Different Regulation of Insulin Receptors in Intracellular (Golgi) And

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Proc. Nati. Acad. Sci. USA Vol. 75, No. 7, pp.3302-3306, July 1978 Cell Biology Different regulation of insulin receptors in intracellular (Golgi) and plasma membranes from livers of obese and lean micea (prolactin/Golgi cistemae/Golgi vesicles/plasmalemma) BARRY I. POSNERb, D. RAQUIDANb, ZEEV JOSEFSBERGb, AND JOHN J. M. BERGERONc b McGill University Clinic, Royal Victoria Hospital, and c Department of Anatomy, McGill University, Montreal, Quebec, Canada H3A 2B2 Communicated by E. R. Stadtrman, April 14, 1978

ABSTRACT Insulin binding to Golgi and plasmalemma Plasmalemma was prepared by a modified Neville procedure fractions from the liver of obese (ob/ob and db/db) and lean (9), as described (6). Golgi fractions were prepared from normal control mice was studied. The specificity of binding in Golgi loading and from fractions (Golgi vesicles and Golgi cisternae) was identical to and lean control mice 90 min after alcohol that observed previousl in rat liver Golgi fractions and plas- obese mice without alcohol loadingd by the method of Ehren- malemma. Binding to piasmalemma was much lower in obese reich et al. (10). In this procedure the Golgi apparatus is sub- compared to lean mice, but binding to Golgi cisternae was either fractionated on the basis of a discontinuous sucrose gradient into not changed (ob/ob) or slightly increased (db/db) in obese three major fractions (Golgi light, GI; Golgi intermediate, Gi; compared to lean littermates. These observations suggest that and Golgi heavy, A smooth vesicle fraction (residual load intracellular (Golgi cisternae) insulin receptors are regulated GO). differently from those of the plasmalemma. We propose that zone), which appeared immediately below the Gh on the gra- insulin affects hepatic receptor content in two different ways. dient, and the pellet, consisting largely of rough endoplasmic On the one han asmalemma receptor loss is accelerated, and reticulum (residual pellet), were also obtained (6). The sub- on the other hand intracellular receptor production is increased. cellular fractions were fixed and studied by electron microscopy This model can explain in one framework the effect of insulin as described (10). in reducing and prolactin in augmenting the level of their re- All subcellular. fractions were frozen and thawed four times spective hepatic receptors. before hormone binding was assayed. This procedure breaks A number of studies have described specific binding of 125I- open the Golgi vesicles and maximizes hormone binding to this labeled insulin (125I-insulin) to subcellular fractions (1-5). In- subcellular fraction (6). In the standard binding assay, 125I- sulin binding to Golgi fractions from rat liver does not reflect insulin (80-140 X 103 cpm), prepared with chloramine T as contamination of these fractions by plasmalemma but involves described (11), was incubated with 150 ,ug of protein of each binding sites on morphologically defined Golgi elements (6). subcellular fraction, in a final volume of 0.5 ml of 25 mM The Golgi sites are very similar to those of the plasmalemma Tris.HCl/10 mM MgCl2/0.1% (wt/vol) bovine serum albumin that the Golgi receptors may be at pH 7.4, for 20-24 hr at 40 with constant shaking. All incu- (7), and it has been suggested bations were in triplicate and were terminated by adding 3 ml precursors for those of the plasmalemma (2, 7). In several strains of ice-cold buffer followed by the separation of bound and free of genetically obese mice (ob/ob and db/db), characterized by radioactivity by filtration (7). Parallel incubations were per- obesity, hyperglycemia, hyperinsulinemia, and insulin resis- insulin. tance, it has been observed that the insulin-receptor concen- formed in the presence of excess (5 ,ug) unlabeled tration of the hepatic plasmalemma is reduced (4, 8). In the Specific binding was the difference between radioactivity present study we demonstrate specific insulin-binding sites in bound to the membranes in the presence of excess unlabeled the Golgi subcellular fractions from mouse liver and show that hormone and that bound in its absence, and is expressed as a of a intracellular percent of the total radioactivity in the incubation. Nonspecific the insulin-receptor concentration specific binding (that observed in the presence of excess unlabeled compartment (the Golgi cisternae) was not suppressed in obese of the total compared to lean mice. On the basis of this observation, we hormone) was btween 1.0 and 3.5% radioactivity propose a general mechanism for polypeptide hormone re- added. ceptor regulation. RESULTS MATERIALS AND METHODS The ultrastructural appearancee of the Golgi apparatus of lean Male ob/ob and db/db mice, their lean littermate controls, and littermate mice in situ (Fig. 1A) resembled that of normal rat C57BL/6J mice were purchased from the Jackson Laboratory, liver (10). In contrast, in both ob/ob and db/db mice consid- Bar Harbor, ME. Porcine insulin (24.4 U/mg) was a gift from the Connaught Laboratories, Willowdale, ON. Porcine proin- Abbreviations: 125I-insulin, 25I-labeled insulin; VLDL, very low sulin, desalanine insulin, desoctapeptide insulin, and rat insulin density lipoprotein; Golgi fractions: light, Gi; intermediate, G1; and were from the Eli Lilly Co., by courtesy of R. Chance. Rat heavy, Gh. somatotropin (growth hormone) and rat prolactin were gen- a Presented in part at the Annual Meeting of the American Diabetes Hormone Pro- Association, June 5-7, 1977, St. Louis, MO. erously supplied by the Pituitary Distribution d Alcohol loading of obese mice resulted in a high mortality; Golgi gram of the National Institute of Arthritis, Metabolism, and fractions could be prepared from the obese animals in good yield Digestive Diseases. Glucagon and corticotropin (ACTH) were without alcohol pretreatment. purchased from Sigma Chemical Co. e Liver tissue was fixed by immersion in the fixative of Karnovsky (13), postfixed for 90 min in 2% OS04 in 0.1 M cacodylate buffer, stained The costs of publication of this article were defrayed in part by the en bloc with galloyl glucose (14), and dehydrated in ascending al- payment of page charges. This article must therefore be hereby marked cohols before it was embedded in Epon. Thin sections were grid- "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate stained with lead citrate before they were viewed in a Siemens 101 this fact. electron microscope. 3302 Cell Biology: Posner etal. Proc. Natl. Acad. Sci. USA 75 (1978) 3303

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* It/i' _-' * :S ' A FIG. 1. Comparison of Golgi apparatus in hepatocytes of lean littermate and ob/ob mice. (A) Golgi apparatus of the lean littermate mice shows stacked cisternae (sc) generally empty of very low density lipoprotein (VLDL), although some was occasionally seen in a distended rim (arrow). (x30,000.) (B) Accumulation of VLDL particles in trans Golgi vesicles (tv) and bulbous rims of the Golgi apparatus is marked in hepatocytes of the obese mice. The stacked cisternae (sc) have fewer particles. (X30,000.) erable accumulation of VLDL particles could be seen in both from the latter was the abundance, in these fractions from obese Golgi vesicles (trans elements) and cisternae (cis elements) (Fig. mice, of small lipid-containing, smooth-surfaced vesicles. In 2), resembling the in situ appearance of liver from alcohol contrast, the heavy Golgi fraction (Fig. 2C) from the obese preloaded rats (10). animals was identical to that described previously in rat liver The light and intermediate Golgi fractions obtained from as probably derived from the stacked Golgi cisternae (10). both ob/ob and db/db mice contained Golgi vesicles filled with In preliminary studies on fractions from normal mice it was VLDL (Fig. 2 A and B) as seen in similar fractions from rats that shown that 125I-insulin binding to Golgi fractions (GI, Gi, and had been loaded with alcohol (6, 10). An important difference GO) reached a plateau after 20-30 hr of incubation at 4°. Spe-

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-.0 AMt|m TI Ab voth *LN-Gov *i>'_-J -1'_1 1 -W- N _ I_ ]o~w 5.Ad FIG. 2. Golgi fractions from the liver homogenate of ob/ob mice (fields at middle of sectioned pellets). (A) Golgi light fraction (G1). The fraction contains large vesicles (cl) with VLDL content characteristic ofGolgi vesicles ofthe hepatocyte. The predominant component consists ofsmaller smooth-surfaced vesicles (c2) with a pale-staining lipoid content (1). Rare lysosomal contaminants (ly) are occasionally seen. (X20,000.) (B) Golgi intermediate fraction (G,). As in the lighter fraction, the major component consists of small smooth-surfaced vesicles (c2) with a lipoid content (1). Small (C3) and large (C4) VLDL-containing vesicles and tubules are also seen. (X20,000.) (C) Golgi heavy fraction (Gh). The fraction consists predominantly of small flattened saccules. Small vesicles are also present. (X20,000.) 330400 Cell Biology: Posner et al. Proc. Nati. Acad. Sci. USA 75 (1978)

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I 0 L )i 5- -00 0.5 5 50 500 5000 C).5 5 50 500 5000 0.5 5 50 500 5000 Unlabeled hormone, ng per tube FIG. 3. Specificity of binding of 1251-insulin to Golgi fractions from normal mouse liver. The fractions were prepared, frozen and thawed, and incubated as described under Materials and Methods. Inhibition was studied in the presence of insulin (-), desalanine insulin (M), proinsulin (0), and desoctapeptide insulin (A). Structurally unrelated hormones studied were rat growth hormone (o), rat prolactin (-), corticotropin (v), and glucagon (v). cific binding varied directly with protein between 30 and 160 The specific binding of 125I-insulin to plasmalemma from ,ug per tube, and increased with increasing amounts of 125I- obese and lean littermate mice was determined in three separate insulin. Fig. 3 depicts the specificity with which unlabeled in- assays on membranes prepared from 10-12 mice of each type. sulin and insulin analogues, as well as structurally unrelated The percent specific binding obtained were: ob/ob, 1.5 i 0.2; hormones, inhibited the binding of 125I-insulin to each of the ob control, 7.2 + 0.4; db/db, 1.3 + 0.3; and db control, 9.8 ± prepared Golgi fractions. The pattern of inhibition was similar 0.1. This confirms previous observations showing that the level in all three fractions and compares favorably with similar ob- of 125I-insulin binding to plasmalemma from obese was much servations on the binding of 125I-insulin to plasmalemma and lower than that to plasmalemma from littermate controls (4). Golgi fractions from rat liver (7). Insulin analogues inhibited The binding of 125I-insulin to Golgi vesicles (trans elements, in each instance, with an efficacy paralleling their biological GI and Gj), Gh, and the smooth vesicle fraction was studied more potency (7, 11), while unrelated hormones had no inhibitory extensively in 60- to 75-day-old mice. In three separate ex- effect. The inhibition curves with unlabeled insulin were re- periments (Table 1) the binding of 125I-insulin was decreased markably similar in all three fractions, with 50% inhibition of in the Golgi vesicles from obese compared to lean animals. The 125I-insulin binding seen with amounts of insulin (ng/tube) of: extent of decrease was comparable in the GI and Gi fractions GI, 11.4; Gi, 10.0; and Gh, 7.2. studied separately (data not shown). In contrast, no such de- Table 1. Percent specific binding of 1251-insulin to subcellular fractions from livers of obese and lean control mice Membrane Exp. ob/ob db/db fraction no. Obese Control Obese Control Golgi vesicles 1 3.5 + 0.2 11.6 + 0.6 1.7 I 0.7 9.0 + 1.2 2 6.7 + 0.6 16.2 + 0.9 3.9 ± 0.3 16.4 + 0.8 3 6.8 + 0.2 16.3 0.3 3.9 ± 0.5 10.4 0.3 Golgi cisternae 1 18.7 I 0.9 18.4 ± 3.2 21.3 ± 1.6 16.2 ± 0.3 2 13.7 + 0.5 17.1 + 0.9 21.0 i 1.0 18.4 ± 1.5 3 23.3 + 0.8 19.4 + 1.5 29.1 + 0.9 24.6 ± 0.4 Smooth vesicles 1 2.4 0.1 6.2 0.1 2.9 ± 0.5 5.8 ± 1.1 2 3.6 0.3 8.1 : 0.1 3.6 ± 0.5 9.4 ± 0.6 3 1.9 + 0.4 4.7 ± 0.4 1.6 i 0.1 3.4 ± 0.5 Insulin, ng/ml 20.9 + 6.6 0.9 ± 0.1 14.6 ± 7.3 1.5 ± 0.5 Binding of 125I-insulin in each experiment was carried out simultaneously on all the fractions in triplicate. Each value is the mean k SEM of three determinations. Nine to twelve animals of each cat- egory were used for membrane preparation in each experiment. The Golgi vesicles were always prepared as separate G1 and Gi fractions but were combined for assay after preliminary study showed comparable changes in each fraction. Insulin values are the mean i SEM of the determinations ofthe pooled serum from each of the three experiments. Cell Biology: Posner et al. Proc. Natl. Acad. Sci. USA 75 (1978) 3305

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-I r (n 0 "° c FIG. 5. Postulated effects consequent to the hormone-receptor in interaction. The mechanism of loss is not necessarily a dissociation N from the surface of the cell but may involve interiorization of the hormone-receptor complex. The effect on biosynthesis may not be ).5 5 50 500 0.5 5 50 500 direct, but may involve the facilitation of flow from the sites of syn- Unlabeled hormone, ng per tube thesis to the Golgi cisternae. FIG. 4. Inhibition of 1251-insulin binding to the Golgi vesicle (G1 and G,) and Golgi heavy (Gh) fractions by unlabeled insulin. 0, ob/ob; 0, control. gradient (6, 10), since specific binding in the smooth vesicle fraction paralleled that in the plasmalemma and was much crease was seen in the binding of 125I-insulin to Golgi cisternae lower than that in the Gh fraction. These observations applied elements (Gh) of obese mice. In fact the binding to Gh from over a range of insulin concentrations (Fig. 4 right), indicating db/db was significantly greater than that to Gh from lean lit- that they reflect changes in receptor number, not affinity. The termates (P < 0.025) by Student's t test for paired comparisons data suggest that the insulin-receptor concentration in a par- (12). Fig. 4 depicts the binding to fractions from ob/ob and lean ticular intracellular pool is regulated differently from that of littermate mice, studied in Table 1, over a range of unlabeled the plasmalemma. The changes in the Golgi vesicle fractions insulin concentrations. Binding of 125I-insulin to the Golgi (trans elements) paralleled that in plasmalemma and would vesicle fraction from obese mice was less than that to the same appear to reflect changes in receptor number rather than af- fraction from lean mice at all insulin concentrations. The con- finity (Fig. 4 left). The interpretation of this is more difficult centrations at which specific binding was inhibited by 50% are since the vesicle fractions from obese mice contained an comparable for obese and lean mice. In contrast, the levels of abundance of lipid-containing smooth-surfaced vesicles of 125I-insulin binding to Gh from obese and lean mice were very uncertain derivation (Fig. 2 A and B) but which are probably similar. The same observations apply equally to db/db mice and related to the numerous lipid-containing vesicles in the livers their lean litermates (data not depicted). of the obese mice. It has been shown that the Golgi apparatus participates in the DISCUSSION secretory process. Products synthesized on the rough endoplasmic reticulum pass through the Golgi cisternae prior to In previous studies on rat liver subcellular fractions we observed concentration and are transported by Golgi secretory vesicles that the properties of insulin binding to the Golgi sites were to the plaalemma, where exocytosis occurs (15, 16). We have strikingly similar to those of the plasmalemma (7). In this suggested that intracellular (Golgi) receptors are precursors for communication we have demonstrated 125I-insulin binding to those in the plasmalemma and that the passage of receptors Golgi fractions from normal mouse liver and have shown that from their sites of synthesis to the plasmalemma might follow the binding specificity for each Golgi subfraction was similar the same intracellular process (2, 7). The observation that, on to that previously reported for fractions from rat liver. Fur- induction, the rat liver prolactin receptor appears in the Golgi thermore, 125I-insulin binding to the mouse liver Golgi fractions elements prior to appearing in the plasmalemma is compatible was inhibited by amounts of unlabeled insulin that were com- with this suggestion (unpublished data). This implies that re- parably effective in rat liver Golgi and plasmalemma fractions ceptors in the Golgi cisternae elements are more intimately (7) as well as mouse liver plasmalemma (4). associated with the sites of biosynthesis than those in the plas- We have examined the level of insulin binding in Golgi malemma. subcellular fractions as well as plasmalemma from two genet- Our observation that insulin-receptor levels are reduced in ically distinct types of obese mice and their lean littermate plasmalenma while remaining unchanged or slightly increased controls. In confirmation of previous studies (4), 125I-insulin in Golgi cisternae can be explained in several different ways. binding was substantially reduced in plasmalemma from obese The suggestion we favor is depicted in Fig. 5. As outlined pre- compared to lean littermate mice. In contrast, the specific viously, we propose that the binding of hormone to its receptor binding of insulin to morphologically defined Golgi cisternae results in two distinctive changes (17). On the one hand there fractions (Fig. 2C) was no different in ob/ob mice and the lean littermates and significantly greater in this fraction from db/db compared to the lean control mice (Table 1).f The distinctive Table 2. Postulated influence of ligand on receptor pattern in the Gh fraction was not due to contamination of this fraction by smooth vesicles from the residual load zone, located Production Loss Net tissue level immediately beneath the Golgi heavy elements on the sucrose Lactogen tt Increased Insulin t tt Decreased f It has been observed (4) that less purified liver fractions from lean and obese mice showed smaller differences in insulin binding than seen Broken arrows indicate processes that have not been directly in purified plasmalemma. demonstrated. 3306 Cell Biology: Posner et al. Proc. Natl. Acad. Sci. USA 75 (1978) is accelerated loss (possibly by interiorization) of receptor from Health Service (IR01 AM 19573-01), and le Conseil de la Recherche plasmalemma and a concomitant increase in production of new en Sante du Quebec. receptors within the cell. Thus, despite accelerated loss of insulin 1. Lee, N. D. & Williams, R. H. (1954) Endocrinology 54,5-19. receptors from the plasmalemma, the concentration of receptors 2. Bergeron, J. J. M., Evans, W. H. & Geschwind, I. I. (1973) J. Cell within Golgi cisternae elements might be sustained or even Biol. 59, 771-776. augmented by accelerated production of new receptors.5 Ob- 3. Horvat, A., Li, E. & Katsoyannis, P. G. (1975) Biochim. Biophys. servations on lymphoblasts in vitro, suggesting that insulin Acta 382, 609-620. promotes an accelerated loss of insulin receptors,h are com- 4. Kahn, C. R., Neville, D. M., Jr. & Roth, J. (1973) J. Biol. Chem. patible with this hypothesis. 248,244-250. 5. Goldfine, I. D., & Smith, C. J. (1976) Proc. Natl. Acad. Sci. USA An attractive feature of our hypothesis is that it provides a 73, 1427-1431. general explanation for the regulation of polypeptide hor- 6. Bergeron, J. J. M., Posner, B. I., Josefsberg, Z. & Sikstrom, R. mone-receptor levels. Thus, prolactin has been shown to induce (1978) J. Biol. Chem., 253,4058-4066. its own receptor (18), whereas several different hormones, in- 7. Posner, B. I., Josefsberg, Z. & Bergeron, J. J. M. (1978) J. Biol. cluding insulin (19), luteinizing hormone (20), thyrotropin re- Chem., 253, 4067-4073. leasing hormones (21), and catecholamines (22), decrease their 8. Soll, A. H., Kahn, C. R., Neville, D.M., Jr. & Roth, J. (1975) J. Clin. respective receptors. If each hormone causes both accelerated Invest. 56, 769-780. receptor loss from the plasmalemma and augmented receptor 9. Neville, D. M., Jr. (1968) Biochim. Biophys. Acta 154, 540- production within the cell, then the net effect observed would 552. 10. Ehrenreich, J. H., Bergeron, J. J. M., Siekevitz, P. & Palade, G. be a consequence of which of the two processes (loss or pro- E. (1973) J. Cell Biol. 59, 45-72. duction) is more markedly affected (Table 2). In this manner 11. Posner, B. I. (1974) Diabetes 23,209-217. the apparent qualitative difference in regulatory processes 12. Huntsberger, D. V. (1967) Elements of Statistical Inference might be explained on kinetic grounds. These observations on (Allyn and Bacon Inc., Boston, MA), pp. 211-215. polypeptide hormone receptors bear some similarity to what 13. Karnovsky, M. L. (1965) J. Cell Biol. 27, 137 (abstr.). has been observed in the immune response (23). The ability to 14. Simionescu, N., & Simionescu, M. (1976) J. Cell Biol. 70,608- analyze the concentration of polypeptide hormone receptors 621. in different subcellular compartments should permit more 15. Palade, G. E. (1975) Science 189,347-358. stringent testing of the present hypothesis. 16. Bergeron, J. J. M., Borts, D. & Cruz, J. (1978) J. Cell Biol. 76, 87-97. 17. Posner, B. I. (1976) Fifth Int. Congr. of Endocrinology, ed. This study was supported by grants from the Medical Research James, V. H. T. (Excerpta Medica, Amsterdam Netherlands), Vol. Council of Canada (MT-4182 to B.P.; MA-5605 to J.B.), the U.S. Public 2, pp. 178-185. 18. Posner, B. I., Kelly, P. A., & Friesen, H. G. (1975) Science 187, 57-59. Assuming that the insulin-receptor concentration in the Golgi vesicle 19. Gavin, J. R., Roth, J., Neville, D. M., Jr., DeMeyts, P. & Buell, D. fractions reflects true Golgi elements, the fall in receptor concen- mice N. (1974) Proc. Natl. Acad. Sci. USA 71, 84-88. tration in the obese hyperinsulinemic might be accomplished, A. M. L. & within the framework of the proposed hypothesis, on the basis of 20. Conti, M., Harwood, J. P., Hsueh, J. W., Dufau, Catt, shuttling of Golgi vesicles, as suggested by Palade (15). In this case K. J. (1976) J. Biol. Chem. 251, 7729-7731. the loss of receptor from Golgi vesicles to the plasmalemma would 21. Hinkle, P. M., & Tashijian, A. H., Jr., (1975) Biochemistry 14, have to exceed the flow of receptor from Golgi cisternae elements 3845-3851. to returning vesicles, with a tendency of vesicle-receptor concen- 22. Mickey, J., Tate, R. & Lefkowitz, R. J. (1975) J. Biol. Chem. 250, tration to approach that of the plasmalemma. 5727-5729. h Kosmakos, F. & Roth, J. (1976) Endocrine Society, 58th Annual 23. Ault, K. A. & Unanue, E. R. (1974) J. Exp. Med. 139, 1110- Meeting, June, 1976, Abstr. 26. 1124.