[Main issues and contributions of the evaluation of health care networks]. / Les problématiques et les apports de l'evaluation d'un réseau de santé.

Health care networks correspond to a form of horizontal and flexible organisation, which consists in dealing with complex problems of health and long-term care. Networks rest entirely on the mechanisms of coordination between the various actors of care, whose keystone is the medical information flow. The challenge of the health networks evaluation is to prove the added value of a network organization, instead of a conventional disease-centred health care. The evaluation of health care networks is legally required but not sustained by specialists. Evaluation must ensure the existence of a real management; measure the interest of health care professionals and the impact of the network on professional practices. Moreover, evaluation accompanies the development of the network and offers the advisability to the decision maker of obtaining a tool for control of management, i.e. an information tool and a decision-making help, with the aim of a continuous improvement of the quality of care. However the absence of formation of the medical coordinators to management, makes networks management problematic. Thus, health care networks cannot make the saving in a good administrative and management base. In the same way health care networks must obtain an information system adapted to their type of coordination, allowing the evaluation required by the financier.

Endotoxin suppresses the oltipraz-mediated induction of major hepatic glutathione transferases and cytochromes P450 in the rat.

The effect of Escherichia coli lipopolysaccharide (LPS), a classic inducer of the acute-phase response, has been analyzed on both constitutive and oltipraz (a chemoprotective agent)-inducible messenger RNAs (mRNAs) and enzyme activities of major cytochromes P450 (CYPs) and glutathione transferases (rGSTs) in rat liver. At the dose administered (1 mg/kg) and the time studied (6 and 24 hours), endotoxin had no effect on the expression of either CYPs and GSTs with the exception of CYP1A2, which was reduced at both mRNA and activity levels. A strong increase of rGSTA1/2, rGSTM1, rGSTP1, CYP1A2, CYP2B1/2, and CYP2E1 was observed after 3 days of treatment with oltipraz (0.075%, wt/wt). Oltipraz induction of these enzymes (with the exception of CYP2E1) was found to be suppressed at both mRNA, protein, and activity levels during the acute-phase response to endotoxin. Moreover, it is shown that oltipraz induction of CYP1A2 and CYP2B1/2 and its suppression by E. coli LPS occurred at a transcriptional level. These data support the idea that the chemoprotective effect of oltipraz is altered in the course of inflammation and that adaptation in chemoprotective strategies should be considered in certain physiopathologic situations.

Oltipraz stimulates the transcription of the manganese superoxide dismutase gene in rat hepatocytes.

Oltipraz (4-methyl-5-(2-pyrazinyl)-1,2-dithiole-3-thione) (OPZ) is recognized as a potent chemoprotective agent against chemical-induced carcinogenesis in several animal models and is thought to act mainly by inducing phase II conjugating together with inhibiting phase I detoxication enzymes. The present study was undertaken to determine whether oltipraz can also influence expression of genes encoding antioxidant enzymes. In rat hepatocytes in primary culture, this compound was found to selectively induce the transcription of the manganese superoxide dismutase (Mn-SOD) gene while it had no effect on copper/zinc-SOD and glutathione peroxidase genes. Oltipraz increased Mn-SOD gene expression in a time- and dose-dependent manner by 2- to 3-fold and enhanced the binding activity of the nuclear factor kappa B within 30 min. Moreover, the increase in Mn-SOD gene transcription was associated with a 2- to 3-fold increase of free malondialdehyde and conjugated dienes, two markers of lipid peroxidation, an index of oxidative stress. These results suggest that in rat hepatocytes, oltipraz induced a production of reactive oxygen species that probably acted as second messengers in order to trigger the transcription of many genes. Such a mechanism of action of OPZ and other dithiolethiones would account for the broad spectrum of action of these anticarcinogenic compounds.

Transcriptional and posttranscriptional mechanisms of glucocorticoid-mediated repression of phosphoenolpyruvate carboxykinase gene expression in adipocytes.

Glucocorticoids exert pleiotropic effects, among which negative regulation of transcription has been recognized as of crucial importance. While glucocorticoids induce phosphoenolpyruvate carboxykinase (PEPCK) gene expression in liver cells, it represses gene activity in adipose cells. We used the 3T3-F442A adipocytes to analyze the underlying mechanisms in these cells, the synthetic glucocorticoid dexamethasone exerts a dominant repression either on basal or on beta-agonist stimulation of PEPCK gene expression. To determine whether glucocorticoid action required protein synthesis, we employed cycloheximide, anisomycin, and puromycin, three different translation inhibitors. None of these affected induction by isoprenaline or repression by dexamethasone of isoprenaline stimulation. In contrast, dexamethasone inhibitory action on basal PEPCK mRNA was totally prevented by the three translation inhibitors. Time courses of glucocorticoid action on basal and on induction by beta-agonist were similar. Half-maximal effect of dexamethasone on isoprenaline-induced PEPCK mRNA was obtained at about 10 nM, a tenfold higher concentration than that observed for the reduction of basal mRNA. Using the transcription inhibitor DRB, we showed that dexamethasone did not alter mRNA half-life, while isoprenaline strongly stabilized mRNA. In a 3T3-F442A stable transfectant bearing -2,100 base pairs of the PEPCK promoter fused to the chloramphenicol acetyltransferase (CAT) gene, isoprenaline stimulated CAT activity, whereas dexamethasone reduced basal and isoprenaline-induced CAT expression. Hence, beta-agonists exert both transcriptional and posttranscriptional regulation, while glucocorticoid action is purely transcriptional. However, mechanisms of glucocorticoid repression of basal and of beta-agonist stimulation appear different.

Modulation of glutathione S-transferase subunits A2, M1, and P1 expression by interleukin-1beta in rat hepatocytes in primary culture.

The influence of various cytokines on the expression of glutathione S-transferases (GSTs) was investigated in rat hepatocytes in primary culture. Only treatment of hepatocytes with interleukin-1beta (IL-1) was effective, resulting in a marked decrease in GSTs. Steady-state mRNA levels of rGSTA2 and M1 were strongly down-regulated by IL-1 in a dose-dependent manner after a 24-h exposure while rGSTP1 mRNA level was increased by a 48-h treatment. Similar effects of IL-1 were observed at the protein level. The response to IL-1 appeared to be specific for each subunit within GST gene families. In addition, IL-1 strongly suppressed the induction of rGSTA2 by 3-methylcholanthrene, oltipraz (a synthetic derivative of 1, 2-dithiole-3-thione), and phenobarbital and that of rGSTM1 by oltipraz and phenobarbital, whereas it was ineffective on rGSTP1 induction by these compounds. Using in vitro nuclear run-on transcription assay and Northern blot analysis of alpha-amanitin-treated cells, IL-1-mediated rGSTM1 mRNA decrease was found to result from mRNA destabilization. These results provide the first demonstration that IL-1 regulates some major GST subunits in hepatocytes by a post-transcriptional mechanism.

Dexamethasone differently modulates TNF-alpha- and IL-1beta-induced transcription of the hepatic Mn-superoxide dismutase gene.

The effects of cytokines, tumor necrosis factor-alpha (TNF-alpha), interleukin-1beta (IL-1beta), and the synthetic glucocorticoid dexamethasone on the gene expression of antioxidant enzymes have been investigated in rat hepatocytes in primary culture. First, we observed that the hepatocyte culture process induced a strong but transient induction of manganese superoxide dismutase (Mn-SOD) gene expression, whereas copper-zinc superoxide dismutase, glutathione peroxidase and catalase genes were down-regulated. IL-1beta and TNF-alpha both stimulated specifically Mn-SOD gene expression in a time-dependent manner. TNF-alpha rapidly induced Mn-SOD gene expression while IL-1beta was a strong but slow inducer of this gene. Both cytokines acted at the transcriptional level as shown by nuclear run on assays. Dexamethasone prevented the TNF-alpha- but not the IL-1beta induced up-regulation of Mn-SOD gene transcription by a mechanism likely to involve the glucocorticoid receptor. Moreover this glucocorticoid did not suppress the TNF-alpha-induced increase of NF-kappaB binding activity. These results suggest that IL-1beta and TNF-alpha regulate Mn-SOD gene transcription by different pathways.

Fatty acids and fibrates are potent inducers of transcription of the phosphenolpyruvate carboxykinase gene in adipocytes.

Cytosolic phosphoenolpyruvate carboxykinase (PEPCK) plays a critical role in adipose tissue glyceroneogenesis. We have previously shown that transcription of the PEPCK gene was stimulated by isoprenaline and retinoic acid in 3T3-F442A adipocytes. We also showed that oleate increased PEPCK mRNA. Here, we analysed the effect that fatty acids of various chain lengths and unsaturation degrees exerted on PEPCK gene expression in 3T3-F442A adipocytes. When maintained in serum-free, glucose-free medium, differentiated cells responded to unsaturated long-chain fatty acids by a large increase in PEPCK mRNA whereas saturated fatty acids were inefficient. A maximum fivefold stimulation by oleate was attained at 4 h of treatment with 1 mM fatty acid bound to albumin in a 6:1 ratio. The poly-unsaturated very long-chain fatty acid all-cis-4,7,10,13,16,19-docosahexaenoic acid (C22:6) was even more potent and produced a tenfold increase. The expression of the genes encoding glycerol-3-phosphate dehydrogenase, hormone-sensitive lipase or actin remained unaffected by oleate exposure. A 4-h treatment by the hypolipidemic drug clofibrate, 0.5-2 mM, also produced a large (3-9-fold) increase in PEPCK mRNA. When used at non-saturating concentrations, oleate and clofibrate acted in an additive manner. At maximally effective concentrations, additivity was lost, suggesting that fatty acids and fibrates might act through similar mechanisms. Nuclear transcription experiments showed that oleate and clofibrate stimulated the transcription rate of the gene. 3T3-F442A cells were stably transfected with a plasmid containing the base pairs -2100 to +69 of the PEPCK gene promoter fused to the chloramphenicol acetyltransferase gene. These differentiated stable transfectants responded to oleate and clofibrate by a specific increase in chloramphenicol acetyltransferase activity. Adipocytes express various isoforms of peroxisome-proliferator-activated receptors that can be activated by fibrates and fatty acids. Potential recognition sequences for peroxisome-proliferator-activated receptors are present in the -2100 to +69 fragment of the PEPCK gene promoter. Thus, this gene represents an ideal molecular target for understanding the complex transcriptional control exerted by fatty acids and peroxisome proliferators.

Expression of the phosphoenolpyruvate carboxykinase gene in 3T3-F442A adipose cells: opposite effects of dexamethasone and isoprenaline on transcription.

The enzyme phosphoenolpyruvate carboxykinase (PEPCK) plays a key role in gluconeogenesis in liver and in glyceroneogenesis in adipose tissue. These processes, and PEPCK, are regulated by a number of hormones, some of which have different effects on the enzyme in liver and adipose tissue. To explore this phenomenon, PEPCK gene expression was studied in 3T3-F442A adipocytes maintained in a serum-free medium. The beta-adrenergic agonist isoprenaline (isoproterenol) and a cyclic AMP analogue (8-CPT-cAMP) increased PEPCK mRNA. A maximal 3-fold induction occurred in 2 h. Dexamethasone decreased PEPCK mRNA by 80% in 4 h. Dexamethasone also counteracted the inductive effects of isoprenaline and 8-CPT-cAMP. Run-on transcription experiments showed that the isoprenaline and dexamethasone actions were, at least in part, exerted at the level of PEPCK gene transcription. These effects were further analysed by using transient and stable transfection of adipocytes with a plasmid containing bp -2100 to 69 of the PEPCK gene promoter fused to the chloramphenicol acetyltransferase (CAT) gene. In such cells isoprenaline stimulated CAT expression, an effect that was prevented if the cells were also exposed to dexamethasone.

Expression of the phosphoenolpyruvate carboxykinase gene in 3T3-F442A adipose cells: effects of retinoic acid and differentiation.

3T3-F442A adipocytes express the gene encoding cytosolic phosphoenolpyruvate carboxykinase (GTP) (PEPCK). Retinoic acid (RA) caused a 5-fold induction of PEPCK mRNA within 6 h in these cells with a half-maximal effective concentration of approximately 75 microM. This effect was independent of cycloheximide and inhibited by actinomycin D. In vitro run-on experiments using isolated nuclei confirmed that the RA-induced increase was mainly due to an increased rate of transcription of the gene. Stable transfectants bearing either the region of the PEPCK promoter from -2100 to +69 fused to the chloramphenicol acetyltransferase (CAT) gene (pPL1-CAT) or -600 to +69 fused to CAT (pPL9-CAT) were used to study PEPCK gene regulation during differentiation. The same transfected cells were used to analyse the RA effect. Preadipocytes containing pPL1-CAT expressed a much lower level of CAT activity than did adipocytes. pPL9-CAT was not expressed in either preadipocytes or adipocytes. RA induced the expression of CAT activity in preadipocytes and adipocytes transfected with pPL1-CAT, but had no effect in cells transfected with pPL9-CAT. These results suggest that one or more DNA sequences located between -2100 and -600 bp of the PEPCK promoter is required for adipocyte-specific expression of this gene. RA action is independent of the state of differentiation and appears to require different elements in fat cells from those required in liver.

Stimulation of phosphoenolpyruvate carboxykinase gene expression by fatty acids.

We used the 3T3-F442A adipocytes and the FAO hepatoma cells to analyze the effect of oleate on phosphoenolpyruvate carboxykinase (PEPCK) gene expression. In serum-deprived, glucose-free medium, 1 mM oleate, bound to albumin in a 6:1 ratio, specifically stimulated PEPCK mRNA. In 3T3-F442A adipocytes, the maximum 5-fold increase occurred in 4 hours then rapidly declined to reach the basal level 20 hours later. This increase was cycloheximide-independent and actinomycin D-dependent, suggesting a direct, transcriptional effect of oleate. FAO cells also responded to oleate with a transient induction of PEPCK mRNA, although the extent of stimulation was lower. Thus, the PEPCK gene provides a useful molecular tool for studying the mechanisms by which fatty acids stimulate gene expression.

Forskolin induces the reorganization of extracellular matrix fibronectin and cytoarchitecture in 3T3-F442A adipocytes: its effect on fibronectin gene expression.

We studied the effect of forskolin on fibronectin and actin gene expression in 3T3-F442A adipogenic cell line. The structural organizations of extracellular matrix fibronectin and actin cytoskeleton were investigated in parallel. Immunofluorescence experiments showed that preadipocytes treated for 48 h with 10 microM forskolin exhibited an intensified network of both actin and fibronectin when compared to control. A similar picture was obtained with adipocytes given long-term exposure to forskolin. As determined by Western analysis, fibronectin protein levels were increased by 50-75% over control, both in preadipocytes and adipocytes. A parallel increase of fibronectin mRNA content was observed in forskolin-treated cells. In contrast, forskolin treatment of preadipocytes and adipocytes did not elicit any change in the steady-state level of either actin mRNA or protein. Nuclear run-on experiments showed that forskolin increased the fibronectin gene transcription rate but left that of the actin gene in adipocytes unchanged. These findings suggest the reorganization of the actin network in forskolin-treated adipocytes to be a consequence of fibronectin-enhanced biosynthesis and reorganization.

Glucocorticoids antagonize retinoic acid stimulation of PEPCK gene transcription in 3T3-F442A adipocytes.

Cytosolic phosphoenolpyruvate carboxykinase (GTP) (PEPCK) is a key glyceroneogenic enzyme in adipose tissue. The regulation of PEPCK gene expression by retinoic acid (RA) and dexamethasone (DEX) was studied in 3T3-F442A adipocytes maintained in a serum-free medium. RA induced whereas DEX reduced PEPCK mRNA steady-state level. RA stimulation was about 4-fold and DEX repression was of 80% in 4 hrs. In addition to reducing basal mRNA level, DEX was able to counteract RA induction in a dominant manner. The use of the glucocorticoid antagonist RU 38486 indicated that the DEX effect was mediated by the glucocorticoid receptor. Stable transfectants bearing the region of the PEPCK promoter from -2100 to +69 fused to the chloramphenicol acetyltransferase (CAT) gene (pPL1-CAT) were used to study PEPCK gene regulation in differentiated adipocytes. In such cells, RA stimulated CAT expression 3 to 5.5 fold. DEX had no effect on basal CAT activity whereas it inhibited the stimulation induced by RA. Thus, in adipocytes, the PEPCK gene regulatory region between -2100 and +69 bp mediates both stimulation by RA and repression by DEX of RA action.