Direct effect of glucocorticoids on glucose-activated adult rat ß-cells increases their cell number and their functional mass for transplantation.

Compounds that increase ß-cell number can serve as ß-cell replacement therapies in diabetes. In vitro studies have identified several agents that can activate DNA synthesis in primary ß-cells but only in small percentages of cells and without demonstration of increases in cell number. We used whole well multiparameter imaging to first screen a library of 1,280 compounds for their ability to recruit adult rat ß-cells into DNA synthesis and then assessed influences of stimulatory agents on the number of living cells. The four compounds with highest ß-cell recruitment were glucocorticoid (GC) receptor ligands. The GC effect occurred in glucose-activated ß-cells and was associated with increased glucose utilization and oxidation. Hydrocortisone and methylprednisolone almost doubled the number of ß-cells in 2 wk. The expanded cell population provided an increased functional ß-cell mass for transplantation in diabetic animals. These effects are age dependent; they did not occur in neonatal rat ß-cells, where GC exposure suppressed basal replication and was cytotoxic. We concluded that GCs can induce the replication of adult rat ß-cells through a direct action, with intercellular differences in responsiveness that have been related to differences in glucose activation and in age. These influences can explain variability in GC-induced activation of DNA synthesis in rat and human ß-cells. Our study also demonstrated that ß-cells can be expanded in vitro to increase the size of metabolically adequate grafts.

Glucocorticoids and checkpoint tyrosine kinase inhibitors stimulate rat pancreatic beta cell proliferation differentially.

Cell therapy for diabetes could benefit from the identification of small-molecule compounds that increase the number of functional pancreatic beta cells. Using a newly developed screening assay, we previously identified glucocorticoids as potent stimulators of human and rat beta cell proliferation. We now compare the stimulatory action of these steroid hormones to a selection of checkpoint tyrosine kinase inhibitors that were also found to activate the cell cycle-in beta cells and analyzed their respective effects on DNA-synthesis, beta cell numbers and expression of cell cycle regulators. Our data using glucocorticoids in combination with a receptor antagonist, mifepristone, show that 48h exposure is sufficient to allow beta cells to pass the cell cycle restriction point and to become committed to cell division regardless of sustained glucocorticoid-signaling. To reach the end-point of mitosis another 40h is required. Within 14 days glucocorticoids stimulate up to 75% of the cells to undergo mitosis, which indicates that these steroid hormones act as proliferation competence-inducing factors. In contrast, by correlating thymidine-analogue incorporation to changes in absolute cell numbers, we show that the checkpoint kinase inhibitors, as compared to glucocorticoids, stimulate DNA-synthesis only during a short time-window in a minority of cells, insufficient to give a measurable increase of beta cell numbers. Glucocorticoids, but not the kinase inhibitors, were also found to induce changes in the expression of checkpoint regulators. Our data, using checkpoint kinase-specific inhibitors further point to a role for Chk1 and Cdk1 in G1/S transition and progression of beta cells through the cell cycle upon stimulation with glucocorticoids.

Peroxisome proliferator-activated receptor alpha-retinoid X receptor agonists induce beta-cell protection against palmitate toxicity.

Fatty acids can stimulate the secretory activity of insulin-producing beta-cells. At elevated concentrations, they can also be toxic to isolated beta-cells. This toxicity varies inversely with the cellular ability to accumulate neutral lipids in the cytoplasm. To further examine whether cytoprotection can be achieved by decreasing cytoplasmic levels of free acyl moieties, we investigated whether palmitate toxicity is also lowered by stimulating its beta-oxidation. Lower rates of palmitate-induced beta-cell death were measured in the presence of L-carnitine as well as after addition of peroxisome proliferator-activated receptor alpha (PPARalpha) agonists, conditions leading to increased palmitate oxidation. In contrast, inhibition of mitochondrial beta-oxidation by etomoxir increased palmitate toxicity. A combination of PPARalpha and retinoid X receptor (RXR) agonists acted synergistically and led to complete protection; this was associated with enhanced expression levels of genes involved in mitochondrial and peroxisomal beta-oxidation, lipid metabolism, and peroxisome proliferation. PPARalpha-RXR protection was abolished by the carnitine palmitoyl transferase 1 inhibitor etomoxir. These observations indicate that PPARalpha and RXR regulate beta-cell susceptibility to long-chain fatty acid toxicity by increasing the rates of beta-oxidation and by involving peroxisomes in fatty acid metabolism.

Essential role of chicken ovalbumin upstream promoter-transcription factor II in insulin secretion and insulin sensitivity revealed by conditional gene knockout.

Chicken ovalbumin upstream promoter-transcription factor II (COUP-TFII) has been implicated in the control of blood glucose by its potent effect on expression and signaling of various nuclear receptors. To understand the role of COUP-TFII in glucose homeostasis, conditional COUP-TFII-deficient mice were generated and crossed with mice expressing Cre under the control of rat insulin II gene promoter, resulting in deletion of COUP-TFII in pancreatic beta-cells. Homozygous mutants died before birth for yet undetermined reasons. Heterozygous mice appeared healthy at birth and showed normal growth and fertility. When challenged intraperitoneally, the animals had glucose intolerance associated with reduced glucose-stimulated insulin secretion. Moreover, these heterozygous mice presented a mild increase in fasting and random-fed circulating insulin levels. In accordance, islets isolated from these animals exhibited higher insulin secretion in low glucose conditions and markedly decreased glucose-stimulated insulin secretion. Their pancreata presented normal microscopic architecture and insulin content up to 16 weeks of study. Altered insulin secretion was associated with peripheral insulin resistance in whole animals. It can be concluded that COUP-TFII is a new, important regulator of glucose homeostasis and insulin sensitivity.

Infliximab and Dexamethasone Attenuate the Ductular Reaction in Mice.

Chronic hepatic injury is accompanied by a ductular response that is strongly correlated with disease severity and progression of fibrosis. To investigate whether anti-inflammatory drugs can modulate the ductular response, we treated mice suffering from a steatotic or cholestatic injury with anti-TNF-α antibodies (Infliximab) or glucocorticoids (Dexamethasone). We discovered that Dexamethasone and Infliximab can both modulate the adaptive remodeling of the biliary architecture that occurs upon liver injury and limit extracellular matrix deposition. Infliximab treatment, at least in these steatotic and cholestatic mouse models, is the safer approach since it does not increase liver injury, allows inflammation to take place but inhibits efficiently the ductular response and extracellular matrix deposition. Infliximab-based therapy could, thus, still be of importance in multiple chronic liver disorders that display a ductular response such as alcoholic liver disease or sclerosing cholangitis.

Glucose regulates rat beta cell number through age-dependent effects on beta cell survival and proliferation.

BACKGROUND: Glucose effects on beta cell survival and DNA-synthesis suggest a role as regulator of beta cell mass but data on beta cell numbers are lacking. We examined outcome of these influences on the number of beta cells isolated at different growth stages in their population. METHODS: Beta cells from neonatal, young-adult and old rats were cultured serum-free for 15 days. Their number was counted by automated whole-well imaging distinguishing influences on cell survival and on proliferative activity. RESULTS: Elevated glucose (10-20 versus 5 mmol/l) increased the number of living beta cells from 8-week rats to 30%, following a time- and concentration-dependent recruitment of quiescent cells into DNA-synthesis; a glucokinase-activator lowered the threshold but did not raise total numbers of glucose-recruitable cells. No glucose-induced increase occurred in beta cells from 40-week rats. Neonatal beta cells doubled in number at 5 mmol/l involving a larger activated fraction that did not increase at higher concentrations; however, their higher susceptibility to glucose toxicity at 20 mmol/l resulted in 20% lower living cell numbers than at start. None of the age groups exhibited a repetitively proliferating subpopulation. CONCLUSIONS: Chronically elevated glucose levels increased the number of beta cells from young-adult but not from old rats; they interfered with expansion of neonatal beta cells and reduced their number. These effects are attributed to age-dependent differences in basal and glucose-induced proliferative activity and in cellular susceptibility to glucose toxicity. They also reflect age-dependent variations in the functional heterogeneity of the rat beta cell population.

Omentum is better site than kidney capsule for growth, differentiation, and vascularization of immature porcine ß-cell implants in immunodeficient rats.

BACKGROUND: Rapid revascularization of islet cell implants is important for engraftment and subsequent survival and function. Development of an adequate vascular network is expected to allow adaptive growth of the ß-cell mass. The present study compares omentum and kidney capsule as sites for growth and differentiation of immature ß-cell grafts. METHODS: Perinatal porcine islet cell grafts were implanted in omentum or under kidney capsule of nondiabetic nude rats. Implants were compared over 10 weeks for their respective growth, cellular composition, number and size of ß cells, their proliferative activity, and implant blood vessel density. RESULTS: In both sites, the ß-cell volume increased fourfold between weeks 1 and 10 reflecting a rise in ß-cell number. In the omental implants, however, the cellular insulin reserves and the percent of proliferating cells were twofold higher than in kidney implants. In parallel, the blood vessel density in omental implants increased twofold, reaching a density comparable with islets in adult pig pancreas. A positive correlation was found between the percent bromodeoxyuridine-positive ß cells and the vessel density. CONCLUSIONS: Growth of the ß-cell volume proceeds similarly in the omentum and under the kidney capsule. However, the omentum leads to higher insulin reserves and an increased pool of proliferating cells, which might be related to a more extended vascular network. Our observations support the omentum as an alternative site for immature porcine islet cells, with beneficial effects on proliferation and implant revascularization.

Clusters of conserved beta cell marker genes for assessment of beta cell phenotype.

BACKGROUND AND METHODOLOGY: The aim of this study was to establish a gene expression blueprint of pancreatic beta cells conserved from rodents to humans and to evaluate its applicability to assess shifts in the beta cell differentiated state. Genome-wide mRNA expression profiles of isolated beta cells were compared to those of a large panel of other tissue and cell types, and transcripts with beta cell-abundant and -selective expression were identified. Iteration of this analysis in mouse, rat and human tissues generated a panel of conserved beta cell biomarkers. This panel was then used to compare isolated versus laser capture microdissected beta cells, monitor adaptations of the beta cell phenotype to fasting, and retrieve possible conserved transcriptional regulators. PRINCIPAL FINDINGS: A panel of 332 conserved beta cell biomarker genes was found to discriminate both isolated and laser capture microdissected beta cells from all other examined cell types. Of all conserved beta cell-markers, 15% were strongly beta cell-selective and functionally associated to hormone processing, 15% were shared with neuronal cells and associated to regulated synaptic vesicle transport and 30% with immune plus gut mucosal tissues reflecting active protein synthesis. Fasting specifically down-regulated the latter cluster, but preserved the neuronal and strongly beta cell-selective traits, indicating preserved differentiated state. Analysis of consensus binding site enrichment indicated major roles of CREB/ATF and various nutrient- or redox-regulated transcription factors in maintenance of differentiated beta cell phenotype. CONCLUSIONS: Conserved beta cell marker genes contain major gene clusters defined by their beta cell selectivity or by their additional abundance in either neural cells or in immune plus gut mucosal cells. This panel can be used as a template to identify changes in the differentiated state of beta cells.

Functional interactions between pancreatic beta cells and (pre)adipocytes.

Type 2 diabetes is causally related to obesity and characterized by dysfunctional pancreatic beta cells. It is so far unclear whether direct interactions exist between adipocytes and beta cells and possibly raise any pathogenic relevance. In this study, we examined whether 9-day co-cultured 3T3-F442A (pre)adipocytes and primary rat pancreatic beta cells exert an influence on each other's function. In the presence of beta cells, 3T3-F442A cells became lipid-storing cells expressing markers of differentiated adipocytes and releasing adiponectin. This effect was attributed to the medium insulin levels (around 0.1 µM) and was associated with an elevated glucose consumption by the 3T3-F442A cells. The subsequent decrease in medium glucose concentration reduced the rate of insulin release by beta cells cultured at 10 mM glucose, and thus suppressed their degranulation during culture. These changes in beta cell function did not occur at 20 mM glucose and were reversible upon removal of the 3T3-F422A cells. They could not be reproduced by 3T3-F422A-conditioned medium containing varying adiponectin concentrations. These data indicate that insulin secreted by beta cells is sufficient to induce differentiation of preadipocytes without addition of exogenous adipogenic factors. Over 9 days culture, (pre)adipocytes did not directly and irreversibly affect beta cell functions.

Protein markers for insulin-producing beta cells with higher glucose sensitivity.

BACKGROUND AND METHODOLOGY: Pancreatic beta cells show intercellular differences in their metabolic glucose sensitivity and associated activation of insulin production. To identify protein markers for these variations in functional glucose sensitivity, rat beta cell subpopulations were flow-sorted for their level of glucose-induced NAD(P)H and their proteomes were quantified by label-free data independent alternate scanning LC-MS. Beta cell-selective proteins were also identified through comparison with rat brain and liver tissue and with purified islet alpha cells, after geometrical normalization using 6 stably expressed reference proteins. PRINCIPAL FINDINGS: All tissues combined, 943 proteins were reliably quantified. In beta cells, 93 out of 467 quantifiable proteins were uniquely detected in this cell type; several other proteins presented a high molar abundance in beta cells. The proteome of the beta cell subpopulation with high metabolic and biosynthetic responsiveness to 7.5 mM glucose was characterized by (i) an on average 50% higher expression of protein biosynthesis regulators such as 40S and 60S ribosomal constituents, NADPH-dependent protein folding factors and translation elongation factors; (ii) 50% higher levels of enzymes involved in glycolysis and in the cytosolic arm of the malate/aspartate-NADH-shuttle. No differences were noticed in mitochondrial enzymes of the Krebs cycle, beta-oxidation or respiratory chain. CONCLUSIONS: Quantification of subtle variations in the proteome using alternate scanning LC-MS shows that beta cell metabolic glucose responsiveness is mostly associated with higher levels of glycolytic but not of mitochondrial enzymes.

Susceptibility of pancreatic beta cells to fatty acids is regulated by LXR/PPARalpha-dependent stearoyl-coenzyme A desaturase.

Chronically elevated levels of fatty acids-FA can cause beta cell death in vitro. Beta cells vary in their individual susceptibility to FA-toxicity. Rat beta cells were previously shown to better resist FA-toxicity in conditions that increased triglyceride formation or mitochondrial and peroxisomal FA-oxidation, possibly reducing cytoplasmic levels of toxic FA-moieties. We now show that stearoyl-CoA desaturase-SCD is involved in this cytoprotective mechanism through its ability to transfer saturated FA into monounsaturated FA that are incorporated in lipids. In purified beta cells, SCD expression was induced by LXR- and PPARalpha-agonists, which were found to protect rat, mouse and human beta cells against palmitate toxicity. When their SCD was inhibited or silenced, the agonist-induced protection was also suppressed. A correlation between beta cell-SCD expression and susceptibility to palmitate was also found in beta cell preparations isolated from different rodent models. In mice with LXR-deletion (LXRbeta(-/-) and LXRalphabeta(-/-)), beta cells presented a reduced SCD-expression as well as an increased susceptibility to palmitate-toxicity, which could not be counteracted by LXR or PPARalpha agonists. In Zucker fatty rats and in rats treated with the LXR-agonist TO1317, beta cells show an increased SCD-expression and lower palmitate-toxicity. In the normal rat beta cell population, the subpopulation with lower metabolic responsiveness to glucose exhibits a lower SCD1 expression and a higher susceptibility to palmitate toxicity. These data demonstrate that the beta cell susceptibility to saturated fatty acids can be reduced by stearoyl-coA desaturase, which upon stimulation by LXR and PPARalpha agonists favors their desaturation and subsequent incorporation in neutral lipids.

Specificity in beta cell expression of L-3-hydroxyacyl-CoA dehydrogenase, short chain, and potential role in down-regulating insulin release.

A loss-of-function mutation of the mitochondrial beta-oxidation enzyme l-3-hydroxyacyl-CoA dehydrogenase, short chain (HADHSC), has been associated with hyperinsulinemic hypoglycemia in man. It is still unclear whether loss of glucose homeostasis in these patients (partly) results from a dysregulation of beta cells. This study examines HADHSC expression in purified rat beta cells and investigates whether its selective suppression elevates insulin release. Beta cells expressed the highest levels of HADHSC mRNA and protein of all examined tissues, including those with high rates of mitochondrial beta-oxidation. On the other hand, beta cells expressed relatively low levels of other beta-oxidation enzymes (acyl-CoA dehydrogenase short, medium, and long chain and acetyl-coenzyme A acyltransferase 2). HADHSC expression was sequence-specifically silenced by RNA interference, and the effects were examined on glucose-stimulated insulin secretion following 48-72 h of suppression. In both rat beta cells and in the beta cell line INS1 832-13, HADHSC silencing resulted in elevated insulin release at low and at high glucose concentrations, which appeared not to be caused by increased rates of glucose metabolism or an inhibition in fatty acid oxidation. These data indicate that the normal beta cell phenotype is characterized by a high expression of HADHSC and a low expression of other beta-oxidation enzymes. Down-regulation of HADHSC causes an elevated secretory activity suggesting that this enzyme protects against inappropriately high insulin levels and hypoglycemia.

Plasticity of the beta cell insulin secretory competence: preparing the pancreatic beta cell for the next meal.

It is well established that the acute rise in plasma glucose and in the incretin hormones glucose-dependent insulinotropic peptide (GIP) and glucagon-like peptide-1 (7-36) amide (GLP-1), as occurs during a meal, is of pivotal importance in regulating the minute-to-minute output of insulin from pancreatic beta cells. In addition to this well studied acute effect, both glucose and incretin hormones have been recently observed to determine the future secretory responsiveness of the cells. Such plasticity of the insulin secretory competence would imply that glucose and incretins not only act during the present meal, but also help to prepare the beta cells to function during the subsequent meal. Evidence supporting this hypothesis is growing as a result of physiological studies of cultured beta cells (either primary cells or beta cell lines), as well as from an increasing number of large-scale gene expression studies, exploring transcriptional and post-transcriptional events in genes regulated by glucose and incretins. On the basis of this hypothesis, one can speculate that genetic or environmental disturbances of plasticity of the insulin secretory competence is one aspect of beta cell dysfunction that can contribute to the aetiology of type 2 diabetes.

Effect of HMG-CoA reductase inhibitors on proliferation and protein synthesis by rat hepatic stellate cells.

BACKGROUND/AIMS: 3-Hydroxy-3-methylglutaryl coenzyme A reductase inhibitors called statins, have besides their cholesterol-lowering function, therapeutic value in conditions such as neo-angiogenesis and atherosclerosis. We investigated the effect of two statins on the proliferation rate and protein steady state levels of hepatic stellate cells (HSC). METHODS: Cellular DNA synthesis under the influence of statins and/or platelet derived growth factor (PDGF) and mevalonate was evaluated by measuring BrdU incorporation. Synthesis of collagens type I, III, IV and fibronectin was quantified by ELISA. Additionally, we examined the influence of simvastatin on isoprenylation of Ras and RhoA proteins. RESULTS: Lovastatin and simvastatin induced a dose-dependent inhibition of the proliferation rate of HSC. Subsequent addition of PDGF and/or mevalonate, after long-term exposure of simvastatin to HSC, did not reverse simvastatins' antiproliferative effect. Lovastatin and simvastatin reduced the protein steady state level of collagens type I (-40%), III (-45%) and IV (-27%). Membrane bound Ras steady state levels decreased under the influence of simvastatin. Membrane bound RhoA remained unaltered, whereas, cytosolic RhoA protein level was strongly reduced. CONCLUSIONS: Our data showed that lovastatin and simvastatin inhibited HSC proliferation and collagen steady state levels by mechanisms independent of their lipid reducing activities.

Differential modulation of rat hepatic stellate phenotype by natural and synthetic retinoids.

Activation of hepatic stellate cells (HSC) is a central event in the pathogenesis of liver fibrosis during chronic liver injury. We examined the expression of retinoic acid (RAR) and retinoid X receptors (RXR) during HSC activation and evaluated the influence of natural and synthetic retinoic acids (RA) on the phenotype of culture-activated HSC. The expression of the major RAR/RXR subtypes and isoforms was analyzed by Northern hybridization. Presence of functional receptor proteins was established by gel shift analysis. Retinoic acids, RAR, and RXR selective agonists and an RAR antagonist were used to evaluate the effects of retinoid signalling on matrix synthesis by Northern blotting and immunoprecipitation, and on cell proliferation by BrdU incorporation. The 9-cisRA and synthetic RXR agonists reduced HSC proliferation and synthesis of collagen I and fibronectin. All-trans RA and RAR agonists both reduced the synthesis of collagen I, collagen III, and fibronectin, but showed a different effect on cell proliferation. Synthetic RAR agonists did not affect HSC proliferation, indicating that ATRA inhibits cell growth independent of its interaction with RARs. In contrast, RAR specific antagonists enhance HSC proliferation and demonstrate that RARs control proliferation in a negative way. In conclusion, natural RAs and synthetic RAR or RXR specific ligands exert differential effects on activated HSC. Our observations may explain prior divergent results obtained following retinoid administration to cultured stellate cells or to animals subjected to fibrogenic stimuli.

Trichostatin A, a histone deacetylase inhibitor, suppresses collagen synthesis and prevents TGF-beta(1)-induced fibrogenesis in skin fibroblasts.

Excessive production of collagens by alpha-smooth muscle actin (alpha-SMA)-positive myofibroblasts leads to fibrotic skin diseases, such as hypertrophic scarring. This process is characterized by an imbalance between extracellular matrix (ECM) synthesis and degradation, while transforming growth factor beta (TGF-beta(1)), known to be a key mediator of fibrogenesis, is up-regulated. In this study we have investigated the possible antifibrogenic effect of Trichostatin A (TSA), a histone deacetylase inhibitor, on rat skin fibroblasts in culture. mRNA steady-state levels and de novo protein synthesis of procollagen types I and III and alpha-SMA were inhibited when skin fibroblasts were treated with 100 nM TSA with or without TGF-beta(1). While the transcription rate of the procollagen alpha1(I) gene was increased following TSA or TGF-beta(1) treatment, TSA abrogated the stimulatory effect of TGF-beta(1) on procollagen alpha1(I) transcription when both compounds were added simultaneously. The reduction of procollagen alpha1(I) and alpha1(III) mRNA steady-state levels by TSA did not require de novo protein synthesis, while the effect of TSA on alpha-SMA mRNA steady-state levels was cycloheximide-sensitive. Interestingly, TSA affected TGF-beta(1) and its downstream mediators, i.e., the Smad family proteins. TSA strongly induced in a biphasic way the expression of 5'TG3' interacting factor (TGIF), a known endogenous corepressor molecule of the TGF-beta(1) signaling pathway. Addition of exogenous TGF-beta(1) did not interfere with the effect of TSA on the TGIF mRNA level. Our study shows that inhibition of histone deacetylases by TSA reduces expression of fibrosis-related genes in skin fibroblasts and this coincides by alterations in the TGF-beta(1) signaling pathway.

Actin filament formation, reorganization and migration are impaired in hepatic stellate cells under influence of trichostatin A, a histone deacetylase inhibitor.

BACKGROUND/AIMS: Previously, trichostatin A (TSA), a histone deacetylase inhibitor, has been shown to exhibit strong antifibrotic characteristics in hepatic stellate cells (HSC), which are known to play a central role in chronic liver diseases. TSA retained a more quiescent phenotype in spite of culture conditions that favor transdifferentiation into activated HSC. METHODS: To identify TSA-sensitive genes, differential mRNA display, Northern and Western blot analysis were used and genes were functionally validated by using contraction and motility assays. RESULTS: TSA prevented new actin filament formation by down-regulation of two nucleating proteins, actin related protein 2 (Arp2) and Arp3, and by up-regulation of adducin like protein 70 (ADDL70) and gelsolin, two capping proteins. RhoA, a key mediator in the development of the actin cytoskeleton, decreased following TSA exposure. Expression of proteins of Class III intermediate filaments was affected by TSA. Furthermore, F-actin and G-actin were expressed heterogeneously under influence of TSA. Functionally, TSA treatment abrogated migration of quiescent HSC, while migration was reduced in transitional HSC. The endothelin-1-induced contractility properties of HSC was not affected by TSA. CONCLUSIONS: These data indicate that TSA affects the development of the actin cytoskeleton in quiescent HSC and thereby abrogates the process of HSC transdifferentiation.

Peroxisome proliferator-activated receptor-beta signaling contributes to enhanced proliferation of hepatic stellate cells.

BACKGROUND & AIMS: The peroxisome proliferator-activated nuclear receptors (PPAR-alpha, PPAR-beta, and PPAR-gamma), which modulate the expression of genes involved in energy homeostasis, cell cycle, and immune function, may play a role in hepatic stellate cell activation. Previous studies focused on the decreased expression of PPAR-gamma in hepatic stellate cell activation but did not investigate the expression and role of the PPAR-alpha and -beta isotypes. The aim of this study was to evaluate the expression of the different PPARs during hepatic stellate cell activation in vitro and in situ and to analyze possible factors that might contribute to their expression. In a second part of the study, the effect of a PPAR-beta agonist on acute liver injury was evaluated. METHODS: The effects of PPAR isotype-specific ligands on hepatic stellate cell transition were evaluated by bromodeoxyuridine incorporation, gel shifts, immunoprecipitation, and use of antisense PPAR-beta RNA-expressing adenoviruses. Tumor necrosis factor alpha-induced PPAR-beta phosphorylation and expression was evaluated by metabolic labeling and by using specific P38 inhibitors. RESULTS: Hepatic stellate cells constitutively express high levels of PPAR-beta, which become further induced during culture activation and in vivo fibrogenesis. No significant expression of PPAR-alpha or -gamma was found. Stimulation of the P38 mitogen-activated protein kinase pathway modulated the expression of PPAR-beta. Transcriptional activation of PPAR-beta by L165041 enhanced hepatic stellate cell proliferation. Treatment of rats with a single bolus of CCl(4) in combination with L165041 further enhanced the expression of fibrotic markers. CONCLUSIONS: PPAR-beta is an important signal-transducing factor contributing to hepatic stellate cell proliferation during acute and chronic liver inflammation.

PPARbeta regulates vitamin A metabolism-related gene expression in hepatic stellate cells undergoing activation.

Activation of cultured hepatic stellate cells correlated with an enhanced expression of proteins involved in uptake and storage of fatty acids (FA translocase CD36, Acyl-CoA synthetase 2) and retinol (cellular retinol binding protein type I, CRBP-I; lecithin:retinol acyltransferases, LRAT). The increased expression of CRBP-I and LRAT during hepatic stellate cells activation, both involved in retinol esterification, was in contrast with the simultaneous depletion of their typical lipid-vitamin A (vitA) reserves. Since hepatic stellate cells express high levels of peroxisome proliferator activated receptor beta (PPARbeta), which become further induced during transition into the activated phenotype, we investigated the potential role of PPARbeta in the regulation of these changes. Administration of L165041, a PPARbeta-specific agonist, further induced the expression of CD36, B-FABP, CRBP-I, and LRAT, whereas their expression was inhibited by antisense PPARbeta mRNA. PPARbeta-RXR dimers bound to CRBP-I promoter sequences. Our observations suggest that PPARbeta regulates the expression of these genes, and thus could play an important role in vitA storage. In vivo, we observed a striking association between the enhanced expression of PPARbeta and CRBP-I in activated myofibroblast-like hepatic stellate cells and the manifestation of vitA autofluorescent droplets in the fibrotic septa after injury with CCl4 or CCl4 in combination with retinol.