Peroxisome proliferator-activated receptor-α agonists modulate CXCL9 and CXCL11 chemokines in Graves' ophthalmopathy fibroblasts and preadipocytes

Peroxisome proliferator-activated receptors (PPAR)α have been shown to exert immunomodulatory effects in autoimmune disorders; no study evaluated the effect of PPARα activation in Graves’ ophthalmopathy (GO).We show the presence of PPARα, δ and γ in GO fibroblasts and preadipocytes. PPARα activators have a potent inhibitory action on the secretion of CXCL9 and CXCL11 chemokines (induced by IFNγ and TNFα) in fibroblasts and preadipocytes. The potency of the used PPARα agonists was maximum on the secretion of CXCL11 (67% inhibition by fenofibrate) in fibroblasts. The relative potency of the compounds in GO fibroblasts was different with each chemokine. PPARα agonists were stronger inhibitors of CXCL9 and CXCL11 (in GO fibroblasts and preadipocytes) than PPARγ activators.This study first shows that PPARα activators inhibit CXCL9 and CXCL11 chemokines in normal and GO fibroblasts and preadipocytes, suggesting that PPARα may be involved in the modulation of the immune response in GO.     

Age-related differences in the effects of α and γ peroxisome proliferator-activated receptor subtype agonists on endothelial vasodilation in human microvessels

Endothelial vasodilation in human vessels is impaired by aging and other cardiovascular risk factors (CVRF) but the differential impact of aging and CVRF in human endothelial function is not completely elucidated. The aim of this work was to evaluate the influence of aging on the effects of peroxisome proliferator-activated receptor (PPAR)-α and -γ subtype agonists on endothelium-dependent vasodilation of isolated human vessels from subjects with or without CVRF. Human mesenteric microarteries were dissected from omentum specimens obtained from subjects younger or older than 60years having or not CVRF and mounted in wire myographs to evaluate endothelium-dependent relaxation to bradykinin (BK). Aging and CVRF independently reduced endothelium-dependent relaxations. An additional impairment was produced when aging and CVRF co-existed (p<0.001). In vessels from adult subjects PPARγ agonist, GW1929 (1μM) improved BK-induced responses only in those obtained from subjects with CVRF. By contrast, GW1929 improved the responses in vessels from elderly subjects having or not CVRF. PPARα agonist, GW7647 (1μM), enhanced endothelial vasodilation in adults with CVRF (p<0.001) but lack any effect in vessels from older subjects having or not CVRF. In vessels from subjects with CVRF, superoxide dismutase (SOD; 100U/ml) improved BK-induced responses only in elderly subjects (p<0.001). Vascular aging negatively impacts endothelial function independently of the presence of additional CVRF through specific molecular mechanisms involving superoxide generation. While PPARγ activation remains effective, the improving effects of PPARα agonists on endothelial responses disappear in aged human vessels.     

Effects of peroxisome proliferator-activated receptor (PPAR)-α and PPAR-γ agonists on glucose and lipid metabolism in patients with type 2 diabetes mellitus

Aims/hypothesis The aim of the study was to examine the effects of pioglitazone (PIO), a peroxisome proliferator-activated receptor (PPAR)-γ agonist, and fenofibrate (FENO), a PPAR-α agonist, as monotherapy and in combination on glucose and lipid metabolism. Subjects and methods Fifteen type 2 diabetic patients received FENO (n = 8) or PIO (n = 7) for 3 months, followed by the addition of the other agent for 3 months in an open-label study. Subjects received a 4 h hyperinsulinaemic–euglycaemic clamp and a hepatic fat content measurement at 0, 3 and 6 months. Results Following PIO, fasting plasma glucose (FPG) (p < 0.05) and HbA_1c (p < 0.01) decreased, while plasma adiponectin (AD) (5.5 ± 0.9 to 13.8 ± 3.5 μg/ml [SEM], p < 0.03) and the rate of insulin-stimulated total-body glucose disposal (R _d) (23.8 ± 3.8 to 40.5 ± 4.4 μmol kg⁻¹ min⁻¹, p < 0.005) increased. After FENO, FPG, HbA_1c, AD and R _d did not change. PIO reduced fasting NEFA (784 ± 53 to 546 ± 43 μmol/l, p < 0.05), triacylglycerol (2.12 ± 0.28 to 1.61 ± 0.22 mmol/l, p < 0.05) and hepatic fat content (20.4 ± 4.8 to 10.2 ± 2.5%, p < 0.02). Following FENO, fasting NEFA and hepatic fat content did not change, while triacylglycerol decreased (2.20 ± 0.14 to 1.59 ± 0.13 mmol/l, p < 0.01). Addition of FENO to PIO had no effect on R _d, FPG, HbA_1c, NEFA, hepatic fat content or AD, but triacylglycerol decreased (1.61 ± 0.22 to 1.00 ± 0.15 mmol/l, p < 0.05). Addition of PIO to FENO increased R _d (24.9 ± 4.4 to 36.1 ± 2.2 μmol kg⁻¹ min⁻¹, p < 0.005) and AD (4.1 ± 0.8 to 13.1 ± 2.5 μg/ml, p < 0.005) and reduced FPG (p < 0.05), HbA_1c (p < 0.05), NEFA (p < 0.01), hepatic fat content (18.3 ± 3.1 to 13.5 ± 2.1%, p < 0.03) and triacylglycerol (1.59 ± 0.13 to 0.96 ± 0.9 mmol/l, p < 0.01). Muscle adenosine 5′-monophosphate-activated protein kinase (AMPK) activity did not change following FENO; following the addition of PIO, muscle AMPK activity increased significantly (phosphorylated AMPK:total AMPK ratio 1.2 ± 0.2 to 2.2 ± 0.3, p < 0.01). Conclusions/interpretation We conclude that PPAR-α therapy has no effect on NEFA or glucose metabolism and that addition of a PPAR-α agonist to a PPAR-γ agent causes a further decrease in plasma triacylglycerol, but has no effect on NEFA or glucose metabolism.     

Short-term inhibition of peroxisome proliferator-activated receptor-γ coactivator-1α expression reverses diet-induced diabetes mellitus and hepatic steatosis in mice

Aims/hypothesis The coactivator of nuclear receptors, peroxisome proliferator-activated receptor- coactivator-1 (PGC-1) has been implicated in a series of events that contribute to the control of glucose metabolism. We have recently reported the use of a PGC-1 antisense oligonucleotide (PGC-1AS) that inhibits up to 60% of PGC-1 expression in pancreatic islets, leading to increased insulin secretion. This oligonucleotide was used in this study to try to ameliorate diet-induced type 2 diabetes in a genetically predisposed mouse strain (Swiss mice).Materials and methods Glucose and insulin tolerance tests, euglycaemic–hyperinsulinaemic clamp, immunoprecipitation assays, immunoblotting assays and immunohistochemistry were used in this investigation.Results Swiss mice became obese and overtly diabetic after 8 weeks of feeding with chow containing 24% saturated fat. One daily dose (1.0 nmol) of PGC-1AS significantly reduced glucose and increased insulin blood levels without affecting food intake and body weight. These effects were accompanied by a reduced area under the glucose curve during an intraperitoneal glucose tolerance test, an increased constant of glucose decay (K_itt) during an insulin tolerance test, and an increased glucose consumption rate during a euglycaemic–hyperinsulinaemic clamp. Moreover, mice treated with PGC-1AS presented an outstanding reduction of macroscopic and microscopic features of hepatic steatosis. These effects were accompanied by reduced expression or function of a series of proteins involved in lipogenesis.Conclusions/interpretation PGC-1 is an attractive target for pharmacological therapeutics in type 2 diabetes mellitus and diet-induced hepatic steatosis.     

Anxiolytic effects and neuroanatomical targets of estrogen receptor-β (ERβ) activation by a selective ERβ agonist in female mice

The dichotomous anxiogenic and anxiolytic properties of estrogens have been reported to be mediated by two distinct neural estrogen receptors (ER), ERα and ERβ, respectively. Using a combination of pharmacological and genetic approaches, we confirmed that the anxiolytic actions of estradiol are mediated by ERβ and extended and these observations to demonstrate the neuroanatomical targets involved in ERβ activation in these behavioral responses. We examined the effects of the biologically active S-enantiomer of diarylpropionitrile (S-DPN) on anxiety-related behavioral measures, the corresponding stress hormonal response to hypothalamo-pituitary-adrenal axis reactivity, and potential sites of neuronal activation in mutant female mice carrying a null mutation for ERβ gene (βERKO). S-DPN administration significantly reduced anxiety-like behaviors in the open field, light-dark exploration, and the elevated plus maze (EPM) in ovariectomized wild-type (WT) mice, but not in their βERKO littermates. Stress-induced corticosterone (CORT) and ACTH were also attenuated by S-DPN in the WT mice but not in the βERKO mice. Using c-fos induction after elevated plus maze, as a marker of stress-induced neuronal activation, we identified the anterodorsal medial amygdala and bed nucleus of the stria terminalis as the neuronal targets of S-DPN action. Both areas showed elevated c-fos mRNA expression with S-DPN treatment in the WT but not βERKO females. These studies provide compelling evidence for anxiolytic effects mediated by ERβ, and its neuroanatomical targets, that send or receive projections to/from the paraventricular nucleus, providing potential indirect mode of action for the control of hypothalamo-pituitary-adrenal axis function and behaviors.     

Interactive influences of peroxisome proliferator-activated receptor α activation and glucocorticoids on pancreatic beta cell compensation in insulin resistance induced by dietary saturated fat in the rat

Aims/hypothesis We sought to elucidate whether excess glucocorticoids and increased dietary lipids act synergistically to impair glucose tolerance and, if so, whether activation of peroxisome proliferator-activated receptor (PPAR) has an adverse or beneficial effect on glucose tolerance.Methods Dexamethasone (100 g kg^–1 body weight day^–1; 5 days) was administered to insulin-resistant rats fed a high-saturated-fat (HF) diet for 4weeks. The PPAR agonist WY14643 was administered (50 mg kg^–1 body weight intraperitoneally) 24 h before sampling. Glucose-stimulated insulin secretion (GSIS) was assessed in vivo after an acute glucose bolus injection, and in vitro using step-up and step-down islet perifusions.Results Although neither PPAR activation nor dexamethasone alone affected fasting glycaemia in the HF group, dexamethasone in combination with PPAR activation elicited marked postabsorptive hyperglycaemia. Dexamethasone treatment of HF rats had little effect on GSIS after an acute glucose challenge in vivo, but induced glucose intolerance. PPAR activation augmented GSIS in dexamethasone-treated HF rats in vivo, restoring glucose tolerance. Contrasting with data obtained in vivo, greatly enhanced peak rates of GSIS were observed ex vivo in perifusions of islets from dexamethasone-treated HF rats compared with those from untreated HF rats, an effect attenuated by antecedent PPAR activation.Conclusions/interpretation The study demonstrates that glucocorticoid excess precipitates the development of glucose intolerance in rats maintained on a high-saturated-fat diet. It does this by interrupting the negative feedback loop between insulin sensitivity and secretion in vivo, such that further enhancement of compensatory insulin secretion is not possible. PPAR activation restores the coupling between insulin secretion and action.     

Peroxisome proliferator-activated receptor α (PPARα) activation advances locomotor activity and feeding daily rhythms in mice

Peroxisome proliferator-activated receptors (PPARs) are key mediators of energy homeostasis, and lipid and glucose metabolism that exhibit circadian expression. PPAR activating drugs are used clinically as lipid and glucose-lowering drugs. We evaluated the effect of long-term (11 weeks) PPARα and PPARγ activation using bezafibrate and rosiglitazone, respectively, on metabolism, locomotor activity and feeding rhythms of non-obese mice. We found that bezafibrate, but not rosiglitazone, led to no weight gain and a slight weight loss with reduced epididymal fat pads. Although rosiglitazone had a minor effect on 24-h food intake rhythm, bezafibrate treatment was accompanied by increased amplitude and an advanced acrophase of the 24-h feeding rhythm. Similarly, unlike rosiglitazone, bezafibrate treatment was accompanied by a significantly advanced acrophase of locomotor activity rhythm under constant darkness conditions. As disrupted circadian rhythms lead to obesity, PPARα activation can serve as a clinical target for the modulation of both circadian rhythms and metabolism.     

Mutation analysis of peroxisome proliferator-activated receptor-γ coactivator-1 (PGC-1) and relationships of identified amino acid polymorphisms to Type II diabetes mellitus

Aim/hypothesis: This study aimed to investigate if variability in the peroxisome proliferator-activated receptor-γ coactivator-1 (PGC-1) gene is associated with Type II (non-insulin-dependent) diabetes mellitus. Methods: The PGC-1 gene was examined in 53 Type II diabetic patients applying single strand conformational polymorphism analysis followed by nucleotide sequencing. Identified variants were genotyped in an association study comprising 483 Type II diabetic patients and 216 glucose-tolerant control subjects. A replication study was done in an additional 201 Type II diabetic patients and 293 glucose-tolerant subjects. Furthermore, a potential interaction between the Pro12Ala polymorphism of PPAR- γ2 and the PGC-1 Gly482Ser variant on risk of Type II diabetes was investigated. Results: A total of seven variants (Ser74Leu, IVS2 + 52C→A, Thr394Thr, Asp475Asp, Gly482Ser, Thr528Thr, and Thr612Met) were identified and investigated in an association study. Six of the variants showed no association with Type II diabetes in the initial study. However, the Gly482Ser polymorphism, was more frequent among Type II diabetic patients (37.0 %) than among glucose-tolerant subjects (30.8 %) (p = 0.032). In a replication study the difference in allele frequencies of the Gly482Ser variant remained significant (p = 0.0135). The combined study yielded an allele frequency of 37.3 % (34.7–39.9) for Type II diabetic patients and 30.5 % (27.7–33.4) for glucose-tolerant subjects (p = 0.0007). No interaction between this variant and the Pro12Ala polymorphism of PPAR- γ2 was observed. Conclusion/interpretation: A widespread Gly482Ser polymorphism of PGC-1 is associated with a 1.34 genotype relative risk of Type II diabetes. [Diabetologia (2001) 44: 2220–2226] Received: 4 April 2001 and in revised form: 19 July 2001     

Common polymorphisms of the peroxisome proliferator-activated receptor-γ (Pro12Ala) and peroxisome proliferator-activated receptor-γ coactivator-1 (Gly482Ser) and the response to pioglitazone in Chinese patients with type 2 diabetes mellitus

We investigated the effects of the common polymorphisms in the peroxisome proliferator-activated receptor-γ (PPAR-γ; Pro12Ala) and in PPAR-γ coactivator-1(PGC-1; Gly482Ser) genes on the response to pioglitazone in Chinese with type 2 diabetes mellitus. A total of 250 patients with type 2 diabetes mellitus were treated with pioglitazone (30 mg/d) for 24 weeks without a change in previous medications. All patients were genotyped for the PPAR-γ Pro12Ala and PGC-1 Gly482Ser polymorphisms. The Ala12Ala and Pro12Ala genotypes (26.0% vs 13.5%, P = .025) and Ala allele (15.6% vs 7.3%, P = .008) were significantly more frequent in pioglitazone responders than in nonresponders. The distribution of PGC-1 genotypes and alleles was not significantly different between responders and nonresponders. The decrease in fasting glucose (50.4 ± 52.2 vs 43.3 ± 51.7 mg/dL, P < .001) and hemoglobin A_1c (0.57% ± 1.44% vs 0.35% ± 1.10%, P = .004) levels was significantly greater in subjects with the Ala12 carriers (Pro12Ala and Ala12Ala) than in those without the allele (Pro12Pro). Baseline fasting glucose and triglyceride levels were related to the response of pioglitazone. Only the PPAR-γ Pro12Ala polymorphism was found to be associated with the response of pioglitazone by multiple logistic regression analysis. The PPAR-γ Pro12Ala gene polymorphism is associated with the response to pioglitazone in Chinese patients with type 2 diabetes mellitus. These findings may be helpful for targeted treatment of diabetes by identifying patients who are likely to respond to pioglitazone.     

Cytokines (interferon-γ and tumor necrosis factor-α)-induced nuclear factor-κB activation and chemokine (C-X-C motif) ligand 10 release in Graves disease and ophthalmopathy are modulated by pioglitazone

Until now, the following are not known: (1) the mechanisms underlying the induction of chemokine (C-X-C motif) ligand 10 (CXCL10) secretion by cytokines in thyrocytes; (2) if pioglitazone is able, like rosiglitazone, to inhibit the interferon (IFN)-γ-induced chemokine expression in Graves disease (GD) or ophthalmopathy (GO); and (3) the mechanisms underlying the inhibition by thiazolidinediones of the cytokines-induced CXCL10 release in thyrocytes. The aims of this study were (1) to study the mechanisms underlying the induction of CXCL10 secretion by cytokines in GD thyrocytes; (2) to test the effect of pioglitazone on IFNγ-inducible CXCL10 secretion in primary thyrocytes, orbital fibroblasts, and preadipocytes from GD and GO patients; and (3) to evaluate the mechanism of action of thiazolidinediones on nuclear factor (NF)-κB activation. The results of the study (1) demonstrate that IFNγ + TNFα enhanced the DNA binding activity of NF-κB in GD thyrocytes, in association with the release of CXCL10; (2) show that pioglitazone exerts a dose-dependent inhibition on IFNγ + TNFα-induced CXCL10 secretion in thyrocytes, orbital fibroblasts, and preadipocytes, similar to the effect observed with rosiglitazone; and (3) demonstrate that thiazolidinediones (pioglitazone and rosiglitazone) act by reducing the IFNγ + TNFα activation of NF-κB in Graves thyrocytes. To the best of our knowledge, this is the first study showing that cytokines are able to activate NF-κB in Graves thyrocytes and a parallel inhibitory effect of pioglitazone both on CXCL10 chemokine secretion and NF-κB activation. Future studies will be needed to verify if new targeted peroxisome proliferator-activated receptor-γ activators may be able to exert the anti-inflammatory effects without the risk of expanding retrobulbar fat mass.     

Peroxisome proliferator-activated receptor (PPAR)α and -γ regulate IFNγ and IL-17A production by human T cells in a sex-specific way

Women develop certain autoimmune diseases more often than men. It has been hypothesized that this may relate to the development of more robust T-helper (Th)1 responses in women. To test whether women exhibit a Th1 bias, we isolated naïve cluster of differentiation (CD)4⁺ T cells from peripheral blood of healthy women and men and measured the proliferation and cytokine production by these cells in response to submaximal amounts of anti-CD3 and anti-CD28. We observed that CD4⁺ T cells from women produced higher levels of IFNγ as well as tended to proliferate more than male CD4⁺ T cells. Intriguingly, male CD4⁺ T cells instead had a predilection toward IL-17A production. This sex dichotomy in Th cytokine production was found to be even more striking in the Swiss/Jackson Laboratory (SJL) mouse. Studies in mice and humans indicated that the sexual dimorphism in Th1 and Th17 cytokine production was dependent on the androgen status and the T-cell expression of peroxisome proliferator activated receptor (PPAR)α and PPARγ. Androgens increased PPARα and decreased PPARγ expression by human CD4⁺ T cells. PPARα siRNA-mediated knockdown had the effect of increasing IFNγ by male CD4⁺ T cells, while transfection of CD4⁺ T cells with PPARγ siRNAs increased IL-17A production uniquely by female T cells. Together, our observations indicate that human T cells exhibit a sex difference in the production of IFNγ and IL-17A that may be driven by expressions of PPARα and PPARγ.     

NF-κB-dependent synergistic regulation of CXCL10 gene expression by IL-1β and IFN-γ in human intestinal epithelial cell lines

Background and aims Little is known about the intestinal epithelial expression and secretion of CXCL10 (IP-10), a chemokine involved in recruiting T cells and monocytes. We aimed to study CXCL10 gene expression and regulation by the pro-inflammatory cytokines interleukin (IL)-1β, interferon (IFN)-γ and tumour necrosis factor (TNF)-α in intestinal epithelial cell lines. Materials and methods CXCL10 expression and secretion kinetics were assessed in Caco-2, HT-29 and DLD1 human colon epithelial cells, treated with IL-1β, TNF-α, IFN-γ alone or in combination with each other by real-time polymerase chain reaction (PCR), Northern blotting and enzyme-linked immunoabsorbent assay (ELISA). Transient transfections with TGL-IP10 (CXCL10 promoter) and TGL-IP10-κB2 mutant promoter and gelshifts and supershifts for nuclear factor (NF)-κB were also performed. Results Real-time PCRs and ELISA experiments revealed that IL-1β was the strongest and earliest inducer of CXCL10 messenger ribonucleic acid (mRNA) expression and protein secretion in Caco-2 cell line, whereas INF-γ had a delayed kinetics. There was a strong synergistic effect of either TNF-α or IL-1β with IFN-γ both on CXCL10 mRNA expression and protein secretion in all three cell lines. Real-time PCR and ELISA experiments using a specific NF-κB inhibitor and transfection experiments with a NF-κB-binding defective CXCL10 promoter construct revealed that the induction of CXCL10 by IL-1β and its synergism with IFN-γ is NF-κB dependent. Conclusion These data demonstrate that in colonic epithelial cells, depending on the cellular context and utilizing the NF-κB pathway, IL-1β alone and/or in synergism with IFN-γ may play a major role in the induction of CXCL10.