Resveratrol Regulates the Quiescence-Like Induction of Activated Stellate Cells by Modulating the PPARγ/SIRT1 Ratio.

The activation of hepatic stellate cell (HSC), from a quiescent cell featuring cytoplasmic lipid droplets to a proliferative myofibroblast, plays an important role in liver fibrosis development. The GRX line is an activated HSC model that can be induced by all-trans-retinol to accumulate lipid droplets. Resveratrol is known for activating Sirtuin1 (SIRT1), a NAD(+)-dependent deacetylase that suppresses the activity of peroxisome proliferator-activated receptor gamma (PPARγ), an important adipogenic transcription factor involved in the quiescence maintenance of HSC. We evaluated the effects of 0.1 µM of resveratrol in retinol-induced GRX quiescence by investigating the interference of SIRT1 and PPARγ on cell lipogenesis. GRX lipid accumulation was evaluated through Oil-red O staining, triacylglycerides quantification, and [(14)C] acetate incorporation into lipids. mRNA expression and protein content of SIRT1 and PPARγ were measured by RT-PCR and immunoblotting, respectively. Resveratrol-mediated SIRT1 stimuli did not induce lipogenesis and reduced the retinol-mediated fat-storing capacity in GRX. In order to support our results, we established a cell culture model of transgenic super expression of PPARγ in GRX cells (GRXPγ). Resveratrol reduced lipid droplets accumulation in GRXPγ cells. These results suggest that the PPARγ/SIRT1 ratio plays an important role in the fate of HSC. Thus, whenever the PPARγ activity is greater than SIRT1 activity the lipogenesis is enabled.

Obesity associated with type 2 diabetes mellitus is linked to decreased PC1/3 mRNA expression in the Jejunum.

BACKGROUND: Bariatric surgery is the most effective therapeutic option for obesity and its complications, especially in type 2 diabetes. The aim of this study was to investigate the messenger RNA (mRNA) gene expression of proglucagon, glucose-dependent insulinotropic peptide (GIP), prohormone convertase 1/3 (PC1/3), and dipeptidyl peptidase-IV (DPP-IV) in jejunum cells of the morbidly obese (OB) non type 2 diabetes mellitus (NDM2) and type 2 diabetes mellitus (T2DM), to determine the molecular basis of incretin secretion after bariatric surgery. METHODS: Samples of jejunal mucosa were obtained from 20 NDM2 patients: removal of a section of the jejunum about 60 cm distal to the ligament of Treitz and 18 T2DM patients: removal of a section of the jejunum about 100 cm distal to the ligament of Treitz. Total RNA was extracted using TRIzol. Reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) was carried out. Samples were sequenced to PC1/3 by ACTGene Análises Moleculares Ltd. Immuno content was quantified with a fluorescence microscope. RESULTS: T2DM showed decreased PC1/3 mRNA expression in the primers tested (primer a, p=0.014; primer b, p=0.048). Many patients (36.5 %) did not express PC1/3 mRNA. NDM2 and T2DM subjects showed nonsignificantly different proglucagon, GIP, and DPP-IV mRNA expression. The immuno contents of glucagon-like peptide-1 and GIP decreased in T2DM jejunum, but incubation with high glucose stimulated the immuno contents. CONCLUSIONS: The results suggest that bioactivation of pro-GIP and proglucagon could be impaired by the lower expression of PC1/3 mRNA in jejunum cells of obese patients with T2DM. However, after surgery, food could activate this system and improve glucose levels in these patients.

Parallel down-regulation of FOXO1, PPARγ and adiponectin mRNA expression in visceral adipose tissue of class III obese individuals.

OBJECTIVE: Adipose tissue is responsible for secretion of several cytokines that mediate systemic effects on obesity and insulin resistance. Subcutaneous abdominal adipose tissue (SAT) and visceral adipose tissue (VAT) are metabolically different and have differences in their gene expression profile. Our study evaluated the expression of adiponectin, FOXO1, PPARγ, and SIRT1 in VAT and SAT of non-obese and class III obese subjects. METHODS: The adipose tissue samples were obtained by surgery. Reverse transcripts of studied genes were determined by quantitative real-time polymerase chain reaction (qRT-PCR). RESULTS: Comparing the different lipid depots, adiponectin expression was lower only in VAT of obese individuals (p = 0.043); FOXO1 and PPARγ levels were decreased in VAT of both groups. When non-obese and obese were compared, only adiponectin expression was lower in SAT and in VAT of obese subjects (p = 0.004 and p = 0.002, respectively). No difference was found with regard to SIRT1 levels in VAT or SAT in both groups. FOXO1 expression in SAT of obese subjects had a negative correlation with age (r = -0.683; p = 0.029) and triglyceride serum levels (r = -0.794; p = 0.006). CONCLUSION: The decrease mRNA expression of this genes in VAT, responsible for central adiposity, may be associated with an increased risk of obesity and co-morbidities.

Resveratrol upregulated SIRT1, FOXO1, and adiponectin and downregulated PPARγ1-3 mRNA expression in human visceral adipocytes.

BACKGROUND: The SIRT1 enzyme is involved in adipose tissue (AT) lipolysis. FOXO1 is a protein that plays a significant role in regulating metabolism. Adiponectin is an adipokine, secreted by the AT, which has been considered to have an antiobesity function. PPARγ is one of the key actors in adipocytes differentiation. This study was undertaken to investigate whether resveratrol can regulate SIRT1, FOXO1, adiponectin, PPARγ1-3, and PPARß/δ in human AT. METHODS: The effects of resveratrol were analyzed in freshly isolated adipocytes prepared from visceral fat tissue samples obtained during bariatric surgery. Genes messenger ribonucleic acid (mRNA) levels were determined by qRT-PCR. RESULTS: Ours results show that resveratrol modulates the studied genes, increasing SIRT1 (p = 0.021), FOXO1 (p = 0.001), and adiponectin (p = 0.025) mRNA expression and decreasing PPARγ1-3 (p = 0.003) mRNA in human visceral adipocytes. CONCLUSIONS: Resveratrol, in vitro and at low concentration, modulates genes that are related to lipid metabolism, possibly preventing metabolic disease in human visceral adipose tissue (VAT).

SIRT1 transcription is decreased in visceral adipose tissue of morbidly obese patients with severe hepatic steatosis.

BACKGROUND: Visceral adipose tissue is known to release greater amounts of adipokines and free fatty acids into the portal vein, being one of the most predictive factors of nonalcoholic fatty liver disease (NAFLD). Our study has the purpose to evaluate sirtuin 1 (SIRT1), adiponectin, Forkhead/winged helix (FOXO1), peroxisome proliferator-activated receptor (PPAR)gamma1-3, and PPARbeta/delta mRNA expression in morbidly obese patients in three different lipid depots: visceral (VAT), subcutaneous (SAT), and retroperitoneal (RAT). Recent studies suggest that SIRT1, a NAD(+)-dependent deacetylase, protects rats from NAFLD. METHODS: We divided the patients in two groups: those with slight or moderate steatosis (hepatic steatosis, HS) and other comprising individuals with severe steatosis associated or not with necroinflammation and fibrosis (severe hepatic steatosis, SHS). The adipose tissue depots were obtained during bariatric surgery. Total RNAs were extracted using TRIzol. The amount of genes of interest was determined by quantitative real-time polymerase chain reaction. RESULTS: When comparing the two groups of patients, a decrease in SIRT1 was observed in VAT of morbidly obese patients in SHS group (p = 0.006). The mRNA expression of the other genes showed no differences in VAT. No difference was found either in SAT or in RAT for all genes in the study. In addition, the homeostasis model assessment for insulin resistance (HOMA-IR) value was higher in SHS group compared to HS (p = 0.006). Also, our results show that the mRNA expression of SIRT1 and the value of HOMA-IR were positively correlated in VAT of SHS patients (r = 0.654; p = 0.048). CONCLUSIONS: Downregulation of SIRT1 mRNA expression in VAT of SHS could be possible impairing mitochondria biogenesis and fatty acid oxidation, promoting severe steatosis in obese patients. Our results provide a possible proof of SIRT1 protective potential in VAT against NAFLD in humans.

Resveratrol inhibits cell growth by inducing cell cycle arrest in activated hepatic stellate cells.

Resveratrol (RSV) exerts anti-proliferative and pro-apoptotic actions in different cell lines. Hepatic stellate cells (HSCs) are major fibrogenic cell types that contribute to collagen accumulation during chronic liver disease. In the present study, the inhibitory effects of RSV on cell proliferation, cell cycle, and apoptosis were evaluated in the mouse hepatic stellate cell line GRX. Cells treated with 1 nM-1 muM of RSV demonstrated a decrease in cell growth of about 35% after 5 days. GRX cells, treated with RSV (100 nM or 1 muM), were analyzed by flow cytometry; RSV induced an increase in the number of GRX cells in the S- and sub-G1 phases. The increase in sub-G1 phase cells and the nuclear condensation and fragmentation shown by DAPI staining identified a possible pro-apoptotic effect of RSV on GRX cells. Furthermore, the RSV anti-proliferative effects could be explained by an S-phase accumulation caused by a decrease in the progression through the cell cycle or an inhibition of S or G2 phase transition. It is notable that these RSV actions are mediated at nanomolar levels, compatible with the concentrations of free RSV in biological fluids after ingestion of polyphenol-rich foods, suggesting a possible effect of these foods as an adjuvant treatment in chronic liver diseases.

Activity and expression of ecto-5'-nucleotidase/CD73 are increased during phenotype conversion of a hepatic stellate cell line.

Hepatic stellate cells (HSC) play a crucial role in the development of liver fibrosis and are important targets in liver disease therapy. Adenosine acts as an extracellular signaling molecule in various tissues and in liver this nucleoside exerts protective effects. Ecto-5'-nucleotidase/CD73 is a marker for the plasma membrane and is considered to be a key enzyme in the generation of adenosine in the extracellular medium, by transforming AMP into adenosine. In addition, adenosine production from AMP is also catalyzed by alkaline phosphatase. We compared the extracellular metabolism of AMP and transcriptional levels of the ecto-5'-nucleotidase/CD73 and tissue non-specific alkaline phosphatase (TNALP) in activated and quiescent HSC of the mouse hepatic stellate cell line GRX. This cell line expresses a myofibroblast phenotype in basal medium and both retinol and indomethacin treatment induced a phenotypic change of GRX cells to quiescent HSC. Ecto-5'-nucleotidase activity and its mRNA expression were found to be higher in quiescent HSC than in activated HSC. During phenotype conversion, mediated by retinol, the AMP decay was accelerated with adenosine accumulation in extracellular medium, likely due to the decrease in adenosine deaminase activity also observed in quiescent HSC. The treatment with retinol also involves transcriptional activation of TNALP. Taken together, these data suggest that ecto-5'-nucleotidase-dependent adenosine generation may play a role in the regulation of quiescent HSC functions.

Hepatic stellate cell line modulates lipogenic transcription factors.

BACKGROUND/AIMS: Pre-adipocyte differentiation into adipocyte is a terminal differentiation process triggered by a cascade of transcription factors. Conversely, hepatic stellate cells (HSC) can switch between lipid storing and the myofibroblast phenotype in association with liver fibrotic processes. Here, adipogenic/lipogenic-related transcription factors and downstream-regulated genes were evaluated in a murine HSC cell line. GRX-HSC cells are transitional myofibroblasts that differentiate into lipocytes following retinol or indomethacin treatment. METHODS/RESULTS: Specific mRNAs were quantified by a real-time polymerase chain reaction after 24 h or 7 days of cell culture with indomethacin or retinol. Proliferator-activated receptorgamma and Pex16 transcripts were increased either by retinol or indomethacin. Retinol induced a minor increase in C/enhancer binding proteinalpha transcripts, while only indomethacin increased adipsin transcripts. CONCLUSIONS: Our results showed that the myofibroblast to lipocyte phenotype switch follows partially different transcriptional pathways, according to the effector. Retinol induces lipid synthesis and storage without affecting characteristic adipocytic genes, while indomethacin treatment restores the lipocytic phenotype with increased adipisin expression.

Adipose tissue distribution and quantification of PPARbeta/delta and PPARgamma1-3 mRNAs: discordant gene expression in subcutaneous, retroperitoneal and visceral adipose tissue of morbidly obese patients.

BACKGROUND: Adipose tissue (AT) metabolism is altered in obese subjects, and the reestablishment of energy homeostasis requires the identification and regulation of genes with altered patterns. The aim of this study was to compare mRNA expression of PPARbeta/delta and PPARgamma1-3 in morbidly obese and nonobese patients. The expression pattern of these receptors in various abdominal adipose tissues, subcutaneous (SAT), retroperitoneal (RAT) and visceral (VAT), was also evaluated. METHODS: The AT depots were obtained by surgery. Total RNAs were extracted using TRIzol. PPARs reverse transcripts were determined by quantitative polymerase chain reaction (qRT-PCR). RESULTS: The amounts of PPARP/8 mRNA in different depots of morbidly obese AT showed a significant decrease in VAT (P < 0.05). In the non-obese group, the level of PPARbeta/delta was higher in SAT (P < 0.05), but PPARgamma1-3 was not differentially expressed in obese and non-obese depots. When comparing obese and non-obese, the results revealed a decrease in PPARPbeta/delta expression in SAT (P = 0.058) and VAT (P = 0.094) of the morbidly obese. PPARgamma1-3 mRNA expression was increased significantly in SAT (P = 0.022) and decreased in RAT (P = 0.034) in morbidly obese subjects. PPARbeta/delta expression in SAT and VAT correlated negatively with hip size and insulin serum respectively. PPARgamma1-3 expression in RAT correlated negatively with waist and hip circumference and in VAT correlated positively with waist size. CONCLUSIONS: The present study demonstrates that PPARbeta/delta and PPARgamma1-3 mRNAs are quantitatively different in AT of morbidly obese individuals compared to non-obese, and that PPARbeta/delta mRNA levels are characteristic for each AT depot.

Variations of ganglioside biosynthetic pathways in the phenotype conversion from myofibroblasts to lipocytes in murine hepatic stellate cell line.

GRX cell line represents hepatic stellate cell and can be transformed from an actively proliferation myofibroblast phenotype into a quiescent fat-storing lipocyte phenotype. Both express the same gangliosides (GM3, GM2, GM1 and GD1a), which are resolved as doublets on HPTLC. Upper/lower band ratio is increased in lipocyte-like cells and the upper band is composed by ceramides with long-chain fatty acids. This study evaluated the contribution of de novo synthesis, sphingosine and Golgi recycling pathways on ganglioside biosynthesis, in both phenotypes. Cells were preincubated with 5 mM beta-chloroalanine (SPT: serine palmitoyltransferase inhibitor) or with 25 muM fumonisin B1 (ceramide synthase inhibitor) and then radiolabeled with [U-(14)C]galactose in the continued presence of inhibitors. Gangliosides were extracted, purified and analyzed by HPTLC. In myofibroblast-like cells, simple gangliosides use the de novo pathway while complex gangliosides are mainly synthesized by recycling pathways. In lipocyte-like cells, de novo pathway has a lesser contribution and this is in agreement with the lower activity of the committed enzyme of sphingolipid synthesis (SPT) detected in this phenotype. SPT mRNA has an identical expression in both phenotypes. It was also observed that gangliosides doublets from myofibroblast-like cells have the same distribution between triton soluble and insoluble fractions (upper band > lower band) while the gangliosides doublets from lipocyte-like cells show an inversion in the insoluble fraction (lower band > upper band) in comparison to soluble fraction. These results indicate that myofibroblast- and lipocyte-like cells have important differences between the glycosphingolipid biosynthetic pathways, which could contribute with the respective glycosphingolipid-enriched membrane microdomain's composition.

Lipídio: fator de risco e prevenção do câncer de mama / Lipid: risk factor and breast cancer prevention

A hipótese de que uma dieta rica em gordura promova o desenvolvimento do câncer de mama na menopausa é fortalecida por estudos caso-controle, que mostram forte associação positiva entre uma dieta rica em lipídios e as taxas de incidência de câncer de mama. Por outro lado, a ingestão dietética de gordura não parece estar relacionada com o risco de câncer de mama em estudos de coorte. Em vista desses achados conflitantes, tem sido difícil propor qualquer recomendação nutricional para a prevenção do câncer de mama. Estudos com animais e observações recentes em humanos, entretanto, têm mostrado evidências de que a dieta rica em ácido graxo linoléico estimula vários estágios no desenvolvimento de câncer mamário. Alguns estudos ainda mostram que o óleo de peixe, constituído de ácidos graxos mega-3, parece prevenir o câncer pela influência sobre a atividade de enzimas e proteínas relacionadas à proliferação celular. Assim, são necessários estudos epidemiológicos que integrem as interações de ácidos graxos específicos com o catabolismo hormonal, fatores nutricionais protetores e de risco relacionados com o câncer de mama. Nesse trabalho, abordaremos os fatores protetores, de risco e as implicações quali e quantitativas dos ácidos graxos da dieta sobre o câncer de mama.

Higher content of trans fatty acids in abdominal visceral fat of morbidly obese individuals undergoing bariatric surgery compared to non-obese subjects.

BACKGROUND: The purpose of this study was to determine the total content of trans fatty acids (TFA) in subcutaneous, retroperitoneal and visceral fat of morbidly obese and non-obese patients submitted to bariatric surgery or plastic and abdominal surgery. METHODS: The adipose tissues were obtained by surgery; lipids were extracted, saponified and esterified. TFA were measured by FTIR-ATR spectroscopy. RESULTS: The TFA average in obese patients was 6.3% for retroperitoneal and 8.7% for visceral fat. For non-obese patients, the figures were 6.9% (subcutaneous) and 9.3% (visceral). There was no difference between the groups. However, the TFA depot in visceral fat was higher than other fatty tissues for morbidly obese (P<0.001) and non-obese (P<0.05) patients. CONCLUSIONS: Our values for TFA content in all adipose tissues analyzed are higher than reported in other countries (3-6%). We showed more TFA in visceral adipose tissue than in other abdominal fat (subcutaneous and retroperitoneal) stores. The visceral adipose tissue level is worrisome because the higher rate of lipolysis in this tissue appears to be an important indicator of metabolic alterations and the levels of TFA found in adipose tissue presumably reflect the higher dietary intake of TFA by Brazilians.

Beta-carotene storage, conversion to retinoic acid, and induction of the lipocyte phenotype in hepatic stellate cells.

Hepatic stellate cells (HSCs) are the major site of retinol (ROH) metabolism and storage. GRX is a permanent murine myofibroblastic cell line, derived from HSCs, which can be induced to display the fat-storing phenotype by treatment with retinoids. Little is known about hepatic or serum homeostasis of beta-carotene and retinoic acid (RA), although the direct biogenesis of RA from beta-carotene has been described in enterocytes. The aim of this study was to identify the uptake, metabolism, storage, and release of beta-carotene in HSCs. GRX cells were plated in 25 cm(2) tissue culture flasks, treated during 10 days with 3 micromol/L beta-carotene and subsequently transferred into the standard culture medium. beta-Carotene induced a full cell conversion into the fat-storing phenotype after 10 days. The total cell extracts, cell fractions, and culture medium were analyzed by reverse phase high-performance liquid chromatography for beta-carotene and retinoids. Cells accumulated 27.48 +/- 6.5 pmol/L beta-carotene/10(6) cells, but could not convert it to ROH nor produced retinyl esters (RE). beta-Carotene was directly converted to RA, which was found in total cell extracts and in the nuclear fraction (10.15 +/- 1.23 pmol/L/10(6) cells), promoting the phenotype conversion. After 24-h chase, cells contained 20.15 +/- 1.12 pmol/L beta-carotene/10(6) cells and steadily released beta-carotene into the medium (6.69 +/- 1.75 pmol/ml). We conclude that HSC are the site of the liver beta-carotene storage and release, which can be used for RA production as well as for maintenance of the homeostasis of circulating carotenoids in periods of low dietary uptake.

Effect of pentoxifylline on arachidonic acid metabolism, neutral lipid synthesis and accumulation during induction of the lipocyte phenotype by retinol in murine hepatic stellate cell.

In liver fibrosis, the quiescent hepatic stellate cells (HSC) are activated to proliferate and express the activated myofibroblast phenotype, losing fat droplets and the stored vitamin A, and depositing more extracellular matrix. Therapeutic strategies for liver fibrosis are focused on HSC. Pentoxifylline (PTF), an analog of the methylxanthine, prevents the biochemical and histological changes associated with animal liver fibrosis. The aim of the present study was to investigate the phenotypic change of myofibroblasts into quiescent lipocytes by PTF and/or retinol, using a permanent cell line GRX that represents murine HSC. We studied the action of both drugs on the synthesis of neutral lipids, activity of phospholipase A2 (PLA2), release of arachidonic acid (AA) and prostaglandins synthesis. Accumulation and synthesis of neutral lipids was dependent upon association of retinol with PTF. PTF (0.5 mg/mL) alone did not induce lipid accumulation and synthesis, but in cells induced by physiologic concentration of retinol (1-2.5 microM), it increased the quantity of stored lipids. Retinol and PTF (5 microM and 0.1 mg/mL, respectively) had a synergistic effect on neutral lipid synthesis and accumulation. In higher PTF concentrations (0.5 and 0.7 mg/ml), the synthesis was stimulated but accumulation decreased. Membrane-associated PLA2 activity decreased after PTF treatment, which increased the AA release 8 fold, and significantly increased the production of PGE2, but not of PGF2. However, when in presence of retinol, we observed a slightly higher increase in PGE2 and PGF2a production. In conclusion, PTF treatment generated an excess of free AA. We propose that retinol counteracts the action of PTF on the AA release and PGs production, even though both drugs stimulated the lipocyte induction in the HSC.

Hepatic stellate cell activation in vitro: cell cycle arrest at G2/M and modification of cell motility.

Hepatic fibrosis is a common response to chronic liver injury and is characterized by increased production of extracellular matrix components, whose major part is produced by hepatic stellate cells activated by inflammatory mediators to proliferate and migrate into the injured regions. GRX cells are a model of hepatic stellate cells characterized as myofibroblasts by morphological and biochemical criteria. We have recently shown that they respond to inflammatory mediators and cytokines present in the concanavalin A-activated spleen cell supernatant (SCS) by quantitative changes in the expression of intermediate filaments. The present study investigated the effects of SCS and TNF-alpha on the GRX cell proliferation and on the organization of the actin cytoskeleton. SCS and TNF-alpha diminished the culture cell density, with an increase of cell [(3)H]thymidine incorporation and of cellular protein content, indicating an arrest in the G2/M phase of the cell cycle, which was reversible 48 h after removal of SCS. This effect was abrogated by dibutiryl-cAMP. Actin cytoskeleton reorganization was observed after 24 h treatment, indicating increased cell motility. Our results suggest that inflammation-dependent activation of stellate cells occurs in ordered interaction and coordination of proinflammatory agents. The increase of cAMP levels activates the conversion of lipocytes into myofibroblasts and increases the number of cells that can participate in repair. Since cAMP retains cells in the G1 phase, cytokines of the TNF-alpha group are required for cell proliferation inducing the entry into the S phase. The progression through the G2/M checkpoint is mediated again by increased cAMP levels.

Changes of sphingolipid species in the phenotype conversion from myofibroblasts to lipocytes in hepatic stellate cells.

Sphingolipids play a relevant role in cell-cell interaction, communication, and migration. We studied the sphingolipid content in the murine hepatic stellate cell line GRX, which expresses the myofibroblast phenotype, and can be induced in vitro to display the fat-storing phenotype. Lipid modifications along this induction were investigated by labeling sphingolipids with [(14)C]galactose, [(14)C]serine, or [(14)C]choline, and determination of fatty acid composition of sphingomyelin. The total ganglioside content and the GM2 synthase activity were lower in myofibroblasts. Both phenotypes presented similar gangliosides of the a-pathway: GM2, GM1, and GD1a as well as their precursor GM3. Sphingomyelin and all the gangliosides were expressed as doublets; the upper/lower band ratio increased in lipocytes, containing more long-chain fatty acids in retinol-induced lipocytes as compared to the insulin/indomethacin induced ones. Time-course experiments indicated a transfer of metabolic precursors from phosphatidylcholine to sphingomyelin in the two phenotypes. Taken together, these results indicate that myofibroblast and lipocytes can use distinct ceramide pools for sphingolipid synthesis. Differential ganglioside expression and presence of the long-chain saturated fatty acids suggested that they may participate in formation of distinct membrane microdomains or rafts with specific functions on the two phenotypes of GRX-cells.