Biomedical subjects

Ralf Paschke

Publications and source records attributed to Ralf Paschke.

10 recordsLinked to original sources

PubMed2003

Functional significance of the thyrotropin receptor germline polymorphism D727E.

In a toxic thyroid adenoma we identified a novel somatic mutation that constitutively activates the thyrotropin receptor (TSHR). Two heterozygous point mutations at adjacent nucleotides led to a substitution of alanine with asparagine at codon 593 (A593N) in the fifth transmembrane helix of TSHR. This somatic mutation resided on the same TSHR allele with the germline polymorphism D727E. The functional characteristics of the single TSHR mutants A593N and D727E and of the double mutant A593N/D727E were studied in transiently transfected COS-7 cells. The TSHR mutants A593N and A593N/D727E constitutively activated the cAMP cascade, whereas the D727E mutant did not differ from the wild-type TSHR. Surprisingly, the double mutant's specific constitutive activity was 2.3-fold lower than the A593N mutant. Thus, the polymorphism significantly ameliorates G(alphas) protein activation in the presence of the gain-of-function mutation A593N, although it is functionally inert in the context of the wild-type TSHR.

Adenoma↗

PubMed2003

Adiponectin gene expression and secretion is inhibited by interleukin-6 in 3T3-L1 adipocytes.

Recently, it has been shown that adiponectin is an important insulin-sensitizing fat-derived protein which is downregulated in insulin resistance and obesity, and replenishment of which improves insulin sensitivity. In contrast, interleukin (IL)-6 appears as an adipocytokine serum concentrations of which are elevated in these states. However, it has not been determined whether IL-6 might impact on expression and secretion of adiponectin. To clarify this, 3T3-L1 adipocytes were treated with different concentrations of IL-6 for various periods of time. Adiponectin mRNA was measured by quantitative real-time reverse transcription-polymerase chain reaction and secretion was determined by radioimmunoassays. Interestingly, treatment of 3T3-L1 cells with 30 ng/ml IL-6 significantly decreased adiponectin secretion to 75% of control levels. Adiponectin secretion was also inhibited between 25% and 45% by chronic treatment with forskolin (50 microM), tumor necrosis factor alpha (100 ng/ml), and dexamethasone (100 nM). Furthermore, adiponectin mRNA expression was downregulated by up to 50% in a time- and dose-dependent manner, with significant inhibition detectable at concentrations as low as 3 ng/ml IL-6 and as early as 8h after effector addition. The inhibitory effect of IL-6 was partially reversed by pretreatment of 3T3-L1 cells with pharmacological inhibitors of a p44/42 mitogen-activated protein (MAP) kinase. Moreover, the negative effect of IL-6 on adiponectin mRNA expression could be reversed by withdrawal of the hormone for 24h. Taken together, our results suggest that adiponectin gene expression is reversibly downregulated by IL-6 and support the concept of adiponectin being an important selectively controlled modulator of insulin sensitivity.

3T3 Cells↗

PubMed2003

Cold thyroid nodules show a marked increase in proliferation markers.

Thyroid follicular adenomas and adenomatous thyroid nodules are a frequent finding in geographical areas with iodine deficiency. They occur as hypofunctioning (scintigraphically cold) or hyperfunctioning (scintigraphically hot) nodules. Their predominant clonal origin suggests that they result from clonal expansion of a single cell, which is very likely the result of a prolonged increase in proliferation compared with non-affected surrounding cells. To test whether increased cell proliferation is detectable in cold thyroid nodules, we studied paraffin-embedded tissue from 40 cold thyroid nodules and their surrounding normal thyroid tissue for the occurrence of the proliferating cell nuclear antigen (PCNA) and Ki-67 (MIB-1 antibody) epitopes as markers for cell proliferation. All 40 thyroid nodules were histologically well characterized and have been studied for molecular characteristics before. The labeling index (number of labeled cells versus total cell number) for nodular and surrounding tissue was calculated. In 33 cold thyroid nodules a significant (p < or = 0.05) increase in the labeling index for PCNA was detectable. In 19 cold thyroid nodules a significant (p < or = 0.05) increase in the labeling index for Ki-67 was detectable. Moreover, surrounding tissues with lymphocyte infiltration showed a significantly higher labeling index for both PCNA and Ki-67 compared with normal surrounding tissue. These findings are first evidence that an increased thyroid epithelial cell proliferation is a uniform feature common to most cold nodules. However, the increase of proliferation markers shows a heterogeneity that is not correlated with histopathologic, molecular, or clinical characteristics.

Biomarkers↗

PubMed2003

Analysis of the relationship between the Pro12Ala variant in the PPAR-gamma2 gene and the response rate to therapy with pioglitazone in patients with type 2 diabetes.

OBJECTIVE: To investigate the influence of peroxisome proliferator-activated receptor-gamma (PPAR-gamma) gene variants on the response rate to therapy with the thiazolidinedione (TZD) pioglitazone, because in vitro studies have suggested that genetic variants of the PPAR-gamma gene may influence the drug efficacy of TZD. RESEARCH DESIGN AND METHODS: A total of 131 patients were treated in an open-label, randomized, multicenter study with pioglitazone (45 mg o.d.) during a course of >or=26 weeks. Response to the pioglitazone therapy was defined by either a >20% decrease in fasting plasma glucose or a >15% decrease in HbA(1c) values after 26 weeks of pioglitazone treatment. We evaluated the association between the PPAR-gamma genotype and the response rate to pioglitazone treatment. RESULTS: The Pro12Ala and the Pro12Pro variants in the PPAR-gamma gene are not associated with the response rate to pioglitazone treatment in patients with type 2 diabetes. However, we identified initial fasting plasma glucose level >11.0 mmol/l, HbA(1c) value >9.0%, BMI >32 kg/m(2), and fasting C-peptide concentrations at baseline >2.5 pmol/l as predominant confounding factors for the responder frequency to pioglitazone treatment. CONCLUSIONS: The Pro12Ala variant in the PPAR-gamma gene does not affect the therapy efficacy of pioglitazone, suggesting that the drug-treatment response is independent from pharmacogenetic effects between PPAR-gamma and its ligand pioglitazone. Whether the Ala12Ala genotype plays a role in the response rate to TZD therapy remains to be determined.

Aged↗

PubMed2002

Hormonal regulation of adiponectin gene expression in 3T3-L1 adipocytes.

Recently, it has been demonstrated that the fat-derived protein adiponectin is an important insulin-sensitizing adipocytokine which is downregulated in insulin resistance and obesity and replenishment of which in adiponectin-deficient states improves insulin sensitivity. To clarify the regulation of adiponectin gene expression, 3T3-L1 adipocytes were treated with various hormones known to induce insulin resistance in vivo and adiponectin mRNA was measured by quantitative real-time reverse transcription-polymerase chain reaction. Interestingly, treatment of 3T3-L1 cells with 100 nM insulin, 10 ng/ml tumor necrosis factor (TNF) alpha, or 100 nM dexamethasone for 16 h suppressed adiponectin gene expression by about 50 to 85% while angiotensin 2, growth hormone, and triiodothyronine did not have any effect. Furthermore, insulin reduced the level of adiponectin mRNA in a dose- and time-dependent fashion with inhibition detectable at concentrations as low as 10 nM insulin and as early as 4 h after effector addition. The inhibitory effect of insulin was partially reversed by pretreatment of 3T3-L1 cells with pharmacological inhibitors of p44/42 mitogen-activated protein (MAP) kinase, phosphatidylinositol (PI) 3-kinase, and p70S6 kinase. Moreover, the negative effects of insulin, TNFalpha, and dexamethasone on adiponectin gene expression could be completely reversed by withdrawal of the hormones for 24 h. Taken together, our results suggest that adiponectin gene expression is reversibly downregulated by insulin, TNFalpha, and dexamethasone. The data support the concept of adiponectin being an important selectively controlled modulator of insulin sensitivity.

3T3 Cells↗

PubMed2002

Somatic mutations in thyroid nodular disease.

Thyroid nodules can be found in up to 50% of inhabitants of iodine-deficient areas and are classified as hot or cold thyroid nodules according to their scintigraphic characteristics. Studies of hot thyroid nodules with comparable mutation detection methods and screening at least exon 10 of the TSH receptor reported frequencies for somatic TSH-receptor mutations ranging from 20 to 82% in patients with similar iodine supply. We have recently screened 75 hot thyroid nodules for somatic TSH-receptor mutations with the more sensitive DGGE method and found somatic TSH-receptor mutations in 57% and Gsalpha mutations in 3%. As 50% of the mutation-negative nodules from female patients are of monoclonal origin when tested for X-chromosome inactivation somatic mutations in other genes are likely to cause the development of hot thyroid nodules. Scintigraphically nonsuppressible areas have been identified in up to 40% of euthyroid goiters in iodine-deficient areas. We recently identified somatic TSH-receptor mutations in microscopic autonomous areas with increased 125T uptake in euthyroid goiters studied by autoradiography 20 years ago. These constitutively activating somatic TSH-receptor mutations in minute autoradiographically hot areas of euthyroid goiters are very likely starting foci which most likely lead to toxic thyroid nodules in iodine-deficient goiters. Therefore iodine deficiency does not only lead to euthyroid goiters but also to thyroid autonomy. The latter is also suggested by epidemiologic studies. Similar mechanisms induced by iodine deficiency and the subsequent hyperplasia, mutagenesis, and selection of cell clones could also lead to cold thyroid nodules by somatic mutations that only initiate growth but not hyperfunction of the affected thyroid epithelial cell. Somatic ras mutations have frequently been detected in histologically characterized thyroid adenomas or adenomatous nodules. However, they seem to be rare in cold thyroid nodules. Since the majority of these latter nodules and 60% of the cold thyroid nodules are monoclonal other somatic mutations are likely in these nodules.

Animals↗

PubMed2002

Evaluation of a computer based interactive diabetes education program designed to train the estimation of the energy or carbohydrate contents of foods.

The objective of this study was to evaluate a computer based interactive diabetes education program designed to train patients with diabetes to correctly estimate the energy or carbohydrate contents of foods. The acceptance and the operator convenience of the program were evaluated with an integrated program and written questionnaires. The training success was calculated for each user from the data sets after multiple repetitions of the same exercise. Age, weight, type of diabetes, previous training and computer experience of 126 patients with type 1 or 2 diabetes from three different German diabetes centres were documented. The evaluation of the computer program indicated a good acceptance and operator convenience. Moreover, there was a significant training success in patients who have never used a structured diabetes education program before. Therefore, the potential impact of the program is to support but not to replace structured diabetes education programs and to further motivate and attract patients to diabetes education programs.

Computer-Assisted Instruction↗

PubMed2002

Expression of G(alpha)(s) proteins and TSH receptor signalling in hyperfunctioning thyroid nodules with TSH receptor mutations.

OBJECTIVE: Constitutively activating mutations of the thyrotrophin receptor (TSHR) are the main molecular cause of hyperfunctioning thyroid nodules (HTNs). The G protein coupling is an important and critical step in the TSHR signalling which mainly includes G(alpha)(s), G(alpha)(i) and G(alpha)(q)/11 proteins. DESIGN: We investigated the in vitro consequences of overexpressing G(alpha) proteins on signalling of the wild-type (WT) or mutated TSHR. Moreover, we investigated whether changes in G(alpha) protein expression are pathophysiologically relevant in HTNs or cold thyroid nodules (CTNs). METHODS: Wild-type TSH receptor and mutated TSH receptors were coexpressed with G(alpha)(s), G(alpha)(i) or G(alpha)(q)/11, and cAMP and inositol phosphate (IP) production was measured after stimulation with TSH. The expression of G(alpha)(s), G(alpha)(i) and G(alpha)(q)/11 proteins was examined by Western blotting in 28 HTNs and 14 CTNs. RESULTS: Coexpression of G(alpha)(s) with the WT TSH receptor in COS 7 cells significantly increased the basal and TSH-stimulated cAMP accumulation while coexpression of the G(alpha)(q) or G(alpha)11 protein significantly increased the production of cAMP and inositol triphosphate (IP(3)). The coexpression of the TSH receptor mutants (I486F, DEL613-621), known to couple constitutively to G(alpha)(s) and G(alpha)(q) with G(alpha)(s) and G(alpha)(q)/11, significantly increased the basal and stimulated cAMP and IP(3) accumulation. Coexpression of the TSH receptor mutant V556F with G(alpha)(s) only increased the basal and stimulated cAMP production while its coexpression with G(alpha)(q)/11 increased the basal and stimulated IP(3) signalling. The expression of G(alpha)(s) protein subunits determined by Western blotting was significantly decreased in 14 HTNs with a constitutively activating TSH receptor mutation in comparison with the corresponding surrounding tissue, while in 14 HTNs without TSH receptor or G(alpha)(s) protein mutation and in 14 CTNs the expression of G(alpha)(s) protein was not different compared with the surrounding tissue. The expression of G(alpha)(i) and G(alpha)(q)/11 proteins in HTNs or CTNs was not significantly different compared with the surrounding tissue. CONCLUSIONS: The reduced expression of G(alpha)(s) protein subunits in HTNs with TSHR mutations could act as a feedback mechanism to desensitise the chronically stimulated cAMP cascade. As G(alpha) protein expression was not significantly increased in the majority of CTNs and HTNs an influence of G(alpha) overexpression on TSH signalling could be excluded in these nodules.

Animals↗

PubMed2002

Negative regulation of adipose-expressed galectin-12 by isoproterenol, tumor necrosis factor alpha, insulin and dexamethasone.

OBJECTIVE: Galectin-12 has recently been shown to be a predominantly adipocyte-expressed protein which is stimulated by insulin-sensitizing thiazolidinediones and possesses apoptosis-inducing activity. METHODS: To further clarify galectin-12 regulation and its potential involvement in the development of insulin resistance, 3T3-L1 adipocytes were chronically treated with various hormones known to impair insulin sensitivity, and galectin-12 mRNA was measured by quantitative real-time reverse transcription-polymerase chain reaction. RESULTS: Treatment of 3T3-L1 cells for 16 h with 10 micromol/l isoproterenol, 100 nmol/l insulin, 0.6 nmol/l tumor necrosis factor alpha (TNFalpha), and 100 nmol/l dexamethasone reduced galectin-12 gene expression between 47% and 85%. These negative effects were dose-dependent with significant inhibition detectable at concentrations as low as 10 nmol/l isoproterenol, 0.06 nmol/l TNFalpha, and 1 nmol/l dexamethasone. Furthermore, the inhibitory effect of isoproterenol could be almost completely reversed by pretreatment with the beta-adrenergic antagonist propranolol and mimicked by stimulation of G(S)-proteins with cholera toxin or by activation of adenylyl cyclase with forskolin and dibutyryl-cAMP. CONCLUSIONS: Our results suggest that galectin-12 is an adipocyte-expressed protein which is downregulated by various insulin resistance-inducing hormones. These findings imply a role for galectin-12 in the pathogenesis of insulin resistance.

Adenylyl Cyclases↗

PubMed2002

Thyroglobulin mRNA quantification in the peripheral blood is not a reliable marker for the follow-up of patients with differentiated thyroid cancer.

BACKGROUND: The detection of serum thyroglobulin (Tg) by immunoassay is widely used to detect residual, recurring or metastatic thyroid carcinoma tIssue in patients with differentiated thyroid cancer (DTC) after total thyroidectomy and radioiodine therapy. However, this method requires thyroid hormone withdrawal to increase sensitivity and is limited by the interference of anti-Tg antibodies. To solve these problems, the detection of Tg mRNA from circulating thyroid cells by reverse transcription (RT)-PCR has been suggested as an alternative method. However, different previous reports show discrepant conclusions as to the clinical usefulness of Tg mRNA quantification. METHODS: We compared three methods of blood collection and RNA extraction, and quantified Tg mRNA (by real time RT-PCR) in the peripheral blood of a) probands without thyroid disease (n=42), patients with b) thyroid autonomy (n=15), c) Graves' disease (n=22), d) euthyroid goiter (n=6), and in DTC-patients after thyroidectomy and radioiodine therapy e) with (n=16) and f) without (n=37) metastasis. As the use of citrate blood in combination with a subsequent separation of mononuclear cells showed a significantly better RNA yield than the extraction of RNA from EDTA or citrate blood without the separation of mononuclear cells, this was the method used. Total RNA was reverse transcribed with random hexamer primers and Tg mRNA was amplified by real time RT-PCR using specific primers and hybridization probes. The Tg mRNA concentrations were normalized to beta-actin mRNA concentrations. RESULTS: Mean circulating Tg mRNA for each group detailed above, expressed as the ratio of Tg to beta-actin concentrations x 1000, were: a) 2.3 (range 0.03-70.89), b) 0.25 (range 0.02-0.55), c) 0.31 (range 0.05-1.36), d) 0.18 (range 0.08-0.35), e) 0.57 (range 0.03-3.03) and f) 0.17 (range 0.02-0.60). Furthermore, we found no correlation between serum Tg and Tg mRNA. CONCLUSIONS: In summary, our data do not show significant differences in Tg mRNA expression between the investigated groups. Therefore, the detection and quantification of Tg mRNA in peripheral blood is unlikely to be suitable for the follow-up of DTC.

Biomarkers, Tumor↗