acyl-CoA Synthetase Medium-chain Family Research Articles

Acyl-CoA Synthetase Medium-Chain Family Member 5–Mediated Fatty Acid Metabolism Dysregulation Promotes the Progression of Hepatocellular Carcinoma

Hepatocellular carcinoma (HCC) is the most common primary liver cancer, with high incidence and mortality worldwide. Despite diagnostic and therapeutic advancements, HCC remains poorly responsive to treatment with a poor prognosis. Understanding the molecular mechanisms driving HCC is crucial for developing effective therapies. Emerging evidence indicates that dysregulated fatty acid metabolism contributes to HCC. Acyl-CoA medium-chain synthetase 5 (ACSM5), involved in fatty acid metabolism, is downregulated in HCC, however, its role is not well understood. In the study, we analyzed ACSM5 expression in HCC patient samples and cell lines. Using newly established ACSM5-overexpressing HCC cell lines, Huh7-ACSM5 and Hepa1-6-ACSM5, we investigated the effects and regulatory mechanisms of ACSM5. Our results showed that ACSM5 was significantly downregulated in HCC tumor tissues compared to non-tumor tissues. ACSM5 expression was regulated by DNA methylation, with a DNMT1 inhibitor effectively increasing ACSM5 expression and reducing promoter region methylation. Overexpression of ACSM5 in Huh7 cells reduced fatty acid accumulation, decreased cell proliferation, migration, and invasion in vitro, and inhibited tumor growth in mouse xenografts. Furthermore, ACSM5 overexpression also decreased STAT3 phosphorylation, subsequently affecting downstream cytokine TGFB and FGF12 mRNA levels. Our findings suggest that ACSM5 downregulation contributes to HCC progression, providing insights into its oncogenic role and highlighting its potential as a biomarker and therapeutic target for HCC.

Delineation of renal protein profiles in aristolochic acid I-induced nephrotoxicity in mice by label-free quantitative proteomics.

Introduction: Aristolochic acid nephropathy (AAN) is a kidney injury syndrome caused by aristolochic acids exposure. Our study used label-free quantitative proteomics to delineate renal protein profiles and identify key proteins after exposure to different doses of aristolochic acid I (AAI). Methods: Male C57BL/6 mice received AAI (1.25mg/kg/d, 2.5mg/kg/d, or 5mg/kg/d) or vehicle for 5 days. Results and discussion: The results showed that AAI induced dose-dependent nephrotoxicity. Differences in renal protein profiles between the control and AAI groups increased with AAI dose. Comparing the control with the low-, medium-, and high-dose AAI groups, we found 58, 210, and 271 differentially expressed proteins, respectively. Furthermore, protein-protein interaction network analysis identified acyl-CoA synthetase medium-chain family member 3 (Acsm3), cytochrome P450 family 2 subfamily E member 1 (Cyp2e1), microsomal glutathione S-transferase 1 (Mgst1), and fetuin B (Fetub) as the key proteins. Proteomics revealed that AAI decreased Acsm3 and Cyp2e1 while increasing Mgst1 and Fetub expression in mice kidneys, which was further confirmed by Western blotting. Collectively, in AAI-induced nephrotoxicity, renal protein profiles were dysregulated and exacerbated with increasing AAI dose. Acsm3, Cyp2e1, Mgst1, and Fetub may be the potential therapeutic targets for AAN.

ACSM1 and ACSM3 Regulate Fatty Acid Metabolism to Support Prostate Cancer Growth and Constrain Ferroptosis.

Solid tumors are highly reliant on lipids for energy, growth, and survival. In prostate cancer, the activity of the androgen receptor (AR) is associated with reprogramming of lipid metabolic processes. Here, we identified acyl-CoA synthetase medium chain family members 1 and 3 (ACSM1 and ACSM3) as AR-regulated mediators of prostate cancer metabolism and growth. ACSM1 and ACSM3 were upregulated in prostate tumors compared with nonmalignant tissues and other cancer types. Both enzymes enhanced proliferation and protected prostate cancer cells from death in vitro, whereas silencing ACSM3 led to reduced tumor growth in an orthotopic xenograft model. ACSM1 and ACSM3 were major regulators of the prostate cancer lipidome and enhanced energy production via fatty acid oxidation. Metabolic dysregulation caused by loss of ACSM1/3 led to mitochondrial oxidative stress, lipid peroxidation, and cell death by ferroptosis. Conversely, elevated ACSM1/3 activity enabled prostate cancer cells to survive toxic levels of medium chain fatty acids and promoted resistance to ferroptosis-inducing drugs and AR antagonists. Collectively, this study reveals a tumor-promoting function of medium chain acyl-CoA synthetases and positions ACSM1 and ACSM3 as key players in prostate cancer progression and therapy resistance. Significance: Androgen receptor-induced ACSM1 and ACSM3 mediate a metabolic pathway in prostate cancer that enables the utilization of medium chain fatty acids for energy production, blocks ferroptosis, and drives resistance to clinically approved antiandrogens.

Liver ACSM3 deficiency mediates metabolic syndrome via a lauric acid-HNF4α-p38 MAPK axis.

Metabolic syndrome combines major risk factors for cardiovascular disease, making deeper insight into its pathogenesis important. We here explore the mechanistic basis of metabolic syndrome by recruiting an essential patient cohort and performing extensive gene expression profiling. The mitochondrial fatty acid metabolism enzyme acyl-CoA synthetase medium-chain family member 3 (ACSM3) was identified to be significantly lower expressed in the peripheral blood of metabolic syndrome patients. In line, hepatic ACSM3 expression was decreased in mice with metabolic syndrome. Furthermore, Acsm3 knockout mice showed glucose and lipid metabolic abnormalities, and hepatic accumulation of the ACSM3 fatty acid substrate lauric acid. Acsm3 depletion markedly decreased mitochondrial function and stimulated signaling via the p38 MAPK pathway cascade. Consistently, Acsm3 knockout mouse exhibited abnormal mitochondrial morphology, decreased ATP contents, and enhanced ROS levels in their livers. Mechanistically, Acsm3 deficiency, and lauric acid accumulation activated nuclear receptor Hnf4α-p38 MAPK signaling. In line, the p38 inhibitor Adezmapimod effectively rescued the Acsm3 depletion phenotype. Together, these findings show that disease-associated loss of ACSM3 facilitates mitochondrial dysfunction via a lauric acid-HNF4a-p38 MAPK axis, suggesting a novel therapeutic vulnerability in systemic metabolic dysfunction.

Transcriptome analysis of ovarian tissues highlights genes controlling energy homeostasis and oxidative stress as potential drivers of heterosis for egg number and clutch size in crossbred laying hens

Heterosis is the major benefit of crossbreeding and has been exploited in laying hens breeding for a long time. This genetic phenomenon has been linked to various modes of nonadditive gene action. However, the molecular mechanism of heterosis for egg production in laying hens has not been fully elucidated. To fill this research gap, we sequenced mRNAs and lncRNAs of the ovary stroma containing prehierarchical follicles in White Leghorn, Rhode Island Red chickens as well as their reciprocal crossbreds that demonstrated heterosis for egg number and clutch size. We further delineated the modes of mRNAs and lncRNAs expression to identify their potential functions in the observed heterosis. Results showed that dominance was the principal mode of nonadditive expression exhibited by mRNAs and lncRNAs in the prehierarchical follicles of crossbred hens. Specifically, low-parent dominance was the main mode of mRNA expression, while high-parent dominance was the predominant mode of lncRNA expression. Important pathways enriched by genes that showed higher expression in crossbreds compared to either one or both parental lines were cell adhesion molecules, tyrosine and purine metabolism. In contrast, ECM-receptor interaction, focal adhesion, PPAR signaling, and ferroptosis were enriched in genes with lower expression in the crossbred. Protein network interaction identified nonadditively expressed genes including apolipoprotein B (APOB), transferrin, acyl-CoA synthetase medium-chain family member (APOBEC) 3, APOBEC1 complementation factor, and cathepsin S as hub genes. Among these potential hub genes, APOB was the only gene with underdominance expression common to the 2 reciprocal crossbred lines, and has been linked to oxidative stress. LncRNAs with nonadditive expression in the crossbred hens targeted natriuretic peptide receptor 1, epidermal differentiation protein beta, spermatogenesis-associated gene 22, sperm-associated antigen 16, melanocortin 2 receptor, dolichol kinase, glycine amiinotransferase, and prolactin releasing hormone receptor. In conclusion, genes with nonadditive expression in the crossbred may play crucial roles in follicle growth and atresia by improving follicle competence and increasing oxidative stress, respectively. These 2 phenomena could underpin heterosis for egg production in crossbred laying hens.

#3467 INHIBITION OF ACYL-COA SYNTHETASE MEDIUM CHAIN FAMILY MEMBER 3 PROMOTES RENAL FIBROSIS BY ACCELERATING CELLULAR SENESCENCE IN TUBULAR EPITHELIAL CELLS

Abstract Background and Aims Cellular senescence is associated with renal disease progression. Accelerated tubular cell senescence promotes the pathogenesis of renal fibrosis no matter caused by natural aging or disease. However, the underlying mechanism is unknown. Method Proteomic analysis of natural aging kidney from 20-week mice was used to find potential target protein which is acyl-CoA synthetase medium chain family member 3 (Acsm3). After finding it, two Acsm3 inhibitor (4-Methylsalicylic acid and 2-hydroxyoctanoic acid) were used separately to treat natural aging and pathological aging (unilateral ureteral obstruction induced renal fibrosis) mice. 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 analysed by western blot and qPCR assay for the expression of Acsm3, fibrosis and cellular senescence related proteins and genes. In order to avoid drug toxicity, tubular epithelial cells were treated with Acsm3 SiRNA to explore the effects of Acsm3 in vitro. Results Firstly, we confirmed that cellular senescence happened in both elderly mice and UUO mice and found SA-β-gal positive cells were mainly tubular epithelial cells. After analysing proteomic sequencing data, Acsm3 is the protein which increased dramatically as the top one protein in elderly mice. We also found Acsm3 increased in tubular cells specifically in adult mice compared with fetal mice from KIT single cell database, which suggested that Acsm3 might have effects for kidney development. After validating changes and location of Acsm3, we treated natural aging and UUO mice with two Acsm3 inhibitor to check the function of Acsm3, separately. Administration of Acsm3 inhibitor (no matter 4-Methylsalicylic acid or 2-hydroxyoctanoic acid) could induce renal histological injuries, renal fibrosis, with significantly increased COL6, α-SMA and FN expression in both natural aging and UUO induced renal fibrosis. The cellular senescence was also observed to increase after Acsm3 inhibitor treatment with growth P21 and P16 expression. In addition, pro-inflammatory senescence-associated secretory phenotype (SASP) including IL6 and MCP1 increased as well. In order to avoid kidney injury induced by drug toxicity instead of by inhibition of Acsm3, we transfected Acsm3 SiRNA in tubular epithelial cells to check in vitro. Similarly, the inhibition of Acsm3 augmented cellular senescence and SASP of tubular epithelial cells as what we found in vivo. It supported that inhibition of Acsm3 could induce senescence of tubular epithelial cells and thus to promote kidney fibrosis. Conclusion These findings suggested that Acsm3 could be an important protector for renal fibrosis by inhibiting cellular senescence of tubular epithelial cells. The underlying mechanism needs to explore more.

Impaired fatty acid metabolism perpetuates lipotoxicity along the transition to chronic kidney injury.

Energy metabolism failure in proximal tubule cells (PTCs) is a hallmark of chronic kidney injury. We combined transcriptomic, metabolomic, and lipidomic approaches in experimental models and patient cohorts to investigate the molecular basis of the progression to chronic kidney allograft injury initiated by ischemia/reperfusion injury (IRI). The urinary metabolome of kidney transplant recipients with chronic allograft injury and who experienced severe IRI was substantially enriched with long chain fatty acids (FAs). We identified a renal FA-related gene signature with low levels of carnitine palmitoyltransferase 2 (Cpt2) and acyl-CoA synthetase medium chain family member 5 (Acsm5) and high levels of acyl-CoA synthetase long chain family member 4 and 5 (Acsl4 and Acsl5) associated with IRI, transition to chronic injury, and established chronic kidney disease in mouse models and kidney transplant recipients. The findings were consistent with the presence of Cpt2-Acsl4+Acsl5+Acsm5- PTCs failing to recover from IRI as identified by single-nucleus RNA-Seq. In vitro experiments indicated that ER stress contributed to CPT2 repression, which, in turn, promoted lipids' accumulation, drove profibrogenic epithelial phenotypic changes, and activated the unfolded protein response. ER stress through CPT2 inhibition and lipid accumulation engaged an auto-amplification loop leading to lipotoxicity and self-sustained cellular stress. Thus, IRI imprints a persistent FA metabolism disturbance in the proximal tubule, sustaining the progression to chronic kidney allograft injury.

The Ginsenoside Rg1 Rescues Mitochondrial Disorders in Aristolochic Acid-Induced Nephropathic Mice.

Chronic exposure to aristolochic acid (AA) leads to renal interstitial fibrosis and nephropathy. In this study, we aimed to investigate the renoprotective effects of Panax ginseng extract (GE) and ginsenoside saponin (GS) on AA-induced nephropathy (AAN) in mice. Eighty female C3H/He mice were randomly divided into eight groups, including normal; AA (3 μg/mL for 56 days); AA with GE (125, 250, or 500 mg/kg/d for 14 days); and AA with important GE ingredients, Rg1, Rb1, or Rd (5 mg/kg/d for 14 days). Compared with the AA group, renal injuries were significantly decreased in the GE (250 mg/kg/d), Rb1, and Rg1 treatment groups. Rg1 exhibited the best renoprotection among all GS-treated groups. There were 24 peaks significantly altered among normal, AA, and AA + Rg1 groups, and four mitochondrial proteins were identified, including acyl-CoA synthetase medium-chain family member 2, upregulated during skeletal muscle growth 5 (Usmg5), mitochondrial aconitase 2 (ACO2), and cytochrome c oxidase subunit Va preprotein (COX5a). We demonstrated for the first time that the AAN mechanism and renoprotective effects of Rg1 are associated with expression of mitochondrial proteins, especially ACO2, Usmg5, and COX5a.

Hepatic Transcriptome Profiling Reveals Lack of Acsm3 Expression in Polydactylous Rats with High-Fat Diet-Induced Hypertriglyceridemia and Visceral Fat Accumulation.

Metabolic syndrome (MetS) is an important cause of worldwide morbidity and mortality. Its complex pathogenesis includes, on the one hand, sedentary lifestyle and high caloric intake, and, on the other hand, there is a clear genetic predisposition. PD (Polydactylous rat) is an animal model of hypertriglyceridemia, insulin resistance, and obesity. To unravel the genetic and pathophysiologic background of this phenotype, we compared morphometric and metabolic parameters as well as liver transcriptomes among PD, spontaneously hypertensive rat, and Brown Norway (BN) strains fed a high-fat diet (HFD). After 4 weeks of HFD, PD rats displayed marked hypertriglyceridemia but without the expected hepatic steatosis. Moreover, the PD strain showed significant weight gain, including increased weight of retroperitoneal and epididymal fat pads, and impaired glucose tolerance. In the liver transcriptome, we found 5480 differentially expressed genes, which were enriched for pathways involved in fatty acid beta and omega oxidation, glucocorticoid metabolism, oxidative stress, complement activation, triacylglycerol and lipid droplets synthesis, focal adhesion, prostaglandin synthesis, interferon signaling, and tricarboxylic acid cycle pathways. Interestingly, the PD strain, contrary to SHR and BN rats, did not express the Acsm3 (acyl-CoA synthetase medium-chain family member 3) gene in the liver. Together, these results suggest disturbances in fatty acid utilization as a molecular mechanism predisposing PD rats to hypertriglyceridemia and fat accumulation.

Abstract PO-036: ACSM1 and ACSM3 regulate fatty acid oxidation in prostate cancer to promote growth and protect against oxidative stress

Abstract Prostate tumours are highly reliant on lipids for growth and survival. Emerging evidence suggests that the androgen receptor (AR) is a major regulator of lipid metabolism in prostate cancer (PCa), but the molecular mechanisms underlying such regulation are largely unknown. By integrating cistromic and transcriptomic data, we identified Acyl-CoA Synthetase Medium Chain Family Members 1 and 3 (ACSM1 and ACSM3) as putative new AR-regulated genes in prostate cancer. These factors are thought to function to activate fatty acids for their utilisation in energy production via mitochondrial beta-oxidation. AR regulation was validated by demonstrating modulation of ACSM1 and ACSM3 by androgen and anti-androgen treatment and confirming direct binding of AR to proximal cis-regulatory elements by chromatin immunoprecipitation (ChIP). ACSM1 and ACSM3 are upregulated in prostate tumours compared to non-malignant prostate tissues and are expressed more highly in prostate cancer than other cancer types. We subsequently applied metabolomics, lipidomics and functional assays to decipher the roles of ACSM1 and ACSM3 in prostate cancer cells. Knockdown of ACSM1 and ACSM3 in PCa cells resulted in growth arrest and ATP depletion, supporting a key role for both factors in energy production from fatty acids. Furthermore, lipidomic analysis of cells showed that poly-unsaturated fatty acids accumulate in response to loss of ACSM1 and ACSM3. Metabolomics revealed that cells adapt to loss of ACSM1 and ACSM3 by switching to a glycolytic phenotype. The metabolic dysregulation induced by knockdown of ACSM1 and ACSM3 caused mitochondrial oxidative stress and subsequent lipid peroxidation, eventually resulting in cell death. Accumulation of mitochondrial reactive oxygen species was abrogated by ferrostatin-1 (an iron chelator), suggesting that cell death was due to an iron-dependent form of apoptosis termed ferroptosis. Supporting this concept, over-expression of ACSM1 and ACSM3 elicited resistance to the ferroptosis inducers Erastin and ML210. Our study has revealed a novel mechanism by which AR regulates lipid metabolism in prostate cancer cells. Importantly, the critical role of ACSM1 and ACSM3 as key regulators of growth and protectors against ferroptosis emphasises their potential as novel therapeutic targets Citation Format: Raj Kumar Shrestha, Scott Townley, Adrienne Hanson, Marie Pickering, Zeyad D. Nassar, Chui Yan Mah, Mohammadreza A. Ghodsi, Andrew J. Hoy, Lake-Ee Quek, Wayne D. Tilley, Lisa M. Butler, Luke A. Selth. ACSM1 and ACSM3 regulate fatty acid oxidation in prostate cancer to promote growth and protect against oxidative stress [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-036.

Expression of Acsm2, a kidney-specific gene, parallels the function and maturation of proximal tubular cells

The acyl-CoA synthetase medium-chain family member 2 (Acsm2) gene was first identified and cloned by our group as a kidney-specific "KS" gene. However, its expression pattern and function remain to be clarified. In the present study, we found that the Acsm2 gene was expressed specifically and at a high level in normal adult kidneys. Expression of Acsm2 in kidneys followed a maturational pattern: it was low in newborn mice and increased with kidney development and maturation. In situ hybridization and immunohistochemistry revealed that Acsm2 was expressed specifically in proximal tubular cells of adult kidneys. Data from the Encyclopedia of DNA Elements database revealed that the Acsm2 gene locus in the mouse has specific histone modifications related to the active transcription of the gene exclusively in kidney cells. Following acute kidney injury, partial unilateral ureteral obstruction, and chronic kidney diseases, expression of Acsm2 in the proximal tubules was significantly decreased. In human samples, the expression pattern of ACSM2A, a homolog of mouse Acsm2, was similar to that in mice, and its expression decreased with several types of renal injuries. These results indicate that the expression of Acsm2 parallels the structural and functional maturation of proximal tubular cells. Downregulation of its expression in several models of kidney disease suggests that Acms2 may serve as a novel marker of proximal tubular injury and/or dysfunction.

Nephrotoxicity Profile of Cadmium Revealed by Proteomics in Mouse Kidney.

Cadmium (Cd) is a highly toxic metal and kidney is its main target. However, the molecular effects and associated potential impacts of Cd-accumulated kidney have not been well investigated. In this study, mouse was used as a model to investigate the Cd-induced proteomic profile change in kidney, and a total of 34 differentially expressed proteins were detected by two-dimensional gel electrophoresis (2-DE) and further identified by matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS). Through Gene Ontology analysis and KEGG pathway annotation, it showed that Cd-regulated kidney metabolism and promoted renal damage and cell migration. By validation of Western blotting and RT-qPCR, metastasis-related proteins LIM and SH3 domain protein 1 (LASP1) and phosphoenolpyruvate carboxykinase/cytosolic [GTP] (PEPCK1) were confirmed to be upregulated; Acyl-CoA synthetase medium-chain family member 3 (ACSM3) was downregulated. Furthermore, carcinoma development-related proteins initiation factor 4A (eIF4A) and pyridoxine-5'-phosphate oxidase (PNPO) were upregulated, and pyridoxal kinase (PK) was downregulated. The downregulation of Na(+)/H(+) exchange regulatory cofactor (NHERF3) might promote renal damage which associated with decrease of transferrin (TRF) in kidney. Taken together, our results revealed proteomic profile of Cd-induced nephrotoxicity and provided data for further insights into the mechanisms of Cd toxicity.

Abstract 4641: Metabolic reprogramming of the breast contributes to a cancer promoting milieu

Abstract In addition to the accumulation of pro-oncogenic mutations in the epithelial cells, the tumorigenic process involves the dysregulation of the interactions between the epithelial cells and their microenvironment, as well as alterations within the microenvironment itself. The latter is composed of endothelial cells, immune cells, fibroblasts, unaffected epithelium, and adipocytes. Increasing evidences support that cell transformation progresses in a potentially cancer-promoting microenvironment context, also known as field cancerization model. Therefore, it is critical to investigate changes occurring in both the epithelial compartment and the surrounding microenvironment to understand how breast cancer initiates. In our preliminary study, we analyzed the transcriptome profile of microdissected breast tissue compartments (epithelium, stroma and adipose tissue) from tissue biopsies obtained from women who donated their histologically normal tissue two-to-five years before breast cancer diagnosis (here labeled pre-cancer), and matched healthy control donors. In the pre-cancer breast epithelium we detected a significant increase in lipid metabolism-related genes including lipases (HSL, LPL) and perilipins (PLIN1, PLIN4), which mediate the release of fatty acids from triacylglycerol storage, and ELOVL5 and ELOVL7, which generate oleic acid. Moreover, Acyl-CoA Synthetase Medium Chain Family Member 1, ACSM1, involved in the activation of lipoic acid, an essential cofactor for mitochondrial metabolism, is also upregulated in pre-cancer breast tissue. Upregulation of genes involved in metabolic activation is observed also in the stroma and adipose tissue compartments. Interestingly, the transcriptome profiling of the pre-cancer breast stroma shows the downregulation of genes coding for several immune cell markers as compared with the healthy controls. Immunohistochemical staining of CD45 confirms a significant reduction in immune cells in the pre-cancer versus matched healthy control breasts. This finding suggests the prevalence of an immune-suppressive environment in the breast long before the clinical diagnosis of breast cancer. Understanding the changes occurring in the cancerized field is critical for elucidating 1) the involvement of microenvironment in cancer initiation 2) the role of histologically normal yet genetically altered surgical margins in risk of disease recurrence upon lumpectomy for early breast cancer diagnosis, and 3) the mechanisms of field cancerization in the contralateral breast. Citation Format: Natascia Marino, Rana German, Xi Rao, George Sandusky, Max Jacobsen, Sha Cao, Anna Maria Storniolo. Metabolic reprogramming of the breast contributes to a cancer promoting milieu [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4641.

Dynamics of fatty acid regulatory genes during ovarian development in Penaeus monodon.

The delay in ovarian maturation in farmed black tiger shrimp Penaeus monodon has resulted in the widespread practice of feeding broodstock with the polychaete Perinereis nuntia and their unilateral eyestalk ablation. Although this practice alters fatty acid content in shrimp ovaries and hepatopancreas, its effects on fatty acid regulatory genes are yet to be systematically examined. Here, microarray analysis was performed on hepatopancreas and ovary cDNA collected from P. monodon at different ovarian maturation stages, revealing that 72 and 58 genes in fatty acid regulatory pathways were differentially expressed in hepatopancreas and ovaries respectively. Quantitative real-time PCR analysis revealed that ovarian maturation was associated with higher expression levels of acetyl-CoA acetyltransferase, acyl-CoA dehydrogenase, acyl-CoA oxidase 3 and long-chain fatty acid transport protein 4 in hepatopancreas, whereas the expression levels of 15 fatty acid regulatory genes were increased in shrimp ovaries. To distinguish the effects of different treatments, transcriptional changes were examined in P. monodon with stage 1 ovaries before polychaete feeding, after 1 month of polychaete feeding and after eyestalk ablation. Polychaete feeding resulted in lower expression levels of enoyl-CoA hydratase and acyl-CoA synthetase medium-chain family member 4, while the expression level of phosphatidylinositide phosphatase SAC1 was higher in shrimp hepatopancreas and ovaries. Additionally, eyestalk ablation resulted in a higher expression level of long-chain fatty acid-CoA ligase 4 in both tissues. Together, our findings describe the dynamics of fatty acid regulatory pathways during crustacean ovarian development and provide potential target genes for alternatives to eyestalk ablation in the future.

Poldip2 is an Oxygen-sensitive Mitochondrial Protein that Controls Oxidative/glycolytic Metabolism Balance and Proteasome Activity

The polymerase delta interacting protein 2 (Poldip2) is a nuclear-encoded mitochondrial protein of unknown function. We recently reported that Poldip2 is repressed under hypoxia and that its deficiency results in repressed mitochondrial function and increased glycolytic activity. However, the mechanisms responsible for this metabolic reprograming and its consequences are unknown. In this study, we show that Poldip2 controls the lipoylation and consequently the activity of the pyruvate and α-ketoglutarate dehydrogenase complexes by a mechanism that involves Clp-protease complex-mediated degradation of Acyl-CoA Synthetase Medium-Chain Family Member 1 (ACSM1), a lipoate-activating enzyme required for the utilization of lipoic acid derived from the salvage pathway. With regards to the increased glycolytic activity, we found that Poldip2 deficiency inhibits PHDs inducing HIF-1α-dependent upregulation of Hexokinase 2. As a result, we observed an increase in the flux through the Hexosamine biosynthetic pathway (HBP), a glucose metabolic pathway that produces uridine diphosphate N-acetylglucosamine, the sugar donor for the O-GlcNAc transferase (OGT). The OGT-catalyzed transfer of O-GlcNAc is a post-translational modification implicated in cell signaling. Interestingly we found that Poldip2 deficiency induces the inhibition of the ubiquitin proteasome system (UPS) by OGT-dependent O-GlcNAcylation of the 19S regulatory unit of the 26S proteasome regulatory subunit 4 (PRS4). Additionally, we demonstrated that Poldip2-mediated inhibition of the UPS is required for the hypoxia-induced expression of connective tissue growth factor (CTGF), a cytokine that participates in multiple pathologies including fibrotic diseases and cancer. In all, we demonstrate that Poldip2 is an oxygen sensitive protein that regulates the activity of key enzymes of the Krebs cycle by coupling proteasome activity to mitochondrial function and fibrotic responses under hypoxia.

Integrative transcriptome analysis of liver cancer profiles identifies upstream regulators and clinical significance of ACSM3 gene expression.

Hepatocellular carcinoma (HCC) is one of the most common human malignancies. It has frequently been associated with metabolic perturbations and liver damages. Various members of the family of acyl-CoA synthetases are known to be involved in the production of bioactive fatty acids, and altered expression of its encoding genes has been found to be involved in metabolic perturbations. For the development of novel diagnostic and therapeutic HCC options, a fundamental understanding of the mechanisms associated with the deregulation of candidate genes involved in metabolic perturbation is required. A meta-analysis of multiple HCC mRNA profiles was performed to identify consistently deregulated genes. Expression of the acyl-CoA synthetase medium chain family member 3 (ACSM3) gene was subsequently assessed in different HCC tumor stages and correlated with various clinicopathological features. Transcription regulation, survival and pathway-associated features of the ACSM3 gene were investigated using integrative functional genomic and molecular cell biological methods. We found that expression of the ACSM3 gene was significantly reduced in HCC tissues and was frequently downregulated in patients exhibiting high alpha-fetoprotein (AFP) levels, high alanine aminotransferase (ALT) levels, multiple nodules and large tumors. Loss of ACSM3 expression was found to correlate with advanced HCC stages and a poor survival. In addition, HNF4α was found to positively regulate the expression of the ACSM3 gene, while PPARγ was found to transcriptionally repress it. Downregulation of ACSM3 expression was perceived upon activation of the TGFβ, WNT, AKT and MYC signalling pathways. In addition, we found that ACSM3 expression correlates with fatty acid oxidation in HCC. Our data provide evidence for a differential expression and regulation of the ACSM3 gene in HCC, and may lay a foundation for therapeutically targeting fatty acid metabolism in these tumors.

307. Hitting the Kidney from All Angles: Using Direct Intrarenal Pelvis Injection Combined with Serotype and Promoter Targeting to Achieve Renal AAV Gene Delivery

Using adeno-associated virus (AAV)-based gene therapy to treat renal diseases is an underdeveloped area. Renal anatomy and physiology make gene delivery to this organ especially difficult. Multiple AAV serotypes and routes of entry in rodents have been tried, with the majority of combinations targeting the renal tubules. To improve upon the current methods of AAV renal transduction for future use in the clinical setting, we employed direct intrarenal pelvis injections of AAV2, 6, 8 and 9 with the luciferase (Luc) or GFP transgene under the control of the cytomegalovirus (CMV) or putative kidney-specific promoters from myo-inositol oxygenase(MIOX), acyl-CoA synthetase medium-chain family member 2 (Acsm2), uromodulin (UMOD), kidney androgen-regulated protein (Kap) and solute carrier family 34 (sodium phosphate), member 1 (Slc34a1) mouse genes in C57Bl/6J mice. Renal luciferase transgene expression was strongest with AAV6, 8 and 9-CMV-Luc. AAV6 and 8-CMV-Luc resulted in stronger expression in the injected kidney, whereas AAV9-CMV-Luc showed equal, bilateral renal expression. AAV9 with the UMOD promoter gave the strongest and most highly renal-specific luciferase expression among all serotype and kidney promoter combinations. Using the GFP transgene we further established AAV transduction of multiple cell types in the kidney. AAV8-CMV-GFP transduced both glomerular and tubular renal cells, while AAV9-CMV-GFP most strikingly transduced periglomerular tubule cells. Both AAV8 and AAV9-UMOD-GFP solely transduced renal tubule cells. We are currently testing AAV variants isolated from clinical urine samples to determine their ability to transduce renal cells in vivo. Our results demonstrate efficient renal transduction by direct intrarenal pelvis injection of AAV vectors. Coupling specific renal cell tropisms of AAV serotypes and renal cell-type-specific promoters, along with the intrarenal pelvis injection strategy would pave the way for a renal-targeted AV gene therapy.

Novel somatic mutations identified by whole-exome sequencing in muscle-invasive transitional cell carcinoma of the bladder.

Transitional cell carcinoma (TCC) is the one of the most commonly observed types of cancer globally. The identification of novel disease-associated genes in TCC has had a significant effect on the diagnosis and treatment of bladder cancer; however, there may be a large number of novel genes that have not been identified. In the present study, the exomes of two individuals who were diagnosed with muscle-invasive TCC (MI-TCC) were sequenced to investigate potential variants. Subsequently, following algorithm and filter analysis, Sanger sequencing was used to validate the results of deep sequencing. Immunohistochemistry (IHC) was employed to observe the differences in HECT, C2 and WW domain-containing E3 ubiquitin protein ligase 1 (HECW1) protein expression between tumor tissues and para-carcinoma tissues. A total of 6 nonsynonymous mutation genes were identified in MI-TCC, identified as copine VII, RNA binding motif protein, X-linked-like 3, acyl-CoA synthetase medium-chain family member 2A, HECW1, zinc finger protein 273 and trichohyalin. Furthermore, 5 cases were identified to possess a HECW1 gene mutation in 61 MI-TCC specimens, and all of these were point mutations located at exon 11 on chromosome 7. The mutation categories of HECW1 had 4 missense mutations and 1 nonsense mutation. IHC revealed that HECW1 protein was expressed at significantly increased levels in MI-TCC compared with normal bladder urothelium (P<0.001). The present study provided a novel approach for investigating genetic changes in the MI-TCC exome, and identified the novel mutant gene HECW1, which may possess a significant role in the pathogenesis of TCC.

Comparative metabolic pathway analysis with special reference to nucleotide metabolism-related genes in chicken primordial germ cells

Metabolism provides energy and nutrients required for the cellular growth, maintenance, and reproduction. When compared with genomics and proteomics, metabolism studies provide novel findings in terms of cellular functions. In this study, we examined significant and differentially expressed genes in primordial germ cells (PGCs), gonadal stromal cells, and chicken embryonic fibroblasts compared with blastoderms using microarray. All upregulated genes (1001, 1118, and 974, respectively) and downregulated genes (504, 627, and 1317, respectively) in three test samples were categorized into functional groups according to gene ontology. Then all selected genes were tested to examine their involvement in metabolic pathways through Kyoto Encyclopedia of Genes and Genomes pathway database using overrepresentation analysis. In our results, most of the upregulated and downregulated genes were involved in at least one subcategory of seven major metabolic pathways. The main objective of this study is to compare the PGC expressed genes and their metabolic pathways with blastoderms, gonadal stromal cells, and chicken embryonic fibroblasts. Among the genes involved in metabolic pathways, a higher number of PGC upregulated genes were identified in retinol metabolism, and a higher number of PGC downregulated genes were identified in sphingolipid metabolism. In terms of the fold change, acyl-CoA synthetase medium-chain family member 3 (ACSM3), which is involved in butanoate metabolism, and N-acetyltransferase, pineal gland isozyme NAT-10 (PNAT10), which is involved in energy metabolism, showed higher expression in PGCs. To validate these gene changes, the expression of 12 nucleotide metabolism-related genes in chicken PGCs was examined by real-time polymerase chain reaction. The results of this study provide new information on the expression of genes associated with metabolism function of PGCs and will facilitate more basic research on animal PGC differentiation and function.

Genetic variants in nuclear-encoded mitochondrial genes influence AIDS progression.

BackgroundThe human mitochondrial genome includes only 13 coding genes while nuclear-encoded genes account for 99% of proteins responsible for mitochondrial morphology, redox regulation, and energetics. Mitochondrial pathogenesis occurs in HIV patients and genetically, mitochondrial DNA haplogroups with presumed functional differences have been associated with differential AIDS progression.Methodology/Principal FindingsHere we explore whether single nucleotide polymorphisms (SNPs) within 904 of the estimated 1,500 genes that specify nuclear-encoded mitochondrial proteins (NEMPs) influence AIDS progression among HIV-1 infected patients. We examined NEMPs for association with the rate of AIDS progression using genotypes generated by an Affymetrix 6.0 genotyping array of 1,455 European American patients from five US AIDS cohorts. Successfully genotyped SNPs gave 50% or better haplotype coverage for 679 of known NEMP genes. With a Bonferroni adjustment for the number of genes and tests examined, multiple SNPs within two NEMP genes showed significant association with AIDS progression: acyl-CoA synthetase medium-chain family member 4 (ACSM4) on chromosome 12 and peroxisomal D3,D2-enoyl-CoA isomerase (PECI) on chromosome 6.ConclusionsOur previous studies on mitochondrial DNA showed that European haplogroups with presumed functional differences were associated with AIDS progression and HAART mediated adverse events. The modest influences of nuclear-encoded mitochondrial genes found in the current study add support to the idea that mitochondrial function plays a role in AIDS pathogenesis.