Diabetologia Springer-Verlag 1986

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1 Diabetoloia (1986) 29: Diabetoloia Spriner-Verla 1986 In vivo quantifiation of removal of asialo-orosomuoid from the irulation in anaesthetized streptozotoin-diabeti rats M. Appel 1, P. Potrat 1, J. Feer l, C. Mas-Chamberlin 2 and G. Durand 1 1 Laboratoire de Biohimie, Centre d'etudes Pharmaeutiques et Bioloiques, Universit6 Paris-Sud, and z Clinial Researh Department, Laboratoire d'etude et de Reherhe Synthalabo, Paris, Frane Summary. The in vivo kineti of removal of 3H asialo-orosomuoid from plasma was investiated in ontrol and streptozotoin-diabeti rats after intravenous injetion of 1 m of asialo-orosomuoid/100 body wt. Mihaelis-Menten kinetis of disappearane were observed. In diabeti rats the maxi-' mal rate (Vmax) of disappearane of 3H asialo-orosomuoid was dereased by 30% with no modifiation of Mihaelis onstant. Sine no aumulation of desialylated orosomuoid in the irulation was observed, the slower rate of removal of 3H asialo-orosomuoid was attributed to a derease in the num- ber of hepati asialolyoprotein reeptors whih are larely involved in the atabolism of asialolyoproteins. Our estimate on in vivo maximal rates was 10- to 20-fold reater than our previous in vitro estimate of the maximal rate of endoytosis. In ontrast, the values of the Mihaelis onstant obtained in vivo and in vitro were very similar. Key words: Asialo-orosomuoid, streptozotoin-diabetes, lyoprotein. A speifi reeptor whih binds and mediates the endoytosis of desialylated lyoproteins is present at the outer surfae of hepatoytes [1, 2]. This reeptor-mediated endoytosis is a eneral phenomenon whih onerns all the lyoproteins, provided the siali aid residues are removed; therefore the alatose residues are at the ultimate position. However, the effiieny of removal from the irulation depends on the struture of the lyan moiety. Asialo-orosomuoid (ASOR), whih ontains a hih perentae of arbohydrates (40%), is most rapidly taken up by liver. Furthermore, the asialolyoproteins reeptor expression is affeted by the state of an animal and partiularly by the rowth state of ells [2]. We have reently reported that the bindin and uptake of 3H ASOR by hepatoytes from streptozotoindiabeti rats were dramatially altered when ompared to their removal by normal rat hepatoytes. This was related to a derease in the number of ell surfae reeptors with no modifiation in the apparent affinity onstants [3]. The number of intraellular reeptors was also dereased [4]. Sine the reported number of ell surfae reeptors is hihly variable, dependin on experimental onditions used in reard to liver ell isolation and inubation [5], it seems diffiult to predit the in vivo behaviour of injeted 3H ASOR from our in vitro results [3]. To this purpose, 3H ASOR was injeted intravenously in normal and diabeti rats, and its disappearane from plasma was measured. The presene of partially desialylated orosomuoid in plasma was also investiated. Materials and methods Experimental animals Spraue-Dawley rats (male, days old) were used for all experiments. Diabetes was indued by a sinle intravenous injetion (dorsal tail vein) of streptozotoin (65 m/k body wt.) dissolved immediately before use in 0.2 tool/1 itrate buffer isotoni, ph 4.6. Control animals were injeted with buffer alone. Fourteen to 20 days after streptozotoin injetion, diabetes was determined by a 24% weiht loss, plasmaluose levels between 5.80 /1 to 6.79 /1 and lyosuria. No protein and no ketone bodies were deteted in urine usin albustix (sensitivity 50 ra/l) and ketostix (sensitivity 50 m/1) respetively (Ames, Paris, Frane). Hematorit was and natraemia was 140_+ 2 mmol/1 for ontrol and diabeti rats respetively. Hene the diabeti rats were free of severe aute or hroni ompliations suh as nephropathy and dehydration. The rats had free aess to food until the mornin of the experiment. Reaents Human orosomuoid was a ift from Dr. Wikerhauser (Amerian Red Cross, NIH, Bethesda, MD, USA). Aarose-immobilized neuraminidase type X.A and streptozotoin were purhased from Sima

2 384 M. Appel et al.: Removal of asialo-orosomuoid in diabeti rats Chemial Co. (St Louis, MO, USA), tritiated sodium borohydride (speifi ativity 60 Ci/mmol) from Commissariat de l'enerie Atomique (Gif-sur-yvette, Frane), Dynael from Comptoir Lyonnais de Verreries Interbio (Lyon, Frane) and Sephadex G 25 from Pharmaia (Uppsala, Sweden). Spraue-Dawley rats were purhased from Charles River (St Aubin Les Elbeuf, Frane). All other reaents were laboratory rade and obtained from loal soures. Determination of serum orosomuoid and its desialylation by immunoloial methods Before the injetion of 3H ASOR, a blood sample was drawn by the juular vein and serum orosomuoid was measured by two immunoloial methods: the radial immunodiffusion method (RID) of Manini [6] and the eletroimmunodiffusion method (EID) of Laurell [7] as desribed by Biou et al. [8]. The perentae of the underevaluation by eletroimmunodiffusion relative to radial immunodiffusion was alulated. Then, usin previously established standard urves of the relation between the perentae of desialylation and the perentae of underevaluation, the deree of desialylation of orosomuoid was alulated [8]. Desialylation and radiolabellin of orosomuoid Human orosomuoid was desialylated with aarose-imrnobilized neuraminidase. The redutive methylation of Wilder [9] was used to enerate 3H ASOR. Typially 10 m of protein in 1 ml of 0.2 mol/1 sodium borate buffer ph 9 were mixed on ie with 18 ixl of 3.5% aqueous formaldehyde and added to a freshly opened ampoule ontainin 25 mci of tritiated sodium borohydride. After 30 min, the labelled protein was purified by el filtration on Sephadex G25, the elutin buffer bein mol/1 NaHCO3/0.15 mol/1 NaC1, ph 7. 3H ASOR an be stored at - 20 C for months with no loss of ativity and no deradation. The speifi ativity was 4 Ci/mmol. 40 Ixl of plasma. After 30 min at room temperature, the mixtures were entrifued. Non-protein radioativity was ounted in 40 p~l of the supematant fluid. Protein-bound radioativity was alulated from the differene between total and non-protein radioativity. Statistial analysis Results from n experiments were expressed as mean + standard error (mean+sem). Student's t-test for unpaired samples was used for analysis, with a level of sinifiane at p < A visual inspetion of the labelled plasma onentration versus time in linear and semi-loarithmi sales shows a profile typial for a Mihaelis-Menten elimination kineti. This is the reason why the disappearane kinetis of 3H ASOR were fitted to the model: dy dt Vm,) KM + S' where Vm is the maximum elimination rate and KM is the onentration of3h ASOR orrespondin to an elimination rate equal to half to Vm. The dispersion error on the measured onentrations was not fully known, so we deided to estimate the Vm and Krvl, minimizin the lo likelihood funtion: L = Z (yi@i)2 + l Vi Vi where Vi is the error variane of the i-th observation yi. Note that usually weihted least squares are appropriately used when it is assumed that the error variane is known up to a proportional fator. In our ase the error variane is not known, so aordin to Skeiner [10] the followin variane model was assumed. Vi=@ b where a and b are variane model parameters. These parameters as well as Vm and KM were estimated usin the PHARM proram Ill]. Catheterization and blood samplin The animals were anaesthetized with sodium pentobarbital (50 m/k body wt.). Juular vein and penial vein were then exposed and annulated with silasti Dow Comin tubin and PE 50 tubin filled with heparinized saline respetively. A pretest blood sample was taken for the determination of hematorit and plasma luose. Doses of 1000 of ASOR per 100 of body weiht were injeted. At time 0, the 3H ASOR at a speifi radioativity of 4 Ci/mmol, mixed with unlabelled asialolyoprotein in a total volume of about 0.5 ml 0.9% NaCl, was rapidly injeted via the penial atheter. The atheter was flushed throuh one time with 0.5 ml of saline and heparin to remove any trae of radioativity, Blood samples (200 Ixl) were taken at samplin times of I, 2, 3, 4, 5, 8, 10, 15, 20, 30, 45 and 60 min. Blood was aspirated via the juular atheter first to dispel the heparinized saline and the sample was then drawn into a lean syrine. On the last blood sample, hematorit was measured. There was no sinifiant haematorit differene between ontrol and streptozotoin-treated rats, nor between the first and the last samples of the experiment. The amount of radioativity deteted in the urine formed durin the experiment represented lp100 of the dose injeted in ontrol as well as diabeti rats. Determination of 3 H asialo-orosomuoid in plasma Eah blood sample was transferred to a mirofue tube, and plasma was separated by entrifuation. All speimens were assayed in dupliates. Total radioativity was determined in a Kontron liquid sintillation ounter (Betamati Intertehnique, Trappes, Frane) usin 30 p.l of plasma and 10 ml of Dynael (Comptoir Lyonnais de Verreries, Interbio, Lyon, Frane) as the sintillant. Deproteinization was arried out with 4091 of 10% (w/v) phosphotunsti aid in 2N HCI and ResuRs Evaluation of the orosomuoid level and the perentae of its desialylation in normal and diabeti rat sera The orosomuoid ontent of normal rat sera was 152 _+ 23 m/1 (n---8) and m/1 (n =8), by EID and RID respetively, whereas in diabeti rats the serum orosomuoid ontent was m/1 (n =7)by both methods. Compared to normal rats the orosomuoid level was sinifiantly hiher (p < 0.001) in diabeti rats. In ontrast, the perentae of underevaluation by EID relative to RID indiatin the deree of desialylation of orosomuoid was found to be within the normal rane in both normal and diabeti rats. No aumulation of partially desialylated orosomuoid oured in diabeti rat serum. Relationship between dose and removal of 3 H asialo-orosomuoid in ontrol and diabeti rats In the first set of experiments, inreasin quantities of 3H ASOR were injeted to find the dose at whih the liver ould no loner eliminate the protein in a matter of

3 M. Appel et al.: Removal of asialo-orosomuoid in diabeti rats 385 o 00i v 270~ 3_ oo. E \ 8 \ O z 10- \ "6 ~x X \\\\\ _ 0ot (73 "6 9 D G 0 20 &O 60 Time Gfter injetion (rain) 8 E 01 I ~ ~ i i &O Time after injetion (minl Fi. 1. Protein-bound radioativity in the plasma of ontrol rats (-) and diabeti rats (- - -) whih reeived different quantities of 3H asialo-orosomuoid (ASOR). The doses, expressed per 100 body wt. were: 75 I.t (11), 100 tt (tt), 400 lx ([]) and 1000 lx (O) 3 u~ a ry 0 U~ <~ I m! minutes. The behaviour of 3H ASOR in plasma is illustrated with some representative urves of the proteinbound radioativity as a funtion of the dose (Fi. 1). The 50% of 3H ASOR remainin in blood was reahed at later intervals as the dose of injeted 3H ASOR inreased (e.. with normal rats about 50% of injeted pm were present in blood at 2 min for 75 Ix/100, 2 min 30 s for 100 ~/100, 8 min for 400 ~t/100 and 15 min for 1000 lx/100 after injetion). The kinetis of plasma disappearane of 3H ASOR were dose-dependent. By administratin 1000 p of 3H ASOR/100 of body weiht in a sinle dose, we ould see that the liver of normal rats maintained maximal disappearane rates for at least 20 min. Plasma disappearane of 3 H asialo-orosomuoid in the presene of native orosomuoid Unlabelled native (sialylated) orosomuoid (1000p.) was simultaneously injeted with 100 of 3HASOR in normal and diabeti rats (data not shown). No derease in the oeffiient of extration of the labelled asialolyoprotein was observed in either roup of rats. The lak of ompetition between ASOR and orosomuoid indiates that these lyoproteins were leared by different proesses in both normal [12] and diabeti rats. 8 l o 2; 2o b Time after injetion (rain) Fi.2. Representative urves in linear (a) and loarithmi (b) sales of disappearane of the labelled protein from plasma of a ontrol (@) and a diabeti rat ([]). The dots are experimental dots and the lines are the result of the modelin aordin to the Mihaelis-Menten equation Plasma disappearane of 3 H asialo-orosomuoid in ontrol and streptozotoin-diabeti rats In this set of experiments a standard dose of 1000 tt 3H ASOR per 100 of body weiht was administered to ontrol (n = 7) and streptozotoin-treated rats (n = 6). The kineti of disappearane was measured durin 60 min. In Fiure 2, the urves are plotted in linear and semi-loarithmi sales. The dots are experimental dots and the lines are the results of the modelin aordin to the Mihaelis-Menten equation. The kineti analysis provided the final parameter estimates Vmax and KM of ASOR removal from plasma in normal and diabeti rats (Table 1). ASOR was rapidly removed from bloodstream in normal rats, whereas in diabeti rats the asialolyoprotein was leared muh more slowly. Mean values of Vmax = ~t/ min per 100 body wt. and ~t/min per 100 body wt. were obtained for normal and streptozotoindiabeti rats respetively, and were statistially sinifi-

4 386 M. Appel et al.: Removal of asialo-orosomuoid in diabeti rats Table 1. Comparison of the maximal rates (Vmax) and the Mihaelis onstants (KM) for the ontrol and diabeti rats for our in vivo study and for our previous in vitro study [1, 12] In vivo studies In vitro studies Control rats Diabeti rats Control rats Diabeti rats (n = 7) (n = 6) (n = 6) (n = 6) Vmax 9.60 ± 0.72 a 6.70 _ a a ~ (a/min per 100 body wt.) KM (nmol/1) 1.88 _ _ a ])<0.05 ant at p <0.05. In ontrast, the estimates of KM, 1.88_+0.31 nmol/1 and 1.44_+0.31nmoi/1 for normal and diabeti rats respetively were not sinifiantly different. Phosphotunsti aid-nonpreipitable radioativity was simultaneously evaluated and expressed as a perentae of the total radioativity. Within the initial 30min, no sinifiant protein-unbound radioativity was deteted in the plasma of both normal and diabeti rats. The perentae then inreased and reahed 50-60% at 60rain and remained onstant in both roups of rats. This findin seems to indiate that, after the uptake of ASOR by tissues [1, 12, 13], the intraellular atabolism of ASOR and the subsequent elimination of radiolabelled metabolites were not apparently altered in diabeti rats [4]. Disussion The present study provides in vivo estimates of the kineti parameters of ASOR removal from plasma of diabeti rats. The effets of insulin treatment were not studied here. In fat, we have previously shown that insulin therapy of streptozotoin-diabeti rats restored the ability of isolated hepatoytes to bind and take up 3H ASOR, and that the withdrawal of insulin led aain to a dereased bindin and uptake of 3H ASOR [3]. Several aspets of these in vivo studies are in areement with our in vitro studies [3]. First, the maximal rate of removal of ASOR (Vmax = 6.70 p~/min per 100 of body wt.) from plasma of diabeti rats was sinifiantly lower than that of normal rats (Vmax = 9.60 ~/min per 100 of body wt.). Seond, no modifiation of Mihaelis onstant was noted in diabeti rats. These data ould be explained either by the presene of desialylated orosomuoid in the plasma, whih ompetes with 3H ASOR, or by a dereased blood flow or by a dereased uptake of ASOR by some tissues, mainly the liver. The first hypothesis is ruled out sine, despite an inreased amount of plasma orosomuoid whih was previously desribed [5, 6], no asialo-orosomuoid was deteted. The seond explanation is not retained, with a dereased blood flow never bein desribed in diabeti rats. The third suestion is the most relevant, takin into aount our previous data showin a dereased num- ber of asialolyoprotein reeptors with diabeti rat hepatoytes. Sine the major site of atabolism of injeted asialolyoproteins is the liver with the kidney and the ut partiipatin to a muh lower extent [12, 13, 14], it appears that the disappearane of ASOR from plasma approximately represents the apaity of liver to bind and internalize asialolyoproteins. In order to ompare adequately the maximal rate of removal of 3H ASOR from plasma to the maximal rate of endoytosis of 3H ASOR by hepatoytes, the last parameter was expressed as 100 of body weiht. Assumin a rat liver is 1.6 x 108 hepatoytes/ and a standard weiht for a liver of 250 rats is 7, the maximal rates of endoytosis of 3H ASOR beome 0.80_+0.09 and ~/min per 100 of body weiht in normal and diabeti rats respetively, whih is ten- to twentyfold the maximal rate of removal of 3H ASOR from plasma. This disrepany between in vitro and in vivo studies was first desribed by Pardride et al. [17], who suested that, durin the isolation proedure of hepatoytes, hepati asialolyoprotein reeptors an undero internalization. In diabeti rats, the dereased rate of hepati learane of 3H ASOR is not due to a redued affinity of ASOR for its reeptor but is rather related to a dereased number of funtional reeptors. Sine the presene of siali aid residues at the ultimate position of the reeptor is needed to bind the alatose-terminated lyoproteins, an alteration of the arbohydrate moiety of membrane lyoprotein whih ours in experimental diabetes [18, 19, 20] ould be evoked. We previously demonstrated that the impairment of the uptake of ASOR was not related to a dereased membrane siali aid ontent [20]. On the other hand, the variations in the number of asialolyoprotein reeptors ould be related to the reulatory mehanism of insulin on protein synthesis [21] and/or breakdown [22, 23]. Diabetes mellitus leads to a dereased rate of synthesis of extra and intrahepati proteins as well as to an inreased rate of protein atabolism. The alterations in the number of asialolyoprotein reeptors we observed with the in vitro model are markedly onfirmed by the present in vivo experiments. Aknowledments. We wish to thank Dr. Wikerhauser (Amerian Red Cross, NIH, Bethesda, MD, USA) for his enerous ift of human

5 M. Appel et al.: Removal of asialo-orosomuoid in diabeti rats 387 orosomuoid, and Professor D. Durand for his trainin in the proedure of atheterization. This work was supported by a rant from the Centre National de la Reherhe Sientifique UA Referenes 1. Ashwell G, Morell AG (1974) The role of surfae arbohydrates in the hepati reonition and transport of irulatin lyoproteins. Adv Enzymol 41: Shwartz AL (1985) The hepati asialolyoprotein reeptor. CRC Crit Rev Biohem 16: Dodeur M, Durand D, Dumont J, Durand G, F6er J, Aneray J (1982) Effets of streptozotoin-indued diabetes mellitus on the bindin and uptake of asialo-orosomuoid by isolated hepatoytes from rats. Eur J Biohem 123: Dodeur M, Coumoul S, Durand D, Durand G, F6er J, Aneray J (1983) Diabetes indued variation in hepati bindin protein. Biohem Biophys Res Comm 115: Zeitlin PL, Hubbard AL (1982) Cell surfae distribution and intraellular fate of asialolyoproteins: a morpholoial and biohemial study of isolated rat hepatoytes and mono-layer ultures. J Cell Biol 92: Manini G, Carbonara AO, Heremans JF (1965) Immunohemial quantitation of antiens by sinle radial immunodiffusion. Immunohemistry 2: Laurell CB (1966) Quantitative estimation of proteins by eletrophoresis in aarose el ontainin antibodies. Anal Biohem 15: Biou D, Monnet D, Millet F, F6er J, Durand G (1984) An immunohemial proedure to evaluate the deree of desialylation of alphal-lyoprotein in rat serum. J Immunol Methods 74: Wilder RL, Yven CC, Subbarao B, Woods VL, Alexander CB, Mae RG (1979) Tritium radio-labellin of protein A and antibody to hih speifi ativity: appliation to ell surfae antien radioimmunoassays. J Immunol Methods 28: Skeiner LB, (1981) ELSFIT Users Manual. Division of Clinial Pharmaoloy, University of California, San Franiso 11. Gomeni R (1984) PHARM, an interative raphi proram for individual and population pharmaokineti parameter estimation. Comput Biol Med 14: Clarenbur R (1983) Asialolyoprotein reeptor is univolved in learin intat lyoproteins from rat blood. Am J Physiol 244: G247-G Reoezi E, Debanne MT, Hatton MWC, Koj A (1978) Elimination of asialofetuin and asialo-orosomuoid by the intat rat. Quantitative aspets of the hepati learane mehanism. Biohim Biophys Ata 541: Dodeur M, Coumoul S, Sarmato P, Durand G, F6er J, Aneray J (1984) Asialo-orosomuoid deradation by normal and diabeti rat hepatoytes. Eur J Biohem 140: Jonsson A, Wales JK (1976) Blood lyoprotein levels in diabetes mellitus. Diabetoloia 12: Guzdek A (1979) The influene of experimental diabetes on the serum and urinary lyopeptide ompounds in rats. Endorinoloy 74: Pardride WM, Van Herle AJ, Naruse RT, Fierer G, Costin A (1981) In vivo quantifiation of reeptor-mediated uptake of asialol)oproteins by rat liver. J Biol Chem 258: Chandramouli V, Williams S, Marshall JS, Carter JR (1977) Cell surfae hanes in diabeti rats. Studies of letin bindin to liver ell plasma membranes. Biohim Biophys Ata 465: Nassar K, Chen S, Levy D (1981) The effet of diabetes on hepatoyte plasma membrane fluidity and Conanavalin A-indued alutination. Exp Cell Res 132: Durand G, Dumont JP, Appel M, Durand D, Davy J, F6er J, Aneray J (1980) Effet of streptozotoin diabetes on siali aid and lyoprotein bindin of isolated hepatoytes. Horm Metab Res 12: Jefferson LS (1980) Role of insulin in the reulation of protein synthesis. Diabetes 29: Draznin B, Trowbride M (1982) Inhibition of intraellular proteolysis by insulin in isolated rat hepatoytes. Possible role of internalized hormone. J Biol Chem 257: Hutson N J, Lloyd CE, Mortimore GE (1982) Deradation of intra and extrahepati protein by livers of normal and diabeti mie: differential responses to starvation. Pro Natl Aad Si USA 79: Reeived: l 5 Otober 1985 and in revised form: 22 April 1986 Professor G. Durand UER des Sienes Pharmaeutiques Laboratoire de Biohimie 5 rue J. B. C16ment Chfitenay-Malabry C6dex Frane