Personalised modelling of clinical heterogeneity between medium-chain acyl-CoA dehydrogenase patients.

BACKGROUND: Monogenetic inborn errors of metabolism cause a wide phenotypic heterogeneity that may even differ between family members carrying the same genetic variant. Computational modelling of metabolic networks may identify putative sources of this inter-patient heterogeneity. Here, we mainly focus on medium-chain acyl-CoA dehydrogenase deficiency (MCADD), the most common inborn error of the mitochondrial fatty acid oxidation (mFAO). It is an enigma why some MCADD patients-if untreated-are at risk to develop severe metabolic decompensations, whereas others remain asymptomatic throughout life. We hypothesised that an ability to maintain an increased free mitochondrial CoA (CoASH) and pathway flux might distinguish asymptomatic from symptomatic patients. RESULTS: We built and experimentally validated, for the first time, a kinetic model of the human liver mFAO. Metabolites were partitioned according to their water solubility between the bulk aqueous matrix and the inner membrane. Enzymes are also either membrane-bound or in the matrix. This metabolite partitioning is a novel model attribute and improved predictions. MCADD substantially reduced pathway flux and CoASH, the latter due to the sequestration of CoA as medium-chain acyl-CoA esters. Analysis of urine from MCADD patients obtained during a metabolic decompensation showed an accumulation of medium- and short-chain acylcarnitines, just like the acyl-CoA pool in the MCADD model. The model suggested some rescues that increased flux and CoASH, notably increasing short-chain acyl-CoA dehydrogenase (SCAD) levels. Proteome analysis of MCADD patient-derived fibroblasts indeed revealed elevated levels of SCAD in a patient with a clinically asymptomatic state. This is a rescue for MCADD that has not been explored before. Personalised models based on these proteomics data confirmed an increased pathway flux and CoASH in the model of an asymptomatic patient compared to those of symptomatic MCADD patients. CONCLUSIONS: We present a detailed, validated kinetic model of mFAO in human liver, with solubility-dependent metabolite partitioning. Personalised modelling of individual patients provides a novel explanation for phenotypic heterogeneity among MCADD patients. Further development of personalised metabolic models is a promising direction to improve individualised risk assessment, management and monitoring for inborn errors of metabolism.

Fifty years of research on mitochondrial fatty acid oxidation disorders: The remaining challenges.

Since the identification of the first disorder of mitochondrial fatty acid oxidation defects (FAOD) in 1973, more than 20 defects have been identified. Although there are some differences, most FAOD have similar clinical signs, which are mainly due to energy depletion and toxicity of accumulated metabolites. However, some of them have an unusual clinical phenotype or specific clinical signs. This manuscript focuses on what we have learnt so far on the pathophysiology of these disorders, which present with clinical signs that are not typical of categorical FAOD. It also highlights that some disorders have not yet been identified and tries to make assumptions to explain why. It also deals with new treatments under consideration in FAOD, including triheptanoin and similar anaplerotic substrates, ketone body treatments, RNA and gene therapy approaches. Finally, it suggests challenges for the diagnosis of FAOD in the coming years, both for symptomatic patients and for those diagnosed through newborn screening. The ultimate goal would be to identify all the patients born with FAOD and ensure for them the best possible quality of life.

Peroxisomal oxidation of erucic acid suppresses mitochondrial fatty acid oxidation by stimulating malonyl-CoA formation in the rat liver.

Feeding of rapeseed (canola) oil with a high erucic acid concentration is known to cause hepatic steatosis in animals. Mitochondrial fatty acid oxidation plays a central role in liver lipid homeostasis, so it is possible that hepatic metabolism of erucic acid might decrease mitochondrial fatty acid oxidation. However, the precise mechanistic relationship between erucic acid levels and mitochondrial fatty acid oxidation is unclear. Using male Sprague-Dawley rats, along with biochemical and molecular biology approaches, we report here that peroxisomal ß-oxidation of erucic acid stimulates malonyl-CoA formation in the liver and thereby suppresses mitochondrial fatty acid oxidation. Excessive hepatic uptake and peroxisomal ß-oxidation of erucic acid resulted in appreciable peroxisomal release of free acetate, which was then used in the synthesis of cytosolic acetyl-CoA. Peroxisomal metabolism of erucic acid also remarkably increased the cytosolic NADH/NAD+ ratio, suppressed sirtuin 1 (SIRT1) activity, and thereby activated acetyl-CoA carboxylase, which stimulated malonyl-CoA biosynthesis from acetyl-CoA. Chronic feeding of a diet including high-erucic-acid rapeseed oil diminished mitochondrial fatty acid oxidation and caused hepatic steatosis and insulin resistance in the rats. Of note, administration of a specific peroxisomal ß-oxidation inhibitor attenuated these effects. Our findings establish a cross-talk between peroxisomal and mitochondrial fatty acid oxidation. They suggest that peroxisomal oxidation of long-chain fatty acids suppresses mitochondrial fatty acid oxidation by stimulating malonyl-CoA formation, which might play a role in fatty acid-induced hepatic steatosis and related metabolic disorders.

Fluxomic assay-assisted diagnosis orientation in a cohort of 11 patients with myopathic form of CPT2 deficiency.

Carnitine palmitoyltransferase type 2 (CPT2) deficiency, a mitochondrial fatty acid oxidation disorder (MFAOD), is a cause of myopathy in its late clinical presentation. As for other MFAODs, its diagnosis may be evocated when blood acylcarnitine profile is abnormal. However, a lack of abnormalities or specificity in this profile is not exclusive of CPT2 deficiency. Our retrospective study reports clinical and biological data in a cohort of 11 patients with circulating acylcarnitine profile unconclusive enough for a specific diagnosis orientation. In these patients, CPT2 gene studies was prompted by prior fluxomic explorations of mitochondrial ß-oxidation on intact whole blood cells incubated with pentadeuterated ([16-2H3, 15-2H2])-palmitate. Clinical indication for fluxomic explorations was at least one acute rhabdomyolysis episode complicated, in 5 of 11 patients, by acute renal failure. Major trigger of rhabdomyolysis was febrile infection. In all patients, fluxomic data indicated deficient CPT2 function showing normal deuterated palmitoylcarnitine (C16-Cn) formation rates associated with increased ratios between generated C16-Cn and downstream deuterated metabolites (Σ deuterated C2-Cn to C14-Cn). Subsequent gene studies showed in all patients pathogenic gene variants in either homozygous or compound heterozygous forms. Consistent with literature data, allelic frequency of the c.338C > T[p.Ser113Leu] mutation amounted to 68.2% in our cohort. Other missense mutations included c.149C > A[p.Pro50His] (9%), c.200C > G[p.Ala200Gly] (4.5%) and previously unreported c.1171A > G[p.ser391Gly] (4.5%) and c.1420G > C[p.Ala474Pro] (4.5%) mutations. Frameshift c.1666-1667delTT[p.Leu556val*16] mutation (9%) was observed in two patients unknown to be related.

Living on the edge: substrate competition explains loss of robustness in mitochondrial fatty-acid oxidation disorders.

BACKGROUND: Defects in genes involved in mitochondrial fatty-acid oxidation (mFAO) reduce the ability of patients to cope with metabolic challenges. mFAO enzymes accept multiple substrates of different chain length, leading to molecular competition among the substrates. Here, we combined computational modeling with quantitative mouse and patient data to investigate whether substrate competition affects pathway robustness in mFAO disorders. RESULTS: First, we used comprehensive biochemical analyses of wild-type mice and mice deficient for medium-chain acyl-CoA dehydrogenase (MCAD) to parameterize a detailed computational model of mFAO. Model simulations predicted that MCAD deficiency would have no effect on the pathway flux at low concentrations of the mFAO substrate palmitoyl-CoA. However, high concentrations of palmitoyl-CoA would induce a decline in flux and an accumulation of intermediate metabolites. We proved computationally that the predicted overload behavior was due to substrate competition in the pathway. Second, to study the clinical relevance of this mechanism, we used patients' metabolite profiles and generated a humanized version of the computational model. While molecular competition did not affect the plasma metabolite profiles during MCAD deficiency, it was a key factor in explaining the characteristic acylcarnitine profiles of multiple acyl-CoA dehydrogenase deficient patients. The patient-specific computational models allowed us to predict the severity of the disease phenotype, providing a proof of principle for the systems medicine approach. CONCLUSION: We conclude that substrate competition is at the basis of the physiology seen in patients with mFAO disorders, a finding that may explain why these patients run a risk of a life-threatening metabolic catastrophe.

From genome to phenome-Simple inborn errors of metabolism as complex traits.

Sporadically, patients with a proven defect in either mFAO or OXPHOS are described presenting with a metabolic profile and clinical phenotype expressing concurrent defects in both pathways. Biochemical linkages between both processes are tight. Therefore, it is striking that concurrent dysfunction of both systems occurs so infrequent. In this review, the linkages between OXPHOS and mFAO and the hypothesized processes responsible for concurrent problems in both systems are reviewed, both from the point of view of primary biochemical connections and secondary cellular responses, i.e. signaling pathways constituting nutrient-sensing networks. We propose that affected signaling pathways may play an important role in the phenomenon of concurrent defects. Recent data indicate that interference in the affected signaling pathways may resolve the pathological phenotype even though the primary enzyme deficiency persists. This offers new (unexpected) prospects for treatment of these inborn errors of metabolism. This article is part of a Special Issue entitled: From Genome to Function.

Triclocarban induces lipid droplet accumulation and oxidative stress responses by inhibiting mitochondrial fatty acid oxidation in HepaRG cells.

Mitochondrial fatty acid oxidation (mtFAO) plays an important role in hepatic energy metabolism. Severe mtFAO injury leads to nonalcoholic fatty liver disease (NAFLD) and liver failure. Several drugs have been withdrawn owing to safety issues, such as induction of fatty liver disease through mtFAO disruption. For instance, the antimicrobial triclocarban (TCC), an environmental contaminant that was removed from the market due to its unknown safety in humans, induces NAFLD in rats and promotes hepatic FAO in mice. Therefore, there are no consistent conclusions regarding the effects of TCC on FAO and lipid droplet accumulation. We hypothesized that TCC induces lipid droplet accumulation by inhibiting mtFAO in human hepatocytes. Here, we evaluated mitochondrial respiration in HepaRG cells to investigate the effects of TCC on fatty acid-driven oxidation in cells, electron transport chain parameters, lipid droplet accumulation, and antioxidant genes. The results suggest that TCC increases oxidative stress gene expression (GCLM, p62, HO-1, and NRF2) through lipid droplet accumulation via mtFAO inhibition in HepaRG cells. The results of the present study provide further insights into the effect of TCC on human NAFLD through mtFAO inhibition, and further in vivo studies could be used to validate the mechanisms.

Impact of newborn screening for fatty acid oxidation disorders on neurological outcome: A Belgian retrospective and multicentric study.

Fatty acid oxidation (FAO) disorders are autosomal recessive genetic disorders affecting either the transport or the oxidation of fatty acids. Acute symptoms arise during prolonged fasting, intercurrent infections, or intense physical activity. Metabolic crises are characterized by alteration of consciousness, hypoglycemic coma, hepatomegaly, cardiomegaly, arrhythmias, rhabdomyolysis, and can lead to death. In this retrospective and multicentric study, the data of 54 patients with FAO disorders were collected. Overall, 35 patients (64.8%) were diagnosed after newborn screening (NBS), 17 patients on clinical presentation (31.5%), and two patients after family screening (3.7%). Deficiencies identified included medium-chain acyl-CoA dehydrogenase (MCAD) deficiency (75.9%), very long-chain acyl-CoA dehydrogenase (VLCAD) deficiency (11.1%), long-chain hydroxyacyl-CoA dehydrogenase (LCHAD) deficiency (3.7%), mitochondrial trifunctional protein (MTP) deficiency (1.8%), and carnitine palmitoyltransferase 2 (CPT 2) deficiency (7.4%). The NBS results of 25 patients were reviewed and the neurological outcome of this population was compared with that of the patients who were diagnosed on clinical presentation. This article sought to provide a comprehensive overview of how NBS implementation in Southern Belgium has dramatically improved the neurological outcome of patients with FAO disorders by preventing metabolic crises and death. Further investigations are needed to better understand the physiopathology of long-term complications in order to improve the quality of life of patients and to ensure optimal management.

UHPLC-MS/MS method for chiral separation of 3-hydroxy fatty acids on amylose-based chiral stationary phase and its application for the enantioselective analysis in plasma and platelets.

3-Hydroxyfatty acids (3-OH-FAs) are formed in the hydration step during mitochondrial ß-oxidation of saturated straight-chain fatty acids, which is a catabolic pathway that involves several enzymes. For an unbiased biological interpretation, an enantioselective analysis of 3-OH-FAs including their stereoisomers is necessary, which may contribute to the elucidation of enzymatic mechanisms in the biological pathways. In this work, an enantioselective gradient UHPLC-MS/MS method based on 1.6 µm particle polysaccharide column (Chiralpak IA-U) for chiral separation of 3-hydroxyfatty acids was developed which covers carbon chain length from C8 to C18 with a good resolution of R and S enantiomers. The method is fast and sensitive for detecting enantiomers of 3-OH-FAs by using a triple quadrupole instrument as a detector in a targeted, selected reaction monitoring (SRM) mode. A matrix matched-calibration strategy was applied for quantification of individual 3-OH-FA enantiomers. The method allows the simultaneous quantification of each enantiomer of 3-OH-FAs from C8-C18. One-phase liquid extraction with 2-propanol showed good extraction recoveries with over 90% on average. Further, the validated method was applied to investigate the alteration of 3-OH-FA enantiomers in platelets and plasma samples from human donors with different diagnoses of cardiovascular disease (acute coronary syndrome ACS, chronic coronary syndrome CCS). Both R and S enantiomers were detected in platelets and plasma samples with different predominance for R or S in dependence on carbon chain length, which might be associated with different functional enzymes of mitochondrial and peroxisomal ß-oxidation. Finally, our study provides a new strategy for chiral separation and enantioselective analysis, showing great potential for targeted metabolomics in clinical biomarker discovery.

Novel treatment and insight for irradiation-induced injuries: Dibucaine ameliorates irradiation-induced testicular injury by inhibiting fatty acid oxidation in primary Leydig cells.

BACKGROUND: Male infertility is a worldwide problem but few treatments, especially irradiation-induced testicular injury. The aim of this research was to investigate novel drugs for the treatment of irradiation-induced testicular injury. METHODS: We administered dibucaine (0.8 mg/kg) intraperitoneally to male mice (6 mice per group) after five consecutive daily 0.5 Gy whole-body irradiation, and evaluated its ameliorating efficacy by testicular HE staining and morphological measurements. Drug affinity responsive target stability assay (Darts) were used to find target protein and pathway; mouse primary Leydig cells were isolated and to explore the mechanism (Flow cytometry, Western blot, and Seahorse palmitate oxidative stress assays); finally rescue experiments were completed by combining dibucaine with fatty acid oxidative pathway inhibitors and activators. RESULTS: The testicular HE staining and morphological measurements in dibucaine treatment group was significantly better than that in irradiation group (P < 0.05); sperm motility and mRNA levels of spermatogenic cell markers were also higher than those in the latter (P < 0.05). Darts and Western blot results showed that dibucaine targets CPT1A and downregulate fatty acid oxidation. Flow cytometry, Western blot, and Palmitate oxidative stress assays of primary Leydig cells demonstrated that dibucaine inhibits fatty acid oxidation in Leydig cells. Dibucaine combined with etomoxir/baicalin confirmed that its inhibition of fatty acid oxidation was beneficial in ameliorating irradiation-induced testicular injury. CONCLUSIONS: In conclusion, our data suggest that dibucaine ameliorates irradiation-induced testicular injury in mice by inhibiting fatty acid oxidation in Leydig cells. This will provide novel ideas for the treatment of irradiation-induced testicular injury.

C3aR Antagonist Alleviates C3a Induced Tubular Profibrotic Phenotype Transition via Restoring PPARα/CPT-1α Mediated Mitochondrial Fatty Acid Oxidation in Renin-Dependent Hypertension.

BACKGROUND: Renin-dependent hypertension with tubulointerstitial injury remains a problem with high prevalence in the clinic. However, whether and how renin participates in tubulointerstitial injury remains incompletely understood. New evidence suggests that renin cleaves C3 into C3a and C3b. In the present study, we aimed to explore the role of renin-mediated C3a/C3a receptor (C3aR) signaling in renin-dependent hypertension-induced kidney injury and illustrate the detailed mechanisms. METHODS: C3a concentration changes in serum from healthy volunteers incubated with recombinant renin were detected by ELISA. C3aR expression in human tubular epithelial cells was evaluated in renal biopsy sections from malignant arteriolonephrosclerosis and benign arteriolonephrosclerosis patients. C3aR changes in human kidney 2 (HK2) cells were detected after the cells were treated with human serum, renin and aliskiren. The C3a analogue and C3aR antagonist SB290157 were used to stimulate HK2 cells to explore the downstream signaling of C3a/C3aR activation. For in vivo studies, two-kidney, one-clipped (2K1C) hypertensive rat model was established to simulate renin-dependent hypertension conditions. C3a and C3aR expression was detected in the clipped kidneys. SB290157 was injected intraperitoneally to block C3a/C3aR signaling in 2K1C rats. RESULTS: The results showed that renin cleaved C3 into C3a and activated C3a/C3aR signaling in tubular epithelial cells (TECs) from both humans and rats. In vitro results demonstrated that C3a/C3aR activation impaired peroxisome proliferator-activated receptor alpha (PPARα)/carnitine palmitoyltransterase-1alpha (CPT-1α)-mediated mitochondrial fatty acid oxidation (Mito FAO) in HK2 cells and induced HK2 cell transition to a profibrotic phenotype, which was inhibited by treatment with the C3aR antagonist SB290157. In vivo results showed that renin mRNA levels, C3a concentrations, C3aR levels and tubulointerstitial fibrosis increased concurrently in the clipped kidney cortex of 2K1C rats. Treatment with the C3aR antagonist SB290157 significantly mitigated the effect of renin induction of C3aR expression and alleviated renin-dependent hypertension-induced tubulointerstitial fibrosis by improving PPARα/CPT-1α-mediated Mito FAO in TECs, as well as inhibiting tubular profibrotic phenotype transition. CONCLUSIONS: Our results prove that renin activates C3a/C3aR signaling to promote renal tubulointerstitial fibrosis by impairing PPARα/CPT-1α-mediated tubular Mito FAO. SB290157 confers a potential therapeutic approach for renin-dependent hypertension-induced kidney injury.

Over-Reduced State of Mitochondria as a Trigger of "ß-Oxidation Shuttle" in Cancer Cells.

A considerable amount of data have accumulated in the last decade on the pronounced mitochondrial fatty acid oxidation (mFAO) in many types of cancer cells. As a result, mFAO was found to coexist with abnormally activated fatty acid synthesis (FAS) and the mevalonate pathway. Recent studies have demonstrated that overactivated mitochondrial ß-oxidation may aggravate the impaired mitochondrial redox state and vice versa. Furthermore, the impaired redox state of cancerous mitochondria can ensure the continuous operation of ß-oxidation by disconnecting it from the Krebs cycle and connecting it to the citrate-malate shuttle. This could create a new metabolic state/pathway in cancer cells, which we have called the "ß-oxidation-citrate-malate shuttle", or "ß-oxidation shuttle" for short, which forces them to proliferate. The calculation of the phosphate/oxygen ratio indicates that it is inefficient as an energy source and must consume significantly more oxygen per mole of ATP produced when combined with acetyl-CoA consuming pathways, such as the FAS and mevalonate pathways. The "ß-oxidation shuttle" is an unconventional mFAO, a separate metabolic pathway that has not yet been explored as a source of energy, as well as a source of cataplerosis, leading to biomass accumulation, accelerated oxygen consumption, and, ultimately, a source of proliferation. The role of the "ß-oxidation shuttle" and its contribution to redox-altered cancer metabolism provides a new direction for the development of future anticancer strategies. This may represent the metabolic "secret" of cancer underlying hypoxia and genomic instability.

Chicken Protein Hydrolysates Have Anti-Inflammatory Effects on High-Fat Diet Induced Obesity in Mice.

Background: Studies have shown that dietary source of protein and peptides can affect energy metabolism and influence obesity-associated diseases. This study aimed to investigate the impact of different chicken protein hydrolysates (CPHs) generated from chicken rest raw materials in a mouse obesity model. Methods: Male C57BL/6 mice were fed a high-fat, high-sucrose diet with casein or CPHs generated using Papain + Bromelain, Alcalase, Corolase PP, or Protamex for 12 weeks (n = 12). Body weight, feed intake, and intraperitoneal glucose tolerance was determined, and plasma and liver and adipose tissues were collected at sacrifice. Results: The average feed intake and body weight did not differ between the groups and white adipose tissue depots were unchanged, except for a reduction in the subcutaneous depot in mice fed the Protamex CPH diet. Moreover, the CPH diets did not prevent increased fasting glucose and insulin levels. Interestingly, the hepatic mitochondrial fatty acid ß-oxidation was increased in mice fed Alcalase and Corolase PP CPHs. All CPH diets reduced plasma interleukine (IL)-1ß, interferon-γ, tumor necrosis factor α, and monocyte chemotactic protein 1 compared to control, indicating anti-inflammatory effects. In addition, Corolase PP and Protamex CPHs significantly reduced plasma levels of IL-1α, IL-2, IL-6, IL-10, and granulocyte macrophage colony-stimulating factor. Conclusions: CPH diets were not able to counteract obesity and glucose intolerance in a mouse obesity model, but strongly reduced inflammatory parameters associated with obesity. Alcalase and Corolase PP CPHs also stimulated mitochondrial fatty acid ß-oxidation. The possibility that hydrolysates from chicken rest raw materials could alleviate obesity-associated metabolic disease should be investigated further.

Mice with an Oncogenic HRAS Mutation are Resistant to High-Fat Diet-Induced Obesity and Exhibit Impaired Hepatic Energy Homeostasis.

Costello syndrome is a "RASopathy" that is characterized by growth retardation, dysmorphic facial appearance, hypertrophic cardiomyopathy and tumor predisposition. >80% of patients with Costello syndrome harbor a heterozygous germline G12S mutation in HRAS. Altered metabolic regulation has been suspected because patients with Costello syndrome exhibit hypoketotic hypoglycemia and increased resting energy expenditure, and their growth is severely retarded. To examine the mechanisms of energy reprogramming by HRAS activation in vivo, we generated knock-in mice expressing a heterozygous Hras G12S mutation (HrasG12S/+ mice) as a mouse model of Costello syndrome. On a high-fat diet, HrasG12S/+ mice developed a lean phenotype with microvesicular hepatic steatosis, resulting in early death compared with wild-type mice. Under starvation conditions, hypoketosis and elevated blood levels of long-chain fatty acylcarnitines were observed, suggesting impaired mitochondrial fatty acid oxidation. Our findings suggest that the oncogenic Hras mutation modulates energy homeostasis in vivo.

Advances in the Understanding and Treatment of Mitochondrial Fatty Acid Oxidation Disorders.

PURPOSE OF REVIEW: This review focuses on advances made in the past three years with regards to understanding the mitochondrial fatty acid oxidation (FAO) pathway, the pathophysiological ramifications of genetic lesions in FAO enzymes, and emerging therapies for FAO disorders. RECENT FINDINGS: FAO has now been recognized to play a key energetic role in pulmonary surfactant synthesis, T-cell differentiation and memory, and the response of the proximal tubule to kidney injury. Patients with FAO disorders may face defects in these cellular systems as they age. Aspirin, statins, and nutritional supplements modulate the rate of FAO under normal conditions and could be risk factors for triggering symptoms in patients with FAO disorders. Patients have been identified with mutations in the ACAD9 and ECHS1 genes, which may represent new FAO disorders. New interventions for long-chain FAODs are in clinical trials. Finally, post-translational modifications that regulate fatty acid oxidation protein activities have been characterized that represent important new therapeutic targets. SUMMARY: Recent research has led to a deeper understanding of FAO. New therapeutic avenues are being pursued that may ultimately cause a paradigm shift for patient care.

Accumulation of 3-hydroxytetradecenoic acid: Cause or corollary of glucolipotoxic impairment of pancreatic ß-cell bioenergetics?

OBJECTIVES: Hyperglycemia and elevated blood lipids are the presumed precipitating causes of ß-cell damage in T2DM as the result of a process termed "glucolipotoxicity". Here, we tested whether glucolipotoxic pathophysiology is caused by defective bioenergetics using islets in culture. METHODS: Insulin secretion, respiration, ATP generation, fatty acid (FA) metabolite profiles and gene expression were determined in isolated islets treated under glucolipotoxic culture conditions. RESULTS: Over time, chronic exposure of mouse islets to FAs with glucose leads to bioenergetic failure and reduced insulin secretion upon stimulation with glucose or amino acids. Islets exposed to glucolipotoxic conditions displayed biphasic changes of the oxygen consumption rate (OCR): an initial increase in baseline and Vmax of OCR after 3 days, followed by decreased baseline and glucose stimulated OCR after 5 days. These changes were associated with lower islet ATP levels, impaired glucose-induced ATP generation, a trend for reduced mitochondrial DNA content and reduced expression of mitochondrial transcription factor A (Tfam). We discovered the accumulation of carnitine esters of hydroxylated long chain FAs, in particular 3-hydroxytetradecenoyl-carnitine. CONCLUSIONS: As long chain 3-hydroxylated FA metabolites are known to uncouple heart and brain mitochondria [53], [54], [55], we propose that under glucolipotoxic condition, unsaturated hydroxylated long-chain FAs accumulate, uncouple and ultimately inhibit ß-cell respiration. This leads to the slow deterioration of mitochondrial function progressing to bioenergetics ß-cell failure.

Apolipoprotein E (APOE) genotype regulates body weight and fatty acid utilization-Studies in gene-targeted replacement mice.

SCOPE: Of the three human apolipoprotein E (APOE) alleles, the ε3 allele is most common, which may be a result of adaptive evolution. In this study, we investigated whether the APOE genotype affects body weight and energy metabolism through regulation of fatty acid utilization. METHODS AND RESULTS: Targeted replacement mice expressing the human APOE3 were significantly heavier on low- and high-fat diets compared to APOE4 mice. Particularly on high-fat feeding, food intake and dietary energy yields as well as fat mass were increased in APOE3 mice. Fatty acid mobilization determined as activation of adipose tissue lipase and fasting plasma nonesterified fatty acid levels were significantly lower in APOE3 than APOE4 mice. APOE4 mice, in contrast, exhibited higher expression of proteins involved in fatty acid oxidation in skeletal muscle. CONCLUSION: Our data suggest that APOE3 is associated with the potential to more efficiently harvest dietary energy and to deposit fat in adipose tissue, while APOE4 carriers tend to increase fatty acid mobilization and utilization as fuel substrates especially under high-fat intake. The different handling of dietary energy may have contributed to the evolution and worldwide distribution of the ε3 allele.

Triglyceride Mobilization from Lipid Droplets Sustains the Anti-Steatotic Action of Iodothyronines in Cultured Rat Hepatocytes.

Adipose tissue, dietary lipids and de novo lipogenesis are sources of hepatic free fatty acids (FFAs) that are stored in lipid droplets (LDs) as triacylglycerols (TAGs). Destiny of TAGs stored in LDs is determined by LD proteomic equipment. When adipose triglyceride lipase (ATGL) localizes at LD surface the lipid mobilization is stimulated. In this work, an in vitro model of cultured rat hepatocytes mimicking a mild steatosis condition was used to investigate the direct lipid-lowering action of iodothyronines, by focusing, in particular, on LD-associated proteins, FFA oxidation and lipid secretion. Our results demonstrate that in "steatotic" hepatocytes iodothyronines reduced the lipid excess through the recruitment of ATGL on LD surface, and the modulation of the LD-associated proteins Rab18 and TIP47. As an effect of ATGL recruitment, iodothyronines stimulated the lipid mobilization from LDs then followed by the up-regulation of carnitine-palmitoyl-transferase (CPT1) expression and the stimulation of cytochrome-c oxidase (COX) activity that seems to indicate a stimulation of mitochondrial function. The lipid lowering action of iodothyronines did not depend on increased TAG secretion. On the basis of our data, ATGL could be indicated as an early mediator of the lipid-lowering action of iodothyronines able to channel hydrolyzed FFAs toward mitochondrial beta-oxidation rather than secretion.

Favorable outcome after physiologic dose of sodium-D,L-3-hydroxybutyrate in severe MADD.

Multiple acyl coenzyme A dehydrogenase deficiency (MADD) is a severe inborn error of metabolism. Experiences with sodium-D,L-3-hydroxybutyrate (3-HB) treatment are limited although positive; however, the general view on outcome of severely affected patients with MADD is relatively pessimistic. Here we present an infant with MADD in whom the previously reported dose of 3-HB did not prevent the acute, severe, metabolic decompensation or progressive cardiomyopathy in the subsequent months. Only after a physiologic dose of 2600 mg/kg of 3-HB per day were ketone bodies detected in blood associated with improvement of the clinical course, N-terminal prohormone of brain natriuretic peptide and echocardiographic parameters. Long-term studies are warranted on 3-HB treatment in patients with MADD.