Volker Bähr

Novel Inactivating Mutations of the Calcium-Sensing Receptor: The Calcimimetic NPS R-568 Improves Signal Transduction of Mutant Receptors

CONTEXT AND OBJECTIVEnInactivating mutations in the calcium-sensing receptor (CaSR) gene cause neonatal severe hyperparathyroidism and familial hypocalciuric hypercalcemia (FHH). The aims of the present study were the functional characterization of novel mutations of the CaSR found in FHH patients, the comparison of in vitro receptor function with clinical parameters, and the effect of the allosteric calcimimetic NPS R-568 on the signaling of mutant receptors.nnnMETHODSnWild-type and mutant CaSRs (W530G, C568Y, W718X, M734R, L849P, Q926R, and D1005N) were expressed in human embryonic kidney 293 cells. Receptor signaling was studied by measuring intracellular free calcium in response to different concentrations of extracellular calcium ([Ca(2+)](o)).nnnRESULTSnFour CaSR mutations (C568Y, W718X, M734R, and L849P) demonstrated a complete lack of a [Ca(2+)](o)-induced cytosolic Ca(2+) response up to 30 mm [Ca(2+)](o), whereas the CaSR mutants W530G, Q926R, and D1005N retained some sensitivity to [Ca(2+)](o). There was no significant relation between the in vitro calcium sensitivity, serum calcium, and intact PTH levels in the patients. Patients with C-terminal CaSR mutations had a calcium to creatine ratio above the established diagnostic threshold of 0.01 for FHH. The calcimimetic NPS R-568 enhanced the responsiveness to [Ca(2+)](o) in CaSR mutants of the extracellular domain (W530G and C568Y) as well as the intracellular C-terminal domain (Q926R and D1005N).nnnCONCLUSIONnTherefore, calcimimetics might offer medical treatment for symptomatic FHH patients, and more important, for patients with neonatal severe hyperparathyroidism that harbor calcimimetic-sensitive CaSR mutants.

Rosiglitazone decreases 11β-hydroxysteroid dehydrogenase type 1 in subcutaneous adipose tissue

Objectiveu2002 The peroxisome proliferator‐activated receptor‐γ (PPARγ) agonist rosiglitazone increases insulin sensitivity, which, in animal models, is comparable to the effect of a reduction in 11β‐hydroxysteroid dehydrogenase type 1 (11β‐HSD1) activity. We therefore investigated whether rosiglitazone‐induced insulin sensitivity is associated with changes in 11β‐HSD1 activity in different tissues.

The flavones apigenin and luteolin induce FOXO1 translocation but inhibit gluconeogenic and lipogenic gene expression in human cells.

The flavones apigenin (4′,5,7,-trihydroxyflavone) and luteolin (3′,4′,5,7,-tetrahydroxyflavone) are plant secondary metabolites with antioxidant, antiinflammatory, and anticancer activities. We evaluated their impact on cell signaling pathways related to insulin-resistance and type 2 diabetes. Apigenin and luteolin were identified in our U-2 OS (human osteosarcoma) cell screening assay for micronutrients triggering rapid intracellular translocation of the forkhead box transcription factor O1 (FOXO1), an important mediator of insulin signal transduction. Insulin reversed the translocation of FOXO1 as shown by live cell imaging. The impact on the expression of target genes was evaluated in HepG2 (human hepatoma) cells. The mRNA-expression of the gluconeogenic enzymes phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase (G6Pc), the lipogenic enzymes fatty-acid synthase (FASN) and acetyl-CoA-carboxylase (ACC) were down-regulated by both flavones with smaller effective dosages of apigenin than for luteolin. PKB/AKT-, PRAS40-, p70S6K-, and S6-phosphorylation was reduced by apigenin and luteolin but not that of the insulin-like growth factor receptor IGF-1R by apigenin indicating a direct inhibition of the PKB/AKT-signaling pathway distal to the IGF-1 receptor. N-acetyl-L-cysteine did not prevent FOXO1 nuclear translocation induced by apigenin and luteolin, suggesting that these flavones do not act via oxidative stress. The roles of FOXO1, FOXO3a, AKT, sirtuin1 (SIRT1), and nuclear factor (erythroid-derived2)-like2 (NRF2), investigated by siRNA knockdown, showed differential patterns of signal pathways involved and a role of NRF2 in the inhibition of gluconeogenic enzyme expression. We conclude that these flavones show an antidiabetic potential due to reduction of gluconeogenic and lipogenic capacity despite inhibition of the PKB/AKT pathway which justifies detailed investigation in vivo.

In vivo activity of 11β-hydroxysteroid dehydrogenase type 1 and free fatty acid-induced insulin resistance

Introductionu2002 Free fatty acids (FFAs) induce hepatic insulin resistance and enhance hepatic gluconeogenesis. Glucocorticoids (GCs) also stimulate hepatic gluconeogenesis. The aim of this study was to investigate whether the FFA‐induced hepatic insulin resistance is mediated by increased activity of hepatic 11β‐hydroxysteroid dehydrogenase type 1 (11β‐HSD1), accompanied by elevated hepatic cortisol levels.

Arginine vasopressin-dependent and AVP-independent mechanisms of renal fluid absorption during thirsting despite glucocorticoid-mediated vasopressin suppression

Glucocorticoids seem to modify the release and effects of plasma arginine vasopressin (pAVP). However, underlying processes are not well understood. This study aimed to evaluate the mechanism of the modulating effects of glucocorticoids on pAVP and renal water reabsorption.

Cell-type specific regulation of the human 11beta-hydroxysteroid dehydrogenase type 1 promoter

Abstract The intracellular enzyme 11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1) converts cortisone into the more active metabolite cortisol. Overexpression of 11beta-HSD1 was associated with features of the metabolic syndrome such as obesity or impaired glucose tolerance. Despite this considerable impact of 11beta-HSD1, the human 11beta-HSD1 promoter has not been described in detail yet. We therefore cloned eight different promoter fragments of the 5′-upstream region of the known transcription/translation-start up to −3034 bp into the luciferase-reporter vector pGL3. A low-cost in-house assay was developed and validated to detect firefly and renilla luciferase activity. Promoter fragments were analysed in human HepG2 and undifferentiated and differentiated murine 3T3-L1 cells. A differential regulation of the human 11beta-HSD1 promoter depending upon the cell type was observed. Specifically, a strong repressor of the basal promoter activity was found between −85 and −172 bp in HepG2 cells only, while an additional repressor appeared to be active between −342 and −823 bp in both, the hepatic and the adipose cell line. The presented data suggest a cell-type specific regulation of the 11beta-HSD1 promoter, which is in agreement with existing expression data from animal and human studies. The described promoter constructs will allow subsequent studies about the role of specific hormonal, metabolic and transcription factors to finally characterise the regulation of the human 11beta-HSD1-promoter in more detail.

Cooperation And Liaison Between Universities And Editors (CLUE): Recommendations On Best Practice

Journals and research institutions have common interests regarding the trustworthiness of research publications but their specific roles and responsibilities differ. These draft recommendations aim to address issues surrounding cooperation and liaison between journals and institutions about possible and actual problems with reported research. The proposals will be discussed at various meetings including the World Conference on Research Integrity in May 2017. We will also consider comments and suggestions posted on this preprint. The main recommendations are that: National registers of individuals or departments responsible for research integrity at institutions should be created. Institutions should develop mechanisms for assessing the validity of research reports that are independent from processes to determine whether individual researchers have committed misconduct. Essential research data and peer review records should be retained for at least 10 years. While journals should normally raise concerns with authors in the first instance, they also need criteria to determine when to contact the institution before, or at the same time as, alerting the authors in cases of suspected data fabrication or falsification to prevent the destruction of evidence. Anonymous or pseudonymous allegations made to journals or institutions should be judged on their merit and not dismissed automatically. Institutions should release relevant sections of reports of research trustworthiness or misconduct investigations to all journals that have published research that was the subject of the investigation.

Glucagon Decreases IGF-1 Bioactivity in Humans, Independently of Insulin, by Modulating Its Binding Proteins

Context: Depending on its lipolytic activity, glucagon plays a promising role in obesity treatment. Glucagon‐induced growth hormone (GH) release can promote its effect on lipid metabolism, although the underlying mechanisms have not been well‐defined. Objective: The present study highlights the glucagon effect on the GH/insulinlike growth factor 1 (IGF‐1)/IGF‐binding protein (IGFBP) axis in vivo and in vitro, taking into consideration insulin as a confounding factor. Materials and Methods: In a double‐blind, placebo‐controlled study, we investigated changes in GH, IGFBP, and IGF‐1 bioactivity after intramuscular glucagon administration in 13 lean controls, 11 obese participants, and 13 patients with type 1 diabetes mellitus (T1DM). The effect of glucagon on the transcription factor forkhead box protein O1 (FOXO1) translocation, the transcription of GH/IGF‐1 system members, and phosphorylation of protein kinase B (Akt) was further investigated in vitro. Results: Despite unchanged total IGF‐1 and IGFBP‐3 levels, glucagon decreased IGF‐1 bioactivity in all study groups by increasing IGFBP‐1 and IGFBP‐2. The reduction in IGF‐1 bioactivity occurred before the glucagon‐induced surge in GH. In contrast to the transient increase in circulating insulin in obese and lean participants, no change was observed in those with T1DM. In vitro, glucagon dose dependently induced a substantial nuclear translocation of FOXO1 in human osteosarcoma cells and tended to increase IGFBP‐1 and IGFBP‐2 gene expression in mouse primary hepatocytes, despite absent Akt phosphorylation. Conclusions: Our data point to the glucagon‐induced decrease in bioactive IGF‐1 levels as a mechanism through which glucagon induces GH secretion. This insulin‐independent reduction is related to increased IGFBP‐1 and IGFBP‐2 levels, which are most likely mediated via activation of the FOXO/mTOR (mechanistic target of rapamycin) pathway.