Ann Meulemans

In vitro differentiated adult human liver progenitor cells display mature hepatic metabolic functions : a potential tool for in vitro pharmacotoxicological testing.

The potential use of stem/progenitor cells as alternative cell sources to mature hepatocytes remains basically dependent on their ability to exhibit some, if not all, the metabolic liver functions. In the current study, four major liver functions were investigated in adult derived human liver stem/progenitor cell (ADHLSCs) populations submitted to in vitro hepatogenic differentiation: gluconeogenesis, ammonia detoxification, and activity of phase I and phase II drug-metabolizing enzymes. These acquired hepatic activities were compared to those of primary adult human hepatocytes, the standard reference. Amino acid content was also investigated after hepatogenic differentiation. Differentiated ADHLSCs display higher de novo synthesis of glucose correlated to an increased activity of glucose-6 phosphatase and mRNA expression of key related enzymes. Differentiated ADHLSCs are also able to metabolize ammonium chloride and to produce urea. This was correlated to an increase in the mRNA expression of relevant key enzymes such arginase. With respect to drug metabolism, differentiated ADHLSCs express mRNAs of all the major cytochromes investigated, among which the CYP3A4 isoform (the most important drug-metabolizing enzyme). Such increased expression is correlated to an enhanced phase I activity as independently demonstrated using fluorescence-based assays. Phase II enzyme activity and amino acid levels also show a significant enhancement in differentiated ADHLSCs. The current study, according to data independently obtained in different labs, demonstrates that in vitro differentiated ADHLSCs are able to display advanced liver metabolic functions supporting the possibility to develop them as potential alternatives to primary hepatocytes for in vitro settings.

A mitochondrial tRNA aspartate mutation causing isolated mitochondrial myopathy

Several mutations in mitochondrial transfer RNA (tRNA) genes can cause mitochondrial myopathy. We describe a young girl who presented with pronounced exercise intolerance. The anaerobic threshold and the maximal oxygen consumption were decreased. She had decreased complex I and IV enzyme activity and ragged red fibers on muscle biopsy. An A to G transition at nucleotide position 7526 in tRNA Aspartate (tRNAAsp) gene was heteroplasmic in several of the patients tissues. We were unable to detect the mutation in muscle tissue from the patients mother. This case adds a new genetic etiology for mitochondrial myopathy. It also illustrates for patients with combined deficiency of the complex I and IV enzyme activity the value of sequencing in the affected tissue muscle, and not only in blood, all mitochondrial tRNA genes including those not commonly affected, such as in this case mt tRNAAsp.

Neonatal Thyroid-Stimulating Hormone Concentrations in Belgium: A Useful Indicator for Detecting Mild Iodine Deficiency?

It has been proposed that neonatal thyroid-stimulating hormone (TSH) concentrations are a good indicator of iodine deficiency in the population. A frequency of neonatal TSH concentrations above 5 mU/L below 3% has been proposed as the threshold indicating iodine sufficiency. The objective of the present study was to evaluate feasibility and usefulness of nation-wide neonatal TSH concentration screening results to assess iodine status in Belgium. All newborns born in Belgium during the period 2009–2011 (n = 377713) were included in the study, except those suffering from congenital hypothyroidism and premature neonates. The frequency of neonatal TSH concentrations above 5 mU/L from 2009 to 2011 in Belgium fluctuated between 2.6 and 3.3% in the centres using the same TSH assay. There was a significant inverse association between neonatal TSH level and birth weight. The longer the duration between birth and screening, the lower the TSH level. Neonatal TSH levels were significantly lower in winter than in spring or autumn and significantly lower in spring and summer than in autumn while significantly higher in spring compared to summer. In conclusion, despite that pregnant women in Belgium are mildly iodine deficient, the frequency of neonatal TSH concentrations above 5 mU/L was very low, suggesting that the neonatal TSH threshold proposed for detecting iodine deficiency needs to be re-evaluated. Although neonatal TSH is useful to detect severe iodine deficiency, it should not be recommended presently for the evaluation of iodine status in mildly iodine deficient regions.

Subcomplexes of mitochondrial complex V reveal mutations in mitochondrial DNA

Complex V, site of the final step in oxidative phosphorylation, uses the proton gradient across the inner mitochondrial membrane for the production of ATP. It is a multi‐subunit complex composed of a catalytic domain (F1) and a membrane domain (F0) linked by two stalks. Subcomplexes of complex V containing the F1 domain have previously been reported in small series of patients. We report the results in tissue samples and/or cultured skin fibroblasts studied by blue native PAGE followed by activity staining in the gel. Catalytically active subcomplexes of complex V were detected in 66 tissues originating from 53 patients. In 29 of the latter (55%), a mitochondrial DNA (mtDNA) defect was identified. Twelve patients had a pathogenic point mutation in a mitochondrial tRNA, one a large mtDNA deletion, 12 showed mtDNA depletion and four had a mutation in the MT‐ATP6 gene. We conclude that the presence of subcomplexes of complex V is a valuable indicator in the detection of mtDNA defects.

Diagnostic Value of Immunostaining in Cultured Skin Fibroblasts from Patients with Oxidative Phosphorylation Defects

In the last decades, a large variety of oxidative phosphorylation (OXPHOS) defects have been reported, expressed as an increasing variety of clinical phenotypes. With the expanding number of genes and proteins involved, new screening techniques leading to more effective diagnostic routes are in ever-increasing demand. Cultured skin fibroblasts from a cohort of patients with various OXPHOS defects, previously recognized by enzyme activity studies and blue native PAGE, were investigated with an immunocytochemical technique. Cytospins of cultured fibroblasts were air dried, fixed, and stained with antibodies specifically directed against subunits of each OXPHOS complex. Control cells stained homogeneously and strongly. In fibroblasts from five out of seven patients with a severe deficiency of one of the OXPHOS complexes, a homogeneous reduction of cytoimmunoreactivity of the affected complex was observed. In five out of seven fibroblast strains harboring a mitochondrial tRNA mutation, a mosaic pattern of staining was observed for both complexes I and IV, reflecting the heteroplasmic nature of the defect. The proportion of deficient fibroblasts varied considerably between cell strains from different subjects. The method described offers a convenient and rapid approach to first-line screening of OXPHOS defects. In association with routine assays of enzyme activity, the technique is helpful in orienting molecular investigation further.

At similar weight loss, dietary composition determines the degree of glycemic improvement in diet-induced obese C57BL/6 mice

Background Achieving weight loss is the cornerstone of the treatment of the metabolic consequences of obesity, in particular of glucose intolerance. Objective To determine whether improvement in glucose control depends on dietary macronutrient composition of the diet at identical weight loss. Materials and methods Twenty-two weeks old diet-induced obese C57BL/6 mice lost weight through caloric restriction on normal chow (R-NC) or high fat diet (R-HF). Control mice were fed normal chow (LEAN) or high fat diet (OBESE) ad libitum. Body weight and composition were assessed after 8 weeks of dietary intervention. Glucose homeostasis was evaluated by intraperitoneal glucose tolerance tests (IPGTT). Epididymal white adipose (eWAT) and hepatic tissues were analyzed by immunohistochemistry and RT-qPCR. Results By 30 weeks of age, the body weight of the mice on R-NC (31.6±1.7g, mean±SEM) and R-HF (32.3±0.9g) was similar to LEAN mice (31.9±1.4g), while OBESE mice weighed 51.7±2.4g. Glucose tolerance in R-NC was better than in LEAN mice (69% AUC IPGTT, P 0.0168) whereas R-HF mice remained significantly less glucose tolerant (125% AUC IPGTT, P 0.0279 vs LEAN), despite identical weight loss. The eWAT pads and adipocyte size were similar in LEAN and R-NC mice, while the eWAT pad size of R-HF was 180% of R-NC (P < 0.0001) and the average adipocyte size of R-HF mice was 134% of R-NC fed mice (P 0.0285). No LEAN or R-NC mice had hepatic steatosis, in contrast to 28.6% of R-HF mice. Compared to OBESE mice, inflammatory markers were lower in eWAT and liver tissue of R-NC, but not in R-HF mice. Measures of visceral adiposity correlated well with glucose tolerance parameters. Conclusions In mice, caloric restriction on a normal chow diet improved glucose tolerance significantly more when identical weight loss was achieved on a high fat diet.

1255 Maternal Betamethason Administration is An Indicator But Not An Independent Risk Factor for Raised 17-Hydroxyprogesterone at Neonatal Screening

1255 Maternal Betamethason Administration is An Indicator But Not An Independent Risk Factor for Raised 17-Hydroxyprogesterone at Neonatal Screening