The natural history of classic galactosemia: lessons from the GalNet registry.

BACKGROUND: Classic galactosemia is a rare inborn error of carbohydrate metabolism, caused by a severe deficiency of the enzyme galactose-1-phosphate uridylyltransferase (GALT). A galactose-restricted diet has proven to be very effective to treat the neonatal life-threatening manifestations and has been the cornerstone of treatment for this severe disease. However, burdensome complications occur despite a lifelong diet. For rare diseases, a patient disease specific registry is fundamental to monitor the lifespan pathology and to evaluate the safety and efficacy of potential therapies. In 2014, the international Galactosemias Network (GalNet) developed a web-based patient registry for this disease, the GalNet Registry. The aim was to delineate the natural history of classic galactosemia based on a large dataset of patients. METHODS: Observational data derived from 15 countries and 32 centers including 509 patients were acquired between December 2014 and July 2018. RESULTS: Most affected patients experienced neonatal manifestations (79.8%) and despite following a diet developed brain impairments (85.0%), primary ovarian insufficiency (79.7%) and a diminished bone mineral density (26.5%). Newborn screening, age at onset of dietary treatment, strictness of the galactose-restricted diet, p.Gln188Arg mutation and GALT enzyme activity influenced the clinical picture. Detection by newborn screening and commencement of diet in the first week of life were associated with a more favorable outcome. A homozygous p.Gln188Arg mutation, GALT enzyme activity of ≤ 1% and strict galactose restriction were associated with a less favorable outcome. CONCLUSION: This study describes the natural history of classic galactosemia based on the hitherto largest data set.

[SREBP-1 and fatty liver. Clinical relevance for diabetes, obesity, dyslipidemia and atherosclerosis]. / SREBP-1 und Fettleber. Klinische Bedeutung für Diabetes, Adipositas, Dyslipidämie und Atherosklerose.

Insulin resistance and visceral fat distribution usually play a major role in the development of clinical aspects of the metabolic syndrome, such as dyslipidemia, diabetes and atherosclerosis. In this review, the focus will be on some novel relationships with a fatty liver, for which susceptibility appears to be mediated by the activity of transcription factors, such as sterol regulatory element-binding protein 1 (SREBP-1). In addition to this molecular aspect therapeutic life-style modifications, such as weight reduction which are associated with increased insulin sensitivity and a decrease of fat in the liver will be discussed.

[Combined analyses of paraoxonase-1 and IGF-2 polymorphism in polycystic ovary syndrome]. / Kombinierte Analyse von Paraoxonase-1- und IGF-2-Polymorphismen bei polyzystischem Ovarsyndrom.

BACKGROUND: Apart from impaired reproductive function patients with polycystic ovary syndrome (PCOS) also have signs and symptoms belonging to the metabolic syndrome. A genetic basis for PCOS is likely as the syndrome clusters in families. Putative candidate genes are paraoxonase (PON)-1 gene and the IGF-2 INS1/VTR IGF cluster, which have been shown to be genetically linked to lipid metabolism o insulin sensitivity, two major aspects of the PCOS phenotype. PATIENTS AND METHODS: The ApaI polymorphism (rs:680) in the IGF-2 cluster and the -108 polymorphism (rs:705 379) in PON-1 were evaluated in a collective of 153 PCOS patients and 178 age and BMI matched controls for an association to PCOS. RESULTS: The polymorphism in the IGF-2 cluster was identified in both groups in comparable frequencies (PCOS/control: A: 0.351/0.325; G: 0.648/0.674; OR: 0.8886, 95 %CI 0,648-1.2236) and equal genotype distribution (PCOS/control: GG: 0.399/0.461; AG: 0.4962/0.4277; AA: 0.1042/0.111). Frequencies of the PON-1 polymorphism were also comparable (PCOS/control: T: 0.493/0.483; C: 0.5633/0.5168; OR: 0.9569 95 % CI: 0.707-1.43024), but the distribution (PCOS/control: CC: 0.2679/0.2032; CT: 0.4768/0.628; TT: 0.258/0.169) was significantly different. The combined analyses of both polymorphism revealed that the genotypes IGF-2 (GG)/ PON-1 (TT) with OR 1.64741 (95 % CI 0.7388 - 3.6735) and IGF-2 (AA)/ PON-1 (TT) with OR 2.6733 (95 % CI 0.7579 - 9.4291) were more frequent in the PCOS group, whereas the genotype IGF-2 (AA)/ PON-1 (CC) did not occur in the PCOS group at all. According to the molecular analyses significant differences in serum parameters were identified. CONCLUSION: This investigation indicates, that only the combined analyses of putative candidate genes allowed a genotype-phenotype correlation in PCOS.

Increased low grade inflammatory serum markers in patients with Polycystic ovary syndrome (PCOS) and their relationship to PPARgamma gene variants.

The Polycystic ovary syndrome (PCOS) is the most frequent endocrine disorder in premenopausal women and is associated with features of the insulin resistance syndrome, altered glucose homeostasis, and central obesity. Inflammation appears to be a link between obesity and insulin resistance, because adipose tissue is one major source of proinflammatory cytokines. Since peroxisome proliferator-activated receptor (PPAR)gamma affects adipocyte differentiation as well as insulin sensitivity, we investigated whether the levels of proinflammatory factors in PCOS patients are related to sequence variations of the PPAR gamma gene. Proinflammatory cytokine levels, i.e. IL-1 beta, IL-6, IL-7, IL-8, IL-17 and TNFalpha, were evaluated in PCOS patients (n=21) in comparison to obese controls (n=120). Next to this the complete coding sequence of the PPAR gamma gene was investigated by resequencing all probands. We show that the levels of IL-8 and IL-17 were unchanged, IL-1 beta, IL-6 and TNFalpha were elevated and the level of IL-7 was decreased in PCOS patients compared to obese controls. Sequence analyses of the PPAR gamma gene indicated that neither the common polymorphisms P12A or H478 H, nor novel polymorphisms (E79Q, V32G, -39 T>C, c.480 +33 t > g,) or unique sequence variations (S22S, A23A, T41A, S226C, K272 T, I484I, c.819 +24 a>c) detected in this investigation revealed evidence for a direct association of PPAR gamma with altered IL-7, IL-1beta, IL-6 and TNFalpha levels in PCOS patients. So, alterations in inflammatory serum markers appear to be a feature of PCOS per se, and are independent of PPAR gamma variants.

[LDL cholesterol--why lower and lower?]. / Wie weit sollen wir die Cholesterinwerte noch senken? Für Hochrisikopatienten kann das LDL nicht niedrig genug sein.

Large interventional studies with statins have confirmed that drug-lowering of LDL cholesterol by at least 25-30% is associated with a significant reduction in the cardiovascular risk. In high-risk patients, the plasma LDL cholesterol level should be < 100 mg/dl. If the levels are initially low, the aim is to achieve a reduction of at least 30-40%. More recent studies have shown that reducing the LDL cholesterol to < 70 mg/dl can stop the further growth of plaques, and can possibly even bring about their regression.

Reduction of postchallenge hyperglycaemia prevents acute endothelial dysfunction in subjects with impaired glucose tolerance.

OBJECTIVES: To investigate whether selective reduction of postchallenge hyperglycaemia influences acute endothelial dysfunction, a very early manifestation of vascular disease, in patients with impaired glucose tolerance. METHODS: In a randomized, double-blind, placebo-controlled, cross-over study the acute effect of 200-mg acarbose was investigated in 28 subjects with diagnosed impaired glucose tolerance. Flow-mediated dilation (FMD) of the brachial artery was determined as a measure of endothelial function before and 2 and 3 h after ingestion of 100-g saccharose. Asymmetrical dimethylarginine (ADMA) was measured by high-performance liquid chromatography. RESULTS: A negative correlation was observed between the changes of glucose and FMD (r = 0.416, P = 0.0018) 2 h after ingestion of saccharose. At 3 h, neither blood glucose nor FMD were different from baseline. Changes of both blood glucose (P = 0.0007) and FMD (P = 0.046) were significantly lower after administration of acarbose. Subgroup analysis revealed that the effect of acarbose was restricted to those subjects with an increase of blood glucose above the median increase of glycaemia. No changes of plasma ADMA were observed. CONCLUSIONS: Our data clearly demonstrate that the postchallenge alteration of vascular function in patients with impaired glucose tolerance is caused by the acute elevation of glycaemia but not mediated by ADMA.

Reduced phosphorylation of transcription factor Elk-1 in cultured fibroblasts of a patient with premature aging syndrome and insulin resistance.

The effect of insulin and growth factor mediated signaling to gene regulation was investigated in cultured fibroblasts of a patient with a premature aging syndrome (metageria) and severe insulin resistance. Insulin receptor structure and function as well as major pathways activated by insulin, i.e. phosphatidyl inositol-3 kinase (PI-3 K) cascade or mitogen-activated protein kinase (MAPK) cascades, were functional. Inducibility of the proto-oncogene cfos, a representative endpoint of signaling pathways related to gene expression, by growth factors or insulin was reduced in patient cells. This reduced induction persisted in cfos promoter reporter gene studies indicating that the post receptor defect is localized proximal to the cfos promoter itself. Abundances of the transcription factors Elk-1 and SRF being major players in coupling of MAPKs to cfos promoter activation were not altered. However, basal and inducible phosphorylation of Elk-1 was impaired. In addition, basal and stimulated transcriptional activity mediated by Elk-1 was almost abolished in patient cells. Therefore these results identify a post receptor defect in cFos induction, which appears to be related to a functional alteration of Elk-1. A possible relation of this signal transduction defect to the specific premature aging syndrome remains to be elucidated.

The Pro387Leu variant of protein tyrosine phosphatase-1B is not associated with diabetes mellitus type 2 in a German population.

OBJECTIVES: Diabetes mellitus type 2 (DM-2) is a complex disorder with a strong genetic background. Protein tyrosine phosphatase-1B (PTP-1B) dephosphorylates various receptor protein kinases in vitro, including the beta subunit of the insulin receptor, therefore representing a potential candidate to be involved in the polygenic pathogenesis of DM-2. Recently a Pro387Leu variant of the PTP-1B gene has been associated with an increased risk of DM-2 in a Danish population. Reports from China and Finland failed to confirm this association. DESIGN, SETTING AND SUBJECTS: The purpose of the present study was to examine the possible association between the presence of DM-2 and the Pro387Leu polymorphism in a German Caucasian population. A total of 836 subjects (age 20-92 years) participated in the study. The presence of the Pro387Leu variant of the PTP-1B gene was investigated using polymerase chain reaction (PCR) restriction fragment-length polymorphism in 402 subjects with DM-2 (231 men, 171 women, age 63.1 +/- 10.8 years, BMI 28.7 +/- 5.1 kg m(-2)) and in 434 normoglycemic age- and sex-matched control subjects (248 men, 186 women, age 64.4 +/- 6.5 years, BMI 26.5 +/- 3.7 kg m(-2)). RESULTS: Nine subjects in the control group and nine in the diabetic group (allelic frequency 0.99% in both groups) carried the Pro387Leu polymorphism. A meta-analysis on published data of >3000 subjects including our own data did not find an association between the polymorphism and DM-2. In addition, the polymorphism had no significant influence on the presence of atherosclerotic disease, whilst the influence of other known cardiovascular risk factors was confirmed. Furthermore, the impact of the mutation on metabolic and anthropometric parameters in both groups was examined. Amongst the controls there were no significant differences in BMI, HDL and LDL cholesterol or blood pressure between the two groups with or without the Pro387Leu polymorphism. The same was true for the diabetic group. Interestingly, in both diabetics and controls, Pro387Leu carriers had significantly higher triglycerides. In a logistic regression model only BMI and family history but not polymorphism were predictors of DM-2. CONCLUSIONS: In conclusion, the present data suggest that in a German Caucasian population the Pro387Leu polymorphism of the PTP-1B gene is not associated with DM-2 but may play a role in other metabolic phenotypes.

Correction of insulin resistance and the metabolic syndrome.

Insulin resistance is a common phenomenon of the metabolic syndrome, which is clinically characterized by a clustering of various cardiovascular risk factors in a single individual and a higher prevalence of respective complications, such as coronary heart disease and stroke. At the cellular level, insulin resistance is defined as a reduced insulin action, which can affect not only glucose uptake, but also gene regulation. Elucidation of novel signaling networks within the cell which are mediating and affecting insulin action will reveal many new genes and drug targets that are potentially of clinical relevance in the future. In this chapter, we propose that the metabolic syndrome might be a clinical consequence of altered gene regulation. This is illuminated in the context of transcription factors, e.g., sterol regulatory element binding proteins (SREBPs), coupling signals from nutrients, metabolites, and hormones at the gene regulatory level with pathobiochemical features of increased lipid accumulation in lean nonadipose tissues. The phenomenon of ectopic lipid accumulation (lipotoxicity) appears to be a novel link between insulin resistance, obesity, and possibly other features of the metabolic syndrome. Therefore, the investigation of specific gene regulatory networks and their alterations might be a clue to understanding the development and clustering of different cardiovascular risk factors in different individuals. As cellular sensors transcription factors--as common denominators of gene regulatory networks--might thereby also determine the susceptibility of individuals to cardiovascular risk factors and their complications.

A novel nonsense mutation in GCK exon 9 co-segregates with diabetes phenotype.

Maturity-onset diabetes of the young is an autosomal dominant form of non-insulin dependent diabetes mellitus and is caused by mutations in at least six different genes. In the most common forms, i.e. MODY2 and MODY3, the glucokinase (GCK) and the hepatocyte nuclear factor (HNF)-1alpha gene is affected, respectively. We have screened the GCK gene and HNF-1alpha gene by direct sequencing in three German families with early onset type-2-diabetes, possibly MODY. Next to known polymorphisms we have identified two novel intronic insertions in GCK and a novel non-sense mutation in exon 9 (C364 X). The latter mutation has an autosomal dominant inheritance pattern. Accordingly, this novel mutation segregates with diabetes phenotype in this family.

Identification of a novel mutation in the arginine vasopressin-neurophysin II gene in familial central diabetes insipidus.

Familial central diabetes insipidus is an inherited disease of predominant autosomal dominant trait characterized by a deficiency of arginine vasopressin. The arginine vasopressin-neurophysin II ( AVP-NPII) gene consists of three exons and is located on chromosome 20p13 encoding for the precursor protein of AVP. We investigated two Caucasian families with a typical autosomal dominant trait of familial central diabetes insipidus, defined by deficiency of arginine vasopressin. After PCR amplification of exon 1 and exon 2/3, fragments were pooled and purified. Nucleotide sequencing was performed with the Taq DyeDeoxy-terminator cycle sequencing method using nested primers. Two mutations in the coding region of NPII were identified. In family C we found a heterozygous G ==> C missense mutation (AA61) in exon 2 leading to the substitution of cystein with serine. In family D a novel heterozygous nonsense mutation in exon 3 (AA 83, GAG ==> TAG) was indentified, leading to a stop codon instead of glutamine. Both mutations were confirmed by restriction analysis and were found in all affected but not in healthy family members or control subjects. We therefore have identified a missense mutation of the AVP-NPII gene and a novel mutation predicting a truncated protein.

Insulin-regulated transcription factors: molecular link between insulin resistance and cardiovascular risk factors.

Patients with insulin resistance and/or type 2 diabetes have a 5-fold increase in cardiovascular mortality rate. Therefore, it is a current issue of discussion that arterial hypertension, lipid disorders as well as visceral obesity are coronary risk factors, which might belong to a syndrome that is caused by decreased insulin sensitivity. Concerning a possible molecular link between insulin resistance, atherosclerosis and obesity, we focus in our research on questions looking for a molecular link between lipid metabolism, insulin action, and obesity at a gene regulatory level. Alterations in the structure, function and regulation of transcription factors appear to be such signalling steps which might play an essential role in the pathogenesis and therapy of cardiovascular risk factors associated with insulin resistance, eg the so called metabolic syndrome. Recent examples are members of the nuclear hormone receptor superfamily, eg peroxisome proliferator-activated receptor (PPAR) isoforms and sterol regulatory element-binding proteins (SREBPs). Beside their regulation by different metabolites, these transcription factors are also targets of hormones, like insulin and leptin, growth factors, and inflammatory signals. Therefore, they appear to be a point of signalling convergence at a gene regulatory level. Major signalling pathways coupling receptors at the cell surface for hormones, growth factors as well as cytokines to gene regulatory events in the nucleus are the MAP-kinase cascades. We have recently defined different postreceptor defects in these pathways in patients with clinical phenotypes corresponding to congenital lipoatrophy. Therefore, these studies may identify novel pathways which play a role in the control of body weight, insulin sensitivity and cardiovascular risk.

Identification of major tyrosine phosphorylation sites in the human insulin receptor substrate Gab-1 by insulin receptor kinase in vitro.

Gab-1 (Grb2-associated binder-1), which appears to play a central role in cellular growth response, transformation, and apoptosis, is a member of the insulin receptor substrate (IRS) family. IRS proteins act downstream in the signaling pathways of different receptor tyrosine kinases, including the insulin receptor (IR). In this paper, we characterize the phosphorylation of recombinant human Gab-1 (hGab-1) by IR in vitro. Kinetic phosphorylation data revealed that hGab-1 is a high affinity substrate for the IR (K(M): 12.0 microM for native IR vs 23.3 microM for recombinant IR). To elucidate the IR-specific phosphorylation pattern of hGab-1, we used phosphopeptide mapping by two-dimensional HPLC analysis. Phosphorylated tyrosine residues were subsequently identified by sequencing the separated phosphopeptides by matrix assisted laser desorption ionization mass spectrometry (MALDI-MS) and Edman degradation. Our results demonstrate that hGab-1 was phosphorylated by IR at eight tyrosine residues (Y242, Y285, Y373, Y447, Y472, Y619, Y657, and Y689). Seventy-five percent of the identified radioactivity was incorporated into tyrosine residues Y447, Y472, and Y619 exhibiting features (NYVPM motif) of potential binding sites for the regulatory subunit (p85) of phosphatidylinositol (PI)-3 kinase. Accordingly, pull down assays with human HepG2 cell lysates showed that IR-specific phosphorylation of wild-type hGab-1 strongly enhanced PI-3 kinase binding. This is still the case when a single tyrosine residue in the NYVPM motif was mutated to phenylalanine. In contrast, phosphorylation-dependent binding of PI-3 kinase was completely abolished by changing a second tyrosine residue in a NYVPM motif independent from its location. Recently, we identified a similar cohort of tyrosine phosphorylation sites for the epidermal growth factor receptor (EGFR) with a predominant phosphorylation of tyrosine residue Y657 and binding of Syp [Lehr, S. et al. (1999) Biochemistry 38, 151-159]. These differences in the phosphorylation pattern of hGab-1 may contribute to signaling specificity by different tyrosine kinase receptors engaging distinct SH2 signaling molecules.

MAP kinases Erk1/2 phosphorylate sterol regulatory element-binding protein (SREBP)-1a at serine 117 in vitro.

Sterol regulatory element-binding protein (SREBP)-1a is a transcription factor sensing cellular cholesterol levels and integrating gene regulatory signals mediated by MAP kinase cascades. Here we report the identification of serine 117 in SREBP-1a as the major phosphorylation site of the MAP kinases Erk1/2. This site was identified by nanoelectrospray mass spectrometry and peptide sequencing of recombinant fusion proteins phosphorylated by Erk1/2 in vitro. Serine 117 was verified as the major phosphorylation site by in vitro mutagenesis. Mutation of serine 117 to alanine abolished Erk2-mediated phosphorylation in vitro and the MAP kinase-related transcriptional activation of SREBP-1a by insulin and platelet-derived growth factor in vivo. Our data indicate that the MAP kinase-mediated effects on SREBP-1a-regulated target genes are linked to this phosphorylation site.

Ribosomal subunit kinase-2 is required for growth factor-stimulated transcription of the c-Fos gene.

Ribosomal subunit kinases (Rsk) have been implicated in the regulation of transcription by phosphorylating and thereby activating numerous transcription factors, such as c-Fos, cAMP responsive element binding protein (CREB), and nuclear receptors. Here we describe the generation and characterization of immortalized embryonic fibroblast cell lines from mice in which the Rsk-2 gene was disrupted by homologous recombinant gene targeting. Rsk-2-deficient (knockout or KO) cell lines have no detectable Rsk-2 protein, whereas Rsk-1 expression is unaltered as compared with cell lines derived from wild-type control mice. KO cells exhibit a major reduction in platelet-derived growth factor (PDGF) and insulin-like growth factor (IGF)-1-stimulated expression of the immediate-early gene c-Fos. This results primarily from a reduced transcriptional activation of the ternary complex factor Elk-1 and reduced activation of the serum response factor. The reduced Elk-1 activation in KO cells occurs despite normal activation of the mitogen-activated protein kinase pathway and normal PDGF- and IGF-1-stimulated Elk-1 phosphorylation. By contrast, PDGF- and IGF-1-stimulated phosphorylation and transcriptional activation of CREB is unaltered in KO cells. Thus Rsk-2 is required for growth factor-stimulated expression of c-Fos and transcriptional activation of Elk-1 and the serum response factor, but not for activation of CREB or the mitogen-activated protein kinase pathway in response to PDGF and IGF-1 stimulation.

Characterization of a postreceptor signaling defect that impairs cfos expression in cultured fibroblasts of a patient with insulin resistance.

Induction of cfos expression is a definite end point of signal transduction by receptor tyrosine kinases via MAPK cascades. We have examined signal transduction to transcription factor cFos in isolated fibroblasts of a patient with an inherited syndrome of insulin resistance. MAPK phosphorylation and activity were unaltered, but inducibility of cfos transcription was strongly impaired by insulin and reduced by PDGF. Induction of the cfos promoter via MAPK is mediated by activation of the ternary complex. Abundance of SRF or Elk-1 was unaltered, but Elk-1 phosphorylation following stimulation was reduced. Transient transfections with reporter genes under control of the Elk-1 binding ets/sre cis element or expression plasmids coding for the regulatory domain of Elk-1 fused to heterologous DNA binding domains revealed a defect of Elk-1 activation in the patient cells. These data identify a novel postreceptor defect of insulin and growth factors involving activation of transcription.

Sterol regulatory element binding proteins (SREBP)-1a and SREBP-2 are linked to the MAP-kinase cascade.

The classic sterol regulatory cis element (sre-1) in the LDL receptor promoter mediates sterol regulatory element binding protein (SREBP)-binding and the effects of insulin and platelet derived growth factor (PDGF). To elucidate whether SREBP-1a and SREBP-2 play a direct role in insulin and PDGF action, stable cell lines of HepG2 deficient in either SREBP-1 or SREBP-2 were used. Transfection of these cells with the wild-type promoter fragment of the low density lipoprotein (LDL) receptor gene showed that the effects of insulin and PDGF were significantly reduced in both, SREBP-1- as well as SREBP-2-deficient cells. Insulin and PDGF action could be reconstituted again in these deficient cell lines by reintroducing SREBP-1a or SREBP-2. Preincubation of cells with either the phosphatidylinositol (PI)-3 kinase inhibitor wortmannin or the mitogen-activated protein (MAP) kinase cascade inhibitor PD 98059 showed that the latter abolished the stimulatory effects of insulin and PDGF on LDL receptor promoter activity completely, whereas wortmannin had no effect. Overexpression of upstream activators of the MAP kinases, like MEKK1 or MEK1, stimulated LDL receptor promoter activity several fold in an sre-1 related manner. These effects could be enhanced by coexpression of the transcriptional active N-terminal domains of SREBP-1a and SREBP-2. Using the heterologous Gal-4 system, we could show that intracellular activation of the MAP kinase cascade by ectopic expression of MEKK1 or MEK1 has a direct stimulatory effect on the transcriptional activity of SREBP-1a and SREBP-2. Experimental evidence for a direct link between MAP kinases and SREBPs was obtained due to the MAP kinases ERK1 and ERK2 phosphorylating recombinant GST-fusion proteins of SREBP-1a and SREBP-2, in vitro. We conclude that SREBP-1a and SREBP-2 mediate different regulatory effects converging at sre-1 and that they appear to be linked to the MAP kinase cascade, possibly being direct substrates of ERK1 and ERK2.