Fatty Acid Β-oxidation Pathway Research Articles

Candidatus Thiothrix phosphatis SCUT-1: A novel polyphosphate-accumulating organism abundant in the enhanced biological phosphorus removal system

A novel coccus Thiothrix-related polyphosphate-accumulating organism (PAO) was enriched in an acetate-fed enhanced biological phosphorus removal (EBPR) system. High EBPR performance was achieved for an extended period (>100 days). A high-quality draft genome (completeness 97.2 %, contamination 3.26 %) was retrieved, representing a novel Thiothrix species (with similarity<93.2 % to known Thiothrix species), and was denoted as 'Candidatus Thiothrix phosphatis SCUT-1'. Its acetate uptake rate (6.20 mmol C/g VSS/h) surpassed most Ca. Accumulibacter and known glycogen-accumulating organisms (GAOs), conferring their predominance in the acetate-fed system. Metatranscriptomic analysis suggested that Ca. Thiothrix phosphatis SCUT-1 employed both low- and high-affinity pathways for acetate activation, and both the conventional (PhaABC) pathway and the fatty acid β-oxidation pathway for PHA synthesis; additionally, a much more efficient FAD-dependent malate: quinone oxidoreductase (MQO) were encoded and employed than the traditional malate dehydrogenase (MDH) to oxidize malate to oxaloacetate in the TCA and glyoxylate cycle, collectively contributing to a higher acetate utilization and processing rate of this microorganism. Batch tests further demonstrated the versatile ability of this PAO in using VFA (acetate, propionate, and butyrate), lactate, amino acids (aspartate and glutamate), and glucose as carbon sources for EBPR, showing a partially overlapped but unique ecological niche of this microorganism comparing to Ca. Accumulibacter and known GAOs. A metabolic model was built for Ca. Thiothrix phosphatis SCUT-1 using the above-mentioned carbon sources for EBPR. Overall, this study represents the first comprehensive characterization of the physiology and metabolic characteristics of representative coccus Thiothrix-related PAOs, which are expected to provide new insights into PAO microbiology in EBPR systems.

Exploring Fatty Acid β-Oxidation Pathways in Bacteria: From General Mechanisms to DSF Signaling and Pathogenicity in Xanthomonas.

Fatty acids (FAs) participate in extensive physiological activities such as energy metabolism, transcriptional control, and cell signaling. In bacteria, FAs are degraded and utilized through various metabolic pathways, including β-oxidation. Over the past ten years, significant progress has been made in studying FA oxidation in bacteria, particularly in E. coli, where the processes and roles of FA β-oxidation have been comprehensively elucidated. Here, we provide an update on the new research achievements in FAs β-oxidation in bacteria. Using Xanthomonas as an example, we introduce the oxidation process and regulation mechanism of the DSF-family quorum sensing signal. Based on current findings, we propose the specific enzymes required for β-oxidation of several specific FAs. Finally, we discuss the future outlook on scientific issues that remain to be addressed. This paper supplies theoretical guidance for further study of the FA β-oxidation pathway with particular emphasis on its connection to the pathogenicity mechanisms of bacteria.

2,3,5,4′-tetrahydroxy-stilbene-2-O-β-D-glucoside promotes liver regeneration after partial hepatectomy in mice: The potential involvement of PPARα-mediated fatty acid metabolism

Ethnopharmacological relevance2,3,5,4′-tetrahydroxy-stilbene-2-O-β-D-glucoside (TSG) is the principal bioactive compound contained in Polygonum multiflorum Thunb. (PMT), which is traditionally recorded to possess tonic and anti-aging efficacy. Aim of the studyTo identify the TSG-provided promotion on liver regeneration (LR) following partial hepatectomy (PHx) in mice and to explicate its involved mechanism. Materials and methodsThe promotion of TSG on LR was evaluated by hematoxylin and eosin (H&E), 5-bromodeoxyuridinc (BrdU) and Ki-67 staining, and measuring the level of proliferating cell nuclear antigen (PCNA) and Cyclin D1 in mice with PHx at different time points. Gene Expression Omnibus (GEO, GSE15239) database and the label-free quantitative proteomics from liver of mice at 24 h after PHx were integrated to identify potential involved critical proteins, which were verified by Western-blot, Real-time polymerase chain reaction (RT-PCR), molecular docking and luciferase activity assay. Primary hepatocytes isolated from mice were used to investigate the TSG-provided promotion on proliferation in vitro. ResultsTSG (20 mg/kg) promoted LR in mice after PHx. Results from RNA expression data from clinical samples and proteomic analysis from liver tissues indicated that peroxisome proliferator-activated receptor α (PPARα)-mediated fatty acid metabolism pathway were crucially associated with the TSG-provided promotion on LR. TSG enhanced the nuclear translocation of PPARα and the mRNA expression of a series of PPARα-regulated downstream genes. In addition, TSG lowered hepatic triglyceride (TG) and non-esterified fatty acid (NEFA) amounts and increased hepatic adenosine triphosphate (ATP) level in mice after PHx. TSG up-regulated the transcriptional activity of PPARα in vitro. Next results displayed that TSG promoted cell proliferation as well as ATP level in mice primary hepatocytes, which were abolished when PPARα was suppressed. Meanwhile, the cell viability was also elevated in mice primary hepatocytes treated with ATP. ConclusionActivating PPARα-mediated fatty acid β-oxidation (FAO) pathway led to the production of ATP, which contributed to the TSG-provided promotion on LR after PHx in mice.

EHHADH deficiency regulates pexophagy and accelerates tubulointerstitial injury in diabetic kidney disease

Peroxisomal l-bifunctional enzyme (EHHADH) plays a role in the classic peroxisomal fatty acid β-oxidation pathway; however, the relationship between EHHADH expression and diabetic kidney disease has not been well understood. Here, we found that endogenous EHHADH levels were strongly correlated with the progression and severity of diabetic nephropathy in T2D patients. EHHADH knockout mice exhibited worsened renal tubular injury in diabetic mice. Furthermore, EHHADH is a modulator of pexophagy. In renal tubular epithelial cells (RTECs) in vitro, the knockdown of EHHADH induced a dramatic loss of peroxisomes. The loss of peroxisomes in EHHADH-deficient RTECs was restored by either an autophagic inhibitor 3-methyladenine or bafilomycin A1 both in vitro and in vivo. NBR1 was required for pexophagy in EHHADH-knockdown cells, where the level of reactive oxygen species (ROS) was increased, while inhibition of ROS blocked pexophagy. In summary, our findings revealed EHHADH deficiency accelerated renal injury in DKD as a modulator of pexophagy.

Long-chain acyl-CoA synthetase-4 regulates endometrial decidualization through a fatty acid β-oxidation pathway rather than lipid droplet accumulation

ObjectiveLipid metabolism plays an important role in early pregnancy, but its effects on decidualization are poorly understood. Fatty acids (FAs) must be esterified by fatty acyl-CoA synthetases to form biologically active acyl-CoA in order to enter the anabolic and/or catabolic pathway. Long-chain acyl-CoA synthetase 4 (ACSL4) is associated with female reproduction. However, whether it is involved in decidualization is unknown. MethodsThe expression of ACSL4 in human and mouse endometrium was detected by immunohistochemistry. ACSL4 levels were regulated by the overexpression of ACSL4 plasmid or ACSL4 siRNA, and the effects of ACSL4 on decidualization markers and morphology of endometrial stromal cells (ESCs) were clarified. A pregnant mouse model was established to determine the effect of ACSL4 on the implantation efficiency of mouse embryos. Modulation of ACSL4 detects lipid anabolism and catabolism. ResultsThrough examining the expression level of ACSL4 in human endometrial tissues during proliferative and secretory phases, we found that ACSL4 was highly expressed during the secretory phase. Knockdown of ACSL4 suppressed decidualization and inhibited the mesenchymal-to-epithelial transition induced by MPA and db-cAMP in ESCs. Further, the knockdown of ACSL4 reduced the efficiency of embryo implantation in pregnant mice. Downregulation of ACSL4 inhibited FA β-oxidation and lipid droplet accumulation during decidualization. Interestingly, pharmacological and genetic inhibition of lipid droplet synthesis did not affect FA β-oxidation and decidualization, while the pharmacological and genetic inhibition of FA β-oxidation increased lipid droplet accumulation and inhibited decidualization. In addition, inhibition of β-oxidation was found to attenuate the promotion of decidualization by the upregulation of ACSL4. The decidualization damage caused by ACSL4 knockdown could be reversed by activating β-oxidation. ConclusionsOur findings suggest that ACSL4 promotes endometrial decidualization by activating the β-oxidation pathway. This study provides interesting insights into our understanding of the mechanisms regulating lipid metabolism during decidualization.

Enhancement of acyl-CoA precursor supply for increased avermectin B1a production by engineering meilingmycin polyketide synthase and key primary metabolic pathway genes.

Avermectins (AVEs), a family of macrocyclic polyketides produced by Streptomyces avermitilis, have eight components, among which B1a is noted for its strong insecticidal activity. Biosynthesis of AVE "a" components requires 2-methylbutyryl-CoA (MBCoA) as starter unit, and malonyl-CoA (MalCoA) and methylmalonyl-CoA (MMCoA) as extender units. We describe here a novel strategy for increasing B1a production by enhancing acyl-CoA precursor supply. First, we engineered meilingmycin (MEI) polyketide synthase (PKS) for increasing MBCoA precursor supply. The loading module (using acetyl-CoA as substrate), extension module 7 (using MMCoA as substrate) and TE domain of MEI PKS were assembled to produce 2-methylbutyrate, providing the starter unit for B1a production. Heterologous expression of the newly designed PKS (termed Mei-PKS) in S. avermitilis wild-type (WT) strain increased MBCoA level, leading to B1a titer 262.2 μg/mL - 4.36-fold higher than WT value (48.9 μg/mL). Next, we separately inhibited three key nodes in essential pathways using CRISPRi to increase MalCoA and MMCoA levels in WT. The resulting strains all showed increased B1a titer. Combined inhibition of these key nodes in Mei-PKS expression strain increased B1a titer to 341.9 μg/mL. Overexpression of fatty acid β-oxidation pathway genes in the strain further increased B1a titer to 452.8 μg/mL - 8.25-fold higher than WT value. Finally, we applied our precursor supply strategies to high-yield industrial strain A229. The strategies, in combination, led to B1a titer 8836.4 μg/mL - 37.8% higher than parental A229 value. These findings provide an effective combination strategy for increasing AVE B1a production in WT and industrial S. avermitilis strains, and our precursor supply strategies can be readily adapted for overproduction of other polyketides.

ACOX1 deficiency-induced lipid metabolic disorder facilitates chronic interstitial fibrosis development in renal allografts

Chronic interstitial fibrosis presents a significant challenge to the long-term survival of transplanted kidneys. Our research has shown that reduced expression of acyl-coenzyme A oxidase 1 (ACOX1), which is the rate-limiting enzyme in the peroxisomal fatty acid β-oxidation pathway, contributes to the development of fibrosis in renal allografts. ACOX1 deficiency leads to lipid accumulation and excessive oxidation of polyunsaturated fatty acids (PUFAs), which mediate epithelial–mesenchymal transition (EMT) and extracellular matrix (ECM) reorganization respectively, thus causing fibrosis in renal allografts. Furthermore, activation of Toll-like receptor 4 (TLR4)-nuclear factor kappa-B (NF-κB) signaling induced ACOX1 downregulation in a DNA methyltransferase 1 (DNMT1)-dependent manner. Overconsumption of PUFA resulted in endoplasmic reticulum (ER) stress, which played a vital role in facilitating ECM reorganization. Supplementation with PUFAs contributed to delayed fibrosis in a rat model of renal transplantation. The study provides a novel therapeutic approach that can delay chronic interstitial fibrosis in renal allografts by targeting the disorder of lipid metabolism.

Biorefinery of volatile fatty acids for the synthesis of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) using Paracoccus sp. TOH: Fermentation performance and metabolic pathway

The residual microbes and uncertain volatile fatty acids (VFAs) components in anaerobic fermentation hydrolysate cause microbial contamination and multi-type polyhydroxyalkanoates (PHA) generation in pure microbial synthesis systems. Meanwhile, the regulation of biosynthesis of poly(3-hydroxybuturate-co-3-hydroxyvalerate) (PHBV), one type of excellent PHA, by re-utilization of waste-organics as carbon substrates is still elusive. In this study, four pretreatment methods for sludge hydrolysate containing 4746 mg-COD·L−1 of VFAs were established to assess the accumulation of PHBV by using Paracoccus sp. TOH as the chassis. Results showed that the autoclaving system exhibited an optimal PHBV concentration of 202.9 mg L−1 with the highest relative abundance of Paracoccus sp. TOH (92.4%). Valeric acid was identified as the primary substrate for 3-hydroxyvalerate accumulation, and the highest PHBV production of 3.76 g L−1 was achieved using 3 g L−1 valeric acid and 5 g L−1 glycerol as co-substrates. The increased carbon metabolism flows through the sequential action of acyl-coenzyme A dehydrogenase and enoyl-coenzyme A hydratase in the fatty acid β-oxidation pathway facilitated PHBV accumulation. This study provides a blueprint for the microbial synthesis of PHBV with oriented acid production from organic wastes.

Alginate oligosaccharides improve high-fat induced hepatic steatosis via PGC-1α-mediated lipophagy and fatty acid β-oxidation pathway

This study explored the lipid-lowering effect and intervention mechanism of alginate oligosaccharides (AOS) on non-alcoholic fatty liver disease (NAFLD). Results showed that both unsaturated (UAOS) and saturated (SAOS) AOS improved the hepatic steatosis of NAFLD mice fed a high-fat diet (HFD). AOS promoted lipid catabolism in vivo and in vitro by activating the lipophagy and free fatty acid (FFA) β-oxidation pathways. Peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α) represented the up-stream target of the FFA β-oxidation pathway and upregulated both in in vivo and in vitro, suggesting it might be the AOS intervention target. By activating or inhibiting PGC-1α, AOS modulated the PGC-1α activity to reduce hepatic lipid accumulation. Therefore, the results indicated that AOS exhibited a lipid-lowering effect via the PGC-1α-mediated lipophagy-FFA β-oxidation transduction signal axis. These findings provided insight into using food-derived ingredients to intervene and treat chronic diseases involving lipid metabolism disorders.

Oral Intake of Inosine 5'-Monophosphate in Mice Promotes the Absorption of Exogenous Fatty Acids and Their Conversion into Triglycerides though Enhancing the Phosphorylation of Adenosine 5'-Monophosphate-Activated Protein Kinase in the Liver, Leading to Lipohyperplasia.

Inosine 5'-monophoaphate (IMP) is a food additive that promotes serious lipohyperplasia in the liver of C57/KsJ-db/db (db/db) mice. Thus, IMP taken orally by healthy mice might also damage their health. To date, how IMP affects health after being taken by healthy animals is still unclear. Therefore, we investigated the health of C57BL/6J mice affected by IMP intake. Our data revealed that C57BL/6J mice administered 255 μM IMP daily via oral gavage for 4 months caused hyperlipidemia and an increase in body fat rate. The expressions of acetyl-CoA carboxylase 1 (ACC1) and phosphorylated acetyl-CoA carboxylase 2 (ACC2) in hepatocytes increased though the administration of IMP, promoting the phosphorylation of adenosine 5'-monophosphate-activated protein kinase (AMPK). The conversion of acetyl-CoA into triglycerides (TGs) was promoted by ACC1. These TGs were transported from the hepatocytes to avoid the development of non-alcoholic fatty liver disease (NAFLD), causing a deficiency of acetyl-CoA in the liver, and then, the increased phosphorylated ACC2 promoted the cytoplasm fatty acids entering the mitochondria and conversion into acetyl-CoA through the fatty acid β-oxidation pathway, causing a deficiency in fatty acids. Therefore, the liver showed enhanced absorption of exogenous fatty acids, which were converted into TGs, causing lipohyperplasia. In conclusion, an excessive IMP intake promotes metabolic dysfunction in adipose tissue.

Integrated Metabolome and Transcriptome Analyses Reveal the Efficacy of Steroidal Saponins for Glucose and Lipid Metabolism in Hybrid Grouper (♀Epinephelus fuscoguttatus × ♂Epinephelus lanceolatu) Fed Higher-Lipid Diets.

An analysis of the extent of the effect of steroidal saponin addition on glucose and lipid metabolism in hybrid grouper liver was performed at the transcriptomic and metabolomic levels. Feeds (52% crude protein, 14% crude lipid) were prepared containing 0% (S0), 0.1% (S0.1), and 0.2% (S0.2) steroidal saponins. After eight weeks of feeding trial, compared to the S0 group, the activities of serum albumin, alanine aminotransferase, and aspartate transaminase were significantly lower and the activities of lysozyme, acid phosphatase, and alkaline phosphatase were significantly higher in the S0.1 group (p < 0.05). The superoxide dismutase, catalase, and glutathione peroxidase activities in the livers of the S0.1 group were significantly higher than those of the S0 group, while the malondialdehyde content was significantly lower than that of the S0 group (p < 0.05). There were forty-two differentially expressed genes and thirty-two differential metabolites associated with glucose and lipid metabolism enriched using KEGG and GO. In the S0 group, the expression of prostaglandin-endoperoxide synthase 1, prostaglandin E synthase 1, and thromboxane-2 synthase mRNA was significantly higher than in the S0.1 group (p < 0.05). The expression levels of genes in the S0 group were significantly higher than those in the S0.1 group (p < 0.05), including for glycogen synthase kinase, glucose-6-phosphatase catalytic subunit 2, fructose-1,6-bisphosphatase, phosphoenolpyruvate carboxykinase, glucose transporter 4, and malate dehydrogenase. The expression of mRNA such as fatty acid synthase, acetyl-CoA carboxylase, and sterol regulatory element-binding protein 1 was significantly lower in the S0.1 group than in the S0 group, while the expression of carnitine acyltransferase 1, acyl-CoA synthetase, and acyl-CoA dehydrogenase genes was significantly higher in the S0 group (p < 0.05). In summary, glycogen synthesis, gluconeogenesis, and the arachidonic acid metabolism pathway were inhibited by 0.1% steroidal saponins, and glycogenolysis, glycolysis, the tricarboxylic acid cycle, and the fatty acid β-oxidation pathway were activated. This study aims to provide a reference for the formulation of grouper feeds with a higher crude-lipid level.

Peroxisomal NAD(H) Homeostasis in the Yeast Debaryomyces hansenii Depends on Two Redox Shuttles and the NAD+ Carrier, Pmp47.

Debaryomyces hansenii is considered an unconventional yeast with a strong biotechnological potential, which can produce and store high amounts of lipids. However, relatively little is known about its lipid metabolism, and genetic tools for this yeast have been limited. The aim of this study was to explore the fatty acid β-oxidation pathway in D. hansenii. To this end, we employed recently developed methods to generate multiple gene deletions and tag open reading frames with GFP in their chromosomal context in this yeast. We found that, similar as in other yeasts, the β-oxidation of fatty acids in D. hansenii was restricted to peroxisomes. We report a series of experiments in D. hansenii and the well-studied yeast Saccharomyces cerevisiae that show that the homeostasis of NAD+ in D. hansenii peroxisomes is dependent upon the peroxisomal membrane protein Pmp47 and two peroxisomal dehydrogenases, Mdh3 and Gpd1, which both export reducing equivalents produced during β-oxidation to the cytosol. Pmp47 is the first identified NAD+ carrier in yeast peroxisomes.

Energy metabolic mechanisms for high altitude sickness: Downregulation of glycolysis and upregulation of the lactic acid/amino acid-pyruvate-TCA pathways and fatty acid oxidation

Hypobaric hypoxia is often associated with the plateau environment and can lead to altitude sickness or death. The underlying cause is a lack of oxygen, which limits energy metabolism and leads to a compensatory stress response. Although glycolysis is commonly accepted as the primary energy source during clinical hypoxia, our preliminary experiments suggest that hypobaric hypoxia may depress glycolysis. To provide a more comprehensive understanding of energy metabolism under short-term hypobaric hypoxia, we exposed mice to a simulated altitude of 5000 m for 6 or 12 h. After the exposure, we collected blood and liver tissues to quantify the substrates, enzymes, and metabolites involved in glycolysis, lactic acid metabolism, the tricarboxylic acid cycle (TCA), and fatty acid β-oxidation. We also performed transcriptome and enzymatic activity analyses of the liver. Our results show that 6 h of hypoxic exposure significantly increased blood glucose, decreased lactic acid and triglyceride concentrations, and altered liver enzyme activities of mice exposed to hypoxia. The key enzymes in the glycolytic, TCA, and fatty acid β-oxidation pathways were primarily affected. Specifically, the activities of key glycolytic enzymes, such as glucokinase, decreased significantly, while the activities of enzymes in the TCA cycle, such as isocitrate dehydrogenase, increased significantly. Lactate dehydrogenase, pyruvate carboxylase, and alanine aminotransferase were upregulated. These changes were partially restored when the exposure time was extended to 12 h, except for further downregulation of phosphofructokinase and glucokinase. This study demonstrates that acute high altitude hypoxia upregulated the lactic acid/amino acid-pyruvate-TCA pathways and fatty acid oxidation, but downregulated glycolysis in the liver of mice. The results obtained in this study provide a theoretical framework for understanding the mechanisms underlying the pathogenesis of high-altitude sickness in humans. Additionally, these findings have potential implications for the development of prevention and treatment strategies for altitude sickness.

The Effect of Glyoxylate Shunt Inactivation on Biosynthesis of Adipic Acid through Inverted Fatty Acid β-Oxidation by Escherichia coli Strains

Using Escherichia coli MG1655 lacIQ, ∆ackA-pta, ∆poxB, ∆ldhA, ∆adhE, ∆fadE, PL-SDφ10-atoB, Ptrc-ideal-4-SDφ10-fadB, PL-SDφ10-tesB, ∆yciA as a core strain, derivatives capable of synthesizing adipic acid from glucose through the inverted fatty acid β-oxidation pathway were obtained. Biosynthesis of the target compound by recombinants was ensured by the primary condensation of acetyl-CoA and succinyl-CoA by 3-oxoacyl-CoA thiolase PaaJ and the catalysis of the final reaction of the cycle by acyl-CoA dehydrogenases FadE and FabI. Deletion in the strains of sucCD genes encoding components of succinyl-CoA synthase did not increase the relative intracellular availability of succinyl-CoA for target biosynthetic reactions and did not lead to an increase in adipic acid accumulation by the recombinants. The secretion of succinic and malic acids by the strains with an impaired tricarboxylic acid cycle remained almost unchanged, indicating the activity in the cells of glyoxylate shunt reactions that compete with the cycle reactions for isocitrate, required for succinyl-CoA formation. When isocitrate lyase, malate synthases A and G, and bifunctional kinase/phosphatase of isocitrate dehydrogenase were inactivated in strains due to deletion of the aceBAK operon genes and glcB, adipic acid synthesis by recombinants increased three-fold and reached 0.33 mM.

Effect of the Glyoxylate Shunt Inactivation on Biosynthesis of Adipic Acid Through the Inverted Fatty Acid β-oxidation by &lt;i&gt;Escherichia coli&lt;/i&gt; Strains

Using Escherichia coli MG1655 lacIQ, ∆ackA-pta, ∆poxB, ∆ldhA, ∆adhE, ∆fadE, PL-SDφ10-atoB, Ptrc-ideal-4-SDφ10-fadB, PL-SDφ10-tesB, ∆yciA as a core strain, the derivatives capable of synthesizing adipic acid from glucose through the inverted fatty acid β-oxidation pathway were obtained. Biosynthesis of the target compound by recombinants was ensured upon the primary condensation of acetyl-CoA and succinyl-CoA by 3-oxoacyl-CoA thiolase PaaJ and the catalysis of the final reaction of the cycle by acyl-CoA dehydrogenases FadE and FabI. Deletion in the strains of sucCD genes encoding components of succinyl-CoA synthase did not increase the relative intracellular availability of succinyl-CoA for target biosynthetic reactions and did not lead to an increase in adipic acid accumulation by the recombinants. The secretion of succinic and malic acids by the strains with an impaired tricarboxylic acid cycle remained almost unchanged, indicating the activity in the cells of glyoxylate shunt reactions that compete with the cycle reactions for isocitrate, required for succinyl-CoA formation. When isocitrate lyase, malate synthases A and G, and bifunctional kinase/phosphatase isocitrate dehydrogenase were inactivated in strains due to deletion of the aceBAK operon genes and glcB, adipic acid synthesis by recombinants increased threefold and reached 0.33 mM.

Abstract 294: Tumor concentration of metformin is a determinant factor of its regulation of fatty acid β-oxidation and c-Src pathway in triple-negative breast cancer

Abstract Biguanides such as metformin are one of the most widely administered anti-diabetic drugs. Biguanides can suppress OXPHOS by inhibiting the complex-I activity of the mitochondrial electron transport chain (ETC). Though metformin recently generated lots of hope in cancer therapy, several clinical trials showed only limited advantages for metformin in breast cancer patients. Considering its long-term observed and widely accepted safety parameters, we investigated the possible mechanisms of the lack of in vivo anticancer effect of metformin and its potential combination therapies. While metformin is an ETC inhibitor, metformin can activate AMPK, leading to ACC phosphorylation, independently of ETC inhibition. Since ACC is the upstream regulator of mitochondrial fatty acid β-oxidation (FAO), lower concentrations of biguanides can also activate FAO. Thus, biguanides can play opposing roles in mitochondrial metabolism by FAO activation, and ETC inhibition. Considering the hybrid metabolic phenotype and increased dependency of TNBC on FAO, we analyzed the effect of biguanides in TNBC progression and metastasis. While higher concentrations of biguanides suppress ETC complex-1 function, their lower concentrations activate AMPK-FAO pathway-driven ETC activity in TNBC. When the mice with TNBC tumors were treated with a clinically relevant dose of metformin, the tumor concentration of metformin after a few hours of treatment was much lower to inhibit ETC effectively. Since low concentrations of metformin can activate FAO, we did in vitro analysis of the combination of metformin and FAO inhibitor, etomoxir, in TNBC cell lines. As expected, this combination synergistically inhibited tumor properties in TNBC cells. We have published that FAO activates c-Src, one of the frequently upregulated oncopathways in TNBC. Thus, we analyzed the role of metformin concentration in Src activation in TNBC. In alignment with FAO, lower metformin concentrations activate, and high concentrations inhibit Src phosphorylation. We then investigated the significance of the combination of Src inhibitor dasatinib with metformin. As observed with FAO inhibitor, Src inhibitor synergistically enhanced the anticancer effect of metformin. To understand the translational significance of this combination, we performed ex vivo studies using TNBC organoids and PDX models. Interestingly, the combination of metformin and dasatinib synergistically inhibited the tumor growth, and metastasis and enhanced survival potential. Our results suggest that the combination of metformin and dasatinib significantly inhibits lung metastasis and modulates the immune microenvironment of the lungs. Since both dasatinib and metformin are clinically approved drugs, our findings provide a clinically translatable option for treating TNBC patients who currently lack targeted therapy. Citation Format: Junhyoung Park, Kwang Hwa Jung, Dongya Jia, Sukjin Yang, Kuldeep S. Attri, Songyeon Ahn, Divya Murthy, Meron Ghidey, Somik Chatterjee, Diego A. Pedroza, Abha Tiwari, Suna Kim, Chad C. Creighton, Nagireddy Putluri, Jeffrey M. Rosen, José N. Onuchic, Andrei Goga, Benny A. Kaipparettu. Tumor concentration of metformin is a determinant factor of its regulation of fatty acid β-oxidation and c-Src pathway in triple-negative breast 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 294.

The metabolite alpha-ketobutyrate extends lifespan by promoting peroxisomal function in C. elegans

Metabolism is intimately linked to aging. There is a growing number of studies showing that endogenous metabolites may delay aging and improve healthspan. Through the analysis of existing transcriptome data, we discover a link between activation of the transsulfuration pathway and a transcriptional program involved in peroxisome function and biogenesis in long-lived glp-1(e2141ts) mutant Caenorhabditis elegans worms. Subsequently, we show that supplementation with α-ketobutyrate, an intermediate of the transsulfuration pathway, extends lifespan in wild-type worms. Alpha-ketobutyrate augments the production of NAD+ via the lactate dehydrogenase LDH-1, leading to SIR-2.1/SIRT1-mediated enhanced peroxisome function and biogenesis, along with a concomitant increase in the expression of acox-1.2/ACOX1 in the peroxisomal fatty acid β-oxidation pathway. ACOX-1.2/ACOX1 promotes H2O2 formation, thereby resulting in activation of SKN-1/NRF2. This transcription factor in turn extends the lifespan of worms by driving expression of autophagic and lysosomal genes. Finally, we show that α-ketobutyrate also delays the cellular senescence in fibroblast cells through the SIRT1-ACOX1-H2O2-NRF2 pathway. This finding uncovers a previously unknown role for α-ketobutyrate in organismal lifespan and healthspan by coordinating the NAD+-SIRT1 signaling and peroxisomal function.

Effects of overfeeding on liver lipid metabolism in mule ducks based on transcriptomics and metabolomics

ABSTRACT 1. In this study, transcriptomics and metabolomics were used to analyse changes in gene expression and metabolites in the liver of 70-d-old mule ducks after 10 and 20 d of continuous overfeeding. 2. In the free-feeding group, 995 differentially expressed genes and 51 metabolites (VIP >1, P < 0.05) were detected in the early stage, and 3,448 differentially expressed genes and 55 metabolites (VIP >1, P < 0.05) were detected in the later stage. There were 775 differentially expressed genes and 47 metabolites (VIP >1, P < 0.05) detected in the early stage of the overfeeding group, and 6,719 differentially expressed genes and 57 metabolites (VIP >1, P < 0.05) detected in the later stage. 3. There were no significant differences between the early stage in the overfeeding and free-feeding groups at the transcriptional and metabolic levels. Oleic acid and palmitic acid synthesis increased in the early stage of the overfeeding and free-feeding groups, however, these were inhibited in the late stage. Fatty acid oxidation and β-oxidation pathways were inhibited and insulin resistance was enhanced significantly in the late overfeeding stage. 4. In the early stage, the digestion and absorption of fat in the overfeeding and free-feeding groups were enhanced. In the later stage, the ability to store triglyceride in the overfeeding group was greater than in the free-feeding group. 5. The expression of nuclear factor κB (NFκB), a key inflammatory factor, was inhibited in the late stage of overfeeding, while arachidonic acid (AA), a metabolite with anti-inflammatory properties, increased in the late stage of overfeeding to inhibit the inflammatory effects caused by excessive lipid accumulation. These results add to the understanding of the mechanism of production of fatty liver in mule ducks and facilitate the development of treatments for non-alcoholic fatty liver disease.

Nutrient sensing pathways regulating adult reproductive diapause in C. elegans.

Genetic and environmental manipulations, such as dietary restriction, can improve both health span and lifespan in a wide range of organisms, including humans. Changes in nutrient intake trigger often overlapping metabolic pathways that can generate distinct or even opposite outputs depending on several factors, such as when dietary restriction occurs in the lifecycle of the organism or the nature of the changes in nutrients. Due to the complexity of metabolic pathways and the diversity in outputs, the underlying mechanisms regulating diet-associated pro-longevity are not yet well understood. Adult reproductive diapause (ARD) in the model organism Caenorhabditis elegans is a dietary restriction model that is associated with lengthened lifespan and reproductive potential. To explore the metabolic pathways regulating ARD in greater depth, we performed a candidate-based genetic screen analyzing select nutrient-sensing pathways to determine their contribution to the regulation of ARD. Focusing on the three phases of ARD (initiation, maintenance, and recovery), we found that ARD initiation is regulated by fatty acid metabolism, sirtuins, AMPK, and the O-linked N-acetyl glucosamine (O-GlcNAc) pathway. Although ARD maintenance was not significantly influenced by the nutrient sensors in our screen, we found that ARD recovery was modulated by energy sensing, stress response, insulin-like signaling, and the TOR pathway. Further investigation of downstream targets of NHR-49 suggest the transcription factor influences ARD initiation through the fatty acid β-oxidation pathway. Consistent with these findings, our analysis revealed a change in levels of neutral lipids associated with ARD entry defects. Our findings identify conserved genetic pathways required for ARD entry and recovery and uncover genetic interactions that provide insight into the role of OGT and OGA.

Improved liver lipid catabolism and utilization in growth hormone transgenic common carp (Cyprinus carpio L.) through enhanced lipolytic and fatty acid β-oxidation pathways.

Growth hormone (GH) transgenic common carp (Cyprinus carpio L.) show desirable aquaculture traits. Their specific growth rate (SGR) and feed efficiency (FE) are approximately 12% and 17% higher than the wild-type (WT) common carp, respectively. However, the mechanisms of lipid catabolism (lipolysis and fatty acid β-oxidation) and utilization in GH transgenic common carp are still unclear. In this study, we firstly compared the lipid metabolism of GH transgenic (initial weight 3.72 ± 0.32 g) and WT (initial weight 3.30 ± 0.28 g) common carp fed with a normal fat level diet (6% lipid, 33% protein) for two months, then compared the growth performance of GH transgenic (initial weight 3.65 ± 0.33 g) and WT (initial weight 3.27 ± 0.26 g) common carp fed with different fat levels diets (6% lipid and 12% lipid, 33% protein) for two months. We found that the lipid content in serum, liver and whole body was significantly reduced in GH transgenic common carp, the hepatic activities of the lipolytic enzymes hormone-sensitive lipase and adipose triglyceride lipase were enhanced, and the hepatic expression level of hormone-sensitive lipase was upregulated. In addition, the mitochondrion numbers were increased, and the expression level of carnitine palmitoyltransferase-1a and carnitine palmitoyltransferase-1b was upregulated in the liver of GH transgenic common carp. GH transgenic common carp showed higher weight gain and SGR than that in WT carp when fed with a normal-fat diet as they did when fed with a high-fat diet, and GH transgenic common carp showed higher FE than that in WT carp when fed with a high-fat diet. These results suggested that the lipid catabolism and utilization was improved in the GH transgenic common carp liver through enhanced lipolytic and fatty acid β-oxidation pathways. Our study provides new insights into improving lipid utilization in some aquaculture fish species.