Troglitazone induces cytotoxicity in part by promoting the degradation of peroxisome proliferator-activated receptor �� co-activator-1�� protein

BACKGROUND AND PURPOSE

Troglitazone (Tro), rosiglitazone (Rosi) and pioglitazone (Pio) are anti-diabetic thiazolidinediones that function as ligands for peroxisome proliferator-activated receptor γ (PPARγ); however, Tro has been withdrawn from the market due to liver toxicity issues. Mitochondrial dysfunction induced by Tro has been suggested to be an important mechanism behind its cytotoxicity. Constitutively active nuclear hormone receptors, oestrogen-related receptor α and γ are thought to regulate mitochondrial mass and oxidative phosphorylation together with their co-activators PPARγ co-activator-1α and -1β (PGC-1α and PGC-1β). Hence, in this study, we investigated whether Tro affects the expression and activity levels of these regulators.

EXPERIMENTAL APPROACH

Cellular viability was measured by an ATP-based assay. Mitochondrial mass and reactive oxygen species (ROS) were quantified by two different fluorogenic probes. Apoptosis was measured by an Annexin-V-based kit. Gene expression at the levels of mRNA and protein was measured by quantitative RT-PCR and Western analysis. Over-expression of PGC-1α was mediated by an adenovirus.

KEY RESULTS

Tro, but not Rosi or Pio, selectively stimulated PGC-1α protein degradation. As a result, Tro reduced mitochondrial mass, and superoxide dismutases 1 and 2 expressions, but induced ROS to initiate apoptosis. Using a ubiquitin–proteasome inhibitor MG132, it was established that blocking PGC-1α degradation partially suppressed the reduction of mitochondrial mass. Importantly, over-expressing PGC-1α partially restored the Tro-suppressed mitochondrial mass and attenuated the cytotoxic effects of Tro.

CONCLUSIONS AND IMPLICATIONS

Collectively, these results suggest that PGC-1α degradation is an important mechanism behind the cytotoxic effects of Tro in the liver.     

Activation of peroxisome proliferator-activated receptor γ in brain inhibits inflammatory pain, dorsal horn expression of Fos, and local edema

Systemic administration of thiazolidinediones reduces peripheral inflammation in vivo, presumably by acting at peroxisome proliferator-activated receptor γ (PPARγ) in peripheral tissues. Based on a rapidly growing body of literature indicating the CNS as a functional target of PPARγ actions, we postulated that brain PPARγ modulates peripheral edema and the processing of inflammatory pain signals in the dorsal horn of the spinal cord. To test this in the plantar carrageenan model of inflammatory pain, we measured paw edema, heat hyperalgesia, and dorsal horn expression of the immediate-early gene c-fos after intracerebroventricular (ICV) administration of PPARγ ligands or vehicle. We found that ICV rosiglitazone (0.5-50 μg) or 15d-PGJ₂ (50-200 μg), but not vehicle, dose-dependently reduced paw thickness, paw volume and behavioral withdrawal responses to noxious heat. These anti-inflammatory and anti-hyperalgesia effects result from direct actions in the brain and not diffusion to other sites, because intraperitoneal and intrathecal administration of rosiglitazone (50 μg) and 15d-PGJ₂ (200 μg) had no effect. PPARγ agonists changed neither overt behavior nor motor coordination, indicating that non-specific behavioral effects do not contribute to PPAR ligand-induced anti-hyperalgesia. ICV administration of structurally dissimilar PPARγ antagonists (either GW9662 or BADGE) reversed the anti-inflammatory and anti-hyperalgesic actions of both rosiglitazone and 15d-PGJ₂. To evaluate the effects of PPARγ agonists on a classic marker of noxious stimulus-evoked gene expression, we quantified Fos protein expression in the dorsal horn. The number of carrageenan-induced Fos-like immunoreactive profiles was less in rosiglitazone-treated rats as compared to vehicle controls. We conclude that pharmacological activation of PPARγ in the brain rapidly inhibits local edema and the spinal transmission of noxious inflammatory signals.     

Regulation of adipogenesis through retinoid X receptor and/or peroxisome proliferator-activated receptor by designed lignans based on natural products in 3T3–L1 cells

Journal of Natural Medicines - We previously synthesized two retinoid X receptor (RXR) agonists, 4′-hydroxy-3′-propyl-[1,1′-biphenyl]-3-propanoic acid ethyl ester (4′OHE)...     

Rosiglitazone, a peroxisome proliferator-activated receptor γ stimulant, abrogates diabetes-evoked hypertension by rectifying abnormalities in vascular reactivity

In addition to insulin sensitization, rosiglitazone exhibits favourable circulatory effects. In the present study, we tested the hypothesis that rosiglitazone protects against hypertension and vascular derangements caused by diabetes. Diabetes was induced by a single bolus injection of streptozotocin (50 mg/kg, i.p.). After 2 weeks, rats were started on a treatment regimen of 5 mg/kg rosiglitazone daily for a period of 6 weeks. The control group consisted of rats treated with vehicle (distilled water) for the same period of time. After 6 weeks treatment, blood pressure (BP) was recorded and serum levels of glucose, advanced glycation end-products (AGE), triglycerides, total cholesterol and low-density lipoprotein-cholesterol (LDL-C) were determined. In in vitro experiments, concentration-response curves were constructed to phenylephrine (PE), KCl and acetylcholine (ACh) in thoracic aorta rings. In addition, ACh-induced nitric oxide (NO) generation and KCl-induced intracellular Ca accumulation were determined in the aorta. Compared with values in control rats, both diastolic and systolic BP were increased in diabetic rats. Rosiglitazone treatment of diabetic rats abolished the increase in diastolic BP and significantly reduced the increased systolic BP without affecting the development of hyperglycaemia. The possibility that changes in vascular reactivity and/or lipid profile contributed to the effects of rosiglitazone on BP in diabetic rats was investigated. In aortic rings from diabetic rats, contractile responses to KCl were increased, whereas the relaxant responses to ACh were decreased. In rings from diabetic rosiglitazone-treated rats, the exaggerated response to KCl and the impaired response to ACh were abolished. Furthermore, rosiglitazone abrogated impaired ACh-stimulated NO generation in aortas isolated from diabetic rats. Diabetes in rats was accompanied by elevated levels of triglycerides, total cholesterol, LDL-C and AGE. Rosiglitazone treatment abrogated the increased levels of triglycerides, total cholesterol and LDL-C, but only partially reduced AGE levels. Collectively, these observations indicate that rosiglitazone abrogates diabetes-evoked hypertension by ameliorating detrimental changes in vascular reactivity and lipid profiles.     

Telmisartan increases localization of glucose transporter 4 to the plasma membrane and increases glucose uptake via peroxisome proliferator-activated receptor γ in 3T3-L1 adipocytes

Angiotensin II is a peptide hormone with strong vasoconstrictive action, and recent reports have shown that Angiotensin II receptor type 1 antagonists (angiotensin II receptor blockers) also improve glucose metabolism. The angiotensin II receptor blocker telmisartan acts as an agonistic ligand of the peroxisome proliferator-activated receptor gamma (PPARγ). In this study, we investigated the effects of telmisartan on glucose uptake and insulin sensitivity in 3T3-L1 adipocytes and compared it with the action of other angiotensin II receptor blockers. Telmisartan treatment dose-dependently increased (from 1 μM) protein expression of PPARγ-regulated molecules such as fatty acid binding protein 4 (FABP4), insulin receptor, and glucose transporter 4 (GLUT4). Telmisartan increased glucose uptake both with and without insulin stimulation in 3T3-L1 adipocytes. Telmisartan increased the up-regulation of phosphorylated insulin receptor, insulin receptor substrate-1 (IRS-1) and Akt by insulin, suggesting that telmisartan increases insulin sensitivity. Furthermore, in the absence of insulin, telmisartan, but not candesartan, increased GLUT4 levels at the plasma membrane. These effects by 10 μM telmisartan were similar potency to those of 1 μM troglitazone, an activator of PPARγ. In addition, up-regulation of glucose uptake by telmisartan was inhibited by a PPARγ antagonist, T0070907 (2-chloro-5-nitro-N-4-pyridinyl-benzamide). These results indicate that telmisartan acts via PPARγ activation in adipose tissue and may be an effective therapy for the metabolic syndrome.     

GSK3β regulates D1 dopamine receptor phosphorylation and function

Many protein kinases have been shown to regulate the phosphorylation of G - protein - coupled receptors. These kinases, together with associated protein phosphatases form complexes that regulate the function of G - protein - coupled receptors. In brain, protein phosphatasel （ PP1 ） exists as a complex with PP1 inhibitor - 2 （ I - 2）. Phosphorylation of I - 2 by cdk5 or GSK3β results in activation of PP1. The present study was designed to investigate the role of GSK3β in the regulation of D1 dopamine receptor function. In rat and rabbit frontal cortex （FCX）, we found that GSK3β co -precipitates with D1A dopamine receptor. Furthermore, GSK3w is physically associated with I -2, indicating that GSK3β/I -2/PP1/D1 receptor form a receptor - kinase - phosphatase complex. In prenatal cocaine - exposed rabbit, we reported a selective impairment of D1 dopamine receptor function in FCX that is the result of D1 receptor hyper - phosphorylation that is due to decreased PP1 activity. Interestingly, we detected a significant decrease in GSK3β activity in FCX of cocaine- exposed rabbits. Moreover, decreased association between GSK3β and I -2 was also observed, suggesting that GSK3β activity is nee ded for the association between these two molecules. Indeed, preincubation of GSK3β inhibitor 1 with FCX membranes induced a decrease in association between the kinase and I -2. In accord with the decrease in FCX GSK3β activity that is found in prenatal cocaine - exposed rabbit, PP1 activity, and specifically, Dt receptor - associated PP1 activity, was also decreased. Taken together, the present results indicate that GSK3β forms a complex with Dt dopamine receptor, PP1 and I - 2 that regulates D1 receptor function via modulation of PP1 activity. Inhibition of GSK3β activity, as is the case in prenatal cocaine - exposed brain, results in reduced PP1 activity and hyper - phosphorylation of the D1 dopamine receptor and ultimately leads to the dysfunction of the D1 receptor. In addition, in HEK293 cell line stably expressing D1 receptor,     

Tobacco smoke affects expression of peroxisome proliferator-activated receptor-γ in monocyte/macrophages of patients with coronary heart disease

Background and purpose:

Tobacco smoke represents a relevant risk factor for coronary heart disease (CHD). Although peroxisome proliferator-activated receptor (PPAR)γ activation reduces inflammation and atherosclerosis, expression of PPARγ in cells and its modulation by smoking are poorly investigated. We previously reported that monocyte/macrophages from healthy smokers exhibited an enhanced constitutive expression of PPARγ. Here, we evaluated PPARγ expression and basal cytokine release in monocytes and monocyte-derived macrophages (MDMs) from 85 CHD patients, classified by their smoking habit (smokers, non-smokers and ex-smokers), and assessed the role of PPARγ ligands in this context.

Experimental approach:

PPARγ protein was detected by Western blot and semi-quantified by PPARγ/β-actin ratio; cytokine release was measured by elisa and nuclear factor-kappaB (NF-κB) translocation by electrophoretic mobility shift assays.

Key results:

As compared to the other groups, MDMs from smoker CHD patients exhibited a reduced PPARγ/β-actin ratio and an increased spontaneous release of tumour necrosis factor-α (TNF-α) and interleukin-6, but with no major variations in monocytes. In cells from selected CHD patients, rosiglitazone inhibited TNF-α release and NF-κB translocation induced by phorbol-12-myristate 13-acetate. The selective PPARγ antagonist GW9662 reversed these effects, with some variations related to smoking habit.

Conclusions and implications:

In CHD patients, exposure to tobacco smoke profoundly affected PPARγ expression, and this was related to levels of secretion of pro-inflammatory cytokines. MDMs from CHD smokers showed the lowest PPARγ expression and released more inflammatory cytokines. Moreover, rosiglitazone''s ability to inhibit cytokine release and its reversal by GW9662 clearly indicated PPARγ involvement in these changes in CHD patients.     

Effects of peroxisome proliferator-activated receptor γ agonists on Na+ transport and activity of the kinase SGK1 in epithelial cells from lung and kidney

Background and purpose:

Peroxisome proliferator-activated receptor γ (PPARγ) agonists, such as rosiglitazone and pioglitazone, sensitize cells to insulin, and are therefore used to treat type 2 diabetes. However, in some patients, these drugs induce oedema, and the present study tests the hypothesis that this side effect reflects serum and glucocorticoid-inducible kinase 1 (SGK1)-dependent enhancement of epithelia Na⁺ absorption.

Experimental approach:

Na⁺ absorbing epithelial cells (H441 cells, mpkCCD cells) on permeable membranes were mounted in Ussing chambers, and the effects of rosiglitazone (2 µM) and pioglitazone (10 µM) on transepithelial Na⁺ absorption were quantified electrometrically. Changes in SGK1 activity were assessed by monitoring phosphorylation of residues within an endogenous protein.

Key results:

Both cell types absorbed Na⁺ via an electrogenic process that was enhanced by insulin. In mpkCCD cells, this stimulation of Na⁺ transport was associated with increased activity of SGK1, whereas insulin regulated Na⁺ transport in H441 cells through a mechanism that did not involve activation of this kinase. Rosiglitazone and pioglitazone had no discernible effect on transepithelial Na⁺ absorption in unstimulated or insulin-stimulated cells and failed to alter cellular SGK1 activity.

Conclusions and implications:

Our results do not support the view that PPARγ agonists stimulate epithelial Na⁺ absorption or alter the control of cellular SGK1 activity. It is therefore likely that other mechanisms are involved in PPARγ-mediated fluid retention, and a better understanding of these mechanisms may help with the identification of patients likely to develop oedema or heart failure when treated with these drugs.     

Triclosan affects the expression of nitric oxide synthases (NOSs), peroxisome proliferator-activated receptor gamma (PPARγ), and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) in mouse neocortical neurons in vitro

Triclosan (TCS) is a well-known compound that can be found in disinfectants, personal care products. There is one publication concerning the involvement of PPARγ in the mechanism of action of TCS. It is known that activation of PPARγ regulates the expression of the NF-κB mediated inflammation by acting on nitric oxide synthase (NOS) genes. However, there are no studies demonstrating a relationship between the effects of TCS on the PPARγ signaling pathway, changes in NF-κB expression, and NOS isoform synthesis. Therefore, the aim of this study was to evaluate the effect of TCS on the expression of PPARγ, NF-κB, nNOS, iNOS, and eNOS in mouse neocortical neurons. In addition, the effects of co-administration of synthetic alpha-naphthoflavone (αNF) or beta-naphthoflavone (βNF) flavonoids and triclosan were investigated. Our results show that TCS alters PPARγ, NF-κB, iNOS, and eNOS expression in mouse neurons in vitro. After 48 h of exposure, TCS increased PPARγ expression and decreased NF-κB expression. Moreover, under the influence of TCS, the expression of iNOS was increased and at the same time the expression of nNOS was decreased, which was probably caused by high levels of ROS. The experiments have shown that both αNF and βNF are able to modulate the effects of TCS in primary cultures of mouse cortical neurons.     

Sigma receptor antagonists attenuate acute methamphetamine-induced hyperthermia by a mechanism independent of IL-1β mRNA expression in the hypothalamus

Methamphetamine is currently one of the most widely abused drugs worldwide, with hyperthermia being a leading cause of death in methamphetamine overdose situations. Methamphetamine-induced hyperthermia involves a variety of cellular mechanisms, including increases in hypothalamic interleukin-1 beta (IL-1β) expression. Methamphetamine also interacts with sigma receptors and previous studies have shown that sigma receptor antagonists mitigate many of the behavioral and physiological effects of methamphetamine, including hyperthermia. The purpose of the current study was to determine if the attenuation of methamphetamine-induced hyperthermia by the sigma receptor antagonists, AZ66 and SN79, is associated with a concomitant attenuation of IL-1β mRNA expression, particularly in the hypothalamus. Methamphetamine produced dose- and time-dependent increases in core body temperature and IL-1β mRNA expression in the hypothalamus, striatum, and cortex in male, Swiss Webster mice. Pretreatment with the sigma receptor antagonists, AZ66 and SN79, significantly attenuated methamphetamine-induced hyperthermia, but further potentiated IL-1β mRNA in the mouse hypothalamus when compared to animals treated with methamphetamine alone. These findings suggest sigma receptor antagonists attenuate methamphetamine-induced hyperthermia through a different mechanism from that involved in the modulation of hypothalamic IL-1β mRNA expression.