Effects of SH-Reagents of Different Molecular Size the adipose tissue mass. Eds.: Vague, J., J. Boyer. Excerpta med.Amst. Internat. Congr.Ser. 315: 158-173 (1974) Lemonnier, D., J. Winand, J. Furnelle, J. Christophe: Effect of a high-fat diet on obese-hyperglycaemic and non-obese Bar-Harbor-mice. Diabetologia 7: 328-333 (1971) Malaisse, W.J., D. Lemonnier, F. Malizisse·Lagae, I.M. Mandel· baum: Secretion of and sensitivity to insulin in obese rats fed a high-fat diet. Horm.Metab.Res. 1: 9-13 (1969) Murphy, B.E.: Sephadex column chromatography as an adj unct to competitive pro tein binding assays of steroids. Nature New Biol. 232: 21-24 (1971) Naeser, P.: Effects of adrenalectomy on the obese-hyperglycemic syndrome in mice (gene symbol ob). Diabetologia 9: 376·379 (1973) Naeser, P.: Function of the adrenal cortex on obese-hyper-

415

glycemic mice (~ene symbol ob). Diabetologia 10: 449 453 (1974) Schemmel, R., O. Miekelsen, U. Mostoky: Influence of body weight, age, diet and sex on fat depots in rats. Anat.Rec. 166: 437-445 (1970) Sehemmel, R., O. Miekelsen, Z. Tolgay: Dietary obesity in rats: influence of diet, weight, age and sex on body composition. Amer.J.Physiol. 216: 373-379 (1969) Shigeta, Y., W.E. Shreeve: Fatty acid synthesis from glucoseI-H3 and glucose-lC-C 14 in obese-hyperglycemic mice. Amer.J.Physiol. 206: 1085-1090 (1964) Westphal, U.: Assay and properties of corticosteroid-binding globulin and other steroid-binding serum proteins. Methods Enzymol. 15: 761-796 (1969) Yen, T. T. T., L. Lowry, J. Steinmetz: Obese locus in mus musculus: A gene dosage etTect. Biochem.Biophys.Res. Comm. 33: 883-887 (1968)

Horm. Metab. Res. 7 (1975) 415-418

© Georg Thieme Verlag Stuttgart

Effects of SH-Reagents of Different Molecular Size upon Glucose Metabolism in lsolated Rat Fat Cells* H. Kather and B. Simon Klinisches Institut zur Erforschung des Herzinfarktes an der Medizinischen Universitätsklinik, Heidelberg, Germany

Summary To study the role of membrane SH-groups in glucose tran:r port of isolated rat fat cells we compared the etTects of a small organic mercurial reagent p-CMB with those of a !arge p-CMB-derivative - p-CMB-Dextran, MW - 10.000 -. It could be shown that both compounds were of alrnost identical reactivity on fat cell homogenate metabolism. When applied to intact fat cells uncoupled p-CMB showed an (I) insulin like enhancement of 14C incorporation from (U_ 14 C) glucose into CO 2 and triglyceride, (2) inhibition of the insulin-stimulatory effect on these parameters and (3) inhibition of basal glucose uptake dependent on the concentrations used. Identical concentrations of p-CMB-Dextran, however, faiied to influence basal glucose uptake as weil as the insulin mediated increase in glucose metabolism. Key-Words: Isolizted Fat Cells - p-CMB - p-CMB-Dextran - Membrane SH-Groups - Glucose Metabolism - Insulin Action

The concept that different honnones are first bound to individual receptors thereby gene rating a signal which is propagated by a transducer to the effector system has been widely accepted. Small organic mercurial reagents such as p-CMB (Diüt and Lazarow 1972) and p-CMBS (Minemura and Crofford 1969) as weil as thiols theirselves like cystein~ (Lavis and Williams 1970) have been shown to exhibit insulin like effects on glucose metabolism of adipocytes. To define more clearly the localization of such critical membrane SH-groups we compared the action of uncoupled p-CMB on isolated fat cells with the effects of a large derivative of this compound - p-CMBDextran. This reagent has a molecular weight of - 10.000.

Introduction

Methods and Materials

The search for the primary site of honnone action has lead to the plasma membrane of the target cell.

Isolated rat fat cells were obtained from the epididymal fat pads of 150-200 gm male Sprague Dawley rats (Süddeutsche Versuchstierfarm, Tuttlingen, Germany) maintained on a Standard pellet chow (Altromin®) prior to decapidation.

·Parts of this work has been published as Short Communica· tion in this Journal (Vol. 7: 98-99, 1975)

Isolated fat cells and fat free homogenate were prepared according to Rodbell (1964, 1966). Adipose tissue pieces were incubated for 60 min in Krebs-Ringer-bicarbonate buffer, pH 7.4 containing 2% bovine serum albumin (Behring,

Received: 15 Mar. 1975

Accepted: 18 June 1975

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Requests for reprints should be addressed to: Dr. Dr. Lieselotte Herberg, Diabetes-Forschungsinstitut an der Universität Dilsseldorf, D-4 Düsseldorf, Aurm Hennekamp 65 (Germany)

H. Kather and B. Simon

Marburg, recrystallised) and crude collagenase 1 mg/mi (Worthington, Biochem.Corporation Freehold, New Jersey) and washed three times. Washed adipocytes were preincubated for 30 min in the presence of various concentrations of uncoupled p-CMB and p-CMB-Dextran (7x 10-6 to 7x10-4M). The p-CMB concentrations of the coupled polymer were calculated from the Hg content. One portion of the cell suspension treated under identical conditions but without p-CMB (p-CMB-Dextran) was used as a contro!. After repeated washing (3x) with 2% Albumin-Krebs-Ringerbicarbonate buffer, pH 7.4, the cell suspension was diluted to a final concentration of about 40 loImol fat cell triglyceride/ ml, dispensed in stoppered plastic scintillation vials containing (U_14C) glucose (4 loImol/0.5 lJCi per mll (AmershamBuchler, Braunschweig, Germany) with and without 0.5 U Insulin per ml and incubated for 60 min. The total volume of the incubation mixture was 2.0 m!. The incubations were carried out in an atmosphere of 95% O2 and 5% C02 at 370C and were terminated by the addition of 0.3 ml of 4 N perchloric acid.

Assay of compounds 14C02 from 14C glucose was trapped by 0.3 ml phenylethylamine injected into plastic vials hanging from the rubber stopper. Phenylethylamine was dissolved in 15 ml of a ~om­ mercially available scintillation fluid (Unisolve 1, Kochhght, Lab. Colnbrook, Bucks, England) and counted for radioactivity. The insoluble residue containing the lipid fraction was freed from the medium by filtration using millipore filter (pore size 0.45 IJ) and extracted with Chloroform/Methanol (2: 1, v/v). The lipid extract was purified according to Foich, Lees and Stanley (1957) and counted for radioactivity.

0",

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Fig. 1a 0'0

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80

60

40

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Fat cell trigijcceride content was determined enzymatically by using a routine assay (Boehringer, Mannheim). Crystalline bovine insulin was a gift of Farbwerke Hoechst, Frankfurt. p-CMB-Dextran was synthesized according to Simon, Zimmerschied, Kinne-Saffran and Kinne (1973): p-CMB was covalently bound to aminoethyldextran via a peptide bond and purified by column chromatography. p-CMB-Dextran was stable for at least 3 months as verified by rechromatography. The final product contained 1.5 mole p-CMB per mole of Dextran.

Results Fig. 1 shows the effects of various concentrations of (a) p-CMB and (b) p-CMB-Dextran on glucose metabolism of fat cell homogenate. Increasing concentrations of p-CMB (from 7 x 10- 6 to 7 x lO-4M) caused a .r.rogressive inhibition of homogenate metabolism. I COl production was alm ost completely inhibited at a p-CMB concentration of 7 x 1O-4 M.

control

h1O-6

7»:10- 5

7.10-4 M

Fig. 1b Fig. 1. Effeet of inereasing concentrations of a) p-CMB and b) p-CMB-Dextran on 14C02 production of fat ceU homogenate. The concentrations of p-CMB and p-CMB-Dextran refer to the mercury content of the sampies. Fat free cell hom~genate was fortified with 2.5 mM ATP, 0.75 mM NADP, and mcubated for 1 hr. The me an values ± SEM of 3 experiments, each in triplicate, are given.

The first was an increase of basal glucose conversion to CO 2 and triglyceride i.e. insulin like stimulation of glucose metabolism. This effect occurred at p-CMB concentrations of 7 x 10- 6 M to 7 x 10-5 M and, although variable, can stimulate glucose metabolism up to 3 times the basal level.

The second effect of p-CMB on intact adipocytes was the inhibition of the insulin stimulatory action: At The large p-CMB derivative - p-CMB-Dextran - inp-CMB concentrations of 3.5 x 1O-4 M insulin stimulahibited the metabolie activities of fat cell homogenate tion was alm ost completely abolished whereas basal to about the same extent as uncoupled p-CMB indiglucose metabolism was virtually not affected. cating that both reagents are of almost identical reFinally, at concentrations above 3.5 x 1O-4 M p-CM~ activity. caused an alm ost complete inhibition of basal and 10When intact adipocytes were preloaded with unsuHn stimulated glucose metaboHsm. coupled p-CMB, however, three distinct and separate In contrast to the influence of p-CMB-Dextran on fat effects of p-CMB were observed (see Figure 2). cell homogenate, however, the metabolism of intact

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416

Effects of SH-Reagents of Different Molecular Size

417

The sites of action can be restricted to the membrane level by eoupling of p-CMB to a soluble macromoleFig. 2. Effect of various concentrations of pCMB on 14C0 2 eular support such as Dextran. Coneentrations of pproduction and triglyceride synthesis in intact adipocytes. CMB-Dextran whieh strongly inhibited homogenate Fat cells were preloaded for 30 min (37 0 C) with various metabolism did not alter the metabolic activities of concentrations of p-CMB, washed thoroughly and incubated intact adipocytes suggesting that the cell membrane with and without insulin (0.5 U/ml) for 60 min. Values are given in nmoles of glucose converted to CO 2 and triglyceride is impervious for this large SH-reagent. This lacking per 100 IJMol fat cell triglyceride in 1 hr. effeet of p-CMB-Dextran further indicates that the The mean values ± SEM of 3 experiments, each in triplicate, reaetive ligand was not Iiberated from the Dextran are given. carrier under the eonditions employed. fat cells was not substantially altered after preloading with this large SH-reagent: Neither insulin Iike nor inhibitory effects were observed up to concentrations of 7 x 10-4 M (Table 1).

Our results demonstrate that the insulin like effects of uncoupled p-CMB are not caused by bloeking of superficial membrane SH-groups in contrast to the proposals of Dixit and Lazarow (1972).

Our conclusions are in good agreement with reeent studies on the insulin reeeptor interaction showing not inhibited over the wh oie eoneentration range that membrane SH-groups are not funetionally imtested. portant for the binding of insulin to the membrane Discussion (Cuatrecasas 1971). Since the plasma membrane of isolated adipoeytes appears to be impermeable to The effeets of small SH-reagents on the metabolie eompounds of comparable moleeular size as insulin, aetivities of isolated fat cells have been previously our results support the eoneept that all effeets of studied (Diloit and Lazarow 1972, Minemura and insulin are mediated by the interaction of the horCrofford 1969, Carter and Martin 1969). Those remone with the plasma membrane rather than by disults are qualitatively similar to these obtained in reet alteration of eertain metabolie steps inside the our experiments. eell. The weil known insulin Iike aetions of these reagents

In addition, insulin responsiveness of the eells was

Table 1. Effects of pCMB-Dextran on basal and insulin stimulated glucose metabolism of isolated rat adipocytes pCMB-Dextran (M)

14C incorporation (nmol U_14C glucose/hr per 100 /JIT!01 triglyceride) with insulin (0.5 U/ml) no insulin CO 2

triglyceride

C02

triglyceride

± 10 ± 9 160 ± 14 148 ± 8 142 ± 12

± 9 ± 12 162 ± 12 174 ± 9 175 ± 15

± 10 ± 15 591 ± 18 577 ± 20 563 ± 25

± 25 ± 10 805 ± 25 755 ± 35 811 ± 22

140

7 x 10-6 7 x 10- s 3.5 x 10"" 7 x 10""

141

171

167

598

523

795

775

Isolated fat cells were incubated in Krebs-Henseleit-HC0 3 buffer, pH 7.4 containing 2% bovine serum albumin for 60 min at 370C. The cell concentration corresponded to about 40 /JIT!01 fat cell triglyceride/ml. The incubation media contained 4 mM U_14C glucose and pCMB-Dextran in concentration as listed above. Values are expressed as nmol U_14C glucose converted to 14C0 2 and 14C-triglycerides per /JIT!01 fat cell triglyceride in 60 min. The mean values ± SEM of three determinations, each in triplicate, are given.

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have been interpreted as suggesting that they react with sulphydryl groups on the cell surface and initiate the same sequence of events as those generated by insulin (Minemura and Crofford 1969). The similar degree of inhibition caused by p-CMB eoncentrations above 3.5 x 10-4 on homogenate metabolism and intact fat cells, however, indicates that this small eompound ean attack enzymes involved in the intermediary metabolism. One may assume that p-CMB aets on a large number of topographieally distinet sites. Its usefulness in evaluating defined membrane effeets is, therefore, impaired.

418

H. Vetter and W. Vetter

References

Carter, J.R., D.8. Martin: The effect of sulfhydryl blockage on insulin action and glucose transport in isolated adipose tissue cells. Biochim.Biophys.Acta 177: 521-526 (1969) Cuatrecasas, P.: Properties of the insulin receptor of isolated fat cell membrane. J.BioI.Chem. 246: 7265-7274 (1971) Dixit, R.K., A. Lazarow: Effects of metal ion and sulfhydryl inhibitors on glucose metabolism by adipose tissue. AmerJ.Physiol. 213: 849-856 (1972) Folch, J., M. Lees, G.H. Stanley: A simple method for the isolation and purification of total lipids from animal tissues. J.BioI.Chem. 226: 497-509 (1957) Lavis, R. V., R.H. Wi/liams: Studies of the insulin like action

of thiols upon isolated fat cells. J.BioI.Chem. 245: 23-41 (1970) Minemura, T., 0.8. Cro[[ord: Insulin-receptor interaction in iso la ted fat cells. I. The insulin-like properties of p-chloromercuribenzene sulfonic acid. J .BioI.Chem. 244: 5181518B (1969) Rodbell, M.: Effects of hormones on glucose metabolism and Iipolysis. J.BioI.Chem. 239: 375-380 (1964) Rodbell, M.: The similar effects of phospholipase C (Clostridium perfringens toxin) and of insulin on glucose and amino acid metabolism. J.BioI.Chem. 241: 130-139 (1966) Simon, 8., G. Zimmerschied, E.M. Kinne-Sa[[ran, R. Kinne: A new synthetic plasma membrane marker: FluorescentMercury-Dextran. J.Membr.Biol. 14: 85-93 (1973)

Horm. Metab. Res. 7 (1975) 418-424

© Georg Thieme Verlag Stuttgart

Regulation of Aldosterone Secretion in Primary Aldosteronism H. Vetter and W. Vetter* Medizinische Poliklinik. University of Bonn. Germany

Summary

Introduction

Plasma aldosterone, plasma renin activity and plasma cortisol were determined in patients with primary aldosteronism in response to posture and at short-time intervals overnight while the patients were supine. In the 5 patients with an aldosterone-producing adenoma postural changes in plasma aldosterone were paralleled by those in cortisol while plasma renin activity was generally undetectable indicating an ACTHdependent secretion of aldosterone. This concept was supported by the observation that in 3 of these patients who were tested overnight 1. episodic secretion of plasma aldosterone was paralleled by those of cortisol and 2. episodic secretion of plasma aldosterone could be blunted by dexamethasone. In the patient with idiopathic adrenal hyperplasia concomittant changes in plasma aldosterone and plasma renin activity occurred. The assumption that in this patient the fluctuations in plasma aldosterone were media ted tbrough changes in renal renin secretion was supported by the finding that episodic secretion of plasma aldosterone persisted under suppression of ACTH-secretion by dexamethasone. Qur results indicate, that the described procedures may all serve as diagnostic criteria to differentiate between aldosterone-producing adenoma and idiopathic adrenal hyperplasia.

The syndrome of primary aldosteronism may be caused generally either by an aldosterone-producing adenoma or by idiopathic adrenal hyperplasia. AIthough this disease has been extensively studied controversial results exist ab out the role of ACTH and renal renin secretion on adrenal aldosterone release (Kaplan and Silah 1964, Newton and Laragh 1968, Horton 1969, Slaton, Schambelan and Big/ieri 1969, George, Wright, Bell and Bartter 1970, Cain, Tuck, Williams, Dluhy and Rosenoff 1972, Ganguly, Melada, Luetscher and Dowdy 1973, Kem, Weinberger, Gomez-Sanchez, Kramer, Lerman, Furuyama and Nugent 1973, Biglieri, Schambelan, Brust, Chang and Hogan 1974, Vetter, Berger, Armbruster, Siegenthaler, Werning and Vetter 1974a).

Key-Words: Aldosterone - Adrenal Glands - Primary AIdosteronism

·Present address: Department of Internal Medicine, Kantonsspital, University of Zürich, Switzerland. Received: 10 Nov. 1974

Accepted: 18 June 1975

This study was performed to investigate the influence of endogenous ACTH- and renin secretion on adrenal aldosterone release in patients with primary aldosteronism. Materials and Methods Six patients suffering from primary aldosteronism (aldosterone-producing adenoma n z 5, idiopathic adrenal hyperplasia n = I) were examined in this study. In all cases the diagnosis was conflImed by surgery. Blood was taken from the patients at different days under normal sodium intake (120-150 meq sodium/day) at 8 a.m. after the patients were recumbent overnight and two hours

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Requests for reprints should be addressed to: Dr. H. Kather , Dr. B. Simon, Klinisches Institut zur Erforschung des Herzinfarktes an der Medizinischen Universitätsklinik Heidelberg, D-69 Heidelberg, Bergheimerstr. 58 (Germany)

Effects of SH-reagents of different molecular size upon glucose metabolism in isolated rat fat cells.

To study the role of membrane SH-groups in glucose transport of isolated rat fat cells we compared the effects of a small organic mercurial reagent p-...
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