In vitro and mouse in vivo characterization of the potent free fatty acid 1 receptor agonist TUG-469.

Activation of the G protein-coupled free fatty acid receptor 1 (FFA1; formerly known as GPR40) leads to an enhancement of glucose-stimulated insulin secretion from pancreatic ß-cells. TUG-469 has previously been reported as a potent FFA1 agonist. This study was performed to confirm the higher in vitro potency of TUG-469 compared to the reference FFA1 agonist GW9508 and to prove in vivo activity in a pre-diabetic mouse model. The in vitro pharmacology of TUG-469 was studied using Ca(2+)-, cAMP-, and impedance-based assays at recombinant FFA1 and free fatty acid receptor 4, formerly known as GPR120 (FFA4) expressing 1321N1 cells and the rat insulinoma cell line INS-1. Furthermore, we investigated the systemic effect of TUG-469 on glucose tolerance in pre-diabetic New Zealand obese (NZO) mice performing a glucose tolerance test after intraperitoneal administration of 5 mg/kg TUG-469. In comparison to GW9508, TUG-469 showed a 1.7- to 3.0-times higher potency in vitro at 1321N1 cells recombinantly expressing FFA1. Both compounds increased insulin secretion from rat insulinoma INS-1 cells. TUG-469 is > 200-fold selective for FFA1 over FFA4. Finally, a single dose of 5 mg/kg TUG-469 significantly improved glucose tolerance in pre-diabetic NZO mice. TUG-469 turned out as a promising candidate for further drug development of FFA1 agonists for treatment of type 2 diabetes mellitus.

HLA-A2 phenotype may be protective against Graves' disease but not against Hashimoto's thyroiditis in Caucasians.

Graves' disease (GD) and Hashimoto's thyroiditis (HT) are the most common autoimmune thyroid diseases (AITDs) affecting up to 5% of the general population. In Caucasians HT has a prevalence of up to 4.60% and GD a prevalence of 1-2%. The aim of this study was to investigate the association between HLA-A2 and the AITDs GD and HT among Caucasians. HLA alleles of 33 patients with GD and 75 patients with HT were determined by serological typing. The frequency of HLA A2 was significantly reduced in GD (p=0.033) but not in HT (p=n.s.) as compared to control samples. In individuals positive for HLA-A2 odds ratio for protection from GD was found to be 2.8. This study supports the hypothesis that genetic predisposition to GD is not restricted to MHC class II molecules. The significant negative association between HLA A2 and GD supports the hypothesis that MHC class I genes may be relevant for the protection from GD. In contrast the nonsignificant results for HT indicate that this association may not apply to AITDs in general.

Evidence for an exercise induced increase of TNF-α and IL-6 in marathon runners.

Regular physical activity of moderate intensity improves cardiovascular risk factors including low-grade inflammation. However, acute vigorous exercise such as marathon running results in marked increases of circulating pro-inflammatory markers. Up to now, the origin of this pro-inflammatory boost is still debated equivocally. We analyzed the change of interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-α), and leptin from pre- to immediately post-race in 15 male runners (age 43 ± 10.9 years and body mass index 24.5 ± 2.7 kg/m(2) ) both on the protein level in the plasma and on the messenger ribonucleic acid (mRNA) level in blood mononuclear cells (BMNC). We observed a significant increase of IL-6 (prerace 2.08 ± 0.10 ng/L and postrace 40.14 ± 24.85 ng/L, P < 0.001) and TNF-α (prerace 8.14 ± 1.38 ng/L and postrace 12.40 ± 3.15 ng/L, P < 0.001) and a decrease of leptin (prerace 1.64 ± 2.64 µg/L and postrace 0.80 ± 1.70 µg/L, P = 0.04) serum levels after the marathon race. Furthermore, TNF-α, IL-6, and leptin were expressed (mRNA level) in BMNC. However no significant differences in mRNA levels were seen before and after the run in these cells. We found an up-regulation of TNF-α and IL-6 in the plasma during vigorous exercise. This increase is not attributable to BMNC. We assume a local production in, or release from, exercised tissues.

ERK1/2 MAPKs and Wnt signaling pathways are independently involved in adipocytokine-mediated aldosterone secretion.

Obesity is one major risk factor for the development of arterial hypertension, and the development of obesity-related hypertension has been associated with increased plasma aldosterone levels. Our previous work shows a direct stimulatory effect of adipokines on aldosterone secretion from human adrenocortical cells, mediated via ERK1/2-dependent upregulation of steroid acute regulatory protein (StAR) activity. Recent evidence also indicates the involvement of the Wnt-signaling pathway in fat cell-mediated aldosterone secretion. Wnt-signaling molecules are secreted by adipocytes and regulate the activity of SF-1, a key transcription factor in adrenal steroidogenesis. The goal of this study was to investigate the cellular mechanisms of adipocyte-induced aldosterone secretion in detail, and to evaluate effects and possible interactions of the ERK1/2 MAPK- and the Wnt-signaling pathways on adipocyte-induced adreno-cortical aldosterone secretion. Our results show that, similar to adipocyte-conditioned medium (ACM), ß-catenin, which is an intracellular mediator of canonical Wnt-signaling, induced StAR promotor activity in human NCI-H295R adrenocortical cells, and ACM-induced StAR promotor activity depended on intact SF-1 binding sites. Wnt antagonist sFRP-1 inhibited adipokine-mediated StAR activity, but did not affect ERK1/2 MAPK activation. Accordingly, Wnt did not stimulate ERK1/2 phosphorylation in adrenocortical cells, indicating that ERK1/2 MAPK and Wnt signaling pathways are independently involved in adipocyte-mediated aldosterone secretion.

Subclinical hypothyroidism and the prevalence of the metabolic syndrome.

There is currently controversial data regarding the prevalence of MS among SCH patients. The aim of the study was to investigate the prevalence of MS in an adult population with SCH. A cross-sectional study was conducted among 6,998 adults in China. Epidemiological information and medical data were obtained (fasting plasma glucose, triglycerides, high density lipoprotein-C, and thyroid function). The IDF criteria were applied for the diagnosis of MS. SCH was defined as TSH more than 4.5 mIU/l with normal values for FT3 and FT4. Among the 6,560 participants, 21.5% were diagnosed with MS and 8.2% suffered from SCH. MS was found in 21.3% in the euthyroidism (EUT) group and in 25.7% in the SCH group (p<0.05). However, this difference between EUT and SCH in MS prevalence was not statistically significant after adjusting for age. Of note, the prevalence of MS increased with age. The proportions of systemic hypertension, hypertriglyceridemia, impaired fasting glucose, and low HDL-C were 86.7, 59.7, 45.1 and 65.9% in EUT while the corresponding values in SCH were 89.9, 63.8, 39.9 and 72.5%, respectively (the differences, however, did not reach statistical significance). The prevalence of high BP, high TG, elevated FBP, and low HDL-C as well as the percentage of patients fulfilling the IDF criteria for the metabolic syndrome are not significantly different among SCH and EUT after adjusting for age. Therefore, subclinical hypothyroidism appears not as an independent risk factor for the metabolic syndrome in this population.

A new mutation in the menin gene causes the multiple endocrine neoplasia type 1 syndrome with adrenocortical carcinoma.

Multiple endocrine neoplasia type 1 (MEN1) is an autosomal dominant tumor syndrome that may be caused by mutations in the MEN1 gene on 11q13. Loss of function of the tumor suppressor gene MEN1 leads to synchronous or metachronous appearance of neuroendocrine tumors arising from neuroendocrine cells of the parathyroid and pituitary glands, the duodenum and pancreatic islets, and other endocrine organs such as the adrenal cortex. We here present a patient with MEN1 who developed hyperparathyroidism, multiple well differentiated functionally inactive neuroendocrine tumors of the pancreas and an adrenal carcinoma. We describe a new mutation at codon 443 in the coding region of exon 9 in the MEN1 gene, where a cytosine residue was exchanged for adenosine (TCC > TAC) and, consequently, serine for tyrosine (p.Ser443Tyr; c.1328C > A). [corrected] Also, we provide clinical data that may add to the genotype-phenotype discussion. We conclude that the novel mutation in the MEN1 gene described herein was clinically relevant.

The CB-1 receptor antagonist rimonabant modulates the interaction between adipocytes and pancreatic beta-cells in vitro.

BACKGROUND: Adipocytes produce signalling molecules which can act on target cells including pancreatic beta-cells. In previous studies we found adipocytes to directly stimulate insulin secretion and the proliferation of pancreatic beta-cells in vitro. Rimonabant acts as an antagonist at the cannabinoid-1 (CB-1) receptor which is expressed on adipocytes. Rimonabant decreases insulin levels in vivo. This effect can either be explained by improving insulin sensitivity or by effects on beta-cells including the modulation of adipocyte - beta-cell interactions. OBJECTIVES: To test how pre-treatment of primary human adipocytes with rimonabant affects the cross-talk between adipocytes and pancreatic beta-cells in vitro. RESULTS: Rimonabant had no direct effect on insulin secretion or beta-cell proliferation at a concentration range from 1 nM to 1 µM. This is in line with previous findings showing that in the murine pancreas CB-1 receptors are preferentially expressed on non-beta-cells, while rimonabant is a selective blocker of CB-1 receptors. We found fat-cell conditioned-medium without (FCCM) and after pre-treatment for 24 h with 100 nM rimonabant (FCCM-RB) to induce insulin secretion from primary murine beta-cells to a similar extent. Proliferation of a pancreatic beta-cell line was enhanced by FCCM to 219%, while FCCM-RB inhibited proliferation to 53%. As we previously found Wnt-signalling to mediate effects of adipocytes on beta-cell proliferation we tested the ability of FCCM and FCCM-RB to activate canonical Wnt-signalling in target cells. However, there was no significant difference between the groups: FCCM and FCCM-RB stimulated Wnt reporter gene activity to 181% and 179%, respectively. In addition, there was no significant difference in adiponectin levels between FCCM and FCCM-RB (56.8 vs. 58.1 ng/ml), showing that adiponectin does not mediate the differential effects on beta-cell proliferation by FCCM and FCCM-RB. CONCLUSION: Our data show that rimonabant modulates the adipocyte - beta-cell interaction with respect to beta-cell proliferation and indicate that signalling molecules other than adiponectin and components of the Wnt pathway mediate this cross-talk.

[Therapy of dyslipidemia in rheumatic diseases]. / Therapie der Dyslipidämie bei entzündlich-rheumatischen Erkrankungen.

Cardiovascular diseases are the main cause of death worldwide and dyslipidemia constitutes a substantial risk factor. Patients with rheumatic diseases, especially active inflammatory arthritis and systemic lupus erythematosus, show unfavorable lipid profiles, which, however, do not account for the total excess cardiovascular morbidity. Effective disease management, life-style changes and cholesterol-lowering agents can ameliorate the lipid profile and lower cardiovascular mortality. Due to their anti-inflammatory and potent cholesterol-lowering properties, statins are the pharmacological agents of first choice. National and international guidelines on cardiovascular risk prevention differ concerning appraisal of the individual risk and lipid targets. The EULAR recently released recommendations for cardiovascular risk management in patients with inflammatory arthritis.

The effects of the endothelium on adrenal steroidogenesis and growth are mainly mediated by proteins other than endothelin-1.

The endothelium releases factors stimulating the adrenal cortex. It is also known that endothelin-1 (ET-1) promotes generation of cortisol and aldosterone, and proliferation of adrenocortical cells. The aim of the study was to find out whether the effect of the endothelium on adrenocortical cells is dominated by the action of ET-1. The effects of endothelial cell-conditioned medium (ECCM), obtained during growth of human umbilical cord vein endothelial cells, on aldosterone and cortisol release by cells of the adrenocortical cancer cell-line NCI-H295R and the promoter activity of steroidogenic acute-regulatory protein (StAR) were studied. The effect of ECCM on proliferation of human primary normal adrenocortical and NCI-H295R cells was also investigated. Concentration-dependent increases in cortisol release that reached 192.7 ± 62.8 in percent of basal secretion, in aldosterone release that reached 188.2 ± 52.3 in percent of basal secretion, and in proliferation after stimulation with ECCM at concentrations of 10-50% were found. ECCM significantly activated the StAR promoter 3-fold in NCI-H295R cells if the ECCM was not pretreated with pronase. These effects of the endothelium were not reversed after co-incubation with endothelin receptor antagonists and could not be mimicked by incubation with endothelin-1. In conclusion, the cultured endothelial cells secrete a protein that stimulates steroidogenesis in adrenal cells and their growth. It was also shown that the ET-1 does not mediate the effect of ECCM on the NCI-H295R cell line.

Reproducibility of Elecsys anti-TSHR test results in a lot-to-lot comparison.

Most recently, a new rapid and fully-automated TSH receptor autoantibody (TRAb) assay has been established. This assay system uses the M22 human monoclonal antibody for competing against the patient's TSH receptor autoantibodies (TRAb) to be detected. The aim of our present study was to compare the reproducibility of TRAb values based on measurements with different TSH receptor preparations in a lot-to-lot comparison. For TRAb values > 2 IU/l the relative differences ranged from -9.0 to +10.0%. The mean difference was 0.28 +/- 8%. For TRAb values around the cutoff for positivity (1.75 IU/l) a higher range of relative differences from -20 up to +15% was obtained. The overall mean of differences was -0.8+/-14%. The data clearly demonstrate that the automated TRAb assay has a high stability in regard to TSH receptor preparations.

Role of the novel mTOR inhibitor RAD001 (everolimus) in anaplastic thyroid cancer.

Activation of the phosphatidylinositol-3-kinase (PI3K) signaling cascade is increasingly recognized as a common feature of thyroid follicular neoplasms. Among the PI3K downstream effectors, the main kinase, directly responsible for the increased cell growth and proliferation, is called mammalian target of rapamycin (mTOR). This central kinase might be directly inhibited via rapamycin and its derivatives. The aim of the present study was to examine whether RAD001 (everolimus) can selectively suppress the proliferation of different anaplastic thyroid cancer (ATC) cells. Five different human ATC cell lines were exposed to different concentrations of RAD001. Importantly, we found a dose-dependent growth inhibition in two ATC cell lines at concentrations of 43.5 and 94.5 nM although not as intensive as within the RAD001 responding K562cell line. The other cell lines revealed a GI (50) between 168 to 234 nM. In parallel, quantitative PCR of PCNA displayed a reduced expression of PCNA within the responding cell lines, respectively. In summary, we found a good responding effect in a part of ATC cell lines, which may have a clinical impact.

Inhibition of human insulin gene transcription by peroxisome proliferator-activated receptor gamma and thiazolidinedione oral antidiabetic drugs.

BACKGROUND AND PURPOSE: The transcription factor peroxisome proliferator-activated receptor gamma (PPARgamma) is essential for glucose homeostasis. PPARgamma ligands reducing insulin levels in vivo are used as drugs to treat type 2 diabetes mellitus. Genes regulated by PPARgamma have been found in several tissues including insulin-producing pancreatic islet beta-cells. However, the role of PPARgamma at the insulin gene was unknown. Therefore, the effect of PPARgamma and PPARgamma ligands like rosiglitazone on insulin gene transcription was investigated. EXPERIMENTAL APPROACH: Reporter gene assays were used in the beta-cell line HIT and in primary mature pancreatic islets of transgenic mice. Mapping studies and internal mutations were carried out to locate PPARgamma-responsive promoter regions. KEY RESULTS: Rosiglitazone caused a PPARgamma-dependent inhibition of insulin gene transcription in a beta-cell line. This inhibition was concentration-dependent and had an EC(50) similar to that for the activation of a reporter gene under the control of multimerized PPAR binding sites. Also in normal primary pancreatic islets of transgenic mice, known to express high levels of PPARgamma, rosiglitazone inhibited glucose-stimulated insulin gene transcription. Transactivation and mapping experiments suggest that, in contrast to the rat glucagon gene, the inhibition of the human insulin gene promoter by PPARgamma/rosiglitazone does not depend on promoter-bound Pax6 and is attributable to the proximal insulin gene promoter region around the transcription start site from -56 to +18. CONCLUSIONS AND IMPLICATIONS: The human insulin gene represents a novel PPARgamma target that may contribute to the action of thiazolidinediones in type 2 diabetes mellitus.

Wnt-signalling and the metabolic syndrome.

The Wnt-signalling pathway plays a well-established role in embryogenesis and tumourigenesis. However, recent data puts Wnt-signalling in the context of metabolic disease. In vitro and in vivo data characterised the role of Wnt-signalling molecules in the regulation of adipocyte differentiation (adipogenesis). Furthermore, Wnts play a pivotal role in regulating pancreatic beta-cell function and mass. In addition, studies found polymorphisms within the gene encoding TCF7L2, a Wnt-regulated transcription factor, to contribute an increased risk to develop type 2 diabetes mellitus in humans. This review will summarise recent aspects of Wnt-signalling in these pathophysiologic events and discuss the contributions of dysregulation in Wnt-signalling to features of the metabolic syndrome.

Association of impaired glucose metabolism in morbid obesity with hypoadiponectinaemia.

BACKGROUND: Increased circulating levels of cytokines and chemokines and decreased adiponectin levels are associated with impaired glucose tolerance (IGT) and type 2 diabetes mellitus (T2DM). As obesity is the major risk factor for T2DM it is not clear why many patients with morbid obesity remain normoglycaemic and if this protection can be attributed to a lower grade of inflammation or higher adiponectin levels. MATERIALS AND METHODS: Glucose tolerance of morbidly obese patients (n=2 754, body mass index > or =40 kg/m2) was assessed by oral glucose tolerance tests. In a case-control design we compared levels of eight immune mediators and adiponectin from patients with IGT/T2DM (n=52) and normal glucose tolerance (NGT; n=59). Gene expression in peripheral blood was determined by quantitative RT-PCR, and serum concentrations of immune mediators and adiponectin were measured by ELISA and bead-based multiplex technology. RESULTS: About 54% of the patients in our morbidly obese cohort were normoglycaemic, while 14% were diagnosed with IGT and 32% with T2DM. There was no statistically significant difference in mRNA expression or serum levels of proinflammatory markers. Interestingly, we could demonstrate an association of NGT with higher adiponectin levels (p=0.039). Adiponectin levels were negatively correlated with interleukin (IL)-6 and macrophage chemoattractant protein (MCP)-1, but independent the other immune mediators. CONCLUSIONS: We found an association of lower adiponectin levels with IGT/T2DM, but no further increase in inflammatory markers in morbid obesity. This suggests that in addition to chronic, low-grade inflammation, adiponectin is an important factor in the development of, or protection against, T2DM in obesity.

The endothelium secretes interleukin-6 (IL-6) and induces IL-6 and aldosterone generation by adrenocortical cells.

Endothelial cells have been shown to induce adrenal steroidogenesis and to enhance aldosterone secretion via angiotensin II and endothelin 1-independent mechanisms. It has been demonstrated that endothelial cells and adrenocortical cells are capable of producing interleukin-6 (IL-6) and IL-6 is a factor known to stimulate adrenal cortisol secretion. We therefore asked whether endothelial cells have an effect on adrenal IL-6 generation and whether IL-6 mediates biosynthesis of aldosterone as is observed after exposure of adrenocortical cells to endothelial cell-conditioned medium (ECCM). Cells from the adrenocortical cancer cell line NCI-H295R were incubated with ECCM produced from human umbilical vein endothelial cells at increasing concentrations. As detected by an enzyme-linked immunosorbent assay, pure ECCM significantly increased IL-6 protein secretion by cultured adrenocortical cells in a dose-dependent fashion, to a 18.0+/-2.0 pg/mL (mean+/-SEM). This was paralleled by an enhanced IL-6 promoter activity as determined with the transfection of an IL-6-promoter-luciferase reporter gene construct. Pure ECCM also induced aldosterone secretion by adrenocortical cells more than three times that of controls with serum-free medium. ECCM PER SE contains significant amounts of IL-6 protein. However, blockade of IL-6 signal transduction did not interfere with aldosterone synthesis. These data suggest that endothelial cells secrete IL-6 and that endothelial cell-derived factors regulate adrenal IL-6 synthesis which does not alter adrenal aldosterone secretion. Our findings support the hypothesis that the endothelium and the adrenal gland may play a role in the development of some forms of hypertension and - more speculative - inflammation.

Intensive glycemic control lowers plasma visfatin levels in patients with type 2 diabetes.

Visfatin is an independent association factor for type 2 diabetes mellitus (T2DM). In order to evaluate the plasma visfatin levels and investigate whether plasma visfatin concentrations are altered by intensive glycemic control in patients with diabetes, we determined plasma visfatin concentrations and metabolic parameters in 53 newly diagnosed type 2 diabetic patients and 35 healthy controls. Visfatin levels were also investigated before and after intensive glycemic control for three months in subgroup of patients with T2DM. Plasma visfatin levels were significantly elevated in diabetic patients compared with healthy controls (p<0.001). Circulating visfatin concentration was associated with fasting plasma glucose (FPG), 2-hour OGTT plasma glucose (2hPG), HOMA-beta indexes (r=0.338, p=0.001; r=0.340, p=0.002; r=-0.296, p=0.006, respectively), but not with insulin sensitivity (HOMA-IR) or other metabolic or anthropometric parameters in all subjects. In addition, visfatin levels were also correlated with HbA1c levels in diabetic patients. Furthermore, visfatin concentrations reduced from 25.0+/-6.5 ng/ml at baseline to 20.3+/-4.7 ng/ml (p<0.01) after 3 months of intensive glycemic control, while HbA1c levels decreased from 9.0+/-1.8% to 6.2+/-0.7% (p<0.01). We conclude that the change of visfatin concentration may be a compensatory mechanism to ameliorate insulin deficiency due to pancreatic beta-cell dysfunction.

New mechanisms to control aldosterone synthesis.

Arterial hypertension is a frequent and leading cardiovascular risk factor, and primary aldosteronism is a well-recognized cause of secondary hypertension. Aldosterone is the basic regulator of extracellular fluid volume and electrolyte balance. Alterations in plasma aldosterone levels significantly contribute to the development and the severity of hypertension. Adrenal steroidogenesis is controlled by two major feedback loops: the hypothalamic-pituitary-adrenal axis, which regulates cortisol synthesis, and the renin-angiotensin-aldosterone system, which directs aldosterone production. In addition to angiotensin, potassium, and corticotropin-which belong to the classic stimulators of aldosterone-neuropeptides, catecholamines, and prostaglandins are also known to stimulate aldosterone synthesis. Recently, several new mechanisms have been characterized that control the release of aldosterone by adrenocortical cells, among them endothelial cell-derived factors and adipokines. Further identification and characterization of these factors may help in the development of novel therapies for the treatment of arterial hypertension, various metabolic diseases, and other disorders.

Characterization of monocyte-derived IFNalpha-generated dendritic cells.

The antitumor effects of IFNalpha is mainly mediated by the activation of cytotoxic T lymphocytes (CTLs), the activation of natural killer (NK) cells, and the generation of highly potent antigen-presenting dendritic cells (IFN-DCs). Recently, we demonstrated that these cells partially express the NK cell marker CD56 and reveal a direct cytotoxic immunity towards tumor cells. The aim of the present study was to explore these cells in more detail with respect to their phenotypical and functional characteristics. Flowcytometric analyses revealed that a 5-day incubation time of CD14+ monocytes with IFNalpha results in a steady increase of CD56 surface expression of these cells from 25% (+/-2%) on day 1 up to 68% (+/-11%) on day 5. Interestingly, additional culturing of negatively selected CD56- IFN-DCs also resulted in a partial CD56 surface expression. By comparing both cell types in more detail we found a significant decrease of CD14 expression on CD56+ IFN-DCs (66+/-6%) compared to CD56- IFN-DCs (76+/-6%). On the basis of functional tests, CD56+ IFN-DCs revealed a slightly increased phagocytosis capacity compared to CD56- IFN-DCs as only 82% of CD56- IFN-DCs showed a positive intracytoplasmatic signal after 60 minutes coculturing with FITC-labeled albumin, whereas 91% of CD56+ IFN-DCs were positive. Moreover, CD56+ IFN-DCs revealed a stronger T cell stimulation capacity compared to CD56- IFN-DCs. These results together with our previously described data suggest that CD56+ IFN-DCs and CD56- IFN-DCs may represent one identical cell population with different maturation status rather than two separate cell entities. Because of their high stimulating capacity and their direct cytolytic effects these cells represent a new promising tool for cellular anticancer therapy.