Chronic prednisolone treatment aggravates hyperglycemia in mice fed a high-fat diet but does not worsen dietary fat-induced insulin resistance

Synthetic glucocorticoids such as prednisolone have potent antiinflammatory actions. Unfortunately, these drugs induce severe adverse effects in patients, many of which resemble features of the metabolic syndrome, such as insulin resistance. In this study, we investigated whether adverse effects of prednisolone on glucose homeostasis are aggravated in mice with compromised insulin sensitivity due to a high-fat diet by applying various methods to analyze changes in insulin sensitivity in mice. C57BL/6J micewerefed a high-fat diet for 6wkandtreated with either prednisolone (10 mg/kg · d) or vehicle for the last 7 d. Insulin sensitivity and blood glucose kinetics were analyzed with state-of-the-art stable isotope procedures in different experimental conditions. Prednisolone treatment aggravated fasting hyperglycemia and hyperinsulinemia caused by high-fat feeding, resulting in a higher homeostatic assessment model of insulin resistance. In addition, prednisolone-treated high-fat diet-fed mice appeared less insulin sensitive by detailed analysis of basal glucose kinetics. Remarkably, using hyperinsulinemic-euglycemic or hyperglycemic clamp techniques, neither hepatic nor peripheral insulin resistance was worsened in the group that was treated with prednisolone. Yet analysis of hepatic glucose metabolism revealed that prednisolone did alter glycogen balance by reducing glycogen synthase flux under hyperinsulinemic as well as hyperglycemic conditions. In addition to elevated insulin levels, prednisolone-treated mice showed a major rise in plasma leptin and fibroblast growth factor 21 levels. Our data indicate that prednisoloneinduced adverse effects on glucose metabolism in high-fat diet-fed mice do not reflect impaired insulin sensitivity but may be caused by other changes in the hormonal regulatory network controlling glucose metabolism such as fibroblast growth factor 21 and leptin. Copyrigh

Org 214007-0: a novel non-steroidal selective glucocorticoid receptor modulator with full anti-inflammatory properties and improved therapeutic index

Contains fulltext : 103595.pdf (publisher's version ) (Open Access)Glucocorticoids (GCs) such as prednisolone are potent immunosuppressive drugs but suffer from severe adverse effects, including the induction of insulin resistance. Therefore, development of so-called Selective Glucocorticoid Receptor Modulators (SGRM) is highly desirable. Here we describe a non-steroidal Glucocorticoid Receptor (GR)-selective compound (Org 214007-0) with a binding affinity to GR similar to that of prednisolone. Structural modelling of the GR-Org 214007-0 binding site shows disturbance of the loop between helix 11 and helix 12 of GR, confirmed by partial recruitment of the TIF2-3 peptide. Using various cell lines and primary human cells, we show here that Org 214007-0 acts as a partial GC agonist, since it repressed inflammatory genes and was less effective in induction of metabolic genes. More importantly, in vivo studies in mice indicated that Org 214007-0 retained full efficacy in acute inflammation models as well as in a chronic collagen-induced arthritis (CIA) model. Gene expression profiling of muscle tissue derived from arthritic mice showed a partial activity of Org 214007-0 at an equi-efficacious dosage of prednisolone, with an increased ratio in repression versus induction of genes. Finally, in mice Org 214007-0 did not induce elevated fasting glucose nor the shift in glucose/glycogen balance in the liver seen with an equi-efficacious dose of prednisolone. All together, our data demonstrate that Org 214007-0 is a novel SGRMs with an improved therapeutic index compared to prednisolone. This class of SGRMs can contribute to effective anti-inflammatory therapy with a lower risk for metabolic side effects

Org 214007-0 is equally effective as prednisolone in the mouse CIA model.

<p>A) Inhibition of arthritis in the CIA model. Mean clinical score of each group (n = 12), shown as area under the curve (AUC) of the arthritis score monitored every other day during 3 weeks, corrected for baseline, is indicated (± SEM). **  =  significantly different from placebo (p<0.01; ANOVA-test). B) Reduction of bone destruction in the CIA model as measured by X-ray. Mean radiological score (sum of the X-ray scores of left and right hindpaws and knees) of each group of mice (n = 12) at the end of the CIA experiment is indicated (± SEM). **  =  significantly different from placebo (p<0.01; ANOVA-test).</p

Org 214007-0 does not effect rates of hepatic enzyme fluxes.

<p>Mass Isotopomer Distribution Analysis (MIDA), as described in detail in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048385#s4" target="_blank">Materials and Methods</a>, was performed in mice treated p.o., once daily, for 7 days with either vehicle, prednisolone (10 mg/kg) or Org 214007-0 (1.5 mg/kg). These doses of each compound are equi-efficacious in suppression of CIA. Neither the glucose-6-phosphatase flux (A) nor the glycogen phosphorylase flux (B) were affected by treatment with prednisolone or Org 214007-0. The glucokinase flux rate (C) was not changed by Org 214007-0, but significantly differed from the effect by prednisolone (##: p = 0.01 <i>vs</i> prednisolone). The glycogen synthase flux rate (D) was significantly decreased by prednisolone (**: p = 0.005 <i>vs</i> vehicle), whereas Org 214007-0 had no significant effect on this flux, but differed significantly from prednisolone (##: p = 0.002 <i>vs</i> prednisolone). Neither Org 214007-0 nor prednisolone, at equi-efficacious dosages, effects the gluconeogenic flux (<i>de novo</i> synthesis of glucose-6-phophate) (E).</p

Org 214007-0 has a relatively lower impact on induction than on respression of genes.

<p>Fold changes for the top 25 genes either induced (A) or repressed (B) by 1 μM prednisolone and 1 μM Org 214007-0 in THP-1 cells. NB. Scales are in 2log. Example of an induced gene, FK506 binding protein 51 (FKBP51) (C) and a repressed gene, interleukin 6 (IL-6) (D) in comparison to the vehicle control under either non-stimulated or stimulated (IFNγ/TNFα) condition. Fold changes for the top 25 genes either induced (E) or repressed (F) by 1.5 mg/kg prednisolone and 0.3 mg/kg Org 214007-0 in muscle tissue from arthritic mice. NB. Scales are in 2log. Example of an induced gene (Per-2) (G) and a repressed gene (Ccl8) (H) in comparison to vehicle treated arthritic mice and vehicle treated healthy mice.</p

Structure and predicted binding mode of Org 214007-0.

<p>A) The structure of ORG 214007-0. This compound, [(-)-N-(2S,10S,14bS)]-N-(8-cyano-1,2,3,4,10,14b-hexahydro-10-methyl dibenzo<i>[c,f]</i>pyrido[1,2-<i>a</i>]azepin-2-yl)-4-methyl-1,2,3-thiadiazole-5-carboxamide] has a molecular weight of 430 g/mole (C₂₄H₂₃N₅OS) B) The predicted binding mode of Org 214007-0 modeled in complex with the glucocorticoid receptor and demonstrating conservation of interactions typical to steroidal glucocorticoids (Gln564, Asn570, Arg611 and Gln642).</p

Org 214007-0 behaves as a partial agonist in vitro.

<p>A) In a co-factor recruitment assay, Org 214007-0, in comparison to prednisolone, shows potent but partial recruitment of a 0.1 μM peptide presenting TIF2 -3. On the Y-axis average fluorescence counts (+/− SD) are shown. EC50 values (%CV) and percentages maximal efficacy (%CV) for Org 214007-0 versus prednisolone were 10 (7.4) nM versus 48 (3.2) nM and 67 (7.1) % vs 100% respectively. B) In THP1 cells Org 214007-0 shows partial induction of FKBP51 protein expression and C) under inflammatory conditions represses the IL-6 protein expression almost as good as prednisolone does.</p

Summary of studies to define the therapeutic index (TI) of Org 214007-0.

<p>A) <i>THP-1</i>: Microarray (<i>m.a</i>.) analysis of mRNA isolated from THP-1 cells incubated for 6 hours with either 1 μM prednisolone or 1 μM Org 214007-0 without (<i>ind</i>) or with IFNγ (220 ng/ml)/TNFα (374 ng/ml) ( = <i>I/T</i>), (<i>rep</i>) The mean percentage repression or induction of genes compared to that by prednisolone (set at 100%) is indicated. B) <i>THP-1 – rep:</i> Repression of gene expression in THP-1 cells. <i>I/T – MCP-1, IL-6, IL-8</i>  =  TNFα (60 ng/ml)/IFNγ (40 ng/ml) induced MCP-1, IL-6 or IL-8 release. C) <i>THP-1 – ind</i>: Induction of FK506 binding protein 51 (<i>FKBP51</i>), glucocorticoid induced leucine zipper (<i>GILZ</i>) and dual specificity phosphatase 1 (<i>DUSP1</i>) in THP-1 cells. D) <i>hWB</i> – <i>rep:</i> Inhibition of LPS-induced (<i>LPS</i>) TNFα release or PMA/anti-CD28 (<i>P/28</i>) induced IL-5 release by primary human whole blood cells and <i>hWB – ind</i>: enhancement of PMA/anti-CD28/compound (<i>P/28</i>) induced G-CSF release by primary human whole blood cells. E) <i>CASM3C – rep</i>: Inhibition of MCP-1 release of coronary artery smooth muscle cells ( = <i>CASM3C</i>) stimulated with a cytokine mixture of IL-1β (1 ng/ml), IFNγ (100 ng/ml) and TFNα (5 ng/ml) ( = <i>1/I/T</i>) by 1 μM prednisolone or 1 μM Org 214007-0. <i>CASM3C – ind</i>: Induction of serum amyloid A (<i>SAA</i>) of the cells mentioned above by 1 μM prednisolone or 1 M Org 214007-0. F) <i>HDF3CGF – rep</i>: inhibition of matrix metalloproteinase (<i>MMP-1</i>) release of human neonatal foreskin fibroblasts stimulated with the cytokine mixture mentioned above plus required growth factors ( = <i>HDF3CGF</i>). <i>HDF3CGF – ind</i>: Activation of plasminogen activator inhibitor-1 (<i>PAI-1</i>) by cells mentioned above by 1 μM prednisolone or 1 μM Org 214007-0. G) <i>CIA mus</i>.: Microarray (<i>m.a</i>.) analysis of mRNA isolated from muscle cells isolated at day 21, 2.5 hours after the final oral administration of either 1.5 mg/kg prednisolone or 0.3 mg/kg Org 214007-0 in the mouse CIA experiment. The mean percentage repression (<i>rep</i>) or induction (<i>ind</i>) of genes compared to that by prednisolone (set at 100%) is indicated.</p><p>IC50 or EC50 values represent the mean concentration of compound (±SD) required to resp. inhibit or effect the response to 50%. Maximal efficacy (Max. eff.) is expressed as the mean relative maximal effect (±SD) compared to the maximal effect by prednisolone (set at 100%). A relative therapeutic index (TI_rel) is calculated by the ratio of (the mean) % maximal efficacy in repression and (the mean) % maximal efficacy in induction of genes by Org 214007, where that of prednisolone is set at 1 (100%/100%). All assays (except for the microarray experiments) are performed at least two times.</p

Org 214007-0 leads to relatively less GR occupancy.

<p>A) Ratio of the read count for clusters induced by 1 μM prednisolone or 1 μM Org 214007-0 in a ChIP-Seq analysis. If Org 214007-0 and prednisolone would induce an equal number of reads, the histogram would be centered on Log0, indicated by the dotted line. There is a clear shift to the right from this line (P-value (mean  = 0) <0.000001), indicating that prednisolone leads to more GR occupancy than Org 214007-0. B) Example profile of the tag clusters in the GR response gene FKBP51. Multiple binding sites are found within this gene, each showing denser clusters after treatment with 1 μM prednisolone than after treatment with 1 μM Org 214007-0. The inset highlights an intronic region at 87 kb downstream of the transcription start site. This region that was identified by Paakinaho et al. (2010) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048385#pone.0048385-Paakinaho1" target="_blank">[54]</a>, as a major intronic enhancer in human A459 lung cancer cells and was shown to be occupied by GR after treatment with the GR ligand dexamethasone.</p

Summary of <i>in vitro</i> studies.

<p>Prednisolone (Pred) and Org 214007-0 (Org) were tested in different <i>in vitro</i> studies. A) <i>GR binding</i>: binding to recombinant human glucocorticoid receptor (GR) assessed by a fluorescence polarization competitor binding assay. Ki  =  inhibition constant or concentration of compound in the competitive binding assay which would occupy 50% of GR if no ligand was present. B) <i>CHO – ind</i>: Induction of gene expression measured in CHO cells stably co-transfected with human GR and a MMTV promoter – luciferase construct. C) <i>HepG2 – ind</i>: Induction of gene expression measured by microarray analysis of mRNA isolated from HepG2 cells incubated with either 1 μM prednisolone or 1 μM Org 214007-0. D) <i>U2OS – rep</i>: Repression of gene expression in U2OS cells overexpressing human GR. INFγ/TNFα – MCP-1  =  IFNγ (100 ng/ml)/TNFα (50 ng/ml) induced MCP-1 release.</p><p>IC50 or EC50 values represent the mean concentration of compound (±SD) required to resp. inhibit or effect the response to 50%. Maximal efficacy (Max. eff.) is expressed as the mean relative maximal effect (±SD) compared to the maximal effect by prednisolone (set at 100%). All assays (except for the micro array experiments) are performed at least two times.</p