Medium-chain Acyl-coenzyme A Dehydrogenase Research Articles

Estrogen-Related Receptor Agonism Reverses Mitochondrial Dysfunction and Inflammation in the Aging Kidney

A gradual decline in renal function occurs even in healthy aging individuals. In addition to aging, per se, concurrent metabolic syndrome and hypertension, which are common in the aging population, can induce mitochondrial dysfunction and inflammation, which collectively contribute to age-related kidney dysfunction and disease. This study examined the role of the nuclear hormone receptors, the estrogen-related receptors (ERRs), in regulation of age-related mitochondrial dysfunction and inflammation. The ERRs were decreased in both aging human and mouse kidneys and were preserved in aging mice with lifelong caloric restriction (CR). A pan-ERR agonist, SLU-PP-332, was used to treat 21-month-old mice for 8 weeks. In addition, 21-month-old mice were treated with a stimulator of interferon genes (STING) inhibitor, C-176, for 3 weeks. Remarkably, similar to CR, an 8-week treatment with a pan-ERR agonist reversed the age-related increases in albuminuria, podocyte loss, mitochondrial dysfunction, and inflammatory cytokines, via the cyclic GMP-AMP synthase-STING and STAT3 signaling pathways. A3-week treatment of 21-month-old mice with a STING inhibitor reversed the increases in inflammatory cytokines and the senescence marker, p21/cyclin dependent kinase inhibitor 1A (Cdkn1a), but also unexpectedly reversed the age-related decreases in PPARG coactivator (PGC)-1α, ERRα, mitochondrial complexes, and medium chain acyl coenzyme A dehydrogenase (MCAD) expression. These studies identified ERRs as CR mimetics and as important modulators of age-related mitochondrial dysfunction and inflammation. These findings highlight novel druggable pathways that can be further evaluated to prevent progression of age-related kidney disease.

Augmentation and Evaluation of an Olive Oil Based Polyherbal Combination against Diabetic Cardiomyopathy in Experimental Model of Rodents

Diabetes mellitus is a metabolic disorder that is prima facie a cause for numerous macro and micro vascular complications. A common macroscopic complication associated with diabetes is cardiomyopathy. Cardiomyopathy refers to diseases of the heart muscle, where the heart muscle becomes enlarged, thick, or rigid. As cardiomyopathy worsens, the heart becomes weaker and is unable to conduct the right amount of blood through the body and maintain a normal electrical rhythm. This can lead to heart failure or arrhythmias. Chronic diabetes is one of the instigating factors behind the etiology of this cardiac complication. Type-II diabetes is associated with impaired glucose metabolism that increases the dependence of a diabetic heart on fatty acid oxidation to meet its functional demands, resulting in mitochondrial uncoupling, glucotoxicity, lipotoxicity and initially subclinical cardiac dysfunction that finally gives way to heart failure. The increasing diabetic population with cardiac disorders and the ironically decreasing trend in newer medications to counter this complication leave us at a crossroads for pharmacological management of diabetic cardiomyopathy. Keeping this in view, the present study proclaims a newly developed polyherbal combination (PHC) with three herbs, namely Tinospora cordifolia, Withania somnifera and Boerhavia diffusa based in olive oil and administered in fixed dose (PHC-6 and PHC-10) to screen its cardioprotective potential against a well-established experimental model for diabetic cardiomyopathy. The three herbs mentioned have been known through the traditional literature for their antidiabetic and cardioprotective roles, hence they became the obvious choice. The study follows an experimental model proposed by Reed et al., where the capacity of the β-cell is unobtrusively impeded without totally compromising insulin release, bringing about a moderate disability in glucose resilience. Various sophisticated parameters, namely intraventricular septum thickness of hearts, Western blot of α/β- MHC monoclonal antibody (Ab), cardiac pyruvate dehydrogenase (PDH) activity, medium chain acyl coenzyme A dehydrogenase (MCAD) enzyme, etc. showed promising results where treatment with PHC (PHC-6 and PHC-10) significantly (*** p < 0.001 and **** p < 0.0001) prevented the symptoms of cardiomyopathy in subsequent groups when compared to disease control group.

Sudden neonatal death in individuals with medium-chain acyl-coenzyme A dehydrogenase deficiency: limit of newborn screening

Medium-chain acyl-coenzyme A dehydrogenase (MCAD) deficiency is the most common disorder of mitochondrial β-oxidation of fatty acids resulting in hypoketotic hypoglycemia, hepatopathy, and often fatal outcome in undiagnosed children. Introduction of tandem mass spectrometry–based newborn screening programs in the late 1990s has significantly reduced morbidity and mortality in MCAD deficiency; however, neonatal death in individuals with early disease manifestation and severe hypoglycemia may still occur. We describe the fatal disease course in eight newborns with MCAD deficiency, aiming to raise awareness for early clinical symptoms and the life-saving treatment, and promote systematic post-mortem protocols for biochemical and genetic testing, necessary for correct diagnosis and counselling of the family if unexpected death occurred in the neonatal period.Conclusion: Early newborn screening and awareness for clinical symptoms is lifesaving in MCAD deficiency, which may present with fatal neonatal crisis. Systematic post-mortem diagnostic protocols are needed for sudden neonatal deaths.What is Known:• Medium-chain acyl-coenzyme A dehydrogenase (MCAD) deficiency identified by newborn screening has an excellent outcome.• Fatal neonatal crises occur in the first days prior to screening.What is New:• Poor feeding, no monitoring of blood glucose, and homozygosity of the common gene variant (c.985A > G) are major risk factors for fatal neonatal crisis in MCAD deficiency.• Post-mortem diagnostic protocols are indispensable for correct diagnosis and counselling of the family if unexpected death occurred in the neonatal period.

MCAD deficiency caused by compound heterozygous pathogenic variants in ACADM

Medium-chain acyl-coenzyme A dehydrogenase (MCAD) deficiency is an autosomal recessive disease caused by biallelic pathogenic ACADM variants. We report a case of an asymptomatic Japanese girl with MCAD deficiency caused by compound heterozygous pathogenic variants (NM_000016.5:c.1040G > T (p.Gly347Val) and c.449_452delCTGA (p.Thr150ArgfsTer4)). Because the MCAD residual activity in lymphocytes of the patient was below the limit of quantification, both variants are likely to cause complete loss of MCAD enzymatic activity.

A Class I Histone Deacetylase Inhibitor Attenuates Insulin Resistance and Inflammation in Palmitate-Treated C2C12 Myotubes and Muscle of HF/HFr Diet Mice.

Histone deacetylase (HDAC) inhibitors, which regulate gene expression by inhibiting the deacetylation of histones and nonhistone proteins, have been shown to exert a wide array of biological effects; these include anti-cancer, anti-obesity, and anti-diabetes effects, as well as cardiovascular-protective activity. However, the effects of class I HDAC inhibition on lipotoxicity in C2C12 myotubes and skeletal muscle tissue remain poorly understood. In this study, we investigated the molecular mechanism underlying the protective effect of class I HDAC inhibition under lipotoxic conditions, i.e., in palmitate (PA)-treated C2C12 myotubes and skeletal muscle tissue in high fat (HF)/high fructose (HFr) diet mice. PA treatment of C2C12 myotubes increased HDAC3 protein expression and impaired mitochondrial oxidation, resulting in increased mitochondrial ROS generation and an accumulation of intracellular triglycerides (TG). Prolonged exposure led to increased inflammatory cytokine expression and insulin resistance. In contrast, MS-275, a class I HDAC inhibitor, dramatically attenuated lipotoxicity, preventing PA-induced insulin resistance and inflammatory cytokine expression. Similar beneficial effects were also seen following HDAC3 knockdown. In addition, MS-275 increased the mRNA expression of peroxisome proliferator activator receptor γ-coactivator 1α (PGC1α) and mitochondrial transcription factor A (TFAM), which serve as transcriptional coactivators in the context of mitochondrial metabolism and biogenesis, and restored expression of peroxisome proliferator-activated receptor alpha (PPARα), medium-chain acyl-coenzyme A dehydrogenase (MCAD), enoyl-CoA hydratase, and 3-hydroxyacyl CoA dehydrogenase (EHHADH). In vivo, treatment of HF/HFr-fed mice with MS-275 ameliorated hyperglycemia, insulin resistance, stress signals, and TNF-α expression in skeletal muscle. Taken together, these results suggest that HDAC3 inhibition rather than HDAC1/2 inhibition by MS-275 protects against lipotoxicity in C2C12 myotubes and skeletal muscle, and may be effective for the treatment of obesity and insulin resistance.

Transcriptome analysis suggests a compensatory role of the cofactors coenzyme A and NAD+ in medium-chain acyl-CoA dehydrogenase knockout mice

During fasting, mitochondrial fatty-acid β-oxidation (mFAO) is essential for the generation of glucose by the liver. Children with a loss-of-function deficiency in the mFAO enzyme medium-chain acyl-Coenzyme A dehydrogenase (MCAD) are at serious risk of life-threatening low blood glucose levels during fasting in combination with intercurrent disease. However, a subset of these children remains asymptomatic throughout life. In MCAD-deficient (MCAD-KO) mice, glucose levels are similar to those of wild-type (WT) mice, even during fasting. We investigated if metabolic adaptations in the liver may underlie the robustness of this KO mouse. WT and KO mice were given a high- or low-fat diet and subsequently fasted. We analyzed histology, mitochondrial function, targeted mitochondrial proteomics, and transcriptome in liver tissue. Loss of MCAD led to a decreased capacity to oxidize octanoyl-CoA. This was not compensated for by altered protein levels of the short- and long-chain isoenzymes SCAD and LCAD. In the transcriptome, we identified subtle adaptations in the expression of genes encoding enzymes catalyzing CoA- and NAD(P)(H)-involving reactions and of genes involved in detoxification mechanisms. We discuss how these processes may contribute to robustness in MCAD-KO mice and potentially also in asymptomatic human subjects with a complete loss of MCAD activity.

Metabolic regulation by the PGC-1α and PGC-1β coactivators in larval zebrafish (Danio rerio)

The peroxisome proliferator activated receptor γ coactivator-1 (PGC-1) family is composed of three coactivators whose role in regulating mammalian bioenergetics regulation is clear, but is much less certain in other vertebrates. Current evidence suggests that in fish, PGC-1α and PGC-1β may exhibit much less redundancy in the control of fatty acid oxidation and mitochondrial biogenesis compared to mammals. To assess these roles directly, we knocked down PGC-1α and PGC-1β expression with morpholinos in zebrafish embryos, and we investigated the resulting molecular and physiological phenotypes. First, we found no effects of either morpholinos on larval hatching, heart rates and oxygen consumption over the first few days of development. Next, at 3 days post fertilization (dpf), we confirmed by real time PCR a specific knock down of both coactivators, that resulted in a significant reduction in the transcript levels of citrate synthase (CS), 3-hydroxyacyl-CoA dehydrogenase (HOAD), and medium-chain acyl-coenzyme A dehydrogenase (MCAD) in both morphant groups. However, there was no effect on transcription factors' gene expression except for a marked reduction in estrogen related receptor α (ERRα) transcripts in PGC-1α morphants. Finally, we assessed whole embryonic enzyme activity for CS, cytochrome oxidase (COX), HOAD and carnitine palmitoyltransferase I (CPT-1) at 4 dpf. The only significant effect of the knockdown was a reduced CS activity in PGC-1α morphants and a counterintuitive increase of cytochrome oxidase activity in PGC-1β morphants. Overall, our results indicate that in larval zebrafish, PGC-1α and PGC-1β both play a role in regulating expression of important mitochondrial genes potentially through ERRα.

Effects of Fish Oil on Lipid Metabolism and Its Molecular Biological Regulators in Chronic Ethanol-Fed Rats.

The purpose of this study was to clarify the hepatoprotective mechanisms of fish oil in ethanol-fed rats based on lipid metabolism. Thirty eight-week-old male Wistar rats were divided into six groups: C (control), CF25 (control diet with 25% fish oil substitution), CF57 (control diet with 57% fish oil substitution), E (ethanol-containing diet) group, EF25 (ethanol-containing diet with 25% fish oil substitution), and EF57 (ethanol-containing diet with 57% fish oil substitution) groups. All of the groups were pair-fed an isoenergetic diet based on E group. Rats were sacrificed after eight weeks. When compared with C group, the plasma aspartate transaminase (AST) activity and hepatic steatosis and inflammatory cell infiltration were significantly higher, while plasma adiponectin level and hepatic AMP-activated protein kinase α (AMPKα) protein expression was significantly lower in the E group. However, the hepatic damage, including steatosis and inflammation were ameliorated in the EF25 and EF57 groups. Moreover, mRNA levels of fatty acid-oxidative enzymes, such as medium-chain acyl-coenzyme A dehydrogenase (MCAD) and carnitine palmitoyltransferase I (CPT-1) were significantly elevated in the EF57 group than those in E group. Partial replacement with fish oil might improve the fatty acid oxidation by raising mRNA levels of downstream transcription factors, finally inhibit the ethanol-induced hepatic steatosis in rats.

Loss of the Mitochondrial Fatty Acid \u03b2-Oxidation Protein Medium-Chain Acyl-Coenzyme A Dehydrogenase Disrupts Oxidative Phosphorylation Protein Complex Stability and Function

Medium-chain acyl-Coenzyme A dehydrogenase (MCAD) is involved in the initial step of mitochondrial fatty acid β-oxidation (FAO). Loss of function results in MCAD deficiency, a disorder that usually presents in childhood with hypoketotic hypoglycemia, vomiting and lethargy. While the disruption of mitochondrial fatty acid metabolism is the primary metabolic defect, secondary defects in mitochondrial oxidative phosphorylation (OXPHOS) may also contribute to disease pathogenesis. Therefore, we examined OXPHOS activity and stability in MCAD-deficient patient fibroblasts that have no detectable MCAD protein. We found a deficit in mitochondrial oxygen consumption, with reduced steady-state levels of OXPHOS complexes I, III and IV, as well as the OXPHOS supercomplex. To examine the mechanisms involved, we generated an MCAD knockout (KO) using human 143B osteosarcoma cells. These cells also exhibited defects in OXPHOS complex function and steady-state levels, as well as disrupted biogenesis of newly-translated OXPHOS subunits. Overall, our findings suggest that the loss of MCAD is associated with a reduction in steady-state OXPHOS complex levels, resulting in secondary defects in OXPHOS function which may contribute to the pathology of MCAD deficiency.

Abstract 394: Medium Chain Acyl-Coenzyme a Dehydrogenase Gene Therapy Induces Physiological Cardiac Hypertrophy and Protects Against Pathological Remodeling

Introduction: Gene-based therapies represent a realistic and feasible avenue for the treatment of cardiac disease. We previously demonstrated that activation of phosphoinositide 3-kinase [P13K(p110α)] was beneficial in heart failure mouse models and identified PI3K(p110α)-regulated mRNAs. We found that gene expression of medium chain acyl-coenzyme A dehydrogenase (MCAD) to be i) elevated in hearts with increased PI3K activity, ii) reduced in hearts with decreased PI3K activity, iii) reduced in settings of cardiac injury, and iv) positively correlated with cardiac function. Aim: The aim of this study was to determine whether gene delivery of MCAD could attenuate pressure overload-induced pathological remodeling. Methods: Recombinant adeno-associated viral vectors (rAAV) encoding MCAD or a control (2x10 11 vector genomes) were delivered to normal adult mice (n=9) and mice with pre-existing pathological hypertrophy and cardiac dysfunction due to pressure overload, induced by transverse aortic constriction (TAC, n=16). Cardiac function was assessed by echocardiography. Molecular/histological analyses were performed on heart tissue. Results: rAAV6:MCAD delivery promoted physiological growth of the heart in normal mice. Treated mice had larger hearts (Heart weight/tibia length [HW/TL] of rAAV6:control 8.3±0.5 mg/mm vs. HW/TL of rAAV6:MCAD 10.1±0.5 mg/mm, n=4-5/group, P<0.05), thicker left ventricular walls (rAAV6:control 0.82±0.03mm vs. rAAV6:MCAD 1.00±0.05mm, n=3-4/group, P<0.05), preserved systolic function, and no evidence of cardiac fibrosis, lung congestion or re-expression of the fetal genes atrial- and B- type natriuretic peptides. In a setting of pressure overload, mice that received rAAV:MCAD had less left ventricular fibrosis (3.4-fold lower vs. control, n=6-10/group, P<0.05), higher levels of SERCA2a gene expression, and a more favorable metabolic gene expression profile (increased gene expression of Pgc1α , Cpt1b and Glut4 , n=6-10/group, P<0.05) than control mice at the end of the treatment period. Conclusion: In summary, we show that MCAD can promote physiological cardiac hypertrophy in normal mice, and can attenuate fibrosis in a mouse model of cardiac injury.

Gene Delivery of Medium Chain Acyl-coenzyme A Dehydrogenase (MCAD) Induces Physiological Cardiac Hypertrophy and Protects Against Pathological Remodelling

Gene Delivery of Medium Chain Acyl-coenzyme A Dehydrogenase (MCAD) Induces Physiological Cardiac Hypertrophy and Protects Against Pathological Remodelling

Resveratrol prevents renal lipotoxicity in high-fat diet-treated mouse model through regulating PPAR-α pathway.

Resveratrol (RSV) has beneficial effects on renal diseases, but its underlying mechanisms are still unclear. In the present study, we investigate the renoprotective effects of RSV on obesity-related renal diseases and clarify the potential mechanisms. Male C57BL/6J mice were fed with high-fat diet (HFD) with or without 400mg/kg RSV treatment for 12weeks. Feeding HFD induced renal injuries, but treating them with RSV significantly decreased glomerular volume (p<0.05), glycogen (p<0.01) and collagen (p<0.05) in renal tissues. Although slightly changed body weight and fasting blood glucose, RSV attenuated renal dysfunction, including decreased levels of blood urea nitrogen (p<0.05), urea protein (p<0.01), and microalbuminuria (p<0.01). Furthermore, RSV treatment markedly reduced gene expression of tumor necrosis factor (TNF)-α, interleukin (IL)-6 and inducible nitric oxide synthase (iNOS) (all p<0.05), 4-Hydroxynonenal expression (p<0.01), and lipid accumulation. Mechanistically, RSV enhanced the expression of lipolytic genes, peroxisome proliferator-activated receptor (PPAR)-α (p<0.001), carnitine palmitoyltransferase (CPT)-1 (p<0.05), and medium-chain acyl-coenzyme A dehydrogenase (MCAD) (p<0.01), but had no effect on lipogenic genes, PPAR-γ and sterol regulatory element-binding protein (SREBP)-1c. RSV also obviously increased renal PPAR-α protein expression (p<0.001) and the phosphorylation of AMPK level. Collectively, these results support the therapeutic effects of RSV on high-fat diet-induced renal damages at least partially through targeting on PPAR-α signaling pathway.

PPARα Protein Expression Was Increased by Four Weeks of Intermittent Hypoxic Training via AMPKα2-Dependent Manner in Mouse Skeletal Muscle.

Peroxisome proliferator-activated receptor α (PPARα) is critical for muscle endurance due to its role in the regulation of fatty acid oxidation. The 5’-AMP-activated protein kinase (AMPK) is an energy sensor in cells, but its role in PPARα regulation in vivo remains unknown. In this study, we examined PPARα expression in the skeletal muscle of AMPKα2 overexpression (OE), knockout (KO) and wild-type (WT) mice after four weeks of exercise under intermittent hypoxia. WT, OE and KO mice were used at 40 mice/strain and randomly subdivided into four subgroups: control (C), running (R), hypoxia (H), and running plus hypoxia (R+H) at 10 mice/group. The treadmill running was performed at the speed of 12 m/min, 60 min/day with a slope of 0 degree for four weeks. The hypoxia treatment was performed in daytime with normobaric hypoxia (11.20% oxygen, 8 hours/day). In the R+H group, the treadmill running was conducted in the hypoxic condition. AMPKα2, phosphor-AMPKα (p-AMPKα) (Thr172), nuclear PPARα proteins were measured by Western blot and the medium chain acyl coenzyme A dehydrogenase (MCAD) mRNA, the key enzyme for fatty acid oxidation and one of the PPARα target genes, was also measured in skeletal muscles after the interventions. The results showed that nuclear PPARα protein was significantly increased by R+H in WT muscles, the increase was enhanced by 41% (p<0.01) in OE mice, but was reduced by 33% (p<0.01) in KO mice. The MCAD mRNA expression was increased after four weeks of R+H intervention. The change in MCAD mRNA followed a similar trend as that of PPARα protein in OE and KO mice. Our data suggest that the increase in nuclear PPARα protein by four-week exercise training under the intermittent hypoxia was dependent on AMPK activation.

Recurrent Ventricular Tachycardia in Medium-Chain Acyl-Coenzyme A Dehydrogenase Deficiency.

We report a baby with medium-chain acyl-coenzyme A dehydrogenase (MCAD) deficiency who presented on day 2 with poor feeding and lethargy. She was floppy with hypoglycaemia (1.8mmol/l) and hyperammonaemia (182μmol/l). Despite correction of these and a continuous intravenous infusion of glucose at 4.5-6.2mg/kg/min, she developed generalised tonic clonic seizures on day 3. She also suffered two episodes of pulseless ventricular tachycardia, from which she was resuscitated successfully. Unfortunately, she died on day 5, following a third episode of pulseless ventricular tachycardia. Arrhythmias are generally thought to be rarer in MCAD deficiency than in disorders of long-chain fatty acid oxidation. This is, however, the sixth report of ventricular tachyarrhythmias in MCAD deficiency. Five of these involved neonates and it may be that patients with MCAD deficiency are particularly prone to ventricular arrhythmias in the newborn period. Three of the patients (including ours) had normal blood glucose concentrations at the time of the arrhythmias and had been receiving intravenous glucose for many hours. These cases suggest that arrhythmias can be induced by medium-chain acylcarnitines or other metabolites accumulating in MCAD deficiency. Ventricular tachyarrhythmias can occur in MCAD deficiency, especially in neonates.

Exendin-4 regulates lipid metabolism and fibroblast growth factor 21 in hepatic steatosis

ObjectiveHepatokine fibroblast growth factor (FGF) 21 takes part in the regulation of lipid metabolism in the liver and adipose tissue. We investigated whether exendin-4 regulates the expression of FGF21 in the liver, and whether the effects of exendin-4 on the regulation of FGF21 expression are mediated via silent mating type information regulation 2 homolog (SIRT) 1 or SIRT6. Materials/methodsThe C57BL/6J mice were fed a low fat diet, high fat diet, or high fat diet with 1nmol/kg/day exendin-4 intraperitoneal injection for 10weeks. HepG2 used in vitro study was treated with palmitic aicd (0.4mM) with or without exendin-4 (100 nM) and FGF21 (50 nM) for 24hours. The change of FGF21 and its receptors expression by exendin-4 were measured using quantitative real-time RT-PCR and Western blot. The intracellular lipid content in HepG2 cells was evaluated by Oil Red O staining. Inhibition of FGF21, SIRT1 and SIRT6, by 10 nM siRNA was performed to establish the signaling pathway of exendin-4 action in hepatic lipid metabolism. ResultsExendin-4 increased the expression of FGF21 and its receptors in high fat diet-induced obese mice. In addition, recombinant FGF21 treatment reduced lipid content in palmitic acid-treated HepG2 cells. We also observed significantly decreased expression of peroxisomal proliferator-activated receptor (PPAR) α and medium-chain acyl-coenzyme A dehydrogenase (MCAD) in hepatocytes transfected with FGF21 siRNA. In cells treated with exendin-4, inhibition of SIRT1, but not SIRT6, by siRNA significantly repressed the expression of FGF21 mRNA, whereas decreased SIRT1 expression by inhibition of FGF21 was not observed. ConclusionsThese data suggest that exendin-4 could improve fatty liver by increasing SIRT1-mediated FGF21.

Experimental evidence for protein oxidative damage and altered antioxidant defense in patients with medium‐chain acyl‐CoA dehydrogenase deficiency

The objective of this study was to test whether macromolecule oxidative damage and altered enzymatic antioxidative defenses occur in patients with medium-chain acyl coenzyme A dehydrogenase (MCAD) deficiency. We performed a cross-sectional observational study of in vivo parameters of lipid and protein oxidative damage and antioxidant defenses in asymptomatic, nonstressed, MCAD-deficient patients and healthy controls. Patients were subdivided into three groups based on therapy: patients without prescribed supplementation, patients with carnitine supplementation, and patients with carnitine plus riboflavin supplementation. Compared with healthy controls, nonsupplemented MCAD-deficient patients and patients receiving carnitine supplementation displayed decreased plasma sulfhydryl content (indicating protein oxidative damage). Increased erythrocyte superoxide dismutase (SOD) activity in patients receiving carnitine supplementation probably reflects a compensatory mechanism for scavenging reactive species formation. The combination of carnitine plus riboflavin was not associated with oxidative damage. These are the first indications that MCAD-deficient patients experience protein oxidative damage and that combined supplementation of carnitine and riboflavin may prevent these biochemical alterations. Results suggest involvement of free radicals in the pathophysiology of MCAD deficiency. The underlying mechanisms behind the increased SOD activity upon carnitine supplementation need to be determined. Further studies are necessary to determine the clinical relevance of oxidative stress, including the possibility of antioxidant therapy.

Forkhead Box Transcription Factor Regulation and Lipid Accumulation by Hepatitis C Virus

We have previously shown that hepatitis C virus (HCV) infection modulates the expression of forkhead box transcription factors, including FoxO1 and FoxA2, which play key roles in gluconeogenesis and β-oxidation of fatty acid, respectively. The aim of the present study was to determine the role of forkhead box transcription factors in modulating lipid metabolism. HCV infection or core protein expression alone in transfected Huh7.5 cells increased expression of sterol regulatory element binding protein 1c (SREBP-1c) and its downstream target, fatty acid synthase (FASN), which are key proteins involved in lipid synthesis. Knockdown of FoxO1 by small interfering RNA in HCV-infected cells significantly decreased SREBP-1c and FASN expression. Further, HCV infection or core protein expression in Huh7.5 cells significantly decreased the expression of medium-chain acyl coenzyme A dehydrogenase (MCAD) and short-chain acyl coenzyme A dehydrogenase (SCAD), involved in the regulation of β-oxidation of fatty acids. Ectopic expression of FoxA2 in HCV-infected cells rescued the expression of MCAD and SCAD. Oil red O and neutral lipid staining indicated that HCV infection significantly increases lipid accumulation compared to that in the mock-infected control. This was further verified by the increased expression of perilipin-2 and decreased activity of hormone-sensitive lipase (HSL) in HCV-infected hepatocytes, implying increased accumulation of neutral lipids. Knockdown of FoxO1 and ectopic expression of FoxA2 significantly decreased HCV replication. Taken together, these results suggest that HCV modulates forkhead box transcription factors which together increase lipid accumulation and promote viral replication. Hepatic steatosis is a frequent complication associated with chronic HCV infection. Its presence is a key prognostic indicator associated with the progression to hepatic fibrosis and hepatocellular carcinoma. Several mechanisms have been proposed to account for the development of steatosis and fatty liver during HCV infection. We observed that HCV infection increases expression of both SREBP-1c and FASN. Further investigation suggested that the expression of SREBP-1c and FASN is controlled by the transcription factor FoxO1 during HCV infection. In addition, HCV infection significantly decreased both MCAD and SCAD expression, which is controlled by FoxA2. HCV infection also increased lipid droplet accumulation, increased perilipin-2 expression, and decreased HSL activity. Thus, knockdown of FoxO1 (decreased lipogenesis) and overexpression of FoxA2 (increased β-oxidation) resulted in a significant disruption of the platform and, hence, a decrease in HCV genome replication. Thus, targeting of FoxO1 and FoxA2 might be useful in developing a therapeutic approach against HCV infection.

Production and Release of Acylcarnitines by Primary Myotubes Reflect the Differences in Fasting Fat Oxidation of the Donors

Acylcarnitines are biomarkers of incomplete β-oxidation and mitochondrial lipid overload but indicate also high rates of mitochondrial fatty acid oxidation. It is unknown whether the production of acylcarnitines in primary human myotubes obtained from lean, metabolically healthy subjects reflects the fat oxidation in vivo. Our objective was to quantify the acylcarnitine production in myotubes obtained from subjects with low and high fasting respiratory quotient (RQ). Fasting RQ was determined by indirect calorimetry. Muscle biopsies from the vastus lateralis muscle were taken from 6 subjects with low fasting RQ (mean 0.79 ± 0.03) and 6 with high fasting RQ (0.90 ± 0.03), and satellite cells were isolated, cultured, and differentiated to myotubes. Myotubes were cultivated with 125 μM (13)C-labeled palmitate for 30 minutes and 4 and 24 hours. Quantitative profiling of 42 intracellular and 31 extracellular acylcarnitines was performed by stable isotope dilution-based metabolomics analysis by liquid chromatography coupled to mass spectrometry. Myotubes from donors with high fasting RQ produced and released significant higher amounts of medium-chain acylcarnitines. High (13)C8 and (13)C10 acylcarnitine levels in the extracellular compartment correlated with high fasting RQ. The decreased expression of medium-chain acyl-coenzyme A dehydrogenase (MCAD) in these myotubes can explain the higher rate of incomplete fatty acid oxidation. A lower intracellular [(13)C]acetylcarnitine to carnitine and lower intracellular (13)C16/(13)C18 acylcarnitine to carnitine ratio indicate reduced fatty acid oxidation capacity in these myotubes. Mitochondrial DNA content was not different. Acylcarnitine production and release from primary human myotubes of donors with high fasting RQ indicate a reduced fatty acid oxidation capacity and a higher rate of incomplete fatty acid oxidation. Thus, quantitative profiling of acylcarnitine production in human myotubes can be a suitable tool to identify muscular determinants of fat oxidation in vivo.

Improvement of myocardial lipid accumulation and prevention of PGC-1α induction by fenofibrate

We recently demonstrated that fenofibrate induces the activities of citrate synthase and NADH oxidase in cardiac mitochondria. To further determine the molecular mechanisms underlying fenofibrate action, 8-week-old mice were administered fenofibrate (100 mg/kg/day) for 7 and 14 days, and the expression of genes involved in cardiac mitochondrial function, such as nuclear respiratory factor 1 transcript variant 2 (NRF-1-L) and 6 (NRF-1-S), mitochondrial outer membrane protein 40 (Tom40), lipoic acid synthetase (Lias), cytochrome b, medium-chain acyl-coenzyme A dehydrogenase (MCAD) and peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α) was determined. Expression of PGC-1α, a key regulator of the entire fatty acid oxidation system, was significantly downregulated after 14 days of fenofibrate administration. Moreover, ventricular triglycerides were also accumulated following 14 days of fenofibrate administration. Thus, fenofibrate functions to improve myocardial lipid accumulation and to prevent PGC-1α induction, which is crucial for understanding the molecular mechanisms underlying fenofibrate action on the heart.

Risk stratification by residual enzyme activity after newborn screening for medium-chain acyl-CoA dehyrogenase deficiency: data from a cohort study

BackgroundSince the introduction of medium-chain acyl coenzyme A dehydrogenase (MCAD) deficiency in population newborn bloodspot screening (NBS) programs, subjects have been identified with variant ACADM (gene encoding MCAD enzyme) genotypes that have never been identified in clinically ascertained patients. It could be hypothesised that residual MCAD enzyme activity can contribute in risk stratification of subjects with variant ACADM genotypes.MethodsWe performed a retrospective cohort study of all patients identified upon population NBS for MCAD deficiency in the Netherlands between 2007–2010. Clinical, molecular, and enzymatic data were integrated.ResultsEighty-four patients from 76 families were identified. Twenty-two percent of the subjects had a variant ACADM genotype. In patients with classical ACADM genotypes, residual MCAD enzyme activity was significantly lower (median 0%, range 0-8%) when compared to subjects with variant ACADM genotypes (range 0-63%; 4 cases with 0%, remainder 20-63%). Patients with (fatal) neonatal presentations before diagnosis displayed residual MCAD enzyme activities <1%. After diagnosis and initiation of treatment, residual MCAD enzyme activities <10% were associated with an increased risk of hypoglycaemia and carnitine supplementation. The prevalence of MCAD deficiency upon screening was 1/8,750 (95% CI 1/7,210–1/11,130).ConclusionsDetermination of residual MCAD enzyme activity improves our understanding of variant ACADM genotypes and may contribute to risk stratification. Subjects with variant ACADM genotypes and residual MCAD enzyme activities <10% should be considered to have the same risks as patients with classical ACADM genotypes. Parental instructions and an emergency regimen will remain principles of the treatment in any type of MCAD deficiency, as the effect of intercurrent illness on residual MCAD enzyme activity remains uncertain. There are, however, arguments in favour of abandoning the general advice to avoid prolonged fasting in subjects with variant ACADM genotypes and >10% residual MCAD enzyme activity.