Long-chain acyl-CoA Research Articles

1630-P: Reduced Fatty Acid Oxidation Does Not Prevent Intralipid-Induced Peripheral Insulin Resistance in Humans

Mitochondrial handling of excess lipid substrate is thought to be an important determinant of insulin resistance. Evidence from animal models support insulin signaling defects resulting from either intra-cellular lipid accumulation or by mitochondrial dysfunction and altered fatty acid oxidation (FAO) flux. To explore the relationship between FAO and insulin sensitivity, we measured the change in peripheral glucose disposal (Rd) during a 5-hour hyperinsulinemic euglycemic clamp with co-infusion of a control glycerol solution versus a 20% intralipid solution in 18 subjects with an inherited defect in FAO (FAOD; including Carnitine Palmitoyltransferase 2, Very long-chain, medium-chain or long-chain 3-hydroxy AcylcoA Dehydrogenase Deficiencies) and 18 age, sex, and weight-matched normal healthy controls. Rd was similar between groups during the glycerol control clamp. Intramyocellular lipid (IMCL) content as measured by MRS increased after the 5 hour intralipid infusion in controls but not in participants with FAOD. Plasma free fatty acids during intralipid infusion were higher in FAOD compared to controls suggesting infused lipid remained in circulation. Muscle fiber type and expression of lipid transporters CD36 and FATP1 were similar in biopsy samples between groups and cannot explain decreased lipid uptake into muscle among FAOD subjects. However, intralipid suppressed Rd equally in subjects with FAOD and controls inducing peripheral insulin resistance in both groups. Impaired FAO decreased lipid accumulation in the muscle during intralipid infusion, but despite lower IMCL, subjects with FAOD developed peripheral insulin resistance similar to controls. Increased IMCL is not essential for, nor is reduced FAO flux protective against, the development of peripheral insulin resistance during intralipid infusion. Disclosure M.B.Gillingham: None. J.Q.Purnell: Advisory Panel; Boehringer Ingelheim International GmbH, Novo Nordisk. A.N.Gregor: None. O.Varlamov: None. J.J.Robino: None. C.E.Mccurdy: None. K.T.Greyslak: None. D.Choi: None. E.Baetscher: Consultant; Biogen. W.Rooney: Consultant; Ultragenix, Research Support; Revalesio. Funding National Institutes of Health (DK102813)

287-LB: Thiazolidinedione Ameliorates Lipotoxicity-Induced Pancreatic ß-Cell Ferroptosis Partly via ACSL4

Pancreatic β cells death is a major factor driving the deterioration of glucose control in type 2 diabetes mellitus (T2DM). Ferroptosis is a non-apoptotic form of lipid peroxidation-induced cell death. The long-chain acyl-CoA synthetase 4 (ACSL4) contributes significantly to lipid metabolism and determines ferroptosis sensitivity. Accounting for still limited understanding of lipotoxicity-related ferroptosis in β cells, we aim to identify pancreatic β cells ferroptosis in lipotoxicity-induced diabetes and the role of ACSL4 plays in this process. In this study, we found that fatty acids could induce apoptosis in pancreatic β cells, along with a new form of death called ferroptosis. Pancreatic islets of 16-week HFD-fed mice showed reduced mitochondrial cristae, decreased mitochondrial cristae, and increased bilayer membrane density compared to LFD-fed mice. Iron fluorescence staining showed increased intracellular Fe2+ levels in pancreatic islets. Ferroptosis inhibitor fer-1 had a specific protective effect of on HFD-induced β-cell ferroptosis. Through using the Cre-LoxP system, we performed a ACSL4 knockout model of the HFD-induced metabolic diseases, we found that lipid metabolism dictates ferroptosis in pancreatic β cells through ACSL4, which appear to be related to pancreatic β cells dysfunction. ACSL4 knockdown attenuated fatty acids-induced ferroptosis in pancreatic β cells in vivo and in vitro. ACSL4 overexpression induces ferroptosis in MIN6 cells. Moreover, we observed that troglitazone attenuated AA, DHA, PA and erastin induced ferroptosis in MIN6 cells. These results together provide definitive evidence linking β cells with lipotoxicity-induced ferroptosis and suggest the anti-ferroptosis effect of ACSL4 may be a new therapeutic target for the targeted therapy of T2DM. Disclosure M. Zhang: None. L. Qin: None. Y. Qin: None. Q. Wei: None. X. Chen: None. Funding National Natural Science Foundation of China (81974103)

266-LB: SGLT2 Inhibition Improved Endurance Performance of db/db Mice Accompanied by an Increased Endogenous AMPK Activator in Skeletal Muscles

Background: Diabetes is associated with loss of skeletal muscle mass and function. Specifically, diabetes may affect the function of oxidative muscle fibers with lower fatigability and higher oxidative capacity in skeletal muscle. The effects of SGLT2 inhibition on skeletal muscle metabolism and function in patients with type 2 diabetes remain unclear. Methods: Male db/db (diabetic) and db/+ (non-diabetic) mice at 8 weeks of age were fed a normal chow diet or a diet containing canagliflozin (CANA) for 4 weeks. Grip strength and running distance were assessed during CANA treatment. At the end of CANA treatment, we measured muscle weight and analyzed metabolites in skeletal muscle. Results: The weights of the oxidative soleus and glycolytic extensor digitorum longus muscles were reduced by approximately 50% in the diabetic group compared to the non-diabetic group, but neither was altered by CANA treatment. Grip strength did not differ between the CANA-treated diabetic, diabetic, and non-diabetic groups. However, the running distance decreased in the diabetic group compared to the non-diabetic group (89 ± 60 m vs. 1980 ± 276 m). This was partially restored by CANA treatment (89 ± 60 m vs. 415 ± 140 m). Metabolomic analysis revealed that long-chain acyl-CoA increased and medium-chain and short-chain acyl-CoA decreased in diabetic soleus muscle. In contrast, CANA treatment increased medium-chain and short-chain acyl-CoA. Furthermore, CANA increased 5-aminoimidazole-4-carboxamide-1-β-D-ribofuranosyl 5’-monophosphate (AICARP), an endogenous AMPK activator, by approximately 3-fold in diabetic soleus muscle with increased levels of AMPK phosphorylation (Thr172). Conclusion: These results suggest that SGLT2 inhibitors may activate the AICARP/AMPK pathway and improve impaired oxidative muscle function in patients with type 2 diabetes. Disclosure S. Nakamura: None. Y. Miyachi: None. H. Yokomizo: None. H. Otsuka: None. R. Sakamoto: None. M. Takahashi: None. Y. Izumi: None. T. Miyazawa: None. T. Bamba: None. Y. Ogawa: None.

The ACSL4 Network Regulates Cell Death and Autophagy in Diseases.

Lipid metabolism, cell death, and autophagy are interconnected processes in cells. Dysregulation of lipid metabolism can lead to cell death, such as via ferroptosis and apoptosis, while lipids also play a crucial role in the regulation of autophagosome formation. An increased autophagic response not only promotes cell survival but also causes cell death depending on the context, especially when selectively degrading antioxidant proteins or organelles that promote ferroptosis. ACSL4 is an enzyme that catalyzes the formation of long-chain acyl-CoA molecules, which are important intermediates in the biosynthesis of various types of lipids. ACSL4 is found in many tissues and is particularly abundant in the brain, liver, and adipose tissue. Dysregulation of ACSL4 is linked to a variety of diseases, including cancer, neurodegenerative disorders, cardiovascular disease, acute kidney injury, and metabolic disorders (such as obesity and non-alcoholic fatty liver disease). In this review, we introduce the structure, function, and regulation of ACSL4; discuss its role in apoptosis, ferroptosis, and autophagy; summarize its pathological function; and explore the potential implications of targeting ACSL4 in the treatment of various diseases.

Identification of a novel crustacean vascular endothelial growth factor b-like in the mud crab Scylla paramamosain, and examination of its role in lipid accumulation

Vascular endothelial growth factor B (VEGFB), unlike other members of the VEGF family such as VEGFA, shows only limited potential in angiogenesis in mammals, but is involved in lipid metabolism, especially in transporting dietary lipids present in circulation through the vascular endothelium to peripheral tissues. However, the role of Vegfb in invertebrates such as decapod crustaceans with an open circulatory system is still poorly understood. Here, cDNA of a vegfb-like gene was cloned and characterized for the first time from a decapod crustacean, mud crab Scylla paramamosain. The mud crab vegfb-like gene encoded a polypeptide of 386 amino acids that possessed all the typical features of the Vegf family, and shared high homology with other crustacean Vegf orthologs. Relative expression levels of the vegfb-like gene were higher in metabolically active tissues such as gill, heart and muscle, and increased in ovary of female crabs as ovary matured. Knockdown of the vegfb-like gene in vivo reduced the contents of triglyceride in hemolymph and hepatopancreas, as well as total lipid in hepatopancreas. In addition, knockdown of the vegfb-like gene increased tissue expression levels of genes related to fatty acid transportation, allocation and hydrolysis, such as long-chain acyl-CoA synthetase 3/4 (acsl3/4), fatty acid transport protein 1 (fatp1), and hormone-sensitive lipase (hsl), and decreased the expression levels of the genes related to fatty acid synthesis like acetyl-CoA carboxylase (acc). These results in mud crab suggest that Vegfb might play important roles in regulating fatty acid absorption, utilization and allocation in crustaceans.

Novel Effect of p-Coumaric Acid on Hepatic Lipolysis: Inhibition of Hepatic Lipid-Droplets.

p-coumaric acid (p-CA), a common plant phenolic acid with multiple bioactivities, has a lipid-lowering effect. As a dietary polyphenol, its low toxicity, with the advantages of prophylactic and long-term administration, makes it a potential drug for prophylaxis and the treatment of nonalcoholic fatty liver disease (NAFLD). However, the mechanism by which it regulates lipid metabolism is still unclear. In this study, we studied the effect of p-CA on the down-regulation of accumulated lipids in vivo and in vitro. p-CA increased a number of lipase expressions, including hormone-sensitive lipase (HSL), monoacylglycerol lipase (MGL) and hepatic triglyceride lipase (HTGL), as well as the expression of genes related to fatty acid oxidation, including long-chain fatty acyl-CoA synthetase 1 (ACSL1), carnitine palmitoyltransferase-1 (CPT1), by activating peroxisome proliferator-activated receptor α, and γ (PPARα and γ). Furthermore, p-CA promoted adenosine 5'-monophosphate (AMP)-activated protein kinase (AMPK) phosphorylation and enhanced the expression of the mammalian suppressor of Sec4 (MSS4), a critical protein that can inhibit lipid droplet growth. Thus, p-CA can decrease lipid accumulation and inhibit lipid droplet fusion, which are correlated with the enhancement of liver lipases and genes related to fatty acid oxidation as an activator of PPARs. Therefore, p-CA is capable of regulating lipid metabolism and is a potential therapeutic drug or health care product for hyperlipidemia and fatty liver.

Homologous Overexpression of Acyl-CoA Thioesterase 8 Enhanced Free Fatty Acid Accumulation in Oleaginous Fungus Mucor circinelloides WJ11

Thioesterases play an essential role in the metabolism of fatty acids since they are considered one of the key enzymes to change the total amount and composition of fatty acid in an organism’s cells. Acyl-coenzyme A thioesterase 8 (ACOT8) exhibits substrate specificity mainly for short- to long-chain acyl-CoA. To identify and characterize the ACOT8 enzyme’s superfamily in Mucor circinelloides, three genes were characterized and homologously expressed in M. circinelloides WJ11 which has been used as a model organism to investigate the mechanism of lipid accumulation. Multiple sequence alignment showed that McACOT8s had significant conserved motifs in the ACOT8 family. Experimental data indicated that the biomass of the three recombinant strains (McACOT8a, McACOT8b and McACOT8c) was slightly lower than that of the control strain Mc2075, but the total fatty acid (TFA) content was significantly increased by 30.3, 21.5 and 23.9%, respectively, and the free fatty acid production increased from 12.9% (control strain) to 19.2, 25.2 and 26.2% (recombinant types), respectively, with a maximum increase of 103.1% in McACOT8c. The three strains did not show any significant improvement in fatty acid composition compared to the control strain. However, in terms of fatty acid composition of free fatty acid, McACOT8b and McACOT8c contained γ-linolenic acid (C18:3), which was not detected in the control strain or in McACOT8a, indicating that ACOT8b and ACOT8c had substrate specificity for C18:3. These results displayed that ACOT8 can increase TFA accumulation and that it may be an important target of genetic manipulation for microbial oil production.

Fetal Bowel Dilatation Associated With LCHAD Deficiency: A New Sonographic Finding

A long-chain 3-hydroxyacyl-CoA dehydrogenase (LCHAD) deficiency constitutes a very rare autosomal recessive disease, affecting long fatty acids’ metabolism. In the prenatal period, it is difficult to suspect this diagnosis in the absence of family history, as there are no specific sonographic signs of this disease. A 32-year-old pregnant woman was receiving follow-up care, as she was a carrier for Duchenne muscular dystrophy (DMD). The fetus was female, and the pregnancy progressed normally until 29th week of gestation. At that time, the fetal bowel had noted dilatation with mild hyperechogenicity. Fetal surveillance was conducted with sequential sonograms. Labor was induced at 36 weeks, due to pre-eclampsia. The newborn presented with poor weight gain and hypoglycemic episodes. Neonatal screening revealed the diagnosis of an LCHAD deficiency. Prenatal diagnosis of LCHAD is usually performed by genetic testing when there is a family history. However, only a few sonographic signs have been described in association with this disease, such as fetal bowel hyperechogenicity or cardiomyopathy. This is the first case study to report a prenatal sonographic finding of bowel dilatation in a fetus with LCHAD deficiency, and one of the few to report fetal bowel hyperechogenicity.

Transcriptomic analysis of the metabolic regulatory mechanism of Schizochytrium limacinum B4D1 using sodium acetate to produce DHA

The aim of the study was to evaluate the potential of acetate as a carbon source by examining the fermentation performance of Schizochytrium limacinum B4D1 and its effects on metabolism using physiology and transcriptomics studies. The results demonstrated that, while the metabolic rate of acetate in S. limacinum B4D1 was lower than glucose, it increased the content of docosahexaenoic acid (DHA) in the total fatty acids. Transcriptomic analysis indicated that the use of acetate as a carbon source significantly upregulated the acetyl-CoA synthetase gene, which increased the source of acetyl-CoA for fatty acid biosynthesis. Moreover, the upregulation of polyunsaturated fatty acid enzymes promoted the polyketide synthase (PKS) pathway of fatty acid synthesis, resulting in an increase in DHA content. Meanwhile, the upregulation of genes such as long-chain acyl-CoA synthetase and 3-hydroxyacyl-CoA dehydrogenase enhanced the β-oxidation process of saturated fatty acids in mitochondria, while the downregulation of acyl-CoA oxidase and catalase genes resulted the β-oxidation process of polyunsaturated fatty acids in peroxisomes was inhibited, leading to an increased proportion of DHA in the total fatty acids. Thus, these changes in gene expression contributed to the increased proportion of DHA in the total fatty acids.

START domain-mediated nuclear translocation of Them1 regulates the expression of metabolic genes relative to thermogenesis in brown adipocytes

Thioesterase superfamily member 1 (Them1) is a long-chain fatty acyl-CoA thioesterase that is highly expressed in brown adipose tissue (BAT), where it limits energy expenditure. Them1 is comprised of tandem enzymatic domains and a C-terminal lipid-binding steroidogenic acute regulatory protein (StAR)-related lipid transfer (START) domain. Fatty acid binding to the START domain stabilizes the enzymatic domains and increases activity, whereas lysophosphatidylcholine (LPC) acts in the opposite manner. In brown adipocytes, Them1 localizes to puncta within the cytoplasm, but diffuses in response to β-adrenergic stimulation after which it is also found in the nucleus. The aim of this study was to determine the mechanism of Them1 nuclear trafficking and the metabolic consequences. METHODS: Identification of a putative nuclear localization signal (NLS) was determined by computational analysis. Full-length, NLS-deleted, or truncated Them1 cDNA constructs were cloned and linked to EGFP. Plasmid or adenoviral vectors were transfected into immortalized brown adipose cells (iBAs) that do not express Them1. Them1 was visualized by live cell confocal imaging via EGFP and with NucSpot nuclear staining. Signal quantification was performed with Imaris software. Them1 nuclear localization was evaluated in the presence or absence of norepinephrine (NE), leptomycin B, cycloheximide, or lipids that bind the START domain. Them1 nuclear localization was also quantified in BAT from mice treated with a β3-adrenergic receptor agonist, CL316,243. mRNA expression in iBAs expressing full-length or NLS-mutant Them1 in response to NE was determined by RNA-Seq. RESULTS: In the diffuse state, Them1 localized to the nucleus in a time-dependent manner. The NLS resides in a hinge region between the thioesterase domains and the START domain. Deletion of either the NLS or the START domain blocked nuclear targeting. Conversely, truncated Them1 containing only the START domain was targeted to the nucleus. Entry into the nucleus was balanced by trafficking out of the nucleus and new protein synthesis was not required, as evidenced by using leptomycin B to block nuclear efflux and cycloheximide to block protein synthesis, respectively. Incubation of NE-stimulated iBAs with LPC promoted nuclear Them1 translocation. Them1 protein was also detected in the nucleus of BAT from wild-type mice in response to CL administration. Nuclear Them1 localization in iBAs was associated with down-regulated mRNA expression of genes that control lipid and glucose metabolism during cold adaptation. CONCLUSIONS: Our data suggest that LPC generated during thermogenesis binds and destabilizes the START domain, leading to exposure of the NLS and nuclear trafficking of Them1 in response to β-adrenergic stimulation. Once inside the nucleus, Them1 regulates the transcription of lipid and glucose metabolic enzymes that are required for chronic cold adaptation. This work was supported by NIH DK103046 to DEC, SJH, and EAO This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Skeletal Muscle Consequences of Phosphatidylethanolamine Synthesis Deficiency.

The maintenance of phospholipid homeostasis is increasingly being implicated in metabolic health. Phosphatidylethanolamine (PE) is the most abundant phospholipid on the inner leaflet of cellular membranes, and we have previously shown that mice with a heterozygous ablation of the PE synthesizing enzyme, Pcyt2 (Pcyt2+/-), develop obesity, insulin resistance, and NASH. Skeletal muscle is a major determinant of systemic energy metabolism, making it a key player in metabolic disease development. Both the total PE levels and the ratio of PE to other membrane lipids in skeletal muscle are implicated in insulin resistance; however, the underlying mechanisms and the role of Pcyt2 regulation in this association remain unclear. Here, we show how reduced phospholipid synthesis due to Pcyt2 deficiency causes Pcyt2+/- skeletal muscle dysfunction and metabolic abnormalities. Pcyt2+/- skeletal muscle exhibits damage and degeneration, with skeletal muscle cell vacuolization, disordered sarcomeres, mitochondria ultrastructure irregularities and paucity, inflammation, and fibrosis. There is intramuscular adipose tissue accumulation, and major disturbances in lipid metabolism with impaired FA mobilization and oxidation, elevated lipogenesis, and long-chain fatty acyl-CoA, diacylglycerol, and triacylglycerol accumulation. Pcyt2+/- skeletal muscle exhibits perturbed glucose metabolism with elevated glycogen content, impaired insulin signaling, and reduced glucose uptake. Together, this study lends insight into the critical role of PE homeostasis in skeletal muscle metabolism and health with broad implications on metabolic disease development.

SLC27 family of fatty acid transporters in GtoPdb v.2023.1

Fatty acid transporter proteins (FATPs) are a family (SLC27) of six transporters (FATP1-6). They have at least one, and possibly six [6, 13], transmembrane segments, and are predicted on the basis of structural similarities to form dimers. SLC27 members have several structural domains: integral membrane associated domain, peripheral membrane associated domain, FATP signature, intracellular AMP binding motif, dimerization domain, lipocalin motif, and an ER localization domain (identified in FATP4 only) [4, 10, 11]. These transporters are unusual in that they appear to express intrinsic very long-chain acyl-CoA synthetase (EC 6.2.1.- , EC 6.2.1.7) enzyme activity. Within the cell, these transporters may associate with plasma and peroxisomal membranes. FATP1-4 and -6 transport long- and very long-chain fatty acids, while FATP5 transports long-chain fatty acids as well as bile acids [9, 13, 1].

The origin of long-chain fatty acids required for de novo ether lipid/plasmalogen synthesis

Peroxisomes are single-membrane bounded organelles that in humans play a dual role in lipid metabolism, including the degradation of very long-chain fatty acids and the synthesis of ether lipids/plasmalogens. The first step in de novo ether lipid synthesis is mediated by the peroxisomal enzyme glyceronephosphate O-acyltransferase, which has a strict substrate specificity reacting only with the long-chain acyl-CoAs. The aim of this study was to determine the origin of these long-chain acyl-CoAs. To this end, we developed a sensitive method for the measurement of de novo ether phospholipid synthesis in cells and, by CRISPR-Cas9 genome editing, generated a series of HeLa cell lines with deficiencies of proteins involved in peroxisomal biogenesis, beta-oxidation, ether lipid synthesis, or metabolite transport. Our results show that the long-chain acyl-CoAs required for the first step of ether lipid synthesis can be imported from the cytosol by the peroxisomal ABCD proteins, in particular ABCD3. Furthermore, we show that these acyl-CoAs can be produced intraperoxisomally by chain shortening of CoA esters of very long-chain fatty acids via beta-oxidation. Our results demonstrate that peroxisomal beta-oxidation and ether lipid synthesis are intimately connected and that the peroxisomal ABC transporters play a crucial role in de novo ether lipid synthesis.

Metagenomic characterization of biomethane transformation by lipid-catalyzed anaerobic fermentation of lignite

The present study aims at using lipid in a novel way to improve the efficiency of methane production from lignite anaerobic digestion. The obtained results showed an increase by 3.13 times of the cumulative biomethane content of lignite anaerobic fermentation, when 1.8 g lipid was added. The gene expression of functional metabolic enzymes was also found to be enhanced during the anaerobic fermentation. Moreover, the enzymes related to fatty acid degradation such as long-chain Acyl-CoA synthetase and Acyl-CoA dehydrogenase were increased by 1.72 and 10.48 times, respectively, which consequently, accelerated the conversion of fatty acid. Furthermore, the addition of lipid enhanced the carbon dioxide trophic and acetic acid trophic metabolic pathways. Hence, the addition of lipids was argued to promote the production of methane from lignite anaerobic fermentation, which provided a new insight for the conversion and utilization of lipid waste.

ACADL suppresses PD-L1 expression to prevent cancer immune evasion by targeting Hippo/YAP signaling in lung adenocarcinoma.

Lung cancer is the leading cause of cancer-related death. Cancer immune evasion is a key barrier in the treatment of lung cancer and the development of effective anticancer therapeutics. Long-chain Acyl-CoA dehydrogenase (ACADL), a key enzyme that regulates β-oxidation of long-chain fatty acyl-CoAs, has been found to act as a tumor suppressor in cancers. However, the role of ACADL in lung adenocarcinoma (LUAD) has not been explored. In the current study, we find that ACADL functions as a tumor suppressor in LUAD to inhibit proliferation and enhanced chemotherapeutic drug-induced apoptosis. Interestingly, ACADL prevents tumor immune evasion by suppressing PD-L1 expression in LUAD. ACADL is critical for Hippo/YAP pathway-mediated PD-L1 regulation. Moreover, YAP activation is essential for ACADL suppression of PD-L1 transcription. In addition, ACADL increases the protein stability and kinase activity of LATS kinase to inhibit YAP activation and PD-L1 transcription. Furthermore, we show that ACADL expression is positively correlated with a better OS and FP in LUAD. Our data reveals that ACADL could be a promising target for regulating Hippo/YAP pathway to prevent tumor immune evasion in LUAD.

Lipid metabolism in dopaminergic neurons influences light entrainment.

Dietary lipids, particularly omega-3 polyunsaturated fatty acids, are speculated to impact behaviors linked to the dopaminergic system, such as movement and control of circadian rhythms. However, the ability to draw a direct link between dopaminergic omega-3 fatty acid metabolism and behavioral outcomes has been limited to the use of diet-based approaches, which are confounded by systemic effects. Here, neuronal lipid metabolism was targeted in a diet-independent manner by manipulation of long-chain acyl-CoA synthetase 6 (ACSL6) expression. ACSL6 performs the initial reaction for cellular fatty acid metabolism and prefers the omega-3 polyunsaturated fatty acid, docosahexaenoic acid (DHA). The loss of Acsl6 in mice (Acsl6-/- ) depletes neuronal membranes of DHA content and results in phenotypes linked to dopaminergic control, such as hyperlocomotion, impaired short-term spatial memory, and imbalances in dopamine neurochemistry. To investigate the role of dopaminergic ACSL6 on these outcomes, a dopaminergic neuron-specific ACSL6 knockout mouse was generated (Acsl6DA-/- ). Acsl6DA-/- mice demonstrated hyperlocomotion and imbalances in striatal dopamine neurochemistry. Circadian rhythms of both the Acsl6-/- and the Acsl6DA-/- mice were similar to control mice under basal conditions. However, upon light entrainment, a mimetic of jet lag, both the complete knockout of ACSL6 and the dopaminergic-neuron-specific loss of ACSL6 resulted in a longer recovery to entrainment compared to control mice. In conclusion, these data demonstrate that ACSL6 in dopaminergic neurons alters dopamine metabolism and regulation of light entrainment suggesting that DHA metabolism mediated by ACSL6 plays a role in dopamine neuron biology.

1,25-Dihydroxyvitamin D3 alleviates hyperandrogen-induced ferroptosis in KGN cells

PurposeHyperandrogenism, one of the most frequent causes of anovulation in women, increases the risk of metabolic disorders in patients with polycystic ovary syndrome (PCOS). Ferroptosis, characterized by iron-dependent lipid peroxidation, has provided new insight into the progression of PCOS. 1,25-dihydroxyvitamin D3 (1,25D3) may play a role in reproduction because its receptor, VDR, which contributes to the inhibition of oxidative stress, is primarily located in the nuclei of granulosa cells. This study has therefore investigated whether 1,25D3 and hyperandrogenism affect granulosa-like tumor cells (KGN cells) through ferroptosis.MethodsKGN cells were treated with dehydroepiandrosterone (DHEA) or pretreated with 1,25D3. Cell viability was evaluated with the cell counting kit-8 (CCK-8) assay. The mRNA and protein expression levels of ferroptosis-related molecules, including glutathione peroxidase 4 (GPX4), solute carrier family 7 member (SLC7A11), and long-chain acyl-CoA synthetase 4 (ACSL4), were assessed via qRT–PCR and western blot. The concentration of malondialdehyde (MDA) was measured by ELISA. The rates of reactive oxygen species (ROS) production and lipid peroxidation were assessed via photometric methods.ResultsDecreased cell viability, suppression of GPX4 and SLC7A11 expression, increased expression of ACSL4, elevated levels of MDA, accumulation of ROS, and increased lipid peroxidation, which are changes representative of ferroptosis, were observed in KGN cells after treatment with DHEA. Pretreatment with 1,25D3 in KGN cells significantly prevented these changes.ConclusionsOur findings demonstrate that 1,25D3 attenuates hyperandrogen-induced ferroptosis of KGN cells. This finding might lead to new insights into the pathophysiology and therapy of PCOS and provides new evidence for the treatment of PCOS with 1,25D3.

Rats with Long-Term Cholestasis Have a Decreased Cytosolic but Maintained Mitochondrial Hepatic CoA Pool.

Previous studies showed that rats with long-term bile duct ligation have reduced coenzyme A stores per g of liver but maintained mitochondrial CoA stores. Based on these observations, we determined the CoA pool in the liver homogenate, liver mitochondria, and liver cytosol of rats with bile duct ligation for 4 weeks (BDL rats, n = 9) and sham-operated control rats (CON rats, n = 5). In addition, we tested the cytosolic and mitochondrial CoA pools by assessing the metabolism of sulfamethoxazole and benzoate in vivo and of palmitate in vitro. The hepatic total CoA content was lower in BDL than CON rats (mean ± SEM; 128 ± 5 vs. 210 ± 9 nmol/g), affecting all subfractions equally (free CoA (CoASH), short- and long-chain acyl-CoA). In BDL rats, the hepatic mitochondrial CoA pool was maintained, and the cytosolic pool was reduced (23.0 ± 0.9 vs. 84.6 ± 3.7 nmol/g liver; CoA subfractions were affected equally). The urinary excretion of hippurate after i.p. benzoate administration (measuring mitochondrial benzoate activation) was reduced in BDL rats (23.0 ± 0.9 vs. 48.6 ± 3.7% of dose/24 h), whereas the urinary elimination of N-acetylsulfamethoxazole after i.p. sulfamethoxazole administration (measuring the cytosolic acetyl-CoA pool) was maintained (36.6 ± 3.0 vs. 35.1 ± 2.5% of dose/24 h BDL vs. CON rats). Palmitate activation was impaired in the liver homogenate of BDL rats but the cytosolic CoASH concentration was not limiting. In conclusion, BDL rats have reduced hepatocellular cytosolic CoA stores, but this reduction does not limit sulfamethoxazole N-acetylation or palmitate activation. The hepatocellular mitochondrial CoA pool is maintained in BDL rats. Impaired hippurate formation in BDL rats is explained best by mitochondrial dysfunction.

The KLF7/PFKL/ACADL axis modulates cardiac metabolic remodelling during cardiac hypertrophy in male mice

The main hallmark of myocardial substrate metabolism in cardiac hypertrophy or heart failure is a shift from fatty acid oxidation to greater reliance on glycolysis. However, the close correlation between glycolysis and fatty acid oxidation and underlying mechanism by which causes cardiac pathological remodelling remain unclear. We confirm that KLF7 simultaneously targets the rate-limiting enzyme of glycolysis, phosphofructokinase-1, liver, and long-chain acyl-CoA dehydrogenase, a key enzyme for fatty acid oxidation. Cardiac-specific knockout and overexpression KLF7 induce adult concentric hypertrophy and infant eccentric hypertrophy by regulating glycolysis and fatty acid oxidation fluxes in male mice, respectively. Furthermore, cardiac-specific knockdown phosphofructokinase-1, liver or overexpression long-chain acyl-CoA dehydrogenase partially rescues the cardiac hypertrophy in adult male KLF7 deficient mice. Here we show that the KLF7/PFKL/ACADL axis is a critical regulatory mechanism and may provide insight into viable therapeutic concepts aimed at the modulation of cardiac metabolic balance in hypertrophied and failing heart.

Heterologous Production of Calendic Acid Naturally Found in Calendula officinalis by Recombinant Fission Yeast.

Calendic acid (CA) is a conjugated fatty acid with anti-cancer properties that is widely present in seed oil of Calendula officinalis. Using the co-expression of C. officinalis fatty acid conjugases (CoFADX-1 or CoFADX-2) and Punica granatum fatty acid desaturase (PgFAD2), we metabolically engineered the synthesis of CA in the yeast Schizosaccharomyces pombe without the need for linoleic acid (LA) supplementation. The highest CA titer and achieved accumulation were 4.4 mg/L and 3.7 mg/g of DCW in PgFAD2 + CoFADX-2 recombinant strain cultivated at 16 °C for 72 h, respectively. Further analyses revealed the accumulation of CA in free fatty acids (FFA) and downregulation of the lcf1 gene encoding long-chain fatty acyl-CoA synthetase. The developed recombinant yeast system represents an important tool for the future identification of the essential components of the channeling machinery to produce CA as a high-value conjugated fatty acid at an industrial level.