PPARgamma mRNA Levels Research Articles

Bovine muscle n−3 fatty acid content is increased with flaxseed feeding

We examined the ability of n-3 FA in flaxseed-supplemented rations to increase the n-3 FA content of bovine muscle. Two groups of animals were used in each of two separate trials: (i) Hereford steers supplemented (or not) with ground flaxseed (907 g/d) for 71 d, and (ii) Angus steers supplemented (or not) with ground flaxseed (454 g/d for 3 d followed by 907 g/d for 110 d). For the Hereford group, flaxseed-supplemented rations increased 18:3n-3 (4.0-fold), 20:5n-3 (1.4-fold), and 22:5n-3 (1.3-fold) mass as compared with the control, and increased total n-3 mass about 1.7-fold. When these data were expressed as mol%, the increase in 18:3n-3 was 3.3-fold and in 20:5n-3 was 1.3-fold in the phospholipid fraction, and 18:3n-3 was increased 4-fold in the neutral lipid fraction. For the Angus group, flaxseed ingestion increased masses and composition of n-3 FA similarly to that for the Herefords and doubled the total n-3 FA mass. The effect of cooking to a common degree of doneness on FA composition was determined using steaks from a third group of cattle, which were Angus steers. We demonstrated no adverse effects on FA composition by grilling steaks to an internal temperature of 64 degrees C. Because n-3 FA may affect gene expression, we used quantitative real-time reverse transcriptase-polymerase chain reaction to quantify the effect of feeding flaxseed on heart-FA binding protein, peroxisome proliferator activated receptor gamma (PPARgamma) and alpha (PPARalpha) gene expression in the muscle tissue. PPARgamma mRNA level was increased 2.7-fold in the flaxseed-fed Angus steers compared with the control. Thus, we demonstrate a significant increase in n-3 FA levels in bovine muscle from cattle fed rations supplemented with flaxseed and increased expression of genes that regulate lipid metabolism.

Peroxisome Proliferator-Activated Receptor α (PPARα) down-regulation in cystic fibrosis lymphocytes

BackgroundPPARs exhibit anti-inflammatory capacities and are potential modulators of the inflammatory response. We hypothesized that their expression and/or function may be altered in cystic fibrosis (CF), a disorder characterized by an excessive host inflammatory response.MethodsPPARα, β and γ mRNA levels were measured in peripheral blood cells of CF patients and healthy subjects via RT-PCR. PPARα protein expression and subcellular localization was determined via western blot and immunofluorescence, respectively. The activity of PPARα was analyzed by gel shift assay.ResultsIn lymphocytes, the expression of PPARα mRNA, but not of PPARβ, was reduced (-37%; p < 0.002) in CF patients compared with healthy persons and was therefore further analyzed. A similar reduction of PPARα was observed at protein level (-26%; p < 0.05). The transcription factor was mainly expressed in the cytosol of lymphocytes, with low expression in the nucleus. Moreover, DNA binding activity of the transcription factor was 36% less in lymphocytes of patients (p < 0.01). For PPARα and PPARβ mRNA expression in monocytes and neutrophils, no significant differences were observed between CF patients and healthy persons. In all cells, PPARγ mRNA levels were below the detection limit.ConclusionLymphocytes are important regulators of the inflammatory response by releasing cytokines and antibodies. The diminished lymphocytic expression and activity of PPARα may therefore contribute to the inflammatory processes that are observed in CF.

Lutein and Eicosapentaenoic Acid Interact to Modify iNOS mRNA Levels through the PPARγ/RXR Pathway in Chickens and HD11 Cell Lines

Two experiments were conducted to investigate the effect of lutein and fat or eicosapentaenoic acid (EPA) interaction on inducible nitric oxide synthase (iNOS), PPARs alpha, beta, and gamma, and retinoic acid X receptor (RXR) alpha and gamma mRNA levels. In Expt. 1, macrophages were collected from broiler chicks fed 3 or 6% dietary fat (g/100 g) with 0, 25, and 50 mg lutein/kg feed for 23 d. In Expt. 2, using a 3 x 3 factorial, eicosapentaenoic acid (EPA) at 0, 15 and 50 micromol/L and lutein at 0, 10 and 100 micromol/L were applied to HD11 cell culture for 24 h. In both experiments, cells were stimulated with lipopolysaccharide before RNA isolation. Lutein interacted with fat in Expt. 1 and with EPA in Expt. 2 to affect mRNA levels of iNOS, PPARgamma, and RXRalpha in chicken macrophages and HD11 cells, respectively (P < 0.05). At 3% dietary fat or up to 15 micromol/L EPA in the medium, increasing lutein increased the iNOS mRNA. However, at 6% dietary fat or 50 micromol/L EPA, lutein did not cause a rise in iNOS mRNA. Increasing lutein in the medium from 0 to 100 micromol/L decreased iNOS mRNA. Increasing lutein with high fat (6%) or EPA (15 micromol/L EPA) increased PPARgamma and RXRalpha mRNA levels. Lutein increased PPARalpha mRNA levels in both macrophages (P < 0.01) and HD11 (P = 0.01) cells and RXRgamma (P < 0.01) mRNA levels in macrophages. GW9662, a PPARgamma antagonist, prevented (P < 0.01) the lutein-induced iNOS mRNA downregulation in HD11 cells. LG101208, a RXR antagonist, prevented (P < 0.01) iNOS upregulation induced by 10 micromol/L lutein and iNOS mRNA downregulation induced by 100 micromol/L lutein. We conclude that lutein and EPA interact through the PPARgamma and RXR pathways to modulate iNOS mRNA.

Peroxisome Proliferator‐Activated Receptor gamma (PPARgamma) ligand, Rosiglitazone, attenuates vascular oxidative stress in a mouse model of Type 2 Diabetes

Purpose The current study was designed to determine if PPARgamma ligands modulate vascular endothelial superoxide generation in vivo through suppressing expression of selected subunits of NADPH oxidase and/or by increasing the expression and activity of Cu/Zn superoxide dismutase. Methods Lean control (db+/db−) and obese, leptin receptor-deficient (db−/db−) mice were treated with either vehicle or rosiglitazone (3 mg/kg/day) by gavage for 7 days. Results Compared to db+/db− mice, obese, db−/db− mice had higher serum glucose, insulin, leptin, triglyceride, and fatty acid levels and lower adiponectin levels. Rosiglitazone had no effect on these metabolic derangements. Aortic superoxide generation measured with ESR spectroscopy was significantly increased in db−/db− mice. Aortic tissue from these mice also demonstrated higher relative mRNA levels of the NADPH oxidase subunits, Nox-1 and Nox-4, as measured by real time PCR analysis and lower mRNA levels of PPARgamma. Rosiglitazone treatment decreased superoxide generation and mRNA levels of Nox homologues in db−/db− mice. Conclusions These data indicate that short-term treatment with the rosiglitazone suppressed vascular NADPH oxidase expression and superoxide production in an animal model of vascular oxidative stress. This work is supported by grants from the Veterans Affairs Research Service

Topical ER36009, a RARγ-Selective Retinoid, Decreases Abdominal White Adipose Tissue and Elicits Changes in Expression of Genes Related to Adiposity and Thermogenesis

Chronic topical treatment of rats with a new RARgamma-selective retinoid, ER36009, resulted in a significant reduction of epididymal white adipose tissue and a significant increase of interscapular brown adipose tissue without affecting food intake. ER36009 markedly decreased PPARgamma, 11beta-HSD1, and Bcl-2 mRNA levels, and increased Bax mRNA in white adipose tissue, while it upregulated UCP1 and UCP3 mRNAs in brown adipose tissue and UCP3 mRNA in gastrocnemial muscle. These results suggest that ER36009 has multiple effects on adipose tissue biology and the energy balance. Topically applied ER36009 may have potential for the treatment of obesity.

Up‐Regulation of Peroxidase Proliferator‐Activated Receptorγ in Cholesteatoma

To evaluate the localization and expression of peroxidase proliferator-activated receptor (PPAR)gamma in cholesteatoma epithelium. Experimental study. Reverse-transcription polymerase chain reaction was performed on cholesteatoma tissues from 10 adult patients undergoing tympanomastoid surgery for middle ear cholesteatoma and on 10 samples of normal external auditory canal skin tissue. The expression levels of PPARgamma to glyceraldehyde-3-phosphate dehydrogenase transcripts were semiquantified by densitometry. We also characterized the cellular localization of the PPARgamma protein immunohistochemically. Ki-67 was also localized to compare the proliferative activity of cells in cholesteatoma epithelium and in normal external auditory canal skin. PPARgamma mRNA and protein were detected in normal external auditory canal skin and in cholesteatoma epithelium. The expression level of PPARgamma mRNA in cholesteatoma was significantly increased compared with that in normal external auditory canal skin. PPARgamma protein was expressed in cells mainly in the granular and prickle cell layers. However, the intensity of its expression was generally decreased in the parabasal layer of the cholesteatoma epithelium. Ki-67 was expressed in the nuclei of cells in the basal and parabasal layers, and a greater number of cells were Ki-67 immunopositive in cholesteatoma epithelium. PPARgamma is up-regulated in the cholesteatoma epithelium compared with normal external auditory canal skin. These results suggest that PPARgamma may play an important role in the pathogenesis of cholesteatoma.

PPARγ activity in subcutaneous abdominal fat tissue and fat mass gain during short-term overfeeding

As the peroxisome proliferator-activated receptor gamma (PPARgamma) plays a central role in fat mass regulation, we investigated whether initial subcutaneous PPARgamma activity is related to fat mass generation during overfeeding. Fourteen healthy female subjects (age 25 +/- 4 years, BMI 22.1 +/- 2.3 kg/m2). Subjects were overfed with a diet supplying 50% more energy than baseline energy requirements for 14 days. Fasting blood samples were analyzed for leptin, insulin and glucose. Fasting subcutaneous abdominal fat biopsies were obtained for analysis of PPARgamma1, PPARgamma2, aP2 and UCP2 mRNAs. Initial PPARgamma1 and 2, aP2 and UCP2 mRNAs were not related to fat gain (P > 0.12). However, PPARgamma1, PPARgamma2 and aP2 mRNA changes were positively related to changes in plasma leptin (P < 0.05) and, except aP2 (P = 0.06), to fat gain (P < 0.05). PPARgamma and aP2 mRNA changes were positively related (P<0.01), indicating that PPARgamma mRNA levels reflected PPARgamma activity. These data suggest that the ability to increase PPARgamma activity might be involved in the susceptibility to gain weight during a positive energy balance.

Evaluation of Peroxisome Proliferator-Activated Receptor-γ Expression in Benign and Malignant Thyroid Pathologies

Impairment of peroxisome proliferator-activated receptor-gamma (PPAR-gamma) function through a dominant negative PAX-8/PPAR-gamma fusion gene or other events resulting in wild-type PPAR-gamma downregulation has been implicated in malignant thyroid cell transformation. The aim of our study was to perform a systematic evaluation of PPAR-gamma mRNA and protein expression in normal thyroid tissue as opposed to benign thyroid pathologies of different functional status and thyroid malignancy, to gain further insights into a putative physiological role of PPAR-gamma in the thyroid and to define whether PPAR-gamma could serve as a marker of thyroid cell differentiation. Ten cold benign (CTN) and 10 toxic (TTN) thyroid nodules and corresponding normal thyroid tissues, 10 follicular thyroid cancers (FTC), 10 papillary thyroid cancers (PTC) and 8 Graves' disease (GD) thyroids were studied by real-time polymerase chain reaction (PCR), immunohistochemistry and reverse transcriptase (RT)-PCR (PAX-8/PPAR-gamma fusion gene). PPAR-gamma mRNA expression was demonstrated in all samples. When comparing benign nodular and normal thyroid tissue of the same patient no significant difference in PPAR-gamma mRNA expression was observed. PPAR-gamma mRNA levels were similar in CTN and FTC. In contrast, PPAR-gamma mRNA expression was downregulated in 9 of 10 PTC and all GD samples, whereby at least 4 fold downregulation (compared with normal and benign nodular thyroid tissues) was observed in the latter. Immunohistochemistry showed an increased, patchy PPAR-gamma nuclear staining in CTNs and TTNs and only faint staining in the corresponding normal thyroid tissues. A diffuse and weak PPAR-gamma staining pattern was observed in all GD samples. No PAX-8/PPAR-gamma rearrangements were detected in any of the 68 thyroid tissue samples. In conclusion PPAR-gamma mRNA and protein expression levels are not concordant in benign thyroid nodular disease. Furthermore there is no clear-cut association of PPAR-gamma mRNA expression with follicular thyroid tumorigenesis. Absence of a PAX-8/PPAR-gamma fusion gene in the series of 68 thyroid samples is in agreement with the suggestion of PAX-8/PPAR-gamma rearrangement being restricted to a subset of follicular thyroid cancers. The marked downregulation of PPAR-gamma in GD warrants further investigation and could be linked, for example, with changes in apoptosis.

Transcriptional synergy and the regulation of Ucp1 during brown adipocyte induction in white fat depots.

Induction of brown adipocytes in white fat depots by adrenergic stimulation is a complex genetic trait in mice that affects the ability of the animal to regulate body weight. An 80-fold difference in expression of the mitochondrial uncoupling gene (Ucp1) at the mRNA and protein levels between A/J and C57BL/6J (B6) mice is controlled by allelic interactions among nine quantitative trait loci (QTLs) on eight chromosomes. Overlapping patterns of these QTLs also regulate expression levels of Pgc-1alpha, Pparalpha, and type 2 deiodinase. Independent validation that PPARalpha is associated with Ucp1 induction was obtained by treating mice with the PPARalpha agonist clofibrate, but not from the analysis of PPARalpha knockout mice. The most upstream sites of regulation for Ucp1 that differed between A/J and B6 were the phosphorylation of p38 mitogen-activated protein kinase and CREB and then followed by downstream changes in levels of mRNA for PPARgamma, PPARalpha, PGC-1alpha, and type 2 deiodinase. However, compared to Ucp1 expression, the two- to fourfold differences in the expression of these regulatory components are very modest. It is proposed that small variations in the levels of several transcriptional components of the Ucp1 enhanceosome interact synergistically to achieve large differences in Ucp1 expression.

A maternal cafeteria diet during gestation and lactation promotes adiposity and impairs skeletal muscle development and metabolism in rat offspring at weaning.

We examined the effects of a maternal cafeteria diet on skeletal muscle and adipose tissue development in the offspring at weaning. Rats born to mothers fed the cafeteria diet either during gestation alone or during both gestation and lactation exhibited a 25% reduction in muscle cross-sectional area with approximately 20% fewer fibres compared with pups fed a balanced chow diet. Maintaining the cafeteria diet during lactation increased intramuscular lipid content and fat pad weights characterized by adipocyte hypertrophy but not hyperplasia. These pups also had elevated muscle IGF-1, IGF-1 receptor, and PPARgamma mRNA levels, which may indicate an attempt to maintain normal insulin sensitivity. The increased adiposity and elevated IGF-1, IGF-1 receptor and PPARgamma mRNAs were not seen in the pups rehabilitated to the balanced diet during lactation. However, these pups exhibited reduced muscle cell proliferation (PCNA) with reduced insulin receptor and a trend towards reduced glucose transporter (GLUT)-4 mRNAs when compared with pups fed a balanced chow diet, indicating possible alterations in glucose uptake by muscle tissue. Therefore, rats born to mothers fed a cafeteria diet during gestation alone or during both gestation and lactation exhibited impaired skeletal muscle development and metabolic disorders normally associated with insulin resistance as early as the weaning stage.

Hypochlorite-oxidized low-density lipoprotein upregulates CD36 and PPARγ mRNA expression and modulates SR-BI gene expression in murine macrophages

The uptake of oxidized low-density lipoprotein (oxLDL) by scavenger receptors of macrophages with resulting foam cell formation is considered a critical event in atherogenesis. Since hypochlorite-oxidized LDL (HOCl-LDL) has been shown to be recognized by macrophages and evidence was provided that HOCl-LDL is internalized via class B scavenger receptors CD36 and SR-BI, the regulatory relationships between CD36, SR-BI, and the nuclear transcription factor PPARgamma in murine macrophages (RAW 264.7) on exposure to HOCl-LDL were examined. Using the highly sensitive real-time RT-PCR we could demonstrate that HOCl-LDL upregulated CD36 and PPARgamma levels dose- and time dependently while modulating SR-BI message levels differently in dependence on HOCl-LDL concentration and incubation time. On exposure of macrophages to HOCl-LDL but not native LDL in varying concentrations, a significant positive correlation between CD36 and PPARgamma (rho = 0.603, p = 0.001) was observed indicating the presence of a positive feedback mechanism by which HOCl-LDL could promote its own uptake. The transcriptional expression of SR-BI in macrophages was not significantly related to PPARgamma mRNA levels after treatment with HOCl-LDL suggesting a differential regulation of the two members of the scavenger receptor class B family in response to HOCl-LDL.

Leptin down-regulates peroxisome proliferator-activated receptor γ (PPARγ) mRNA levels in primary human monocyte-derived macrophages

Increased leptin levels are associated with cardiovascular disease in obesity although the mechanism is unknown. Peroxisome proliferator-activated receptor gamma (PPARgamma) is a key regulator of macrophage lipid metabolism and its activation by thiazolidinediones protects against atherosclerosis. The aim of this study was to assess the effects of human recombinant leptin on PPARgamma mRNA levels in primary human macrophages and macrophage-derived foam cells. Leptin treatment (100 ng/ml) for 24 h caused a 41% reduction (p < 0.01) in PPARgamma transcript levels in human-derived macrophages. This fall was accompanied by a reduction in the mRNA expression of carnitine palmitoyltransferase (CPT-I) (36%, p < 0.05) and ABCA1 (62%, p < 0.05), whereas CD36 mRNA reduction (34%) was not significant. In macrophage-derived foam cells, leptin at 20 ng/ml reduced PPARgamma mRNA levels by 33% (p < 0.01) and CPT-I by 27% (p < 0.05). At this concentration, leptin did not modify the expression of either ABCA1 or CD36. In agreement with these results, intracellular cholesterol ester accumulation was not altered in macrophage-derived foam cells by leptin at 20 ng/ml. We propose that the reduction in PPARgamma expression in both macrophages and foam cells may be one of the factors linking high leptin levels and cardiovascular disease.

Contenido de vitamina A en dieta de cafetería y expresion de receptores nucleares en tejido adiposo de rata

The aim of this study was to determine the effects of cafeteria diet containing control or elevated level of vitamin A on the expression of nuclear receptors in adipose tissue. Male Wistar rats were submitted to 3 experimental diets during 8 weeks, a standard diet and two hyper-energetic, hyperlipidic "cafeteria" diets containing normal (Caf) or higher (Caf+) vitamin A level. During the experiment, body weights and energy intakes were measured. At the end of the experimental period, subcutaneous adipose tissue (Swat) and all the fat mass were removed and weighted. Nuclear receptors mRNA levels of RARalpha, RARgamma, RXRalpha, PPARgamma were measured in the Swat by a real-time semi-quantitative RT-PCR method. We observed that energy intake of Caf+ and Caf groups was significantly higher than that of the control group. Despite a higher increase of the energy intake in the Caf group compared to the Caf+ group, no significant difference was observed in the body weight gain of the Caf+ compared to the Caf group. The Caf+ and Caf diets led to a significant increase of adipose tissue in cafeteria groups as observed in the Swat depot. The mRNA levels of PPARgamma and RXRalpha were significantly increased in the Caf+ group as compared to control group, with a significant positive correlation between these two parameters. Expressions of RARalpha and RARgamma were not modified in experimental groups compared to controls. In conclusion, 8-week exposure to cafeteria diets with normal and higher levels of vitamin A led to an increase of adiposity in rats, associated, only in the group fed with the higher vitamin A level cafeteria diet, with an increase of PPARgamma and RXRalpha expressions in subcutaneous adipose tissue.

Regulation of Peroxisome Proliferator Activated Receptor‐γ in Rat Pituitary

Peroxisome proliferator activated-receptor gamma (PPARgamma) is a member of the nuclear receptor superfamily and, in addition to its relation with obesity and insulin sensitivity, it has recently been localized in human and mice pituitary, indicating a functional significance of PPARgamma in adenopituitary tumours. In the present study, we localized the PPARgamma mRNA and protein in different cell types of rat pituitary. Moreover, using the real-time polymerase chain reaction, we assessed the mRNA expression of PPARgamma in different physiological and pathological settings known to be associated with alterations in anterior pituitary cell proliferation and/or function. Our experiments have shown that PPARgamma mRNA levels were repressed by oestrogen through an oestrogen receptor-alpha effect. However, PPARgamma protein levels were only modified in males but not in females. On the other hand, PPARgamma mRNA expression was increased in dwarf rats in comparison with Lewis rats. Finally, nutritional, thyroid status or pregnancy did not change PPARgamma expression. Taken together, we provide new data regarding the regulation of pituitary PPARgamma mRNA by hormonal and metabolic status.

Histone Deacetylase 1 Contributes to Cell Cycle and Apoptosis

Histone deacetylases (HDACs) are generally thought to play important roles in human disease. However, little information is available concerning the specific functions of individual HDACs. We previously reported on transgenic mice that expressed human HDAC1 and experienced steatosis and nuclear pleomorphism in their hepatic tissues. To find out if the over-expression of HDAC1 contributes to the expression of genes related to the cell cycle, apoptosis, and lipid metabolism that eventually contribute to the pathological changes in the livers of the transgenic mice, the expression profiles of the related genes in liver tissues were determined by reverse transcription-polymerase chain reaction (RT-PCR) and Western blot analysis. The activated human HDAC1 significantly induced the expression levels of mRNA for p53, PPAR-gamma and Bak and reduced the p21 expression level compared with the levels in control littermates. However, the protein levels of p53 and PPAR-gamma were significantly decreased. In conclusion, our results indicate that HDAC1 can regulate gene expression at the mRNA and protein levels independently and that this may be a potential cytopathic factor for hepatic tissue in transgenic mice that over-express HDAC1.

Pioglitazone Prevents Acute Liver Injury Induced by Ethanol and Lipopolysaccharide Through the Suppression of Tumor Necrosis Factor-??

Peroxisome proliferator-activated receptor-gamma (PPAR-gamma), which is a ligand-dependent transcriptional factor, forms a heterodimer with retinoid X receptor (RXR) and controls many genes that are relevant to the regulation of lipid metabolism and insulin sensitization. Recent studies have shown that stimulation of PPAR-gamma inhibits the production of inflammatory cytokines in monocytes and macrophages. Alcohol and lipopolysaccharide (LPS) have already been shown to induce liver injury through the activation of many inflammatory cytokines. Thus, the activation of PPAR-gamma by its ligand may represent a potential effect causing liver injury. In this study, we investigated the effects of pioglitazone, a ligand for PPAR-gamma, on acute liver injury induced by ethanol and LPS. Female Sprague-Dawley rats that weighed 300 g were given ethanol (5 g/kg body weight) intragastrically and received an intraperitoneal injection of LPS 24 hr later. Subsequently, pioglitazone (1 mg/kg body weight) or vehicle alone was injected intraperitoneally 10 min and 24 hr after ethanol administration. Plasma levels of aspartate transaminase and alanine aminotransferase were measured by spectrophotometer. Plasma levels of tumor necrosis factor-alpha (TNF-alpha) were also determined using an enzyme-linked immunosorbent assay. Plasma and hepatic levels of lipid peroxide were measured, and the histologic findings of the liver were examined. Reverse transcription-polymerase reaction analysis of TNF-alpha, PPAR-gamma, RXR-alpha, and beta-actin mRNA was performed. Western blot analysis using the p65 subunit of NF-kappaB was also performed. Pioglitazone prevented increase in plasma aspartate transaminase, alanine aminotransferase, and TNF-alpha levels but had no effect on plasma and hepatic levels of lipid peroxide. Pioglitazone also prevented hepatic inflammation and necrosis induced by ethanol and LPS. Ethanol and LPS induction of TNF-alpha mRNA in the liver was blunted by pioglitazone; however, RXR-alpha mRNA was not affected. PPAR-gamma mRNA levels were suppressed by ethanol and LPS but recaptured by pioglitazone. Western blot analysis showed that pioglitazone did not inhibit translocation of NF-kappaB to nuclei. These results suggest that pioglitazone may prevent liver injury induced by ethanol and LPS through the suppression of TNF-alpha.

Inhibition of Histone Deacetylase Activity by Valproic Acid Blocks Adipogenesis

Adipogenesis is dependent on the sequential activation of transcription factors including the CCAAT/enhancer-binding proteins (C/EBP), peroxisome proliferator-activated receptor gamma (PPARgamma), and steroid regulatory element-binding protein (SREBP). We show that the mood stabilizing drug valproic acid (VPA; 0.5-2 mm) inhibits mouse 3T3 L1 and human preadipocyte differentiation, likely through its histone deacetylase (HDAC) inhibitory properties. The HDAC inhibitor trichostatin A (TSA) also inhibited adipogenesis, whereas the VPA analog valpromide, which does not possess HDAC inhibitory effects, did not prevent adipogenesis. Acute or chronic VPA treatment inhibited differentiation yet did not affect mitotic clonal expansion. VPA (1 mm) inhibited PPARgamma induced differentiation but does not activate a PPARgamma reporter gene, suggesting that it is not a PPARgamma ligand. VPA or TSA treatment reduced mRNA and protein levels of PPARgamma and SREBP1a. TSA reduced C/EBPalpha mRNA and protein levels, whereas VPA only produced a decrease in C/EBPalpha protein expression. Overall our results highlight a role for HDAC activity in adipogenesis that can be blocked by treatment with VPA.

Peroxisome proliferator activated receptor-gamma in pathogenesis of experimental fatty liver disease.

To study the expression of peroxisome proliferator activated receptor-gamma (PPARgamma) in the liver of rats with fatty liver disease (FLD) and to explore the role of PPARgamma in the pathogenesis of FLD to provide the basis for using PPARgamma ligand to treat patients with FLD. Forty Wistar rats were divided into 4 groups of ten rats each randomly: normal group (group A), alcohol group (group B), fat-rich diet group (group C), alcohol and fat-rich diet group (group D). The rats were sacrificed at the end of the 16th week from the feeding day. Alanine aminotransferase (ALT), tumor necrosis factor-alfa (TNFalpha) in serum and malondialdehyde (MDA) in liver homogenate were determined; livers were collected for observing pathologic changes by HE, Sudan IV, Masson stain under microscope. The morphologic results were analyzed by picture quantitative analysis technique. The changes of ultrastructure were also examined under electron microscope. The expression of PPARgamma in liver was detected by immunohistochemistry and RT-PCR. The correlations between the expression of PPARgamma and biochemical indexes, and liver histology were analyzed. The steatosis, inflammation, necrosis and fibrosis were present in livers of different experimental groups, especially in livers of alcohol and fat-rich diet group. The content of immunodetectable PPARgamma was decreased remarkably in the livers of model rats (group B-D); the level in alcohol and fat-rich diet group (3.43+/-1.48) was significantly lower than that in normal group (18.34+/-3.73), alcohol group (8.82+/-2.52) and fat-rich diet group (11.73+/-2.51) (all P<0.01). The level of PPARgamma mRNA was also lower in the livers of model rats (group B-D) than in livers of controls. The expression of PPARgamma in rat liver correlated negatively with the degree of its inflammation, necrosis and fibrosis, as well as the level of serum TNFalpha and the content of MDA in liver homogenates, but not with steatosis or serum ALT. Decreased expression of PPARgamma may play an important role in the development of hepatocellular inflammation, necrosis and fibrosis of rats with FLD. Thus, activating PPARgamma by its ligand can be anticipated to provide a therapy target for FLD.

Induction of differentiation and peroxisome proliferator-activated receptor gamma expression in colon cancer cell lines by troglitazone.

We investigated the relationship between the effects of troglitazone (TGZ) on cellular growth, differentiation and apoptosis induction, and the induction of peroxisome proliferator-activated receptor (PPAR) gamma in three human colon cancer cell lines, HCT-15, DLD-1and LoVo. Viable cell number was evaluated by the Alamar blue assay and apoptotic cell death by TUNEL methods. Expression of PPARgamma mRNA and protein was examined by reverse transcription-polymerase chain reaction (RT-PCR) and Western blot, respectively. The differentiation markers of colonic mucosa, villin and MUC2 mRNAs, were analyzed by real-time RT-PCR. HCT-15 and DLD-1 cells proliferated rapidly while LoVo cells grew slowly. TGZ dose-dependently inhibited the proliferation of all the cell lines, and also induced apoptotic cell death. High expression of PPARgamma mRNA and protein was demonstrated in DLD-1 and LoVo cells before TGZ treatment. After the treatment, PPARgamma mRNA and protein levels were increased in HCT-15 and LoVo cells. Villin and MUC2 mRNAs were increased by TGZ treatment in HCT-15 cells while villin mRNA was repressed in LoVo cells. Changes in expression of PPARgamma, villin or MUC2 mRNAs were not observed in DLD-1 cells. These results suggest that PPARgamma levels are not correlated with the rates of cell proliferation. Differentiation induction by TGZ was only observed in the cell lines with enhanced PPARgamma expression.

Decreased fatty acid esterification compensates for the reduced lipolytic activity in hormone-sensitive lipase-deficient white adipose tissue

It has been observed previously that hormone-sensitive lipase-deficient (HSL-ko) mice have reduced white adipose tissue (WAT) stores compared to control mice. These findings contradict the expectation that the decreased lipolytic activity in WAT of HSL-ko mice would cause accumulation of triglycerides (TGs) in that tissue. Here we demonstrate that the cellular TG synthesis in HSL-deficient WAT is markedly reduced due to downregulation of the enzymatic activities of glycerophosphate acyltransferase, dihydroxyacetonphosphate acyltransferase, lysophosphatidate acyltransferase, and diacylglycerol acyltransferase. Fatty acid de novo synthesis is also decreased due to reduced cellular glucose uptake, reduced glucose incorporation into adipose tissue lipids, and reduced activities of acetyl:CoA carboxylase and fatty acid synthase. Finally, the activities of phosphoenolpyruvate carboxykinase (PEPCK), acyl:CoA synthetase (ACS), and glucose 6-phosphate dehydrogenase, the enzymes that provide glycerol-3-phosphate, acyl-CoA, and NADPH for TG synthesis, respectively, are decreased in HSL-ko mice. The reduced expression of the peroxisome proliferator-activated receptor gamma (PPAR gamma) target genes PEPCK, ACS, and aP2, as well as reduced mRNA levels of PPAR gamma itself, suggest the involvement of this transcription factor in the downregulation of lipogenesis. Taken together, these results establish that in the absence of HSL, the reduced NEFA production is counteracted by a drastic reduction of NEFA reesterification that provides sufficient quantities of NEFA for release into the circulation. These metabolic adaptations result in decreased fat mass in HSL-ko mice.