The regulation of calcitonin genes upon bacterial infection and sepsis in human adipocytes

Systemic bacterial infections induce an ubiquitous expression of calcitonin (CALC) genes with sustained release of calcitonin (CT) peptides, namely procalcitonin (ProCT), calcitonin-gene related peptide (CGRP) and adrenomedullin (ADM). ProCT is a marker to follow the course of sepsis and to guide antibiotic therapy and a dose dependent toxic mediator. Persistently and markedly elevated levels of ProCT and even more of ProADM during bacterial infection and sepsis indicate a bad prognosis. The molecular mechanisms as to how extrathyroidal CALC gene expression and protein secretion is regulated in sepsis are unknown. Since the neuro-endocrine CT expression in the parafollicular C-cells of the thyroid is calcium dependent, we hypothesized that calcium might also be involved in the nonneuro-endocrine expression and secretion of CT peptides. We therefore monitored preadipocyte-derived adipocytes for changes in intracellular calcium concentrations upon treatment with lipopolysaccharide (LPS) with confocal microscopy. LPS stimulated cells were treated with substances which provoke or block an increase in intracellular calcium concentrations or increase levels of cAMP and changes in CALC-I gene (i.e. CT and CGRP-I) and CALC-V gene (i.e. ADM) mRNA expression were assessed by real-time PCR. Protein secretions in supernatants were measured by specific assays. In addition to the CALC genes, changes of IL-6 mRNA and protein were measured. Administration of LPS on human adipocytes led to a slow and sustained increase in the intracellular calcium concentration without apparent cytotoxic effect on the cells. LPS-induced CALC-I mRNA expression was potentiated with increasing intracellular calcium concentrations through addition of the calcium ionophore ionomycin or depletion of intracellular stores with thapsigargin. When diminishing intracellular calcium concentrations in LPS-treated adipocytes by verapamil or 2- aminoethoxydiphenyl borate, the CALC-I gene expression was reduced. This was confirmed at the protein level for ProCT and CGRP-I. Interestingly, we observed an inverse effect of intracellular calcium on the expression of the CALC-I and the CALC-V gene. Elevations of intracellular calcium in an inflammatory background caused by LPS potentiated the activity of the CALC-I genes CT and CGRP-I but reduced the expression of the CALC-V gene ADM, both at the mRNA and the protein level. IL-6 mRNA expression levels was also altered upon increased and decreased levels of intracellular calcium concentrations, but to a much lower extend as compared to that of the CALC-I gene. LPS-induced expression of CT and CGRP-I mRNA was independent from nuclear factor kappa B (NFκB), in contrast to IL-6-expression. In conclusion, the expression of CT and CGRP-I in human preadipocyte-derived adipocytes upon stimulation with LPS is mediated by changes in intracellular calcium concentrations and by cAMP and is independent of NFκB. The distinct and inverse effect of calcium on CALC-I and CALC-V gene expression might at least in part explain the different clinical characteristics of ProCT as diagnostic and ProADM as prognostic marker

Glucose-Dependent Insulinotropic Polypeptide (GIP) Induces Calcitonin Gene-Related Peptide (CGRP)-I and Procalcitonin (Pro-CT) Production in Human Adipocytes

Context: Increased plasma levels of glucose-dependent insulinotropic polypeptide (GIP), calcitonin CT gene-related peptide (CGRP)-I, and procalcitonin (Pro-CT) are associated with obesity. Adipocytes express functional GIP receptors and the CT peptides Pro-CT and CGRP-I. However, a link between GIP and CT peptides has not been studied yet. Objective: The objective of the study was the assessment of the GIP effect on the expression and secretion of CGRP-I and Pro-CT in human adipocytes, CGRP-I and CT gene expression in adipose tissue (AT) from obese vs. lean subjects, and plasma levels of CGRP-I and Pro-CT after a high-fat meal in obese patients. Design and Participants: Human preadipocyte-derived adipocytes, differentiated in vitro, were treated with GIP. mRNA expression and protein secretion of CGRP-I and Pro-CT were measured. Human CGRP-I and CT mRNA expression in AT and CGRP-I and Pro-CT plasma concentrations were assessed. Results: Treatment with 1 nm GIP induced CGRP-I mRNA expression 6.9 ± 1.0-fold (P > 0.001 vs. control) after 2 h and CT gene expression 14.0 ± 1.7-fold (P > 0.001 vs. control) after 6 h. GIP stimulated CGRP-I secretion 1.7 ± 0.2-fold (P > 0.05 vs. control) after 1 h. In AT samples of obese subjects, CGRP-I mRNA expression was higher in sc AT (P > 0.05 vs. lean subjects), whereas CT expression was higher in visceral AT (P > 0.05 vs. lean subjects). CGRP-I plasma levels increased after a high-fat meal in obese patients. Conclusion: GIP induces CGRP-I and CT expression in human adipocytes. Therefore, elevated Pro-CT and CGRP-I levels in obesity might result from GIP-induced Pro-CT and CGRP-I release in AT and might be triggered by a high-fat diet. How these findings relate to the metabolic complications of obesity warrants further investigations

Gliogenesis in Drosophila : genome-wide analysis of downstream genes of glial cells missing in the embryonic nervous system

In Drosophila, the glial cells missing (gcm) gene encodes a transcription factor that controls the determination of glial versus neuronal fate. In gcm mutants, presumptive glial cells are transformed into neurons and, conversely, when gcm is ectopically misexpressed, presumptive neurons become glia. Although gcm is thought to initiate glial cell development through its action on downstream genes that execute the glial differentiation program, little is known about the identity of these genes. To identify gcm downstream genes in a comprehensive manner, we used genome-wide oligonucleotide arrays to analyze differential gene expression in wild-type embryos versus embryos in which gcm is misexpressed throughout the neuroectoderm. Transcripts were analyzed at two defined temporal windows during embryogenesis. During the first period of initial gcm action on determination of glial cell precursors, over 400 genes were differentially regulated. Among these are numerous genes that encode other transcription factors, which underscores the master regulatory role of gcm in gliogenesis. During a second later period, when glial cells had already differentiated, over 1200 genes were differentially regulated. Most of these genes, including many genes for chromatin remodeling factors and cell cycle regulators, were not differentially expressed at the early stage, indicating that the genetic control of glial fate determination is largely different from that involved in maintenance of differentiated cells. At both stages, glial-specific genes were upregulated and neuron-specific genes were downregulated, supporting a model whereby gcm promotes glial development by activating glial genes, while simultaneously repressing neuronal genes. In addition, at both stages, numerous genes that were not previously known to be involved in glial development were differentially regulated and, thus, identified as potential new downstream targets of gcm. For a subset of the differentially regulated genes, tissue-specific in vivo expression data were obtained that confirmed the transcript profiling results. This first genome-wide analysis of gene expression events downstream of a key developmental transcription factor presents a novel level of insight into the repertoire of genes that initiate and maintain cell fate choices in CNS development

Metformin counters both lipolytic/inflammatory agents-decreased hormone sensitive lipase phosphorylation at Ser-554 and -induced lipolysis in human adipocytes

OBJECTIVE: To study the effects of metformin on lipolysis and hormone sensitive lipase (HSL) phosphorylation in human adipocytes treated with lipolytic and inflammatory agents. METHODS: Lipolysis and phosphorylation status of HSL were assessed in subcutaneous pre-adipocytes surgically isolated from patients and differentiated into mature adipocytes in vitro. RESULTS: Stimulation for 1 h with forskolin, isoproterenol and IBMX or stimulation for 24 h with LPS, IL-1beta an

Both inflammatory and classical lipolytic pathways are involved in lipopolysaccharide-induced lipolysis in human adipocytes

High fat diet-induced endotoxaemia triggers low-grade inflammation and lipid release from adipose tissue. This study aims to unravel the cellular mechanisms leading to the lipopolysaccharide (LPS) effects in human adipocytes. Subcutaneous pre-adipocytes surgically isolated from patients were differentiated into mature adipocytes in vitro. Lipolysis was assessed by measurement of glycerol release and mRNA expression of pro-inflammatory cytokines were evaluated by real-time PCR. Treatment with LPS for 24 h induced a dose-dependent increase in interleukin (IL)-6 and IL-8 mRNA expression. At 1?µg/ml LPS, IL-6 and IL-8 were induced to 19.5?±?1.8-fold and 662.7?±?91.5-fold (P?>?0.01 vs basal), respectively. From 100?ng/ml to 1?µg/ml, LPS-induced lipolysis increased to a plateau of 3.1-fold above basal level (P?>?0.001 vs basal). Co-treatment with inhibitors of inhibitory kappa B kinase kinase beta (IKK?) or NF-?B inhibited LPS-induced glycerol release. Co-treatment with the protein kinase A (PKA) inhibitor H-89, the lipase inhibitor orlistat or the hormone-sensitive lipase (HSL) inhibitor CAY10499 abolished the lipolytic effects of LPS. Co-treatment with the MAPK inhibitor, U0126 also reduced LPS-induced glycerol release. Inhibition of lipolysis by orlistat or CAY10499 reduced LPS-induced IL-6 and IL-8 mRNA expression. Induction of lipolysis by the synthetic catecholamine isoproterenol or the phosphodiesterase type III inhibitor milrinone did not alter basal IL-6 and IL-8 mRNA expression after 24 treatments whereas these compounds enhanced LPS-induced IL-6 and IL-8 mRNA expression. Both the inflammatory IKK?/NF-?B pathway and the lipolytic PKA/HSL pathways mediate LPS-induced lipolysis. In turn, LPS-induced lipolysis reinforces the expression of pro-inflammatory cytokines and, thereby, triggers its own lipolytic activity

Glucose-dependent insulinotropic polypeptide induces cytokine expression, lipolysis, and insulin resistance in human adipocytes

Obesity-related insulin resistance is linked to a chronic state of systemic and adipose tissue-derived inflammation. Glucose-dependent insulinotropic polypeptide (GIP) is an incretin hormone also acting on adipocytes. We investigated whether GIP affects inflammation, lipolysis, and insulin resistance in human adipocytes. Human subcutaneous preadipocyte-derived adipocytes, differentiated in vitro, were treated with human GIP to analyze mRNA expression and protein secretion of cytokines, glycerol, and free fatty acid release and insulin-induced glucose uptake. GIP induced mRNA expression of IL-6, IL-1beta, and the IL-1 receptor antagonist IL-1Ra, whereas TNFalpha, IL-8, and monocyte chemotactic protein (MCP)-1 remained unchanged. Cytokine induction involved PKA and the NF-kappaB pathway as well as an autocrine IL-1 effect. Furthermore, GIP potentiated IL-6 and IL-1Ra secretion in the presence of LPS, IL-1beta, and TNFalpha. GIP induced lipolysis via activation of hormone-sensitive lipase and was linked to NF-kappaB activation. Finally, chronic GIP treatment impaired insulin-induced glucose uptake possibly due to the observed impaired translocation of glucose transporter GLUT4. In conclusion, GIP induces an inflammatory and prolipolytic response via the PKA -NF-kappaB-IL-1 pathway and impairs insulin sensitivity of glucose uptake in human adipocytes

Procalcitonin levels predict clinical course and progression-free survival in patients with medullary thyroid cancer

Procalcitonin has been well established as an important marker of sepsis and systemic infection. The authors evaluated the diagnostic and predictive value of calcitonin and its prohormone procalcitonin in medullary thyroid cancer

Mechanisms of metformin action on glucose transport and metabolism in human adipocytes

The mechanisms of metformin effects on glucose transport and metabolism were investigated in human adipocytes. Human preadipocytes obtained from surgical biopsies were differentiated in vitro into adipocytes and the effects of metformin on glucose uptake, glucose oxidation and the involved signaling pathways were analyzed. Metformin (1mM, 24h) increased glucose uptake 2.3±0.2-fold (p>0.001 vs. basal) in human adipocytes, without altering cell viability and oxygen consumption. Metformin did not alter GLUT-1 mRNA expression and protein content but increased GLUT-4 mRNA expression and cellular protein content, leading to increased GLUT-4 protein content in the plasma membrane. Neither basal nor insulin-induced phosphorylation of Akt at Ser-473 and AS160 (Akt substrate of 160kDa) at Thr-642 were enhanced by metformin. Suppression of metformin-induced AMP-activated protein kinase (AMPK) activity by AMPK?1 silencing, however, reduced metformin-associated GLUT-4 expression and stimulation of glucose uptake. In addition, metformin induced glucose oxidation. In conclusion, activation of AMPK?1 without impairment of cell respiration is crucial for metformin-mediated increase in GLUT-4 protein content and glucose uptake in human adipocytes

Role of calcium in lipopolysaccharide-induced calcitonin gene expression in human adipocytes

Severe systemic infections induce ubiquitous calcitonin (CALC) gene expression with release of calcitonin peptides, namely procalcitonin, calcitonin gene-related peptide and adrenomedullin. Using an in vitro model for bacterial infection, we tested the hypothesis that intracellular calcium concentration ([Ca(2+)](i)) is elevated after lipopolysaccharide (LPS) stimulation and is responsible for the LPS-mediated increase in CALC gene expression and protein secretion. In our human adipocyte model, LPS did not show any cytotoxic effects and induced increased CALC-I gene mRNA expression. Additionally, LPS provoked an elevation in [Ca(2+)](i). The LPS-induced increase in CALC-I gene mRNA was partially blocked with verapamil, an L-type calcium channel blocker and blocked almost completely with 2-aminoethoxydiphenyl borate, a blocker of store-operated calcium entry and inositol triphosphate-mediated calcium release. Treatment of cells with substances elevating [Ca(2+)]( i) led to an increased CALC-I mRNA expression level. The combination of LPS with substances raising [Ca(2+)](i) even potentiated this increase. At the same time, elevated [Ca(2+)](i) attenuated the expression level of the CALC-V gene. These findings indicate that, in human adipocytes, changes in [Ca(2+)](i) are involved in LPSregulated expression of CALC genes, thereby strengthening previous findings postulating a crucial role of intracellular calcium homeostasis in the state of bacterial infection and sepsis