Hexose-6-phosphate dehydrogenase modulates 11beta-hydroxysteroid dehydrogenase type 1-dependent metabolism of 7-keto- and 7beta-hydroxy-neurosteroids

BACKGROUND: The role of 11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1) in the regulation of energy metabolism and immune system by locally reactivating glucocorticoids has been extensively studied. Experiments determining initial rates of enzyme activity revealed that 11beta-HSD1 can catalyze both the reductase and the dehydrogenase reaction in cell lysates, whereas it predominantly catalyzes the reduction of cortisone to cortisol in intact cells that also express hexose-6-phosphate dehydrogenase (H6PDH), which provides cofactor NADPH. Besides its role in glucocorticoid metabolism, there is evidence that 11beta-HSD1 is involved in the metabolism of 7-keto- and 7-hydroxy-steroids; however the impact of H6PDH on this alternative function of 11beta-HSD1 has not been assessed. METHODOLOGY: We investigated the 11beta-HSD1-dependent metabolism of the neurosteroids 7-keto-, 7alpha-hydroxy- and 7beta-hydroxy-dehydroepiandrosterone (DHEA) and 7-keto- and 7beta-hydroxy-pregnenolone, respectively, in the absence or presence of H6PDH in intact cells. 3D-structural modeling was applied to study the binding of ligands in 11beta-HSD1. PRINCIPAL FINDINGS: We demonstrated that 11beta-HSD1 functions in a reversible way and efficiently catalyzed the interconversion of these 7-keto- and 7-hydroxy-neurosteroids in intact cells. In the presence of H6PDH, 11beta-HSD1 predominantly converted 7-keto-DHEA and 7-ketopregnenolone into their corresponding 7beta-hydroxy metabolites, indicating a role for H6PDH and 11beta-HSD1 in the local generation of 7beta-hydroxy-neurosteroids. 3D-structural modeling offered an explanation for the preferred formation of 7beta-hydroxy-neurosteroids. CONCLUSIONS: Our results from experiments determining the steady state concentrations of glucocorticoids or 7-oxygenated neurosteroids suggested that the equilibrium between cortisone and cortisol and between 7-keto- and 7-hydroxy-neurosteroids is regulated by 11beta-HSD1 and greatly depends on the coexpression with H6PDH. Thus, the impact of H6PDH on 11beta-HSD1 activity has to be considered for understanding both glucocorticoid and neurosteroid action in different tissues.

Identification of polymorphisms in the human 11beta-hydroxysteroid dehydrogenase type 2 gene promoter: functional characterization and relevance for salt sensitivity

Reduced activity of 11beta-hydroxysteroid dehydrogenase type 2 (11beta-HSD2) plays a role in essential hypertension and the sensitivity of blood pressure to dietary salt. Nonconservative mutations in the coding region are extremely rare and do not explain the variable 11beta-HSD2 activity. We focused therefore on the 5'-regulatory region and identified and characterized the first promoter polymorphisms. Transfections of variants G-209A and G-126A into SW620 cells reduced promoter activity and affinity for activators nuclear factor 1 (NF1) and Sp1. Chromatin immunoprecipitation revealed Sp1, NF1, and glucocorticoid receptor (GR) binding to the HSD11B2 promoter. Dexamethasone induced expression of mRNA and activity of HSD11B2. GR and/or NF1 overexpression increased endogenous HSD11B2 mRNA and activity. GR complexes cooperated with NF1 to activate HSD11B2, an effect diminished in the presence of the G-209A variant. When compared to salt-resistant subjects (96), salt-sensitive volunteers (54) more frequently had the G-209A variant, higher occurrence of alleles A4/A7 of polymorphic microsatellite marker, and higher urinary ratios of cortisol to cortisone metabolites. First, we conclude that the mechanism of glucocorticoid-induced HSD11B2 expression is mainly mediated by cooperation between GR and NF1 on the HSD11B2 promoter and, second, that the newly identified promoter variants reduce activity and cooperation of cognate transcription factors, resulting in diminished HSD11B2 transcription, an effect favoring salt sensitivity.

11beta-Hydroxysteroid dehydrogenase type 2 in pregnancy and preeclampsia

Cortisol availability is controlled by 11beta-hydroxysteroid dehydrogenase type 2 (11beta-HSD2), which inactivates cortisol in cortisone, unable to bind to the glucocorticoid receptor. The 11beta-HSD2 enzyme activity limits either intracellular cortisol concentrations or within the uteroplacental compartment the transfer of cortisol into the fetal circulation. Mechanisms, by which 11beta-HSD2 activity is controlled, include transcriptional control, posttranscriptional modifications of 11beta-HSD2 transcript half-life, epigenetic regulation via methylation of genomic DNA and direct inhibition of enzymatic activity. The 11beta-HSD2 expression and activity is reduced in preeclampsia and the enzyme activity correlates with factors associated with increased vasoconstriction, such as an increased angiotensin II receptor subtype 1 expression, and notably fetal growth. Numerous signals such as proinflammatory cytokines known to be present and/or elevated in preeclampsia regulate 11beta-HSD2 activity. Shallow trophoblast invasion with the resulting hypoxemia seems to critically reduce available 11beta-HSD2 activity. A positive feedback exists as activated glucocorticoid receptors do enhance 11beta-HSD2 mRNA transcription and mRNA stability. No data are currently available on pregnancy and either epigenetic or direct effects on the activity of the translated enzyme.

Sodium retention and ascites formation in a cholestatic mice model: role of aldosterone and mineralocorticoid receptor?

Renal sodium retention in experimental liver cirrhosis originates from the distal nephron sensitive to aldosterone. The aims of this study were to (1) determine the exact site of sodium retention along the aldosterone-sensitive distal nephron, and (2) to evaluate the role of aldosterone and mineralocorticoid receptor activation in this process. Liver cirrhosis was induced by bile duct ligation in either adrenal-intact or corticosteroid-clamped mice. Corticosteroid-clamp was achieved through adrenalectomy and corticosteroid supplementation with aldosterone and dexamethasone via osmotic minipumps. 24-hours renal sodium balance was evaluated in metabolic cages. Activity and expression of sodium- and potassium-dependent adenosine triphosphatase were determined in microdissected segments of nephron. Within 4-5 weeks, cirrhosis induced sodium retention in adrenal-intact mice and formation of ascites in 50% of mice. At that time, sodium- and potassium-dependent adenosine triphosphatase activity increased specifically in cortical collecting ducts. Hyperaldosteronemia was indicated by increases in urinary aldosterone excretion and in sgk1 (serum- and glucocorticoid-regulated kinase 1) mRNA expression in collecting ducts. Corticosteroid-clamp prevented induction of sgk1 but not cirrhosis-induced sodium retention, formation of ascites and stimulation of sodium- and potassium-dependent adenosine triphosphatase activity and expression (mRNA and protein) in collecting duct. These findings demonstrate that sodium retention in cirrhosis is independent of hyperaldosteronemia and of the activation of mineralocorticoid receptor. CONCLUSION: Bile duct ligation in mice induces cirrhosis which, within 4-5 weeks, leads to the induction of sodium- and potassium-dependent adenosine triphosphatase in cortical collecting ducts, to renal sodium retention and to the formation of ascites. Sodium retention, ascites formation and induction of sodium- and potassium-dependent adenosine triphosphatase are independent of the activation of mineralocorticoid receptors by either aldosterone or glucocorticoids.

Characterization of the regulation of renal Na+/H+ exchanger NHE3 by insulin

Insulin receptors are widely distributed in the kidney and affect multiple aspects of renal function. In the proximal tubule, insulin regulates volume and acid-base regulation through stimulation of the Na(+)/H(+) exchanger NHE3. This paper characterizes the signaling pathway by which insulin stimulates NHE3 in a cell culture model [opossum kidney (OK) cell]. Insulin has two distinct phases of action on NHE3. Chronic insulin (24 h) activates NHE3 through the classic phosphatidylinositol 3-kinase-serum- and glucocorticoid-dependent kinase 1 (PI3K-SGK1) pathway as insulin stimulates SGK1 phosphorylation and the insulin effect can be blocked by the PI3K inhibitor wortmannin or a dominant-negative SGK1. We showed that SGK1 transcript and protein are expressed in rat proximal tubule and OK cells. We previously showed that glucocorticoids augment the effect of insulin on NHE3 (Klisic J, Hu MC, Nief V, Reyes L, Fuster D, Moe OW, Ambuhl PM. Am J Physiol Renal Physiol 283: F532-F539, 2002). Part of this can be mediated via induction of SGK1 by glucocorticoids, and indeed the insulin effect on NHE3 can also be amplified by overexpression of SGK1. We next addressed the acute effect of insulin (1-2 h) on NHE3 by systematically examining the candidate signaling cascades and activation mechanisms of NHE3. We ruled out the PI3K-SGK1-Akt and TC10 pathways, increased surface NHE3, NHE3 phosphorylation, NHE3 association with calcineurin homologous protein 1 or megalin as mechanisms of acute activation of NHE3 by insulin. In summary, insulin stimulates NHE3 acutely via yet undefined pathways and mechanisms. The chronic effect of insulin is mediated by the classic PI3K-SGK1 route.

Chondrogenic potential of human synovial mesenchymal stem cells in alginate

OBJECTIVE: In a recent study, we demonstrated that mesenchymal stem cells (MSCs) derived from the synovial membranes of bovine shoulder joints could differentiate into chondrocytes when cultured in alginate. The purpose of the present study was to establish the conditions under which synovial MSCs derived from aging human donors can be induced to undergo chondrogenic differentiation using the same alginate system. METHODS: MSCs were obtained by digesting the knee-joint synovial membranes of osteoarthritic human donors (aged 59-76 years), and expanded in monolayer cultures. The cells were then seeded at a numerical density of 4x10(6)/ml within discs of 2% alginate, which were cultured in serum-containing or serum-free medium (the latter being supplemented with 1% insulin, transferrin, selenium (ITS). The chondrogenic differentiation capacity of the cells was tested by exposing them to the morphogens transforming growth factor-beta1 (TGF-beta1), TGF-beta2, TGF-beta3, insulin-like growth factor-1 (IGF-1), bone morphogenetic protein-2 (BMP-2) and BMP-7, as well as to the synthetic glucocorticoid dexamethasone. The relative mRNA levels of collagen types I and II, of aggrecan and of Sox9 were determined quantitatively by the real-time polymerase chain reaction (PCR). The extracellular deposition of proteoglycans was evaluated histologically after staining with Toluidine Blue, and that of type-II collagen by immunohistochemistry. RESULTS: BMP-2 induced the chondrogenic differentiation of human synovial MSCs in a dose-dependent manner. The response elicited by BMP-7 was comparable. Both of these agents were more potent than TGF-beta1. A higher level of BMP-2-induced chondrogenic differentiation was achieved in the absence than in the presence of serum. In the presence of dexamethasone, the BMP-2-induced expression of mRNAs for aggrecan and type-II collagen was suppressed; the weaker TGF-beta1-induced expression of these chondrogenic markers was not obviously affected. CONCLUSIONS: We have demonstrated that synovial MSCs derived from the knee joints of aging human donors possess chondrogenic potential. Under serum-free culturing conditions and in the absence of dexamethasone, BMP-2 and BMP-7 were the most potent inducers of this transformation process.

SFRP-4 abrogates Wnt-3a-induced beta-catenin and Akt/PKB signalling and reverses a Wnt-3a-imposed inhibition of in vitro mammary differentiation

ABSTRACT: BACKGROUND: Conserved Wnt ligands are critical for signalling during development; however, various factors modulate their activity. Among these factors are the Secreted Frizzled-Related Proteins (SFRP). We previously isolated the SFRP-4 gene from an involuting rat mammary gland and later showed that transgenic mice inappropriately expressing SFRP-4 during lactation exhibited a high level of apoptosis with reduced survival of progeny. RESULTS: In order to address the questions related to the mechanism of Wnt signalling and its inhibition by SFRP-4 which we report here, we employed partially-purified Wnt-3a in a co-culture model system. Ectopic expression of SFRP-4 was accomplished by infection with a pBabepuro construct. The co-cultures comprised Line 31E mouse mammary secretory epithelial cells and Line 30F, undifferentiated, fibroblast-like mouse mammary cells. In vitro differentiation of such co-cultures can be demonstrated by induction of the beta-casein gene in response to lactogenic hormones.We show here that treatment of cells with partially-purified Wnt-3a initiates Dvl-3, Akt/PKB and GSK-3beta hyperphosphorylation and beta-catenin activation. Furthermore, while up-regulating the cyclin D1 and connexin-43 genes and elevating transepithelial resistance of Line 31E cell monolayers, Wnt-3a treatment abrogates differentiation of co-cultures in response to the lactogenic hormones prolactin, insulin and glucocorticoid. Cells which express SFRP-4, however, are largely unaffected by Wnt-3a stimulation. Since a physical association between Wnt-3a and SFRP-4 could be demonstrated with immunoprecipitation/Western blotting experiments, this interaction, presumably owing to the Frizzled homology region typical of all SFRPs, explains the refractory response to Wnt-3a which was observed. CONCLUSION: This study demonstrates that Wnt-3a treatment activates the Wnt signalling pathway and interferes with in vitro differentiation of mammary co-cultures to beta-casein production in response to lactogenic hormones. Similarly, in another measure of differentiation, following Wnt-3a treatment mammary epithelial cells could be shown to up-regulate the cyclin D1 and connexin-43 genes while phenotypically they show increased transepithelial resistance across the cell monolayer. All these behavioural changes can be blocked in mammary epithelial cells expressing SFRP-4. Thus, our data illustrate in an in vitro model a mechanism by which SFRP-4 can modulate a differentiation response to Wnt-3a.

Tumor necrosis factor-alpha upregulates 11beta-hydroxysteroid dehydrogenase type 1 expression by CCAAT/enhancer binding protein-beta in HepG2 cells

The enzyme 11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1) catalyzes the conversion of inactive to active glucocorticoids. 11beta-HSD1 plays a crucial role in the pathogenesis of obesity and controls glucocorticoid actions in inflammation. Several studies have demonstrated that TNF-alpha increases 11beta-HSD1 mRNA and activity in various cell models. Here, we demonstrate that mRNA and activity of 11beta-HSD1 is increased in liver tissue from transgenic mice overexpressing TNF-alpha, indicating that this effect also occurs in vivo. To dissect the molecular mechanism of this increase, we investigated basal and TNF-alpha-induced transcription of the 11beta-HSD1 gene (HSD11B1) in HepG2 cells. We found that TNF-alpha acts via p38 MAPK pathway. Transient transfections with variable lengths of human HSD11B1 promoter revealed highest activity with or without TNF-alpha in the proximal promoter region (-180 to +74). Cotransfection with human CCAAT/enhancer binding protein-alpha (C/EBPalpha) and C/EBPbeta-LAP expression vectors activated the HSD11B1 promoter with the strongest effect within the same region. Gel shift and RNA interference assays revealed the involvement of mainly C/EBPalpha, but also C/EBPbeta, in basal and only of C/EBPbeta in the TNF-alpha-induced HSD11B1 expression. Chromatin immunoprecipitation assay confirmed in vivo the increased abundance of C/EBPbeta on the proximal HSD11B1 promoter upon TNF-alpha treatment. In conclusion, C/EBPalpha and C/EBPbeta control basal transcription, and TNF-alpha upregulates 11beta-HSD1, most likely by p38 MAPK-mediated increased binding of C/EBPbeta to the human HSD11B1 promoter. To our knowledge, this is the first study showing involvement of p38 MAPK in the TNF-alpha-mediated 11beta-HSD1 regulation, and that TNF-alpha stimulates enzyme activity in vivo.

Nimodipine prior to alcohol withdrawal prevents memory deficits during the abstinence phase

Effects of the dihydropyridine, nimodipine, an antagonist at L-type calcium channels, on the memory loss in rats caused by long term alcohol consumption were examined. Either a single dose of nimodipine or 2 weeks of repeated administration was given prior to withdrawal from 8 months of alcohol consumption. Memory was measured by the object recognition test and the T maze. Both nimodipine treatments prevented the memory deficits when these were measured between 1 and 2 months after alcohol withdrawal. At the end of the memory testing, 2 months after cessation of chronic alcohol consumption, glucocorticoid concentrations were increased in specific regions of rat brain without changes in plasma concentrations. Both nimodipine treatment schedules substantially reduced these rises in brain glucocorticoid. The data indicate that blockade of L-type calcium channels prior to alcohol withdrawal protects against the memory deficits caused by prolonged alcohol intake. This shows that specific drug treatments, such as nimodipine, given over the acute withdrawal phase, can prevented the neuronal changes responsible for subsequent adverse effects of long term consumption of alcohol. The results also suggest the possibility that regional brain glucocorticoid increases may be involved in the adverse effects of long term alcohol intake on memory. Such local changes in brain glucocorticoid levels would have major effects on neuronal function. The studies indicate that L-type calcium channels and brain glucocorticoid levels could form new targets for the treatment of cognitive deficits in alcoholics.

Mineralocorticoid receptor is essential for corticosteroid-induced up-regulation of L-type calcium currents in cultured neonatal cardiomyocytes

Despite the fact that mineralocorticoid receptor (MR) antagonist drugs such as spironolactone and eplerenone reduce the mortality in heart failure patients, there is, thus far, no unambiguous demonstration of a functional role of MR in cardiac cells. The aim of this work was to investigate the activation pathway(s) mediating corticosteroid-induced up-regulation of cardiac calcium current (ICa). In this study, using neonatal cardiomyocytes from MR or glucocorticoid receptor (GR) knockout (KO) mice, we show that MR is essential for corticosteroid-induced up-regulation of ICa. This study provides the first direct and unequivocal evidence for MR function in the heart.

Hsp90 transcriptionally and post-translationally regulates the expression of NDRG1 and maintains the stability of its modifying kinase GSK3beta

N-myc downstream-regulated gene 1 (NRDG1) is a stress-induced protein whose putative function is suppression of tumor metastasis. A recent proteonomic study showed NDRG1 interacts with the molecular chaperone heat shock protein 90 (Hsp90). From their reported association, we investigated if NDRG1 is dependent on Hsp90 for its stability and is therefore a yet unidentified Hsp90 client protein. Here, we demonstrate that endogenous NDRG1 and Hsp90 physically associate in hepatocellular cancer cell lines. However, geldanamycin (GA)-mediated inhibition of Hsp90 did not disrupt their interaction or result in NDRG1 protein destabilization. On the contrary, inhibition of Hsp90 led to a transcriptional increase of NDRG1 protein which was associated with cell growth arrest. We also observed that GA inhibited the phosphorylation of NDRG1 by targeting its regulating kinases, serum- and glucocorticoid-induced kinase 1 (SGK1) and glycogen synthase kinase 3 beta (GSK3beta). We demonstrate that in the presence of GA, GSK3beta protein and activity were decreased thus indicating that Hsp90 is necessary for GSK3beta stability. Taken together, our data demonstrate that NDRG1 is not a classic client protein but interacts with Hsp90 and is still dually regulated by Hsp90 at a transcriptional and post-translational level. Finally, we suggest for the first time GSK3beta as a new client protein of Hsp90.

[Insulin and type 2 diabetes mellitus]

Worldwide an increasing number of persons suffers from type 2 diabetes. Often treatment with oral hypoglycemic agents is not sufficient for adequate glycemic control and additional insulin treatment is necessary. Treatment with insulin is recommended if HbA1c levels below 7% cannot be achieved despite lifestyle measures and the proper use of oral hypoglycemic agents. In addition, pregnancy, periods pre and post major operations, treatment in intensive care units, glucocorticoid medication, severe peripheral neuropathy as well as contraindications of oral hypoglycaemic agents may be indications for insulin therapy irrespective of the actual glycemic control. The choice of the appropriate insulin regimen depends on the daily blood glucose profiles and patient needs.

Hyperaldosteronism in pregnancy

Aldosterone is a key regulator of electrolyte and water homeostasis and plays a central role in blood pressure regulation. Hormonal changes during pregnancy, among them increased progesterone and aldosterone production, lead to the required plasma volume expansion of the maternal body as an accommodation mechanism for fetus growth. This review discusses the regulation of aldosterone production by aldosterone synthase (CYP11B2); the impact on aldosterone secretion due to the presence of a chimeric gene originating from a crossover between CYP11B1 and CYP11B2 in glucocorticoid remediable aldosteronism (GRA) - the inherited form of hypertension; enhanced aldosterone production in aldosterone-producing adenoma (APA); and idiopathic hyperaldosteronism (IHA). Features of hyperaldosteronism are also found in patients with apparent mineralocorticoid excess (AME), in which glucocorticoids exacerbate activation of the mineralocorticoid receptor (MR) because of a defect in the 11beta-hydroxysteroid dehydrogenase type 2 enzyme. Regulation of aldosterone production and tissue-specific activation of the mineralocorticoid receptor are prerequisites for optimal control of body fluids and blood pressure during pregnancy and contribute largely to the wellbeing of the mother-to-be.

Inhibition of 11beta-hydroxysteroid dehydrogenase type 1 by plant extracts used as traditional antidiabetic medicines

Elevated glucocorticoids are a key risk factor for metabolic diseases, and the glucocorticoid-activating enzyme 11beta-hydroxysteroid dehydrogenase 1 (11beta-HSD1) represents a promising therapeutic target. We measured the potential of six traditional antidiabetic medicinal plants extracts to inhibit 11beta-HSD1 activity and glucocorticoid receptor (GR) activation in transfected HEK-293 cells. Leave extracts of Eriobotrya japonica preferentially inhibited 11beta-HSD1 over 11beta-HSD2. Extracts of roasted but not native coffee beans preferentially inhibited 11beta-HSD1 over 11beta-HSD2, emphasizing the importance of sample preparation. Thus, natural compounds inhibiting 11beta-HSD1 may contribute to the antidiabetic effect of the investigated plant extracts.

Body composition and fuel metabolism after kidney grafting

Kidney transplant patients display decreased muscle mass and increased fat mass. Whether this altered body composition is due to glucocorticoid induced altered fuel metabolism is unclear. To answer this question, 16 kidney transplant patients were examined immediately after kidney transplantation (12 +/- 4 days, mean +/- SEM) and then during months 2, 5, 11 and 16, respectively, by whole body dual energy X-ray absorptiometry (Hologic QDR 1000W) and indirect calorimetry. Results were compared with those of 16 age, sex and body mass index matched healthy volunteers examined only once. All patients received dietary counselling with a step 1 diet of the American Heart Association and were advised to restrict their caloric intake to the resting energy expenditure plus 30%. Immediately after transplantation, lean mass of the trunk was higher by 7 +/- 1% (P < 0.05) and that of the limbs was lower by more than 10% (P < 0.01) in patients than in controls. In contrast, no difference in fat mass and resting energy expenditure could be detected between patients and controls. During the 16 months of observation, total fat mass increased in male (+4.9 +/- 1.5 kg), but not in female patients (0.1 +/- 0.8 kg). The change in fat mass observed in men was due to an increase in all subregions of the body analysed (trunk, arms+legs as well as head+neck), whereas in women only an increase in head+neck by 9 +/- 2% (P = 0.05) was detected. Body fat distribution remained unchanged in both sexes over the 16 months of observation. Lean mass of the trunk mainly decreased between days 11 and 42 (P < 0.01) and remained stable thereafter. After day 42, lean mass of arms and legs (mostly striated muscle) and head+neck progressively increased over the 14 months of observation by 1.6 +/- 0.6 kg (P < 0.05) and 0.4 +/- 0.1 kg (P < 0.01), respectively. Resting energy expenditure was similar in controls and patients at 42 days (30.0 +/- 0.7 vs. 31.0 +/- 0.9 kcal kg-1 lean mass) and did not change during the following 15 months of observation. However, composition of fuel used to sustain resting energy expenditure in the fasting state was altered in patients when compared with normal subjects, i.e. glucose oxidation was higher by more than 45% in patients (P < 0.01) during the second month after grafting, but gradually declined (P < 0.01) over the following 15 months to values similar to those observed in controls. Protein oxidation was elevated in renal transplant patients on prednisone at first measurement, a difference which tended to decline over the study period. In contrast to glucose and protein oxidation, fat oxidation was lower in patients 42 days after grafting (P < 0.01), but increased by more than 100% reaching values similar to those observed in controls after 16 months of study. Mean daily dose of prednisone per kg body weight correlated with the three components of fuel oxidation (r > 0.93, P < 0.01), i.e. protein, glucose and fat oxidation. These results indicate that in prednisone treated renal transplant patients fuel metabolism is regulated in a dose-dependent manner. Moreover, dietary measures, such as caloric and fat intake restriction as well as increase of protein intake, can prevent muscle wasting as well as part of the usually observed fat accumulation. Furthermore, the concept of preferential upper body fat accumulation as consequence of prednisone therapy in renal transplant patients has to be revised.