Activated Receptor Gamma Research Articles

Rosiglitazone Activates AMPK and Improves Non-Alcoholic Fatty Liver Disease in OLETF Rats

Background: Insulin resistance is very common in patients with nonalcoholic fatty liver disease (NAFLD). Glitazones improve insulin sensitivity by acting as a selective agonist of the peroxisome proliferators -activated receptor gamma (PPAR γ), and were shown to activate AMP-activated protein kinase (AMPK) in skeletal muscle and the liver. Glitazones were also shown to reduce hepatic lipogenesis. The aim of this study was to investigate whether the protective mechanism of rosiglitazone on NAFLD is associated with AMPK activation. Methods: Twelve OLETF rats were divided into 2 groups (control, treatment, n = 6 each). LETO rats served as controls. At 35 weeks of age, treatment group received rosig litazone 4 mg/kg daily for 3 days. Fasting plasma glucose, insulin, free fatty acid, lactate and triglycerides were measured. Liver tissues from each group were processed for histological and hepatic triglyceride content analysis and western blotting. Results: Fasting plasma glucose, insulin and triglycerides levels were significantly lower in treatment group than in control group. Histologic examination disclosed decreased hepatic steatosis in treatment group. Hepatic triglyceride content was also decreased in treatment group. Sterol regulatory binding protein-1c (SREBP-1c) and fatty acid synthase (FAS) expression were increased and AMPK phosphorylation was reduced in OLETF rats compared with LETO rats, and these changes were reversed by rosiglitazone treatment. Conclusion: Rosiglitazone reduced hepatic steatosis in OLETF rats, and activated AMPK in the liver. These results suggest the role of AMPK activation in the protective action of rosiglitazone on NAFLD. (KOREAN DIABETES J 32:141~148, 2008)

Adipogenic potential can be activated during muscle regeneration

Fatty degeneration is observed in various neuromuscular diseases, but the mechanism(s) of its initiation remains unclear. To gain insight into the regulation of fatty degeneration, we employed a freeze-induced model of muscle degeneration/regeneration. Using this model, we examined the distribution of adipocyte-like cells with Oil Red-O staining and the expression pattern of adipogenic transcriptional factors, an adipocyte-terminal differentiation marker, and Wnt10b signaling molecules during muscle regeneration. Mice were subjected to freeze injury, and the gastrocnemius muscles were isolated 1, 3, 5, 7, 10, 14 and 28 days after surgery. Adipocyte-like cells with nuclei were readily observed, but not in normal muscle. Large amount of lipid accumulation was also observed in regenerating muscle. The area of Oil Red-O staining was significantly increased from 3 to 5 days after muscle injury and then rapidly decreased to almost control levels by day 10. Adipogenic transcriptional factors, sterol regulatory element binding protein-1c, CCAAT/enhancer-binding proteins alpha, beta and delta, peroxisome-proliferator activated receptors gamma1 and gamma2, and the terminal differentiation marker, leptin were significantly up-regulated in the early stage of muscle regeneration, suggesting activation of the adipogenic potential. Secreted Frizzled-related protein-2, a Wnt pathway inhibitory protein, was strongly up-regulated 3 days after muscle injury, suggesting active repression of the Wnt10b pathway. In regenerating muscle, expression of CCAAT/enhancer-binding protein alpha and peroxisome-proliferator activated receptor gamma2 proteins were increased 3 days after muscle injury. Taken together, our results suggest that adipogenic potential can be activated during muscle regeneration through increased adipogenic signaling in conjunction with decreased Wnt10b signaling.

Long-term Pharmacological Activation of PPARγDoes not Prevent Left Ventricular Remodeling in Dogs with Advanced Heart Failure

Peroxisome proliferator-activated receptor gamma (PPARgamma) activators affect the myocardium through inhibition of inflammatory cytokines and metabolic modulation but their effect in the progression of heart failure is unclear. In the present study, we examined the effects of the PPARgamma activator, GW347845 (GW), on the progression of heart failure. Heart failure was produced in 21 dogs by intracoronary microembolizations to LV ejection fraction (EF) less than 30% and randomized to 3 months of therapy with high-dose GW (10 mg/Kg daily, n = 7), low-dose GW (3 mg/Kg daily, n = 7), or no therapy (control, n = 7). In control dogs, EF significantly decreased (28 +/- 1 vs. 22 +/- 1%, p < 0.001) and end-diastolic volume (EDV) and end-systolic volume (ESV) increased during the 3 months of the follow-up period (64 +/- 4 vs. 76 +/- 5; p = 0.003, 46 +/- 3 vs. 59 +/- 4 ml, p = 0.002, respectively). In dogs treated with low-dose GW, EDV increased significantly (69 +/- 4 vs.81 +/- 5 ml, p = 0.01), whereas ESV remained statistically unchanged (50 +/- 3 vs. 54 +/- 3 ml, p = 0.10) resulting in modestly increased ejection fraction (27 +/- 1 vs. 32 +/- 3%, p = 0.05). In dogs treated with high-dose GW, both EDV and ESV increased (72 +/- 4 vs. 79 +/- 5 ml, p = 0.04; 53 +/- 3 vs. 62 +/- 5 ml, p = 0.04) and EF decreased (26 +/- 1 vs. 23 +/- 1%, p = 0.04) as with control dogs. There was significantly increased myocardial hypertrophy as evidenced by increased LV weight to body weight ratio and myocyte cross-section area in the GW treated animals compared to controls. Compared to control, treatment with GW had no effect on mRNA expression of PPARgamma, inflammatory cytokines, stretch response proteins, or transcription factors that may induce hypertrophy. Long-term PPARgamma activation with GW did not prevent progressive LV remodeling in dogs with advanced heart failure.

MULTIPLE DIFFERENTIATION POTENTIALS OF NEONATAL DURA MATER-DERIVED CELLS

The involvement of the dura mater in calvarial development and bone healing lead to a hypothesis that progenitor cells with multiple differentiation potentials exist within this tissue. The present study investigated the differentiation potentials of dura mater-derived cells by driving them into several cell-restricted lineages. Dissected dura mater tissue of neonatal rats was washed, finely minced, and enzymatically digested. The harvested cells were exposed to different differentiation (osteogenic, adipogenic, and chondrogenic) and basic media. At defined time points, dura mater-derived cells were observed to differentiate into osteoblastic, adipoblastic, and chondroblastic cells, evidenced by specific biochemical staining. In addition, gene expressions of osteogenesis (alkaline phosphatase, osteocalcin, and osteopontin), chondrogenesis (collagen Type II and aggrecan core protein) and adipogenesis (peroxisome proliferator activated receptor gamma-2) were up-regulated in the differentiated dura mater-derived cells, confirmed by polymerase chain reaction. Preliminarily, it was concluded that a subpopulation of multiple potential mesenchymal cells exists in neonatal dura mater, which explains the function of the dura mater on neurocranium development and calvarial bone healing.

Biomarker Analysis as a Decision-Making Tool in Drug Discovery and Development

Biomarkers are measurable properties that reflect the pathophysiology of disease, mechanism of action of a molecule or the interaction between the two. The early implementation of a biomarker strategy within drug development offers a great opportunity to enhance the efficiency of drug development. More specifically, biomarkers may be used to aid in lead compound selection, dose focusing, patient stratification and defining the mechanism of action of novel therapeutics. Validated biomarkers, especially surrogate endpoints, may have broader utility in both a regulatory context and in clinical practice. Biomarkers may be used to evaluate both the safety and efficacy of new drugs. Biomarker identification is enhanced with a clear understanding of the steps involved in a pathophysiology cascade. Biomarkers discovered with the aid of molecular profiling may have particular utility in approaching complex diseases with poorly characterised pathophysiology, as in type 2 diabetes mellitus. For example, expression profiling experiments enabled the discovery of the 30kD protein adiponectin — a specific biomarker for in vivo activation of peroxisome proliferator-activator receptor gamma (PPARψ) activity. Adiponectin offers great utility in short-term clinical studies in healthy volunteers or patients with type 2 diabetes to assess whether new potential PPAR? agonists are efficacious in humans. In addition to enlightening key underlying pathophysiology, biomarkers such as adiponectin allow for improved decision-making earlier in development. Ultimately biomarkers can be used to optimise development efficiency from discovery through to registration.

Inhibition of peroxisome proliferator‐activated receptor gamma activity in esophageal carcinoma cells results in a drastic decrease of invasive properties

Esophageal cancer is difficult to treat because of its rapid progression, and more effective therapeutic approaches are needed. The PPARgamma is a nuclear receptor superfamily member that is expressed in many cancers. PPARgamma expression is a feature of esophageal cancer cell lines, and in the present investigation, the PPARgamma antagonists T0070907 and GW9662 could induce loss of invasion but could not induce growth reduction or apoptosis at low concentrations (< 10 mM). A high concentration of antagonists (50 microM) inhibited cell growth and induced apoptosis, but these effects did not explain our result at the low concentration. Morphological change, decreased expression of the cell signaling pathway and inhibition of cancer cell invasion were observed in the low concentration. This suggested that PPARgamma antagonists inhibited esophageal cancer cell invasion as well as cell adherence, most likely due to alteration in the FAK-MAPK pathway, and this was independent of apoptosis. These results suggested that PPARgamma plays an important role in cancer cell invasion and that it might be a novel target for therapy of esophageal cancer.

Subchronic exposure to high-dose ACE-inhibitor moexipril induces catalase activity in rat liver

The long-term clinical effects of ACE-inhibitors have similarities with those of both fibrates and glitazones, activators of peroxisome proliferator activator receptor (PPAR) alpha and gamma, respectively. The antioxidant enzyme catalase, a heme protein that degrades hydrogen peroxide, is found at high concentrations in peroxisomes. Catalase activity is one of the recognized surrogate markers indicative of PPAR activation in the rat liver. The purpose of the study was to establish the effect of moexipril on catalase activity and to compare it with the effect of both saline controls and that of the known PPAR agonist clofibrate (positive control). Three groups of seven rats were used. All substances were applied i.p. daily for 5 days, followed by a 2-day break. The cycle was repeated eight times. After the final cycle (day 56) the animals were sacrificed and liver tissue collected. The number of catalase positive cells in both moexipril group (95% CI 57-61) and clofibrate group (95% CI 72-80) is higher than in controls (95% CI 3-16) (p < or = 0.01). The number of catalase positive cells in the clofibrate group is higher than in the moexipril group (p < or = 0.01). High-dose subchronic exposure to the ACE-inhibitor moexipril induces catalase activity in the rat liver to an extent comparable to fibrates. We suggest that some of the long-term advantages of ACE inhibitor use - beyond mere BP lowering - might be due to a PPAR mediated effect.

Review: Molecular-specific effects of angiotensin II antagonists: clinical relevance to treating hypertension?

Angiotensin II receptor blockers (ARBs) may produce a number of molecule-specific effects that appear to be independent of interaction with the angiotensin II type 1 (AT1)-receptor. These include antagonism of the thromboxane A2 receptor, inhibition of platelet aggregation, induction of peroxisome proliferator- activated receptor gamma (PPARgamma) activity, and reduction of serum uric acid levels. However, definitive evidence is lacking that these molecule-specific effects give rise to a therapeutic advantage of one ARB over another. Currently, the possibility of a link between a molecule-specific effect of an ARB and an improvement in clinical outcomes is best illustrated by a reduction in serum uric acid levels with losartan. Data from Losartan Intervention For Endpoint reduction in hypertension (LIFE) study suggest a treatment-induced decrease in serum uric acid may contribute to the treatment benefit of a losartan-based versus atenolol-based therapy on the composite endpoint (death, myocardial infarction, or stroke). This finding should prompt further studies to investigate the long-term cardioprotective benefits issue of reducing hyperuricaemia in hypertensive patients.

Alanine for proline substitution in the peroxisome proliferator-activated receptor gamma-2 (PPARG2) gene and the risk of incident myocardial infarction.

Recent studies have implicated the potential importance of peroxisome proliferator-activated receptors as a molecular mechanism involved in atherothrombosis. A common alanine (A) for proline (P) substitution at codon 12 in the peroxisome proliferator activated receptor gamma-2 gene (PPARG2) has been associated with reduced risk of developing type 2 diabetes mellitus. Because diabetes and atherothrombosis share common antecedents, we sought evidence that this polymorphism might also be associated with reduced risk of myocardial infarction. Using DNA samples collected at baseline in a prospective cohort of 14 916 initially healthy American men, we evaluated a P12A polymorphism in the PPARG2 among 523 individuals who subsequently developed myocardial infarction and among 2092 individuals who remained free of reported cardiovascular disease over a mean follow-up period of 13.2 years. As hypothesized, presence of the A12 allele was associated with significantly reduced risk of myocardial infarction (odds ratio in an age- and smoking-adjusted dominant model of inheritance, 0.77; 95% CI, 0.60 to 0.98; P=0.034). This protective effect remained statistically significant in analyses controlling for traditional cardiovascular risk factors, was present among nondiabetic study participants, was observed to be of similar magnitude in analyses assuming codominant or dominant modes of inheritance, and was seen in fully adjusted post hoc analyses in which we limited our control group to those individuals specifically matched to myocardial infarction cases (OR, 0.71; 95% CI, 0.53 to 0.96; P=0.024). In this cohort, a common A for P substitution at codon 12 in the PPARG2 was associated with reduced incidence of myocardial infarction. If confirmed in other cohorts, these data would have implications for novel treatments of cardiovascular disease, including development of PPARG-targeted therapy.

Extracellular matrix mineralization and osteoblast gene expression by human adipose tissue-derived stromal cells.

Human adipose tissue represents an abundant reservoir of stromal cells with potential utility for tissue engineering. The current study demonstrates the ability of human adipose tissue-derived stromal cells to display some of the hallmarks of osteoblast differentiation in vitro. Following treatment with ascorbate, beta-glycerophosphate, dexamethasone, and 1,25 dihydroxy vitamin D(3), adipose tissue-derived stromal cells mineralize their extracellular matrix based on detection of calcium phosphate deposits using Alizarin Red and von Kossa histochemical stains. Fourier transform infrared analysis demonstrates the apatitic nature of these crystals. Mineralization is accompanied by increased expression or activity of the osteoblast-associated proteins osteocalcin and alkaline phosphatase. These and other osteoblast-associated gene markers are detected based on polymerase chain reaction. In contrast, the adipocyte gene markers--leptin, lipoprotein lipase, and peroxisome proliferator activated receptor gamma2--are reduced under mineralization conditions, consistent with the reciprocal relationship postulated to exist between adipocytes and osteoblasts. The current work supports the presence of a multipotent stromal cell population within human extramedullary adipose tissue. These findings have potential implications for human bone tissue bioengineering.

Trans-10,cis-12 conjugated linoleic acid reduces triglyceride content while differentially affecting peroxisome proliferator activated receptor gamma2 and aP2 expression in 3T3-L1 preadipocytes.

A series of experiments was conducted using 3T3-L1 preadipocytes as the cell model to determine: (i) whether the triglyceride (TG)-lowering effects of a crude mixture of conjugated linoleic acid (CLA) isomers were due to a specific isomer of CLA and the timing of treatment, (ii) if CLA reduced TG content by inhibiting a key regulator of adipogenesis, (iii) if CLA incorporated into either neutral lipid or phospholipid cell fractions, and (iv) whether the effects of CLA treatment were reversible. Trans-10,cis-12 CLA reduced TG content, whereas the cis-9,trans-11 isomer increased TG content compared to vehicle [bovine serum albumin (BSA)] controls. Treatment with 50 microM trans-10,cis-12 CLA during the entire 6 d of differentiation reduced TG content to a greater extent than treatment during either the first 3 d or last 3 d of differentiation. Trans-10,cis-12 CLA treatment of preadipocyte cultures for 48 h increased peroxisome proliferator activated receptor gamma2 (PPARgamma2) protein expression compared to cultures treated with linoleic acid (LA) or the BSA controls. CLA had no effect on adipose P2 (aP2), a fatty acid-binding protein regulated by PPARgamma2. Both the cis-9,trans-11 and the trans-10,cis-12 isomers of CLA were incorporated into neutral lipids and phospholipids. However, cis-9,trans-11 CLA levels were one- to twofold higher than trans-10,cis-12 CLA levels. Moreover, trans-10,cis-12 CLA treatment reduced cis-11 18:1 concentrations in both neutral lipids and phospholipids while increasing cis-9 18:1 and 18:2 concentrations. Palmitoleic acid (16:1) levels were also lower in the neutral lipid fraction of cultures treated with trans-10,cis-12 CLA. Supplementing trans-10,cis-12 CLA-treated cultures (50 microM) with increasing levels of LA resulted in a dose-dependent increase in TG content compared to cultures treated with 50 microM CLA alone. LA supplementation also prevented some of the morphological changes associated with trans-10,cis-12 CLA treatment as seen with scanning electron microscopy. Treatment with 50 microM trans-10,cis-12 CLA for 6 d decreased PPARgamma2 levels, and supplementation of CLA-treated cultures with LA increased PPARgamma2 levels compared with cultures treated with CLA alone. Taken together, these data indicate that in cultures of 3T3-L1 preadipocytes: (i) trans-10,cis-12 CLA is the TG-lowering isomer of CLA, and its effects are dependent on dose, duration of treatment, and the amount of LA in the cultures; (ii) trans-10,cis-12 CLA treatment alters the monounsaturated fatty acid profile of neutral- and phospholipids of the cultures; and (iii) although acute (2-d) trans-10,cis-12 CLA treatment increased PPARgamma2 protein levels, chronic (6-d) treatment decreased PPARgamma2 levels.

Hepatic over-expression of peroxisome proliferator activated receptor gamma2 in the ob/ob mouse model of non-insulin dependent diabetes mellitus.

Studies of the molecular basis of insulin resistance have focused on the peroxisome proliferator activated receptor gamma (PPARgamma, gamma1 and gamma2). The aim of this study was to determine whether the insulin resistance in liver of diabetic animals is associated with abnormal expression of these receptors. PPARgamma mRNA and protein expression levels were quantified in liver of 9-week-old male ob/ob mice as a model of diabetes and compared to age- and gender-matched wild type control animals of the same genetic background. Semi-quantitative reverse transcription-polymerase chain reaction, using 18S rRNA as an internal standard, indicated that PPARgamma2 mRNA was significantly upregulated in ob/ob liver vs. that in wild type mice. Western blotting revealed greater immunoreactivity of PPARgamma2 in liver from ob/ob mice relative to that in wild type mice. An index of insulin resistance (product of serum glucose and insulin concentration) was correlated with liver PPARgamma2 mRNA expression (r = 0.776; p < 0.001). The findings that liver PPARgamma2 expression is (1) significantly elevated in the ob/ob model of diabetes and (2) positively associated with an index of insulin resistance, suggests a possible compensatory response through which type II diabetic and obese organisms strive to maintain insulin sensitivity of the liver.

Hunting for human obesity genes? Look in the adipose tissue!

Over-eating and physical inactivity in combination with genetic factors play the most important roles in the development of over weight in humans. The common genetic components behind excess accumulation of body fat are so far unknown. Studies of candidate genes indicate that most of the genes that associate with obesity control important functions of adipose tissue as well. Furthermore, structural variations in these genes may alter adipose tissue function in a way that promotes obesity. The genes which both are functional in human adipose tissue and associate with obesity are: hormone sensitive lipase, beta2 and beta3-adrenoceptors, tumor necrosis factor alpha, low density lipoprotein receptor, uncoupling protein-1 and peroxisome proliferator activated receptor gamma-2. Other genes are mostly important for obesity among women (for example beta2 -and beta3-adrenoceptors, low density lipoprotein receptor and tumor necrosis factor alpha). Some of these genes may promote obesity by gene-gene interactions (for example beta3-adrenoceptors and uncoupling protein-1) or gene-environmental interactions (for example beta2-adrenoceptors and physical activity). Few genes with no known function in adipose tissue have shown a firm association with excess body fat. The latter suggests that the important human obesity genes also control adipose tissue function. Therefore it might be of value to focus the further hunt for obesity genes on the fat tissue.

Inhibition of adipogenesis by Wnt signaling.

Wnts are secreted signaling proteins that regulate developmental processes. Here we show that Wnt signaling, likely mediated by Wnt-10b, is a molecular switch that governs adipogenesis. Wnt signaling maintains preadipocytes in an undifferentiated state through inhibition of the adipogenic transcription factors CCAAT/enhancer binding protein alpha (C/EBPalpha) and peroxisome proliferator- activated receptor gamma (PPARgamma). When Wnt signaling in preadipocytes is prevented by overexpression of Axin or dominant-negative TCF4, these cells differentiate into adipocytes. Disruption of Wnt signaling also causes transdifferentiation of myoblasts into adipocytes in vitro, highlighting the importance of this pathway not only in adipocyte differentiation but also in mesodermal cell fate determination.

Impact of the Peroxisome Proliferator Activated Receptor gamma2 Pro12Ala polymorphism on adiposity, lipids and non-insulin-dependent diabetes mellitus.

The Pro12Ala polymorphism of the Peroxisome Proliferator Activated Receptor gamma2 (PPARgamma2) gene has been inconsistently associated with body mass index variations and non-insulin-dependent diabetes mellitus (NIDDM). We investigated the impact of this polymorphism on obesity markers, lipid and glucose variables in a sample of French subjects and evaluated its possible role in the onset of NIDDM. Within the framework of the WHO-MONICA project, a population study composed of 1195 subjects aged 35-64 y was randomly sampled from the electoral rolls of the urban community of Lille, in northern France. Subjects receiving medical treatment for hypercholesterolemia, hypertension or diabetes mellitus were excluded for the analyses, to avoid any interferences between medical treatment and biological variables. This resulted in a sample size of 839 subjects (421 men/418 women, age=49.4+/-8.1 y, body mass index (BMI)=25.7+/-4.4 kg/m2). To evaluate the role of the Pro12Ala polymorphism in the onset of NIDDM, we evaluated its distribution in 170 Caucasian NIDDM subjects from a clinical series (117 men/53 women, age=62.3+/-9.0 y, BMI=30.1+/-3.6 kg/m2). The PPARgamma2 Pro12Ala polymorphism genotyping was carried out with allele specific oligonucleotides hybridisation. Data were statistically analysed for association with various obesity markers (body weight (BW), BMI, waist-to-hip ratio (WHR), plasma leptin concentrations, lipid and glucose variables. In the WHO-MONICA population, the Ala allele frequency was 0.11. The presence of the Ala allele was significantly associated with higher body weight (P=0.002), BMI (P=0.02), height (P=0.02) and waist circumference (P=0.04). Increased plasma concentrations of total cholesterol (P=0.01), LDL-cholesterol (P=0.004) and apolipoprotein B (P=0.01) were also detected in Ala allele bearers. The distribution of the Pro12Ala polymorphism was similar in NIDDM subjects (Ala allele frequency: 0.10) and in the WHO-MONICA population subjects. Our results suggest that genetic variability of PPARgamma2 affects body weight control and lipid homeostasis in humans and do not support a significant role for the PPARgamma2 Pro12Ala polymorphism in the aetiology of NIDDM. International Journal of Obesity (2000) 24, 195-199

The effect of activation of peroxisome proliferator-activated receptor gamma (PPARgamma) on human monocyte function: PPARgamma ligands do not inhibit tumor necrosis factor-alpha release in human monocytic cell line THP-1.

Peroxisome proliferator-activated receptor gamma (PPARgamma) activation by its ligands reportedly inhibits monocyte function. However, because the concentrations of PPARgamma ligands used in previous studies were higher than typically expected to activate PPARgamma, we clarified whether PPARgamma ligands influence monocyte function and cell viability of the human monocyte cell line THP-1. We determined tumor necrosis factor-alpha (TNF-alpha) release as a monocyte function and cell viability using 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide. Both troglitazone and 15-deoxy-delta12,14-prostaglandin J2 (15-d-PGJ2) seemed to inhibit phorbol ester-induced TNF-alpha release from THP-1 cells. On the other hand, neither pioglitazone nor rosiglitazone inhibited phorbol ester-induced TNF-alpha release. Because the cytotoxicity of troglitazone and 15-d-PGJ2 was significantly (p<0.05, Tukey-Kramer) stronger than that of pioglitazone and rosiglitazone, the inhibition of TNF-alpha release seemed to parallel the lack of cell viability. We concluded that PPARgamma ligands did not directly inhibit TNF-alpha release in THP-1 cells.