[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.

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.

An association between NIDDM and a GAA trinucleotide repeat polymorphism in the X25/frataxin (Friedreich's ataxia) gene.

Friedreich's ataxia is the most common hereditary ataxia and is frequently associated with disturbances of glucose metabolism. This autosomal recessive disease is caused by the decreased expression of a mitochondrial protein, frataxin, encoded by the X25 gene. Homozygous expansion of a GAA repeat in the first intron of X25 inhibits frataxin expression and is associated with clinical disease. We evaluated whether heterozygous expansions of the triplet repeat in the frataxin gene X25 may be associated with NIDDM in two genetically distinct populations--one in Germany (n = 358) and the other in the U.S. (n = 292)--using a polymerase chain reaction-based assay. Intermediate expansions (10-36 repeats), which are longer than normal but not sufficient for the appearance of the ataxia phenotype, were found in 24.7 and 27.3% of these two NIDDM cohorts compared with 7.6 and 6.3% of the matched control subjects (both P < 0.001). The odds ratios were 3.36 (95% CI 1.72-6.55) for the German group and 4.01 (2.08-7.74) for the U.S. group. Therefore, we conclude that the X25/frataxin GAA repeat polymorphism is associated with NIDDM in a frequency higher than any other mutation heretofore described. Further studies are needed to elucidate the possible role of frataxin in the pathogenesis of NIDDM.

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.

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.

[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.

Metabolic syndrome and hypertension: pathophysiology and molecular basis of insulin resistance.

Several recent studies indicate that type 2 diabetes, arterial hypertension, lipid disorders as well as visceral obesity are coronary risk factors which might belong to a syndrome which is caused by decreased insulin sensitivity with compensatory hyperinsulinaemia. More than 50% of patients with essential hypertension have some degree of insulin resistance, but in contrast to dyslipoproteinaemia and glucose intolerance the causal relation between insulin resistance and elevated arterial blood pressure appears not to be as evident. One explanation is that the link between blood pressure and insulin sensitivity might be mainly related to concomitant obesity. Accordingly, obesity can be associated with an increased activity of the sympathetic nervous system, elevated plasma levels of the vasoconstrictor endothelin-1, and decreased insulin-induced endothelium-dependent vasodilation. Furthermore, adipocytes can secrete vasogenic peptides, such as angiotensinogen. Since insulin resistance is a polygenic disorder, the two basic genetic approaches we follow is to identify genetic defects of insulin action in cells of patients with inherited syndromes of insulin resistance and to characterize molecular mechanisms of insulin regulated gene expression. The results show that insulin can affect the expression rate of various genes, e.g. involved in cholesterol and fatty acid metabolism, by modulating the activity of transcription factors coupled to the MAP kinase cascade and that a genetic postreceptor defect in these intracellular signaling pathways might have a pleiotropic effect on cell metabolism and clinical phenotype.

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.

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.

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.

Defects of insulin and IGF-1 action at receptor and postreceptor level in a patient with type A syndrome of insulin resistance.

The action of insulin and IGF-1 in comparison to non-diabetic controls was studied in cultured fibroblasts of a patient with an inherited syndrome of insulin resistance (Type A syndrome). Insulin binding was reduced due to decreased receptor affinity, but sequence analyses revealed no alterations of splicing or primary insulin receptor (IR) structure. Most likely due to the IR affinity defect analyses of signal transduction pathways showed an impairment of insulin action on glucose uptake, total RNA synthesis and phosphorylation as well as activity of MAP-kinase. In addition inducibility of c-fos mRNA level was strongly impaired by insulin and IGF-1, but comparable to controls by PDGF indicating a postreceptor defect. In conclusion, we provide evidence that genetic syndromes of insulin resistance can be associated with both, receptor and postreceptor defects.

ADD1/SREBP-1c mediates insulin-induced gene expression linked to the MAP kinase pathway.

The aim of this study was to define the role of sterol regulatory element binding protein (SREBP)-1c, the human homologue to ADD1 (adipocyte determination- and differentiation-dependent factor 1), in insulin-induced gene expression. Transfection studies using SREBP-1-deficient cells and a LDL receptor promoter fragment containing the ADD1/SREBP-1c binding side showed that the effects of insulin and PDGF were abolished compared to control cells and completely reconstituted by overexpressing ADD1/SREBP-1c. Overexpression of upstream activators of MAP kinases, like MEKK1 or MEK1, demonstrated that ADD1/SREBP-1c-mediated effects of insulin and PDGF might be linked to the MAP kinase cascade. The recombinant N-terminal domain of ADD1/SREBP-1c was phosphorylated predominantly on serine and slightly on threonine residues by MAP kinases ERK1 and ERK2 in vitro. This was reversible by alkaline phosphatase. We conclude that ADD1/SREBP-1c mediates gene regulatory effects of insulin as well as PDGF and that this signalling is linked to the MAP kinase cascade.

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.

Identification of tyrosine phosphorylation sites in human Gab-1 protein by EGF receptor kinase in vitro.

Grb2-associated binder-1 (Gab-1) has been identified recently in a cDNA library of glioblastoma tumors and appears to play a central role in cellular growth response, transformation, and apoptosis. Structural and functional features indicate that Gab-1 is a multisubstrate docking protein downstream in the signaling pathways of different receptor tyrosine kinases, including the epidermal growth factor receptor (EGFR). Therefore, the aim of the study was to characterize the phosphorylation of recombinant human Gab-1 (hGab-1) protein by EGFR in vitro. Using the pGEX system to express the entire protein and different domains of hGab-1 as glutathione S-transferase proteins, kinetic data for phosphorylation of these proteins by wheat germ agglutinine-purified EGFR and the recombinant EGFR (rEGFR) receptor kinase domain were determined. Our data revealed similar affinities of hGab-1-C for both receptor preparations (KM = 2.7 microM for rEGFR vs 3.2 microM for WGA EGFR) as well as for the different recombinant hGab-1 domains. To identify the specific EGFR phosphorylation sites, hGab-1-C was sequenced by Edman degradation and mass spectrometry. The entire protein was phosphorylated by rEGFR at eight tyrosine residues (Y285, Y373, Y406, Y447, Y472, Y619, Y657, and Y689). Fifty percent of the identified radioactivity was incorporated in tyrosine Y657 as the predominant peak in HPLC analysis, a site exhibiting features of a potential Syp (PTP1D) binding site. Accordingly, GST-pull down assays with A431 and HepG2 cell lysates showed that phosphorylated intact hGab-1 was able to bind Syp. This binding appears to be specific, because it was abolished by changing the Y657 of hGab-1 to F657. These results demonstrate that hGab-1 is a high-affinity substrate for the EGFR and the major tyrosine phosphorylation site Y657 in the C terminus is a specific binding site for the tyrosine phosphatase Syp.