Inactivation of the peroxisomal multifunctional protein-2 in mice impedes the degradation of not only 2-methyl-branched fatty acids and bile acid intermediates but also of very long chain fatty acids

According to current views, peroxisomal beta-oxidation is organized as two parallel pathways: the classical pathway that is responsible for the degradation of straight chain fatty acids and a more recently identified pathway that degrades branched chain fatty acids and bile acid intermediates. Multifunctional protein-2 (MFP-2), also called d-bifunctional protein, catalyzes the second (hydration) and third (dehydrogenation) reactions of the latter pathway. In order to further clarify the physiological role of this enzyme in the degradation of fatty carboxylates, MFP-2 knockout mice were generated. MFP-2 deficiency caused a severe growth retardation during the first weeks of life, resulting in the premature death of one-third of the MFP-2(-/-) mice. Furthermore, MFP-2-deficient mice accumulated VLCFA in brain and liver phospholipids, immature C(27) bile acids in bile, and, after supplementation with phytol, pristanic and phytanic acid in liver triacylglycerols. These changes correlated with a severe impairment of peroxisomal beta-oxidation of very long straight chain fatty acids (C(24)), 2-methyl-branched chain fatty acids, and the bile acid intermediate trihydroxycoprostanic acid in fibroblast cultures or liver homogenates derived from the MFP-2 knockout mice. In contrast, peroxisomal beta-oxidation of long straight chain fatty acids (C(16)) was enhanced in liver tissue from MFP-2(-/-) mice, due to the up-regulation of the enzymes of the classical peroxisomal beta-oxidation pathway. The present data indicate that MFP-2 is not only essential for the degradation of 2-methyl-branched fatty acids and the bile acid intermediates di- and trihydroxycoprostanic acid but also for the breakdown of very long chain fatty acids.     

Depletion of ceramides with very long chain fatty acids causes defective skin permeability barrier function, and neonatal lethality in ELOVL4 deficient mice

Very long chain fatty acids (VLCFA), either free or as components of glycerolipids and sphingolipids, are present in many organs. Elongation of very long chain fatty acids-4 (ELOVL4) belongs to a family of 6 members of putative fatty acid elongases that are involved in the formation of VLCFA. Mutations in ELOVL4 were found to be responsible for an autosomal dominant form of Stargardt's-like macular dystrophy (STGD3) in human. We have previously disrupted the mouse Elovl4 gene, and found that Elovl4+/- mice were developmentally normal, suggesting that haploinsufficiency of ELOVL4 is not a cause for the juvenile retinal degeneration in STGD3 patients. However, Elovl4-/- mice died within several hours of birth for unknown reason(s). To study functions of ELOVL4 further, we have explored the causes for the postnatal lethality in Elovl4-/- mice. Our data indicated that the mutant mice exhibited reduced thickness of the dermis, delayed differentiation of keratinocytes, and abnormal structure of the stratum corneum. We showed that all Elovl4-/- mice exhibited defective skin water permeability barrier function, leading to the early postnatal death. We further showed that the absence of ELOVL4 results in depletion in the epidermis of ceramides with omega-hydroxy very long chain fatty acids (> or = C28) and accumulation of ceramides with non omega-hydroxy fatty acids of C26, implicating C26 fatty acids as possible substrates of ELOVL4. These data demonstrate that ELOVL4 is required for VLCFA synthesis that is essential for water permeability barrier function of skin.     

Hormone-sensitive lipase deficiency disturbs the fatty acid composition of mouse testis

Hormone-sensitive lipase (HSL) is a key enzyme in the mobilization of fatty acids from intracellular stores. In mice, HSL deficiency results in male sterility caused by a major defect in spermatogenesis. The testes contain high concentrations of PUFA and specific PUFA are essential for spermatogenesis. We investigated the fatty acid composition and the mRNA levels of key enzymes involved in fatty acid metabolism in testis of HSL-knockout mice. HSL deficiency altered fatty acid composition in the testis but not in plasma. The most important changes were decreases in the essential n-6 PUFA LNA and the n-3 PUFA ALA, and an increase in the corresponding synthesis intermediates C22:4n-6 and C22:5n-3 without changes in DPAn-6 or DHA acids. Mead acid, which has been associated with an essential fatty acid deficit leading to male infertility, was increased in the testis from HSL-knockout mice. Moreover, the expression of SCD-1, FADS1, and FADS2 was increased while expression of ELOVL2, an essential enzyme for the formation of very-long PUFA in testis, was decreased. Given the indispensability of these fatty acids for spermatogenesis, the changes in fatty acid metabolism observed in testes from HSL-knockout male mice may underlie the infertility of these animals.     

Cerebral Developmental Abnormalities in a Mouse with Systemic Pyruvate Dehydrogenase Deficiency

Pyruvate dehydrogenase (PDH) complex (PDC) deficiency is an inborn error of pyruvate metabolism causing a variety of neurologic manifestations. Systematic analyses of development of affected brain structures and the cellular processes responsible for their impairment have not been performed due to the lack of an animal model for PDC deficiency. METHODS: In the present study we investigated a murine model of systemic PDC deficiency by interrupting the X-linked Pdha1 gene encoding the α subunit of PDH to study its role on brain development and behavioral studies. RESULTS: Male embryos died prenatally but heterozygous females were born. PDC activity was reduced in the brain and other tissues in female progeny compared to age-matched control females. Immunohistochemical analysis of several brain regions showed that approximately 40% of cells were PDH⁻. The oxidation of glucose to CO₂ and incorporation of glucose-carbon into fatty acids were reduced in brain slices from 15 day-old PDC-deficient females. Histological analyses showed alterations in several structures in white and gray matters in 35 day-old PDC-deficient females. Reduction in total cell number and reduced dendritic arbors in Purkinje neurons were observed in PDC-deficient females. Furthermore, cell proliferation, migration and differentiation into neurons by newly generated cells were reduced in the affected females during pre- and postnatal periods. PDC-deficient mice had normal locomotor activity in a novel environment but displayed decreased startle responses to loud noises and there was evidence of abnormal pre-pulse inhibition of the startle reflex. CONCLUSIONS: The results show that a reduction in glucose metabolism resulting in deficit in energy production and fatty acid biosynthesis impairs cellular differentiation and brain development in PDC-deficient mice.     

Increased L-CPT-1 activity and altered gene expression in pancreatic islets of malnourished adult rats: a possible relationship between elevated free fatty acid levels and impaired insulin secretion

Intrauterine growth restriction is associated with chronically elevated levels of serum fatty acids and reduced glucose-stimulated insulin secretion. Lipid metabolism in pancreatic beta cells is critical for the regulation of insulin secretion, and the chronic exposure to fatty acids results in higher palmitate oxidation rates and an altered insulin response to glucose. Using a rat model of isocaloric protein restriction, we examined whether pre- and postnatal protein malnutrition influences the properties of pancreatic islet carnitine palmitoyltransferase-1 (liver isoform, L-CPT-1), a rate-limiting enzyme that regulates fatty acid oxidation in mitochondria. The activity of L-CPT-1 in pancreatic islets increased in the low protein (LP), although the L-CPT-1 mRNA levels were unaffected by malnutrition. The susceptibility of enzyme to inhibition by malonyl-CoA was unaltered and the content of malonyl-CoA was reduced in LP cells. Because the mitochondrial oxidation of fatty acids is related to the altered expression of a number of genes encoding proteins involved in insulin secretion, the levels of expression of insulin and GLUT-2 mRNA were assessed. A reduced expression of both genes was observed in malnourished rats. These results provide further evidence that increased L-CPT-1 activity and changes in gene expression in pancreatic islets may be involved in the reduced insulin secretion seen in malnourished rats.     

Postnatal essential fatty acid deficiency in mice affects lipoproteins, hepatic lipids, fatty acids and mRNA expression

We have previously reported that essential fatty acid deficiency (EFAD) during suckling in mice resulted in an adult lean phenotype and a resistance to diet-induced obesity. We now hypothesized that postnatal EFAD would cause long-term effects on lipid metabolism. C57BL/6 mice were fed an EFAD or a control diet from the 16th day of gestation and throughout lactation. The pups were weaned to standard diet (STD) and at 15 weeks of age given either high fat diet (HFD) or STD. Lipoprotein profiles, hepatic lipids, fatty acids and mRNA expression were analyzed in 3-week-old and 25-week-old offspring. At weaning, the EFAD pups had higher cholesterol levels in both plasma and liver and 6-fold higher concentrations of hepatic cholesterol esters than control pups. Adult EFAD offspring had higher levels of hepatic cholesterol and linoleic acid, but lower levels of dihomo-γ-linolenic acid and Pparg mRNA expression in the liver. In addition, HFD fed EFAD offspring had lower plasma total cholesterol, lower hepatic triglycerides and lower liver weight compared to controls fed HFD. In conclusion, early postnatal EFAD resulted in short-term alterations with increased hepatic cholesterol accumulation and long-term protection against diet-induced liver steatosis and hypercholesterolemia.     

Dietary effects on brain fatty acid composition: the reversibility of n-3 fatty acid deficiency and turnover of docosahexaenoic acid in the brain, erythrocytes, and plasma of rhesus monkeys

Rhesus monkeys given pre- and postnatal diets deficient in n-3 essential fatty acids develop low levels of docosahexaenoic acid (22:6 n-3, DHA) in the cerebral cortex and retina and impaired visual function. This highly polyunsaturated fatty acid is an important component of retinal photoreceptors and brain synaptic membranes. To study the turnover of polyunsaturated fatty acids in the brain and the reversibility of n-3 fatty acid deficiency, we fed five deficient juvenile rhesus monkeys a fish oil diet rich in DHA and other n-3 fatty acids for up to 129 weeks. The results of serial biopsy samples of the cerebral cortex indicated that the changes of brain fatty acid composition began as early as 1 week after fish oil feeding and stabilized at 12 weeks. The DHA content of the phosphatidylethanolamine of the frontal cortex increased progressively from 3.9 +/- 1.2 to 28.4 +/- 1.7 percent of total fatty acids. The n-6 fatty acid, 22:5, abnormally high in the cerebral cortex of n-3 deficient monkeys, decreased reciprocally from 16.2 +/- 3.1 to 1.6 +/- 0.4%. The half-life (t 1/2) of DHA in brain phosphatidylethanolamine was estimated to be 21 days. The fatty acids of other phospholipids in the brain (phosphatidylcholine, -serine, and -inositol) showed similar changes. The DHA content of plasma and erythrocyte phospholipids also increased greatly, with estimated half-lives of 29 and 21 days, respectively. We conclude that monkey cerebral cortex with an abnormal fatty acid composition produced by dietary n-3 fatty acid deficiency has a remarkable capacity to change its fatty acid content after dietary fish oil, both to increase 22:6 n-3 and to decrease 22:5 n-6 fatty acids. The biochemical evidence of n-3 fatty acid deficiency was completely corrected. These data imply a greater lability of the fatty acids of the phospholipids of the cerebral cortex than has been hitherto appreciated.     

Ablation of steroid receptor coactivator-3 resembles the human CACT metabolic myopathy

Oxidation of lipid substrates is essential for survival in fasting and other catabolic conditions, sparing glucose for the brain and other glucose-dependent tissues. Here we show Steroid Receptor Coactivator-3 (SRC-3) plays a central role in long chain fatty acid metabolism by directly regulating carnitine/acyl-carnitine translocase (CACT) gene expression. Genetic deficiency of CACT in humans is accompanied by a constellation of metabolic and toxicity phenotypes including hypoketonemia, hypoglycemia, hyperammonemia, and impaired neurologic, cardiac and skeletal muscle performance, each of which is apparent in mice lacking SRC-3 expression. Consistent with human cases of CACT deficiency, dietary rescue with short chain fatty acids drastically attenuates the clinical hallmarks of the disease in mice devoid of SRC-3. Collectively, our results position SRC-3 as a key regulator of β-oxidation. Moreover, these findings allow us to consider platform coactivators such as the SRCs as potential contributors to syndromes such as CACT deficiency, previously considered as monogenic.     

Lipid and cell metabolic changes associated with essential fatty acid enrichment of articular chondrocytes

Observations of impaired chondrocyte metabolism in essential fatty acid (EFA) deficiency as well as EFA protection against development of osteoarthrosis in inbred mice suggest the existence of a relationship between EFA, chondrocyte metabolism, and cartilage degeneration. To explore this relationship further, the fatty acid content of lipids in normal fetal bovine chondrocytes was manipulated by in vitro exposure to media supplemented with 100 microM arachidonic acid (20:4) or oleic acid (18:1). Chondrocytes rapidly and differentially incorporated both fatty acids into their lipid pools. The predominant acceptor was triacylglycerols. A 980% enrichment of arachidonic acid was associated with increased concentrations of fatty acids, increased 35SO4 and [3H]proline incorporation into matrix macromolecules (170% and 54-103%, respectively), and a 24-fold elevation in chondrocyte prostaglandin synthesis. No metabolic effects elicited observed in cells enriched by 377% with 18:1 oleic acid. The metabolic effects elicited by 20:4 arachidonic acid were abolished by pretreatment of cells with indomethacin, suggesting that the cellular responses to essential fatty acid loading may be associated with induced increases in prostaglandin synthesis. The data indicate that excessive in vitro accumulation of arachidonic acid is associated with an increase in synthetic activity that is causally related to increased prostaglandin synthesis and elevated levels of cellular fatty acids.     

Emerging role of mammalian autophagy in ketogenesis to overcome starvation

Autophagy is essential for the survival of lower organisms under conditions of nutrient depletion. However, whether autophagy plays a physiological role in mammals experiencing starvation is unknown. Ketogenesis is critical for overcoming starvation in mammals. We recently revealed that hepatic and renal autophagy are involved in starvation-induced ketogenesis, by utilizing tissue-specific autophagy-deficient mouse models. The liver is the principal organ to regulate ketogenesis, and a deficiency of liver-specific autophagy partially but significantly attenuates starvation-induced ketogenesis. While deficiency of renal-specific autophagy does not affect starvation-induced ketogenesis, mice with deficiency of both liver and kidney autophagy have even lower blood ketone levels and physical activity under starvation conditions than those lacking autophagy in the liver alone. These results suggest that the kidney can compensate for impaired hepatic ketogenesis. Since ketone bodies are catabolized from fatty acids, the uptake of fatty acids, the formation of intracellular lipid droplets, and fatty acid oxidation are critical for ketogenesis. We found that starvation-induced lipid droplet formation is impaired in autophagy-deficient organs. Thus, hepatic and renal autophagy are required for starvation-induced ketogenesis. This process is essential for maintaining systemic energy homeostasis and physical activity during starvation. Our findings provide a novel insight into mammalian autophagy and the physiology of starvation.     

Dietary long-chain fatty acids and visual response in malnourished nursing infants

Polyunsaturated fatty acids (PUFAs) derived from essential fatty acids (EFAs) play an important role in prenatal visual and neural development. Protein-energy malnutrition affects PUFA supply, and hence the synthesis of structural lipids during growth. Recently, some physiological studies reported abnormalities in the visual function of formula-fed infants relative to breast-fed infants. The purpose of our study was to assess whether fatty acid composition of the malnourished infant diet modifies the visual function and erythrocyte phospholipid fatty acid composition. Three groups of full-term malnourished infants were selected. Two groups received commercial formulas. One of them supplied linoleic and alpha -linolenic acid: Formula I (FI), and the other supplied, in addition, long-chain PUFAs from n-3 and n-6 series: Formula II (FII). A reference group of breast-fed infants was also enrolled. Visual function was assessed using full-field flash electroretinography, and the erythrocyte phospholipid fatty acid composition was determined by gas-liquid chromatography. Those infants receiving the supplemented formula (FII) exhibited a similar retinal function to that of breast-fed infants. However, normal results were not achieved when infants were fed on the FI formula. In all groups, the results were correlated with the proportion of docosahexaenoic acid in erythrocyte phospholipid fatty acid composition. We conclude that in malnourished infants a nutrient formula enriched with long-chain fatty acids of n-6 and n-3 series could be helpful to achieve an erythrocyte fatty acid pattern and a visual function similar to that obtained in breast-fed infants.     

Exposure to omega-3 fatty acids at early age accelerate bone growth and improve bone quality

Omega-3 fatty acids (FAs) are essential nutritional components that must be obtained from foods. Increasing evidence validate that omega-3 FAs are beneficial for bone health, and several mechanisms have been suggested to mediate their effects on bone, including alterations in calcium absorption and urinary calcium loss, prostaglandin synthesis, lipid oxidation, osteoblast formation and inhibition of osteoclastogenesis. However, to date, there is scant information regarding the effect of omega-3 FAs on the developing skeleton during the rapid growth phase. In this study we aim to evaluate the effect of exposure to high levels of omega-3 FAs on bone development and quality during prenatal and early postnatal period. For this purpose, we used the fat-1 transgenic mice that have the ability to convert omega-6 to omega-3 fatty acids and the ATDC5 chondrogenic cell line as models. We show that exposure to high concentrations of omega-3 FAs at a young age accelerates bone growth through alterations of the growth plate, associated with increased chondrocyte proliferation and differentiation. We further propose that those effects are mediated by the receptors G-protein coupled receptor 120 (GPR120) and hepatic nuclear factor 4α, which are expressed by chondrocytes in culture. Additionally, using a combined study on the structural and mechanical bone parameters, we show that high omega-3 levels contribute to superior trabecular and cortical structure, as well as to stiffer bones and improved bone quality. Most interestingly, the fat-1 model allowed us to demonstrate the role of maternal high omega-3 concentration on bone growth during the gestation and postnatal period.     

Sensitivity to alcohol in mice with an altered brain fatty acid composition

Ethanol-induced sleep onset times, sleep times and blood alcohol levels upon awakening were measured in mice fed an essential fatty acid deficient, Purina Chow or unsaturated fat diet for nine months. These values in animals fed the essential fatty acid deficient and Purina Chow diets did not differ, but mice fed the unsaturated fat diet had longer sleep times and lower blood alcohol levels upon awakening than mice fed essential fatty acid deficient or Purina Chow diets. Crude brain mitochondrial fractions isolated from mice fed the essential fatty acid deficient diet had decreased levels of docosahexaenoic [22:6(n-3)] and increased levels of eicosatrienoic [20:3(n-9)], docosatrienoic [22:3(n-9)] and docosapentaenoic [22:5(n-6)] acids compared to mice fed the Purina Chow diet. The unsaturated fat diet decreased 22:6(n-3) and increased 22:5(n-6) compared to the Purina Chow dietary regimen. The longer sleep times and lower blood alcohol levels found in mice fed the unsaturated fat diet probably resulted from an artifact due to the obesity of the mice fed this diet and from the hinderance of obesity to the righting reflex (our measure of ethanol potency). We conclude that the alteration of several polyunsaturated fatty acid components in the brain has little or no influence on the sensitivity of the nervous system to alcohol.     

Nutrition and infectious diseases in developing countries and problems of acquired immunodeficiency syndrome

Infectious diseases are the major causes of death and morbidity in underdeveloped countries, particularly in children. Increasing evidence suggests that malnutrition-both Protein-Energy type Malnutrition (PEM) and essential micronutrient (vitamins, trace minerals, essential amino acids, polyunsaturated fatty acids) type-is the underlying reason for increased susceptibility to infections. On the other hand, certain infectious diseases also cause malnutrition, which results in a vicious cycle. Before its viral origin was known, acquired immunodeficiency syndrome (AIDS) had been termed the thin disease because cachexia was AIDS' main clinical manifestation. The relationship between infection and malnutrition is well documented in the literature. Our experience supports this. Preventive and therapeutic measures are suggested.     

Ultrastructural changes in the neuropile of the sensorimotor cortex during long-term protein-calorie deficiency

The effect of alimentary (protein-caloric) deficiency on the brain sensomotor cortex of mice was studied during their postnatal development. It was found that malnutrition of mice from day 10 to day 40 of postnatal period brought about the most remarkable changes in synaptic junctions located on the dendritic spines. They manifested destruction of the spine apparatus, reduction of the width of synaptic slits and of postsynaptic membranes. Dystrophic and destructive alterations are frequently encountered in dendrites, axon terminals, and myelinized axons, indicating the reduced compensatory functions of the CNS.     

Gestational zinc deficiency amplifies the regulation of metallothionein induction in adult mice

To determine if prenatal zinc deficiency has a persistent effect on metallothionein (MT) regulation, Swiss-Webster mice were mated and fed a diet containing either control (100 micrograms Zn/g) or low levels of zinc (5 micrograms Zn/g) from Day 7 of gestation to parturition. After birth all mice were given the control diet. Liver zinc and MT levels were 50% lower in newborn pups from dams fed the low zinc diets than in control pups. In control pups, liver zinc and MT concentrations were relatively stable during the first week of postnatal life. In contrast, in pups prenatally deprived of zinc, liver levels of zinc and MT increased such that by Day 3 of postnatal life, the levels were not significantly different from controls. At Day 56, serum IgM concentrations were significantly lower in the low zinc offspring. Liver zinc concentrations in the two groups of mice were similar at Day 70 postnatal, and in both groups liver MT levels were below detection limits. However, when Day 70 mice were given zinc injections to stimulate MT synthesis, the prenatally zinc deprived offspring showed markedly higher liver MT levels than did control mice given similar injections, despite similar liver zinc concentrations in the two groups. These results show that prenatal zinc deficiency has pronounced effects on postnatal MT metabolism which can persist into adulthood.     

Associations of protein 4.2 with band 3 and ankyrin

It was shown previously that the intestinal fatty acid binding protein (I-FABP) is not essential for the absorption of dietary fat. One notable feature of I-FABP deficiency was the enhancement of body weight gain in male mice but not in female mice. To explore a possible cause for this gender dimorphic effect, we examined the changes in expression of genes that encode liver fatty acid binding protein (L-FABP) and ileal lipid binding protein in the small intestine resulting from I-FABP deficiency. The results indicate that both L-FABP and ilbp levels are modestly increased in the small intestine of chow-fed mice lacking I-FABP. There was no discernible alteration of overall morphology or histology in the small intestine but changes in liver histology were evident in I-FABP deficient male mice. Glucose tolerance was also investigated in aged mice. I-FABP deficiency had no effect on glucose tolerance in male mice but it appeared to be improved in female mice. Thus, male and female mice clearly respond differently to the loss of I-FABP from the small intestine but the observed changes in the abundance of L-FABP and ilbp protein do not readily account for this phenomenon. (Mol Cell Boichem xxx: 1–8, 2005)     

Maternal Vitamin C Deficiency during Pregnancy Persistently Impairs Hippocampal Neurogenesis in Offspring of Guinea Pigs

While having the highest vitamin C (VitC) concentrations in the body, specific functions of VitC in the brain have only recently been acknowledged. We have shown that postnatal VitC deficiency in guinea pigs causes impairment of hippocampal memory function and leads to 30% less neurons. This study investigates how prenatal VitC deficiency affects postnatal hippocampal development and if any such effect can be reversed by postnatal VitC repletion. Eighty pregnant Dunkin Hartley guinea pig dams were randomized into weight stratified groups receiving High (900 mg) or Low (100 mg) VitC per kg diet. Newborn pups (n = 157) were randomized into a total of four postnatal feeding regimens: High/High (Control); High/Low (Depleted), Low/Low (Deficient); and Low/High (Repleted). Proliferation and migration of newborn cells in the dentate gyrus was assessed by BrdU labeling and hippocampal volumes were determined by stereology. Prenatal VitC deficiency resulted in a significant reduction in postnatal hippocampal volume (P<0.001) which was not reversed by postnatal repletion. There was no difference in postnatal cellular proliferation and survival rates in the hippocampus between dietary groups, however, migration of newborn cells into the granular layer of the hippocampus dentate gyrus was significantly reduced in prenatally deficient animals (P<0.01). We conclude that a prenatal VitC deficiency in guinea pigs leads to persistent impairment of postnatal hippocampal development which is not alleviated by postnatal repletion. Our findings place attention on a yet unrecognized consequence of marginal VitC deficiency during pregnancy.     

Mitochondrial long chain fatty acid β-oxidation in man and mouse

Several mouse models for mitochondrial fatty acid β-oxidation (FAO) defects have been developed. So far, these models have contributed little to our current understanding of the pathophysiology. The objective of this study was to explore differences between murine and human FAO. Using a combination of analytical, biochemical and molecular methods, we compared fibroblasts of long chain acyl-CoA dehydrogenase knockout (LCAD^−/−), very long chain acyl-CoA dehydrogenase knockout (VLCAD^−/−) and wild type mice with fibroblasts of VLCAD-deficient patients and human controls. We show that in mice, LCAD and VLCAD have overlapping and distinct roles in FAO. The absence of VLCAD is apparently fully compensated, whereas LCAD deficiency is not. LCAD plays an essential role in the oxidation of unsaturated fatty acids such as oleic acid, but seems redundant in the oxidation of saturated fatty acids. In strong contrast, LCAD is neither detectable at the mRNA level nor at the protein level in men, making VLCAD indispensable in FAO. Our findings open new avenues to employ the existing mouse models to study the pathophysiology of human FAO defects.