Endocrinology & Diabetes

Exp Clin Endocrinol Diabetes 109 (2001) 374 ± 377

Experimental and Clinical

Endocrinology & Diabetes 2001 Johann Ambrosius Barth

Activities of 11b-hydroxysteroid dehydrogenase 2 in different regions of the intestinal tract of pigs R. Claus 1, S. Raab 2, M. Lacorn 1 2

FG Tierhaltung und Leistungsphysiologie, Institut für Tierhaltung und Tierzüchtung, Universität Hohenheim, Stuttgart, Germany F. Hoffmann-La Roche Ltd., PRBB, Basel, Switzerland

Key words: 11b-hydroxysteroid dehydrogenase, intestinal tract, pig, glucocorticoids Summary: 11b-Hydroxysteroid dehydrogenase 2 (11b-HSD 2) converts active cortisol to inactive cortisone and thus modifies the availability of glucocorticoids for the target tissue. An additional function is the protection of the aldosterone receptor in mineralocorticoid±sensitive tissues such as the kidney and the gut. The occurrence of 11b-HSD 2 activity was investigated in several species. Data for the pig, however, so far are missing. The activity was determined by a radio-enzyme-assay based on the conversion of tritiated cortisol to cortisone under standardized incubation conditions in supernatants of homogenates prepared

Introduction Glucocorticoids play an essential role for the cell cycle regulation in many organs. Their specific effect on cell differentiation and apoptosis also explains their overall catabolic function. In consequence, the glucocorticoid receptor (GR) concentrations are correlated with the fractional turnover rates in different tissues (Claus et al., 1996; Simon, 1989). Because circulating levels of glucocorticoids reach every organ, an organ-specific reaction is additionally ensured by the local expression of 11b-hydroxysteroid dehydrogenases (11b-HSD) which either convert the active glucocorticoid cortisol to inactive cortisone or cortisone to cortisol (e.g. Walker, 1994; Stewart and Krozowski, 1999; Pµcha et al., 1997). Two isoenzymes of the 11b-HSD have been identified. 11b-HSD type 1 is a 34 kDa glycoprotein which acts as a bi-directional oxidoreductase in vitro. It depends on NADP(H) as a cofactor (Monder, 1990) and occurs in many tissues such as in adipose tissue (Jamieson et al., 1999; Stewart and Krozowski, 1999), in the uterine myometrium in late pregnancy (Yang, 1997; Waddell and Burton, 2000), in bone (Cooper et al., 2000), and in the lung (Hundertmark et al., 1993). Its highest expression occurs in the liver where it predominantly catalyses the regeneration of cortisone to cortisol (Jamieson et al., 2000) thus

from tissues of four castrates. Tissues comprised several locations along the intestinal tract and in addition kidney, lung, muscle, heart, spleen and pancreas. Highest values of the enzyme activity were found in kidney and very low activities in lung tissue but no activity in muscle, spleen, heart and pancreas. In the gut, there was a continuous increase in enzyme activity from the duodenum (0.60 pmol minÿ 1 mg proteinÿ 1) towards the colon with maximum values in the colon transversum (23.32 pmol minÿ 1 mg proteinÿ 1). In the colon the activity was 10-fold higher than in jejunum and 3-fold higher compared to ileum. The activities did not differ significantly between the colon transversum and colon descendens.

maintaining levels of circulating glucocorticoids (Krozowski, 1999). 11b-HSD type 2 has a molecular weight of 40 kDa and reveals exclusively a NAD dependent dehydrogenase activity thus protecting tissues against an excess of active cortisol (e.g. Leckie et al., 1998). Such tissues comprise, e.g. the brain (Seckl, 1997), the endometrium (Burton et al., 1998), the placenta (Klemke and Christenson, 1996; Klemke, 2000), and Leydig cells (Pacha et al., 1997; Ge et al., 1997). In such tissues the variation of the enzyme expression explains an individual variation in the susceptibility to circulating glucocorticoids. A peculiar role exists in mineralocorticoid target tissues such as the kidneys and the colon. Due to similar affinities of aldosterone receptors for both aldosterone and cortisol, 11b-HSD 2 protects the receptor against a 100-fold excess of cortisol in peripheral plasma. This mechanism, therefore, plays a key role in maintaining normal salt-water homeostasis and blood pressure (Hermans et al., 1999; Sheppard et al., 1999; Diederich et al., 2000). Whereas the occurrence of 11b-HSD 2 was extensively studied in kidneys of several species, the gut was far less investigated. In rats there was no evidence for 11b-HSD 1 expression, but 11b-HSD 2 was expressed in all regions of the intestine with expression being 10-times higher in the colon than the

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ileum. In pigs, such studies so far were not performed. The objective of this study, therefore, was to determine the activity of 11b-HSD 2 along the intestinal tract of pigs and other tissues for comparison. Materials and methods For the investigation four male-castrated German Landrace pigs with an average age of 7 months and an average weight of 113 kg (96±123 kg) were kept in individual crates (3 2.5 m) and fed with diets containing 14.1 MJ metabolizable energy/kg and 13.6% crude protein at an amount of 2 kg at each feeding time (08.00 and 15.00 h). The mineral content was according to the standards for pigs. The pigs had unlimited access to water. They were killed by intravenous infusion of 25 ml Narcorene (Merial, 85399 Hallbergmoos, Germany, Best.Nr. 03580) and the tissue samples were collected within 10 min post mortem. They were shock-frozen in liquid nitrogen and then stored at ÿ80 8C until enzyme determination. The location of the gut samples removed is schematically shown in Figure 1. In addition samples were taken from the pancreas, kidney, spleen, muscle (M. longissimus dorsi), heart, and lung. The enzyme activity was determined by a radioenzyme-assay as previously described by Stewart and Mason (1995) and Mazzochi et al. (1998). The principle is based on the percentage conversion of tritiated cortisol to cortisone in the presence of a constant amount of unlabelled cortisol and NAD. Pieces of 0.5 g per tissue and individual were homogenized, centrifuged and the supernatant was used for two enzyme determinations. The tissues were homogenized at 4 8C in 2 ml phosphate buffer (80 mM; pH 7.2) and the homogenate centrifuged at 800g for 20 minutes (48C). The supernatants

Fig. 1 Anatomical sites of gut tissue sampling and 11b-HSD 2 activity (pmol minÿ1 mg proteinÿ 1) given as mean SD (1 duodenum; 2 jejunum; 3 ileum; 4 caecum; 5 colon transversum; 6 colon descendens)

375 were taken for measuring the enzyme activity and additionally for the determination of the protein content according to the method of Bradford (1976). For enzyme determination, 100 nCi 3H-cortisol (443 pg; specific activity: 82 Ci/mmol; Amersham International, Aylesbury, UK) were added to 50 ml incubation buffer (100 mM Tris; 0.25 mM NAD; pH 8.3) and combined with an additional volume of 50 ml buffer containing 49.1 ng unlabelled cortisol (100-fold surplus compared to the tracer). To this volume of 100 ml, 10 ml of the tissue supernatant were added to start the reaction which was performed at 37 8C for 30 min. In each supernatant the enzyme activity was determined in duplicate. The reaction was stopped by heating the samples at 958C for 2 minutes in a water bath. Thereafter the samples were extracted with 2 ml dichloromethane. The organic phase was transferred into another reagent tube, evaporated to dryness and redissolved in 100 ml dichloromethane. Steroids were separated by TLC on silica gel 60 F254 plates (Merck, 89081 Ulm, Germany) and developed in chloroform: acetone (70: 30; v/v). Unlabelled standards (cortisol, cortisone) were run in parallel on the plates to identify the substances under UV light at 254 nm. Radioactivity was detected directly on the thinlayer plates using an automatic TLC-linear analyzer (Tracemaster 20, Berthold, Wildbad, Germany). The conversion of tritiated cortisol to cortisone was determined and used to calculate the conversion of the unlabelled steroid. The activity of one unit was defined as the formation of one pmol unlabelled cortisone per minute and mg protein. The assay conditions had been optimized based on preceding experiments where the incubation time (2± 30 min) and the protein content (0.1±0.5 mg/tube) had been varied. The reliability of the determinations was further characterized by determining the procedural losses till TLC. The solvent extracts after the incubation were evaporated, taken up in scintillation cocktail and the radioactivity determined in a liquid scintillation counter (Beckman LS 1801, Palo Alto, USA). To investigate an unspecific conversion of cortisol to cortisone a preheated supernatant (1008C, 5 min) was processed as a sample. The mean recovery of the total activity after solvent extraction (n=10) was 87.9% and no unspecific conversion of cortisol to cortisone occurred. In addition the coefficient of the inter-assay variation was obtained by determining the enzyme activity in a pool of kidney supernatants in consecutive tests (n= 10). Similarly, the intra-assay coefficient was determined by the repeated determination at the same day (n = 5). The inter-assay coefficient of variation was 10.0% and the intraassay variation was 9.4%. The detection limit (0.56 U) was calculated from the doubled noise of the TLC-linear analyzer. Kinetic enzyme constants were determined by variation of the amounts of unlabelled cortisol [S]


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(from 0.25 to 17.5 mM) and calculation of the rate of reaction [V] (pmol cortisoneminÿ1 mgproteinÿ 1) under the conditions described above. From a Lineweaver-Burk plot of 1/[V] versus 1/[S], the apparent Km and vmax values were obtained. The mean values of the two determinations per sampling site were used to calculate the standard deviation between animals. In case of non-detectable enzyme activity the detection limit was taken for these calculations. To compare differences between different sampling sites, the data were analyzed by a one-way analysis of variance (ANOVA) followed by Duncans test for multiple comparisons using the statistical analysis system (SAS; SAS Institute, Cary, NC). Differences were considered significant at p £0.05.

Results The apparent kinetic enzyme constants determined in porcine colon transversum revealed values for Km of 2.88 mM and vmax of 10.13 pmol minÿ 1 mgproteinÿ 1 as shown in Figure 2. Measurements of 11b-HSD 2 activity in samples from intestinal tract are included in Figure 1 and revealed continuously increasing activities from the duodenum to the colon. The pattern of enzyme activities was the same for all animals except one case where only minimal activity was found in the caecum. Very low enzyme activities were observed in the duodenum (0.600.07 U) and slightly elevated but not significantly different activities in the jejunum (2.98 U). The mean duodenal enzyme activity was derived from one measurable value (0.71 U) and three samples below the detection limit. Therefor as descirbed above the detection limit was taken for calculation of means, SD and ANOVA. A 3-fold but not significant increase compared to the jejunum

could be shown in ileum (9.16 U) and caecum (10.97 U). Maximal intestinal values were observed in colon tissues with activities of 23.32 U in colon transversum and 19.39 U in colon descendens. The concentrations in the colon were significantly higher compared to all other intestinal segments (p< 0.05). The slight decrease in colon descendens activities was not significantly different compared to colon transversum. In relation to jejunal enzyme activities, colon activities showed a 10-fold increase. In tissues other than gut 11b-HSD 2 activity was found in the lung (1.450.62 U) and kidney (25.22 0.1 U) so that the latter organ revealed the highest activity of all tissues investigated. No activity could be detected in spleen, muscle, heart and pancreas.

Discussion So far the occurrence of 11b-HSD 2 in pig tissues was only studied in the placenta (Klemcke, 2000; Klemcke and Christenson, 1996) and in a porcine renal cell line LLC-PK1 (Leckie et al., 1995; Mobus et al., 1999). Our data on 11b-HSD 2 in different organs fit to the pattern in other species, where the enzyme was characterized by various methods, including mRNA determination, western blot and immunocytochemistry (Agarwal et al., 1994; Sheppard et al., 1999). The other studies using porcine tissues or cells were performed by enzyme determination which revealed similar vmax values as in our study (e.g. Mobus et al., 1999). The Km value of 11b-HSD 2 in porcine colon transversum, however, is 10 to 100-fold higher compared to other tissues and species (Stewart and Krozowski, 1999; Mobus et al., 1999). This discrepancy probably is mainly explained by species differences. To our knowledge, systematic studies in the intestinal tract were performed only in rats (Sheppard et al., 1999). In this study also the aldosteronereceptor mRNA was determined and it was found that the receptor expression increased significantly from jejunum towards the colon. In the same study the expression of 11b-HSD 2 was 10-times higher in the colon than in ileum and in ileum 3-times higher than in duodenum and jejunum (Sheppard et al., 1999) as similarly found in the pig intestinal tract. Thus the importance of 11b-HSD 2 in pig for ensuring specificity of aldosterone function in the colon apparently is also valid in the pig. Because glucocorticoids play an essential role for cell maturation in the gut the high enzyme activities in the colon and the high inactivation of glucocorticoids in consequence may lead to a deficit of glucocorticoid functions specifically in this compartment. In earlier studies with cytosol-binding of dexamethasone (Claus et al., 1996) and similarly in recent determination of the glucocorticoid receptor by both immuno-staining and


Fig. 2 Lineweaver-Burk plot for kinetic analysis of 11b-HSD 2 in porcine colon transversum. An apparent Km and vmax for cortisol of 2.88 mM and 10.13 pmol minÿ 1 mg proteinÿ 1 respectively, is shown

Exp Clin Endocrinol Diabetes 109 (2001)

western blot (unpublished data) we found much higher glucocorticoid receptor activities in the colon compared to the small intestine. It is likely, therefore, that a loss of cortisol due to the enzymatic inactivation is compensated partly by an increased GRexpression. Alternatively a novel 11-dehydrocorticosterone-preferring receptor that may mediate glucocorticoid effects in tissues with high level of 11b-HSD 2 activity was assumed (Sheppard and Funder, 1996). Acknowledgement: The authors would like to thank Dr. NµrayFejes-Tóth for providing antiserum against rabbit 11b-HSD 2 and S. Knöllinger for her expert technical assistance.

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Prof. Dr. Rolf Claus FG Tierhaltung und Leistungsphysiologie Institut für Tierhaltung und Tierzüchtung (470) Universität Hohenheim Garbenstr. 17 D-70599 Stuttgart Germany Tel.: +49-711-459-2455 Fax: +49-711-459-2498 E-mail: [email protected]


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R. Claus et al., 11b-HSD 2 activity in the pig