Combined Multi-Omics Analysis Reveals the Potential Role of ACADS in Yak Intramuscular Fat Deposition.

The Yak (Bos grunniens) is a special breed of livestock predominantly distributed in the Qinghai-Tibet Plateau of China. Intramuscular fat (IMF) content in beef cattle is a vital indicator of meat quality. In this study, RNA-Seq and Protein-Seq were respectively employed to sequence the transcriptome and proteome of the longissimus dorsi (LD) tissue from 4-year-old yaks with significant differences in IMF content under the same fattening conditions. Five overlapping genes (MYL3, ACADS, L2HGDH, IGFN1, and ENSBGRG00000000-926) were screened using combined analysis. Functional verification tests demonstrated that the key gene ACADS inhibited yak intramuscular preadipocyte (YIMA) differentiation and proliferation, promoted mitochondrial biogenesis gene expression, and increased the mitochondrial membrane potential (MMP). Furthermore, co-transfection experiments further demonstrated that interfering with ACADS reversed the effect of PPARα agonists in promoting lipid differentiation. In conclusion, ACADS potentially inhibits lipid deposition in YIAMs by regulating the PPARα signalling pathway. These findings offer insights into the molecular mechanisms underlying yak meat quality.

ACADM inhibits AMPK activation to modulate PEDV-induced lipophagy and ß-oxidation for impairing viral replication.

Porcine epidemic diarrhea virus (PEDV) belongs to the Alphacoronavirus genus within the Coronavirus family, causing severe watery diarrhea in piglets and resulting in significant economic losses. Medium-chain acyl-CoA dehydrogenase (ACADM) is an enzyme participating in lipid metabolism associated with metabolic diseases and pathogen infections. Nonetheless, the precise role of ACADM in regulating PEDV replication remains uncertain. In this study, we identified ACADM as the host binding partner of NSP4 via immunoprecipitation-mass spectrometry analysis. The interaction between ACADM and NSP4 was subsequently corroborated through coimmunoprecipitation and laser confocal microscopy. Following this, a notable upsurge in ACADM expression was observed during PEDV infection. ACADM overexpression effectively inhibited virus replication, whereas ACADM knockdown facilitated virus replication, suggesting ACADM has negative regulation effect on PEDV infection. Furthermore, we demonstrated fatty acid ß-oxidation affected PEDV replication for the first time, inhibition of fatty acid ß-oxidation reduced PEDV replication. ACADM decreased PEDV-induced ß-oxidation to suppress PEDV replication. Mechanistically, ACADM reduced cellular free fatty acid levels and subsequent ß-oxidation by hindering AMPK-mediated lipophagy. In summary, our results reveal that ACADM plays a negative regulatory role in PEDV replication by regulating lipid metabolism. The present study introduces a novel approach for the prevention and control of PEDV infection.

Medium-chain triglyceride-specific appetite is regulated by the ß-oxidation of medium-chain fatty acids in the liver.

Most studies on fat appetite have focused on long-chain triglycerides (LCTs) due to their obesogenic properties. Medium-chain triglycerides (MCTs), conversely, exhibit antiobesogenic effects; however, the regulation of MCT intake remains elusive. Here, we demonstrate that mice can distinguish between MCTs and LCTs, and the specific appetite for MCTs is governed by hepatic ß-oxidation. We generated liver-specific medium-chain acyl-CoA dehydrogenase (MCAD)-deficient (MCADL-/-) mice and analyzed their preference for MCT and LCT solutions using glyceryl trioctanoate (C8-TG), glyceryl tridecanoate (C10-TG), corn oil, and lard oil in two-bottle choice tests conducted over 8 days. In addition, we used lick microstructure analyses to evaluate the palatability and appetite for MCT and LCT solutions. Finally, we measured the expression levels of genes associated with fat ingestion (Galanin, Qrfp, and Nmu) in the hypothalamus 2 h after oral gavage of fat. Compared with control mice, MCADL-/- mice exhibited a significantly reduced preference for MCT solutions, with no alteration in the preference for LCTs. Lick analysis revealed that MCADL-/- mice displayed a significantly decreased appetite for MCT solutions only while the palatability of both MCT and LCT solutions remained unaffected. Hypothalamic Galanin expression in control mice was elevated by oral gavage of C8-TG but not by LCTs, and this response was abrogated in MCADL-/- mice. In summary, our data suggest that hepatic ß-oxidation is required for MCT-specific appetite but not for LCT-specific appetite. The induction of hypothalamic galanin upon MCT ingestion, dependent on hepatic ß-oxidation, could be involved in the regulation of MCT-specific appetite.NEW & NOTEWORTHY Whether and how medium-chain triglyceride (MCT) intake is regulated remains unknown. Here, we showed that mice can discriminate between MCTs and LCTs. Hepatic ß-oxidation participates in MCT-specific appetite, and hypothalamic galanin may be one of the factors that regulate MCT intake. Because of the antiobesity effects of MCTs, studying MCT-specific appetite may help combat obesity by promoting the intake of MCTs instead of LCTs.

Low ACADM expression predicts poor prognosis and suppressive tumor microenvironment in clear cell renal cell carcinoma.

Clear cell renal cell carcinoma (ccRCC) represents a highly frequent renal cancer subtype. However, medium-chain acyl-CoA dehydrogenase (ACADM) encodes an important enzyme responsible for fatty acid ß-oxidation (FAO) and its association with prognosis and immunity in cancers has rarely been reported. Therefore, the present work focused on exploring ACADM's expression and role among ccRCC cases. We used multiple public databases and showed the hypo levels of ACADM protein and mRNA within ccRCC. Additionally, we found that ACADM down-regulation showed a remarkable relation to the advanced stage, high histological grade, as well as dismal prognostic outcome. As suggested by Kaplan-Meier curve analysis, cases showing low ACADM levels displayed shorter overall survival (OS) as well as disease-free survival (DFS). Moreover, according to univariate/multivariate Cox regression, ACADM-mRNA independently predicted the prognosis of ccRCC. In addition, this work conducted immunohistochemistry for validating ACADM protein expression and its prognostic role in ccRCC samples. KEGG and GO analyses revealed significantly enriched genes related to ACADM expression during fatty acid metabolism. The low-ACADM group with more regulatory T-cell infiltration showed higher expression of immune negative regulation genes and higher TIDE scores, which might contribute to poor response to immunotherapies. In conclusion, our results confirmed that downregulated ACADM predicted a poor prognosis for ccRCC and a poor response to immunotherapy. Our results provide important data for developing immunotherapy for ccRCC.

The male-to-female ratio in late-onset multiple acyl-CoA dehydrogenase deficiency: a systematic review and meta-analysis.

BACKGROUND: Late-onset multiple acyl-CoA dehydrogenase deficiency (MADD) is the most common lipid storage myopathy. There are sex differences in fat metabolism and it is not known whether late-onset MADD affects men and women equally. METHODS: In this systematic review and meta-analysis, the PubMed, Embase, Web of Science, CNKI, CBM, and Wanfang databases were searched until 01/08/2023. Studies reporting sex distribution in patients with late-onset MADD were included. Two authors independently screened studies for eligibility, extracted data, and assessed risk of bias. Pre-specified outcomes of interest were the male-to-female ratio (MFR) of patients with late-onset MADD, the differences of clinical characteristics between the sexes, and factors influencing the MFR. RESULTS: Of 3379 identified studies, 34 met inclusion criteria, yielding a total of 609 late-onset MADD patients. The overall pooled percentage of males was 58% (95% CI, 54-63%) with low heterogeneity across studies (I2 = 2.99%; P = 0.42). The mean onset ages, diagnostic delay, serum creatine kinase (CK), and allelic frequencies of 3 hotspot variants in ETFDH gene were similar between male and female patients (P > 0.05). Meta-regressions revealed that ethnic group was associated with the MFR in late-onset MADD, and subgroup meta-analyses demonstrated that East-Asian patients had a higher percentage of male, lower CK, and higher proportion of hotspot variants in ETFDH gene than non-East-Asian patients (P < 0.05). CONCLUSIONS: Male patients with late-onset MADD were more common than female patients. Ethnicity was proved to be a factor influencing the MFR in late-onset MADD. These findings suggest that male sex may be a risk factor for the disease.

A gene therapy targeting medium-chain acyl-CoA dehydrogenase (MCAD) did not protect against diabetes-induced cardiac pathology.

Diabetic cardiomyopathy describes heart disease in patients with diabetes who have no other cardiac conditions but have a higher risk of developing heart failure. Specific therapies to treat the diabetic heart are limited. A key mechanism involved in the progression of diabetic cardiomyopathy is dysregulation of cardiac energy metabolism. The aim of this study was to determine if increasing the expression of medium-chain acyl-coenzyme A dehydrogenase (MCAD; encoded by Acadm), a key regulator of fatty acid oxidation, could improve the function of the diabetic heart. Male mice were administered streptozotocin to induce diabetes, which led to diastolic dysfunction 8 weeks post-injection. Mice then received cardiac-selective adeno-associated viral vectors encoding MCAD (rAAV6:MCAD) or control AAV and were followed for 8 weeks. In the non-diabetic heart, rAAV6:MCAD increased MCAD expression (mRNA and protein) and increased Acadl and Acadvl, but an increase in MCAD enzyme activity was not detectable. rAAV6:MCAD delivery in the diabetic heart increased MCAD mRNA expression but did not significantly increase protein, activity, or improve diabetes-induced cardiac pathology or molecular metabolic and lipid markers. The uptake of AAV viral vectors was reduced in the diabetic versus non-diabetic heart, which may have implications for the translation of AAV therapies into the clinic. KEY MESSAGES: The effects of increasing MCAD in the diabetic heart are unknown. Delivery of rAAV6:MCAD increased MCAD mRNA and protein, but not enzyme activity, in the non-diabetic heart. Independent of MCAD enzyme activity, rAAV6:MCAD increased Acadl and Acadvl in the non-diabetic heart. Increasing MCAD cardiac gene expression alone was not sufficient to protect against diabetes-induced cardiac pathology. AAV transduction efficiency was reduced in the diabetic heart, which has clinical implications.

Acyl-CoA dehydrogenase substrate promiscuity: Challenges and opportunities for development of substrate reduction therapy in disorders of valine and isoleucine metabolism.

Toxicity of accumulating substrates is a significant problem in several disorders of valine and isoleucine degradation notably short-chain enoyl-CoA hydratase (ECHS1 or crotonase) deficiency, 3-hydroxyisobutyryl-CoA hydrolase (HIBCH) deficiency, propionic acidemia (PA), and methylmalonic aciduria (MMA). Isobutyryl-CoA dehydrogenase (ACAD8) and short/branched-chain acyl-CoA dehydrogenase (SBCAD, ACADSB) function in the valine and isoleucine degradation pathways, respectively. Deficiencies of these acyl-CoA dehydrogenase (ACAD) enzymes are considered biochemical abnormalities with limited or no clinical consequences. We investigated whether substrate reduction therapy through inhibition of ACAD8 and SBCAD can limit the accumulation of toxic metabolic intermediates in disorders of valine and isoleucine metabolism. Using analysis of acylcarnitine isomers, we show that 2-methylenecyclopropaneacetic acid (MCPA) inhibited SBCAD, isovaleryl-CoA dehydrogenase, short-chain acyl-CoA dehydrogenase and medium-chain acyl-CoA dehydrogenase, but not ACAD8. MCPA treatment of wild-type and PA HEK-293 cells caused a pronounced decrease in C3-carnitine. Furthermore, deletion of ACADSB in HEK-293 cells led to an equally strong decrease in C3-carnitine when compared to wild-type cells. Deletion of ECHS1 in HEK-293 cells caused a defect in lipoylation of the E2 component of the pyruvate dehydrogenase complex, which was not rescued by ACAD8 deletion. MCPA was able to rescue lipoylation in ECHS1 KO cells, but only in cells with prior ACAD8 deletion. SBCAD was not the sole ACAD responsible for this compensation, which indicates substantial promiscuity of ACADs in HEK-293 cells for the isobutyryl-CoA substrate. Substrate promiscuity appeared less prominent for 2-methylbutyryl-CoA at least in HEK-293 cells. We suggest that pharmacological inhibition of SBCAD to treat PA should be investigated further.

Functional and structural impact of 10 ACADM missense mutations on human medium chain acyl-Coa dehydrogenase.

Medium chain acyl-CoA dehydrogenase (MCAD) deficiency (MCADD) is associated with ACADM gene mutations, leading to an impaired function and/or structure of MCAD. Importantly, after import into the mitochondria, MCAD must incorporate a molecule of flavin adenine dinucleotide (FAD) per subunit and assemble into tetramers. However, the effect of MCAD amino acid substitutions on FAD incorporation has not been investigated. Herein, the commonest MCAD variant (p.K304E) and 11 additional rare variants (p.Y48C, p.R55G, p.A88P, p.Y133C, p.A140T, p.D143V, p.G224R, p.L238F, p.V264I, p.Y372N, and p.G377V) were functionally and structurally characterized. Half of the studied variants presented a FAD content <65 % compared to the wild-type. Most of them were recovered as tetramers, except the p.Y372N (mainly as dimers). No correlation was found between the levels of tetramers and FAD content. However, a correlation between FAD content and the cofactor's affinity, proteolytic stability, thermostability, and thermal inactivation was established. We showed that the studied amino acid changes in MCAD may alter the substrate chain-length dependence and the interaction with electron-transferring-flavoprotein (ETF) necessary for a proper functioning electron transfer thus adding additional layers of complexity to the pathological effect of ACADM missense mutations. Although the majority of the variant MCADs presented an impaired capacity to retain FAD during their synthesis, some of them were structurally rescued by cofactor supplementation, suggesting that in the mitochondrial environment the levels and activity of those variants may be dependent of FAD's availability thus contributing for the heterogeneity of the MCADD phenotype found in patients presenting the same genotype.

Bulk and single-cell transcriptome profiling reveal extracellular matrix mechanical regulation of lipid metabolism reprograming through YAP/TEAD4/ACADL axis in hepatocellular carcinoma.

Emerging studies have revealed matrix stiffness promotes hepatocellular carcinoma (HCC) development. We studied metabolic dysregulation in HCC using the TCGA-LIHC database (n=374) and GEO datasets (GSE14520). HCC samples were classified into three heterogeneous metabolic pathway subtypes with different metabolic profiles: Cluster 1, an ECM-producing subtype with upregulated glycan metabolism; Cluster 2, a hybrid subtype with partial pathway dysregulation. Cluster 3, a lipogenic subtype with upregulated lipid metabolism; These three subtypes have different prognosis, clinical features and genomic alterations. We identified key enzymes that respond to matrix stiffness and regulate lipid metabolism through bioinformatic analysis. We found long-chain acyl-CoA dehydrogenase (ACADL) is a mechanoreactive enzyme that reprograms HCC cell lipid metabolism in response to extracellular matrix stiffness. ACADL is also regarded as tumor suppressor in HCC. We found that increased extracellular matrix stiffness led to activation of Yes-associated protein (YAP) and the YAP/TEA Domain transcription factor 4 (TEAD4) transcriptional complex was able to directly repress ACADL at the transcriptional level. The ACADL-dependent mechanoresponsive pathway is a potential therapeutic target for HCC treatment.

Messenger RNA rescues medium-chain acyl-CoA dehydrogenase deficiency in fibroblasts from patients and a murine model.

Medium-chain acyl-CoA dehydrogenase (MCAD) deficiency is the most common inherited disorder of mitochondrial fatty acid ß-oxidation (FAO) in humans. Patients exhibit clinical episodes often associated with fasting. Symptoms include hypoketotic hypoglycemia and Reye-like episodes. With limited treatment options, we explored the use of human MCAD (hMCAD) mRNA in fibroblasts from patients with MCAD deficiency to provide functional MCAD protein and reverse the metabolic block. Transfection of hMCAD mRNA into MCAD- deficient patient cells resulted in an increased MCAD protein that localized to mitochondria, concomitant with increased enzyme activity in cell extracts. The therapeutic hMCAD mRNA-lipid nanoparticle (LNP) formulation was also tested in vivo in Acadm-/- mice. Administration of multiple intravenous doses of the hMCAD mRNA-LNP complex (LNP-MCAD) into Acadm-/- mice produced a significant level of MCAD protein with increased enzyme activity in liver, heart and skeletal muscle homogenates. Treated Acadm-/- mice were more resistant to cold stress and had decreased plasma levels of medium-chain acylcarnitines compared to untreated animals. Furthermore, hepatic steatosis in the liver from treated Acadm-/- mice was reduced compared to untreated ones. Results from this study support the potential therapeutic value of hMCAD mRNA-LNP complex treatment for MCAD deficiency.

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 ß-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.

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.

Generation of a human induced pluripotent stem cell line (LZUSHi002-A) from a MADD patient with ETFDH mutation.

Multiple acyl-coenzyme A dehydrogenase deficiency (MADD) is an inborn metabolic disorder that affects fatty acid oxidation and the catabolism of branched-chain amino acids, vitamins B and energy metabolism. In this study, the induced pluripotent stem cell (iPSC) line LZUSHi002-A from PBMCs of a 10-year-old male patient with ETFDH mutations using the episomal plasmids was established, which is an ideal in vitro model to understand the exact pathogenesis of MADD.

MCAD activation by empagliflozin promotes fatty acid oxidation and reduces lipid deposition in NASH.

Medium-chain acyl-CoA dehydrogenase (MCAD) is one of the significant enzymes involved in the ß-oxidation of mitochondrial fatty acids. MCAD deficiency affects the ß-oxidation of fatty acid and leads to lipid deposition in multiple organs, but little is known about its importance in nonalcoholic steatohepatitis (NASH). Empagliflozin is revealed to effectively improve NASH by increasing research, whereas the specific mechanism still has to be explored. Human liver tissues of patients with or without NASH were obtained for proteomic analysis to screen proteins of interest. db/db mice were given empagliflozin by gavage for 8 weeks. The expression of MCAD and signaling molecules involved in hepatic lipid metabolism was evaluated in human liver, mice and HL7702 cells. We found that the MCAD levels in the liver were significantly reduced in NASH patients compared to patients without NASH. Protein-protein interaction network analysis showed that MCAD was highly correlated with forkhead box A2 (FOXA2) and protein kinase AMP-activated catalytic subunit alpha (PRKAA). AMPK/FOXA2/MCAD signaling pathway was detected to be inhibited in the liver of NASH patients. Decreased expression of MCAD was also observed in the livers of db/db mice and hepatocyte treated with palmitic acid and glucose. Of note, empagliflozin could upregulate MCAD expression by activating AMPK/FOXA2 signaling pathway, reduce lipid deposition and improve NASH in vivo and in vitro. This research demonstrated that MCAD is a key player of hepatic lipid deposition and its targeting partially corrects NASH. MCAD thus may be a potential therapeutic target for the treatment of NASH.

Formation of fatty alcohols-components of meibum lipids-by the fatty acyl-CoA reductase FAR2 is essential for dry eye prevention.

Various lipids (mainly meibum lipids secreted by the meibomian glands) are present in the tear film lipid layer and play important roles in tear stability and the health of the cornea and conjunctiva. Many meibum lipids contain fatty alcohols (FAls) with chain lengths ≥C24, but the fatty acyl-CoA reductases (FARs) that produce them remain unclear. Here, using cell-based assays, we found that the two FAR isozymes (FAR1 and FAR2) show different substrate specificities: FAR1 and FAR2 are involved in the production of C16-C18 and ≥C20 FAls, respectively. Next, we generated Far2 knockout (KO) mice and examined their dry eye phenotype and meibum lipid composition. These mice showed a severe dry eye phenotype, characterized by plugged meibomian gland orifices, corneal damage, and tear film instability. The plugging was attributed to an increase in the melting point of the meibum lipids. Liquid chromatography coupled with tandem mass spectrometry revealed that FAl-containing meibum lipids (wax monoesters and types 1ω, 2α, and 2ω wax diesters) with a hydroxyl group at position 1 were almost completely absent in Far2 KO mice. The levels of di-unsaturated (O-acyl)-ω-hydroxy fatty acids were higher in Far2 KO mice than in wild type mice, but those of tri-unsaturated ones were comparable, suggesting the presence of two synthesis pathways for type 1ω wax diesters. These results indicate the importance of FAl-containing meibum lipids in the formation of a functional tear film lipid layer. In addition, our study provides clues to the molecular mechanism of the biosynthesis of meibum lipids.

SGIV Induced and Exploited Cellular De Novo Fatty Acid Synthesis for Virus Entry and Replication.

Considerable attention has been paid to the roles of lipid metabolism in virus infection due to its regulatory effects on virus replication and host antiviral immune response. However, few literature has focused on whether lipid metabolism is involved in the life cycle of lower vertebrate viruses. Singapore grouper iridovirus (SGIV) is the causative aquatic virus that extensively causes fry and adult groupers death. Here, the potential roles of cellular de novo fatty acid synthesis in SGIV infection was investigated. SGIV infection not only increased the expression levels of key enzymes in fatty acid synthesis in vivo/vitro, including acetyl-Coenzyme A carboxylase alpha (ACC1), fatty acid synthase (FASN), medium-chain acyl-CoA dehydrogenase (MCAD), adipose triglyceride lipase (ATGL), lipoprotein lipase (LPL) and sterol regulatory element-binding protein-1 (SREBP1), but it also induced the formation of lipid droplets (LDs), suggesting that SGIV altered de novo fatty acid synthesis in host cells. Using the inhibitor and specific siRNA of ACC1 and FASN, we found that fatty acid synthesis was essential for SGIV replication, evidenced by their inhibitory effects on CPE progression, viral gene transcription, protein expression and virus production. Moreover, the inhibitor of fatty acid ß-oxidation could also reduce SGIV replication. Inhibition of fatty acid synthesis but not ß-oxidation markedly blocked virus entry during the life cycle of SGIV infection. In addition, we also found that inhibition of ACC1 and FASN increased the IFN immune and inflammatory response during SGIV infection. Together, our data demonstrated that SGIV infection in vitro regulated host lipid metabolism and, in that process, cellular fatty acid synthesis might exert crucial roles during SGIV infection via regulating virus entry and host immune response.

miR-669a-5p promotes adipogenic differentiation and induces browning in preadipocytes.

Obesity is a major global health issue that contributes to the occurrence of metabolic disorders. Based on this fact, understanding the underlying mechanisms and to uncover promising therapeutic approaches for obesity have attracted intense investigation. Brown adipose tissue (BAT) can help burns excess calories. Therefore, promoting White adipose tissue (WAT) browning and BAT activation is an attractive strategy for obesity treatment. MicroRNAs (miRNAs) are small, non-coding RNAs, which are involved in regulation of adipogenic processes and metabolic functions. Evidence is accumulating that miRNAs are important regulators for both brown adipocyte differentiation and white adipocyte browning. Here we report that the expression of miR-669a-5p increases during the adipogenic differentiation of 3T3-L1 and C3H10T1/2 adipocytes. miR-669a-5p supplementation promotes adipogenic differentiation and causes browning of 3T3-L1 and C3H10T1/2 cells. Moreover, the expression of miR-669a-5p is upregulated in iWAT of mice exposed to cold. These data demonstrate that miR-669a-5p plays a role in regulating adipocyte differentiation and fat browning.Abbreviations: Acadl: long-chain acyl-Coenzyme A dehydrogenase; Acadm: medium-chain acyl-Coenzyme A dehydrogenase; Acadvl: very long-chain acyl-Coenzyme A dehydrogenase, very long chain; Aco2: mitochondrial  aconitase 2; BAT: brown adipose tissue; Bmper: BMP-binding endothelial regulator; Cpt1-b:carnitine palmitoyltransferase 1b; Cpt2: carnitine palmitoyltransferase 2; Crat: carnitine acetyltransferase; Cs: citrate synthase; C2MC: Chromosome 2 miRNA cluster; DMEM: Dulbecco's modified Eagle medium; eWAT: epididymal white adipose tissue; ETC: electron transport chain; FAO: fatty acid oxidation; Fabp4:fatty acid binding protein 4; FBS: fetal bovine serum; Hadha: hydroxyacyl-CoA dehydrogenase trifunctional multienzyme complex subunit alpha; Hadhb: hydroxyacyl-CoA dehydrogenase trifunctional multienzyme complex subunit beta; HFD: high fat diet; Idh3a: isocitrate dehydrogenase 3 alpha; iWAT: inguinal subcutaneous white adipose tissue; Lpl: lipoprotein lipase; Mdh2: malate dehydrogenase 2; NBCS: NewBorn Calf Serum; mt-Nd1: mitochondrial NADH dehydrogenase 1; Ndufb8:ubiquinone oxidoreductase subunit B8; Nrf1: nuclear respiratory factor 1; Pgc1α: peroxisome proliferative activated receptor gamma coactivator 1 alpha; Pgc1b: peroxisome proliferative activated receptor, gamma, coactivator 1 beta; Pparγ: peroxisome proliferator activated receptor gamma; Prdm16: PR domain containing 16; Rgs4: regulator of G-protein signaling 4; Sdhb: succinate dehydrogenase complex, subunit B; Sdhc: succinate dehydrogenase complex, subunit C; Sdhd: succinate dehydrogenase complex, subunit D; Sh3d21: SH3 domain containing 21; Sfmbt2: Scm-like with four mbt domains 2; TG: triglyceride; TCA: tricarboxylic acid cycle; Tfam: transcription factor A, mitochondrial; TMRE: tetramethylrhodamine, methyl ester; Ucp1: uncoupling protein 1; Uqcrc2: ubiquinol cytochrome c reductase core protein 2; WAT: White adipose tissue.

A Remimazolam and Remifentanil Anesthetic for a Pediatric Patient With a Medium-Chain Acyl-CoA Dehydrogenase Deficiency: A Case Report.

Medium-chain acyl-CoA dehydrogenase (MCAD) deficiency is one of the most common fatty acid oxidation disorders. The choice of anesthetics and blood glucose management are crucial to prevent metabolic decompensation. A 5-year-old Japanese boy with MCAD deficiency was scheduled to undergo surgery for an inguinal hernia. Glucose was continuously infused perioperatively, and his glucose concentrations were within the normal range. Anesthesia was induced and maintained with remimazolam, remifentanil, and intermittent rocuronium. No metabolic decompensation was observed. This case indicates the importance of a continuous intravenous glucose infusion, and that remimazolam can be the first-line anesthetic for a patient with MCAD deficiency.

Comparative Analysis of Bile-Salt Degradation in Sphingobium sp. Strain Chol11 and Pseudomonas stutzeri Strain Chol1 Reveals Functional Diversity of Proteobacterial Steroid Degradation Enzymes and Suggests a Novel Pathway for Side Chain Degradation.

The reaction sequence for aerobic degradation of bile salts by environmental bacteria resembles degradation of other steroid compounds. Recent findings show that bacteria belonging to the Sphingomonadaceae use a pathway variant for bile-salt degradation. This study addresses this so-called Δ4,6-variant by comparative analysis of unknown degradation steps in Sphingobium sp. strain Chol11 with known reactions found in Pseudomonas stutzeri Chol1. Investigations of strain Chol11 revealed an essential function of the acyl-CoA dehydrogenase (ACAD) Scd4AB for growth with bile salts. Growth of the scd4AB deletion mutant was restored with a metabolite containing a double bond within the side chain which was produced by the Δ22-ACAD Scd1AB from P. stutzeri Chol1. Expression of scd1AB in the scd4AB deletion mutant fully restored growth with bile salts, while expression of scd4AB only enabled constricted growth in P. stutzeri Chol1 scd1A or scd1B deletion mutants. Strain Chol11 Δscd4A accumulated hydroxylated steroid metabolites which were degraded and activated with coenzyme A by the wild type. Activities of five Rieske type monooxygenases of strain Chol11 were screened by heterologous expression and compared to the B-ring cleaving KshABChol1 from P. stutzeri Chol1. Three of the Chol11 enzymes catalyzed B-ring cleavage of only Δ4,6-steroids, while KshABChol1 was more versatile. Expression of a fourth KshA homolog, Nov2c228, led to production of metabolites with hydroxylations at an unknown position. These results indicate functional diversity of proteobacterial enzymes for bile-salt degradation and suggest a novel side chain degradation pathway involving an essential ACAD reaction and a steroid hydroxylation step. IMPORTANCE This study highlights the biochemical diversity of bacterial degradation of steroid compounds in different aspects. First, it further elucidates an unexplored variant in the degradation of bile-salt side chains by sphingomonads, a group of environmental bacteria that is well-known for their broad metabolic capabilities. Moreover, it adds a so far unknown hydroxylation of steroids to the reactions Rieske monooxygenases can catalyze with steroids. Additionally, it analyzes a proteobacterial ketosteroid-9α-hydroxylase and shows that this enzyme is able to catalyze side reactions with nonnative substrates.

Differential protein expression during growth on model and commercial mixtures of naphthenic acids in Pseudomonas fluorescens Pf-5.

Naphthenic acids (NAs) are carboxylic acids with the formula (Cn H2n+Z O2 ) and are among the most toxic, persistent constituents of oil sands process-affected waters (OSPW), produced during oil sands extraction. Currently, the proteins and mechanisms involved in NA biodegradation are unknown. Using LC-MS/MS shotgun proteomics, we identified proteins overexpressed during the growth of Pseudomonas fluorescens Pf-5 on a model NA (4'-n-butylphenyl)-4-butanoic acid (n-BPBA) and commercial NA mixture (Acros). By day 11, >95% of n-BPBA was degraded. With Acros, a 17% reduction in intensity occurred with 10-18 carbon compounds of the Z family -2 to -14 (major NA species in this mixture). A total of 554 proteins (n-BPBA) and 631 proteins (Acros) were overexpressed during growth on NAs, including several transporters (e.g., ABC transporters), suggesting a cellular protective response from NA toxicity. Several proteins associated with fatty acid, lipid, and amino acid metabolism were also overexpressed, including acyl-CoA dehydrogenase and acyl-CoA thioesterase II, which catalyze part of the fatty acid beta-oxidation pathway. Indeed, multiple enzymes involved in the fatty acid oxidation pathway were upregulated. Given the presumed structural similarity between alkyl-carboxylic acid side chains and fatty acids, we postulate that P. fluorescens Pf-5 was using existing fatty acid catabolic pathways (among others) during NA degradation.