470
Biochimica et Biophysica Acta, 542 ( 1 9 7 8 ) 4 7 0 - - 4 8 5 © Elsevler/North-Holland Biomedical Press
BBA 28636
HORMONE RECEPTORS 7. CHARACTERISTICS OF INSULIN RECEPTORS IN A NEW LINE OF CLONED NEONATAL RAT HEPATOCYTES
BARBARA
PETERSEN, S U Z A N N E B E C K N E R and M E L V I N B L E C H E R *
Department of Biochemistry, Georgetown University Medical Center, Washington, D.C. 20007 (U.S.A.) (Received J a n u a r y 18th, 1978)
Summary 1. A new line of cloned, differentiated rat hepatocytes (RL-PR-C) was evaluated for its usefulness as an in vitro system for studying the regulation of the insulin receptor. 2. Insulin rapidly reversibly and specifically bound to RL-PR-C hepatocytes. Binding of tracer 12SI-labeled insulin, which was competitively inhibited by native insulin as well as by proinsulin and analogs of insulin and proinsulin in proportion to their biological activity, was not influenced by glucagon, corticotropin, or human growth hormone. Anti-insulin receptor serum from a patient with Acanthosis Nigricans Type B competed with l~SI-labeled insulin for binding to cell surface sites. 3. Trypsinization destroyed insulin binding sites, but these were restored by incubation under growth conditions; a 75% restoration of binding sites was achieved by one cell population doubling. 4. RL-PR~ hepatocytes responded to insulin binding by an increase in glycogen synthesis from glucose. The insulin effect was maximal at 85 nM, but was detectable at lower, more physiological, concentrations. 5. Chronic exposure (for at least 3 h) of hepatocytes to insulin (10 - l ° 10 -s M) reduced by up to 60% the number of binding sites for insulin (downregulation). Down-regulation was prevented by cycloheximide at concentrations (10 #M) sufficient to inhibit markedly protein synthesis from tracer isoleucine. Recovery from down-regulation induced by native insulin at 10 -7 M or lower concentrations was complete by 18 h under growth conditions. 6. Although RL-PR~ hepatocytes spontaneously transform after about 90 population doublings, no significant differences between normal and trans* To whom reprint requests axe to be
addressed.
471 formed cells were observed in insulin binding characteristics and in interaction of cells with anti-insulin receptor serum. However, transformed cells exhibited a substantially reduced (maximum of 20%) down-regulation response to insulin. 7. RL-PR~ rat hepatocytes appear, for these masons, to be a useful model system for studying the regulation of the insulin receptor.
Introduction Insulin mediates a large number of cellular processes, and the evidence strongly suggests that its effects are triggered by its interaction with cell surface protein receptors. The kinetics of this interaction have been extensively studied (for a recent review, see ref. 1), but have not as yet led to an understanding of the mechanism of insulin action. Of particular interest to us have been the biochemical mechanisms which are responsible for disease states in man and animals in which chronic exposure to high concentrations of insulin appear to produce a relative resistance to the effects of insulin.Such mechanisms are nearly impossible to study in intact animals or even in isolated organs due to the relativelysmall differencesbetween concentrations of insulin which produce a normal physiological response and those which produce abnormal responses. Therefore, an in vitro system was sought which would meet the following requirements: (a) relative homogeneity; (b) long-term survivalin culture without de-differentiating;(c) response to insulin in a quantitativemanner; and (d) possessing regulatableinsulinreceptors. Various lines of cells have been used to study insulin binding and/or action: human skin fibroblasts [2,3], chick embryo fibroblasts [4], human lymphoblastoid cells(IM-9 cells) [5], peripheral white blood cells [6] and isolated, non 0.05) and uridine incorporation into R N A at an insulin concentration of 4 ng/ml. Other groups have reported enhanced incorporation of leucine into protein, phosphate uptake, uridine uptake R N A synthesis and glucose uptake, but only at supraphysiological concentrations of insulin (40 ng/ml and greater) [2,3,9-13]. Freshly prepared hepat'ocytes do not survive in culture for a period of time sufficientfor the ttirnover of plasma membrane proteins, a requirement, for example, for studies on the effect of chronic exposure of cellsto elevated concentrations of insulin. Rat liver cells have been cultured in chemically defined media for periods of up to 3 years and shown to respond to insulin by induction of tyrosine aminotransferase [14] and the regulation of pyruvate kinase [15], but those cells were not tested for specificity of the insulin response, nor was the effect of long term exposure to insulin investigated.Fur-
472 thermore, these cells were heteroploid, rather than the normal diploid karyotype. Dr. Warren Schaeffer of the University of Vermont has established a cloned line of hepatocytes derived from neonatal rats, designated RL-PR-C [16]. These cells grow as monolayers and exhibit a diploid karyotype which is stable for at least 60 population doublings. These cells exhibit several of the characteristics of normal hepatocytes, namely, tyrosine aminotransferase, serum albumin synthesis, inducible arylhydrocarbon hydroxylase, and RNAase II [16]. After 90 population doublings the strain spontaneously acquires the cultural characteristics of transformed cells, including tumorigenicity in isogenic animals [17]. This latter property permits one to study the effects of transformation on cell~urface components. The present study was designed to test the suitability of RL-PR-C hepatocytes for the study of the regulation of the insulin receptor. In this paper we describe the kinetics and specificity of binding of insulin to RL-PR-C hepatocytes, the interaction of cell with a human anti-insulin receptor serum derived from a patient with Acanthosis Nigricans Type B, competition for binding sites by insulin and proinsulin analogs and other polypeptide hormones, biological response (increased glycogen synthesis) to insulin, and downregulation of the insulin receptor. Materials and Methods Hormones. Stock solutions of insulin (Lilly Research Labs) and glucagon (Elanco) were prepared in 0.01 M HC1; those for corticotropin (Mann) and human growth hormone (National Institutes of Health) in H20. Insulin and proinsulin analogs were generous gifts of Dr. R. Chance of Lilly Research Labs. Biologically active 12SI-labeled insulin was prepared by the method of DeMeyts [18] and checked for quality by trichloroacetic acid precipitation, thin-layer chromatography on cellulose, and binding to purified rat liver plasma membranes [18]. Cell cultures. RL-PR-C hepatocytes from early (40--76 population doublings) and late (126--135 population doublings) lines were subcultured in T-75 CoStar flasks in a medium composed of 10% fetal bovine serum in Hams F-12 medium with 0.22 pg/ml of cortisol hemisuccinate (Sigma) and penicillin (25 units/ml)-streptomycin (25 pg/ml) in a humidified atmosphere of 5% CO2 in air at 37°C [16]. For subculturing, cells were removed by trypsinization (2.5 mg/ ml of "1 : 250" trypsin in Hanks balanced salt solution without Ca ~÷ and Mg2+), and split 1 : 4 (v/v). Specific insulin binding was abnormally low after trypsinization, but cells recovered full specific binding by 72 h in a complete growth medium (Fig. 1); trypsinization had no significant influence on nonspecific binding. For experiments, cells were seeded on 60- or 100-mm plastic petri dishes at a density of approx. 1.2 • 106 cells/dish unless otherwise indicated; cultures were refed on day 2, and used 24 h later at confluency. Cortisol was omitted from growth media when glycogenesis from glucose was examined. Insulin binding assay. After aspirating the incubation medium, monolayers were washed twice for 5 min each time with 2 ml of Dulbecco's phosphate-buffered saline at 4°C. Generally, cells were then incubated for 1 h at 22°C with 1
473
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~=4 ¸
a}2 ._c ~ p e c i f i c 24 48 72 Time After Trypsinizotion,h
96
Fig. 1. Time course for the " r e p a i r " of insulin receptors following trypsinization. Specific insulin binding was determin ed (as described in Materials and Methods) at various times after t he trypsinization/re-sceding procedure. Each point is the mean +S.D. of triplicate dishes.
ml of 1.5% dialyzed bovine serum albumin in pH 8.0 phosphate-buffered Saline (medium A) containing 12SI-labeled insulin (0.83 nM, 800--1000 cpm/fmol); in the absence (total binding) and presence (non-specific binding) of 1.7/zM native insulin. Following the binding period, the medium was aspirated, and the cells rapidly washed five times with 5 ml of medium A at 4°C. Cells were then dissolved in 1.0 rnl 2 M NaOH, the dishes rinsed with .1.0 ml water, and the pooled solutions counted in a Packard gamma-counter with 58% efficiency. Specific experimental details are given with each figure. Dissociation experiments. Following a standard insulin binding assay, the cells in parallel dishes were washed four times rapidly with medium A at 4 ° C, then incubated with 5 ml medium A at either 22 or 4 ° C. At intervals, the medium in sets of dishes was aspirated, the cells were washed with an additional 5 ml medium A at 4°C, and then dissolved in 2 M NaOH as above for radioactivity determination. Cell counts. In all experiments, cells from paired dishes were removed by scraping into phosphate-buffered saline, and diluted suspensions were counted with a Coulter ZBI Counter fitted with a 100 #m aperture and a size distribution analyzer. Effects o f chronic exposure to elevated concentrations o f insulin. To test for changes in the ability to bind 125I-labeled insulin following exposure to native insulin, i.e. down-regulation, cells were pre-incubated with insulin in medium A for periods of from 1 to 5 h at 37 ° C, washed twice with medium A for 20 min per wash and twice with phosphate-buffered saline for 10 rain per wash, all at 22°C. The standard insulin binding assay was then performed as described above. Where indicated, cycloheximide was included in the down-regulation media. To test for the recovery from the effect of cycloheximide, paired sets of dishes were preincubated, washed, and 2 ml of standard growth medium added to each dish. These cells were incubated at 37°C for recovery periods of 18--42 h. At appropriate times, the cells were washed twice with 2 ml phosphate-buffered saline, followed by a standard i2SI-labeled insulin binding assay at 22°C.
474
Incorporation of isoleucine into protein. After incubation for 1 h with L-[4,53H2(N)]isoleucine (New England Nuclear), dishes were placed on ice, and cells washed with 2 ml aliquots of phosphate-buffered saline. Cells were then removed b y scraping into 1 ml phosphate-buffered saline. Dishes were rinsed with t w o additional 1-ml aliquots of phosphate-buffered saline. The rinses were pooled with the original cell suspension and centrifuged at 1600 × g for 5 min at 4°C. The supernatant fluid was aspirated, and the cells suspended in 1 ml H20. Carrier bovine serum albumin (50 pl of a 10 mg/ml solution) was added, and the proteins were precipitated with 1 ml 20% trichloroacetic acid at 4 ° C. The precipitate was isolated on Whatman GF-C glass fiber filters using a Millipore manifold. The tubes were rinsed with two 2-ml aliquots of cold 5% trichloroacetic acid, and the rinses were poured over the filters. The proteins on the filters were dissolved in Protosol (Amersham-Searle), the solution neutralized with acetic acid, then counted in a mixture composed to 0.4% PPO and 0.01% POPOP in toluene/Triton X-100 ( 9 : 8 , v/v) in a liquid scintillation counter. Incorporation of glucose into glycogen. Cells were seeded in petri dishes and refed on the second day. After 24 h, the medium was changed to an Eagle's minimal essential medium modified to contain only 400 •g/ml glucose. After 24 h in modified minimal essential medium, the medium was aspirated, and fresh modified minimal essential medium with D-[6-~4C]glucose (AmershamSearle 2.0 Ci/mol; 10 pCi), with insulin as indicated, added. Cells were incubated for 1 h at 37°C, unless otherwise indicated. After incubation, the medium was aspirated, and the cells quickly washed with two 2-ml aliquots of phosphate-buffered saline (4°C). Carrier glycogen (0.1 ml of a 20 mg/ml solution) was added to each dish, and 1 ml of warm (37°C) 30% KOH was added to dissolve the cells. Dishes were rinsed twice with 0.5-ml aliquots of 30% KOH, and the extracts pooled in glass centrifuge tubes. The tubes were maintained in a 100°C bath for 5 min, cooled, and the glycogen precipitated b y the method of Van Handel [19]. The precipitated glycogen was collected on Whatman GF-A filters, dissolved in 0.2 ml H20, neutralized with acetic acid, and counted in the same liquid scintillation solution used above. Results
Characteristics of insulin binding Specific and non-specific binding reached maxima in 30 min at 22°C, and these were maintained for at least 90 min (Fig. 2); no differences in binding kinetics were noted between early (normal) and late passage (spontaneously transformed) cells. Specific insulin binding, which was taken as the difference between total and non-specific binding, and which varied substantially among repetitive experiments, averaged 15.0 fmol/5 • 106 hepatocytes in this experiment; non-specific binding never exceeded 2--3% of the total binding (Fig. 2). Binding of 12SI-labeled insulin to hepatocytes was linearly related to cell number b e t w e e n low density ( 0 . 4 . 1 0 6 cells per dish) and confluent ( 1 . 3 - 1 0 4 cells per dish) m o n o l a y e r s contained in 60-mm petri dishes. No degradation of 12SI-labeled insulin during the binding assay could be detected b y the criteria of precipitation b y 10% trichloroacetic acid and rebinding to isolated rat liver
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plasma membranes (data not shown). The binding of 12Sl-labeledinsulin was competitively inhibited by native insulin, as well as b y desnonapeptide proinculin, proinsulin and desoctapeptide insulin, in proportion to their biological activity; binding of 12SI-labeledinsulin was unaffected by corticotropin, human growth hormone, or glucagon (Fig. 3). Insulin, bound to h epatocytes, dissociated rapidly at 22°C, whereas at 4°C dissociation was m u c h slower (Fig. 4).
In contrast to reports that insulin can substitute for serum in supporting the
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growth of chick embryo cells [20--22], insulin could not do so for RL-PR4~ hepatocytes (Fig. 5). Flier et al. [23,24] have shown that sera from patients with Acanthosis Nigricans Type B contain an immunoglobulin (IgG) which is directed against a variety of cell~urface insulin receptors, in addition to those of the patients' own circulating monocytes. Monolayers of RL-PR~ cells were assessed for their capacity to bind a tracer concentration of 12SI4abeled insulin either after or during exposure to the anti-insulin receptor serum; control ceils were exposed to normal human serum. When cells were exposed to the antibody prior to the insulin binding assay (Fig. 6) the serum dilutions (titers) which inhibited insulin binding by 50% were about 1 : 3000 for both late passage (126--135 doublings) and early passage (45--76 doublings) cells. When the antiserum was present during the insulin binding assay (data not shown) titers were somewhat lower, being about 1 : 2500 and 1 : 1500 for late and early passage cells, respectively.
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Time A f t e r Seeding. Days Fig. 5. Fetal bovine serum and insulin as g r o w t h factors f o r cultuzed hepatocytea. Cells were seeded at 1 . 2 • 1 0 fl e e l l s l d i l h . M e d i a w e r e c h a n g e d e v e r y 3 d a y s a n d c e l l n u m b e r s d e t e r m i n e d o n d a y s i n d i c a t e d o n t h e a b s c i s s a . E a c h p o i n t is t h e m e a n ~S.D. o ! t r i p l i c a t e d i s h e s . T h e b a s a l Z n e d i u m w a s H a m s F - 1 2 w i t h eortisol and antibiotics; supplements were either fetal bovine serum (10%), or insulin (1.75 #M) with 1 . 5 % b o v i n e s e r u m albumin, o r 1 . 5 % b o v i n e s e r u m a l b u m i n a l o n e .
Effects of chronic exposure to insulin Chronic exposure to insulin in vivo in humans and laboratory animals has been reported to reduce the number of insulin binding sites, at least on rat fat cells and thyrnocytes, human circulating monocytes and cultured IM-9 lymphocytes [1], This phenomenon, which was initially observed by Gavin et al. [5] in studies with IM-9, has been termed "down-regulation". Pre-incubation of R L - P R ~ hepatocytes with a high concentration of native insulin, followed by extensive washing to remove all bound insulin, reduced by approx. 60% in 5 h the subsequent binding of ~2SI-labeled insulin (Fig. 7). Periods longer than 5 h were not tested since the cells could not be maintained well in the down-regulation medium for longer periods. At 5 h, down-regulation was maximal with 10 -s M insulin (Fig. 8); down-regulation at the more physiological concentration of 10 -1° M was low, but statistically different than control values. It was possible that the observed decreases in binding of 125I-labeled insulin might be due, not to changes in receptor number, but rather, to saturation of the receptor sites with native insulin during the preincuhation period which was not removed during washing. To test this possibility, parallel experiments were performed in which the same high concentrations of native insulin were present with the cells during only the final hour. Cells so treated did n o t subsequently
478
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D i l u t i o n of Patients Serum Fig. 6 . E f f e c t o f h u m a n a n t i 4 n s u l i n r e c e p t o r s e r u m o n t h e b i n d i n g o f 1 2 S i 4 a b e l e d i n s u l i n t o c u l t u r e d h e p a t o c y t e s . T h e a n t i s e r u m w a s f r o m A c a n t h o s i s N i g r i c a n s p a t i e n t B-2 [ 2 3 ] . W a s h e d m o n o l a y e r s w e r e i n c u b a t e d f o r 1 h at 3 7 ° C w i t h 1 m l o f t h e a n t i s e r u m ( d i l u t e d i n 1 . 5 % m e d i u m A ) o r o f n o r m a l h u m a n s e r u m i n c o n t r o l s ; a f t e r a s p i r a t i n g t h e s e r a , cells w e r e w a s h e d o n c e w i t h 2 m l p h o s p h a t e - b u f f e r e d saline p r i o r t o t h e i n s u l i n b i n d i n g a s s a y . C o n t r o l i n s u l i n b i n d i n g w a s 9 . 6 3 ± 1 . 5 9 f m o l / 5 • 1 0 6 cells i n e a r l y p a s sage (o) a n d 8 . 1 8 ± 0 . 4 0 f m o l / 5 • 1 0 6 cells in l a t e p a s s a g e ( e ) h e p a t o c y t e s .
exhibit decreased binding of 12SIqabeled insulin (Table I, top panel). In addition, other washing procedures were tested. Two additional washes with medium A did not alter the subsequent binding of 12SIqabeled insulin (Table II). Neither was binding increased following washes at 37°(3, or at pH 6 instead of 7.5 (Table II), procedures known to strip hormones from cell surfaces. The ability of the hepatocytes to recover from down-regulation was I00 .......
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Biochimica et Biophysica Acta, 542 ( 1 9 7 8 ) 4 7 0 - - 4 8 5 © Elsevler/North-Holland Biomedical Press
BBA 28636
HORMONE RECEPTORS 7. CHARACTERISTICS OF INSULIN RECEPTORS IN A NEW LINE OF CLONED NEONATAL RAT HEPATOCYTES
BARBARA
PETERSEN, S U Z A N N E B E C K N E R and M E L V I N B L E C H E R *
Department of Biochemistry, Georgetown University Medical Center, Washington, D.C. 20007 (U.S.A.) (Received J a n u a r y 18th, 1978)
Summary 1. A new line of cloned, differentiated rat hepatocytes (RL-PR-C) was evaluated for its usefulness as an in vitro system for studying the regulation of the insulin receptor. 2. Insulin rapidly reversibly and specifically bound to RL-PR-C hepatocytes. Binding of tracer 12SI-labeled insulin, which was competitively inhibited by native insulin as well as by proinsulin and analogs of insulin and proinsulin in proportion to their biological activity, was not influenced by glucagon, corticotropin, or human growth hormone. Anti-insulin receptor serum from a patient with Acanthosis Nigricans Type B competed with l~SI-labeled insulin for binding to cell surface sites. 3. Trypsinization destroyed insulin binding sites, but these were restored by incubation under growth conditions; a 75% restoration of binding sites was achieved by one cell population doubling. 4. RL-PR~ hepatocytes responded to insulin binding by an increase in glycogen synthesis from glucose. The insulin effect was maximal at 85 nM, but was detectable at lower, more physiological, concentrations. 5. Chronic exposure (for at least 3 h) of hepatocytes to insulin (10 - l ° 10 -s M) reduced by up to 60% the number of binding sites for insulin (downregulation). Down-regulation was prevented by cycloheximide at concentrations (10 #M) sufficient to inhibit markedly protein synthesis from tracer isoleucine. Recovery from down-regulation induced by native insulin at 10 -7 M or lower concentrations was complete by 18 h under growth conditions. 6. Although RL-PR~ hepatocytes spontaneously transform after about 90 population doublings, no significant differences between normal and trans* To whom reprint requests axe to be
addressed.
471 formed cells were observed in insulin binding characteristics and in interaction of cells with anti-insulin receptor serum. However, transformed cells exhibited a substantially reduced (maximum of 20%) down-regulation response to insulin. 7. RL-PR~ rat hepatocytes appear, for these masons, to be a useful model system for studying the regulation of the insulin receptor.
Introduction Insulin mediates a large number of cellular processes, and the evidence strongly suggests that its effects are triggered by its interaction with cell surface protein receptors. The kinetics of this interaction have been extensively studied (for a recent review, see ref. 1), but have not as yet led to an understanding of the mechanism of insulin action. Of particular interest to us have been the biochemical mechanisms which are responsible for disease states in man and animals in which chronic exposure to high concentrations of insulin appear to produce a relative resistance to the effects of insulin.Such mechanisms are nearly impossible to study in intact animals or even in isolated organs due to the relativelysmall differencesbetween concentrations of insulin which produce a normal physiological response and those which produce abnormal responses. Therefore, an in vitro system was sought which would meet the following requirements: (a) relative homogeneity; (b) long-term survivalin culture without de-differentiating;(c) response to insulin in a quantitativemanner; and (d) possessing regulatableinsulinreceptors. Various lines of cells have been used to study insulin binding and/or action: human skin fibroblasts [2,3], chick embryo fibroblasts [4], human lymphoblastoid cells(IM-9 cells) [5], peripheral white blood cells [6] and isolated, non 0.05) and uridine incorporation into R N A at an insulin concentration of 4 ng/ml. Other groups have reported enhanced incorporation of leucine into protein, phosphate uptake, uridine uptake R N A synthesis and glucose uptake, but only at supraphysiological concentrations of insulin (40 ng/ml and greater) [2,3,9-13]. Freshly prepared hepat'ocytes do not survive in culture for a period of time sufficientfor the ttirnover of plasma membrane proteins, a requirement, for example, for studies on the effect of chronic exposure of cellsto elevated concentrations of insulin. Rat liver cells have been cultured in chemically defined media for periods of up to 3 years and shown to respond to insulin by induction of tyrosine aminotransferase [14] and the regulation of pyruvate kinase [15], but those cells were not tested for specificity of the insulin response, nor was the effect of long term exposure to insulin investigated.Fur-
472 thermore, these cells were heteroploid, rather than the normal diploid karyotype. Dr. Warren Schaeffer of the University of Vermont has established a cloned line of hepatocytes derived from neonatal rats, designated RL-PR-C [16]. These cells grow as monolayers and exhibit a diploid karyotype which is stable for at least 60 population doublings. These cells exhibit several of the characteristics of normal hepatocytes, namely, tyrosine aminotransferase, serum albumin synthesis, inducible arylhydrocarbon hydroxylase, and RNAase II [16]. After 90 population doublings the strain spontaneously acquires the cultural characteristics of transformed cells, including tumorigenicity in isogenic animals [17]. This latter property permits one to study the effects of transformation on cell~urface components. The present study was designed to test the suitability of RL-PR-C hepatocytes for the study of the regulation of the insulin receptor. In this paper we describe the kinetics and specificity of binding of insulin to RL-PR-C hepatocytes, the interaction of cell with a human anti-insulin receptor serum derived from a patient with Acanthosis Nigricans Type B, competition for binding sites by insulin and proinsulin analogs and other polypeptide hormones, biological response (increased glycogen synthesis) to insulin, and downregulation of the insulin receptor. Materials and Methods Hormones. Stock solutions of insulin (Lilly Research Labs) and glucagon (Elanco) were prepared in 0.01 M HC1; those for corticotropin (Mann) and human growth hormone (National Institutes of Health) in H20. Insulin and proinsulin analogs were generous gifts of Dr. R. Chance of Lilly Research Labs. Biologically active 12SI-labeled insulin was prepared by the method of DeMeyts [18] and checked for quality by trichloroacetic acid precipitation, thin-layer chromatography on cellulose, and binding to purified rat liver plasma membranes [18]. Cell cultures. RL-PR-C hepatocytes from early (40--76 population doublings) and late (126--135 population doublings) lines were subcultured in T-75 CoStar flasks in a medium composed of 10% fetal bovine serum in Hams F-12 medium with 0.22 pg/ml of cortisol hemisuccinate (Sigma) and penicillin (25 units/ml)-streptomycin (25 pg/ml) in a humidified atmosphere of 5% CO2 in air at 37°C [16]. For subculturing, cells were removed by trypsinization (2.5 mg/ ml of "1 : 250" trypsin in Hanks balanced salt solution without Ca ~÷ and Mg2+), and split 1 : 4 (v/v). Specific insulin binding was abnormally low after trypsinization, but cells recovered full specific binding by 72 h in a complete growth medium (Fig. 1); trypsinization had no significant influence on nonspecific binding. For experiments, cells were seeded on 60- or 100-mm plastic petri dishes at a density of approx. 1.2 • 106 cells/dish unless otherwise indicated; cultures were refed on day 2, and used 24 h later at confluency. Cortisol was omitted from growth media when glycogenesis from glucose was examined. Insulin binding assay. After aspirating the incubation medium, monolayers were washed twice for 5 min each time with 2 ml of Dulbecco's phosphate-buffered saline at 4°C. Generally, cells were then incubated for 1 h at 22°C with 1
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Fig. 1. Time course for the " r e p a i r " of insulin receptors following trypsinization. Specific insulin binding was determin ed (as described in Materials and Methods) at various times after t he trypsinization/re-sceding procedure. Each point is the mean +S.D. of triplicate dishes.
ml of 1.5% dialyzed bovine serum albumin in pH 8.0 phosphate-buffered Saline (medium A) containing 12SI-labeled insulin (0.83 nM, 800--1000 cpm/fmol); in the absence (total binding) and presence (non-specific binding) of 1.7/zM native insulin. Following the binding period, the medium was aspirated, and the cells rapidly washed five times with 5 ml of medium A at 4°C. Cells were then dissolved in 1.0 rnl 2 M NaOH, the dishes rinsed with .1.0 ml water, and the pooled solutions counted in a Packard gamma-counter with 58% efficiency. Specific experimental details are given with each figure. Dissociation experiments. Following a standard insulin binding assay, the cells in parallel dishes were washed four times rapidly with medium A at 4 ° C, then incubated with 5 ml medium A at either 22 or 4 ° C. At intervals, the medium in sets of dishes was aspirated, the cells were washed with an additional 5 ml medium A at 4°C, and then dissolved in 2 M NaOH as above for radioactivity determination. Cell counts. In all experiments, cells from paired dishes were removed by scraping into phosphate-buffered saline, and diluted suspensions were counted with a Coulter ZBI Counter fitted with a 100 #m aperture and a size distribution analyzer. Effects o f chronic exposure to elevated concentrations o f insulin. To test for changes in the ability to bind 125I-labeled insulin following exposure to native insulin, i.e. down-regulation, cells were pre-incubated with insulin in medium A for periods of from 1 to 5 h at 37 ° C, washed twice with medium A for 20 min per wash and twice with phosphate-buffered saline for 10 rain per wash, all at 22°C. The standard insulin binding assay was then performed as described above. Where indicated, cycloheximide was included in the down-regulation media. To test for the recovery from the effect of cycloheximide, paired sets of dishes were preincubated, washed, and 2 ml of standard growth medium added to each dish. These cells were incubated at 37°C for recovery periods of 18--42 h. At appropriate times, the cells were washed twice with 2 ml phosphate-buffered saline, followed by a standard i2SI-labeled insulin binding assay at 22°C.
474
Incorporation of isoleucine into protein. After incubation for 1 h with L-[4,53H2(N)]isoleucine (New England Nuclear), dishes were placed on ice, and cells washed with 2 ml aliquots of phosphate-buffered saline. Cells were then removed b y scraping into 1 ml phosphate-buffered saline. Dishes were rinsed with t w o additional 1-ml aliquots of phosphate-buffered saline. The rinses were pooled with the original cell suspension and centrifuged at 1600 × g for 5 min at 4°C. The supernatant fluid was aspirated, and the cells suspended in 1 ml H20. Carrier bovine serum albumin (50 pl of a 10 mg/ml solution) was added, and the proteins were precipitated with 1 ml 20% trichloroacetic acid at 4 ° C. The precipitate was isolated on Whatman GF-C glass fiber filters using a Millipore manifold. The tubes were rinsed with two 2-ml aliquots of cold 5% trichloroacetic acid, and the rinses were poured over the filters. The proteins on the filters were dissolved in Protosol (Amersham-Searle), the solution neutralized with acetic acid, then counted in a mixture composed to 0.4% PPO and 0.01% POPOP in toluene/Triton X-100 ( 9 : 8 , v/v) in a liquid scintillation counter. Incorporation of glucose into glycogen. Cells were seeded in petri dishes and refed on the second day. After 24 h, the medium was changed to an Eagle's minimal essential medium modified to contain only 400 •g/ml glucose. After 24 h in modified minimal essential medium, the medium was aspirated, and fresh modified minimal essential medium with D-[6-~4C]glucose (AmershamSearle 2.0 Ci/mol; 10 pCi), with insulin as indicated, added. Cells were incubated for 1 h at 37°C, unless otherwise indicated. After incubation, the medium was aspirated, and the cells quickly washed with two 2-ml aliquots of phosphate-buffered saline (4°C). Carrier glycogen (0.1 ml of a 20 mg/ml solution) was added to each dish, and 1 ml of warm (37°C) 30% KOH was added to dissolve the cells. Dishes were rinsed twice with 0.5-ml aliquots of 30% KOH, and the extracts pooled in glass centrifuge tubes. The tubes were maintained in a 100°C bath for 5 min, cooled, and the glycogen precipitated b y the method of Van Handel [19]. The precipitated glycogen was collected on Whatman GF-A filters, dissolved in 0.2 ml H20, neutralized with acetic acid, and counted in the same liquid scintillation solution used above. Results
Characteristics of insulin binding Specific and non-specific binding reached maxima in 30 min at 22°C, and these were maintained for at least 90 min (Fig. 2); no differences in binding kinetics were noted between early (normal) and late passage (spontaneously transformed) cells. Specific insulin binding, which was taken as the difference between total and non-specific binding, and which varied substantially among repetitive experiments, averaged 15.0 fmol/5 • 106 hepatocytes in this experiment; non-specific binding never exceeded 2--3% of the total binding (Fig. 2). Binding of 12SI-labeled insulin to hepatocytes was linearly related to cell number b e t w e e n low density ( 0 . 4 . 1 0 6 cells per dish) and confluent ( 1 . 3 - 1 0 4 cells per dish) m o n o l a y e r s contained in 60-mm petri dishes. No degradation of 12SI-labeled insulin during the binding assay could be detected b y the criteria of precipitation b y 10% trichloroacetic acid and rebinding to isolated rat liver
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plasma membranes (data not shown). The binding of 12Sl-labeledinsulin was competitively inhibited by native insulin, as well as b y desnonapeptide proinculin, proinsulin and desoctapeptide insulin, in proportion to their biological activity; binding of 12SI-labeledinsulin was unaffected by corticotropin, human growth hormone, or glucagon (Fig. 3). Insulin, bound to h epatocytes, dissociated rapidly at 22°C, whereas at 4°C dissociation was m u c h slower (Fig. 4).
In contrast to reports that insulin can substitute for serum in supporting the
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growth of chick embryo cells [20--22], insulin could not do so for RL-PR4~ hepatocytes (Fig. 5). Flier et al. [23,24] have shown that sera from patients with Acanthosis Nigricans Type B contain an immunoglobulin (IgG) which is directed against a variety of cell~urface insulin receptors, in addition to those of the patients' own circulating monocytes. Monolayers of RL-PR~ cells were assessed for their capacity to bind a tracer concentration of 12SI4abeled insulin either after or during exposure to the anti-insulin receptor serum; control ceils were exposed to normal human serum. When cells were exposed to the antibody prior to the insulin binding assay (Fig. 6) the serum dilutions (titers) which inhibited insulin binding by 50% were about 1 : 3000 for both late passage (126--135 doublings) and early passage (45--76 doublings) cells. When the antiserum was present during the insulin binding assay (data not shown) titers were somewhat lower, being about 1 : 2500 and 1 : 1500 for late and early passage cells, respectively.
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Effects of chronic exposure to insulin Chronic exposure to insulin in vivo in humans and laboratory animals has been reported to reduce the number of insulin binding sites, at least on rat fat cells and thyrnocytes, human circulating monocytes and cultured IM-9 lymphocytes [1], This phenomenon, which was initially observed by Gavin et al. [5] in studies with IM-9, has been termed "down-regulation". Pre-incubation of R L - P R ~ hepatocytes with a high concentration of native insulin, followed by extensive washing to remove all bound insulin, reduced by approx. 60% in 5 h the subsequent binding of ~2SI-labeled insulin (Fig. 7). Periods longer than 5 h were not tested since the cells could not be maintained well in the down-regulation medium for longer periods. At 5 h, down-regulation was maximal with 10 -s M insulin (Fig. 8); down-regulation at the more physiological concentration of 10 -1° M was low, but statistically different than control values. It was possible that the observed decreases in binding of 125I-labeled insulin might be due, not to changes in receptor number, but rather, to saturation of the receptor sites with native insulin during the preincuhation period which was not removed during washing. To test this possibility, parallel experiments were performed in which the same high concentrations of native insulin were present with the cells during only the final hour. Cells so treated did n o t subsequently
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exhibit decreased binding of 12SIqabeled insulin (Table I, top panel). In addition, other washing procedures were tested. Two additional washes with medium A did not alter the subsequent binding of 12SIqabeled insulin (Table II). Neither was binding increased following washes at 37°(3, or at pH 6 instead of 7.5 (Table II), procedures known to strip hormones from cell surfaces. The ability of the hepatocytes to recover from down-regulation was I00 .......
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