THP-1 Macrophages and SGBS Adipocytes – A New Human in vitro Model System of Inflamed Adipose Tissue

Obesity is associated with an accumulation of macrophages in adipose tissue. This inflammation of adipose tissue is a key event in the pathogenesis of several obesity-related disorders, particularly insulin resistance. Here, we summarized existing model systems that mimic the situation of inflamed adipose tissue in vitro, most of them being murine. Importantly, we introduce our newly established human model system which combines the THP-1 monocytic cell line and the preadipocyte cell strain Simpson–Golabi–Behmel syndrome (SGBS). THP-1 cells, which originate from an acute monocytic leukemia, differentiate easily into macrophages in vitro. The human preadipocyte cell strain SGBS was recently introduced as a unique tool to study human fat cell functions. SGBS cells are characterized by a high capacity for adipogenic differentiation. SGBS adipocytes are capable of fat cell-specific metabolic functions such as insulin-stimulated glucose uptake, insulin-stimulated de novo lipogenesis and β-adrenergic-stimulated lipolysis and they secrete typical adipokines including leptin, adiponectin, and RBP4. Applying either macrophage-conditioned medium or a direct co-culture of macrophages and fat cells, our model system can be used to distinguish between paracrine and cell-contact dependent effects. In conclusion, we propose this model as a useful tool to study adipose inflammation in vitro. It represents an inexpensive, highly reproducible human system. The methods described here can be easily extended for usage of primary human macrophages and fat cells

Dibutyltin Disrupts Glucocorticoid Receptor Function and Impairs Glucocorticoid-Induced Suppression of Cytokine Production

BACKGROUND: Organotins are highly toxic and widely distributed environmental chemicals. Dibutyltin (DBT) is used as stabilizer in the production of polyvinyl chloride plastics, and it is also the major metabolite formed from tributyltin (TBT) in vivo. DBT is immunotoxic, however, the responsible targets remain to be defined. Due to the importance of glucocorticoids in immune-modulation, we investigated whether DBT could interfere with glucocorticoid receptor (GR) function. METHODOLOGY: We used HEK-293 cells transiently transfected with human GR as well as rat H4IIE hepatoma cells and native human macrophages and human THP-1 macrophages expressing endogenous receptor to study organotin effects on GR function. Docking of organotins was used to investigate the binding mechanism. PRINCIPAL FINDINGS: We found that nanomolar concentrations of DBT, but not other organotins tested, inhibit ligand binding to GR and its transcriptional activity. Docking analysis indicated that DBT inhibits GR activation allosterically by inserting into a site close to the steroid-binding pocket, which disrupts a key interaction between the A-ring of the glucocorticoid and the GR. DBT inhibited glucocorticoid-induced expression of phosphoenolpyruvate carboxykinase (PEPCK) and tyrosine-aminotransferase (TAT) and abolished the glucocorticoid-mediated transrepression of TNF-alpha-induced NF-kappaB activity. Moreover, DBT abrogated the glucocorticoid-mediated suppression of interleukin-6 (IL-6) and TNF-alpha production in lipopolysaccharide (LPS)-stimulated native human macrophages and human THP-1 macrophages. CONCLUSIONS: DBT inhibits ligand binding to GR and subsequent activation of the receptor. By blocking GR activation, DBT may disturb metabolic functions and modulation of the immune system, providing an explanation for some of the toxic effects of this organotin

Disruption of glucocorticoid action by environmental chemicals: potential mechanisms and relevance

Glucocorticoids play an essential role in the regulation of key physiological processes, including immunomodulation, brain function, energy metabolism, electrolyte balance and blood pressure. Exposure to naturally occurring compounds or industrial chemicals that impair glucocorticoid action may contribute to the increasing incidence of cognitive deficits, immune disorders and metabolic diseases. Potentially, "glucocorticoid disruptors" can interfere with various steps of hormone action, e.g. hormone synthesis, binding to plasma proteins, delivery to target cells, pre-receptor regulation of the ratio of active versus inactive hormones, glucocorticoid receptor (GR) function, or export and degradation of glucocorticoids. Several recent studies indicate that such chemicals exist and that some of them can cause multiple toxic effects by interfering with different steps of hormone action. For example, increasing evidence suggests that organotins disturb glucocorticoid action by altering the function of factors that regulate the expression of 11beta-hydroxysteroid dehydrogenase (11beta-HSD) pre-receptor enzymes, by direct inhibition of 11beta-HSD2-dependent inactivation of glucocorticoids, and by blocking GR activation. These observations emphasize on the complexity of the toxic effects caused by such compounds and on the need of suitable test systems to assess their effects on each relevant step

Coffee inhibits the reactivation of glucocorticoids by 11beta-hydroxysteroid dehydrogenase type 1: a glucocorticoid connection in the anti-diabetic action of coffee?

Recent epidemiological studies demonstrated a beneficial effect of coffee consumption for the prevention of type 2 diabetes, however, the underlying mechanisms remained unknown. We demonstrate that coffee extract, corresponding to an Italian Espresso, inhibits recombinant and endogenous 11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1) activity. The inhibitory component is heat-stable with considerable polarity. Coffee extract blocked 11beta-HSD1-dependent cortisol formation, prevented the subsequent nuclear translocation of the glucocorticoid receptor and abolished glucocorticoid-induced expression of the key gluconeogenic enzyme phosphoenolpyruvate carboxykinase. We suggest that at least part of the anti-diabetic effects of coffee consumption is due to inhibition of 11beta-HSD1-dependent glucocorticoid reactivation

Inhibition of ligand binding to GR by dibutyltin.

<p>HEK-293 cells were transfected with a plasmid for human GR-α, followed by overnight incubation in serum-free medium containing vehicle (0.1% DMSO)(<i>A</i>,<i>B</i>) or in medium containing either vehicle or 50 nM or 100 nM DBT (<i>C</i>). Cells were then incubated for 3 h with 10 nM radiolabeled dexamethasone and various concentrations of DBT (<i>A</i>) or with 10 nM radiolabeled dexamethasone, various concentrations of unlabeled dexamethasone and various concentrations of DBT (<i>B</i>,<i>C</i>) prior to removal of unbound steroids and determination of bound dexamethasone by scintillation counting (<i>A</i>). Data represent mean±SD from three independent experiments. <i>D</i>, cells transiently expressing GR-α were preincubated for 16 h in serum-free medium containing vehicle (0.1% DMSO), 10 nM dexamethasone (Dex) or 500 nM DBT, followed by incubation for another 3 h with vehicle, 10 nM dexamethasone or 500 nM DBT and 10 nM dexamethasone, respectively (lane 1–3). Alternatively, cells preincubated for 16 h in serum-free medium were incubated for 3 h in medium containing 500 nM DBT and 10 nM dexamethasone (lane 4). Cells were lysed and the expression of GR protein and actin control was analyzed by Western blotting.</p

Inhibition of glucocorticoid-mediated expression of hepatic target genes by dibutyltin.

<p><i>A</i>, DBT prevents glucocorticoid-induced stimulation of PEPCK mRNA expression. H4IIE hepatoma cells were preincubated for 16 h in the absence or presence of DBT, followed by adding 10 nM dexamethasone and incubation for another 24 h at 37°C. mRNA levels were determined by real-time RT-PCR using TaqMan technology. Data (mean±S.D. of triplicates from four independent experiments) are relative to the ratio of PEPCK mRNA to β-actin control mRNA from cells treated with dexamethasone in the absence of DBT. <i>B</i>, DBT inhibits glucocorticoid-induced stimulation of TAT activity. H4IIE cells were preincubated for 16 h in the absence or presence of DBT, followed by adding 5 nM dexamethasone and incubation for another 20 h. TAT activity, determined in cell lysates and normalized to protein content, is given as mean±S.D. of triplicates from four independent experiments and normalized to the positive control in the presence of dexamethasone. * p<0.05.</p

Docking of organotins into the dexamethasone-binding site and at an allosteric site in the GR.

<p><i>A</i>, DBT (white), TBT (blue), DPT (orange) and TPT (yellow) fit into the dexamethasone-binding site of the GR. In these unminimized complexes with the GR, DBT and TBT overlap the D-ring and DPT and TPT most of dexamethasone. Distances between dexamethasone and Gln⁵⁷⁰, Arg⁶¹¹, Asn⁵⁶⁴ and Gln⁶⁴² are taken from the crystal structure <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0003545#pone.0003545-Bledsoe1" target="_blank">[21]</a>. <i>B</i>, organotins docked into the GR at an allosteric site close to Glu⁵⁷⁰ and Arg⁶¹¹.</p

Inhibition of GR-mediated transrepression and potentiation of TNF-α-induced activation of NF-κB.

<p>HEK-293 cells were transfected with a plasmid for a luciferase reporter gene under the control of a promoter containing three NF-κB binding sites together with a plasmid for human GR. Cells were preincubated for 16 h with various concentrations of DBT prior to stimulation with TNF-α in the presence of glucocorticoids. Data represent mean±S.D. from three independent experiments, normalized to the TNF-α -stimulated control.</p