Search

Hepatic lipidosis in dairy cows is the result of a disturbed balance between the uptake of non-esterified fatty acids (NEFA), their metabolism in the hepatocytes, and the limited efflux of TG as very-low-density lipoprotein (VLDL). Lipidosis and the associated risk for ketosis represents a consequence of selecting dairy cows primarily for milk production without considering the basic physiological mechanisms of this trait. The overall risk for lipidosis and ketosis possesses a genetic background and the recently released new breeding value of the German Holstein Friesian cows now sets the path for correction of this risk and in that confirms the assumed genetic threat. Ectopic fat deposition in the liver is the result of various steps including lipolysis, uptake of fat by the liver cell, its metabolism, and finally release as very-low-density lipoprotein (VLDL). These reactions may be modulated directly or indirectly and hence, serve as basis for prophylactic measures. The pertaining methods are described in order to support an improved understanding of the pathogenesis of lipidosis and ketosis. They consist of feeding a glucogenic diet, restricted feeding during the close-up time as well as supplementation with choline, niacin, carnitine, or the reduction of milking frequency. Prophylactic measures for the prevention of ketosis are also included in this discussion., Competing Interests: Es liegt kein Interessenkonflikt vor., (Thieme. All rights reserved.)

Objectives: The aim of this study was to evaluate if de novo hepatic lipid synthesis contributes to fatty acid overload in the liver of cats with feline hepatic lipidosis (FHL)., Methods: Lipogenic gene expression of peroxisome proliferator-activated receptor-alpha ( PPAR-α ), peroxisome proliferator-activated receptor-gamma ( PPAR-γ ), fatty acid synthase ( FASN ) and sterol regulatory element-binding factor ( SREBF1 ) were evaluated using quantitative RT-PCR in liver tissue of six cats with FHL and compared with the liver tissue of eight healthy cats., Results: In liver tissue, PPAR-α, PPAR-γ and FASN mRNA expression levels were not significantly different ( P >0.12, P >0.89 and P >0.5, respectively) in the FHL group compared with the control group. SREBF1 gene expression was downregulated around 10-fold in the FHL group vs the control group ( P = 0.039)., Conclusions and Relevance: The downregulation of SREBF1 in the liver tissue of cats with FHL does not support the hypothesis that de novo lipogenesis in the liver is an important pathway of fatty acid accumulation in FHL.

Background: AMP-activated protein kinase (AMPK), particularly AMPKα2 isoform, plays a critical role in maintaining cardiac homeostasis. It was reported that sulforaphane (SFN) prevented type 2 diabetes (T2D)-induced cardiomyopathy accompanied by the activation of AMPK; In this study, AMPK's pivotal role in SFN-mediated prevention against T2D-induced cardiomyopathy was tested using global deletion of AMPKα2 gene (AMPKα2-KO) mice., Methods and Results: T2D was established by feeding 3-month high-fat diet (HFD) to induce insulin resistance, followed by an intraperitoneal injection of streptozotocin (STZ) to induce mild hyperglycemia in both AMPKα2-KO and wild-type (WT) mice. Then both T2D and control mice were subsequently treated with or without SFN for 3 months while continually feeding HFD or normal diet. Upon completion of the 3-month treatment, five mice from each group were sacrificed as a 3-month time-point (3 M). The rest continued normal diet or HFD until terminating study at the sixth month (6 M) of diabetes. Cardiac function was examined with echocardiography before sacrifice at both 3 M and 6 M. SFN prevented T2D-induced progression of cardiac dysfunction, remodeling (hypertrophy and fibrosis), inflammation, and oxidative damage in wild-type diabetic mice, but not in AMPKα2-KO mice. Mechanistically, SFN prevented T2D-induced cardiomyopathy not only by improving AMPK-mediated lipid metabolic pathways, but also enhancing NRF2 activation via AMPK/AKT/GSK3β pathway. However, these improving effects of SFN were abolished in AMPKα2-KO diabetic mice., Conclusions: AMPK is indispensable for the SFN-induced prevention of cardiomyopathy in T2D, and the activation of NRF2 by SFN is mediated by AMPK/AKT/GSK3β signaling pathways., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Background: Non-alcoholic fatty liver disease (NAFLD) is the most common chronic liver disease, and its pathogenesis and mechanism are intricate. In the present study, we aimed to evaluate the role of PPAR δ in LPS associated NAFLD and to investigate the signal transduction pathways underlying PPAR δ treatment in vitro. Material and Methods: L02 cells were exposed to palmitic acid (PA) and/or LPS in the absence or presence of PPAR δ inhibition and/or activation. Results: LPS treatment markedly increased lipid deposition, FFA contents, IL-6 and TNF-α levels, and cell apoptosis in PA treatment (NAFLD model). PPAR δ inhibition protects L02 cells against LPS-induced lipidosis and injury. Conversely, the result of PPAR δ activation showed the reverse trend. LPS+PA treatment group significantly decreases the relative expression level of IRS-1, PI3K, AKT, phosphorylation of AKT, TLR-4, MyD88, phosphorylation of IKKα, NF-κB, Bcl-2 and increases the relative expression level of Bax, cleaved caspase 3 and cleaved caspase 8, compared with the cells treated with NAFLD model. PPAR δ inhibition upregulated the related proteins' expression level in insulin resistance and inflammation pathway and downregulated apoptotic relevant proteins. Instead, PPAR δ agonist showed the reverse trend. Conclusion: Our data show that PPAR δ inhibition reduces steatosis, inflammation and apoptosis in LPS-related NAFLD damage, in vitro. PPAR δ may be a potential therapeutic implication for NAFLD., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)

Phospholipidosis is a metabolic disorder characterized by intracellular accumulation of phospholipids. It can be caused by short-term or chronic exposure to cationic amphiphilic drugs (CADs). These compounds bind to phospholipids, leading to inhibition of their degradation and consequently to their accumulation in lysosomes. Drug-induced phospholipidosis (DIPL) is frequently at the basis of discontinuation of drug development and post-market drug withdrawal. Therefore, reliable human-relevant in vitro models must be developed to speed up the identification of compounds that are potential inducers of phospholipidosis. Here, hepatic cells derived from human skin (hSKP-HPC) were evaluated as an in vitro model for DIPL. These cells were exposed over time to amiodarone, a CAD known to induce phospholipidosis in humans. Transmission electron microscopy revealed the formation of the typical lamellar inclusions in the cell cytoplasm. Increase of phospholipids was already detected after 24 h exposure to amiodarone, whereas a significant increase of neutral lipid vesicles could be observed after 72 h. At the transcriptional level, the modulation of genes involved in DIPL was detected. These results provide a valuable indication of the applicability of hSKP-HPC for the quick assessment of drug-induced phospholipidosis in vitro, early in the drug development process., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Apolipoprotein E deficiency (ApoE(-/-)) combined with a high-fat Western-type diet (WD) is known to activate the toll-like receptor (TLR4) pathway and promote atherosclerosis. However, to date, the pathogenic effects of these conditions on the lung have not been extensively studied. Therefore, the present study examined the effects of ApoE(-/-) and a WD on lung injury and investigated the underlying mechanisms. ApoE(-/-) and wild-type mice were fed a WD or normal chow diet for 4, 12 and 24 weeks. Lung inflammation, lung cholesterol content and cytokines profiles in broncho-alveolar lavage fluid (BALF) were determined. TLR4 and its main downstream molecules were analyzed with western blot analysis. In addition, the role of the TLR4 pathway was further validated using TLR4-targeted gene silencing. The results showed that ApoE(-/-) mice developed lung lipidosis following 12 weeks of receiving a WD, as evidenced by an increased lung cholesterol content. Moreover, dependent on the time period of receiving the diet, those mice exhibited pulmonary inflammation, which was manifested by initial leukocyte recruitment (at 4 weeks), by increased alveolar septal thickness and mean linear intercept as well as elevated production of inflammation mediators (at 12 weeks), and by granuloma formation (at 24 weeks). The expression levels of TLR4, myeloid differentiation primary response 88 (MyD88) and nuclear factor kappa B were markedly upregulated in ApoE(-/-) WD mice at week 12. However, these effects were ameliorated by shRNA-mediated knockdown of TLR4. By contrast, ApoE(-/-) ND or wild-type WD mice exhibited low-grade or no inflammation and mild lipidosis. The levels of TLR4 and MyD88 in those mice showed only minor changes. In conclusion, ApoE deficiency acts synergistically with a WD to trigger lung lipidosis and inflammation at least in part via TLR4 signaling.

Phospholipidosis (PLD) is characterized by an intracellular accumulation of phospholipids in lysosomes and concurrent development of concentric lamellar bodies. It is induced in humans and in animals by drugs with a cationic amphiphilic structure. The purpose of the present study was to identify a set of molecular biomarkers of PLD in rat blood and heart, hypotheticallya pplicable in preclinical screens within the drug development process. A toxicological study was set up in rats orally treated up to 11 days with 300 mg kg(–1) per day(–1) amiodarone (AMD). Light and transmission electron microscopy investigations were performed to confirm the presence of lamellar bodies indicative of phospholipid accumulation. The effects of AMD upon the transcriptome of these tissues were estimated using DNA microarray technology. Microarray data analysis showed that a total of 545 and 8218 genes were modulated by AMD treatment in heart and blood, respectively. Some genes implicated in the phospholipid accumulation in cells, such as phospholipase A2, showed similar alterations of gene expression. After transcriptome criteria of analysis and target selection, including also the involvement in the onset of PLD, 7 genes (Pla2g2a, Pla2g7, Gal, Il1b, Cebpb, Fcgr2b, Acer 2) were selected as candidate biomarkers of PLD in heart and blood tissues, and their potential usefulness as a sensitive screening test was screened and confirmed by quantitative Real-Time PCR analysis. Collectively, these data underscore the importance of transcriptional profiling in drug discovery and development, and suggest blood as a surrogate tissue for possible phospholipid accumulation in cardiomyocytes.

Enterocytes are the only cell type that must balance the de novo synthesis and absorption of cholesterol, although the coordinate regulation of these processes is not well understood. Our previous studies demonstrated that enterocytes respond to the pharmacological blockade of cholesterol absorption by ramping up de novo sterol synthesis through activation of sterol regulatory element-binding protein-2 (SREBP-2). Here, we genetically disrupt both Insig1 and Insig2 in the intestine, two closely related proteins that are required for the feedback inhibition of SREBP and HMG-CoA reductase (HMGR). This double knock-out was achieved by generating mice with an intestine-specific deletion of Insig1 using Villin-Cre in combination with a germ line deletion of Insig2. Deficiency of both Insigs in enterocytes resulted in constitutive activation of SREBP and HMGR, leading to an 11-fold increase in sterol synthesis in the small intestine and producing lipidosis of the intestinal crypts. The intestine-derived cholesterol accumulated in plasma and liver, leading to secondary feedback inhibition of hepatic SREBP2 activity. Pharmacological blockade of cholesterol absorption was unable to further induce the already elevated activities of SREBP-2 or HMGR in Insig-deficient enterocytes. These studies confirm the essential role of Insig proteins in the sterol homeostasis of enterocytes.

Introduction: Metabolically obese but normal-weight (MONW) individuals have metabolic features of overt obesity, and abdominal adiposity is common in them. Animal models of MONW individuals are lacking. We aimed to develop an abdominally obese and normal-weight (AONW) rat model., Methods and Results: Young male Sprague-Dawley rats were fed chow or a modified high-sucrose (HS) diet for 20 weeks. The HS diet induced increased visceral adipose tissue without increased body weight, reduced glucose disposal rates, and increased hepatic glucose output during the hyperinsulinemic-euglycemic clamp, increased plasma glucose during the intraperitoneal glucose tolerance test, and increased plasma free fatty acids. Hepatic lipidosis and hepatocyte mitochondria swelling were found in HS rats through light microscopy and transmission electron microscopy; similar impairments were not observed in muscle. RT-PCR showed that mRNA expression of uncoupling protein 3 and peroxisome proliferator-activated receptor-gamma coactivator 1α increased in muscle of HS rats, while expression of mitochondrial transcription factor A, glucose transporter type 4, and insulin receptor substrate-1 did not change significantly., Conclusion: AONW rats developed metabolic disorders seen in MONW individuals. Steatosis, mitochondrial morphologic changes, and insulin resistance were more serious in liver than in muscle. Genes involved in fatty acid metabolism and mitochondrial function changed in less impaired muscle.

Cytochrome P450 reductase (POR) is a microsomal electron transport protein essential to cytochrome P450-mediated drug metabolism and sterol and bile acid synthesis. The conditional deletion of hepatic POR gene expression in mice results in a marked decrease in plasma cholesterol levels counterbalanced by the accumulation of triglycerides in lipid droplets in hepatocytes. To evaluate the role of cholesterol and bile acid synthesis in this hepatic lipidosis, as well as the possible role of lipid transport from peripheral tissues, we developed a stable, small interfering RNA (siRNA)-mediated cell culture model for the suppression of POR. POR mRNA and protein expression were decreased by greater than 50% in McArdle-RH7777 rat hepatoma cells 10 days after transfection with a POR-siRNA expression plasmid, and POR expression was nearly completely extinguished by day 20. Immunofluorescent analysis revealed a marked accumulation of lipid droplets in cells by day 15, accompanied by a nearly 2-fold increase in cellular triglyceride content, replicating the lipidosis seen in hepatic POR-null mouse liver. In contrast, suppression of CYP51A1 (lanosterol demethylase) did not result in lipid accumulation, indicating that loss of cholesterol synthesis is not the basis for this lipidosis. Indeed, addition of cholesterol to the medium appeared to augment the lipidosis in POR-suppressed cells, whereas removal of lipids from the medium reversed the lipidosis. Oxysterols did not accumulate in POR-suppressed cells, discounting a role for liver X receptor in stimulating triglyceride synthesis, but addition of chenodeoxycholate significantly repressed lipid accumulation, suggesting that the absence of bile acids and loss of farnesoid X receptor stimulation lead to excessive triglyceride synthesis.

Lipid storage myopathies (LSMs) are characterized pathologically by the accumulation of lipid droplets in muscle fibers due to impaired cellular lipid metabolism. The purpose of this study was to determine etiologies and genetic mutations associated with LSMs in ethnic Han Taiwanese. The usefulness of the blood acylcarnitine (AC) profile for diagnosing LSMs in adult patients was also investigated. Nine patients were diagnosed with late-onset LSMs following a review of muscle biopsies and medical records and were recruited retrospectively. Genetic studies were performed to detect mutations in the SLC22A5 for primary carnitine deficiency, PNPLA2 for neutral lipid storage disease with myopathy, ABHD5 for neutral lipid storage disease with ichthyosis, ETFDH for multiple acyl-CoA dehydrogenation deficiency (MADD), and CPT2 for carnitine palmitoyltransferase II deficiency. Blood AC levels were measured by tandem mass spectrometry. The mutation c.250G>A in ETFDH was detected in seven (78%) patients, six of whom were homozygous for the variant. Patients with ETFDH mutations had elevated blood levels of ACs ranging from C8 to C16 species, a pattern consistent with MADD. ETFDH c.250G>A mutation is common in Taiwanese patients with late-onset LSMs. The blood AC profile is a sensitive biochemical marker for diagnosing MADD arising from ETFDH mutations in adults., (© 2010 John Wiley & Sons A/S.)

Lipolysis in adipocytes, the hydrolysis of triacylglycerol (TAG) to release fatty acids (FAs) and glycerol for use by other organs, is a unique function of white adipose tissue. Lipolysis in adipocytes occurs at the surface of cytosolic lipid droplets, which have recently gained much attention as dynamic organelles integral to lipid metabolism. Desnutrin/ATGL is now established as a bona fide TAG hydrolase and mutations in human desnutrin/ATGL/PNPLA2, as well as in its activator, comparative gene identification 58, are associated with Neutral Lipid Storage Disease. Furthermore, recent identification of AdPLA as the major adipose phospholipase A(2), has led to the discovery of a dominant autocrine/paracrine regulation of lipolysis through PGE(2). Here, we review emerging concepts in the key players in lipolysis and the regulation of this process. We also examine recent findings in mouse models and humans with alterations/mutations in genes involved in lipolysis and discuss activation of lipolysis in adipocytes as a potential therapeutic target., (2009 Elsevier Ltd. All rights reserved.)

Introduction: Multiple acyl-CoA dehydrogenase deficiency (MADD) is a rare genetic disease involving fatty acid oxidation. It is due to the deficiency of one of the two electron transporters: electron transfer flavoprotein (ETF) or electron transfer flavoprotein ubiquinone oxydoreductase (ETF-QO). Symptoms begin more often in childhood or in young adulthood with a multisystemic disease with encephalopathy or muscular weakness., Case Reports: We report here two adult cases with ETF-QO deficiency, confirmed by mutation analysis (ETFDH gene), revealed by a muscular weakness associated with muscle lipidosis. One of our patients presented an acute encephalopathy with vomiting ten years before the onset of muscular symptoms. The second patient exhibited a slowly progressive pelvic girdle muscle weakness. Diagnosis was established by characteristic abnormalities of acylcarnitine profile by tandem mass spectrometry. For both patients, a dramatic clinical improvement was observed under treatment with riboflavine and L-carnitine., Conclusion: Since it is a treatable disorder, this diagnosis must be considered by performing an acylcarnitine profile in all patients presenting with an unexplained muscular weakness., (Copyright (c) 2009 Elsevier Masson SAS. All rights reserved.)

Metabolic disorders of energy production characterise the group of rare, mainly autosomal recessively inherited metabolic muscular diseases which are often associated with multi-systemic symptoms. In this report, an update on the clinics, pathophysiology, pathomorphology and current treatment options of metabolic myopathies will be given. Beyond classic phenotypes of these disorders, one should be aware of oligosymptomatic patients who can be easily missed. The relevant gene mutations and the pathophysiology and pathomorphology they cause are now known for almost all these metabolic diseases. Establishing the correct diagnosis has become even more important since highly specific therapy options are now available for at least some of these inherited disorders, e.g. enzyme replacement therapy in Pompe disease.

X-linked genetic diseases include a wide range of disorders such as the dystrophinopathies. Additionally in some rare genetic diseases, severity of expression is gender dependent. Prevention of such disorders usually involves prenatal diagnosis and termination of affected pregnancies, while preimplantation genetic diagnosis (PGD) represents a specialized alternative that avoids pregnancy termination. To preclude the rejection of unaffected male embryos that cannot be differentiated from those affected when using fluorescence in-situ hybridization, a flexible protocol based on multiplex fluorescence polymerase chain reaction (PCR) was standardized and validated for gender determination in single cells, which can potentially incorporate any disease-specific locus. The final panel of nine loci included four loci on the Y chromosome, two on the X chromosome plus up to three microsatellite markers to either support the gender diagnosis or to further monitor extraneous contamination. The protocol, standardized on single lymphocytes, established a PCR efficiency of >93% for all loci with maximum allele dropout rates of 4%. Microsatellite analysis excluded external contamination and confirmed biallelic inheritance. Proof of principle for the simplicity and flexibility of the assay was demonstrated through its application to clinical PGD cycles for lipoid congenital adrenal hyperplasia, which presents a more severe clinical course in males, and Duchenne muscular dystrophy.

Metabolic syndrome (MS) encompasses a clustering of risk factors for cardiovascular disease, including obesity, insulin resistance, and dyslipidemia. We characterized a new mouse model carrying a dominant mutation, C57BL/6J-Nmf15/+ (B6-Nmf15/+), which develops additional complications of MS such as adipose tissue inflammation and cardiomyopathy. A backcross was used to genetically map the Nmf15 locus. Mice were examined in the comprehensive laboratory animal monitoring system, and dual energy X-ray absorptiometry and blood chemistry analyses were performed. Hypothalamic LEPR, SOCS1, and STAT3 phosphorylation were examined. Cardiac function was assessed by echo- and electrocardiography. Adipose tissue inflammation was characterized by in situ hybridization and measurement of Jun kinase activity. The Nmf15 locus mapped to distal mouse chromosome 5 with an LOD (logarithm of odds) score of 13.8. Nmf15 mice developed obesity by 12 weeks of age. Plasma leptin levels were significantly elevated in pre-obese Nmf15 mice at 8 weeks of age and an attenuated STAT3 phosphorylation in the hypothalamus suggests a primary leptin resistance. Adipose tissue from Nmf15 mice showed a remarkable degree of inflammation and macrophage infiltration as indicated by expression of the F4/80 marker and increased phosphorylation of JUN N-terminal kinase 1/2. Lipidosis was observed in tubular epithelial cells and glomeruli of the kidney. Nmf15 mice demonstrate both histological and pathophysiological evidence of cardiomyopathy. The Nmf15 mouse model provides a new entry point into pathways mediating leptin resistance and obesity. It is one of few models that combine many aspects of MS and can be useful for testing new therapeutic approaches for combating obesity complications, particularly cardiomyopathy.

Background: Bovine renal lipofuscinosis (BRL) is an incidental finding in cattle at slaughter. Condemnation of the kidneys as unfit for human consumption was until recently considered the only implication of BRL. Recent studies have indicated a negative influence on the health of affected animals. The present study investigated the prevalence, genetics and effect of BRL on milk yield and weight at slaughter., Methods: BRL status of slaughter cattle was recorded at four abattoirs during a 2-year-period. Data regarding breed, age, genetic descent, milk yield and weight at slaughter were extracted from the Danish Cattle Database. The prevalence of BRL was estimated stratified by breed and age-group. Furthermore, total milk yield, milk yield in last full lactation and weight at slaughter were compared for BRL-affected and non-affected Danish Holsteins and Danish Red cattle., Results: 433,759 bovines were slaughtered and 787 of these had BRL. BRL was mainly diagnosed in Danish Red, Danish Holstein and crossbreds. The age of BRL affected animals varied from 11 months to 13 years, but BRL was rarely diagnosed in cattle less than 2 years of age.The total lifelong energy corrected milk (ECM) yields were 3,136 and 4,083 kg higher for BRL affected Danish Red and Danish Holsteins, respectively. However, the median life span of affected animals was 4.9 months longer, and age-corrected total milk yield was 1,284 kg lower for BRL affected Danish Red cows. These cows produced 318 kg ECM less in their last full lactation. Weight at slaughter was not affected by BRL status.The cases occurred in patterns consistent with autosomal recessive inheritance and several family clusters of BRL were found. Analysis of segregation ratios demonstrated the expected ratio for Danish Red cattle, but not for Danish Holsteins., Conclusion: The study confirmed that BRL is a common finding in Danish Holsteins and Danish Red cattle at slaughter. The disorder is associated with increased total milk yield due to a longer production life. However, a reduced milk yield was detected in the end of the production life in Danish Red. The study supports that BRL is inherited autosomal recessively in the Danish Red breed and Danish Holsteins, but with incomplete penetrance of the genotype in Danish Holsteins.

Context: The steroidogenic acute regulatory protein (StAR) is essential for the production of steroid hormones. The mutations in the StAR gene typically cause congenital lipoid adrenal hyperplasia (lipoid CAH), characterized by severe adrenal insufficiency in both sexes and complete female external genitalia in genetic males. Affected 46, XX females feminize at puberty and menstruate but have progressive hypergonadotropic hypogonadism. It has been hypothesized that the cholesterol accumulation in the steroidogenic cells destroys the residual steroidogenic capacity and progressive ovarian failure occurs (two-hit model). Additionally, ovulation and luteinization in the patients is supposed to be impaired. However, those hypotheses have not been confirmed histologically., Objective: We examined whether pathological findings of the ovary in a patient of lipoid CAH corresponded with two-hit model, and whether ovulation and luteinization occurred or not in the patient., Subject: The ovary in an adult 46, XX female with a homozygous nonsense mutation (Q258X) in the StAR gene was examined. When the patient was age 22 yr, the ovary was resected because of enlargement with polycysts and subsequent torsion., Result: The affected ovary demonstrated remarkable lipoid deposition and changes of the mitochondrial ultrastructure. Immunohistochemical examination showed decrease of steroidogenic enzymes such as P450 cholesterol side-chain cleavage (P450scc). Additionally, we detected corpus albicans in the affected ovary., Conclusion: This is the first detailed report on ovarian histology in an adult 46, XX female with a null type mutation of the StAR gene (Q258X), which indicates the evidence of the impairment of ovarian StAR-independent steroidogenesis by lipoid deposition.

Arylsulfatase A (ASA)-deficient mice represent an animal model for the lysosomal storage disorder metachromatic leukodystrophy (MLD). Although the model has been applied in pathophysiological and therapeutic studies, the behavioural phenotype of ASA(-/-) mice is only partially characterized, and the most decisive outcome measures for therapy evaluation only emerge beyond 1 year of age. Presently, ASA(-/-) mice and ASA(+/-) control mice were studied at 6 and 12 months of age on an extensive battery including tests of neuromotor ability, exploratory behaviour, and learning and memory. Overt signs of ataxia were not observed in 6-month-old ASA(-/-) mice, but quantitative gait analysis during open-field exploration revealed that ASA(-/-) mice displayed increased hind base width and increased stride lengths for all paws. Their covert motor incoordination was evident in a correlation analysis which unveiled decreased harmonisation of concurrent gait parameters. For example, while ASA(+/-) controls demonstrated substantial convergence of front and hind base width (r=0.54), these variables actually diverged in ASA(-/-) mice (r=-0.37). Furthermore, various behavioural observations indicated emotional alterations in ASA(-/-) mice. Six-month-old ASA(-/-) mice also showed decreased response rates in scheduled operant responding. The present findings could provide relevant behavioural outcome measures for further use of this murine MLD model in preclinical studies.

We describe the clinical features, muscle pathology features, and molecular studies of seven patients with Chanarin-Dorfman syndrome (CDS) or neutral lipid storage disease and ichthyosis (NLSDI), a multisystem triglyceride storage disease with massive accumulation of lipid droplets in muscle fibers. All patients presented with congenital ichthyosiform erythroderma, cytoplasmic lipid droplets in blood cells, mild to severe hepatomegaly, and increased serum CK levels and liver enzymes. Three patients showed muscle symptoms and three had steathorrea. Molecular analysis identified five mutations, three of which are novel. These findings expand the clinical and mutational spectrum and underline the genetic heterogeneity of this disease.

Dorfman-Chanarin syndrome is a rare, autosomal recessive inherited lipid storage disease with congenital ichthyotic erythroderma due to an acylglycerol recycling defect. It is characterized by accumulation of neutral lipids in different tissues. Liver, muscle, ear, eye, and central nervous system are generally involved, so we presented a patient with severe ichthyosis, lipid vacuoles in neutrophils, and multiorgan involvement including a very rare complication, renal involvement. A 7-month-old girl was presented with frequent respiratory infection, congenital ichthyotic erithroderma and suspicion for immune deficiency. On her physical examination hepatomegaly, developmental delay, palmar and plantar hyperkeratosis and increased deep tendon reflexes with clonus and high tonus were found. Laboratory investigations revealed elevation at transaminases levels, hypoalbuminemia, hypergammaglobulinemia, presence of autoantibodies and eosinophilia. Vacuolization in leukocytes confirmed Dorfman-Chanarin syndrome, whereas no mutation at RAG1-2 and ARTEMIS genes ruled-out immune deficient status of the patient. At the age of eight months the patient died from severe renal failure. Her necropsies demonstrated microvesicular lipid accumulation not only at the liver but also at the renal species. The variability of involvement of different systems in Dorfman-Chanarin syndrome is well described, however the renal findings has not been reported previously at the literature.

To understand the evolution of human mental activity, we performed population genetic analyses of nucleotide sequences ( approximately 11 kb) from a worldwide sample of 60 chromosomes of the N-acylsphingosine amidohydrolase (ASAH1) gene. ASAH1 hydrolyzes ceramides and regulates neuronal development, and its deficiency often results in mental retardation. In the region ( approximately 4.4 kb) encompassing exons 3 and 4 of this gene, two distinct lineages (V and M) have been segregating in the human population for 2.4 +/- 0.4 million years (MY). The persistence of these two lineages is attributed to ancient population structure of humans in Africa. However, all haplotypes belonging to the V lineage exhibit strong linkage disequilibrium, a high frequency (62%), and small nucleotide diversity (pi = 0.05%). These features indicate a signature of positive Darwinian selection for the V lineage. Compared with the orthologs in mammals and birds, it is only Val at amino acid site 72 that is found exclusively in the V lineage in humans, suggesting that this Val is a likely target of positive selection. Computer simulation confirms that demographic models of modern humans except for the ancient population structure cannot explain the presence of two distinct lineages, and neutrality is incompatible with the observed small genetic variation of the V lineage at ASAH1. On the basis of the above observations, it is argued that positive selection is possibly operating on ASAH1 in the modern human population.

ABCG1, a member of the ATP-binding cassette transporter superfamily, is highly expressed in multiple cells of the lung. Loss of ABCG1 results in severe pulmonary lipidosis in mice, with massive deposition of cholesterol in both alveolar macrophages and type 2 cells and the accumulation of excessive surfactant phospholipids. These observations are consistent with ABCG1 controlling cellular sterol metabolism. Herein, we report on the progressive and chronic inflammatory process that accompanies the lipidosis in the lungs of Abcg1-/- mice. Compared with wild-type animals, the lungs of aged chow-fed mice deficient in ABCG1 show distinctive signs of inflammation that include macrophage accumulation, lymphocytic infiltration, hemorrhage, eosinophilic crystals, and elevated levels of numerous cytokines and cytokine receptors. Analysis of bronchoalveolar lavages obtained from Abcg1-/- mice revealed elevated numbers of foamy macrophages and leukocytes and the presence of multiple markers of inflammation including crystals of chitinase-3-like proteins. These data suggest that cholesterol and/or cholesterol metabolites that accumulate in Abcg1-/- lungs can trigger inflammatory signaling pathways. Consistent with this hypothesis, the expression of a number of cytokines was found to be significantly increased following increased cholesterol delivery to either primary peritoneal macrophages or Raw264.7 cells. Finally, cholesterol loading of primary mouse macrophages induced cytokine mRNAs to higher levels in Abcg1-/-, as compared with wild-type cells. These results demonstrate that ABCG1 plays critical roles in pulmonary homeostasis, balancing both lipid/cholesterol metabolism and inflammatory responses.

Phospholipidosis (PLD) is characterized by an intracellular accumulation of phospholipids in lysosomes and the concurrent development of concentric lamellar bodies. Recently, H. Sawada et al. (2005, Toxicol. Sci. 83, 282-292) identified 17 genes as potential biomarkers of PLD in HepG2 cells. The present study was undertaken to determine if this set of genes measured by quantitative PCR could be validated in the same cell line. The objective was also to investigate the dose-response relationship to further validate the assay and to select the concentrations to use for screening activities. In a first experiment (one concentration tested), out of the 17 genes, the best gene biomarkers of PLD (i.e., 11 genes) were selected for practical screening reasons. Based on these genes, 91.6% (i.e., 11 of 12) of the compounds known to induce PLD were identified as positive and all the negative compounds (i.e., five of five) were also confirmed. When the data obtained in the first experiment were compared to the data by Sawada et al., (2005) the coefficient of correlation calculated was slightly higher than 75%. In the second experiment (26 compounds [all 17 compounds from the first experiment plus 9 other compounds] tested at a minimum of three concentrations), 93.3% (14/15) of the compounds known to induce PLD were identified as such and all the negative controls (six compounds) were also confirmed. Three compounds likely to induce PLD were identified as positive in our assay. Finally, two compounds for which no data are available were also tested. When both experiments 1 and 2 were compared, the coefficient of correlation for 16 compounds tested at the same concentrations reached 87.7%. In conclusion, the present study further confirms the utility of gene expression in HepG2 cells to identify a potential to induce PLD. Finally, based on the data presented, researchers are encouraged to use a range of minimum three concentrations (e.g., 12.5, 25, and 50 microM) to screen for PLD in the human HepG2 cell line.

Neutral lipid storage disease comprises a heterogeneous group of autosomal recessive disorders characterized by systemic accumulation of triglycerides in cytoplasmic droplets. Here we report a neutral lipid storage disease subgroup characterized by mild myopathy, absence of ichthyosis and mutations in both alleles of adipose triglyceride lipase (PNPLA2, also known as ATGL). Three of these mutations are predicted to lead to a truncated ATGL protein with an intact patatin domain containing the active site, but with defects in the hydrophobic domain. The block in triglyceride degradation was mimicked by short interfering RNA directed against ATGL. NLSDM is distinct from Chanarin-Dorfman syndrome, which is characterized by neutral lipid storage disease with ichthyosis, mild myopathy and hepatomegaly due to mutations in ABHD5 (also known as CGI-58).

Background: Dorfman-Chanarin syndrome is a rare, autosomal recessive, inherited, lipid storage disease. It is characterized by nonbullous congenital ichthyosiform erythroderma, leukocyte vacuoles and variable involvement of the liver, muscles and central nervous system, due to errors of triacylglycerol metabolism. To date only 32 cases of this syndrome have been described worldwide., Aims: To report the case of a boy with Dorfman-Chanarin syndrome with rickets., Case Report: A boy of Turkish origin was born of a nonconsanguineous marriage after an uncomplicated perinatal period. On examination the patient had failure to thrive, diffuse erythroderma and ichthyosis and clinical features suggesting rickets. A light-microscopic examination of peripheral smear revealed vacuolated leukocytes typical of Jordans' anomaly. Lipid analysis showed an increase in triacylglycerol and very low density lipoprotein. A radiographic study of the wrist revealed rickets., Conclusion: The clinical progression in these patients ranges from mild to fatal. We believe that patients with ichthyosis should be evaluated bearing in mind the possibility of Dorfman-Chanarin syndrome. This may increase the number of reported Dorfman-Chanarin syndrome cases, and the pathogenesis and progression of the disease will become clearer., (2007 S. Karger AG, Basel)

We previously showed that a toxicogenomics analysis of drug-induced phospholipidosis enabled the identification of 12 specific gene markers and the establishment of an in vitro real-time PCR screening assay for the assessment of the phospholipidosis-inducing potential of compounds. The purpose of this study was to transfer our PCR-based assay into a 96-well microplate-based multiple mRNAs measuring assay (ArrayPlate assay) in order to increase throughput. Specifically, we determined the expression of the 12 marker genes using real-time PCR and ArrayPlate in human hepatoma HepG2 cells that were treated for 24h with each of amiodarone and 80 proprietary compounds. The following three performance criteria were satisfied in the ArrayPlate analysis: 1. Sensitivity-the expression of mRNA for all target genes was detected at quantifiable levels. 2. Repeatability-signal intensities and fold change values of each marker gene were highly repeatable. 3. Correlation-fold change values and their average values, which were used as indices of phospholipidosis induction potential, showed apparent correlation between the ArrayPlate and real-time PCR assays. Thus, the in vitro screening assay for compound-induced phospholipidosis should be transferable from a PCR-based assay to the higher-throughput ArrayPlate-based method.

Most newly synthesized soluble lysosomal proteins are delivered to the lysosome via the mannose 6-phosphate (Man-6-P)-targeting pathway. The presence of the Man-6-P post-translational modification allows these proteins to be affinity-purified on immobilized Man-6-P receptors. This approach has formed the basis for a number of proteomic studies that identified multiple as yet uncharacterized Man-6-P glycoproteins that may represent new lysosomal proteins. Although the presence of Man-6-P is suggestive of lysosomal function, the subcellular localization of such candidates requires experimental verification. Here, we have investigated one such candidate, ependymin-related protein (EPDR). EPDR is a protein of unknown function with some sequence similarity to ependymin, a fish protein thought to play a role in memory consolidation and learning. Using classical subcellular fractionation on rat brain, EPDR co-distributes with lysosomal proteins, but there is significant overlap between lysosomal and mitochondrial markers. For more definitive localization, we have developed a novel approach based upon a selective buoyant density shift of the brain lysosomes in a mutant mouse lacking NPC2, a lysosomal protein involved in lipid transport. EPDR, in parallel with lysosomal markers, shows this density shift in gradient centrifugation experiments comparing mutant and wild type mice. This approach, combined with morphological analyses, demonstrates that EPDR resides in the lysosome. In addition, the lipidosis-induced density shift approach represents a valuable tool for identification and validation of both luminal and membrane lysosomal proteins that should be applicable to high throughput proteomic studies.

We show that mice lacking the ATP-binding cassette transmembrane transporter ABCG1 show progressive and age-dependent severe pulmonary lipidosis that recapitulates the phenotypes of different respiratory syndromes in both humans and mice. The lungs of chow-fed Abcg1(-/-) mice, >6-months old, exhibit extensive subpleural cellular accumulation, macrophage, and pneumocyte type 2 hypertrophy, massive lipid deposition in both macrophages and pneumocytes and increased levels of surfactant. No such abnormalities are observed at 3 months of age. However, gene expression profiling reveals significant changes in the levels of mRNAs encoding key genes involved in lipid metabolism in both 3- and 8-month-old Abcg1(-/-) mice. These data suggest that the lungs of young Abcg1(-/-) mice maintain normal lipid levels by repressing lipid biosynthetic pathways and that such compensation is inadequate as the mice mature. Studies with A-549 cells, a model for pneumocytes type 2, demonstrate that overexpression of ABCG1 specifically stimulates the efflux of cellular cholesterol by a process that is dependent upon phospholipid secretion. In addition, we demonstrate that Abcg1(-/-), but not wild-type macrophages, accumulate cholesterol ester droplets when incubated with surfactant. Together, these data provide a mechanism to explain the lipid accumulation in the lungs of Abcg1(-/-)mice. In summary, our results demonstrate that ABCG1 plays essential roles in pulmonary lipid homeostasis.

The number of people keeping reptiles in captivity has markedly increased, and widespread inappropriate husbandry will result in an increasing incidence of the diagnosis of hepatic lipidosis(HL). It may become apparent that some species of snake have predisposition to develop HL. In the specific instance of the genus Aspidites, alterations to the usual husbandry that limit feeding and thereby body weight should markedly lessen the chance of the animal's developing HL. It is important for clinicians to develop a more aggressive approach to these cases and to build a bank of data with which to promote understanding of this common malady.

The aim of the present study was to investigate ovarian function in a 46,XX female patient with congenital lipoid adrenal hyperplasia (lipoid CAH) during infancy and childhood. We measured serum gonadotropin and estradiol levels regularly from 6 months to 10.9 years of age. Serum FSH levels were clearly elevated during the first 2 years of life; they decreased gradually until the age of 5 years and they then fell into the normal range for a prepubertal female. Serum LH levels were elevated until the age of 2 years and they then fell into the normal range. Serum estradiol levels were always in the normal prepubertal range. Pelvic ultrasonography performed at the age of 9 months and at 9.8 years of age showed normal uterine and ovarian morphology for a prepubertal female. In conclusion, our data suggest that ovarian insufficiency in lipoid CAH begins in infancy.

Phospholipidosis is a lipid storage disorder in which excess phospholipids accumulate within cells. Some cationic amphiphilic compounds are known to have the potential to induce phospholipidosis. This study was undertaken to examine the molecular mechanisms that contribute to the development of phospholipidosis and to identify specific markers that might form the basis of an in vitro screening test. Specifically, we performed a large-scale gene expression analysis using DNA microarrays on human hepatoma HepG2 cells after they were treated with each of 12 compounds known to induce phospholipidosis. In electron microscopy, HepG2 cells developed lamellar myelin-like bodies in their lysosomes, the characteristic change of phospholipidosis, after treatment with these compounds for 72 h. DNA microarray analysis performed 6 and 24 h after treatment showed alterations in gene expression reflecting the inhibition of lysosomal phospholipase activity and lysosomal enzyme transport, and the induction of phospholipid and cholesterol biosynthesis. Seventeen genes that showed a similar expression profile following treatment were selected as candidate markers. Real-time PCR analysis confirmed that 12 gene markers showed significant concordance with lamellar myelin-like body formation. Furthermore, the average fold change values of these markers correlated well with the magnitude of this pathological change. In conclusion, microarray analysis revealed that factors such as alterations in lysosomal function and cholesterol metabolism were involved in the induction of phospholipidosis. Furthermore, comprehensive gene expression analysis enabled us to identify biomarkers of this condition that we then used to develop a rapid and sensitive in vitro screening test for drug-induced phospholipidosis.

Rare diffuse infiltrative lung diseases are a challenge for clinicians, radiologists, and pathologists for at least three reasons: (a) their low incidence and prevalence hamper the acquisition of expertise and frequently the diagnosis is delayed; (b) therapeutic actions are mainly empirical and based on steroid use, and (c) pathogenetic events are difficult to explain and only recently new therapeutic measures taking advantage of innovative genetic and/or immunopathogenetic studies have been suggested. In this review rare diffuse lung disorders are briefly discussed (pulmonary alveolar proteinosis, inherited lipidoses, acute eosinophilic pneumonia, amyloidosis, pulmonary ossification, pulmonary alveolar microlithiasis). The list is obviously not exhaustive and arbitrarily chosen. The intent is, however, to emphasize that in this difficult field multidisciplinary expertise and the knowledge of the most recent pathogenetic mechanisms have the main role in diagnosis and treatment.

Genetic neurometabolic diseases in childhood are multisystemic. Surprisingly, these genetic diseases can manifest for the first time during adolescence and adulthood. In this case, the clinical presentation and evolutivity are very different. In childhood, many neurological systems are touched and their evolution is rapidly lethal. In the adult, their presentation may be that of a degenerative disease of the central nervous system and, according to the disease, the syndrome is very particular and very systematized. From our clinical and biological experience, we would like to suggest a decision tree.

Androgens and estrogens are made from dehydroepiandrosterone (DHEA), which is made from cholesterol via four steps. First, cholesterol enters the mitochondria with the assistance of the steroidogenic acute regulatory protein (StAR). Mutations in the StAR gene cause congenital lipoid adrenal hyperplasia. Second, within the mitochondria, cholesterol is converted to pregnenolone by the cholesterol side chain cleavage enzyme, P450scc. Third, pregnenolone undergoes 17alpha-hydroxylation by microsomal P450c17. Finally, 17-OH pregnenolone is converted to DHEA by the 17,20 lyase activity of P450c17. The ratio of the 17,20 lyase to 17alpha-hydroxylase activity of P450c17 determines the ratio of C21 to C19 steroids produced. This ratio is regulated post-translationally by at least three factors: the abundance of the electron-donating protein P450 oxidoreductase, the presence of cytochrome b(5), and the serine phosphorylation of P450c17. Study of these and related factors may yield important information about the pathophysiology of adrenarche and the polycystic ovary syndrome (PCOS)., (Copyright 2002 Elsevier Science Ireland Ltd.)

Despite progress in molecular characterization, specific diagnoses of disorders belonging to a group of inherited hypoalphalipoproteinemias, i.e., apolipoprotein AI deficiency, lecithin-cholesterol acyltransferase deficiency, Tangier disease (TD), and familial high-density lipoprotein (HDL) deficiency, remain difficult on a purely clinical basis. Several TD patients were recently found to be homozygous for mutations in the ABCA1 gene. We have documented here a clinical variant of TD in a Japanese patient who manifested corneal lipidosis and premature coronary artery disease as well as an almost complete absence of HDL-cholesterol, by identifying a novel homozygous ABCA1 mutation (R1680W). We propose that patients with apparently isolated HDL deficiency who are found to carry ABCA1 mutations may in fact belong to a category of TD patients whose phenotypic features are only partially expressed, and that a number of hidden clinical variants of TD might exist among other HDL deficiency patients who have escaped correct clinical diagnosis.

Carnitine-acylcarnitine translocase (CACT) deficiency is an inherited defect of the co-transport of free and esterified carnitine across the inner mitochondrial membrane. We report a case of CACT deficiency in a newborn who died at 72 h of age from severe, intractable cardiac tachyarrhythmia, despite an improvement in his neurological and biochemical status. Postmortem examination showed marked steatosis of myocardium, liver, and kidney. In addition, electron microscopic studies showed virtually complete elimination of mitochondria from cardiomyocytes. It appears that the correction of the acute metabolic derangements in this condition may not prevent rapid progression to death, suggesting that the rhythm disturbances in CACT deficiency result from prior and ongoing accumulation of toxic metabolites, rather than from an acute metabolic derangement. Furthermore, we speculate that the choice of anti-arrhythmic agent in this patient may paradoxically have contributed to his death.

A child's development generally proceeds along expected pathways, with anticipated levels of function for specific ages. When these levels are not met, the treating physician must determine whether the child has a static or a progressive process. If the child achieves certain levels of development, then loses these skills, the chance is greater that the process is progressive. The technological revolution and progress in molecular genetics in the past 20 to 30 years provide greater avenues for the diagnosis of many of the progressive disorders affecting neurons and central nervous system (CNS) function. Many disorders can be diagnosed in utero, and treatments are available for some. It has become increasingly evident that many of the inherited neurodegenerative disorders have varied clinical phenotypes, and clinical phenotypes may overlap between some of the disorders. This article provides a framework for the primary physician to consider some of these disorders and a rational approach to the evaluation of a child suspected of having a progressive neurodegenerative disorder.

1. Peritoneal macrophages (Mphi) were harvested from non-stimulated 2 month old SHRSP and WKY, and incubated with acetyl LDL or [14C]-oleate-albumin complex to check total, free and esterified cholesterol (Cho) accumulation as well as acy coenzyme A-cholesterol acyltransferase (ACAT) activity and its activation by hyperlipidaemic serum. 2. Total and esterified Cho accumulation as well as ACAT activity were enhanced in Mphi from SHRSP compared with those from WKY. 3. To observe the dietary or pharmacological influence on atherogenesis, arteriolipidosis-prone SHR (ALR) were given a high fat cholesterol diet with 3% taurine or a Ca blocker (Nilvadipine, N; 30 mg/kg), both of which significantly decreased serum Cho level and clearly attenuated arterial fat deposition.

It is estimated that 70-100 children suffering from a lysosomal storage disease are born in Poland every year. From 1975 to 1993, the activity of various lysosomal enzymes was determined in the leukocytes, cultured skin fibroblasts, or hair roots from 5,594 patients, mainly children, in whom the diagnosis of a lipidosis was suspected. In that material 162 cases of a lipidosis were diagnosed. Metachromatic leukodystrophy seems to be the most frequent of the lipidoses; GM1 gangliosidosis is more frequent than GM2 gangliosidosis and Gaucher and Niemann-Pick diseases appear to be almost as frequent as the former.

The drugs used for the treatment of psychiatric disorders sometimes produce neurological symptoms (drug-induced lipidosis) similar to those found in subjects with genetic disorders of lipid metabolism. We report the case of a young man with Niemann-Pick disease first treated for behavioural disorders. As the family accused the Medical Department to be responsible for a disease caused by excess of medication, a detailed study of the patient's biochemical features was used to refute the complaint.

The characterization of mutations in Japanese patients with lipidosis, particularly in metachromatic leukodystrophy (MLD) and Gaucher disease has been studied in detail. Metachromatic leukodystrophy is characterized by an accumulation of sulfatide in nervous tissues and kidney due to a deficiency of arylsulfatase A (ASA). We analyzed the presence of three known mutant arylsulfatase A alleles in Japanese patients with MLD. Among 10 patients of Japanese patients with MLD, we found that allele 445A mutation has moderately high incidence and also homozygosity of this mutation results in the late infantile form. Allele 2381T was not found in Japanese patients. Furthermore, we found novel mutation which is G- to A mutation at the 1070 nucleotide of the ASA gene (designated 1070 A) in Japanese patients with juvenile onset. This mutation results in a amino acid substitution of Gly245 by Arg and found in heterozygote form. Our studies of molecular analysis in 10 Japanese patients with MLD indicate that Japanese MLD patients have unique characteristics of ASA mutations compared with those of Caucasian patients. On the other hand, Gaucher disease is the most prevalent sphingolipidosis, characterized by an accumulation of glucocerebroside in macrophage derived cells due to a deficiency of lysosomal hydrolase glucocerebrosidase. To study the molecular basis of Gaucher disease in Japanese patients, we analyzed the presence of the two known mutations (6433C and 3548A) in the glucocerebrosidase gene of 15 patients with Gaucher disease. We found that the 6433C and 3548A mutations occur in all subtypes of Japanese patients with Gaucher disease.(ABSTRACT TRUNCATED AT 250 WORDS)

The effects of mutation, migration, random drift, and selection on the change in frequency of the alleles associated with Huntington disease, porphyria variegata, and lipoid proteinosis have been assessed in the Afrikaner population of South Africa. Although admixture cannot be completely discounted, it was possible to exclude migration and new mutation as major sources of changes in the frequency of these alleles by limiting analyses to pedigrees descendant from founding families. Calculations which overestimated the possible effect of random drift demonstrated that drift did not account for the observed changes in gene frequencies. Therefore these changes must have been caused by natural selection, and a coefficient of selection was estimated for each trait. For the rare, dominant, deleterious allele associated with Huntington disease, the coefficient of selection was estimated to be .34, indicating that this allele has a selective disadvantage, contrary to some recent studies. For the presumed dominant and probably deleterious allele associated with porphyria variegata, the coefficient of selection lies between .07 and .02. The coefficient of selection for the rare, clinically recessive allele associated with lipoid proteinosis was estimated to be .07. Calculations based on a model system indicate that the observed decrease in allele frequency cannot be explained solely on the basis of selection against the homozygote. Thus, this may be an example of a pleiotropic gene which has a dominant effect in terms of selection even though its known clinical effect is recessive.