Peroxisomal Fatty Acid Oxidation Research Articles

Dietary dicarboxylic acids provide a non-storable alternative fat source that protects mice against obesity.

Dicarboxylic fatty acids are generated in the liver and kidney in a minor pathway called fatty acid ω-oxidation. The effects of consuming dicarboxylic fatty acids as an alternative source of dietary fat have not been explored. Here, we fed dodecanedioic acid, a 12-carbon dicarboxylic (DC12), to mice at 20% of daily caloric intake for nine weeks. DC12 increased metabolic rate, reduced body fat, reduced liver fat, and improved glucose tolerance. We observed DC12-specific breakdown products in liver, kidney, muscle, heart, and brain, indicating that oral DC12 escaped first-pass liver metabolism and was utilized by many tissues. In tissues expressing the "a" isoform of acyl-CoA oxidase-1 (ACOX1), a key peroxisomal fatty acid oxidation enzyme, DC12 was chain shortened to the TCA cycle intermediate succinyl-CoA. In tissues with low peroxisomal fatty acid oxidation capacity, DC12 was oxidized by mitochondria. In vitro, DC12 was catabolized even by adipose tissue and was not stored intracellularly. We conclude that DC12 and other dicarboxylic acids may be useful for combatting obesity and for treating metabolic disorders.

Abstract 4422: Metabolic adaptations of glioblastoma in hypoxia is mediated by acox-1

Abstract Background: Glioblastoma (GBM) is a recurrent, chemoresistant, malignant brain tumor. Hypoxia is a major factor contributing to resistance in GBM which develops due to aggressive tumor growth and chronic anti-angiogenic therapy. Thus, under limited oxygen supply it is unlikely that the high energy consuming pathways are solely able to provide the energy requirement of GBM cells. Thus, based on our metabolomics data stated below we hypothesize that GBM cells adapt to chronic hypoxia utilizing alternate metabolic pathways such as peroxisomal fatty acid oxidation (FAO). Here we have explored the role of Acyl coenzyme A oxidase (ACOX-1), a rate limiting enzyme for peroxisomal FAO in the metabolic adaptations to hypoxia and as a potential drug target. Methods: U251 CRISPR dox inducible ACOX1 (KO) cells and patient derived primary GBM were grown under normoxic and hypoxic conditions (2%O2, 5%CO2, 70% humidity). Metabolic analysis was done using UHPLC-MS and LCMS analysis. RNAseq was done using TopHat2 (genome alignment), HTSeq, and DEseq to study the differential gene expression at cut-off of 2-fold change, and p<0.05. GSEA was used to interpret biological pathways. NCr/SCID (Severe Combined Immunodeficiency) mice (n=5) inoculated with U251 ACOX1 knockout cells expressing luciferase were used and treated with anti-angiogenic agent pazopanib at 30 mg/kg, 5 days a week. The U251 luciferase-labeled tumors were imaged starting weekly for tumor volumes using the IVIS (In Vivo Imaging System) Lumina. Results: In patient derived primary GBM cell lines alterations in metabolites in chronic hypoxia compared to normoxia, such as decrease in citric acid cycle metabolites, increase in glycolytic metabolites, and dysfunctional mitochondrial metabolites. In RNA seq downregulation of genes PCK1, SCD, SREBF1, and PIPOX (p<1x108) involved in PPAR and lipid metabolism pathways in hypoxia versus normoxia were noted. Inhibition of ACOX-1 displayed increased survival in mice administered (p=6.7E-04) with pazopanib compared to the U251 parental control group. Conclusion: There is metabolomic reprogramming in ACOX-1 KO cells channeled predominantly through FAO as seen through the downregulation of genes downstream of the PPAR signaling pathway, mediated through SREBF1 the master regulator of lipid metabolism. Thus, these genes could be future targets for therapy in combination with anti-angiogenic therapies that aggravate hypoxia in GBM. Citation Format: Ayon Bhattacharya, Laura Caflisch, Henriette Balinda, Alessia Lodi, Mei Zhou, Mengxing Li, Jennifer Chiou, Renu Pandey, Gangadhara R. Sareddy, Stefano Tiziani, Yidong Chen, Ratna K. Vadlamudi, Andrew J. Brenner. Metabolic adaptations of glioblastoma in hypoxia is mediated by acox-1 [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 4422.

Interaction between fatty acid oxidation and ethanol metabolism in liver.

Fatty acid oxidation (FAO) releases the energy stored in fat to maintain basic biological processes. Dehydrogenation is a major way to oxidize fatty acids, which needs NAD+ to accept the released H+ from fatty acids and form NADH, which increases the ratio of NADH/NAD+ and consequently inhibits FAO leading to the deposition of fat in the liver, which is termed fatty liver or steatosis. Consumption of alcohol (ethanol) initiates simple steatosis that progresses to alcoholic steatohepatitis, which constitutes a spectrum of liver disorders called alcohol-associated liver disease (ALD). ALD is linked to ethanol metabolism. Ethanol is metabolized by alcohol dehydrogenase (ADH), microsomal ethanol oxidation system (MEOS), mainly cytochrome P450 2E1 (CYP2E1), and catalase. ADH also requires NAD+ to accept the released H+ from ethanol. Thus, ethanol metabolism by ADH leads to increased ratio of NADH/NAD+, which inhibits FAO and induces steatosis. CYP2E1 directly consumes reducing equivalent NADPH to oxidize ethanol, which generates reactive oxygen species (ROS) that lead to cellular injury. Catalase is mainly present in peroxisomes, where very long-chain fatty acids and branched-chain fatty acids are oxidized, and the resultant short-chain fatty acids will be further oxidized in mitochondria. Peroxisomal FAO generates hydrogen peroxide (H2O2), which is locally decomposed by catalase. When ethanol is present, catalase uses H2O2 to oxidize ethanol. In this review, we introduce FAO (including α-, β-, and ω-oxidation) and ethanol metabolism (by ADH, CYP2E1, and catalase) followed by the interaction between FAO and ethanol metabolism in the liver and its pathophysiological significance.

ATM deficiency differentially affects expression of proteins related to fatty acid oxidation and oxidative stress in a sex-specific manner in response to Western-type diet prior to and following myocardial infarction

AimsPublished work has shown that ataxia-telangiectasia mutated kinase (ATM) deficiency is associated with cardioprotective effects in Western-type diet (WD)-fed female mice. This study assessed the expression of proteins related to fatty acid oxidation (FAO) and oxidative stress in WD-fed male and female mouse hearts, and investigated if sex-specific cardioprotective effects in WD-fed female ATM-deficient mice are maintained following myocardial infarction (MI). Main methodsWild-type (WT) and ATM-deficient (hKO) mice (both sexes) were placed on WD for 14 weeks. Myocardial tissue from a subset of mice was used for western blot analyses, while another subset of WD-fed mice underwent MI. Heart function was analyzed by echocardiography prior to and 1 day post-MI. Key findingsCPT1B (mitochondrial FAO enzyme) expression was lower in male hKO-WD, while it was higher in female hKO-WD vs WT-WD. WD-mediated decrease in ACOX1 (peroxisomal FAO enzyme) expression was only observed in male WT-WD. PMP70 (transports fatty acyl-CoA across peroxisomal membrane) expression was lower in male hKO-WD vs WT-WD. Catalase (antioxidant enzyme) expression was higher, while Nox4 (pro-oxidant enzyme) expression was lower in female hKO-WD vs WT-WD. Heart function was better in female hKO-WD vs WT-WD. However, post-MI heart function was not significantly different among all MI groups. Post-MI, CPT1B and catalase expression was higher in male hKO-WD-MI vs WT-WD-MI, while Nox4 expression was higher in female hKO-WD-MI vs WT-WD-MI. SignificanceIncreased mitochondrial FAO and decreased oxidative stress contribute towards ATM deficiency-mediated cardioprotective effects in WD-fed female mice which are abolished post-MI with increased Nox4 expression.

Tubular CPT1A deletion minimally affects aging and chronic kidney injury.

Kidney tubules use fatty acid oxidation (FAO) to support their high energetic requirements. Carnitine palmitoyltransferase 1A (CPT1A) is the rate-limiting enzyme for FAO, and it is necessary to transport long-chain fatty acids into mitochondria. To define the role of tubular CPT1A in aging and injury, we generated mice with tubule-specific deletion of Cpt1a (Cpt1aCKO mice), and the mice were either aged for 2 years or injured by aristolochic acid or unilateral ureteral obstruction. Surprisingly, Cpt1aCKO mice had no significant differences in kidney function or fibrosis compared with wild-type mice after aging or chronic injury. Primary tubule cells from aged Cpt1aCKO mice had a modest decrease in palmitate oxidation but retained the ability to metabolize long-chain fatty acids. Very-long-chain fatty acids, exclusively oxidized by peroxisomes, were reduced in kidneys lacking tubular CPT1A, consistent with increased peroxisomal activity. Single-nuclear RNA-Seq showed significantly increased expression of peroxisomal FAO enzymes in proximal tubules of mice lacking tubular CPT1A. These data suggest that peroxisomal FAO may compensate in the absence of CPT1A, and future genetic studies are needed to confirm the role of peroxisomal β-oxidation when mitochondrial FAO is impaired.

Tissue-specific roles of peroxisomes revealed by expression meta-analysis

Peroxisomes are primarily studied in the brain, kidney, and liver due to the conspicuous tissue-specific pathology of peroxisomal biogenesis disorders. In contrast, little is known about the role of peroxisomes in other tissues such as the heart. In this meta-analysis, we explore mitochondrial and peroxisomal gene expression on RNA and protein levels in the brain, heart, kidney, and liver, focusing on lipid metabolism. Further, we evaluate a potential developmental and heart region-dependent specificity of our gene set. We find marginal expression of the enzymes for peroxisomal fatty acid oxidation in cardiac tissue in comparison to the liver or cardiac mitochondrial β-oxidation. However, the expression of peroxisome biogenesis proteins in the heart is similar to other tissues despite low levels of peroxisomal fatty acid oxidation. Strikingly, peroxisomal targeting signal type 2-containing factors and plasmalogen biosynthesis appear to play a fundamental role in explaining the essential protective and supporting functions of cardiac peroxisomes.

627 - The role of peroxisomal fatty acid oxidation in kidney injury

627 - The role of peroxisomal fatty acid oxidation in kidney injury

ACOX1-mediated peroxisomal fatty acid oxidation contributes to metabolic reprogramming and survival in chronic lymphocytic leukemia.

Chronic lymphocytic leukemia (CLL) is still an incurable disease, with many patients developing resistance to conventional and targeted therapies. To better understand the physiology of CLL and facilitate the development of innovative treatment options, we examined specific metabolic features in the tumor CLL B-lymphocytes. We observed metabolic reprogramming, characterized by a high level of mitochondrial oxidative phosphorylation activity, a low glycolytic rate, and the presence of C2- to C6-carnitine end-products revealing an unexpected, essential role for peroxisomal fatty acid beta-oxidation (pFAO). Accordingly, downmodulation of ACOX1 (a rate-limiting pFAO enzyme overexpressed in CLL cells) was enough to shift the CLL cells' metabolism from lipids to a carbon- and amino-acid-based phenotype. Complete blockade of ACOX1 resulted in lipid droplet accumulation and caspase-dependent death in CLL cells, including those from individuals with poor cytogenetic and clinical prognostic factors. In a therapeutic translational approach, ACOX1 inhibition spared non-tumor blood cells from CLL patients but led to the death of circulating, BCR-stimulated CLL B-lymphocytes and CLL B-cells receiving pro-survival stromal signals. Furthermore, a combination of ACOX1 and BTK inhibitors had a synergistic killing effect. Overall, our results highlight a less-studied but essential metabolic pathway in CLL and pave the way towards the development of new, metabolism-based treatment options.

Dicarboxylic Acid Dietary Supplementation Protects against AKI.

In this study, we demonstrate that a common, low-cost compound known as octanedioic acid (DC 8 ) can protect mice from kidney damage typically caused by ischemia-reperfusion injury or the chemotherapy drug cisplatin. This compound seems to enhance peroxisomal activity, which is responsible for breaking down fats, without adversely affecting mitochondrial function. DC 8 is not only affordable and easy to administer but also effective. These encouraging findings suggest that DC 8 could potentially be used to assist patients who are at risk of experiencing this type of kidney damage. Proximal tubules are rich in peroxisomes, which are damaged during AKI. Previous studies demonstrated that increasing peroxisomal fatty acid oxidation (FAO) is renoprotective, but no therapy has emerged to leverage this mechanism. Mice were fed with either a control diet or a diet enriched with dicarboxylic acids, which are peroxisome-specific FAO substrates, then subjected to either ischemia-reperfusion injury-AKI or cisplatin-AKI models. Biochemical, histologic, genetic, and proteomic analyses were performed. Both octanedioic acid (DC 8 ) and dodecanedioic acid (DC 12 ) prevented the rise of AKI markers in mice that were exposed to renal injury. Proteomics analysis demonstrated that DC 8 preserved the peroxisomal and mitochondrial proteomes while inducing extensive remodeling of the lysine succinylome. This latter finding indicates that DC 8 is chain shortened to the anaplerotic substrate succinate and that peroxisomal FAO was increased by DC 8 . DC 8 supplementation protects kidney mitochondria and peroxisomes and increases peroxisomal FAO, thereby protecting against AKI.

Glucagon Agonism Protects the Kidney from Obesity by Restoring Peroxisomal Catalase and Fatty Acid Oxidation Capacity

Glucagon Agonism Protects the Kidney from Obesity by Restoring Peroxisomal Catalase and Fatty Acid Oxidation Capacity

Proteomics reveals defective peroxisomal fatty acid oxidation during the progression of acute kidney injury and repair

Acute kidney injury (AKI) is characterized by a rapid decrease in renal function with high mortality and risk of progression to chronic kidney disease (CKD). Ischemia and reperfusion injury (IRI) is one of the major causes of AKI. However, the cellular and molecular responses of the kidney to IRI are complex and not fully understood. Herein, we conducted unbiased proteomics and bioinformatics analyses in an IRI mouse model on days 3, 7, and 21, and validated the results using IRI, unilateral ureteral obstruction (UUO), and biopsies from patients with AKI or CKD. The results indicated an obvious temporal expression profile of differentially expressed proteins and highlighted impaired lipid metabolism during the progression of AKI to CKD. Acyl-coenzyme A oxidase 1 (Acox1), the first rate-limiting enzyme of peroxisomal fatty acid beta-oxidation, was then selected, and its disturbed expression in the two murine models validated the proteomic findings. Accordingly, Acox1 expression was significantly downregulated in renal biopsies from patients with AKI or CKD, and its expression was negatively correlated with kidney injury score. Furthermore, in contrast to the decreased Acox1 expression, lipid droplet accumulation was remarkably increased in these renal tissues, suggesting dysregulation of fatty acid oxidation. In conclusion, our results suggest that defective peroxisomal fatty acid oxidation might be a common pathological feature in the transition from AKI to CKD, and that Acox1 is a promising intervention target for kidney injury and repair.

SLC9A5 promotes tumor growth and cell motility via ACOX1-mediated peroxisomal fatty acid oxidation

Growing evidence suggests a strong association between decreased lipid catabolism and the development of cancer. Solute carrier family 9 member A5 (SLC9A5) plays a regulatory role in colorectal function. However, the specific involvement of SLC9A5 in colorectal cancer (CRC) remains unclear, as well as its potential connection to lipid catabolism. We found that SLC9A5 exhibited significantly higher expression in CRC tumor tissues compared to adjacent paratumor tissues, as confirmed through analysis of the TCGA database and validation on a CRC tissue chip using IHC. Furthermore, in vitro experiments showed that knockdown of SLC9A5 resulted in suppressed cell proliferation, migration, and invasion. Then we performed bioinformatics analysis and found that SLC9A5 was significantly enriched in peroxisomal fatty acid oxidation (FAO) pathway and negatively correlated with its first rate-limiting enzyme acyl-CoA oxidases (ACOX). Interestingly, the expression of ACOX1, as well as FAO process indicated by changes in very long chain fatty acid levels, were enhanced upon SLC9A5 knockdown in CRC cells. Moreover, the attenuated tumor growth, migration, invasion, and increased FAO observed after SLC9A5 knockdown could be reversed by simultaneous knockdown of both SLC9A5 and ACOX1. In summary, these findings reveal the oncogenic role of SLC9A5 in CRC, particularly in relation to ACOX1-mediated peroxidation, and might serve as a promising therapeutic target for inhibiting the progression of colorectal cancer.

Abstract 1157: Uncovering a novel role of peroxisomal β-oxidation in advanced, treatment-resistant prostate cancer

Abstract Background and Aims: Prostate cancer (PCa) is highly reliant on lipids for energy, hence targeting fatty acid oxidation (FAO) is a highly promising approach to the treatment of PCa. Recent work by our group and others have shown that FAO is critical for cell viability and survival, and is one of the key drivers of treatment resistance. Although FAO occurs in both the mitochondria and peroxisomes, the functional relevance and therapeutic exploitability of peroxisomal FAO (perFAO) in PCa has not been extensively explored compared to the mitochondria. Herein, we aim to investigate the therapeutic efficacy of targeting perFAO in clinical prostate tumors and PCa cells, and explore its role in PCa progression. Methods and Results: Pharmacological inhibition of perFAO using the clinical agent, thioridazine, significantly suppressed proliferation, migration, and colony formation of a range of advanced and treatment-resistant PCa cells. Further, we evaluated the therapeutic efficacy of thioridazine in a more clinically relevant setting using patient-derived tissues and showed a significant inhibition of cell proliferation (n = 11, p < 0.05). Next, we performed a bioinformatics analysis of clinical RNAseq datasets composed of primary and metastatic PCa tissues and identified DECR2, the auxiliary enzyme of perFAO, as a key target of perFAO in PCa. DECR2 is overexpressed in metastatic castrate-resistant PCa (CRPC) tissues compared to primary PCa and benign tissues, and is significantly associated with shorter relapse-free survival (p < 0.01). DECR2 knockdown significantly suppressed proliferation and migration of CRPC and enzalutamide-resistant PCa cells, and markedly reduced LNCaP tumor growth in vivo. In contrast, overexpression of DECR2 in LNCaP cells significantly increased tumor growth in vivo. Interestingly, analysis of a published proteomics dataset of PCa cells that are either androgen-dependent or resistant to androgen receptor (AR) inhibitors revealed a significant correlation of peroxisomal genes (MSigDB) with acquired resistance to AR inhibition. Notably, DECR2 overexpression was significantly associated with shorter overall survival (p < 0.05) in a metastatic CRPC cohort treated with AR inhibitors. We further showed that DECR2 overexpression in LNCaP cells significantly reduced sensitivity to AR inhibitor treatments and androgen-deprivation, suggesting a role for perFAO in promoting survival and treatment resistance. Conclusion: Our study has revealed a novel role for perFAO in PCa progression and treatment resistance. Importantly, this study also identified DECR2 as a key regulator of tumor cell proliferation and lipid metabolism, representing a potential novel therapeutic target for advanced, treatment-resistant PCa that currently lacks effective targeted therapies. Citation Format: Chui Yan Mah, An D. Nguyen, Takuto Niijima, Madison Helm, Jonas Dehairs, Feargal J. Ryan, Natalie Ryan, Ian G. Mills, Johannes V. Swinnen, David J. Lynn, Zeyad D. Nassar, Lisa M. Butler. Uncovering a novel role of peroxisomal β-oxidation in advanced, treatment-resistant prostate cancer [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 1157.

The deacylase sirtuin 5 reduces malonylation in nonmitochondrial metabolic pathways in diabetic kidney disease

Early diabetic kidney disease (DKD) is marked by dramatic metabolic reprogramming due to nutrient excess, mitochondrial dysfunction, and increased renal energy requirements from hyperfiltration. We hypothesized that changes in metabolism in DKD may be regulated by Sirtuin 5 (SIRT5), a deacylase that removes posttranslational modifications derived from acyl-coenzyme A and has been demonstrated to regulate numerous metabolic pathways. We found decreased malonylation in the kidney cortex (∼80% proximal tubules) of type 2 diabetic BKS db/db mice, associated with increased SIRT5 expression. We performed a proteomics analysis of malonylated peptides and found that proteins with significantly decreased malonylated lysines in the db/db cortex were enriched in nonmitochondrial metabolic pathways: glycolysis and peroxisomal fatty acid oxidation. To confirm relevance of these findings in human disease, we analyzed diabetic kidney transcriptomic data from a cohort of Southwestern American Indians, which revealed a tubulointerstitial-specific increase in Sirt5 expression. These data were further corroborated by immunofluorescence data of SIRT5 from nondiabetic and DKD cohorts. Furthermore, overexpression of SIRT5 in cultured human proximal tubules demonstrated increased aerobic glycolysis. Conversely, we observed reduced glycolysis with decreased SIRT5 expression. These findings suggest that SIRT5 may lead to differential nutrient partitioning and utilization in DKD. Taken together, our findings highlight a previously unrecognized role for SIRT5 in metabolic reprogramming in DKD.

Targeting peroxisomal fatty acid oxidation improves hepatic steatosis and insulin resistance in obese mice

Obesity and diabetes normally cause mitochondrial dysfunction and hepatic lipid accumulation, while fatty acid synthesis is suppressed and malonyl-CoA is depleted in the liver of severe obese or diabetic animals. Therefore, a negative regulatory mechanism might work for the control of mitochondrial fatty acid metabolism that is independent of malonyl-CoA in the diabetic animals. As mitochondrial β-oxidation is controlled by the acetyl-CoA/CoA ratio, and the acetyl-CoA generated in peroxisomal β-oxidation could be transported into mitochondria via carnitine shuttles, we hypothesize that peroxisomal β-oxidation might play a role in regulating mitochondrial fatty acid oxidation and inducing hepatic steatosis under the condition of obesity or diabetes. This study reveals a novel mechanism by which peroxisomal β-oxidation controls mitochondrial fatty acid oxidation in diabetic animals. We determined that excessive oxidation of fatty acids by peroxisomes generates considerable acetyl-carnitine in the liver of diabetic mice, which significantly elevates the mitochondrial acetyl-CoA/CoA ratio and causes feedback suppression of mitochondrial β-oxidation. Additionally, we found that specific suppression of peroxisomal β-oxidation enhances mitochondrial fatty acid oxidation by reducing acetyl-carnitine formation in the liver of obese mice. In conclusion, we suggest that induction of peroxisomal fatty acid oxidation serves as a mechanism for diabetes-induced hepatic lipid accumulation. Targeting peroxisomal β-oxidation might be a promising pathway in improving hepatic steatosis and insulin resistance as induced by obesity or diabetes.

Peroxisomal Fitness: A Potential Protective Mechanism of Fenofibrate against High Fat Diet-Induced Non-Alcoholic Fatty Liver Disease in Mice.

Non-alcoholic fatty liver disease (NAFLD) has been increasing in association with the epidemic of obesity and diabetes. Peroxisomes are single membrane-enclosed organelles that play a role in the metabolism of lipid and reactive oxygen species. The present study examined the role of peroxisomes in high-fat diet (HFD)-induced NAFLD using fenofibrate, a peroxisome proliferator-activated receptor α (PPARα) agonist. Eight-week-old male C57BL/6J mice were fed either a normal diet or HFD for 12 weeks, and fenofibrate (50 mg/kg/day) was orally administered along with the initiation of HFD. HFD-induced liver injury as measured by increased alanine aminotransferase, inflammation, oxidative stress, and lipid accumulation was effectively prevented by fenofibrate. Fenofibrate significantly increased the expression of peroxisomal genes and proteins involved in peroxisomal biogenesis and function. HFD-induced attenuation of peroxisomal fatty acid oxidation was also significantly restored by fenofibrate, demonstrating the functional significance of peroxisomal fatty acid oxidation. In Ppara deficient mice, fenofibrate failed to maintain peroxisomal biogenesis and function in HFD-induced liver injury. The present data highlight the importance of PPARα-mediated peroxisomal fitness in the protective effect of fenofibrate against NAFLD.

CNSC-18. HYPOXIA-INDUCED METABOLIC ADAPTATIONS THROUGH ACOX-1 IN GLIOBLASTOMA

Abstract BACKGROUND Hypoxia is a major driver of invasiveness and resistance in glioblastoma (GBM). We hypothesize that GBM adapts to chronic hypoxic conditions through utilization of peroxisomal fatty acid oxidation (FAO). We explored the contribution of acyl coenzyme A oxidase 1 (ACOX-1), a rate-limiting enzyme for peroxisomal FAO, in metabolic adaptation to hypoxia and as a potential therapeutic target. METHODS A primary cell line from a bevacizumab-resistant tumor (012015) was cultured under chronic hypoxia (2%O2) or normoxia (21% O2). We used UPLC-MS/MS and GC-MS to determine lipid and polar metabolites. RNA seq was performed on U251 ACOX-1 (KO) cells under normoxic and hypoxic conditions. Differential gene expression was obtained using TopHat2 (genome alignment), HTSeq, and DEseq. Gene set enrichment analysis was used to compare biological pathways between conditions. ACOX-1 was deleted using CRISPR-Cas9 and orthotopically implanted in NCr/SCID nude mice. Animals were treated with 30 mg/kg of pazopanib or vehicle daily through oral gavage. RESULTS In 012015 cells we observed altered carnitine metabolites, increase in specific triglycerides, and S-lactoylglutathione indicating dysfunctional mitochondrial FAO, and increase in oxidative stress between hypoxic and normoxic conditions. Also, decrease in citric acid cycle metabolites, increase in glycolytic metabolites were seen in hypoxia. Further RNA seq on U251 ACOX-1 (KO) cells showed downregulated genes such as PCK1, SCD, SREBF1, PIPOX (p< 1E-8) and significant KEGG pathways such as unsaturated fatty acid (p= 0.009), peroxisome (p= 0.025) and PPAR (p= 0.016) between hypoxia and normoxia suggesting increased dependence of these cells on peroxisomal FAO. ACOX-1 (KO) mice showed increased survival when treated (p= 6.7E-04) with pazopanib. CONCLUSION In ACOX-1 (KO) model, we observed metabolomic reprogramming with downregulated genes, dysregulated metabolites, and pathways in peroxisomal FAO in hypoxia. These identified genes could be potential targets for therapy in combination with anti-angiogenic therapies that increase hypoxia in GBM.

Dietary restriction and medical therapy drives PPARα-regulated improvements in early diabetic kidney disease in male rats.

The attenuation of diabetic kidney disease (DKD) by metabolic surgery is enhanced by pharmacotherapy promoting renal fatty acid oxidation (FAO). Using the Zucker Diabetic Fatty and Zucker Diabetic Sprague Dawley rat models of DKD, we conducted studies to determine if these effects could be replicated with a non-invasive bariatric mimetic intervention. Metabolic control and renal injury were compared in rats undergoing a dietary restriction plus medical therapy protocol (DMT; fenofibrate, liraglutide, metformin, ramipril, and rosuvastatin) and ad libitum-fed controls. The global renal cortical transcriptome and urinary 1H-NMR metabolomic profiles were also compared. Kidney cell type-specific and medication-specific transcriptomic responses were explored through in silico deconvolution. Transcriptomic and metabolomic correlates of improvements in kidney structure were defined using a molecular morphometric approach. The DMT protocol led to ∼20% weight loss, normalized metabolic parameters and was associated with reductions in indices of glomerular and proximal tubular injury. The transcriptomic response to DMT was dominated by changes in fenofibrate- and peroxisome proliferator-activated receptor-α (PPARα)-governed peroxisomal and mitochondrial FAO transcripts localizing to the proximal tubule. DMT induced urinary excretion of PPARα-regulated metabolites involved in nicotinamide metabolism and reversed DKD-associated changes in the urinary excretion of tricarboxylic acid (TCA) cycle intermediates. FAO transcripts and urinary nicotinamide and TCA cycle metabolites were moderately to strongly correlated with improvements in glomerular and proximal tubular injury. Weight loss plus pharmacological PPARα agonism is a promising means of attenuating DKD.

Ban-xia-xie-xin-tang ameliorates hepatic steatosis by regulating Cidea and Cidec expression in HFD-fed mice

BackgroundBan-xia-xie-xin-tang (BXXXT) has been applied in treating metabolic diseases, such as nonalcohol fatty liver disease, diabetes mellitus, and obesity. However, the underlying molecular mechanism of BXXXT in treating diabetes mellitus is unknown. PurposeTo clarify the underlying molecular mechanism of BXXXT in alleviating hepatic steatosis in high-fat diet (HFD)-fed mice. MethodsAfter 12 weeks of HFD treatment, mice were administered BXXXT for 4 weeks. The main chemical components of BXXXT were identified by UPLC-TQ-MS/MS. Indicators associated with insulin resistance and lipid metabolism were detected. The effect of improving glucose and lipid metabolism between BXXXT and the different components was compared. Differentially expressed genes (DEGs) were identified by hepatic transcriptomics. Key DEGs and proteins were further detected by real-time quantitative polymerase chain reaction, western blotting, immunohistochemistry, and immunofluorescence staining. LDs and mitochondria were detected by transmission electron microscopy. ResultsFirst of all, our data demonstrated that the capacity to improve glucose and lipid metabolism for BXXXT was significantly superior to different components of BXXXT. BXXXT was found to improve HFD-induced insulin resistance. Moreover, BXXXT decreased weight, serum/hepatic triglycerides, total cholesterol, and FFAs to alleviate HFD-induced hepatic steatosis. According to the results of the hepatic transcription, Cidea and Cidec were identified as critical DEGs for promoting LD fusion and reducing FFAs β-oxidation in mitochondria and peroxisome resulting in hepatic steatosis, which was reversed by BXXXT. ConclusionBXXXT ameliorates HFD-induced hepatic steatosis and insulin resistance by increasing Cidea and Cidec-mediated mitochondrial and peroxisomal fatty acid oxidation, which may provide a potential strategy for therapy of NAFLD and T2DM.

Peroxisomal Fitness: A Potential Protective Mechanism of Fenofibrate against High Fat Diet-Induced Non-Alcoholic Fatty Liver Disease in Mice

Non-alcoholic fatty liver disease (NAFLD) has been increasing in association with the epidemic of obesity and diabetes. Peroxisomes are single membrane-enclosed organelles that play a role in the metabolism of lipid and reactive oxygen species. The present study examined the role of peroxisomes in high-fat diet (HFD)-induced NAFLD using fenofibrate, a peroxisome proliferator-activated receptor α (PPARα) agonist.Eight-week-old male C57BL/6J mice were fed either a normal diet or HFD for 12 weeks, and fenofibrate (50 mg/kg/day) was orally administered along with the initiation of HFD.HFD-induced liver injury as measured by increased alanine aminotransferase, inflammation, oxidative stress, and lipid accumulation was effectively prevented by fenofibrate. Fenofibrate significantly increased the expression of peroxisomal genes and proteins involved in peroxisomal biogenesis and function. HFD-induced attenuation of peroxisomal fatty acid oxidation was also significantly restored by fenofibrate, demonstrating the functional significance of peroxisomal fatty acid oxidation. In Ppara deficient mice, fenofibrate failed to maintain peroxisomal biogenesis and function in HFD-induced liver injury.The present data highlight the importance of PPARα-mediated peroxisomal fitness in the protective effect of fenofibrate against NAFLD.