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The effects of diabetes and insulin on glycoprotein metabolism by rat liver
Journal of Hepatology, 1985; 1:629-638
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Elsevier HEP 0065
The Effects of Diabetes and Insulin on Glycoprotein Metabolism by Rat Liver
N o b u y o s h i T a n a k a 1,*, M a r k Leaning 2, M a u r e e n Taylor 1 and John A. Summerfield 1 Departments of 1Medicine and 2Academic Medical Physics, Royal Free Hospital School of Medicine, Pond Street, London NW3 2QG (U. K.) (Received 12 December, 1984) (Accepted 16 April, 1985)
Summary (1) The hepatic metabolism of [t2SI]agalactoorosomucoid ([125I]AGOR) was studied in normal and streptozotocin-induced diabetic rats. (2) The blood clearance, hepatic transport time and rate of catabolism of [I2SI]AGOR were calculated from data of the blood [12SI]AGOR disappearance rates and the appearance in blood of acid-soluble catabolites. (3) In control rats the blood clearance of [I2SI]AGOR was rapid (8.7 + 0.6 ml/min) and the hepatic transport time of the ligand was 12.8 + 0.7 min. Insulin prolonged the hepatic transport time (18.1 + 1.9 min) and depressed ligand catabolism. Chloroquine had similar effects. (4) Diabetes impaired hepatic [12SI]AGOR uptake as judged by the prolonged blood clearance rate and depressed ligand catabolism but did not alter ligand transport time. The measured parameters returned to normal when diabetic animals were rendered acutely normoglycaemic. Diabetic rats, in which implanted osmotic insulin pumps had maintained normoglycaemia for 3 days, cleared [1251]A G O R from the blood more rapidly than controls. This effect appeared to be due to the lower blood glucose levels in this group. (5) The experiments have shown the complexity of the effects of insulin and dia* Present address: Department of Internal Medicine, School of Medicine, Kanazawa University, Kanazawa, Japan. Address for correspondence and reprint requests: Dr. John A. Summerfield, Department of Medicine, Royal Free Hospital, Pond Street, London NW3 2QG, U.K. 0168-8278/85/$03.30© 1985 Elsevier Science Publishers B.V. (Biomedical Division)
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betes mellitus on the uptake and processing of a glycoprotein by the hepatic mannose receptor.
Introduction
Glycoproteins terminating in mannose (Man) or N-acetylglucosamine (GIcNAc) are rapidly cleared from the circulation by carbohydrate-specific receptor-mediated pinocytosis [1]. The receptors are located on hepatic sinusoidal cells, probably on both endothelial [2-4] and Kupffer cells [3,4]. Uptake of these glycoproteins is inhibited by high glucose concentrations and diabetes mellitus [2,5] and enhanced by fasting [2]. Much remains to be learned of how this ligand uptake and processing system functions and the mechanisms by which physiological and pathological events modulate it. In this paper we report the results of analysis of the in vivo metabolism of radioiodinated agalactoorosomucoid ([125I]AGOR), a GlcNAc-terminated glycoprotein in normal, diabetic and chloroquine-treated rats. The data show that insulin and diabetes have complex effects on the uptake and processing of this glycoprotein by the liver. Materials and Methods
Materials Streptozotocin, human orosomucoid, neuraminidase (type X) (EC 3.2.1.18), crystalline bovine insulin (23.6 IU/mg) and chloroquine (BSA) were obtained from Sigma (Poole, U.K.). fl-Galactosidase (EC 3.2.1.234) was from Miles (Slough, U.K.), and galactose dehydrogenase (EC 1.1.1.48) from Boehringer Mannheim (F.R.G.). Carrier free Na12SI was from Amersham (Amersham, U.K.) and Alzet osmotic minipumps (Model 2001) were obtained from Alza (Palo Alto, CA, U.S.A.).
Preparation of the glycoprotein ligand Agalactoorosomucoid (AGOR) was prepared by the modification of human orosomucoid. The terminal non-reducing carbohydrate residues of orosomucoid were modified by incubation with purified neuraminidase to yield asialoorosomucoid followed by incubation with purified fl-D-galactosidase to yield agalactoorosomucoid [6]. The efficiency of removal of the terminal carbohydrate residues was assessed by estimating released sialic acid using the thiobarbituric acid assay [7] and galacrose using galactose dehydrogenase. At least 17 mol of carbohydrate were removed/mol of orosomucoid at each step. The quality of the AGOR preparation was assessed in 2 further ways. Cross-inhibition studies, in vitro, of the inhibition by [12SI]AGOR of [125I]asialoorosomucoid uptake by isolated rat hepatocytes showed that only 1.4% of the [125I]AGOR preparation was bound by hepatocytes. Injection of excess mannan (1 mg) with [125I]AGOR in vivo reduced the hepatic uptake of [125I]AGOR by approximately 95%. The products were purified by chromatography on Sephacryl $200 (Pharmacia, Uppsala, Sweden). AGOR (200/~g) was radio-
EFFECTS OF DIABETES AND INSULIN ON GLYCOPROTEIN METABOLISM
631
iodinated with carrier-free Na125I by a solid phase Enzymobead system (Bio-Rad, Richmond, CA, U.S.A.) which used immobilized lactoperoxidase and glucose oxidase [2]. The specific activity of [125I]AGOR was 8-17.5/~Ci//zg. The percentage of TCA-soluble radioactivityin the [~25I]AGOR was approximately 2%.
Preparation of animals Male Sprague-Dawley rats (300-350 g) bred in the Comparative Biology Unit of the Royal Free Hospital School of Medicine were used. In control rats the blood glucose concentration was found to increase during the course of the in vivo experiments from 6.8 + 0.4 mmol/l (mean + SEM: n = 3) to 18.1 + 1.3 mmol/I. This hyperglycaemia was probably part of a 'stress' reaction. Glucose is a competitive inhibitor of [125I]AGOR uptake [2,5] and the changing blood glucose concentrations would affect the dynamics of hepatic uptake of ligand. To prevent the blood glucose concentration rising during the experiment, it was clamped at or below 7.7 + 0.3 mmol/l by an insulin injection (2 IU) 30 min before administering ligand in one group of 3 rats. Diabetes mellitus was induced by an intraperitoneal injection of streptozotocin (65 mg/kg in 0.09 M NaCI, 0.01 M citrate buffer, pH 3.5). The animals were used 7 days later when the blood glucose concentrations were greater than 27 mmol/l. A group of diabetic rats was rendered acutely normoglycaemic. The blood glucose concentrations were clamped at or below 7.5 + 0.1 mmol/l (n = 3) by intramuscular injections of large doses of insulin (100-120 IU) commencing 3 h before the experiments. In another group of diabetic rats an osmotic minipump containing insulin (17.6 mg/mi in 0.09 M NaC1, 0.1 M NaHCO 3 buffer, pH 8) was implanted subcutaneously in the subscapular region under ether anaesthesia. The osmotic pump administered 10 IU insulin/24 h for 3 days prior to and duririg the experiment. The blood glucose concentration at the start of the experiment was 3.2 + 0.53 mmol/1 and rose to 5.8 + 0.7 mmol/l (n = 3). Chloroquine-treated rats received an intramuscular injection of 7 mg, 30 min before the experiment and 1.8 mg intravenously just prior to ligand administration. Blood glucose concentrations were estimated before and at 5-min intervals throughout the experiment using the Reflomat system (Boehringer Mannheim, F.R.G.).
In vivo metabolism of the glycoprotein Under ether anaesthesia, [x25I]AGOR (1 ~zg) was injected into a femoral vein and 13 blood samples (0.5 ml) were obtained by repeated cardiac puncture. To obtain sufficient data for mathematical analysis, the samples were collected over 30 rain in normal rats, over 60 min in rats rendered acutely normoglycaemic and over 90 min in rats with uncontrolled diabetes. Because of the relatively large volume of blood removed during the experiments, the [125I]AGOR analysis was performed on the first 6 samples only. Furthermore, measurements of the fall in haematocrit (data not shown) indicated that the overall change in blood volume was small. The use of the same protocol throughout, permitted comparison between the experimental groups. The liver, spleen, kidneys and lungs were then rapidly removed, weighed and the radioactivity content estimated. Portions of tissue (0.5 g) homogenized in water (4 ml), or blood, were mixed with an equal volume of trichloroacetic acid (25 g/100 ml) and centrifuged at 3500 x g for 15 min. This method precipitated approximately 98% of [125I]AGOR in standards or when added to
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serum. Radioactivity in the supernate and pellet was estimated. The hepatic accumulation of ligand at 30 min in uncontrolled diabetes, where the blood disappearance of [t25I]AGOR was assessed over 90 min, was obtained from another group (n = 3) of identically treated rats. The blood disappearance curve of [125I]AGOR was d e t e r m i n e d from the experimental measurements of blood acid-precipitable radioactivity. Catabolism of [125I]AGOR was determined from the experimental m e a s u r e m e n t s of blood acid-soluble radioactivity. The spleen, kidneys and lungs together accounted for less than 4% of the injected dose of [t25I]AGOR.
Data analysis (A) Blood clearance of p251]AGOR was estimated from blood data collected in the first 10 min after ligand injection (the first 6 samples). Later m e a s u r e m e n t s were excluded to avoid interference from larger catabolic products which were acid precipitable (Fig. 1). The data of blood [~25I]AGOR concentration (nM) over the first 10 min were fitted with weighted least s q u a r e s to the sum of two exponentials Yd(t) = A t e x p ( - a t t ) + A 2
100.. 50'~y d 20. (t) i04~ YC (t) /5-] " , . . ! ".~ ..~. !Ligandcataia01jsnl !
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0.5 0"2t 0,1 0
10 tc itJ ICq0 lime (minutes)
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Fig. 1. Analysis of metabolism of [zzSI]AGOR. Data are shown as mean + standard error for the control group. Acid-precipitable data are fitted with the curve Yd(t). The first 10 min of ya(t) are used to derive blood clearance. Acid-soluble data are fitted with the curve yc(t). The initial decline of the curve yc(t) is caused by loss from the circulation of free radioiodine that was present in the [t~I]AGOR. The onset of catabolism was defined as the time tc when the blood level of catabolites yc(t) rose above 2% of the injected dose. Ligand catabolism was defined as the difference between the blood concentration of catabolites at tc and tc + 10 min.
EFFECTSOF DIABETESAND INSULINON GLYCOPROTEINMETABOLISM
633
exp(-a2t), using the MiNurr program [8]. The speed of clearance of [lZSI]AGOR from blood was characterised by the measure Dose CR10 -
(ml/min) Ii
ya(t)dt
where dose is the amount of injected [~zSI]AGOR (nmoles) and the integral is the area under the blood disappearance curve over the first 10 min. The true blood clearance is given by CR®, however, this was not used as the fitted curves could not be reliably extrapolated beyond 10 min. (B) Hepatic transport time of ligand was estimated as the interval between injection of ligand to the point when the blood level of acid-soluble material rose above 2% of the injected dose (Fig. 1). This point is taken to represent the onset of catabolism, and depends not only on the hepatic transport time, but to a small extent on the clearance rate from blood. In order to determine this interval to, the data were interpolated with a least squares cubic spline y¢(t) with three internal knots using a subroutine from the NAG library (NAG Library Manual) [9]. (C) Rate of ligand catabolism was assumed to be proportional to the excess percentage of catabolites in blood 10 min after the onset of catabolism (Fig. 1). This was determined from the cubic spline interpolation, y¢(tc+ 10). The figure is an underestimate of total ligand catabolism since acid-soluble material is continuously exchanging between the circulation and peripheral tissues (mainly muscle, skin and gut), and being excreted. However, this estimate did permit comparisons between different treatments. The parameter reflects total ligand flux and therefore depends on the blood clearance rate, in addition to the catabolic rate constant. When the blood clearance rate for different groups is the same, differences in the parameter must represent differences in the catabolic rate constant.
Statistics The results from different groups of rats were compared using a pooled independent t-test. Results
Table 1 summarises the metabolism of [125I]AGOR for the different groups.
Control group The volume of distribution (Vd) of [125I]AGOR was 23.1 + 0.7 ml and did not differ significantly in the treatment groups. In control rats the blood clearance of [I25I]AGOR was rapid (8.7 + 0.6 ml/min; mean + SEM, n = 3). The hepatic transport time of [I25I]AGOR was 12.8 + 0.7 min. Ligand catabolism was 4.9 _+ 0.3% of the injected dose. By 30 min, the liver contained 36 + 1.6% of the injected dose consisting of both intact [125I]AGOR and catabolites.
N. TANAKA et al.
634 TABLE 1 METABOLISM OF [I~I]AGALACTOOROSOMUCOID Group a
Blood clearance rate (ml/min)
Hepatic transport time (min)
Catabolic rate Hepatic accumulation (% of dose/10 rain) (% of dose at 30 rain)
Control
8.7 + 0.6
12.8 + 0.7
4.9 + 0.3
36 + 1.6
Insulintreated
8.2 + 0.3
18.1 + 1.9
3.4 +_.0.3
54.8 + 2.0
14.5 + 1.2
3.2 + 0.4
43.3 + 3.6
15.2 + 1.4
4.1 _+0.3
ND
13.6+0.1
4.9+0.5
48.7+3.0
15.9 + 1.1
2.9 _ 0.5
44_7 + 3.9
Diabetic uncontrolled 4.6 + 0.5 acute normoglycaemia 9.0 + 0.7 insulin osmotic pump 11.7+1.1 Chloroquinetreated
9_4 + 0.3
a n = 3 in each group. ND = not determined.
Insulin treatment Insulin did not alter the blood clearance of [mSI]AGOR (8.21 + 0.3 ml/min) but had two effects on hepatic A G O R metabolism. Firstly hepatic transport time of ligand was delayed to 18.1 + 1.91 min (P < 0.06) and secondly ligand catabolism was reduced (3.4 + 0.3%; P < 0.02). These changes were reflected in significantly greater ligand accumulation in the liver at 30 min (54.8 + 2.0%; P < 0.01).
Chloroquine Pretreatment of rats with chloroquine did not alter the blood clearance of [125I]AGOR (9.4 + 0.3 ml/min) but prolonged the hepatic transport time of ligand (15.9 + 1.1 ml/min, P < 0.07) and significantly reduced the ligand catabolism (2.9 + 0.5%, P < 0.03) (Table 1). At 30 min the liver contained 44.7 + 3.9% of the ligand.
Diabetes mellitus Diabetes significantly reduced the blood clearance of [125I]AGOR (4.6 + 5 ml/min, P < 0.005) but did not alter the hepatic transport time of ligand (14.5 _+ 1.2 min). However, diabetes markedly depressed ligand catabolism (3.2 + 0.4%, P < 0.03). The result was that the quantity of [lZSI]AGOR in the liver at 30 min (43.3 _+ 3.6%) was not significantly different f r o m the controls. The effect of rendering the diabetic animals acutely normoglycaemic was to return the measured p a r a m e t e r s to normal. In diabetic rats m a d e normoglycaemic with an implanted osmotic insulin pump, the blood clearance of [125I]AGOR was m o r e rapid than normal (11.7 + 1.1 ml/min, P < 0.075). This m a y have been related to the lower blood glucose in this
EFFECTS OF DIABETES AND INSULIN ON GLYCOPROTEIN METABOLISM
635
group (3.2 + 0.5 mmol/1) compared to controls (6.7 + 0.3 mmol/1). The hepatic transport time of ligand and ligand catabolism were similar to the controls. The result was that ligand accumulation in the liver at 30 min (48.7 + 3.0%) was significantly higher (P < 0.05) than in controls. Discussion
Many macromolecules are recognised and bound by specific cell surface receptors and are then internalized via clathrin-coated pits [10,11] to form vesicles [12,13]. The ligand receptor complexes are uncoupled [12,14] by acidification of these vesicles [15-18] and ligand and receptor are then segregated into separate vesicles [19]. Ligand-containing vesicles are usually targeted to secondary lysosomes for degradation while receptor-containing vesicles appear to be recycled to the cell surface where the receptors can be reused [20-26]. A G O R is an N-acetylglucosamine-terminated glycoprotein that is rapidly cleared from the blood by mannose/N-acetylglucosamine- (Man/GlcNAc-) receptors located on hepatic sinusoidal cells [1]. In these studies we have used these concepts of ligand binding, intracellular transport and lysosomal catabolism to analyse dynamic data obtained from in vivo experiments of [~2sI]AGOR metabolism to determine the effects of diabetes mellitus and insulin on this processing. Among the advantages of analysing data derived from an intact animal are optimized cell function and preservation of cell polarity. Studies of another glycoprotein recognition system indicate that uptake rates may be up to 10 times greater in vivo than in vitro [27]. Furthermore hepatic sinusoidal cells which mediate the uptake of [~25I]AGOR are in direct contact with blood so that transfer across an interstitial space could be neglected. The main disadvantage identified was the extrahepatic loss of some of the [~25I]AGOR (approximately 10% in the first 10 min; unpublished data) and the rising blood glucose concentration during the experiment. Extrahepatic loss of ligand to other tissues (mainly bone marrow) was similar in all the experimental groups. It made little difference to the estimates of the blood clearance parameters and did not influence comparisons between the groups. The extrahepatic loss can be reduced using glycoproteins of higher molecular weight (unpublished data) and thus may represent non-specific 'leakage' of ligand from the circulation. The magnitude of the hyperglycaemia was surprising and to our knowledge this important variable influencing the in vivo metabolism of glycoproteins has not been noted before. The hyperglycaemia probably reflected the stress of repeated cardiac puncture and removal of blood in the anaesthetised rat. Although the blood glucose concentration could be 'clamped' by insulin pretreatment, it is possible that other components of this 'stress' reaction might have influenced the metabolism of [125I]AGOR. The blood clearance data confirmed that [125I]AGOR is rapidly removed from the blood by the liver. About 30% of the blood volume (VD) was cleared of ligand each minute. We confirmed that the rate of ligand clearance was slower in the diabetic rat and also demonstrated that rendering the diabetic rat acutely normoglycaemic returned the blood clearance rate of [~25I]AGOR to control values. Further-
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N. TANAKAet al_
more we showed that in diabetic animals, rendered normoglycaemic with an implanted insulin pump, the blood clearance of [125I]AGOR was even greater than in the controls. The mechanism(s) involved could not be resolved although it is likely they were mediated by the blood glucose concentration. Probable mechanisms are that the lower blood glucose levels in rats with an implanted insulin pump could either (a) reduce the inhibitory effect of glucose on ligand uptake [2,5] or (b) increase cell surface expression of mannose receptors (up-regulation) [28]. Although regulation of mannose receptor number on the cell surface was not observed in studies of endothelial cells isolated from diabetic rats [2], it has been demonstrated in macrophages [28]. The discrepancy could reflect either differences in experimental design or different populations of mannose receptors on endothelial and Kupffer cells [3,4]. The data also provided an estimate of the duration of ligand transport to the lysosomes measured from the time of ligand injection until ligand catabolism was observed. This estimate is the sum of several events including ligand internalization, intracellular transport and the time the ligand spends in the lysosome awaiting catabolism. Two agents, insulin and chloroquine, prolonged the hepatic transport time. Chloroquine, a weak base, by raising the pH of endocytic vesicles, inhibits receptor-ligand uncoupling, which seems to be necessary before ligand is delivered to the lysosomes [16,23,24]. Such inhibition of uncoupling would be expected to prolong the hepatic transport time. The effect of insulin on hepatic transport time of ligand was unexpected and even more marked than that of chloroquine in normal rats, although in diabetic rats insulin did not have this effect. It is clear that different ligand-receptor complexes may be internalized together by a single coated pit [11] and that receptors may compete for entry into the coated pit [29]. Thus it is conceivable that insulin delays the intracellular ~ransport time of [125I]AGOR by competing with it for internalization. Other possibilities include effects of insulin on intracellular vesicle transport. The reduction by chloroquine of the ligand catabolic rate is due to this amine inhibiting proteolytic enzymes in the lysosomes [16,23,26]. In contrast the inhibitory effect of diabetes on ligand catabolism is of uncertain mechanism. However, the lower ligand catabolic rate observed in insulin-treated control rats may have been due to reduced availability of ligand in the lysosomes due to the prolonged hepatic transport time. In conclusion, these experiments have demonstrated the complex effects of diabetes mellitus and insulin treatment on the uptake and processing of a glycoprotein by a mannose-mediated glycoprotein system in the liver. These experimental approaches will permit analysis of the mechanisms mediating the endocytosis of proteins by the liver.
Acknowledgements J.A.S. is a Wellcome Trust Senior Clinical Fellow.
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References 1 Neufeld, E. F. and Ashwell, G., Carbohydrate recognition systems for receptor-mediated pinocytosis. In: W_ J. Lennarz (Ed.), The Biochemistry of Glycoproteins and Proteoglycans, Plenum Press, New York, 1980:241-266. 2 Summerfield, J. A., Vergalla, J. and Jones, E. A., Modulation of a glycoprotein recognition system on rat hepatic endothelial cells by glucose and diabetes mellitus, J. Clin. Invest., 1982; 69: 1337-1347_ 3 Hubbard, A_ L., Wilson, G_, Ashwell, G. and Stukenbrok, H., An electron microscope autoradiographic study of the carbohydrate recognition systems in rat liver, J. Cell. Biol., 1979; 83: 47-64. 4 Praaning van Dalen, M. and Knook, D. L., Quantitative determination of in vivo endocytosis by rat liver Kupffer and endothelial cells facilitated by an improved cell isolation method, FEBS Lett., 1982; 141: 229-232. 5 Pizzo, S. V., Lehrman, M. A., Imber, M. J. and Guthrow, C. E., The clearance of glycoproteins in diabetic mice, Biochem. Biophys. Res. Commun., 1981; 101: 704-708. 6 Kawasaki, T. and Ashwell, G., Isolation and characterization of an avian hepatic binding protein specific for N-acetylglucosamine-terminated glyeoproteins, J_ Biol. Chem., 1977; 252: 6536-6543. 7 Warren, L., The thiobarbituric acid assay of sialic acids, J. Biol. Chem., 1959; 234: 1971-1975. 8 James, F. and Roos, M., 'MINuIT', Cern Computer Centre Program Library, No. D506, Data Handling Division, Cern, 1979, Geneva, Switzerland. 9 Nag Library Manual, Mark 10, Numerical Algorithms Group, Oxford, 1982. 10 Goldstein, J. L., Anderson, R_ G. W. and Brown, M. S., Coated pits, coated vesicles, and receptormediated endocytosis, Nature (Lond.), 1979; 279: 679-685. 11 Pastan, I. H. and Willingham, M. C., Journey to the center of the cell - - Role of the receptosome, Science, 1981; 214: 504-509. 12 Bridges, K., Harford, J., Ashwell, G. and Klausner, R. D., Fate of receptor and ligand during endocytosis of asialoglycoproteins by isolated hepatocytes, Proc. Nat. Acad. Sci. (USA), 1982; 79: 350-354. 13 Weigel, P. H., Evidence that the hepatic asialoglycoprotein receptor is internalized during endocytosis at low temperature, Biochem. Biophys. Res. Commun., 1981; 101: 1419-1425. 14 Baenziger, J. U. and Fiete, D., Recycling of the hepatocyte asialoglycoprotein receptor does not require delivery of ligand to lysosomes, J. Biol. Chem., 1982; 257: 6007-6009. 15 Tycko, B. and Maxfield, F. R., Rapid acidification of endocytic vesicles containing alpha2-macroglobulin, Cell, 1981; 28: 643-651. 16 Maxfield, F_ R., Weak bases and ionophores rapidly and reversibly raise the pH of endocytic vesicles in cultured mouse fibroblasts, J. Cell. Biol., 1982; 95: 676-681. 17 Fiete, D., Brownell, M. D. and Baenziger, J. U., Evidence for transmembrane modulation of the ligand-binding site of the hepatocyte galactose/N-acetylgalactosamine-specific receptor, J. Biol. Chem., 1983; 258: 817-823. 18 Forgac, M., Cantley, L., Wiedenmann, B., Altstiel, L. and Branton, D., Clathrin-coated vesicles contain an ATP-dependent proton pump, Proc. Nat. Acad. Sci. (USA), 1983; 80: 1300-1303. 19 Geuze, H. J., Slot, J. W., Strous, G. J. A. M., Lodish, H. F. and Schwartz, A. L., Intracellular site of asialoglycoprotein receptor-ligand uncoupling - - Double-label immunoelectronmicroscopy during receptor-mediated endocytosis, Cell, 1983; 32: 277-287. 20 Anderson, R. G. W., J. L. Goldstein and Brown, M. S., A mutation that impairs the ability of lipoprotein receptor to Iocalise in coated pits on cell surface of human fibroblasts, Nature (Lond_), 1977; 270: 695-699. 21 Tolleshaug, H. and Berg, T., Chloroquine reduces the number of asialoglycoprotein receptors in the hepatocyte plasma membrane, Biochem. Pharmacol., 1979; 28: 2912-2922_ 22 Steer, C. J. and Ashwell, G., Studies on a mammalian hepatic binding protein specific for asialoglycoproteins - - Evidence for receptor recycling in isolated rat hepatocytes, J. Biol. Chem., 1980; 255: 3008-3013. 23 Gonzalez-Noriega, A., Grubb, J. H_, Talkad, V. and Sly, W_ S., Chloroquine inhibits lysosomal en-
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zyme pinocytosis and enhances lysosomal enzyme secretion by impairing receptor recycling, J. Cell. Biol., 1980; 85: 839-852. Stahl, P., Schlesinger, P. H., Sigardson, E., Rodman, J. S. and Lee, Y. C., Receptor-mediated pinocytosis of mannose glycoconjugates by macrophages - - Characterization and evidence for receptor recycling, Cell, 1980; 19: 207-215. Kaptan, J., Evidence for reutilization of surface receptors for ~-macroglobufin - - Protease complexes in rabbit alveolar macrophages, Cell, 1980; 19: 197-205. Van Leuven, F., Cassiman, J. J. and Van Den Berghe, H., Primary amines inhibit recycling of M2 receptors in fibroblasts, Cell, 1980; 20: 37-43. Pardridge, W. M., Van Herle, A. J., Naruse, R_ T., Fierer, O. and Costin, A., In vivo quantification of receptor-mediated uptake of asialoglycoprotein by rat liver, J. Biol. Chem., 1983; 258: 990-994. Weiel, J. E. and Pizzo, S. V., Down-regulation of macrophage mannose/N-acetylglucosamine receptors by elevated glucose concentration, Biochim. Biophys. Acta, 1983; 759: 170-175. Bretscher, M. S. and Pearse, B. M. F., Coated pits in action, Cell, 1984; 38: 3-4.
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The Effects of Diabetes and Insulin on Glycoprotein Metabolism by Rat Liver
N o b u y o s h i T a n a k a 1,*, M a r k Leaning 2, M a u r e e n Taylor 1 and John A. Summerfield 1 Departments of 1Medicine and 2Academic Medical Physics, Royal Free Hospital School of Medicine, Pond Street, London NW3 2QG (U. K.) (Received 12 December, 1984) (Accepted 16 April, 1985)
Summary (1) The hepatic metabolism of [t2SI]agalactoorosomucoid ([125I]AGOR) was studied in normal and streptozotocin-induced diabetic rats. (2) The blood clearance, hepatic transport time and rate of catabolism of [I2SI]AGOR were calculated from data of the blood [12SI]AGOR disappearance rates and the appearance in blood of acid-soluble catabolites. (3) In control rats the blood clearance of [I2SI]AGOR was rapid (8.7 + 0.6 ml/min) and the hepatic transport time of the ligand was 12.8 + 0.7 min. Insulin prolonged the hepatic transport time (18.1 + 1.9 min) and depressed ligand catabolism. Chloroquine had similar effects. (4) Diabetes impaired hepatic [12SI]AGOR uptake as judged by the prolonged blood clearance rate and depressed ligand catabolism but did not alter ligand transport time. The measured parameters returned to normal when diabetic animals were rendered acutely normoglycaemic. Diabetic rats, in which implanted osmotic insulin pumps had maintained normoglycaemia for 3 days, cleared [1251]A G O R from the blood more rapidly than controls. This effect appeared to be due to the lower blood glucose levels in this group. (5) The experiments have shown the complexity of the effects of insulin and dia* Present address: Department of Internal Medicine, School of Medicine, Kanazawa University, Kanazawa, Japan. Address for correspondence and reprint requests: Dr. John A. Summerfield, Department of Medicine, Royal Free Hospital, Pond Street, London NW3 2QG, U.K. 0168-8278/85/$03.30© 1985 Elsevier Science Publishers B.V. (Biomedical Division)
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N. T A N A K A et al.
betes mellitus on the uptake and processing of a glycoprotein by the hepatic mannose receptor.
Introduction
Glycoproteins terminating in mannose (Man) or N-acetylglucosamine (GIcNAc) are rapidly cleared from the circulation by carbohydrate-specific receptor-mediated pinocytosis [1]. The receptors are located on hepatic sinusoidal cells, probably on both endothelial [2-4] and Kupffer cells [3,4]. Uptake of these glycoproteins is inhibited by high glucose concentrations and diabetes mellitus [2,5] and enhanced by fasting [2]. Much remains to be learned of how this ligand uptake and processing system functions and the mechanisms by which physiological and pathological events modulate it. In this paper we report the results of analysis of the in vivo metabolism of radioiodinated agalactoorosomucoid ([125I]AGOR), a GlcNAc-terminated glycoprotein in normal, diabetic and chloroquine-treated rats. The data show that insulin and diabetes have complex effects on the uptake and processing of this glycoprotein by the liver. Materials and Methods
Materials Streptozotocin, human orosomucoid, neuraminidase (type X) (EC 3.2.1.18), crystalline bovine insulin (23.6 IU/mg) and chloroquine (BSA) were obtained from Sigma (Poole, U.K.). fl-Galactosidase (EC 3.2.1.234) was from Miles (Slough, U.K.), and galactose dehydrogenase (EC 1.1.1.48) from Boehringer Mannheim (F.R.G.). Carrier free Na12SI was from Amersham (Amersham, U.K.) and Alzet osmotic minipumps (Model 2001) were obtained from Alza (Palo Alto, CA, U.S.A.).
Preparation of the glycoprotein ligand Agalactoorosomucoid (AGOR) was prepared by the modification of human orosomucoid. The terminal non-reducing carbohydrate residues of orosomucoid were modified by incubation with purified neuraminidase to yield asialoorosomucoid followed by incubation with purified fl-D-galactosidase to yield agalactoorosomucoid [6]. The efficiency of removal of the terminal carbohydrate residues was assessed by estimating released sialic acid using the thiobarbituric acid assay [7] and galacrose using galactose dehydrogenase. At least 17 mol of carbohydrate were removed/mol of orosomucoid at each step. The quality of the AGOR preparation was assessed in 2 further ways. Cross-inhibition studies, in vitro, of the inhibition by [12SI]AGOR of [125I]asialoorosomucoid uptake by isolated rat hepatocytes showed that only 1.4% of the [125I]AGOR preparation was bound by hepatocytes. Injection of excess mannan (1 mg) with [125I]AGOR in vivo reduced the hepatic uptake of [125I]AGOR by approximately 95%. The products were purified by chromatography on Sephacryl $200 (Pharmacia, Uppsala, Sweden). AGOR (200/~g) was radio-
EFFECTS OF DIABETES AND INSULIN ON GLYCOPROTEIN METABOLISM
631
iodinated with carrier-free Na125I by a solid phase Enzymobead system (Bio-Rad, Richmond, CA, U.S.A.) which used immobilized lactoperoxidase and glucose oxidase [2]. The specific activity of [125I]AGOR was 8-17.5/~Ci//zg. The percentage of TCA-soluble radioactivityin the [~25I]AGOR was approximately 2%.
Preparation of animals Male Sprague-Dawley rats (300-350 g) bred in the Comparative Biology Unit of the Royal Free Hospital School of Medicine were used. In control rats the blood glucose concentration was found to increase during the course of the in vivo experiments from 6.8 + 0.4 mmol/l (mean + SEM: n = 3) to 18.1 + 1.3 mmol/I. This hyperglycaemia was probably part of a 'stress' reaction. Glucose is a competitive inhibitor of [125I]AGOR uptake [2,5] and the changing blood glucose concentrations would affect the dynamics of hepatic uptake of ligand. To prevent the blood glucose concentration rising during the experiment, it was clamped at or below 7.7 + 0.3 mmol/l by an insulin injection (2 IU) 30 min before administering ligand in one group of 3 rats. Diabetes mellitus was induced by an intraperitoneal injection of streptozotocin (65 mg/kg in 0.09 M NaCI, 0.01 M citrate buffer, pH 3.5). The animals were used 7 days later when the blood glucose concentrations were greater than 27 mmol/l. A group of diabetic rats was rendered acutely normoglycaemic. The blood glucose concentrations were clamped at or below 7.5 + 0.1 mmol/l (n = 3) by intramuscular injections of large doses of insulin (100-120 IU) commencing 3 h before the experiments. In another group of diabetic rats an osmotic minipump containing insulin (17.6 mg/mi in 0.09 M NaC1, 0.1 M NaHCO 3 buffer, pH 8) was implanted subcutaneously in the subscapular region under ether anaesthesia. The osmotic pump administered 10 IU insulin/24 h for 3 days prior to and duririg the experiment. The blood glucose concentration at the start of the experiment was 3.2 + 0.53 mmol/1 and rose to 5.8 + 0.7 mmol/l (n = 3). Chloroquine-treated rats received an intramuscular injection of 7 mg, 30 min before the experiment and 1.8 mg intravenously just prior to ligand administration. Blood glucose concentrations were estimated before and at 5-min intervals throughout the experiment using the Reflomat system (Boehringer Mannheim, F.R.G.).
In vivo metabolism of the glycoprotein Under ether anaesthesia, [x25I]AGOR (1 ~zg) was injected into a femoral vein and 13 blood samples (0.5 ml) were obtained by repeated cardiac puncture. To obtain sufficient data for mathematical analysis, the samples were collected over 30 rain in normal rats, over 60 min in rats rendered acutely normoglycaemic and over 90 min in rats with uncontrolled diabetes. Because of the relatively large volume of blood removed during the experiments, the [125I]AGOR analysis was performed on the first 6 samples only. Furthermore, measurements of the fall in haematocrit (data not shown) indicated that the overall change in blood volume was small. The use of the same protocol throughout, permitted comparison between the experimental groups. The liver, spleen, kidneys and lungs were then rapidly removed, weighed and the radioactivity content estimated. Portions of tissue (0.5 g) homogenized in water (4 ml), or blood, were mixed with an equal volume of trichloroacetic acid (25 g/100 ml) and centrifuged at 3500 x g for 15 min. This method precipitated approximately 98% of [125I]AGOR in standards or when added to
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serum. Radioactivity in the supernate and pellet was estimated. The hepatic accumulation of ligand at 30 min in uncontrolled diabetes, where the blood disappearance of [t25I]AGOR was assessed over 90 min, was obtained from another group (n = 3) of identically treated rats. The blood disappearance curve of [125I]AGOR was d e t e r m i n e d from the experimental measurements of blood acid-precipitable radioactivity. Catabolism of [125I]AGOR was determined from the experimental m e a s u r e m e n t s of blood acid-soluble radioactivity. The spleen, kidneys and lungs together accounted for less than 4% of the injected dose of [t25I]AGOR.
Data analysis (A) Blood clearance of p251]AGOR was estimated from blood data collected in the first 10 min after ligand injection (the first 6 samples). Later m e a s u r e m e n t s were excluded to avoid interference from larger catabolic products which were acid precipitable (Fig. 1). The data of blood [~25I]AGOR concentration (nM) over the first 10 min were fitted with weighted least s q u a r e s to the sum of two exponentials Yd(t) = A t e x p ( - a t t ) + A 2
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0.5 0"2t 0,1 0
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30
Fig. 1. Analysis of metabolism of [zzSI]AGOR. Data are shown as mean + standard error for the control group. Acid-precipitable data are fitted with the curve Yd(t). The first 10 min of ya(t) are used to derive blood clearance. Acid-soluble data are fitted with the curve yc(t). The initial decline of the curve yc(t) is caused by loss from the circulation of free radioiodine that was present in the [t~I]AGOR. The onset of catabolism was defined as the time tc when the blood level of catabolites yc(t) rose above 2% of the injected dose. Ligand catabolism was defined as the difference between the blood concentration of catabolites at tc and tc + 10 min.
EFFECTSOF DIABETESAND INSULINON GLYCOPROTEINMETABOLISM
633
exp(-a2t), using the MiNurr program [8]. The speed of clearance of [lZSI]AGOR from blood was characterised by the measure Dose CR10 -
(ml/min) Ii
ya(t)dt
where dose is the amount of injected [~zSI]AGOR (nmoles) and the integral is the area under the blood disappearance curve over the first 10 min. The true blood clearance is given by CR®, however, this was not used as the fitted curves could not be reliably extrapolated beyond 10 min. (B) Hepatic transport time of ligand was estimated as the interval between injection of ligand to the point when the blood level of acid-soluble material rose above 2% of the injected dose (Fig. 1). This point is taken to represent the onset of catabolism, and depends not only on the hepatic transport time, but to a small extent on the clearance rate from blood. In order to determine this interval to, the data were interpolated with a least squares cubic spline y¢(t) with three internal knots using a subroutine from the NAG library (NAG Library Manual) [9]. (C) Rate of ligand catabolism was assumed to be proportional to the excess percentage of catabolites in blood 10 min after the onset of catabolism (Fig. 1). This was determined from the cubic spline interpolation, y¢(tc+ 10). The figure is an underestimate of total ligand catabolism since acid-soluble material is continuously exchanging between the circulation and peripheral tissues (mainly muscle, skin and gut), and being excreted. However, this estimate did permit comparisons between different treatments. The parameter reflects total ligand flux and therefore depends on the blood clearance rate, in addition to the catabolic rate constant. When the blood clearance rate for different groups is the same, differences in the parameter must represent differences in the catabolic rate constant.
Statistics The results from different groups of rats were compared using a pooled independent t-test. Results
Table 1 summarises the metabolism of [125I]AGOR for the different groups.
Control group The volume of distribution (Vd) of [125I]AGOR was 23.1 + 0.7 ml and did not differ significantly in the treatment groups. In control rats the blood clearance of [I25I]AGOR was rapid (8.7 + 0.6 ml/min; mean + SEM, n = 3). The hepatic transport time of [I25I]AGOR was 12.8 + 0.7 min. Ligand catabolism was 4.9 _+ 0.3% of the injected dose. By 30 min, the liver contained 36 + 1.6% of the injected dose consisting of both intact [125I]AGOR and catabolites.
N. TANAKA et al.
634 TABLE 1 METABOLISM OF [I~I]AGALACTOOROSOMUCOID Group a
Blood clearance rate (ml/min)
Hepatic transport time (min)
Catabolic rate Hepatic accumulation (% of dose/10 rain) (% of dose at 30 rain)
Control
8.7 + 0.6
12.8 + 0.7
4.9 + 0.3
36 + 1.6
Insulintreated
8.2 + 0.3
18.1 + 1.9
3.4 +_.0.3
54.8 + 2.0
14.5 + 1.2
3.2 + 0.4
43.3 + 3.6
15.2 + 1.4
4.1 _+0.3
ND
13.6+0.1
4.9+0.5
48.7+3.0
15.9 + 1.1
2.9 _ 0.5
44_7 + 3.9
Diabetic uncontrolled 4.6 + 0.5 acute normoglycaemia 9.0 + 0.7 insulin osmotic pump 11.7+1.1 Chloroquinetreated
9_4 + 0.3
a n = 3 in each group. ND = not determined.
Insulin treatment Insulin did not alter the blood clearance of [mSI]AGOR (8.21 + 0.3 ml/min) but had two effects on hepatic A G O R metabolism. Firstly hepatic transport time of ligand was delayed to 18.1 + 1.91 min (P < 0.06) and secondly ligand catabolism was reduced (3.4 + 0.3%; P < 0.02). These changes were reflected in significantly greater ligand accumulation in the liver at 30 min (54.8 + 2.0%; P < 0.01).
Chloroquine Pretreatment of rats with chloroquine did not alter the blood clearance of [125I]AGOR (9.4 + 0.3 ml/min) but prolonged the hepatic transport time of ligand (15.9 + 1.1 ml/min, P < 0.07) and significantly reduced the ligand catabolism (2.9 + 0.5%, P < 0.03) (Table 1). At 30 min the liver contained 44.7 + 3.9% of the ligand.
Diabetes mellitus Diabetes significantly reduced the blood clearance of [125I]AGOR (4.6 + 5 ml/min, P < 0.005) but did not alter the hepatic transport time of ligand (14.5 _+ 1.2 min). However, diabetes markedly depressed ligand catabolism (3.2 + 0.4%, P < 0.03). The result was that the quantity of [lZSI]AGOR in the liver at 30 min (43.3 _+ 3.6%) was not significantly different f r o m the controls. The effect of rendering the diabetic animals acutely normoglycaemic was to return the measured p a r a m e t e r s to normal. In diabetic rats m a d e normoglycaemic with an implanted osmotic insulin pump, the blood clearance of [125I]AGOR was m o r e rapid than normal (11.7 + 1.1 ml/min, P < 0.075). This m a y have been related to the lower blood glucose in this
EFFECTS OF DIABETES AND INSULIN ON GLYCOPROTEIN METABOLISM
635
group (3.2 + 0.5 mmol/1) compared to controls (6.7 + 0.3 mmol/1). The hepatic transport time of ligand and ligand catabolism were similar to the controls. The result was that ligand accumulation in the liver at 30 min (48.7 + 3.0%) was significantly higher (P < 0.05) than in controls. Discussion
Many macromolecules are recognised and bound by specific cell surface receptors and are then internalized via clathrin-coated pits [10,11] to form vesicles [12,13]. The ligand receptor complexes are uncoupled [12,14] by acidification of these vesicles [15-18] and ligand and receptor are then segregated into separate vesicles [19]. Ligand-containing vesicles are usually targeted to secondary lysosomes for degradation while receptor-containing vesicles appear to be recycled to the cell surface where the receptors can be reused [20-26]. A G O R is an N-acetylglucosamine-terminated glycoprotein that is rapidly cleared from the blood by mannose/N-acetylglucosamine- (Man/GlcNAc-) receptors located on hepatic sinusoidal cells [1]. In these studies we have used these concepts of ligand binding, intracellular transport and lysosomal catabolism to analyse dynamic data obtained from in vivo experiments of [~2sI]AGOR metabolism to determine the effects of diabetes mellitus and insulin on this processing. Among the advantages of analysing data derived from an intact animal are optimized cell function and preservation of cell polarity. Studies of another glycoprotein recognition system indicate that uptake rates may be up to 10 times greater in vivo than in vitro [27]. Furthermore hepatic sinusoidal cells which mediate the uptake of [~25I]AGOR are in direct contact with blood so that transfer across an interstitial space could be neglected. The main disadvantage identified was the extrahepatic loss of some of the [~25I]AGOR (approximately 10% in the first 10 min; unpublished data) and the rising blood glucose concentration during the experiment. Extrahepatic loss of ligand to other tissues (mainly bone marrow) was similar in all the experimental groups. It made little difference to the estimates of the blood clearance parameters and did not influence comparisons between the groups. The extrahepatic loss can be reduced using glycoproteins of higher molecular weight (unpublished data) and thus may represent non-specific 'leakage' of ligand from the circulation. The magnitude of the hyperglycaemia was surprising and to our knowledge this important variable influencing the in vivo metabolism of glycoproteins has not been noted before. The hyperglycaemia probably reflected the stress of repeated cardiac puncture and removal of blood in the anaesthetised rat. Although the blood glucose concentration could be 'clamped' by insulin pretreatment, it is possible that other components of this 'stress' reaction might have influenced the metabolism of [125I]AGOR. The blood clearance data confirmed that [125I]AGOR is rapidly removed from the blood by the liver. About 30% of the blood volume (VD) was cleared of ligand each minute. We confirmed that the rate of ligand clearance was slower in the diabetic rat and also demonstrated that rendering the diabetic rat acutely normoglycaemic returned the blood clearance rate of [~25I]AGOR to control values. Further-
636
N. TANAKAet al_
more we showed that in diabetic animals, rendered normoglycaemic with an implanted insulin pump, the blood clearance of [125I]AGOR was even greater than in the controls. The mechanism(s) involved could not be resolved although it is likely they were mediated by the blood glucose concentration. Probable mechanisms are that the lower blood glucose levels in rats with an implanted insulin pump could either (a) reduce the inhibitory effect of glucose on ligand uptake [2,5] or (b) increase cell surface expression of mannose receptors (up-regulation) [28]. Although regulation of mannose receptor number on the cell surface was not observed in studies of endothelial cells isolated from diabetic rats [2], it has been demonstrated in macrophages [28]. The discrepancy could reflect either differences in experimental design or different populations of mannose receptors on endothelial and Kupffer cells [3,4]. The data also provided an estimate of the duration of ligand transport to the lysosomes measured from the time of ligand injection until ligand catabolism was observed. This estimate is the sum of several events including ligand internalization, intracellular transport and the time the ligand spends in the lysosome awaiting catabolism. Two agents, insulin and chloroquine, prolonged the hepatic transport time. Chloroquine, a weak base, by raising the pH of endocytic vesicles, inhibits receptor-ligand uncoupling, which seems to be necessary before ligand is delivered to the lysosomes [16,23,24]. Such inhibition of uncoupling would be expected to prolong the hepatic transport time. The effect of insulin on hepatic transport time of ligand was unexpected and even more marked than that of chloroquine in normal rats, although in diabetic rats insulin did not have this effect. It is clear that different ligand-receptor complexes may be internalized together by a single coated pit [11] and that receptors may compete for entry into the coated pit [29]. Thus it is conceivable that insulin delays the intracellular ~ransport time of [125I]AGOR by competing with it for internalization. Other possibilities include effects of insulin on intracellular vesicle transport. The reduction by chloroquine of the ligand catabolic rate is due to this amine inhibiting proteolytic enzymes in the lysosomes [16,23,26]. In contrast the inhibitory effect of diabetes on ligand catabolism is of uncertain mechanism. However, the lower ligand catabolic rate observed in insulin-treated control rats may have been due to reduced availability of ligand in the lysosomes due to the prolonged hepatic transport time. In conclusion, these experiments have demonstrated the complex effects of diabetes mellitus and insulin treatment on the uptake and processing of a glycoprotein by a mannose-mediated glycoprotein system in the liver. These experimental approaches will permit analysis of the mechanisms mediating the endocytosis of proteins by the liver.
Acknowledgements J.A.S. is a Wellcome Trust Senior Clinical Fellow.
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