Fatty Acid Binding Protein 4 Expression Research Articles

#2743 Impact of fatty acid-binding protein 4 on SUMOylation balance and renal tubular epithelial cell senescence in acute kidney injury

Abstract Background and Aims Dysregulated lipid metabolism and cellular senescence intricately interact in the context of acute kidney injury (AKI). Fatty acid-binding protein 4 (FABP4), a critical enzyme regulating lipid metabolism, potentially induces cell senescence through lipotoxicity in AKI. However, the specific mechanisms remain unclear. Method We established an ischemia-reperfusion-induced AKI (IRI-AKI) model in C57 mice and quantified and localized FABP4 expression in renal tissues using Western blotting (WB), qPCR, and immunofluorescence (IF). Oil Red O staining and beta-galactosidase staining were employed to assess lipid deposition and cellular senescence in tubular cells. The same measurements were repeated in IRI-AKI by knocking out Fabp4. Additionally, bulk mRNA sequencing and single-cell sequencing were conducted on renal tissues to analyze key molecules and related mechanisms. Results In vivo, we observed a significant decline in renal function, severe tubular epithelial damage, and abundant inflammation in the IRI-AKI group, which was noticeably alleviated in the Fabp4 knockout group (Fig. 1). Furthermore, we detected upregulation of FABP4 expression in the kidneys of IRI-AKI, primarily localized in the tubular epithelial cells. Fabp4 knockout effectively reduced tubular lipid accumulation and cellular senescence (Fig. 3). Through integrating mRNA and single-cell sequencing data, UBC9, an E2 enzyme involved in protein SUMOylation, emerged as a potential key regulator of cell senescence and AKI. WB and qPCR both confirmed the upregulation of UBC9 expression in IRI-AKI and its downregulation after Fabp4 knockout. Conversely, we observed that downregulated SUMOylation in IRI-AKI was upregulated in the Fabp4 knockout group (Fig. 4). Conclusion In conclusion, FABP4 modulates the expression of UBC9 and SUMOylation, thereby mediating cellular senescence and causing renal tubular cell damage in IRI-AKI. The mechanisms underlying FABP4’s regulation of UBC9 and the opposing trend between UBC9 and SUMOylation require further elucidation.

Abstract 5451: FOXC2 influences EMT-induced stem cell properties through regulation of FABP4 in breast cancer

Abstract Cancer stem cells (CSCs) pose a significant clinical challenge due to their inherent resistance to therapies and prevalence in cancer recurrence. CSCs have the ability for self-renewal and to adapt to different microenvironments during dissemination and seeding into distal organs, and these properties are critical in metastasis. We and others have shown that epithelial to mesenchymal transition (EMT) drives the acquisition of stem cell properties, particularly via the forkhead box protein C2 (FOXC2) transcription factor. FOXC2 is a master regulator of EMT and is necessary for the induction and maintenance of CSC properties. Importantly, FOXC2 is not expressed in most adult tissues except adipocytes, in which it dictates several aspects of lipid metabolism. Additionally, metabolic changes during EMT can equip cancer cells with enhanced survival mechanisms during tumor progression and metastasis. We hypothesized that FOXC2 regulates metabolism in EMT-induced cells, facilitating stem cell properties and metastatic competence. To determine the downstream metabolic genes regulated by FOXC2 and that enhance stemness properties in cancer cells, we performed gene expression profiling and identified fatty acid binding protein 4 (FABP4), which is known to be involved in lipid metabolism, especially in adipocytes and macrophages. We demonstrate that inhibition of FOXC2 expression using shRNA in EMT-induced cells markedly reduces FABP4 expression. In agreement with this, we found that the overexpression of FOXC2 expression rescues the expression of FABP4. In addition, FOXC2 binds to the FABP4 promoter and regulates its expression suggesting that FABP4 is a direct target of FOXC2. We demonstrate that both lipid uptake and CSC formation is inhibited in a dose-dependent manner using a FABP4-specific small molecule inhibitor. Additional experiments are underway to assess the role of FABP4 in CSC formation in vivo and in contributions to metastasis, using our small molecule inhibitor in mouse models of metastatic breast cancer. Overall, our data indicate that FABP4 is a key regulator of stemness in breast cancer cells and targeting the FOXC2—FABP4 axis may provide therapeutic benefits for breast cancer. This work also provides insights into the metabolic shifts that occur during EMT and CSC formation, which warrants further investigation into these mechanisms. Citation Format: Breanna R. Demestichas, Vinita Shivakumar, Esmeralda Ramirez-Peña, Joseph Taube, Petra den Hollander, Sendurai A. Mani. FOXC2 influences EMT-induced stem cell properties through regulation of FABP4 in breast 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 5451.

Radiomics features based on dual-area CT predict the expression levels of fatty acid binding protein 4 and outcome in hepatocellular carcinoma.

To evaluate the predictive value of tumor and peritumor radiomics in the fatty acid binding protein 4 (FABP4) expression levels and overall survival in patients with hepatocellular carcinoma. The genomic data of HCC patients were obtained from The Cancer Genome Atlas. The Dual-area CT images of corresponding patients were downloaded from The Cancer Imaging Archive, for radiomics feature extraction, model construction and prognosis analysis. Simultaneously, using patients from Sichuan Provincial People's Hospital, the prognostic value of the radiomics model in HCC patients was validated. In the TCIA database, the area under the curve (AUC) values of the volumes of interest (VOI)whole model in the training set and internal validation set were 0.812 and 0.754, respectively, and the AUC value of VOIwhole+periphery in the training set and internal validation set were 0.866 and 0.779, respectively. In the VOIwhole and the VOIwhole+periphery model of the independent cohort, there were significant differences in OS between the high and low rad-score groups (P = 0.009, P = 0.021, respectively). Significant positive correlations can be observed between FABP4 expression and correlations with rad-score of VOIwhole model (r = 0.691) and VOIwhole+periphery model (r = 0.732) in the independent cohort. Radiomics models of tumor and peritumor Dual-area CT images could predict stably the expression levels of FABP4 and may be helping in personalized treatment strategies.

High levels of fatty acid-binding protein 5 excessively enhances fatty acid synthesis and proliferation of granulosa cells in polycystic ovary syndrome.

Polycystic ovary syndrome (PCOS) is one of the most complex endocrine disorders in women of reproductive age. Abnormal proliferation of granulosa cells (GCs) is an important cause of PCOS. This study aimed to explore the role of fatty acid-binding protein 5 (FABP5) in granulosa cell (GC) proliferation in polycystic ovary syndrome (PCOS) patients. The FABP5 gene, which is related to lipid metabolism, was identified through data analysis of the gene expression profiles of GSE138518 from the Gene Expression Omnibus (GEO) database. The expression levels of FABP5 were measured by quantitative real-time PCR (qRT‒PCR) and western blotting. Cell proliferation was evaluated with a cell counting kit-8 (CCK-8) assay. Western blotting was used to assess the expression of the proliferation marker PCNA, and immunofluorescence microscopy was used to detect Ki67 expression. Moreover, lipid droplet formation was detected with Nile red staining, and qRT‒PCR was used to analyze fatty acid storage-related gene expression. We found that FABP5 was upregulated in ovarian GCs obtained from PCOS patients and PCOS mice. FABP5 knockdown suppressed lipid droplet formation and proliferation in a human granulosa-like tumor cell line (KGN), whereas FABP5 overexpression significantly enhanced lipid droplet formation and KGN cell proliferation. Moreover, we determined that FABP5 knockdown inhibited PI3K-AKT signaling by suppressing AKT phosphorylation and that FABP5 overexpression activated PI3K-AKT signaling by facilitating AKT phosphorylation. Finally, we used the PI3K-AKT signaling pathway inhibitor LY294002 and found that the facilitation of KGN cell proliferation and lipid droplet formation induced by FABP5 overexpression was inhibited. In contrast, the PI3K-AKT signaling pathway agonist SC79 significantly rescued the suppression of KGN cell proliferation and lipid droplet formation caused by FABP5 knockdown. FABP5 promotes active fatty acid synthesis and excessive proliferation of GCs by activating PI3K-AKT signaling, suggesting that abnormally high expression of FABP5 in GCs may be a novel biomarker or a research target for PCOS treatment.

Integrated multi-omic analysis identifies fatty acid binding protein 4 as a biomarker and therapeutic target of ischemia–reperfusion injury in steatotic liver transplantation

Background and aimsDue to a lack of donor grafts, steatotic livers are used more often for liver transplantation (LT). However, steatotic donor livers are more sensitive to ischemia–reperfusion (IR) injury and have a worse prognosis after LT. Efforts to optimize steatotic liver grafts by identifying injury targets and interventions have become a hot issue.MethodsMouse LT models were established, and 4D label-free proteome sequencing was performed for four groups: normal control (NC) SHAM, high-fat (HF) SHAM, NC LT, and HF LT to screen molecular targets for aggravating liver injury in steatotic LT. Expression detection of molecular targets was performed based on liver specimens from 110 donors to verify its impact on the overall survival of recipients. Pharmacological intervention using small-molecule inhibitors on an injury-related target was used to evaluate the therapeutic effect. Transcriptomics and metabolomics were performed to explore the regulatory network and further integrated bioinformatics analysis and multiplex immunofluorescence were adopted to assess the regulation of pathways and organelles.ResultsHF LT group represented worse liver function compared with NC LT group, including more apoptotic hepatocytes (P < 0.01) and higher serum transaminase (P < 0.05). Proteomic results revealed that the mitochondrial membrane, endocytosis, and oxidative phosphorylation pathways were upregulated in HF LT group. Fatty acid binding protein 4 (FABP4) was identified as a hypoxia-inducible protein (fold change > 2 and P < 0.05) that sensitized mice to IR injury in steatotic LT. The overall survival of recipients using liver grafts with high expression of FABP4 was significantly worse than low expression of FABP4 (68.5 vs. 87.3%, P < 0.05). Adoption of FABP4 inhibitor could protect the steatotic liver from IR injury during transplantation, including reducing hepatocyte apoptosis, reducing serum transaminase (P < 0.05), and alleviating oxidative stress damage (P < 0.01). According to integrated transcriptomics and metabolomics analysis, cAMP signaling pathway was enriched following FABP4 inhibitor use. The activation of cAMP signaling pathway was validated. Microscopy and immunofluorescence staining results suggested that FABP4 inhibitors could regulate mitochondrial membrane homeostasis in steatotic LT.ConclusionsFABP4 was identified as a hypoxia-inducible protein that sensitized steatotic liver grafts to IR injury. The FABP4 inhibitor, BMS-309403, could activate of cAMP signaling pathway thereby modulating mitochondrial membrane homeostasis, reducing oxidative stress injury in steatotic donors.Graphical

High-expression of FABP4 in Tubules is a Risk Factor for Poor Prognosis in DKD Patients.

Lipid metabolism imbalance is involved in the mechanism of renal tubular injury in diabetic kidney disease (DKD). Fatty acid binding protein 4 (FABP4) has been reported to participate in cellular lipid toxicity. However, the expression of FABP4 in renal tissues of DKD and its correlation with clinical/ pathological parameters and prognosis have not been studied. A retrospective cohort study was conducted in 108 hospitalized Type 2 diabetes (T2D) patients with renal injury, including 70 with DKD and 38 with NDKD (non-DKD). Clinical features, pathological findings, and follow-up parameters were collected. Serum and urine FABP4 were detected by ELISA. An immunohistochemistry stain was used to determine FABP4 in renal tubulointerstitium. A double immunofluorescence stain was employed to assess FABP4- and CD68-positive macrophages. Correlation analysis, logistic regression models, receiver operating characteristic (ROC), and Kaplan-Meier survival curve were performed for statistical analysis. DKD patients had increased expression of FABP4 and ectopic fat deposition in tubules. As shown by correlation analyses, FABP4 expression in renal tubules was positively correlated with UNAG (r=0.589, p=0.044) and ESRD (r=0.740, p=0.004). Multivariate regression analysis revealed that UNAG level was correlated with FABP4 expression level above median value (odds ratio:1.154, 95% confidence interval:1.009-1.321, p=0.037). High-expression of FABP4 in renal tubules of DKD was at an increased risk of ESRD. Increased FABP4 expression in inflammatory cells was also associated with ESRD in DKD. High-expression of FABP4 is involved in the pathogenesis of renal tubular lipid injury and is a risk factor for poor prognosis in DKD patients.

Effect of Semaglutide on High-Fat-Diet-Induced Liver Cancer in Obese Mice.

This study aims to investigate the impact of semaglutide on the expression of liver cancer proteins in obese mice induced by a high-fat diet. Sixteen obese mice were randomly divided into two groups: the high-fat diet group and the semaglutide group, each consisting of eight mice. Additionally, eight normal male mice were included as the control group. Serum samples were collected, and a differential expression analysis of total proteins in adipose tissue was performed using quantitative tandem mass spectrometry (TMT) in combination with liquid chromatography-tandem mass spectrometry (LC-MS/MS). Significant differential proteins were identified and subjected to a bioinformatics analysis. The findings revealed that these differential proteins, namely, integrin αV (ITGAV), laminin γ1 (LAMC1), fatty acid-binding protein 5 (FABP5), and lipoprotein lipase (LPL), regulate the occurrence and development of liver cancer by participating in the extracellular matrix (ECM) signaling pathway and the peroxisome proliferator-activated receptor (PPAR) signaling pathway. Notably, semaglutide can decelerate the progression of liver cancer by inducing the expression of ITGAV, LAMC1, FABP5, and LPL in the adipose tissue of obese mice.

Fatty Acid Binding Protein-4 Silencing Inhibits Ferroptosis to Alleviate Lipopolysaccharide-induced Injury of Renal Tubular Epithelial Cells by Blocking Janus Kinase 2/Signal Transducer and Activator of Transcription 3 Signaling.

Sepsis-induced kidney injury (SAKI) has been frequently established as a prevailing complication of sepsis which is linked to unfavorable outcomes. Fatty acid-binding protein-4 (FABP4) has been proposed as a possible target for the treatment of SAKI. In the current work, we aimed to explore the role and underlying mechanism of FABP4 in lipopolysaccharide (LPS)-induced human renal tubular epithelial cell damage. In LPS-induced human kidney 2 (HK2) cells, FABP4 expression was tested by the reverse transcription-quantitative polymerase chain reaction and Western blot. Cell counting kit-8 method assayed cell viability. Inflammatory levels were detected using the enzyme-linked immunosorbent assay. Immunofluorescence staining measured the nuclear translocation of nuclear factor kappa B p65. Thiobarbituric acid-reactive substances assay and C11 BODIPY 581/591 probe were used to estimate the level of cellular lipid peroxidation. Fe2+ content was examined by the kit. In addition, the expression of proteins related to inflammation-, ferroptosis- and Janus kinase 2 (JAK2)/signal transducer, and activator of transcription 3 (STAT3) signaling was detected by the Western blot analysis. The results revealed that FABP4 was significantly upregulated in LPS-treated HK2 cells, the knockdown of which elevated the viability, whereas alleviated the inflammation and ferroptosis in HK2 cells challenged with LPS. In addition, down-regulation of FABP4 inactivated JAK2/STAT3 signaling. JAK2/STAT3 stimulator (colivelin) and ferroptosis activator (Erastin) partially restored the effects of FABP4 interference on LPS-triggered inflammation and ferroptosis in HK2 cells. Together, FABP4 knockdown inhibited ferroptosis to alleviate LPS-induced injury of renal tubular epithelial cells through suppressing JAK2/STAT3 signaling.

Expression and Significance of FABP4 and miRNA-182 in Plasma and Placental Tissues of Patients with Hypertensive Disorders During Pregnancy

Objective: To investigate the expression and significance of fatty acid binding protein 4 (FABP4) and microRNA-182 (miRNA-182) in the plasma and placental tissues of patients with hypertensive disorders in pregnancy (HDIP). Methods: 141 mothers admitted to The Affiliated Hospital of Hebei University between January 2019 and December 2022 were selected. The patients were divided into the observation group (61 HDIP patients) and control group (80 healthy pregnant women) based on the criteria for determining hypertensive diseases in pregnancy. The differences in the expression of FABP4 and miRNA-182 between the two groups were compared. Results: The expression levels of FABP4 in plasma and placental tissues of the observation group were significantly higher than those of the control group (P&lt; 0.05). Conversely, the expression levels of miRNA-182 in the plasma and placental tissues of the observation group were significantly lower than those of the control group (P &lt; 0.05). A negative correlation was observed between the plasma level of FABP4 and miRNA-182. However, there was no significant correlation between the expression levels of FABP4 and miRNA-182 in placental tissues. While plasma FABP4 levels exhibited a positive correlation with the severity of HDIP, miRNA-182 expression levels did not significantly correlate with the severity of HDIP. Conclusion: The expression of FABP4 and miRNA-182 in plasma and placental tissues of patients with HDIP differed significantly from that of healthy pregnant women. This observation may provide a new perspective on the pathogenesis of HDIP.

FABP5 can substitute for androgen receptor in malignant progression of prostate cancer cells.

Fatty acid‑binding protein 5 (FABP5) and androgen receptor (AR) are critical promoters of prostate cancer. In the present study, the effects of knocking out the FABP5 or AR genes on malignant characteristics of prostate cancer cells were investigated, and changes in the expression of certain key proteins in the FABP5 (or AR)‑peroxisome proliferator activated receptor‑γ (PPARγ)‑vascular endothelial growth factor (VEGF) signaling pathway were monitored. The results obtained showed that FABP5‑ or AR‑knockout (KO) led to a marked suppression of the malignant characteristics of the cells, in part, through disrupting this signaling pathway. Moreover, FABP5 and AR are able to interact with each other to regulate this pathway, with FABP5 controlling the dominant AR splicing variant 7 (ARV7), and AR, in return, regulates the expression of FABP5. Comparisons of the RNA profiles revealed the existence of numerous differentially expressed genes (DEGs) comparing between the parental and the FABP5‑ or AR‑KO cells. The six most abundant changes in DEGs were found to be attributable to the transition from androgen‑responsive to androgen‑unresponsive, castration‑resistant prostate cancer (CRPC) cells. These findings have provided novel insights into the complex molecular pathogenesis of CRPC cells, and have demonstrated that interactions between FABP5 and AR contribute to the transition of prostate cancer cells to an androgen‑independent state. Moreover, gene enrichment analysis revealed that the most highly enriched biological processes associated with the DEGs included those responsive to fatty acids, cholesterol and sterol biosynthesis, as well as to lipid and fatty acid transportation. Since these pathways regulated by FABP5 or AR may be crucial in terms of transducing signals for cancer cell progression, targeting FABP5, AR and their associated pathways, rather than AR alone, may provide a new avenue for the development of therapeutic strategies geared towards suppressing the malignant progression to CRPC cells.

Fatty Acid Binding Protein FABP5 Inhibition Activates Retinoic Acid Signaling and Induces Differentiation in Acute Myeloid Leukemia

Introduction. Accumulation of undifferentiated immature blood cells is a hallmark of myeloid neoplasia (MN), and indicates decoupling of terminal-differentiation from proliferation. Therefore, reinstating to a programmed state of differentiation (lineage-maturation) is a treatment approach, exemplified by all-trans retinoic acid (ATRA) treatment of acute promyelocytic leukemias that contain t(15;17) (q24;q21). This translocation inactivates retinoic acid receptor alpha (RARA) due to its fusion with promyelocytic leukemia PML resulting in PML-RARA. This fusion gene responsible for cellular transformation confers sensitivity to treatment with ATRA. However, ATRA's therapeutic-success in APLs has not extended to other AML phenotypes. We and others have shown that RA is a ligand, not just for RARs, but also pro-survival peroxisome proliferator-activated receptor β/δ (PPARβ/δ), a member of the nuclear hormone receptor superfamily transcription factors that regulate several metabolic pathways. RA is transported by two carrier proteins, cellular retinoid-binding protein CRABP2, or fatty-acid-binding protein 5 (FABP5), depending on cellular context. These carriers preferentially deliver RA to RARs versus PPARβ/δ respectively. Somatic lesions such as loss of function TET2 or Tp53 mutation, NPM1 and ASXL1 mutations, are associated with upregulated FABP5 and downregulated CRABP2. In AML cells with these mutations, therefore, the high FABP5 can be expected to deliver RA to PPARβ/δ instead of RAR, to potentially contribute to decoupling of differentiation from proliferation. We therefore investigated this possibility and its treatment implications. Methods. Overexpression, gene knockdown/knockout (ko/kd) of key regulators of RAR and PPARβ/δ were generated in specific genotypes of AMLs. Small molecule inhibitors of FABP5 were used to further interrogate the pathways in a translationally relevant manner, both in vitro, in both malignant and normal cells, and in pre-clinical in vivo models. Results. Gene expression analysis of AMLs in the Beat AML and TCGA databases demonstrated upregulation of pro-survival PPARβ/δ target genes including FABP5 and downregulation of differentiation promoting RAR pathway genes including CRABP2in AMLs 1. In particular, FABP5 was highly expressed in AMLs containing TET2 NPM1, TP53 and ASXL1 mutations. AML cell lines OCIAML3 (NPM1 ins/DNMT3a R882c), OCIAML5 (ASXL1 Y591*/TET2 S825*), THP1 (TP53 del) and isogenic CRISPR engineered K562 (TET2 +/+/TET2 -/-) demonstrated 4-100 fold higher FABP5 expression compared to normal myeloid precursors. These models did not differentiate in response to pharmacologic concentrations of ATRA. To test if FABP5 is necessary and sufficient to maintain the undifferentiated state of these cells in the presence of ATRA, we genetically inactivated FABP5 either using RNAi (kd) or CRISPR (ko). FABP5 knockdown or knockout cells created sensitivity to ATRA, resulting in terminal-differentiation as observed by flow cytometry for terminal-differentiation markers ( See Figure). FABP5 was then inhibited using highly specific small molecule FABP5 inhibitors with a 56-fold higher affinity compared to ATRA 1: combination of iFABP5 and ATRA induced terminal-differentiation with significant upregulation of the monocyte lineage markers CD14 and granulocyte lineage marker CD11b measured by flow cytometry at 72h. Toinvestigatewhether knocking down FABP5 and ATRA treatment affects gene expression changes we performed transcriptomic profiling of OCIMAL3 Scr, shFABP5 cells treated with or without ATRA using whole exome RNA sequencing. Treatment of FABP5 kd OCIMAL3 cells with ATRA resulted in the upregulation of RAR pathways genes and downregulation of proliferation promoting genes. Conclusion. In sum, recoupling of lineage-maturation to proliferation can be achieved in non-APL AMLs that highly express the RA carrier FABP5, by inhibiting FABP5 using small molecules. This candidate treatment modality is distinct from cytotoxic chemotherapy, and importantly, can spare normal hematopoiesis.

CNSC-23. ROLE OF GLIAL PH-DEPENDENT METABOLIC REGULATION IN GLIOMA PROGRESSION

Abstract Malignant glioma is a devastating brain cancer with barely any improvement in prognosis over decades. One key mechanism for glioma invasiveness is the dynamic metabolic communication between glioma and surrounding cells shaping the tumor microenvironment. While extracellular acidosis is a pathological hallmark of glioma, pH-dependent mechanisms underlying tumor metabolic reprogramming remain poorly defined. Our study focuses on a glial-enriched sodium-bicarbonate cotransporter, Slc4a4, which was previously identified as intracellular and extracellular pH regulator. While Slc4a4-mediated pH regulation is important for physiological brain function and responses after injury, its role in glioma progression is largely undefined. To begin addressing this question, we analyzed The Cancer Genome Atlas (TCGA) database, finding high Slc4a4 expression correlates with prolonged survival in glioma patients. Corroboratively, Slc4a4 gain-of-function (GOF) drastically decreases tumorigenesis and angiogenesis in patient-derived-xenograft and CRISPR-mediated de novo mouse glioma models. These results are further complemented by loss-of-function (LOF) studies using glial-specific Slc4a4 knockout mice, suggesting an inhibitory role of Slc4a4 in glioma growth and associated angiogenesis. A combination of unbiased metabolomic and cancer-targeted proteomic profiling of Slc4a4 GOF/LOF mouse glioma reveals an inverse correlation between Slc4a4 and a key lipid metabolism protein, FABP5 (Fatty Acid Binding Protein 5), which is coupled with dysregulated lipid metabolism. Moreover, analysis of TCGA database shows inverse expression and survival correlation of Slc4a4 and FABP5 in glioma patients. Further functional study shows that overexpression of FABP5 reverses Slc4a4’s inhibitory effects on glioma growth in immunocompetent mouse glioma model, supporting a functional antagonism between Slc4a4 and FABP5. Together, our study indicates that Slc4a4 plays a protective role in glioma progression by regulating metabolic activities, providing critical insights into pH-dependent metabolic reprogramming in glioma.

Whole genome methylation sequencing reveals epigenetic landscape and abnormal expression of FABP5 in extramammary Paget's disease.

Extramammary Paget's disease (EMPD) is a rare cutaneous malignant tumor with a high recurrence rate after surgery. However, the genetic and epigenetic alterations underlying its pathogenesis remain unknown. DNA methylation is an important epigenetic modification involved in many biological processes. In this study, enzymatic methyl-sequencing (EM-seq) technique was used to investigate the landscape of genome-wide DNA methylation from three pairs of tumor tissues and adjacent tissues of patients with EMPD. Additionally, we conducted histopathological examinations to assess the expression of fatty acid-binding protein 5 (FABP5) in another three paired samples from EMPD patients. The cluster analysis showed the good quality of the samples. A differential methylation region (DMR) heat map was used to quantitatively characterize genome-wide methylation differences between tumors and controls. Global DNA methylation level is lower in EMPD tissue compared to matched controls, indicating that DNA methylation discriminates between tumor and normal skin. And the top hypomethylation gene on the promoter region in tumor tissues was FABP5 on chromosome 8 with 38.44% decreased median methylation. We next identified the expression of FABP5 in paired tumors and adjacent tissues in three additional patients with EMPD. Immunofluorescence results showed FABP5 highly expressed in tumor tissues and co-located with CK7, CK20 and EMA. GO and KEGG enrichment analysis showed DMR genes on promoter are mainly enriched in the calcium ion transport, GTPase mediated signal transduction, Rap1 signaling pathway and GnRH signaling pathway. Taken together, our findings provide the first description of the whole genome methylation map of EMPD and identify FABP5 as a pathogenic target of EMPD.

NRF2 Deficiency Attenuates Diabetic Kidney Disease in Db/Db Mice via Down-Regulation of Angiotensinogen, SGLT2, CD36, and FABP4 Expression and Lipid Accumulation in Renal Proximal Tubular Cells.

The role(s) of nuclear factor erythroid 2-related factor 2 (NRF2) in diabetic kidney disease (DKD) is/are controversial. We hypothesized that Nrf2 deficiency in type 2 diabetes (T2D) db/db mice (db/dbNrf2 knockout (KO)) attenuates DKD progression through the down-regulation of angiotensinogen (AGT), sodium-glucose cotransporter-2 (SGLT2), scavenger receptor CD36, and fatty -acid-binding protein 4 (FABP4), and lipid accumulation in renal proximal tubular cells (RPTCs). Db/dbNrf2 KO mice were studied at 16 weeks of age. Human RPTCs (HK2) with NRF2 KO via CRISPR-Cas9 genome editing and kidneys from patients with or without T2D were examined. Compared with db/db mice, db/dbNrf2 KO mice had lower systolic blood pressure, fasting blood glucose, kidney hypertrophy, glomerular filtration rate, urinary albumin/creatinine ratio, tubular lipid droplet accumulation, and decreased expression of AGT, SGLT2, CD36, and FABP4 in RPTCs. Male and female mice had similar results. NRF2 KO attenuated the stimulatory effect of the Nrf2 activator, oltipraz, on AGT, SGLT2, and CD36 expression and high-glucose/free fatty acid (FFA)-stimulated lipid accumulation in HK2. Kidneys from T2D patients exhibited markedly higher levels of CD36 and FABP4 in RPTCs than kidneys from non-diabetic patients. These data suggest that NRF2 exacerbates DKD through the stimulation of AGT, SGLT2, CD36, and FABP4 expression and lipid accumulation in RPTCs of T2D.

FattyAcid-Binding Protein 4 is Essential for the Inflammatory and Metabolic Response of Microglia to Lipopolysaccharide.

Prolonged activation of microglia leads to excessive release of proinflammatory mediators, which are detrimental to brain health. Therefore, there are significant efforts to identify pathways mediating microglial activation. Recent studies have demonstrated that fatty acid-binding protein 4 (FABP4), a lipid binding protein, is a critical player in macrophage-mediated inflammation. Given that we have previously identified FABP4 in microglia, the aim of this study was to assess whether FABP4 activity contributed to inflammation, metabolism and immune function (i.e. immunometabolism) in immortalised mouse microglia (BV-2 cells) using the proinflammatory stimulus lipopolysaccharide (LPS) to induce general microglial activation. Microglial FABP4 expression was significantly increased following exposure to LPS, an outcome associated with a significant increase in microglial proliferation rate. LPS-stimulated BV-2 microglia demonstrated a significant increase in the production of reactive oxygen species (ROS) and tumour necrosis factor-alpha (TNF-α), phosphorylation of c-Jun N-terminal kinase (JNK), increased expression of Toll-like receptor 4 (TLR4), and reduced expression of uncoupling protein 2 (UCP2), all of which were reversed following FABP4 genetic silencing and chemical inhibition with BMS309403. The oxidation rate of 3H-oleic acid and microglial uptake of 3H-2-deoxy-D-glucose were modulated with LPS activation, processes which were restored with genetic and chemical inhibition of FABP4. This is the first study to report on the critical role of FABP4 in mediating the deleterious effects of LPS on microglial immunometabolism, suggesting that FABP4 may present as a novel therapeutic target to alleviate microglia-mediated neuroinflammation, a commonly reported factor in multiple neurodegenerative diseases.

Clinicopathological significance and prognostic implication of nuclear fatty acid-binding protein 4 expression in colorectal cancer

This study aimed to evaluate the clinicopathological significance and prognostic role of fatty acid-binding protein 4 (FABP4) expression in colorectal cancer (CRC). Nuclear expression of FABP4 was investigated by immunohistochemistry for FABP4 on 246 human CRC tissues. The correlations between FABP4 expression, and clinicopathological characteristics and survival, was evaluated in patients with CRC. FABP4 was expressed in 91 of the 246 CRC tissues (37.0%). FABP4 expression was significantly correlated with older age, right-sided colon cancer, perineural invasion, higher pT stage, lymph node metastasis, and higher pTNM stage. However, there was no significant correlation between FABP4 expression and sex, tumor size, tumor differentiation, vascular or lymphatic invasion, or distant metastasis. Nuclear FABP4 expression was not significantly correlated with cytoplasmic FABP4 expression (P = 0.412). FABP4 expression was significantly correlated with nuclear pNF-κB expression (P = 0.001), and was significantly higher in CRC with a low immunoscore than in CRC with a high immunoscore (P < 0.001). There were significant correlations between FABP4 expression and worse overall and recurrence-free survival rates (P < 0.001 and P = 0.007, respectively). FABP4 expression was significantly correlated with aggressive tumor behaviors and pathological characteristics. In addition, patients with CRC with FABP4 expression had worse survival rates.

FABP4 in LSECs promotes CXCL10-mediated macrophage recruitment and M1 polarization during NAFLD progression

Background and aimsNon-alcoholic liver disease (NAFLD) is emerging as the leading cause of end-stage liver disease with a serious threat to global health burden. Fatty acid-binding protein 4 (FABP4) is closely associated with metabolic syndromes. We aimed to explore the potential mechanisms of FABP4 in NAFLD progression. Materials and methodsFor NAFLD mice, animals were fed with high fat diet (HFD) for 20 weeks. The assays of hematoxylin and eosin, Sirius Red, oil red O staining and immunohistology were performed to evaluate hepatic pathology. Flow cytometric analysis was used to distinguish macrophage subtypes. ResultsSerum FABP4 level was positively correlate with the severity of hepatic steatosis in NAFLD patients. FABP4 expression was mainly distributed in liver sinusoidal endothelial cells (LSECs), which was significantly increased in HFD mice. The level of CXCL10 was positively correlated with FABP4 at mRNA and serum level. FABP4 inhibition resulted in decreased expression of CXCL10. The percentage of M1 macrophage and CXCR3+ cells in infiltrated macrophage was increased in liver of HFD mice. Inhibition of FABP4 ameliorated HFD-induced M1 macrophage polarization as well as CXCR3+ macrophages recruitment. Recombinant CXCL10 and co-culturing with TMNK-1 stimulated macrophage toward M1 polarization, which could be reversed by CXCR3 inhibitor. Palmitic acid treatment resulted in increased nuclear P65 expression, which could be reversed by inhibiting FABP4. Cxcl10 expression was dramatically suppressed by NF-κB inhibitor. ConclusionsFABP4 in LSECs may play a pathogenic role in NAFLD course by promoting CXCL10-mediated macrophage M1 polarization and CXCR3+ macrophage infiltration via activating NF-κB/p65 signaling.

FABP4 Regulates Cell Proliferation, Stemness, Apoptosis, and Glycolysis in Colorectal Cancer via Modulating ROS/ERK/mTOR Pathway.

Colorectal cancer is a common digestive tract malignancy. This study aimed to expound the functional role of fatty-acid-binding protein 4 (FABP4) and the potential underlying mechanisms in the development of colorectal cancer. Several techniques were utilized to investigate the role of FABP4 in colorectal cancer. FABP4 mRNA expression was quantified using Real time-quantitative PCR (RT-qPCR). Cell counting kit-8 (CCK-8), 5-ethynyl-2'-deoxyuridine (EdU), sphere formation assays and flow cytometry evaluated cell growth, stemness, and apoptosis in SW480 and HT29 cells. Glycolysis was assessed via extracellular acidification rate (ECAR) , lactate production, glucose uptake, adenosine triphosphate (ATP)/adenosine 5'-diphosphate (ADP) ratio, and Glut1 and Elevated lactate dehydrogenase A (LDHA) protein expression. Reactive oxygen species (ROS) levels were analyzed by flow cytometry. Western blot measured the protein expression of FABP4, Proliferating cell nuclear antigen (PCNA), Bax, Bcl-2, Glut1, LDHA, stemness makers (Sox2, Oct4, and ALDHA1), and extracellular regulated protein kinase (ERK)/mammalian target of rapamycin (mTOR) pathway proteins. In vivo experiments, BALB/c nude mice (n = 12) were inoculated with 200 μL HT29 cells (5 × 106 cells) transfected with sh-FABP4 or short hairpin (sh)-negative control (NC), forming two groups with 6 mice each. The in vivo mice tumor model allowed for evaluating FABP4's impact on tumor growth. FABP4 was significantly upregulated in colorectal cancer tissues and cells (p < 0.05). FABP4 knockdown markedly inhibited cell proliferation, stemness, and glycolysis, while promoting apoptosis in these cells (p < 0.05). Additionally, FABP4 depletion led to a significant increase in ROS level (p < 0.05). However, N-acetyl-L-cysteine (NAC) (p < 0.05), a ROS scavenger, mitigates these effects. Furthermore, the effects of FABP4 depletion on cell growth, stemness, glycolysis, and apoptosis in colorectal cancer cells were also retarded by NAC (p < 0.05). Notably, FABP4 knockdown also suppressed the ERK/mTOR pathway, suggesting its regulation via ROS (p < 0.05). In vivo study results showed, FABP4 depletion significantly curbed tumor growth in colorectal cancer (p < 0.05). These results suggest that FABP4 depletion inhibits colorectal cancer progression by modulating cell growth, stemness, glycolysis and apoptosis. This regulation occurs through the ROS/ERK/mTOR pathway.

61 Altering Fatty Acid Ratios to Regulate Adipocyte HypertrophyIn Vitro

Abstract Adipose tissue deposition in cattle can prove advantageous and detrimental when grading carcasses. Intramuscular fat (IMF) deposition, or marbling, can enhance sensory qualities such as tenderness and flavor, whereas subcutaneous fat deposition reduces carcass red meat yield. Little is known about how different mixtures of fatty acids influence adipocyte hypertrophy in vitro. Primary preadipocytes were isolated from subcutaneous fat harvested from Angus-cross yearling steers. Preadipocytes were propagated and differentiated into adipocytes. Then eight different mixtures with pairs of different fatty acids (C16:0, C18:0, C18:1cis-9, and C18:2) at varying ratios were added to the cultures. Each individual mix was applied at the same total molar concentration. At the end of the 4-day treatment period, cells were harvested for future RNA extraction and fatty acid analysis. The expression of critical adipogenic and lipogenic genes was evaluated. Mixes exhibited differences in total fatty acid content (ug) which would not only confirm that the adipocytes are differentiated but also translates to potential differences in hypertrophic growth between mixes (P &amp;lt; 0.05). The control exhibited significantly different fatty acid binding protein-4 (FABP4) and stearoyl co-A desaturase-1 (SCD-1) expressions in comparison with all other mixes (P &amp;lt; 0.05). The expression of FABP4 in mix 3 (75% C16:0-25% C18:1cis-9) was substantially different from all other treatments aside from a similarly structured mix (50% C16:0-50% C18:1cis-9; P &amp;lt; 0.05). The SCD-1 was upregulated, while peroxisome proliferating-activated receptor gamma (PPARy) was downregulated in the fully saturated fatty acid mix, mix 5 (50% C16:0- 50% C18:0), compared with all other treatments (P &amp;lt; 0.05). All three mixes containing C18:2cis-9 exhibited a greater relative expression of sterol-regulatory element-binding protein (SREB) than all other mixes (P &amp;lt; 0.05). The supplementation of fatty acids at different ratios can affect molecular mechanisms and hypertrophic growth in differentiated bovine adipocytes. Further investigation of this type of fatty acid supplementation could impact the future understanding of hypertrophic growth in the bovine adipocyte and provide further insight into the alteration of fat deposition in cattle.

Inhibition of Fatty Acid β-Oxidation by Fatty Acid Binding Protein 4 Induces Ferroptosis in HK2 Cells Under High Glucose Conditions.

Ferroptosis, which is caused by an iron-dependent accumulation of lipid hydroperoxides, is a type of cell death linked to diabetic kidney disease (DKD). Previous research has shown that fatty acid binding protein 4 (FABP4) is involved in the regulation of ferroptosis in diabetic retinopathy. The present study was constructed to explore the role of FABP4 in the regulation of ferroptosis in DKD. We first detected the expression of FABP4 and proteins related to ferroptosis in renal biopsies of patients with DKD. Then, we used a FABP4 inhibitor and small interfering RNA to investigate the role of FABP4 in ferroptosis induced by high glucose in human renal proximal tubular epithelial (HG-HK2) cells. In kidney biopsies of DKD patients, the expression of FABP4 was elevated, whereas carnitine palmitoyltransferase-1A (CP-T1A), glutathione peroxidase 4, ferritin heavy chain, and ferritin light chain showed reduced expression. In HG-HK2 cells, the induction of ferroptosis was accompanied by an increase in FABP4. Inhibition of FABP4 in HG-HK2 cells changed the redox state, sup-pressing the production of reactive oxygen species, ferrous iron (Fe2+), and malondialdehyde, increasing superoxide dismutase, and reversing ferroptosis-associated mitochondrial damage. The inhibition of FABP4 also increased the expression of CPT1A, reversed lipid deposition, and restored impaired fatty acid β-oxidation. In addition, the inhibition of CPT1A could induce ferroptosis in HK2 cells. Our results suggest that FABP4 mediates ferroptosis in HG-HK2 cells by inhibiting fatty acid β-oxidation.