PPARβ mRNA Research Articles

LPS impairs steroidogenesis and ROS metabolism and induces PPAR transcriptional activity to disturb estrogen/androgen receptor expression in testicular cells

Inflammation can deregulate the testicular functions of steroidogenesis and spermatogenesis, consequently contributing to male infertility. Animals and cells treated with lipopolysaccharide (LPS) exhibit infection- and inflammation-induced testicular dysfunction. However, the precise mechanisms affecting steroidogenesis and spermatogenesis in response to LPS-treatment remain poorly understood. We isolated distinct testicular cells including spermatocytes, round spermatids and late spermatids to analyze distribution of peroxisome proliferator-activated receptor (PPAR) family, plays central roles in the regulation of metabolism. Our results suggested Pparα/Pparγ mRNA was highly expressed in late spermatids, while Pparβ mRNA was highly expressed in round spermatids. To analyze the effect of LPS on testicular cells, we established an LPS infection model using primary Sertoli cells and testicular cell lines (TM4, GC2 and MLTC1). We observed that PPARγ and SIRT1 were concentrated in the nuclear region and that the mRNA expression levels of antioxidative enzymes (Cat and Homx1) and PPARγ were upregulated in primary Sertoli cells after LPS-treatment. Moreover, luciferase reporter gene assays of the testicular cell lines revealed that the activity of the PPAR response element (PPRE) was significantly increased. Importantly, the transcriptional activity of the androgen response element was significantly reduced, whereas activity of estrogen response element was strongly induced in LPS-treated TM4 cells, consistent with the RT-PCR results. Meanwhile, the qRT-PCR results revealed that the LPS-induced upregulation of Ar mRNA in MLTC1 cells and Erβ mRNA in TM4 cells were significantly recovered after treatment with the specific PPARγ-antagonist GW9662. In addition, we also found that LPS induced alterations in enzymes involved in steroidogenesis in testicular cell lines. Taken together, our results revealed that LPS may induce PPAR transcriptional activity to disturb estrogen/androgen receptor expression and impair steroidogenesis and ROS metabolism in testicular cells.

Toll-like receptors control p38 and JNK MAPK signaling pathways in rat astrocytes differently, when cultured in normal or high glucose concentrations

Astrocytes play a vital role in regulating central nervous system inflammation, energy metabolism and brain homeostasis. Unlike macrophages and microglia, which are cells of myeloid ancestry, astrocytes are of ectodermal origin. However, regulatory specificities of signaling pathways connecting inflammatory and metabolic processes are still largely unknown. We analyzed firstly cellular responses to toll-like receptor (TLR) agonists and secondly, modulation of the mRNA of the three isoforms of the transcription factors PPARs (peroxisome proliferator-activated receptors) in primary rat astrocytes exposed to normal glucose (5.5 mM) and high glucose (25 mM). Cell culturing of rat brain astrocytes for 2 days in high glucose did not alter cellular morphology, but i) enhanced the release of TNFα that was induced by TLR4 agonist LPS or TLR3 agonist PIC and the synthesis of prostaglandin E2 (PGE2), ii) changed the signaling pathways of TLR4/MAPK (increase in p38 MAPK, and decrease in JNK activities at early stages of TLR activation) and iii) modulated mRNA expression of PPARs. High glucose cultivation reduced PPARα and PPARβ mRNA levels, without altering PPARγ mRNA level and changed the sensitivity of expressions to agonists of TLR1/2 (PGN), TLR4 (LPS), TLR3 (PIC), and TLR5 (FGN). Differences between low and high glucose-adapted cells were obtained for agonists of TLR1/2 (PPARα, PPARβ), TLR4 (PPAR β), TLR3 (PPARα). In the TLR4/p38/PPARβ signaling pathway, there was a stimulatory connection in normal glucose but an inhibitory connection in high glucose. TLR4/JNK/activated PPARβ, TLR4/JNK/inhibited PPARγ both in cells adapted to normal or high glucose, but PPARα expression was not affected. As PPARs in astrocytes are involved in inflammatory processes in the form of the recently published PPAR triad, the changes in expression revealed here are most likely resulting in implications of high glucose in inflammatory processes. Our data underline the complexity of multiple regulatory interactions between inflammatory responses and energy metabolism in astrocytes.

PPARβ in yellow catfish Pelteobagrus fulvidraco: molecular characterization, tissue expression and transcriptional regulation by dietary Cu and Zn.

Peroxisome proliferator-activated receptor beta (PPARβ) is a ligand-activated transcription factor that plays critical roles in the regulation of many important physiological processes. In this study, PPARβ was cloned and characterized in yellow catfish Pelteobagrus fulvidraco. PPARβ cDNA was 2350bp in length with an open reading frame (ORF) of 1530bp, encoding 509 amino acids, a 5'-untranslated region (UTR) of 474bp, and a 3'-UTR of 346bp. Similar to mammals, PPARβ protein was predicted to consist of four domains, the A/B domain, DNA-binding domain (DBD), D domain, and ligand-binding domain (LBD). The DBD contained two zinc fingers with eight conserved cysteine residues. The predicted secondary structure of LBD consisted of 12 highly conserved α-helices and a small β-sheet of 4 strands. In addition, PPARβ was widely expressed across the tested tissues (liver, heart, muscle, intestine, brain, spleen, kidney, fat, ovary, and gill), but at the variable levels. Furthermore, the transcriptional responses of PPARβ by dietary Cu and Zn levels were also investigated. Dietary Cu levels showed no significant effects on PPARβ mRNA levels in the liver and intestine; in contrast, dietary Zn levels upregulated the hepatic PPARβ mRNA levels, but not in the intestine. The present study serves to increase our understanding into the function of the PPARβ gene in fish.

Anticancer properties of 4-thiazolidinone derivatives depend on peroxisome proliferator-activated receptor gamma (PPARγ)

Peroxisome proliferator-activated receptors (PPARs) play an important role in numerous chronic diseases such as diabetes, obesity, atherosclerosis and cancer, and PPAR modulators are among the approved drugs and drug-candidates for their treatment. The aim of this study was to elucidate the involvement of PPARs in the mechanism of cytotoxic and pro-apoptotic action of novel anticancer 4-thiazolidinone derivatives (Les-2194, Les-3377, Les-3640) and approved 4-thiazolidinones (Rosiglitazone, Pioglitazone) towards the human squamous carcinoma (SCC-15) cell line. Experiments with 4-thiazaolidinone derivatives and PPAR-specific siRNA were conducted and PPARα, PPARβ and PPARγ mRNA expression was studied. Moreover, after PPARα, PPARβ and PPARγ siRNA gene silencing, cell viability, cell metabolism and caspase-3 activity were measured. The results showed a decrease of mRNA expression of the studied PPARs in SCC-15 cells treated with 10 and 50 μM Les-2194, Les-3377 and Les-3640. PPARγ knockdown protected the cells from the cytotoxic effect of the tested compounds (50 μM). It was established that novel anticancer 4-thiazolidinone derivatives act mainly through the PPARγ pathway in SCC-15 cells. Our results suggest that all studied compounds act as PPARs agonists. Interestingly, silencing of PPARγ gene increases the expression of PPARα, PPARβ mRNA in SCC-15 cells. The anticancer potential of new studied compounds was more expressed as compared to Rosiglitazone and Pioglitazone.

Topically administered rhGM-CSF affects PPARβ expression in the stasis zone.

Using a rat comb thermal damage model, we investigated the effects of topically administered recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) on peroxisome proliferator-activated receptor PPARβ expression. We created bilateral comb scald models on the backs of fifty Sprague-Dawley rats. The left sides of the backs served as the experimental group and the right sides served as the control group. The experimental group received topically applied rhGM-CSF hydrogel and the control group did not. The survival situations of the stasis zones were compared between the experimental and control groups on the 1st, 3rd, 7th, 14th and 21st post-burn days. Tissues from the surviving stasis zones of both groups were collected at different time-points. Reverse transcriptase-polymerase chain reaction (RT-PCR) and western blotting were used to detect the PPARβ mRNA and protein expression levels. Immunohistochemical methods were applied to detect the localization of PPARβ protein expression. The results showed that, first, the tissue viability numbers for the stasis zones of the experimental group were significantly increased compared with those of the control group. Second, RT-PCR revealed that the PPARβ mRNA expression first increased and then gradually declined in both groups. At all time-points, the expression level in the experimental group was increased compared with that in the control group and the highest expression levels were observed in both groups on the 3rd post-burn day. Third, western blot analysis revealed that the PPARβ protein expression in both groups increased after thermal damage and then gradually decreased. PPARβ protein expression in the experimental group was greater than that in the control group, and the highest expression quantities in both groups were observed on the 3rd post-burn day. In conclusion, rhGM-CSF hydrogel effectively promotes the expression of PPARβ, and the hydrogel had a specific protective effect for the stasis zone.

Expression of peroxisome proliferator-activated receptors, fatty acid binding protein-4 in placenta and their correlations with the prognosis of pre-eclampsia

Objective: To examine the mRNA and protein expression of peroxisome proliferator-activated receptors (PPAR) and fatty acid binding protein-4 (FABP-4) in placenta, and to investigate their correlation with the prognosis of pre-eclampsia. Methods: The data of 177 women who delivered from January 2013 to December 2015 in Chinese People's Liberation Army No.94 Hospital were collected. Among them, 60 cases were term pregnancy and not in labor (TN); 46 cases were term pregnancy with preeclampsia and not in labor (TPE); 42 cases were preterm pregnancy and not in labor (PN); 29 cases were preterm pregnancy with pre-eclampsia and not in labor (PPE). Real-time PCR and western blot were used to examine the PPAR and FABP-4 mRNA expression and protein expression in placentas. And linear correlation was used to analyze the relationship between PPAR and FABP-4 protein expression and the prognosis of pre-eclampsia. Results: (1) Real-time PCR showed that: ① PPAR-α, PPAR-β mRNA expression were not statistically different between placentas from TN and TPE (P>0.05), but PPAR-γ mRNA level in TPE (0.59±0.17) was significantly lower than that in TN (0.81±0.19, P<0.01). However, FABP-4 mRNA level in TPE (0.70±0.23) was significantly higher than that in TN (0.43±0.18, P<0.01). ②There were no statistical differences in PPAR-α, PPAR-β mRNA expression level between PN and PPE (P>0.05) . However, PPAR-γ mRNA in the PPE group (0.33±0.14) was significantly lower than that in PN (0.52±0.16, P<0.01), and FABP-4 mRNA level in the PPE group (0.80±0.29) was significantly higher than in the PN group (0.63±0.22, P<0.01). (2) Western-blot showed the same tendency as the mRNA results. ①There were no statistical differences in the protein expression of PPAR-α, PPAR-β, not in term groups (TN and TPE) , nor in premature groups (PN and PPE) . PPAR-γ protein level in the TPE group (0.46±0.17) was significantly lower than that in TN (0.65±0.20, P<0.01) and FABP-4 protein level in the TPE group (0.60±0.19) was significantly higher than that in the TN group (0.50±0.21, P<0.05). ②The PPAR-γ protein level in the PPE group (0.30±0.16) was significantly lower than that in the PN group (0.61±0.16, P<0.05), while the FABP-4 protein expression in the PPE group (0.58±0.19) was significantly higher than that in the PN group (0.40±0.19, P<0.05). (3) Linear correlation showed that: ①PPAR-γ protein expression correlated negatively with FABP-4 protein expresssion [P<0.01, R(2)=0.12 in the term groups (TN+TPE) , R(2)=0.15 in the premature groups (PN+PPE) ]. ②PPAR-γ protein expression correlated negatively with blood pressure recovery time, 24-hour urinary protein quantification and blood lipid recovery time (P<0.01, R(2)=0.37, 0.35, 0.18). FABP-4 protein expression correlated positively with lipid recovery time and blood cholesterol concentrations (P<0.01, R(2)=0.13, 0.25). Conclusions: The expression of PPAR-γ decreases in placentas from patients with pre-eclampsia, while the expression of FABP-4 increases. The expression of FABP-4 correlates negatively with the expression of PPAR-γ, and both are associated with the prognosis of pre-eclampsia.

α-Lipoic acid up-regulates expression of peroxisome proliferator-activated receptor β in skeletal muscle: involvement of the JNK signaling pathway.

We hypothesized that α-lipoic acid (α-LA) might interact with the transcriptional control of peroxisome proliferator-activated receptor (PPAR)β in skeletal muscle. Molecular mechanisms were investigated using differentiated C2C12 myotubes treated with α-LA and/or PPARβ agonist GW0742. In vivo studies with 3-mo-old C57Bl6 mice were realized: voluntary wheel running (VWR) training (7 wk), and a 6 wk diet containing (or not) α-LA (0.25% wt/wt). This last condition was combined with (or not) 1 bout of treadmill exercise (18 m/min for 1 h). Using a reporter assay, we demonstrate that α-LA is not an agonist of PPARβ but regulates PPARβ target gene expression through an active PPARβ pathway. GW0742-induced pyruvate dehydrogenase kinase 4 mRNA is potentiated by α-LA. In C2C12, α-LA lowers the activation of the JNK signaling pathway and increases PPARβ mRNA and protein levels (2-fold) to the same extent as with the JNK inhibitor SP600125. Similarly to VWR training effect, PPARβ expression increases (2-fold) in vastus lateralis of animals fed an α-LA-enriched diet. However, α-LA treatment does not further stimulate the adaptive up-regulation of PPARβ observed in response to 1 bout of exercise. We have identified a novel mechanism of regulation of PPARβ expression/action in skeletal muscle with potential physiologic application through the action of α-LA, involving the JNK pathway.

Xenobiotic-contaminated diets affect hepatic lipid metabolism: Implications for liver steatosis in Sparus aurata juveniles

The metabolic effects induced by feed contaminated with a lower or a higher concentration of -nonylpnenol (NP), 4-tert-octylphenol (t-OP) or bisphenol A (BPA), three environmental endocrine disruptors, were assessed in juvenile sea bream liver. Histological analysis demonstrated that all these three xenobiotics induced hepatic lipid accumulation and steatosis. These findings prompted analysis of the expression of the major molecules involved in lipid metabolism: peroxisome proliferator activated receptors (which is encoded by ppars), fatty acid synthase (encoded by fas), lipoprotein lipase (encoded by lpl) and hormone-sensitive lipase (encoded by hsl). The enzymes encoded by ppars and fas are in fact responsible for lipid accumulation, whereas lpl- and hsl- encoded proteins play a pivotal role in fat mobilization. The three xenobiotics modulated ppar mRNA expression: pparα mRNA expression was induced by the higher dose of each contaminant; pparβ mRNA expression was upregulated by the lower doses and in BPA2 fish ppary mRNA overexpression was induced by all pollutants. These data agreed with the lipid accumulation profiles documented by histology. Fas mRNA levels were modulated by the two NP doses and the higher BPA concentration. Lpl mRNA was significantly upregulated in all experimental groups except for BPA1 fish while hsl mRNA was significantly downregulated in all groups except for t-OP2 and BPA1 fish. The plasma concentrations of cortisol, the primary stress biomarker, were correlated with the levels of pepck mRNA level. This gene encodes phosphoenolpyruvate carboxykinase which is one of the key enzymes of gluconeogenesis. Pepck mRNA was significantly overexpressed in fish exposed to NP2 and both t-OP doses. Finally, the genes encoding cyclooxygenase 2 (cox2) and 5-lipoxygenase (5 lox), the products of which are involved in the inflammatory response, transcriptions were significantly upregulated in NP and BPA fish, whereas they were unchanged in t-OP specimens. The present findings suggest that dietary xenobiotic contamination can give rise to metabolic disorders also in fish and highlight the potential for their vertical transfer through the trophic levels and ultimately to humans.

Insulin, IGF-I, and muscle MAPK pathway responses after sustained exercise and their contribution to growth and lipid metabolism regulation in gilthead sea bream

Herein, we studied whether sustained exercise positively affects growth of gilthead sea bream by alterations in a) plasma concentrations of insulin and IGF-I, b) signaling pathways in muscle, or c) regulation of lipid metabolism. Specifically, we evaluated the effects of moderated swimming (1.5 body lengths per second; BL/s) on the circulating concentrations of insulin and IGF-I, morphometric parameters, and expression of genes related to lipid metabolism in gilthead sea bream (80–90 g BW). Exercise increased the specific growth rate (P < 0.05) and reduced the hepatosomatic index (P = 0.006). Plasma IGF-I concentrations increased in exercised fish (P = 0.037), suggesting a role for this endocrine factor in the control of muscular growth and metabolic homeostasis during swimming. The observed decrease in plasma insulin concentrations (P = 0.016) could favor the mobilization of tissue reserves in exercised fish. In this sense, the increase in liver fatty acid content (P = 0.041) and the changes in expression of peroxisome proliferator-activated receptors PPARα (P = 0.017) and PPARγ (P = 0.033) indicated a hepatic lipid mobilization. Concentration of glycogen in both white and red muscles was decreased (P = 0.021 and P = 0.017, respectively) in exercised (n = 12) relative to control (n = 12) gilthead sea bream, whereas concentrations of glucose (P = 0.016) and lactate (P = 0.0007) were decreased only in red muscle, indicating the use of these substrates. No changes in the glucose transporter and in lipoprotein lipase mRNA expression were found in any of the tissues studied. Exercised sea bream had decreased content of PPARβ mRNA in white and red muscle relative to control sea bream expression (P = 0.001 and P = 0.049, respectively). Mitogen-activated protein kinase phosphorylation was significantly down-regulated in both white and red muscles of exercised sea bream (P = 0.0374 and P = 0.0371, respectively). Tumor necrosis factor-α expression of white muscle was down-regulated in exercised gilthead sea bream (P = 0.045). Collectively, these results contribute to the knowledge base about hormonal regulation of growth and lipid metabolism in exercised gilthead sea bream.

PPARβ mRNA expression, reduced by n − 3 PUFA diet in mammary tumor, controls breast cancer cell growth

The effect of numerous anticancer drugs on breast cancer cell lines and rodent mammary tumors can be enhanced by a treatment with long-chain n−3 polyunsaturated fatty acids (n−3 PUFA) such as docosahexaenoic acid (DHA, 22:6n−3) which is a natural ligand of peroxisome proliferator-activated receptors (PPAR). In order to identify the PPAR regulating breast cancer cell growth, we tested the impact of siRNA, selected to suppress PPARα, PPARβ or PPARγ mRNA in MDA-MB-231 and MCF-7 breast cancer cell lines. The siPPARβ was the most effective to inhibit breast cancer cell growth in both cell lines. Using PPARα, PPARβ and PPARγ pharmacological antagonists, we showed that PPARβ regulated DHA-induced inhibition of growth in MDA-MB-231 and MCF-7 cells. In addition, the expressions of all 3 PPAR mRNA were co-regulated in both cell lines, upon treatments with siRNA or PPAR antagonists. PPAR mRNA expression was also examined in the NitrosoMethylUrea (NMU)-induced rat mammary tumor model. The expressions of PPARα and PPARβ mRNAs were correlated in the control group but not in the n−3 PUFA group in which the expression of PPARβ mRNA was reduced. Although PPARα expression was also increased in the n−3 PUFA-enriched diet group under docetaxel treatment, it is only the expression of PPARβ mRNA that correlated with the regression of mammary tumors: those that most regressed displayed the lowest PPARβ mRNA expression. Altogether, these data identify PPARβ as an important player capable of modulating other PPAR mRNA expressions, under DHA diet, for inhibiting breast cancer cell growth and mammary tumor growth.

Regulation of Carnitine Palmitoyltransferase (CPT) I during Fasting in Rainbow Trout (Oncorhynchus mykiss) Promotes Increased Mitochondrial Fatty Acid Oxidation

Periods of fasting, in most animals, are fueled principally by fatty acids, and changes in the regulation of fatty acid oxidation must exist to meet this change in metabolic substrate use. We examined the regulation of carnitine palmitoyltransferase (CPT) I, to help explain changes in mitochondrial fatty acid oxidation with fasting. After fasting rainbow trout (Oncorhynchus mykiss) for 5 wk, the mitochondria were isolated from red muscle and liver to determine (1) mitochondrial fatty acid oxidation rate, (2) CPT I activity and the concentration of malonyl-CoA needed to inhibit this activity by 50% (IC(50)), (3) mitochondrial membrane fluidity, and (4) CPT I (all five known isoforms) and peroxisome proliferator-activated receptor (PPARα and PPARβ) mRNA levels. Fatty acid oxidation in isolated mitochondria increased during fasting by 2.5- and 1.75-fold in liver and red muscle, respectively. Fasting also decreased sensitivity of CPT I to malonyl-CoA (increased IC(50)), by two and eight times in red muscle and liver, respectively, suggesting it facilitates the rate of fatty acid oxidation. In the liver, there was also a significant increase CPT I activity per milligram mitochondrial protein and in whole-tissue PPARα and PPARβ mRNA levels. However, there were no changes in mitochondrial membrane fluidity in either tissue, indicating that the decrease in CPT I sensitivity to malonyl-CoA is not due to bulk fluidity changes in the membrane. However, there were significant differences in CPT I mRNA levels during fasting. Overall, these data indicate some important changes in the regulation of CPT I that promote the increased mitochondrial fatty acid oxidation that occurs during fasting in trout.

Peroxisome proliferator-activated receptors alpha, Beta, and gamma mRNA and protein expression in human fetal tissues.

Peroxisome proliferator-activated receptors (PPARs) regulate lipid and glucose homeostasis, are targets of pharmaceuticals, and are also activated by environmental contaminants. Almost nothing is known about expression of PPARs during human fetal development. This study examines expression of PPARα, β, and γ mRNA and protein in human fetal tissues. With increasing fetal age, mRNA expression of PPARα and β increased in liver, but PPARβ decreased in heart and intestine, and PPARγ decreased in adrenal. Adult and fetal mean expression of PPARα, β, and γ mRNA did not differ in intestine, but expression was lower in fetal stomach and heart. PPARα and β mRNA in kidney and spleen, and PPARγ mRNA in lung and adrenal were lower in fetal versus adult. PPARγ in liver and PPARβ mRNA in thymus were higher in fetal versus adult. PPARα protein increased with fetal age in intestine and decreased in lung, kidney, and adrenal. PPARβ protein in adrenal and PPARγ in kidney decreased with fetal age. This study provides new information on expression of PPAR subtypes during human development and will be important in evaluating the potential for the developing human to respond to PPAR environmental or pharmaceutical agonists.

Ontogenic effects of early feeding of sea bass (Dicentrarchus labrax) larvae with a range of dietary n-3 highly unsaturated fatty acid levels on the functioning of polyunsaturated fatty acid desaturation pathways

Four replicated groups of sea bass (Dicentrarchus labrax) larvae were fed diets containing an extra-high level of highly unsaturated fatty acids (HUFA) (XH; 3.7 % EPA+DHA), a high level of HUFA (HH; 1.7 %), a low level of HUFA (LH; 0.7 %) or an extra-low level of HUFA (XLH; 0.5 %) from day 6 to day 45 (experiment 1; XH1, HH1, LH1, XLH1). After a subsequent 1-month period feeding a commercial diet (2.7 % EPA+DHA), the capacity of the four initial groups to adapt to an n-3 HUFA-restricted diet (0.3 % EPA+DHA; R-groups: XH2R, HH2R, LH2R, XLH2R) was tested for 35 d. Larval dietary treatments had no effect on larval and juvenile survival rates. The wet weight of day 45 larvae was higher in XH1 and HH1 (P < 0.001), but the R-juvenile mass gains were similar in all treatments. Delta-6-desaturase (Delta6D) mRNA level was higher in LH1 and XLH1 at day 45 (P < 0.001), and higher in LH2R and XLH2R, with a significant increase at day 118.Concomitantly, PPARalpha and PPARbeta mRNA levels were higher in XLH1 at day 45, and PPARbeta and gamma mRNA levels were higher in XLH2R at day 118, suggesting possible involvement of PPAR in stimulation of Delta6D expression, when drastic dietary larval conditioning occurred. The low DHA content in the polar lipids (PL) of LH1 and XLH1 revealed an n-3-HUFA deficiency in these groups. Larval conditioning did not affect DHA content in the PL of R-juveniles. The present study showed (i) a persistent Delta6D mRNA enhancement in juveniles pre-conditioned with an n-3 HUFA-deficient larval diet, over the 1-month intermediate period, and (ii) brought new findings suggesting the involvement of PPAR in the Delta6D mRNA level stimulation. However, such nutritional conditioning had no significant effect on juvenile growth and lipid composition.

PPARalpha and PPARbeta are reduced in the aged rat heart following ischemia and reperfusion injury

Background. Ischemia and reperfusion (I/R) injury is increased with age and may be associated with cardiac peroxisome proliferator-activated receptor (PPAR) activation. The role of specific PPAR isoforms in the heart, particularly with differences in gender and aging, is poorly understood. Purpose. To determine the singular and combined effects of gender and age on recovery from I/R injury and PPAR expression in the rat heart. Methods. Adult (Y) and aged (O) male (M) and female (F) F344 rats were subjected to I/R using Langendorff perfusion (31 min global I; 0.4mM palmitate,1.25mM Ca2+). Recovery function was determined as left ventricular developed pressure and +/−dP/dt. RNA was extracted and real time PCR performed using primer sequences for rat PPARα and PPARβ. Results. A reduction in PPARα and PPARβ mRNA was observed in O compared to Y at baseline. OF had a further reduction of both PPARα and PPARβ mRNA after I/R. Summary. These data suggest, for the first time, that changes in PPARα and PPARβ expression during I/R injury are associated with gender and age.

Waterborne gemfibrozil challenges the hepatic antioxidant defense system and down-regulates peroxisome proliferator-activated receptor beta (PPARβ) mRNA levels in male goldfish ( Carassius auratus)

The lipid regulator gemfibrozil (GEM) is one of many human pharmaceuticals found in the aquatic environment. We previously demonstrated that GEM bioconcentrates in blood and reduces plasma testosterone levels in goldfish ( Carassius auratus). In this study, we address the potential of an environmentally relevant waterborne concentration of GEM (1.5 μg/l) to induce oxidative stress in goldfish liver and whether this may be linked to GEM acting as a peroxisome proliferator (PP). We also investigate the autoregulation of the peroxisome proliferator-activated receptors (PPARs) as a potential index of exposure. The three PPAR subtypes (α, β, and γ) were amplified from goldfish liver cDNA. Goldfish exposed to a concentration higher (1500 μg/l) than environmentally relevant for 14 and 28 days significantly reduce hepatic PPARβ mRNA levels ( p < 0.001). Levels of CYP1A1 mRNA were unchanged. GEM exposure significantly induced the antioxidant defense enzymes catalase ( p < 0.001), glutathione peroxidase ( p < 0.001) and glutathione- S-transferase ( p = 0.006) but not acyl-CoA oxidase or glutathione reductase. As GEM exposure failed to increase levels of thiobarbituric reactive substances (TBARS), we conclude that a sub-chronic exposure to GEM upregulates the antioxidant defense status of the goldfish as an adaptive response to this human pharmaceutical.

Effects of Caloric Restriction on the Expression of PGC-1 and PPARs mRNA in Liver of Otsuka Long-Evans Tokushima Fatty Rats

- Abstract Background: Gluconeogenesis is strongly stimulated during fasting and is aberrantly activated in diabetes mellitus. PPARγ-coactivator 1 (PGC-1) and Peroxisome proliferator -activated receptors (PPARs) costimulate the expression of key enzymes of gluconeogenetic pathway. This study was performed to evaluate the response to dietary caloric restriction (CR) on the PPARs and PGC-1 expression in liver of diabetic Otsuka Long-Evans Tokushima Fatty (OLETF) rats. Methods: Diabetic OLETF rats (male, 24 weeks) and Long-Evans Tokushima Otsuka (LETO) rats (male, 24 weeks) were used in this study. Liver PPARs and PGC-1 mRNA, and blood glucose levels were investigated at 1, 2, and 3 weeks after the beginning of 30% CR. PPARs and PGC-1 mRNA were determined by RT-PCR and blood glucose levels were measured by spectrophotometric assay. Results: The liver PGC-1 mRNA expressions were increased to 19% in non-diabetic LETO rats but significant change was not observed in diabetic OLETF rats by 30% CR. The liver PPARγ mRNA expressions were not changed in non-diabetic LETO rats but increased to 23% in diabetic OLETF rats by 30% CR. The difference of PPARα and PPARβ mRNA expressions in liver of OLETF and LETO rats were not observed. Conclusion: The liver PPARγ and PGC-1 expression response to CR are altered in OLETF rats compared to in LETO rats. These findings suggested that PPARγ and PGC-1 expression control system altered in diabetic OLETF rat liver and altered PPARγ and PCG-1 expression may some roles on the aberrantly activated gluconeogenesis in diabetes mellitus. (J Kor Diabetes Assoc 30:161～169, 2006)

Peroxisome proliferator-activated receptor β expression in human breast epithelial cell lines of tumorigenic and non-tumorigenic origin

Peroxisome proliferator-activated receptor β (PPARβ) is a member of the nuclear hormone receptor superfamily and is a ligand activated transcription factor, although the precise genes that it regulates and its physiological and pathophysiological role remain unclear. In view of the association of PPARβ with colon cancer and increased mRNA levels of PPARβ in colon tumours we sought in this study to examine the expression of PPARβ in human breast epithelial cells of tumorigenic (MCF-7 and MDA-MB-231) and non-tumorigenic origin (MCF-10A). Using quantitative RT-PCR we measured PPARβ mRNA levels in MCF-7, MDA-MB-231 and MCF-10A cells at various stages in culture. After serum-deprivation, MDA-MB-231 and MCF-10A cells had a 4.2- and 3.8-fold statistically greater expression of PPARβ compared with MCF-7 cells. The tumorigenic cell lines also exhibited a significantly greater level of PPARβ mRNA after serum deprivation compared with subconfluence whereas such an effect was not observed in non-tumorigenic MCF-10A cells. The expression of PPARβ was inducible upon exposure to the PPARβ ligand bezafibrate. Our results suggest that unlike colon cancer, PPARβ overexpression is not an inherent property of breast cancer cell lines. However, the dynamic changes in PPARβ mRNA expression and the ability of PPARβ in the MCF-7 cells to respond to ligand indicates that PPARβ may play a role in mammary gland carcinogenesis through activation of downstream genes via endogenous fatty acid ligands or exogenous agonists.

Rat PPARs: Quantitative Analysis in Adult Rat Tissues and Regulation in Fasting and Refeeding

PPARs are members of the nuclear hormone receptor superfamily and are primarily involved in lipid metabolism. The expression patterns of all 3 PPAR isotypes in 22 adult rat organs were analyzed by a quantitative ribonuclease protection assay. The data obtained allowed comparison of the expression of each isotype to the others and provided new insight into the less studied PPARβ (NR1C2) expression and function. This isotype shows a ubiquitous expression pattern and is the most abundant of the three PPARs in all analyzed tissues except adipose tissue. Its expression is especially high in the digestive tract, in addition to kidney, heart, diaphragm, and esophagus. After an overnight fast, PPARβ mRNA levels are dramatically down-regulated in liver and kidney by up to 80% and are rapidly restored to control levels upon refeeding. This tight nutritional regulation is independent of the circulating glucocorticoid levels and the presence of PPARα, whose activity is markedly up-regulated in the liver and small intestine during fasting. Finally, PPARγ2 mRNA levels are decreased by 50% during fasting in both white and brown adipose tissue. In conclusion, fasting can strongly influence PPAR expression, but in only a few selected tissues.

Differential Regulation of Macrophage Peroxisome Proliferator–Activated Receptor Expression by Glucose

Peroxisome proliferator-activated receptors (PPARs) are implicated in several metabolic disorders with altered glucose and lipid metabolism, including atherosclerosis and diabetes. In the present study, we evaluated the in vitro and ex vivo effects of high glucose concentrations on macrophage PPAR mRNA expression. Exposition of monocyte-derived macrophages isolated from healthy donors to a high glucose environment led to an increase in PPARalpha and PPARbeta mRNA expression. In contrast, this treatment significantly decreased human macrophage PPARgamma mRNA expression. Overexpression of PPARalpha and PPARbeta mRNA and inhibition of PPARgamma mRNA expression were also observed in monocyte-derived macrophages isolated from patients with type 2 diabetes. Because high glucose and PPARalpha agonists increase lipoprotein lipase (LPL) gene expression, the role of PPARalpha in the glucose-mediated upregulation of macrophage LPL gene expression was next evaluated. Incubation of murine J774 macrophages with high glucose concentrations increased the expression of PPARalpha at the mRNA and protein levels and enhanced nuclear protein binding to the peroxisome proliferator responsive element of the LPL promoter. Incubation of nuclear extracts in the presence of anti-PPARalpha and anti-PPARbeta antibodies decreased glucose-stimulated nuclear protein binding to the peroxisome proliferator responsive element. These results demonstrate that glucose is an important regulator of macrophage PPAR expression and suggest a role of PPARalpha and PPARbeta in the upregulation of macrophage LPL by glucose. Dysregulation of macrophage PPAR expression in type 2 diabetes may contribute, by altering arterial lipid metabolism and inflammatory response, to the accelerated atherosclerosis associated with diabetes.