Mulberry Leaves and Neochlorogenic Acid on Glucotoxicity-Induced Hepatic Steatosis in High Fat Diet Treated db/db Mice.

There are many complications of type 2 diabetes mellitus. Nonalcoholic fatty liver disease (NAFLD) and nonalcoholic steatohepatitis (NASH) are two complications related to the increased lipid accumulation in the liver. Previous studies have shown that mulberry leaf water extract (MLE) has the effect of lowering lipid levels in peripheral blood, inhibiting the expression of fatty acid synthase (FASN) and increasing the activity of liver antioxidant enzymes superoxide dismutase (SOD) and catalase. Our study aimed to investigate the role of MLE and its main component, neochlorogenic acid (nCGA), in reducing serum lipid profiles, decreasing lipid deposition in the liver, and improving steatohepatitis levels. We evaluated the antioxidant activity including glutathione (GSH), glutathione reductase (GRd), glutathione peroxidase (GPx), glutathione S-transferase (GST), and superoxide dismutase (SOD), and catalase was tested in mice fed with MLE and nCGA. The results showed a serum lipid profile, and fatty liver scores were significantly increased in the HFD group compared to the db/m and db mice groups, while liver antioxidant activity significantly decreased in the HFD group. When fed with HFD + MLE or nCGA, there was a significant improvement in serum lipid profiles, liver fatty deposition conditions, steatohepatitis levels, and liver antioxidant activity compared to the HFD group. Although MLE and nCGA do not directly affect the blood sugar level of db/db mice, they do regulate abnormalities in lipid metabolism. These results demonstrate the potential of MLE/nCGA as a treatment against glucotoxicity-induced diabetic fatty liver disease in animal models.

Ecdysone-controlled nuclear receptor ERR regulates metabolic homeostasis in the disease vector mosquito Aedes aegypti.

Hematophagous mosquitoes require vertebrate blood for their reproductive cycles, making them effective vectors for transmitting dangerous human diseases. Thus, high-intensity metabolism is needed to support reproductive events of female mosquitoes. However, the regulatory mechanism linking metabolism and reproduction in mosquitoes remains largely unclear. In this study, we found that the expression of estrogen-related receptor (ERR), a nuclear receptor, is activated by the direct binding of 20-hydroxyecdysone (20E) and ecdysone receptor (EcR) to the ecdysone response element (EcRE) in the ERR promoter region during the gonadotropic cycle of Aedes aegypti (named AaERR). RNA interference (RNAi) of AaERR in female mosquitoes led to delayed development of ovaries. mRNA abundance of genes encoding key enzymes involved in carbohydrate metabolism (CM)-glucose-6-phosphate isomerase (GPI) and pyruvate kinase (PYK)-was significantly decreased in AaERR knockdown mosquitoes, while the levels of metabolites, such as glycogen, glucose, and trehalose, were elevated. The expression of fatty acid synthase (FAS) was notably downregulated, and lipid accumulation was reduced in response to AaERR depletion. Dual luciferase reporter assays and electrophoretic mobility shift assays (EMSA) determined that AaERR directly activated the expression of metabolic genes, such as GPI, PYK, and FAS, by binding to the corresponding AaERR-responsive motif in the promoter region of these genes. Our results have revealed an important role of AaERR in the regulation of metabolism during mosquito reproduction and offer a novel target for mosquito control.

Intervention effect of Erchen Decoction on methionine and choline deficient diet-induced non-alcoholic steatohepatitis in mice

This study investigated the effect of Erchen Decoction(ECD) on the prevention of non-alcoholic steatohepatitis(NASH) in mice and explored its possible mechanism, so as to provide scientific data for the clinical application of ECD in the prevention of NASH. C57BL/6 male mice were randomly divided into normal group(methionine and choline supplement, MCS), model group(methionine and choline deficient, MCD), low-dose ECD group(ECD＿L, 6 g·kg~(-1)), medium-dose ECD group(ECD＿M, 12 g·kg~(-1)), and high-dose ECD group(ECD＿H, 24 g·kg~(-1)), with eight mice in each group. The MCS group was fed with an MCS diet, and the other groups were fed with an MCD diet. The mice in each group were given corresponding diets, but the drug intervention group was given low-, medium-, and high-dose ECD(10 mL·kg~(-1)·d~(-1)) by intragastric administration for six weeks on the basis of MCD diet feeding, and the mice could eat and drink freely during the whole experiment. At the end of the experiment, mice were fasted overnight(12 h) and were anesthetized with 20% urethane. Thereafter, the blood and liver tissue were collected. The serum was used to detect the levels of alanine aminotransferase(ALT), aspartate aminotransaminase(AST), interleukin-1β(IL-1β), interleukin-6(IL-6), interleukin-10(IL-10), and tumor necrosis factor-α(TNF-α). Liver tissue was processed by hematoxylin-eosin(HE) staining and used for hepatic histological analysis and detection of the expression levels of genes and proteins related to nuclear factor erythroid 2-related factor 2/glutathione peroxidase 4(Nrf2/GPX4) pathway by real-time quantitative reverse transcriptase-polymerase chain reaction(RT-qPCR) and Western blot analysis, respectively. The results showed that compared with the MCS group, the MCD group showed higher serum ALT and AST levels; the HE staining exhibited fat vacuoles and obvious inflammatory cell infiltration in liver tissue; serum IL-1β, IL-6, and TNF-α levels were significantly increased, and the serum IL-10 level was significantly decreased. The mRNA expressions of fatty acid synthase(FASN), monocyte chemoattractant protein-1(MCP-1), and IL-1β in liver tissue were significantly up-regulated, while those of GPX4, Nrf2, and NAD(P)H:quinine oxidoreductase(NQO1) were significantly down-regulated. Compared with the MCD group, the serum ALT and AST levels of ECD＿M and ECD＿H groups were significantly decreased, and the AST level in the ECD＿L group was significantly decreased. The number of fat vacuoles and the degree of inflammatory cell infiltration in liver tissue were improved; serum IL-1β, IL-6, and TNF-α levels were significantly decreased, but the serum IL-10 level was significantly increased only in the ECD＿H group. The mRNA expressions of FASN, MCP-1, and IL-1β in liver tissue were significantly down-regulated, and those of GPX4 and NQO1 were significantly up-regulated. The mRNA expressions of Nrf2 in ECD＿M and ECD＿H groups were significantly up-regulated. Western blot results showed that compared with the MCD group, the protein expression levels of Nrf2 and GPX4 in each group were significantly increased after ECD administration, and the protein expression level of FASN was significantly decreased; the protein expression of NQO1 was increased in ECD＿M and ECD＿H groups. In summary, ECD can reduce hepatic lipid accumulation, oxidative stress, liver inflammation, and liver injury in NASH mice, which may be related to the activation of the Nrf2/GPX4 pathway.

Effect of different levels of Lactobacillus added to diets on fat deposition and meat quality of Sunit lambs

In this study, we examined the impact of adding Lactobacillus to the diet on fat distribution and meat quality of Sunit lambs. For 90 days, twenty-four lambs (19.31 ± 0.47 kg) were fed diets that contained 0 (NP), 6 (P1), 12 (P2), or 24 (P3) g of Lactobacillus casei/d. The results suggested that dietary supplementation with Lactobacillus decreased serum triglyceride in Sunit lambs (P < 0.001). The loin muscle area displayed notable increases in the P1 group (P < 0.05). Meanwhile, tail and visceral fat deposition of lambs were reduced when Lactobacillus was added to the diet (P < 0.05). Compared with the NP group, the values of shear force and cooking loss of in the P1 group exhibited a significant reduction, and intramuscular fat content increased significantly (P < 0.05). Additionally, the P1 group showed an increase in polyunsaturated fatty acids and a decrease in saturated fatty acids in the longissimus thoracis and biceps femoris muscles (P < 0.05). The P1 group showed downregulation of protein kinase AMP-activated catalytic subunit alpha 2 (AMPKα2) and carnitine palmitoyltransferase 1B expression in the longissimus thoracis muscle (P < 0.05). However, there was an upregulation of acetyl-CoA carboxylase (ACC), sterol regulatory element binding transcription factor 1 (SREBF1), and fatty acid synthase (FASN) expression (P < 0.05). In conclusion, feeding Sunit lambs 6 g/d of Lactobacillus as a dietary supplement may be a valuable way to improve fat distribution and meat quality. The AMPK/ACC and AMPK/SREBF1/FASN signaling pathways may be involved in this outcome.

Vitamin-C-dependent downregulation of the citrate metabolism pathway potentiates pancreatic ductal adenocarcinoma growth arrest.

In pancreatic ductal adenocarcinoma (PDAC), metabolic rewiring and resistance to standard therapy are closely associated. PDAC cells show enormous requirements for glucose-derived citrate, the first rate-limiting metabolite in the synthesis of new lipids. Both the expression and activity of citrate synthase (CS) are extraordinarily upregulated in PDAC. However, no previous relationship between gemcitabine response and citrate metabolism has been documented in pancreatic cancer. Here, we report for the first time that pharmacological doses of vitamin C are capable of exerting an inhibitory action on the activity of CS, reducing glucose-derived citrate levels. Moreover, ascorbate targets citrate metabolism towards the de novo lipogenesis pathway, impairing fatty acid synthase (FASN) and ATP citrate lyase (ACLY) expression. Lowered citrate availability was found to be directly associated with diminished proliferation and, remarkably, enhanced gemcitabine response. Moreover, the deregulated citrate-derived lipogenic pathway correlated with a remarkable decrease in extracellular pH through inhibition of lactate dehydrogenase (LDH) and overall reduced glycolytic metabolism. Modulation of citric acid metabolism in highly chemoresistant pancreatic adenocarcinoma, through molecules such as vitamin C, could be considered as a future clinical option to improve patient response to standard chemotherapy regimens.

Brown Algae Dictyopteris divaricata Attenuates Adipogenesis by Modulating Adipocyte Differentiation and Promoting Lipolysis through Heme Oxygenase-1 Activation in 3T3-L1 Cells.

The present study aims to explore the probable anti-adipogenesis effect of Dictyopteris divaricata (D. divaricata) in 3T3-L1 preadipocytes by regulating heme oxygenase-1 (HO-1). The extract of D. divaricata retarded lipid accretion and decreased triglyceride (TG) content in 3T3-L1 adipocytes but increased free glycerol levels. Treatment with the extract inhibited lipogenesis by inhibiting protein expressions of fatty acid synthase (FAS) and lipoprotein lipase (LPL), whereas lipolysis increased by activating phosphorylation of hormone-sensitive lipase (p-HSL) and AMP-activated protein kinase (p-AMPK). The extract inhibited adipocyte differentiation of 3T3-L1 preadipocytes through down-regulating adipogenic transcription factors, including peroxisome proliferator-activated receptor gamma (PPARγ) and CCAAT/enhancer-binding protein α (C/EBPα), and sterol regulatory element-binding protein 1 (SREBP1). This is attributed to the triggering of Wnt/β-catenin signaling. In addition, this study found that treatment with the extract activated HO-1 expression. Pharmacological approaches revealed that treatment with Zinc Protoporphyrin (ZnPP), an HO-1 inhibitor, resulted in an increase in lipid accumulation and a decrease in free glycerol levels. Finally, three adipogenic transcription factors, such as PPARγ, C/EBPα, and SREBP1, restored their expression in the presence of ZnPP. Analysis of chemical constituents revealed that the extract of D. divaricata is rich in 1,4-benzenediol, 7-tetradecenal, fucosterol, and n-hexadecanoic acid, which are known to have multiple pharmacological properties.

MiR-122/PPARβ axis is involved in hypoxic exercise and modulates fatty acid metabolism in skeletal muscle of obese rats

Hypoxic exercise is an effective intervention for obesity, because it promotes weight loss by regulating fatty acid (FA) metabolism. The regulation of peroxisome proliferator-activated receptor β (PPARβ) by miR-122 may be involved in this process, but the detailed mechanisms are unknown. In order to address this issue, we probed how miR-122 affected the expression of factors associated with FA metabolism in skeletal muscle of obese rats undergoing hypoxic training. By injecting adeno-associated virus 9 containing miR-122 overexpression vector or miR-122 inhibitor into skeletal muscles of rats with a 4-week hypoxic exercise regimen, the miR-122 expression level can be regulated. Body composition and blood lipid levels were analyzed, and PPARβ, carnitine palmitoyltransferase 1b (CPT1b), acetylCoA carboxylase 2 (ACC2), and FA synthase (FAS) mRNA and protein levels were evaluated using quantitative reverse transcription quantitative PCR(RT-qPCR) and Western blot analysis. We found that miR-122 overexpression increased low-density lipoprotein cholesterol (LDL-C) and triglyceride (TG) levels and decreased PPARβ, ACC2, and FAS expression. Conversely, miR-122 inhibition decreased TG level, increased high-density lipoprotein cholesterol (HDL-C) level, and upregulated PPARβ, ACC2, FAS, and CPT1b. These data indicated that the negative regulation of PPARβ by miR-122 promotes FA metabolism by altering the levels of the factors related to FA metabolism in skeletal muscle of obese rat under hypoxic training, thus providing molecular-level insight into the beneficial effects of this intervention.