acyl-CoA Synthetase Short-chain Family Member Research Articles

ACLY and ACSS2 link nutrient-dependent chromatin accessibility to CD8 T cell effector responses.

Coordination of cellular metabolism is essential for optimal T cell responses. Here, we identify cytosolic acetyl-CoA production as an essential metabolic node for CD8 T cell function in vivo. We show that CD8 T cell responses to infection depend on acetyl-CoA derived from citrate via the enzyme ATP citrate lyase (ACLY). However, ablation of ACLY triggers an alternative, acetate-dependent pathway for acetyl-CoA production mediated by acyl-CoA synthetase short-chain family member 2 (ACSS2). Mechanistically, acetate fuels both the TCA cycle and cytosolic acetyl-CoA production, impacting T cell effector responses, acetate-dependent histone acetylation, and chromatin accessibility at effector gene loci. When ACLY is functional, ACSS2 is not required, suggesting acetate is not an obligate metabolic substrate for CD8 T cell function. However, loss of ACLY renders CD8 T cells dependent on acetate (via ACSS2) to maintain acetyl-CoA production and effector function. Together, ACLY and ACSS2 coordinate cytosolic acetyl-CoA production in CD8 T cells to maintain chromatin accessibility and T cell effector function.

Comprehensive pan-cancer analysis of ACSS3 as a biomarker for prognosis and immunotherapy response

BackgroundACSS3 (acyl-CoA synthetase short-chain family member 3) is found in numerous tissues and is linked to tumor cell type development and metastasis. MethodsWe conducted a comprehensive pan-cancer analysis of ACSS3. The TCGA (Cancer Genome Atlas), CPTAC (Clinical Proteomic Tumor Analysis Consortium), and HPA databases were used to ascertain the connection between ACSS3 and various types of tumors. Genes in the TCGA database would be identified using cBioPortal queries, and their transcriptome expression would then be verified using GEO data. ACSS3 expression and cellular localization in various tumor tissues of most cancer types were analyzed using single-cell sequencing data from the TISCH database. According to HPA and CPTAC databases, we analyzed and evaluated protein expression levels. Predictive analysis based on precise survival data of ACSS3 expression levels for 26 cancer types predicted using the TCGA database. Furthermore, we investigated the relationship between ACSS3 and immune microenvironments in different tumor tissues using the TIMER and TISCH databases. CellMiner, GDSC, and CTRP data would clarify the relationship between ACSS3 and drug resistance and explore the chemicals that affect ACSS3 expression. The final part of our study explored and validated the role ACSS3 played in glioma proliferation, migration, and invasion. ResultsACSS3 is differentially expressed in various tumors and exhibits early diagnostic value. ACSS3 expression is associated with clinical features, and high ACSS expression anticipates a worse prognosis in multiple tumors and may impact drug sensitivity. The changes in the immunosuppressive microenvironment of gliomas are closely related to the upregulation of ACSS3. ConclusionsACSS3 is a novel biomarker for forecasting different human cancer prognoses, as it can influence the biological process by modulating the immune microenvironment. ACSS3 is a critical prognostic factor for glioma and is related to its proliferation, migration, and invasion.

The attenuated hepatic clearance of propionate increases cardiac oxidative stress in propionic acidemia.

Propionic acidemia (PA), arising from PCCA or PCCB variants, manifests as life-threatening cardiomyopathy and arrhythmias, with unclear pathophysiology. In this work, propionyl-CoA metabolism in rodent hearts and human pluripotent stem cell-derived cardiomyocytes was investigated with stable isotope tracing analysis. Surprisingly, gut microbiome-derived propionate rather than the propiogenic amino acids (valine, isoleucine, threonine, and methionine) or odd-chain fatty acids was found to be the primary cardiac propionyl-CoA source. In a Pcca-/-(A138T) mouse model and PA patients, accumulated propionyl-CoA and diminished acyl-CoA synthetase short-chain family member 3 impede hepatic propionate disposal, elevating circulating propionate. Prolonged propionate exposure induced significant oxidative stress in PCCA knockdown HL-1 cells and the hearts of Pcca-/-(A138T) mice. Additionally, Pcca-/-(A138T) mice exhibited mild diastolic dysfunction after the propionate challenge. These findings suggest that elevated circulating propionate may cause oxidative damage and functional impairment in the hearts of patients with PA.

#2394 Inhibition of ACSS2-mediated H3K9 crotonylation alleviates kidney fibrosis via IL-1β-dependent macrophage activation and tubular cell senescence

Abstract Background and Aims Histone lysine crotonylation (Kcr), a novel posttranslational modification, is widespread as acetylation (Kac); however, its roles are largely unknown in kidney fibrosis. Method Human renal tissue, fixed in formaldehyde, and embedded in paraffin, was selected from the files of the Service of Pathology of West China Hospital, Sichuan University.ACSS2 knockout mice and tubular specific knock out of ACSS2 mice in C57BL/6N background and wild-type littermates (WT) were used to induce the FAN and UUO model or as control groups (n = 6 each). In the parallel study, C57BL/6N mice were randomly divided into three groups: control, FAN/UUO and FAN/UUO+ACSS2 inhibitor (n = 6 each). At day 7, mice were sacrificed and the serum creatinine, BUN and urinary albumin/creatinine ratio (ACR) were tested. Renal histological injury was measured by HE and Masson's trichrome staining. SA-β-gal staining was used to check cellular senescence. Renal tissues of the mice were analyzed by western blot and qPCR assay for the expression of ACSS2, H3K9cr, fibrosis and cellular senescence related proteins and genes. RNA-sequencing and ChIP-sequencing combined with in vitro cell experiments were used to figure out the underlying mechanism. Results In this study, we reported that histone Kcr of tubular epithelial cells was abnormally elevated in fibrotic kidneys. By screening these crotonylated/acetylated factors, a crotonyl-CoA-producing enzyme ACSS2 (acyl-CoA synthetase short chain family member 2) was found to remarkably increase histone 3 lysine 9 crotonylation (H3K9cr) level without influencing H3K9ac in kidneys and tubular epithelial cells. The integrated analysis of ChIP-seq and RNA-seq of fibrotic kidneys revealed that the hub proinflammatory cytokine IL-1β, which is regulated by H3K9cr, play crucial roles in fibrogenesis. Furthermore, genetic and pharmacologic inhibition of ACSS2 both suppressed H3K9cr-mediated IL-1β expression, which thereby alleviated IL-1β-dependent macrophage activation and tubular cell senescence to delay renal fibrosis. Conclusion Collectively, our findings uncover that H3K9cr exerts a critical, previously unrecognized role in kidney fibrosis, where ACSS2 represents an attractive drug target to slow fibrotic kidney disease progression.

Inhibition of ACSS2-mediated histone crotonylation alleviates kidney fibrosis via IL-1β-dependent macrophage activation and tubular cell senescence

Histone lysine crotonylation (Kcr), as a posttranslational modification, is widespread as acetylation (Kac); however, its roles are largely unknown in kidney fibrosis. In this study, we report that histone Kcr of tubular epithelial cells is abnormally elevated in fibrotic kidneys. By screening these crotonylated/acetylated factors, a crotonyl-CoA-producing enzyme ACSS2 (acyl-CoA synthetase short chain family member 2) is found to remarkably increase histone 3 lysine 9 crotonylation (H3K9cr) level without influencing H3K9ac in kidneys and tubular epithelial cells. The integrated analysis of ChIP-seq and RNA-seq of fibrotic kidneys reveal that the hub proinflammatory cytokine IL-1β, which is regulated by H3K9cr, play crucial roles in fibrogenesis. Furthermore, genetic and pharmacologic inhibition of ACSS2 both suppress H3K9cr-mediated IL-1β expression, which thereby alleviate IL-1β-dependent macrophage activation and tubular cell senescence to delay renal fibrosis. Collectively, our findings uncover that H3K9cr exerts a critical, previously unrecognized role in kidney fibrosis, where ACSS2 represents an attractive drug target to slow fibrotic kidney disease progression.

Abstract 6609: NRF2/ACSS2 axis regulates alcohol-induced metabolic reprogramming in esophageal squamous epithelial cells

Abstract Esophageal diseases such as gastroesophageal reflux disease (GERD) and esophageal cancer have a strong association with alcohol drinking. However, the molecular mechanisms of alcohol-induced esophageal pathology remain unclear. In our previous study, we identified acyl-CoA synthetase short-chain family member 2 (ACSS2) as a transcriptional target of nuclear factor erythroid 2-related factor 2 (NRF2) in the esophageal epithelial cells in vitro and in vivo. Ethanol exposure enhanced de novo lipogenesis in NRF2high cells and generated a more energetic and invasive phenotype. In this study, we aimed to investigate the role of the NRF2/ACSS2 axis in regulating alcohol-induced metabolic reprogramming in esophageal squamous epithelial cells. We first demonstrated that ethanol exposure activated NRF2 signaling and upregulated the expression of NRF2 and ACSS2 in human esophageal squamous epithelial cells. 13C-ethanol/13C-acetate metabolic flux assay showed a dramatic NRF2-dependent increase of labeling of the TCA cycle intermediates and de novo lipogenesis in NRF2high cells in comparison with NRF2null or NRF2low cells. Long-term ethanol exposure caused metabolic reprogramming, in particular induced de novo lipogenesis, in the mouse esophagus according to metabolomics profiling of 833 metabolites. PET/CT and autoradiography showed increased 11C-acetate uptake in the NRF2high esophagus of a conditional tissue-specific mouse (Sox2CreER;LSL-Nrf2E79Q) due to upregulation of transporters and metabolic enzymes. In summary, Our data demonstrated the functional role of the NRF2-ACSS2 axis in alcohol-induced metabolic reprogramming in esophageal squamous epithelial cells both in vitro and in vivo, suggesting NRF2/ACSS2 inhibition as a novel mechanism-based prevention for alcohol-associated esophageal pathology. Citation Format: Zhaohui Xiong, Boopathi Subramaniyan, Chorlada Paiboonrungruang, Yahui Li, Wentao He, Hong Yuan, Guofang Zhang, Xiaoxin Chen. NRF2/ACSS2 axis regulates alcohol-induced metabolic reprogramming in esophageal squamous epithelial cells [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 6609.

Protein Crotonylation Promotes Osteogenic Differentiation of Periodontal Ligament Stem Cells via the PI3K-AKT Pathway.

Posttranslational modifications (PTMs) are crucial regulatory mechanisms for cellular differentiation and organismal development. Acylation modification is one of the main PTMs that plays a pivotal role in regulating the osteogenic differentiation of mesenchymal stem cells and is a focal point of research in bone tissue regeneration. However, its mechanism remains incompletely understood. This article aims to investigate the impact of protein crotonylation on osteogenic differentiation in periodontal ligament stem cells (PDLSCs) and elucidate its underlying mechanisms. Western blot analysis identified that the modification level of acetylation, crotonylation, and succinylation were significantly upregulated after osteogenic induction of PDLSCs. Subsequently, sodium crotonate (NaCr) was added to the medium and acyl-CoA synthetase short-chain family member 2 (ACSS2) was knocked down by short hairpin RNA plasmids to regulate the total level of protein crotonylation. The results indicated that treatment with NaCr promoted the expression of osteogenic differentiation-related factors in PDLSCs, whereas silencing ACSS2 had the opposite effect. In addition, mass spectrometry analysis was used to investigate the comprehensive analysis of proteome-wide crotonylation in PDLSCs under osteogenic differentiation. The analysis revealed that the level of protein crotonylation related to the PI3K-AKT signaling pathway was significantly upregulated in PDLSCs after osteogenic induction. Treatment with NaCr and silencing ACSS2 affected the activation of the PI3K-AKT signaling pathway. Collectively, our study demonstrates that protein crotonylation promotes osteogenic differentiation of PDLSCs via the PI3K-AKT pathway, providing a novel targeting therapeutic approach for bone tissue regeneration.

Physiology and Mechanism of Milk Fat Globules Secretion in Dairy Animals: A Review

Milk fat globules (MFGs) are a complex compound secreted in the mammary gland, composed of triglycerides; phosphatidylethanolamine (PE) phosphatidylcholine (PC) with unclear mechanism and the fused lipid droplets enclosed by inner layer derived from the endoplasmic reticulum and outer bilayer is directly derived from the apical plasma membrane as the lipid droplets bud out from the cell. The core components of MFGs, triglycerides are synthesized via the de novo fatty acid pathway and LCFA was directly taken from serum. The de novo fatty acid pathway was significantly dependent on the enzymes acetyl-CoA carboxylase (ACACA), fatty acid synthase (FASN) and acyl-CoA synthetase short-chain family member 2 (ACSS2) to synthesize short and medium-chain fatty acids, whereas the bio-hydrogenation pathway is a principal pathway for the synthesis of long-chain fatty acids. Following this, the milk fat globules coated by the membrane are released from the MEC to the lumen with or without other milk components and the membrane that surrounds the fat globule stabilize the milk fat in its dispersed state, prevent flocculation and globule coalescence, and protects against the deleterious effects of lipases. Even though the secretion mechanism of lipid droplets is controversial hormonal and molecular mechanisms are involved. Moreover, the fat globule size and composition are determined by factors such as stage of lactation, physiological state of the animal, Diacylglycerol Acyltransferase1 (DGAT1) activity, and PC/PE ratio along with breed and animal species. While some studies were conducted on milk synthesis and secretion, the molecular mechanism behind lipid droplet fusion, secretion in the apical membrane and how MFGM is formed and the mechanism of MFG synthesis regulation are undiscovered. Therefore, it needs advanced investigation.

Folate-deficiency induced acyl-CoA synthetase short-chain family member 2 increases lysine crotonylome involved in neural tube defects.

Maternal folate deficiency increases the risk of neural tube defects (NTDs), but the mechanism remains unclear. Here, we established a mouse model of NTDs via low folate diets combined with MTX-induced conditions. We found that a significant increase in butyrate acid was observed in mouse NTDs brains. In addition, aberrant key crotonyl-CoA-producing enzymes acyl-CoA synthetase short-chain family member 2 (ACSS2) levels and lysine crotonylation (Kcr) were elevated high in corresponding low folate content maternal serum samples from mouse NTD model. Next, proteomic analysis revealed that folate deficiency led to global proteomic modulation, especially in key crotonyl-CoA-producing enzymes, and dramatic ultrastructural changes in mouse embryonic stem cells (mESCs). Furthermore, we determined that folate deficiency induced ACSS2 and Kcr in mESCs. Surprisingly, folic acid supplementation restored level of ACSS2 and Kcr. We also investigated overall protein post-translational Kcr under folate deficiency, revealing the key regulation of Kcr in glycolysis/gluconeogenesis, and the citric acid cycle. Our findings suggest folate deficiency leads to the occurrence of NTDs by altering ACSS2. Protein crotonylation may be the molecular basis for NTDs remodeling by folate deficiency.

Pyruvate Dehydrogenase Inhibition Leads to Decreased Glycolysis, Increased Reliance on Gluconeogenesis and Alternative Sources of Acetyl-CoA in Acute Myeloid Leukemia

Acute myeloid leukemia (AML) is an aggressive disease characterized by poor outcomes and therapy resistance. Devimistat is a novel agent that inhibits pyruvate dehydrogenase complex (PDH). A phase III clinical trial in AML patients combining devimistat and chemotherapy was terminated for futility, suggesting AML cells were able to circumvent the metabolic inhibition of devimistat. The means by which AML cells resist PDH inhibition is unknown. AML cell lines treated with devimistat or deleted for the essential PDH subunit, PDHA, showed a decrease in glycolysis and decreased glucose uptake due to a reduction of the glucose transporter GLUT1 and hexokinase II. Both devimistat-treated and PDHA knockout cells displayed increased sensitivity to 2-deoxyglucose, demonstrating reliance on residual glycolysis. The rate limiting gluconeogenic enzyme phosphoenolpyruvate carboxykinase 2 (PCK2) was significantly upregulated in devimistat-treated cells, and its inhibition increased sensitivity to devimistat. The gluconeogenic amino acids glutamine and asparagine protected AML cells from devimistat. Non-glycolytic sources of acetyl-CoA were also important with fatty acid oxidation, ATP citrate lyase (ACLY) and acyl-CoA synthetase short chain family member 2 (ACSS2) contributing to resistance. Finally, devimistat reduced fatty acid synthase (FASN) activity. Taken together, this suggests that AML cells compensate for PDH and glycolysis inhibition by gluconeogenesis for maintenance of essential glycolytic intermediates and fatty acid oxidation, ACLY and ACSS2 for non-glycolytic production of acetyl-CoA. Strategies to target these escape pathways should be explored in AML.

The mechanistic target of rapamycin complex 1 pathway involved in hepatic gluconeogenesis through peroxisome-proliferator-activated receptor γ coactivator-1α.

Cattle can efficiently perform de novo generation of glucose through hepatic gluconeogenesis to meet post-weaning glucose demand. Substantial evidence points to cattle and non-ruminant animals being characterized by phylogenetic features in terms of their differing capacity for hepatic gluconeogenesis, a process that is highly efficient in cattle yet the underlying mechanism remains unclear. Here we used a variety of transcriptome data, as well as tissue and cell-based methods to uncover the mechanisms of high-efficiency hepatic gluconeogenesis in cattle. We showed that cattle can efficiently convert propionate into pyruvate, at least partly, via high expression of acyl-CoA synthetase short-chain family member 1 (ACSS1), propionyl-CoA carboxylase alpha chain (PCCA), methylmalonyl-CoA epimerase (MCEE), methylmalonyl-CoA mutase (MMUT), and succinate-CoA ligase (SUCLG2) genes in the liver (P < 0.01). Moreover, higher expression of the rate-limiting enzymes of gluconeogenesis, such as phosphoenolpyruvate carboxykinase (PCK) and fructose 1,6-bisphosphatase (FBP), ensures the efficient operation of hepatic gluconeogenesis in cattle (P < 0.01). Mechanistically, we found that cattle liver exhibits highly active mechanistic target of rapamycin complex 1 (mTORC1), and the expressions of PCCA, MMUT, SUCLG2, PCK, and FBP genes are regulated by the activation of mTORC1 (P < 0.001). Finally, our results showed that mTORC1 promotes hepatic gluconeogenesis in a peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α) dependent manner. Collectively, our results not only revealed an important mechanism responsible for the quantitative differences in the efficiency of hepatic gluconeogenesis in cattle versus non-ruminant animals, but also established that mTORC1 is indeed involved in the regulation of hepatic gluconeogenesis through PGC-1α. These results provide a novel potential insight into promoting hepatic gluconeogenesis through activated mTORC1 in both ruminants and mammals.

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.

Deoxynivalenol exposure inhibits biosynthesis of milk fat and protein by impairing tight junction in bovine mammary epithelial cells

Deoxynivalenol (DON) is one of the most common feed contaminants, and it poses a serious threat to the health of dairy cows. The existing studies of biological toxicity of DON mainly focus on the proliferation, oxidative stress, and inflammation in bovine mammary epithelial cells, while its toxicity on the biosynthesis of milk components has not been well documented. Hence, we investigated the toxic effects and the underlying mechanism of DON on the bovine mammary alveolar cells (MAC-T). Our results showed that exposure to various concentrations of DON significantly inhibited cell proliferation, induced apoptosis, and altered the cell morphology which was manifested by cell distortion and shrinkage. Moreover, the transepithelial electrical resistance (TEER) values of MAC-T cells exposed to DON were gradually decreased in a time- and concentration- dependent manner, but lactate dehydrogenase (LDH) leakage was significantly increased with the maximum increase of 2.4-fold, indicating the cell membrane and tight junctions were damaged by DON. Importantly, DON significantly reduced the synthesis of β-casein and lipid droplets, along with the significantly decreases of phospho-mTOR, phospho-4EBP1, phospho-JAK2, and phospho-STAT5. Gene expression profiles showed that the expressions of several genes related to lipid synthesis and metabolism were changed, including acyl-CoA synthetase short-chain family member 2 (ACSS2), fatty acid binding protein 3 (FABP3), 3-hydroxy-3-methylglutaryl-CoA synthase 1 (HMGCS1), and insulin-induced gene 1 (INSIG1). GO and KEGG enrichment analyses revealed that the differentially expressed genes (DEGs) were significantly enriched in ribosome, glutathione metabolism, and lipid biosynthetic process, which play important roles in the toxicological process induced by DON. Taken together, DON affects the proliferation and functional differentiation of MAC-T cells, which might be related to the cell junction disruption and morphological alteration. Our data provide new insights into functional differentiation and transcriptomic alterations of MAC-T cells after DON exposure, which contributes to a comprehensive understanding of DON-induced toxicity mechanism.

ACSS3 in brown fat drives propionate catabolism and its deficiency leads to autophagy and systemic metabolic dysfunction.

Propionate is a gut microbial metabolite that has been reported to have controversial effects on metabolic health. Here we show that propionate is activated by acyl‐CoA synthetase short‐chain family member 3 (ACSS3), located on the mitochondrial inner membrane in brown adipocytes. Knockout of Acss3 gene (Acss3–/–) in mice reduces brown adipose tissue (BAT) mass but increases white adipose tissue (WAT) mass, leading to glucose intolerance and insulin resistance that are exacerbated by high‐fat diet (HFD). Intriguingly, Acss3–/– or HFD feeding significantly elevates propionate levels in BAT and serum, and propionate supplementation induces autophagy in cultured brown and white adipocytes. The elevated levels of propionate in Acss3–/– mice similarly drive adipocyte autophagy, and pharmacological inhibition of autophagy using hydroxychloroquine ameliorates obesity, hepatic steatosis and insulin resistance of the Acss3–/– mice. These results establish ACSS3 as the key enzyme for propionate metabolism and demonstrate that accumulation of propionate promotes obesity and Type 2 diabetes through triggering adipocyte autophagy.

Dietary threonine deficiency affects expression of genes involved in lipid metabolism in adipose tissues of Pekin ducks in a genotype-dependent manner

Dietary threonine (Thr) deficiency increases hepatic triglyceride content and reduces sebum and abdominal fat percentages in lean type (LT), but not in fatty type (FT) Pekin ducks. However, the molecular changes regarding the role of Thr in lipid metabolism in LT and FT ducks induced by Thr deficiency remains unknown. This study compared differential expression gene profiles related to lipid metabolism in FT and LT Pekin ducks affected by Thr deficiency. We performed transcriptomic profiling and scanned the gene expression in the liver, sebum, and abdominal fat of Pekin ducks fed either Thr-deficient or Thr-adequate diet for 21 days from 14 to 35 days of age. There were 187, 52, and 50 differentially expressed genes (DEGs) identified in the liver, sebum, and abdominal fat of LT ducks affected by Thr deficiency, of which 12, 9, and 5 genes were involved in lipid metabolism, respectively. Thr deficiency altered the expression of 27, 6, and 3 genes in FT ducks’ liver, sebum, and abdominal fat, respectively. None of the DEGs had a relationship with lipid metabolism in FT ducks. KEGG analysis showed that the DEGs in the LT ducks’ livers were enriched in lipid metabolism pathways (linolenic acid metabolism, glycerophospholipid metabolism, and arachidonic acid metabolism) and amino acid metabolism pathways (biosynthesis of amino acids, phenylalanine metabolism, β-alanine metabolism, and glycine, serine and threonine metabolisms). The DEGs in the sebum and abdominal fat of LT ducks were not enriched in lipid and amino acid metabolic pathways. Additionally, DEGs involved in lipid metabolism were found to be upregulated by Thr deficiency in LT ducks, such as malic enzyme 3 (ME3), acyl-CoA synthetase short-chain family member 2 (ACSS2) in liver, and lipase member M (LIPM) in sebum. In summary, dietary Thr deficiency regulated the gene expression involved in lipid metabolism in the liver, sebum, and abdominal fat of Pekin ducks in a genotype-dependent manner.

Hsa-miR-15b-5p regulates the proliferation and apoptosis of human vascular smooth muscle cells by targeting the ACSS2/PTGS2 axis.

A previous bioinformatic analysis from our group predicted that the interaction of microRNA (miRNA/miR)-15b with the acyl-CoA synthetase short chain family member 2 (ACSS2) gene was important for the development of abdominal aortic aneurysm (AAA). Apoptosis of aortic vascular smooth muscle cells (VSMCs) is a pathological feature of AAA. The present study aimed to explain the roles of miR-15b/ACSS2 in AAA by exploring their effects on the proliferation and apoptosis of aortic VSMCs. Human aortic VSMCs (T/G HA-VSMC cell line) were divided into six groups and were transfected with miR-15b-5p mimics, mimic negative control (NC), miR-15b-5p inhibitors, inhibitor NC, miR-15b-5p mimics+pcDNA3.1 and miR-15b-5p mimics+ACSS2-overexpessing vector. CCK-8 assay was used to determine cell proliferation. Annexin V-FITC/PI staining and flow cytometry assays were used to measure cell apoptosis. Dual-luciferase reporter assays were used to confirm the targeted relationship between miR-15b-5p and ACSS2. Reverse transcription-quantitative PCR and/or western blotting were used to examine the expression levels of miR-15b-5p, ACSS2 and prostaglandin-endoperoxide synthase 2 (PTGS2). Following transfection of T/G HA-VSMCs with mimics and inhibitors to respectively upregulate and downregulate miR-15b-5p, the results demonstrated that overexpression of miR-15b-5p inhibited cell proliferation and promoted cell apoptosis; silencing of miR-15b-5p obtained the opposite results. ACSS2 may be a direct target of miR-15b-5p, since the luciferase activity of a ACSS2 wild-type vector, but not that of a ACSS2 mutant reporter, was significantly inhibited by miR-15b-5p mimics compared with controls. Additionally, the expression levels of ACSS2 and its downstream gene PTGS2 were significantly reduced or increased following transfection with miR-15b-5p mimics or inhibitors, respectively. Furthermore, overexpression of ACSS2 reversed the antiproliferative and proapoptotic effects of miR-15b-5p mimics by blocking the production of PTGS2 protein. In conclusion, miR-15b-5p may promote the apoptosis and inhibit the proliferation of aortic VSMCs via targeting the ACSS2/PTGS2 axis. The present study provided preliminary evidence indicating that the miR-15b-5p/ACSS2/PTGS2 axis may be a potential target for the treatment of AAA.

Butyrate enhances CPT1A activity to promote fatty acid oxidation and iTreg differentiation

Inducible regulatory T (iTreg) cells play a crucial role in immune suppression and are important for the maintenance of immune homeostasis. Mounting evidence has demonstrated connections between iTreg differentiation and metabolic reprogramming, especially rewiring in fatty acid oxidation (FAO). Previous work showed that butyrate, a specific type of short-chain fatty acid (SCFA) readily produced from fiber-rich diets through microbial fermentation, was critical for the maintenance of intestinal homeostasis and capable of promoting iTreg generation by up-regulating histone acetylation for gene expression as an HDAC inhibitor. Here, we revealed that butyrate could also accelerate FAO to facilitate iTreg differentiation. Moreover, butyrate was converted, by acyl-CoA synthetase short-chain family member 2 (ACSS2), into butyryl-CoA (BCoA), which up-regulated CPT1A activity through antagonizing the association of malonyl-CoA (MCoA), the best known metabolic intermediate inhibiting CPT1A, to promote FAO and thereby iTreg differentiation. Mutation of CPT1A at Arg243, a reported amino acid required for MCoA association, impaired both MCoA and BCoA binding, indicating that Arg243 is probably the responsible site for MCoA and BCoA association. Furthermore, blocking BCoA formation by ACSS2 inhibitor compromised butyrate-mediated iTreg generation and mitigation of mouse colitis. Together, we unveil a previously unappreciated role for butyrate in iTreg differentiation and illustrate butyrate-BCoA-CPT1A axis for the regulation of immune homeostasis.

Maturation system affects lipid accumulation in bovine oocytes.

This study evaluated the effects of three maturation systems, namely invitro (MatV) and invivo (MatS) systems, as well as intrafollicular transfer of immature oocytes (IFIOT; MatT), on the accumulation of lipid droplets in bovine oocytes. Lipids were evaluated using confocal microscopy and transmission electron microscopy. The expression of genes related to lipid metabolism, namely acyl-CoA synthetase short chain family member 2 (ACSS2), ELOVL fatty acid elongase 1 (ELOVL1) and fatty acid binding protein 3 (FABP3), was quantified by quantitative polymerase chain reaction. The mean (±s.d.) area occupied by lipids in immature oocytes (13±2%) was similar to those matured invivo (MatS, 16±2%; MatT, 12±2%). However, there was a significant increase in lipids in oocytes in the MatV group (24±2%) compared with all other groups (P<0.001). In the ultrastructural evaluations, MatV oocytes also showed the highest lipid content. The expression of ELOVL1 and FABP3 was similar in the MatS and IFIOT groups. However, transcript levels of ACSS2 were lower in IFIOT than MatV oocytes. These results indicate, for the first time, that oocytes matured by IFIOT are similar to those matured invivo with regard to lipid accumulation, which indicates better quality than those matured invitro.

Abstract PO-046: LKB1 loss-associated changes in histone acetylation are potential therapeutic targets in lung adenocarcinoma

Abstract Loss of liver kinase B (LKB1, encoded by STK11) has been observed in about one-third of lung adenocarcinomas (ADCs) and can cause the inactivation of its major target AMP-activated protein kinase (AMPK) and subsequently, activation of the mammalian target of rapamycin (mTOR) pathway. In addition to its inhibitory effects on tumor growth and proliferation, AMPK may affect acetyl-CoA homeostasis and induces histone acetylation through regulating the activity and nuclear translocation of acyl-CoA synthetase short-chain family member 2 (ACSS2) and ATP citrate lyase (ACLY). Nuclear and/or cytoplasmic expression of LKB1/AMPK/mTOR pathway markers (LKB1, p-AMPKα, p-S6), enzymes involved in acetate and lipid metabolism (p-ACC, ACSS2, ACLY), acetyl-histone H3 (ac-H3), and immune regulatory proteins (PD-L1, COX-2) was assessed in lung ADCs (N=100) by immunohistochemistry. The expression of mRNAs coding for proteins involved in antitumor immunity was also analyzed in STK11-mutant and wild-type cases with using TCGA data. Associations with the clinicopathological parameters as well as the antiproliferative effects of mTOR and metabolic inhibitors (rapamycin, metformin, phenformin, and ACSS2 inhibitor) and a histone deacetylase (HDAC) inhibitor (valproic acid) were also studied in ADC cell lines harboring KRAS and/or STK11 mutations. In cases with low or no LKB1 expression, we observed significantly lower expression of p-AMPKα, p-ACC, ac-H3, ACSS2, and ACLY (P &amp;lt; .01). PD-L1 expression was also slightly decreased in these samples. The expression of most of the studied mRNAs, particularly those involved in innate immunity, was also lower in STK11 mutant cases. In our in vitro studies, rapamycin had a significant antiproliferative effect on KRAS- and/or STK11-mutant cell lines with high mTOR activity. The ACSS2 inhibitor and valproic acid were not effective as monotherapies, however, valproic acid significantly increased the antitumoral effects of rapamycin in STK11-mutant cells similarly to phenformin. Our results suggest that histone acetylation levels are lower in ADCs with LKB1 loss, perhaps by lower expression and decreased nuclear translocation of ACSS2 and ACLY. HDAC inhibitors may have the benefit to maintain histone acetylation in STK11-mutant ADCs, therefore, restoring the expression of tumor suppressors or proteins involved in antitumor immunity, which are silenced in cancer cells. Simultaneous inhibition of mTOR and HDACs may exhibit significant antitumor properties, especially in cancer cells with STK11 mutation and high mTOR activity. Supported by the Hungarian Respiratory Foundation, National Bionics Program of National Research, Development and Innovation Fund of Hungary (ED_17-1-2017-0009), and grants of the Hungarian National Research, Development and Innovation Office (NKFI-FK-128404). Citation Format: Ildiko Krencz, Daniel Sztankovics, Titanilla Danko, Gabor Petovari, Eniko Vetlenyi, Regina Raffay, Judit Moldvay, Judit Papay, Anna Sebestyen. LKB1 loss-associated changes in histone acetylation are potential therapeutic targets in lung adenocarcinoma [abstract]. In: Abstracts: AACR Special Virtual Conference on Epigenetics and Metabolism; October 15-16, 2020; 2020 Oct 15-16. Philadelphia (PA): AACR; Cancer Res 2020;80(23 Suppl):Abstract nr PO-046.

Mir-193b Regulates the Differentiation, Proliferation, and Apoptosis of Bovine Adipose Cells by Targeting the ACSS2/AKT Axis.

Simple SummarySeveral studies have shown that miR-193b plays an important role in preadipocyte differentiation. Herein, we explored the role of bta-miR-193b in adipocyte development, using EdU, flow cytometry, CCK-8, RT-qPCR, Western blotting, and oil red O staining. The result showed that bta-miR-193b could regulate the proliferation, differentiation and apoptosis of adipocytes. The dual-fluorescent reporter vector experiments showed that bta-miR-193b directly targeted ACSS2, and the regulatory function of ACSS2 is opposite to that of miR-193b. Meanwhile, we demonstrated that ACSS2 could significantly promote the expression of AKT and pAKT proteins. In conclusion, our research provides new insights that confirm that bta-miR-193b inhibits bovine adipose cell proliferation, and promotes apoptosis by negative regulation of the ACSS2/AKT pathway.The precise functions and molecular mechanisms of microRNAs (miRNAs) in adipocytes are primarily unknown. Studies have demonstrated that miR-193b plays a pivotal role in the differentiation of preadipocytes. Herein, we evaluated the effects of bta-miR-193b on the growth and development of adipocytes, using the EdU cell proliferation method, flow cytometry analysis, CCK-8 assay, RT-qPCR, Western blotting, and oil red O staining. We observed that the overexpression of bta-miR-193b significantly affected the differentiation, proliferation, and apoptosis of adipocytes. The results of the dual-fluorescent reporter vector experiments demonstrated that bta-miR-193b directly targeted Acyl-CoA synthetase short-chain family member 2 (ACSS2). Additionally, the effects of ACSS2 overexpression on the proliferation and apoptosis in adipose cells were the opposite of those induced by bta-miR-193b. We also demonstrated that ACSS2 can significantly promote the expression of AKT and pAKT proteins. Therefore, this study presents a novel mechanism by which bta-miR-193b regulates adipocyte development by targeting ACSS2.