Synthetase Short-chain Family Member Research Articles

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.

#1326 Impairment of ACSS2 aggravates renal fibrosis and cell senescence during aging

Abstract Background and Aims Age-related changes in kidney function, structure, and their relationships have been extensively recognized recently. Here, we report that acyl-CoA synthetase short chain family member 2 (Acss2) as a regulator of lipid metabolism, play an important role in age-associated renal fibrosis. Method 21-month-old Acss2 knockout (Acss2−/−) mice were used as the renal aging models, and H2O2 also was used to induced renal tubular epithelial cells (TCMK-1) senescence. Serum creatinine (Cr) and urinary protein creatinine ratio (UACR) were measured to evaluate kidney function, and renal tissue lesions were observed by HE staining and Masson staining. The expression of gene/protein in relation to inflammation, fibrosis and cell senescence were measured by qPCR, western blot. SA-β-gal staining also used to assess cell senescence. Results The results indicate that Acss2 was decreased in aging mice, Acss2 knockout impaired renal function as detected by increased UACR. Additionally, HE and Masson staining showed that Acss2 deficiency was associated with more glomerular sclerosis and fibrosis (Fig. 1). RT-qPCR and Western blotting also revealed that Acss2 enhanced kidney fibrosis and cell senescence, accompanied by an increase in SA-β-gal-positive senescent cells (Fig. 2). These phenomena were most apparent in the TCMK cells treated with Acss2 siRNA and H2O2 (Fig. 3). What's more, KEGG analysis showed that fatty acid metabolism changes significantly during aging in kidney, and Acss2 knockout could impact the expression of lipid metabolism genes (Fig. 4) Conclusion Acss2 is associated with renal aging, and it may played a role in renal fibrosis and cell senescence by regulating fatty acid metabolism.

Mitochondrial ACSS1-K635 acetylation knock-in mice exhibit altered metabolism, cell senescence, and nonalcoholic fatty liver disease.

Acetyl-CoA synthetase short-chain family member 1 (ACSS1) uses acetate to generate mitochondrial acetyl-CoA and is regulated by deacetylation by sirtuin 3. We generated an ACSS1-acetylation (Ac) mimic mouse, where lysine-635 was mutated to glutamine (K635Q). Male Acss1K635Q/K635Q mice were smaller with higher metabolic rate and blood acetate and decreased liver/serum ATP and lactate levels. After a 48-hour fast, Acss1K635Q/K635Q mice presented hypothermia and liver aberrations, including enlargement, discoloration, lipid droplet accumulation, and microsteatosis, consistent with nonalcoholic fatty liver disease (NAFLD). RNA sequencing analysis suggested dysregulation of fatty acid metabolism, cellular senescence, and hepatic steatosis networks, consistent with NAFLD. Fasted Acss1K635Q/K635Q mouse livers showed increased fatty acid synthase (FASN) and stearoyl-CoA desaturase 1 (SCD1), both associated with NAFLD, and increased carbohydrate response element-binding protein binding to Fasn and Scd1 enhancer regions. Last, liver lipidomics showed elevated ceramide, lysophosphatidylethanolamine, and lysophosphatidylcholine, all associated with NAFLD. Thus, we propose that ACSS1-K635-Ac dysregulation leads to aberrant lipid metabolism, cellular senescence, and NAFLD.

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.

Reconciling host-microbiota metabolic incompatibility safeguards male fertility

The symbiotic relationship between the host and microbiota is widely acknowledged as mutually beneficial. However, due to significant differences in metabolic substrates and products between prokaryotic bacteria and mammalian cells, mechanisms must exist to reconcile the metabolic incompatibility between the host and microbiota. We report that host enzymes are required to detoxify gut microbiota-derived acetate to maintain male fertility in mice. The combined deletion of acetyl-CoA synthetase short-chain family member 1 and 2 (ACSS1 and ACSS2), two enzymes consuming acetate in mammals, leads to excessive accumulation of acetate in circulation. This accumulation causes metabolic acidosis, blocking spermatogenesis and rendering male mice infertile. ACSS1/2-deficient germ cells exhibit comprehensive metabolic alterations with nicotinamide adenine dinucleotide (NAD+) deficiency that impairs betaine production. Supplementation with betaine restores spermatogenesis and fertility in ACSS1/2-deficient mice. Thus, the inevitable production of acetate by gut bacteria and its reproductive toxicity to the host represents an unappreciated metabolic incompatibility between the host and microbiota, which is reconciled by ACSS1/2.

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.

Abstract 1799: Regulation of epidermal stem cell proliferation and adhesion by ACSS2

Abstract Acetyl-CoA Synthetase Short-Chain Family Member 2 (ACSS2) is an enzyme that converts acetate into acetyl-CoA, a crucial metabolite required for protein acetylation and lipogenesis. Acetyl-CoA is membrane-impermeable and must be synthesized in the compartment in which it will be used. Among ACSS enzymes, ACSS2 alone is found in the nucleocytoplasmic compartment rather than the mitochondria. This enzyme shows dynamic localization dependent on cell nutrient status, with nutrient stress promoting nuclear localization where ACSS2 activity promotes cell growth and survival. Emerging evidence suggests ACSS2 may also play roles in promoting metastasis, and has been proposed as a potential therapeutic target. However, the role of ACSS2 in controlling normal tissue homeostasis is understudied. Addressing this knowledge gap is critical to ensure safety and efficacy of cancer therapeutics. As 80% to 90% of human cancers originate from epithelial tissues, we have leveraged human epidermal stem cells to further interrogate the role of ACSS2. As progenitor keratinocytes differentiate, they detach from the basal lamina and migrate from the metabolically privileged basal layer to the more challenging environment at the surface of the epidermis. Using shRNA-mediated knockdown, we first found ACSS2 knockdown dramatically decreases progenitor keratinocyte clonogenicity, abolishing proliferative capacity. Analysis of RNA sequencing data following ACSS2 knockdown with five constructs showed significant increases in expression of genes related to cell adhesion, extracellular matrix organization, collagen catabolism, and cell migration, in addition to keratinocyte differentiation. Downregulated genes were associated with cell division, DNA repair, and DNA replication. Live cell imaging revealed knockdown cells show striking differences in cell adhesion compared to control. These results indicate a role for ACSS2 in regulating keratinocyte proliferation and adhesion during the maintenance of epidermal homeostasis. Citation Format: Nicholas K. Chamberlain, Xiaomin Bao. Regulation of epidermal stem cell proliferation and adhesion by ACSS2 [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 1799.

D-arabinose acts as antidepressant by activating the ACSS2-PPARγ/TFEB axis and CRTC1 transcription

CREB-regulated transcription coactivator 1 (CRTC1), a pivotal synaptonuclear messenger, regulates synaptic plasticity and transmission to prevent depression. Despite exhaustive investigations into CRTC1 mRNA reductions in the depressed mice, the regulatory mechanisms governing its transcription remain elusive. Consequently, exploring rapid but non-toxic CRTC1 inducers at the transcriptional level is important for resisting depression. Here, we demonstrate the potential of D-arabinose, a unique monosaccharide prevalent in edible-medicinal plants, to rapidly enter the brain and induce CRTC1 expression, thereby eliciting rapid-acting and persistent antidepressant responses in chronic restrain stress (CRS)-induced depressed mice. Mechanistically, D-arabinose induces the expressions of peroxisome proliferator-activated receptor gamma (PPARγ) and transcription factor EB (TFEB), thereby activating CRTC1 transcription. Notably, we elucidate the pivotal role of the acetyl-CoA synthetase short-chain family member 2 (ACSS2) as an obligatory mediator for PPARγ and TFEB to potentiate CRTC1 transcription. Furthermore, D-arabinose augments ACSS2-dependent CRTC1 transcription by activating AMPK through lysosomal AXIN-LKB1 pathway. Correspondingly, the hippocampal down-regulations of ACSS2, PPARγ or TFEB alone failed to reverse CRTC1 reductions in CRS-exposure mice, ultimately abolishing the anti-depressant efficacy of D-arabinose. In summary, our study unveils a previously unexplored role of D-arabinose in activating the ACSS2-PPARγ/TFEB-CRTC1 axis, presenting it as a promising avenue for the prevention and treatment of depression.

Cancer-associated fibroblast-derived acetate promotes pancreatic cancer development by altering polyamine metabolism via the ACSS2–SP1–SAT1 axis

The ability of tumour cells to thrive in harsh microenvironments depends on the utilization of nutrients available in the milieu. Here we show that pancreatic cancer-associated fibroblasts (CAFs) regulate tumour cell metabolism through the secretion of acetate, which can be blocked by silencing ATP citrate lyase (ACLY) in CAFs. We further show that acetyl-CoA synthetase short-chain family member 2 (ACSS2) channels the exogenous acetate to regulate the dynamic cancer epigenome and transcriptome, thereby facilitating cancer cell survival in an acidic microenvironment. Comparative H3K27ac ChIP–seq and RNA–seq analyses revealed alterations in polyamine homeostasis through regulation of SAT1 gene expression and enrichment of the SP1-responsive signature. We identified acetate/ACSS2-mediated acetylation of SP1 at the lysine 19 residue that increased SP1 protein stability and transcriptional activity. Genetic or pharmacologic inhibition of the ACSS2–SP1–SAT1 axis diminished the tumour burden in mouse models. These results reveal that the metabolic flexibility imparted by the stroma-derived acetate enabled cancer cell survival under acidosis via the ACSS2–SP1–SAT1 axis.

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.

ACSS2 controls PPARγ activity homeostasis to potentiate adipose-tissue plasticity

The appropriate transcriptional activity of PPARγ is indispensable for controlling inflammation, tumor and obesity. Therefore, the identification of key switch that couples PPARγ activation with degradation to sustain its activity homeostasis is extremely important. Unexpectedly, we here show that acetyl-CoA synthetase short-chain family member 2 (ACSS2) critically controls PPARγ activity homeostasis via SIRT1 to enhance adipose plasticity via promoting white adipose tissues beiging and brown adipose tissues thermogenesis. Mechanistically, ACSS2 binds directly acetylated PPARγ in the presence of ligand and recruits SIRT1 and PRDM16 to activate UCP1 expression. In turn, SIRT1 triggers ACSS2 translocation from deacetylated PPARγ to P300 and thereafter induces PPARγ polyubiquitination and degradation. Interestingly, D-mannose rapidly activates ACSS2-PPARγ-UCP1 axis to resist high fat diet induced obesity in mice. We thus reveal a novel ACSS2 function in coupling PPARγ activation with degradation via SIRT1 and suggest D-mannose as a novel adipose plasticity regulator via ACSS2 to prevent obesity.

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.

ACSS3 regulates the metabolic homeostasis of epithelial cells and alleviates pulmonary fibrosis

Idiopathic pulmonary fibrosis (IPF) is a progressive and fatal interstitial lung disease of unknown etiology. The emerging evidence demonstrates that metabolic homeostatic imbalance caused by repetitive injuries of the alveolar epithelium is the potential pathogenesis of IPF. Proteomic analysis identified that Acetyl-CoA synthetase short chain family member 3 (ACSS3) expression was decreased in IPF patients and mice with bleomycin-induced fibrosis. ACSS3 participated in lipid and carbohydrate metabolism. Increased expression of ACSS3 downregulated carnitine palmitoyltransferase 1A (CPT-1A) and resulted in the accumulation of lipid droplets, while enhanced glycolysis which led to an increase in extracellular lactic acid levels in A549 cells. ACSS3 increases the production of succinyl-CoA through propionic acid metabolism, and decreases the generation of acetyl-CoA and ATP in alveolar epithelial cells. Overexpression of Acss3 inhibited the excessive deposition of ECM and attenuated the ground-glass opacity which determined by micro-CT in vivo. In a nutshell, our findings demonstrate that ACSS3 decreased the fatty acid oxidation through CPT1A deficiency and enhanced anaerobic glycolysis, this metabolic reprogramming deactivate the alveolar epithelial cells by lessen mitochondrial fission and fusion, increase of ROS production, suppression of mitophagy, promotion of apoptosis, suggesting that ACSS3 might be potential therapeutic target in pulmonary fibrosis.

D-mannose is a rapid inducer of ACSS2 to trigger rapid and long-lasting antidepressant responses through augmenting BDNF and TPH2 levels

The potentiation of synaptic plasticity and serotonin generation by brain-derived neurotrophic factor (BDNF) and tryptophan hydroxylase 2 (TPH2) is well characterized to facilitate rapid and long-lasting antidepressant actions. Therefore, the identification of the key protein that simultaneously controls both BDNF and TPH2 is important for the treatment of depression. We show here that a lack of acetyl-CoA synthetase short-chain family member 2 (ACSS2) causes impairments in BDNF-dependent synaptic plasticity and tryptophan hydroxylase 2 (TPH2)-mediated serotonin generation, thereby contributing to spontaneous and chronic restraint stress (CRS)-induced depressive-like behavior in mice. Conversely, D-mannose is identified as a rapid ACSS2 inducer and thus mediates rapid and long-lasting antidepressant-like effects. Mechanistically, acute and chronic D-mannose administration inhibits the phosphorylation of EF2 to increase BDNF levels and reverse the reduction of TPH2 histone acetylation and transcription. We reveal that ACSS2 promotes TPH2 histone acetylation and transcription with the requirement of AMPK activation. To elevate nuclear ACSS2 levels, D-mannose can rapidly and persistently activate AMPK via Ca2+-CAMKK2 and the lysosomal AXIN-LKB1 pathway to facilitate its fast-acting and persistent antidepressant responses. Taken together, the results presented here reveal that ACSS2 functions as a novel target to link rapid and persistent antidepressant actions and further suggest that D-mannose is a potential therapeutic agent to resist depression through its augmentation of the ACSS2 dependent BDNF and TPH2 pathways.

Integration of proteomics and network toxicology reveals the mechanism of mercury chloride induced hepatotoxicity, in mice and HepG2 cells.

Mercury is one heavy metal toxin that could cause severe health impairments. Mercury exposure has become a global environmental issue. Mercury chloride (HgCl2) is one of mercury's main chemical forms, but it lacks detailed hepatotoxicity data. The present study aimed to investigate the mechanism of hepatotoxicity induced by HgCl2 through proteomics and network toxicology at the animal and cellular levels. HgCl2 showed apparent hepatotoxicity after being administrated with C57BL/6 mice (16mg/kg.bw, oral once a day, 28 days) and HepG2 cells (100μmol/L, 12h). Otherwise, oxidative stress, mitochondrial dysfunction and inflammatory infiltration play an important role in HgCl2-induced hepatotoxicity. The differentially expressed proteins (DEPs) after HgCl2 treatment and enriched pathways were obtained through proteomics and network toxicology. Western blot and qRT-PCR results showed acyl-CoA thioesterase 1 (ACOT1), acyl-CoA synthetase short chain family member 3 (ACSS3), epidermal growth factor receptor (EGFR), apolipoprotein B (APOB), signal transducer and activator of transcription 3 (STAT3), alanine--glyoxylate aminotransferase (AGXT), cytochrome P450 3A5 (CYP3A5), CYP2E1 and CYP1A2 may be the major biomarkers for HgCl2-induced hepatotoxicity, which involved chemical carcinogenesis, fatty acid metabolism, CYPs-mediated metabolism, GSH metabolism and others. Therefore, this study can provide scientific evidence for the biomarkers and mechanism of HgCl2-induced hepatotoxicity.

Abstract 4836: Fasting induces greater expression of mitochondrial proteins associated with fatty acid metabolism and non-shivering thermogenesis in brown adipose tissue of knock-in ACSS1K635Q mice

Abstract Introduction: Acyl-CoA Synthetase Short Chain Family Member 1 (ACSS1) is an important mitochondrial enzyme for the production of ATP from short chain fatty acids under energetic stress. ACSS1 is highly expressed in brown adipose tissue, heart, and skeletal muscle. ACSS1 is regulated in part by a post-translational modification of ACSS1, whereby the side chain of Lysine-635 is acetylated. The activity of ACSS1 is downregulated by the acetylation of Lysine-635. The downregulation of ACSS1 activity has been shown to impose changes on fatty acid metabolism and body temperature. Our lab observed that homozygous knock-in ACSS1 mice that fasted for 48 hours were hypothermic with a temperature of 30 °C. Here, we utilized mutant mice to simulate acetylated ACSS1, to investigate the role of ACSS1K635Q in mitochondrial fatty acid metabolism and maintenance of body temperature under fasting conditions. Methods: A knock-in ACSS1 mouse model with a mutation of ACSS1K635Q was designed to model the downregulation of ACSS1. Wild Type mice and Homozygous Knock-in ACSS1K635Q mice were either fasted for 48 hours (F48) or fed with a normal diet (ND). Four experimental groups - Wild Type ND and F48 mice and Homozygous Knock-in ACSS1K635Q ND and F48 mice - were examined. Protein was extracted from 10 samples each containing 20 mg of brown adipose tissue. Tissue samples were lysed and immunoblotted with primary and secondary antibodies. Western Blot Western Blot was performed on the proteins Uncoupling Protein 1 (UCP1), UCP2, ACC, and AMPK. Membranes were imaged using the FluorChem M system, and chemiluminescence was quantified using the NIH ImageJ software. ANOVA and t-test were used to conduct statistical analysis (P < 0.05). Results: Homozygous Ki-ACSS1K635Q F48 mice expressed higher levels of UCP1 and UCP2 in BAT than Homozygous Ki-ACSS1K635Q ND mice. We observed a greater difference in UCP1 expression between Homozygous Ki-CSS1K635Q ND and F48 mice compared to the difference in UCP1 expression between Wild Type ND and F48 mice. In Wild Type and Homozygous Ki-ACSS1K635Q mice, UCP1 expression was lower than UCP2 expression. Fasting for 48 hours significantly upregulated p-AMPK in Wild Type mice. Conclusions: Under fasting conditions, metabolic processes may shift towards a greater proportion of non-shivering thermogenesis due to ACSS1 inactivity. UCP1 likely contributes more to non-shivering thermogenesis than UCP2. Understanding the impact of acetylating ACSS1 provides avenues towards developing personalized treatments that effectively respond to metabolic stressors. Citation Format: Aishani Sivasai Gargapati, Mahboubeh Varmazyad, David Gius. Fasting induces greater expression of mitochondrial proteins associated with fatty acid metabolism and non-shivering thermogenesis in brown adipose tissue of knock-in ACSS1K635Q mice. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4836.

Acss2 Deletion Reveals Functional Versatility via Tissue-Specific Roles in Transcriptional Regulation.

The coordination of cellular biological processes is regulated in part via metabolic enzymes acting to match cellular metabolism to current conditions. The acetate activating enzyme, acyl-coenzyme A synthetase short-chain family member 2 (Acss2), has long been considered to have a predominantly lipogenic function. More recent evidence suggests that this enzyme has regulatory functions in addition to its role in providing acetyl-CoA for lipid synthesis. We used Acss2 knockout mice (Acss2-/-) to further investigate the roles this enzyme plays in three physiologically distinct organ systems that make extensive use of lipid synthesis and storage, including the liver, brain, and adipose tissue. We examined the resulting transcriptomic changes resulting from Acss2 deletion and assessed these changes in relation to fatty acid constitution. We find that loss of Acss2 leads to dysregulation of numerous canonical signaling pathways, upstream transcriptional regulatory molecules, cellular processes, and biological functions, which were distinct in the liver, brain, and mesenteric adipose tissues. The detected organ-specific transcriptional regulatory patterns reflect the complementary functional roles of these organ systems within the context of systemic physiology. While alterations in transcriptional states were evident, the loss of Acss2 resulted in few changes in fatty acid constitution in all three organ systems. Overall, we demonstrate that Acss2 loss institutes organ-specific transcriptional regulatory patterns reflecting the complementary functional roles of these organ systems. Collectively, these findings provide further confirmation that Acss2 regulates key transcription factors and pathways under well-fed, non-stressed conditions and acts as a transcriptional regulatory enzyme.

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.