To safeguard the cell from the accumulation of potentially harmful metabolic intermediates, specific repair mechanisms have evolved. APOA1BP, now renamed NAXE, encodes an epimerase essential in the cellular metabolite repair for NADHX and NADPHX. The enzyme catalyzes the epimerization of NAD(P)HX, thereby avoiding the accumulation of toxic metabolites. The clinical importance of the NAD(P)HX repair system has been unknown. Exome sequencing revealed pathogenic biallelic mutations in NAXE in children from four families with (sub-) acute-onset ataxia, cerebellar edema, spinal myelopathy, and skin lesions. Lactate was elevated in cerebrospinal fluid of all affected individuals. Disease onset was during the second year of life and clinical signs as well as episodes of deterioration were triggered by febrile infections. Disease course was rapidly progressive, leading to coma, global brain atrophy, and finally to death in all affected individuals. NAXE levels were undetectable in fibroblasts from affected individuals of two families. In these fibroblasts we measured highly elevated concentrations of the toxic metabolite cyclic-NADHX, confirming a deficiency of the mitochondrial NAD(P)HX repair system. Finally, NAD or nicotinic acid (vitamin B3) supplementation might have therapeutic implications for this fatal disorder.

Very long-chain acyl-CoA dehydrogenase (VLCAD)-deficiency is the most common long-chain fatty acid oxidation disorder presenting with heterogeneous phenotypes. Similar to many patients with VLCADD, VLCAD-deficient mice (VLCAD(-/-)) remain asymptomatic over a long period of time. In order to identify the involved compensatory mechanisms, wild-type and VLCAD(-/-) mice were fed one year either with a normal diet or with a diet in which medium-chain triglycerides (MCT) replaced long-chain triglycerides, as approved intervention in VLCADD. The expression of the mitochondrial long-chain acyl-CoA dehydrogenase (LCAD) and medium-chain acyl-CoA dehydrogenase (MCAD) was quantified at mRNA and protein level in heart, liver and skeletal muscle. The oxidation capacity of the different tissues was measured by LC-MS/MS using acyl-CoA substrates with a chain length of 8 to 20 carbons. Moreover, in white skeletal muscle the role of glycolysis and concomitant muscle fibre adaptation was investigated. In one year old VLCAD(-/-) mice MCAD and LCAD play an important role in order to compensate deficiency of VLCAD especially in the heart and in the liver. However, the white gastrocnemius muscle develops alternative compensatory mechanism based on a different substrate selection and increased glucose oxidation. Finally, the application of an MCT diet over one year has no effects on LCAD or MCAD expression. MCT results in the VLCAD(-/-) mice only in a very modest improvement of medium-chain acyl-CoA oxidation capacity restricted to cardiac tissue. In conclusion, VLCAD(-/-) mice develop tissue-specific strategies to compensate deficiency of VLCAD either by induction of other mitochondrial acyl-CoA dehydrogenases or by enhancement of glucose oxidation. In the muscle, there is evidence of a muscle fibre type adaptation with a predominance of glycolytic muscle fibres. Dietary modification as represented by an MCT-diet does not improve these strategies long-term.

Medium-chain acyl-CoA dehydrogenase (MCAD) deficiency (OMIM 201450) is the most common inherited disorder of fatty acid metabolism presenting with hypoglycaemia, hepatopathy and Reye-like symptoms during catabolism. In the past, the majority of patients carried the prevalent c.985A>G mutation in the ACADM gene. Since the introduction of newborn screening many other mutations with unknown clinical relevance have been identified in asymptomatic newborns. In order to identify functional effects of these mutant genotypes we correlated residual MCAD (OMIM 607008) activities as measured by octanoyl-CoA oxidation in lymphocytes with both genotype and relevant medical reports in 65 newborns harbouring mutant alleles. We identified true disease-causing mutations with residual activities of 0 to 20%. In individuals carrying the c.199T>C or c.127G>A mutation on one allele, residual activities were much higher and in the range of heterozygotes (31%-60%). Therefore, both mutations cannot clearly be associated with a clinical phenotype. This demonstrates a correlation between the octanoyl-CoA oxidation rate in lymphocytes and the clinical outcome. With newborn screening, the natural course of disease is difficult to assess. The octanoyl-CoA oxidation rate, therefore, allows a risk assessment at birth and the identification of new ACADM genotypes associated with asymptomatic disease variants.

Dietary fat restriction and increased carbohydrate intake are part of treatment in very-long-chain acyl-CoA dehydrogenase (VLCAD)-deficiency, the most common defect of long-chain fatty acid oxidation. The long-term impact of these interventions is unknown. We characterized here the effects of a fat-reduced, carbohydrate-enriched diet and an increased fat intake on energy metabolism in a mouse model of VLCAD-deficiency. Wild-type and VLCAD(-/-) mice were fed one year either with a normal (5.1%), a high fat (10.6%) or a low-fat, carbohydrate-enriched (2.6%) diet. Dietary effects on genes involved in lipogenesis, energy homeostasis and substrate selection were quantified by real-time-PCR. Acylcarnitines as sign of impaired energy production were determined in dried blood spots and tissues. White skeletal muscle was analyzed for muscle fiber type as well as for glycogen and triglyceride content. Both dietary modifications induced enhanced triacylglyceride accumulation in skeletal muscle and inhibition of glucose oxidation. This was accompanied by an up-regulation of genes coding for oxidative muscle fiber type I and a marked accumulation of acylcarnitines, especially prominent in the heart (164±2.8 in VLCAD(-/-) vs. 82.3±2.1 in WT μmol/mg) under a low-fat, carbohydrate-enriched diet. We demonstrate here that both dietary interventions with respect to the fat content of the diet reverse endogenous compensatory mechanisms in muscle that have evolved in VLCAD(-/-) mice resulting in pronounced energy deficiency. In particular, the low-fat carbohydrate-enriched diet was not effective in the long term. Further experiments are necessary to define the optimal energy provision for fatty acid oxidation defects.

Newborn screening for medium- and very long-chain acyl-CoA dehydrogenase (MCAD and VLCAD, respectively) deficiency, using acylcarnitine profiling with tandem mass spectrometry, has increased the number of patients with fatty acid oxidation disorders due to the identification of additional milder, and so far silent, phenotypes. However, especially for VLCADD, the acylcarnitine profile can not constitute the sole parameter in order to reliably confirm disease. Therefore, we developed a new liquid chromatography tandem mass spectrometry (LC-MS/MS) method to rapidly determine both MCAD- and/or VLCAD-activity in human lymphocytes in order to confirm diagnosis.

The endocannabinoid system is involved in the modulation of inflammatory, fibrotic, metabolic, and carcinogenesis-associated signaling pathways via cannabinoid receptor (CB)1 and CB2. We hypothesized that the pharmacologic antagonization of CB1 receptor improves cholestasis in Abcb4-/- mice.

Thrombocytopenia has been described in most patients with acute and chronic liver failure. Decreased platelet production and decreased half-life of platelets might be a consequence of low levels of thrombopoietin (TPO) in these patients. Platelet production is tightly regulated to avoid bleeding complications after vessel injury and can be enhanced under elevated platelet destruction as observed in liver disease. Thrombopoietin (TPO) is the primary regulator of platelet biogenesis and supports proliferation and differentiation of megakaryocytes.

Gunn rats bear a mutation within the uridine diphosphate glucuronosyltransferase-1a1 (Ugt1a1) gene resulting in high serum bilirubin levels as seen in Crigler-Najjar syndrome. In this study, the Gunn rat was used as an animal model for heritable liver dysfunction. Induced mesenchymal stem cells (iMSCs) derived from embryonic stem cells (H1) and induced pluripotent stem cells were transplanted into Gunn rats after partial hepatectomy. The iMSCs engrafted and survived in the liver for up to 2 months. The transplanted iMSCs differentiated into functional hepatocytes as evidenced by partially suppressed hyperbilirubinemia and expression of multiple human-specific hepatocyte markers such as albumin, hepatocyte nuclear factor 4α, UGT1A1, cytokeratin 18, bile salt export pump, multidrug resistance protein 2, Na/taurocholate-cotransporting polypeptide, and α-fetoprotein. These findings imply that transplanted human iMSCs can contribute to liver regeneration in vivo and thus represent a promising tool for the treatment of inherited liver diseases.

Dextromethorphan (DXM) acts as cough suppressant via its central action. Cell-protective effects of this drug have been reported in peripheral tissues, making DXM potentially useful for treatment of several common human diseases, such as type 2 diabetes mellitus (T2DM). Pancreatic islets are among the peripheral tissues that positively respond to DXM, and anti-diabetic effects of DXM were observed in two placebo-controlled, randomized clinical trials in humans with T2DM. Since these effects were associated with central side effects, we here developed chemical derivatives of DXM that pass the blood-brain barrier to a significantly lower extent than the original drug. We show that basic nitrogen-containing residues block central adverse events of DXM without reducing its anti-diabetic effects, including the protection of human pancreatic islets from cell death. These results show how to chemically modify DXM, and possibly other morphinans, as to exclude central side effects, while targeting peripheral tissues, such as pancreatic islets.

Type 2 diabetes is characterized by insulin hypersecretion followed by reduced glucose-stimulated insulin secretion (GSIS). Here we show that acute stimulation of pancreatic islets with the insulin secretagogue dextrorphan (DXO) or glibenclamide enhances GSIS, whereas chronic treatment with high concentrations of these drugs reduce GSIS but protect islets from cell death. Bulk RNA sequencing of islets shows increased expression of genes for serine-linked mitochondrial one-carbon metabolism (OCM) after chronic, but not acute, stimulation. In chronically stimulated islets, more glucose is metabolized to serine than to citrate, and the mitochondrial ATP/ADP ratio decreases, whereas the NADPH/NADP+ ratio increases. Activating transcription factor-4 (Atf4) is required and sufficient to activate serine-linked mitochondrial OCM genes in islets, with gain- and loss-of-function experiments showing that Atf4 reduces GSIS and is required, but not sufficient, for full DXO-mediated islet protection. In sum, we identify a reversible metabolic pathway that provides islet protection at the expense of secretory function.

Retinoid-storing hepatic stellate cells (HSCs) have recently been described as a liver-resident mesenchymal stem cell (MSC) population; however, it is not clear whether these cells contribute to liver regeneration or serve as a progenitor cell population with hepatobiliary characteristics. Here, we purified HSCs with retinoid-dependent fluorescence-activated cell sorting from eGFP-expressing rats and transplanted these GFP(+) HSCs into wild-type (WT) rats that had undergone partial hepatectomy in the presence of 2-acetylaminofluorene (2AAF) or retrorsine, both of which are injury models that favor stem cell-based liver repair. Transplanted HSCs contributed to liver regeneration in host animals by forming mesenchymal tissue, progenitor cells, hepatocytes, and cholangiocytes and elevated direct bilirubin levels in blood sera of GUNN rats, indicating recovery from the hepatic bilirubin-handling defect in these animals. Transplanted HSCs engrafted within the bone marrow (BM) of host animals, and HSC-derived cells were isolated from BM and successfully retransplanted into new hosts with injured liver. Cultured HSCs transiently adopted an expression profile similar to that of progenitor cells during differentiation into bile acid-synthesizing and -transporting hepatocytes, suggesting that stellate cells represent a source of liver progenitor cells. This concept connects seemingly contradictory studies that favor either progenitor cells or MSCs as important players in stem cell-based liver regeneration.

Deficiency of very long-chain acyl-CoA dehydrogenase (VLCAD) is the most common disorder of mitochondrial β-oxidation of long-chain fatty acids. In order to maintain glucose homeostasis, the kidney and liver as the main gluconeogenic organs play an important role under conditions of impaired fatty acid oxidation. However, little is known about how a defective fatty acid oxidation machinery affects renal metabolism and function as well as renal energy supply especially during catabolic situations.

The Th2 cytokine IL-13 is involved in biliary epithelial injury and liver fibrosis in patients as well as in animal models. The aim of this study was to investigate IL-13 as a therapeutic target during short term and chronic intrahepatic cholestasis in an Abcb4-knockout mouse model (Abcb4-/-). Lack of IL-13 protected Abcb4-/- mice transiently from cholestasis. This decrease in serum bile acids was accompanied by an enhanced excretion of bile acids and a normalization of fecal bile acid composition. In Abcb4-/-/IL-13-/- double knockout mice, bacterial translocation to the liver was significantly reduced and the intestinal microbiome resembled the commensal composition in wild type animals. In addition, 52-week-old Abcb4-/-IL-13-/- mice showed significantly reduced hepatic fibrosis. Abcb4-/- mice devoid of IL-13 transiently improved cholestasis and converted the composition of the gut microbiota towards healthy conditions. This highlights IL-13 as a potential therapeutic target in biliary diseases.

Although p38 MAP Kinase α (p38 MAPKα) is generally accepted to play a central role in the cardiac stress response, to date its function in maladaptive cardiac hypertrophy is still not unambiguously defined. To induce a pathological type of cardiac hypertrophy we infused angiotensin II (AngII) for 2 days via osmotic mini pumps in control and tamoxifen-inducible, cardiomyocyte (CM)-specific p38 MAPKα KO mice (iCMp38αKO) and assessed cardiac function by echocardiography, complemented by transcriptomic, histological, and immune cell analysis. AngII treatment after inactivation of p38 MAPKα in CM results in left ventricular (LV) dilatation within 48 h (EDV: BL: 83.8 ± 22.5 µl, 48 h AngII: 109.7 ± 14.6 µl) and an ectopic lipid deposition in cardiomyocytes, reflecting a metabolic dysfunction in pressure overload (PO). This was accompanied by a concerted downregulation of transcripts for oxidative phosphorylation, TCA cycle, and fatty acid metabolism. Cardiac inflammation involving neutrophils, macrophages, B- and T-cells was significantly enhanced. Inhibition of adipose tissue lipolysis by the small molecule inhibitor of adipocytetriglyceride lipase (ATGL) Atglistatin reduced cardiac lipid accumulation by 70% and neutrophil infiltration by 30% and went along with an improved cardiac function. Direct targeting of neutrophils by means of anti Ly6G-antibody administration in vivo led to a reduced LV dilation in iCMp38αKO mice and an improved systolic function (EF: 39.27 ± 14%). Thus, adipose tissue lipolysis and CM lipid accumulation augmented cardiac inflammation in iCMp38αKO mice. Neutrophils, in particular, triggered the rapid left ventricular dilatation. We provide the first evidence that p38 MAPKα acts as an essential switch in cardiac adaptation to PO by mitigating metabolic dysfunction and inflammation. Moreover, we identified a heart-adipose tissue-immune cell crosstalk, which might serve as new therapeutic target in cardiac pathologies.

Major histocompatibility complex I (MHC-I) molecules present epitopes on the cellular surface of antigen-presenting cells to prime cytotoxic clusters of differentiation 8 (CD8)+ T cells (CTLs), which then identify and eliminate other cells such as virus-infected cells bearing the antigen. Human hepatitis virus cohort studies have previously identified MHC-I molecules as promising predictors of viral clearance. However, the underlying functional significance of these predictions is not fully understood. Here, we show that expression of single MHC-I isomers promotes virus-induced liver immunopathology. Specifically, using the lymphocytic choriomeningitis virus (LCMV) model system, we found MHC-I proteins to be highly up-regulated during infection. Deletion of one of the two MHC-I isomers histocompatibility antigen 2 (H2)-Db or H2-Kb in C57Bl/6 mice resulted in CTL activation recognizing the remaining MHC-I with LCMV epitopes in increased paucity. This increased CTL response resulted in hepatocyte death, increased caspase activation, and severe metabolic changes in liver tissue following infection with LCMV. Moreover, depletion of CTLs abolished LCMV-induced pathology in these mice with resulting viral persistence. In turn, natural killer (NK) cell depletion further increased antiviral CTL immunity and clearance of LCMV even in the presence of a single MHC-I isomer. Conclusion: Our results suggest that uniform MHC-I molecule expression promotes enhanced CTL immunity during viral infection and contributes to increased CTL-mediated liver cell damage that was alleviated by CD8 or NK cell depletion.

Cholestatic patients exhibiting high bile acid serum levels were reported to be more susceptible to bacterial and viral infections. Animal studies in bile duct ligated (BDL) mice suggest that cholestasis leads to an aggravation of hepatic bacterial infections. We have investigated the impact of cholestasis on mouse cytomegalovirus (MCMV)-induced immune responses and viral replication. While MCMV did not aggravate BDL-induced liver damage, BDL markedly reduced MCMV-triggered chemokine expression and immune cell recruitment to the liver. MCMV-infected BDL mice showed diminished trafficking of Ly6C+/F4/80+ myeloid cells and NK1.1+ NK cells to the liver compared to MCMV infected control mice. Moreover, virus-driven expression of CCL7, CCL12, CXCL9 and CXCL10 was clearly impaired in BDL- compared to sham-operated mice. Furthermore, production of the anti-inflammatory cytokine IL-10 was massively augmented in infected BDL mice. In contrast, intra- and extrahepatic virus replication was unaltered in BDL-MCMV mice when compared to sham-MCMV mice. Cholestasis in the BDL model severely impaired pathogen-induced chemokine expression in the liver affecting CCR2- and CXCR3-dependent cell trafficking. Cholestasis resulted in reduced recruitment of inflammatory monocytes and NK cells to the liver.

ATAD3 (ATPase family AAA domain-containing protein 3) is a mitochondrial protein, which is essential for cell viability and organismal development. ATAD3 has been implicated in several important cellular processes such as apoptosis regulation, respiratory chain function and steroid hormone biosynthesis. Moreover, altered expression of ATAD3 has been associated with several types of cancer. However, the exact mechanisms underlying ATAD3 effects on cellular metabolism remain largely unclear. Here, we demonstrate that Caenorhabditis elegans ATAD-3 is involved in mitochondrial iron and heme homeostasis. Knockdown of atad-3 caused mitochondrial iron- and heme accumulation. This was paralleled by changes in the expression levels of several iron- and heme-regulatory genes as well as an increased heme uptake. In conclusion, our data indicate a regulatory role of C. elegans ATAD-3 in mitochondrial iron and heme metabolism.

The Fragile X mental retardation (FMR) syndrome is a frequently inherited intellectual disability caused by decreased or absent expression of the FMR protein (FMRP). Lack of FMRP is associated with neuronal degradation and cognitive dysfunction but its role outside the central nervous system is insufficiently studied. Here, we identify a role of FMRP in liver disease.

Induced pluripotent stem cells (iPSCs) are similar to embryonic stem cells and can be generated from somatic cells. We have generated episomal plasmid-based and integration-free iPSCs (E-iPSCs) from human fetal foreskin fibroblast cells (HFF1). We used an E-iPSC-line to model hepatogenesis in vitro. The HLCs were characterized biochemically, i.e. glycogen storage, ICG uptake and release, UREA and bile acid production, as well as CYP3A4 activity. Ultra-structure analysis by electron microscopy revealed the presence of lipid and glycogen storage, tight junctions and bile canaliculi- all typical features of hepatocytes. Furthermore, the transcriptome of undifferentiated E-iPSC, DE, HE and HLCs were compared to that of fetal liver and primary human hepatocytes (PHH). K-means clustering identified 100 clusters which include developmental stage-specific groups of genes, e.g. OCT4 expression at the undifferentiated stage, SOX17 marking the DE stage, DLK and HNF6 the HE stage, HNF4α and Albumin is specific to HLCs, fetal liver and adult liver (PHH) stage. We use E-iPSCs for modeling gene regulatory networks associated with human hepatogenesis and gastrulation in general.

To investigate the relation of two different mutations to the outcome of partial external biliary diversion (PEBD) in severe bile salt export pump (BSEP) deficiency.