0013-7227/78/1034-1440$02.00/0 Endocrinology Copyright © 1978 by The Endocrine Society
Vol. 103, No. 4 Printed in U.S.A.
The Effect of Zinc on Insulin Metabolism* EDWARD R. ARQUILLA, STEVEN PACKER, WARREN TARMAS, AND SUZANNE MIYAMOTO Department of Pathology, University of California Irvine Medical Center, Orange, California 92668 ABSTRACT. Experiments were designed to study the effect of Zn on in vivo and in vitro insulin metabolism. The in vivo experiments involved pretreating mice with either Zn or Na, followed by ip [126I]iodoinsulin injection. Pretreatment of mice with Zn resulted in an accelerated and increased magnitude of binding of [125I]iodoinsulin to the liver compared to mice pretreated with Na. Results are submitted which support the probability that the changes in the amounts of intact and degraded insulin in circulation with time are related to the binding and degradation of insulin in the liver rather than in the kidney. In vivo ip injected insulin was demonstrated to
preferentially bind to the plasma membrane of the liver. Liver plasma membranes isolated from mice pretreated with Zn bound more [125I]iodoinsulin than plasma membranes of Na-pretreated mice. In vitro experiments employing isolated liver plasma membranes demonstrated that added Zn increased the binding and inhibited the degradation of insulin. Evidence is presented that supports the concept that two receptors exist, one at which degradation of [126I]iodoinsulin occurs and another at which degradation does not occur. (Endocrinology 103: 1440, 1978)
I
N MOST mammalian species, insulin is the presence of Zn might have upon insulin stored in the /?-cells of the pancreas as Zn binding and degradation in vivo and in vitro. The results obtained support the interprecrystals (1-3). Therefore, it is likely that insulin is secreted from the pancreas in a Zn tation that pretreatment with Zn causes an form and reaches the liver in a very short accelerated and increased magnitude in the time. The conformation of insulin as it circu- binding and simultaneously inhibits the deglates in the periphery and back to the liver is radation of insulin by liver plasma membranes. In the same experiments, pretreatless certain. The addition of Zn to insulin is known to ment with Zn did not affect the binding and cause conformational changes. These changes degradation of insulin by the kidney. Therehave been detected by near and far ultraviolet fore, it was concluded that ip injected insulin circular dichroic spectroscopy (4-7). Recently, is metabolized differently in mice pretreated Bently et al. have demonstrated a structural with Zn than in those pretreated with Na. The difference of three of the six insulin molecules events which affect this metabolism are cenin 4-Zn insulin crystals compared to 2-Zn crys- trally focused on the binding and degradation tals (8). We have recently demonstrated that of the insulin by receptors in the liver plasma the antigenic determinants of insulin in solu- membranes. The data presented in this report tion at monomeric concentrations are per- support earlier findings that there seems to be turbed by Zn (6, 7). In view of these Zn-in- two types of insulin receptors present on mice duced structural alterations of insulin, exper- liver plasma membranes, one concerned with iments were designed to determine the effect the degradation of insulin and another at which degradation does not take place (6). Received November 7,1977. Address requests for reprints to: Dr. Edward R. Arquilla, California College of Medicine, University of California Irvine Medical Center, Department of Pathology, 101 City Drive South, Orange, California 92668. * Presented in part at the Ninth Congress of the International Diabetes Federation, November, 1976, New Delhi, India (Excerpta Medica Proceedings, in press) and at the 59th Annual Meeting of The Endocrine Society, June 8,1977, Chicago, IL {Endocrinology 100:108,1977).
Materials and Methods Animals Swiss Webster outbred male albino mice (28-40 g; Simonsen Laboratories) were maintained on a standard diet of Purina mouse chow, starved overnight, and fed ad libitum 1 h before the experiments.
1440 The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 08 July 2015. at 14:31 For personal use only. No other uses without permission. . All rights reserved.
EFFECT OF Zn ON INSULIN METABOLISM Chemicals
1441
of the samples was measured (Table 1) by atomic absorbtion by Elek Microanalytical Laboratories (Torrance, CA).
Chemicals were purchased from the following suppliers: l25I (SA, 11-17 mCi/ju,g), AmershamSearle; ZnSO4, A. R. Baker; Na2SO4, A. R. Mallinckrodt Co.; sodium heparin, Riker Laboratories; in- Innh vivo effect ofZn on binding and degradation of sulin (Novo lot YZ0223 AMX), Elanco Products [ I]iodoinsulin by liver fractions Co.; sucrose, Schwarz Mann; Trizma base, Sigma; Weight-matched Swiss Webster male mice were and Ficoll, Pharmacia. injected ip with 5 /xl/g BW 2 X 10~2 M solution of either ZnSO4 or Na2SO4 in saline containing 800 Preparation of monocomponent insulin and car- U/ml sodium heparin. One hour later, all mice were rier-free fl25ljmonoiodotyrosylinsulin injected with 3 x 106 cpm (1.75 pmol) [125I]iodoinsulin in 150 jul RIB-GPS. Five minutes after this Both monocomponent insulin and [125I]monoioinjection, the animals were sacrificed and their 125 dotyrosylinsulin ([ I]iodoinsulin) were prepared livers were excised, weighed, and processed immeby a modification (9) of the method of Hamlin and diately. 125 Arquilla (5). The preparation of [ I]iodoinsulin Isolation of liver plasma membranes was percontained 1 atom iodine/insulin molecule, and had formed by a modification of the method of House a specific activity of approximately 250 mCi/mg and Weidemann (11-14). Livers from five animals (9). were minced and washed three times with medium H (0.25 M sucrose-5 mM Tris, pH 7.4, 4C). The In vivo effect ofZn on the binding and degradation minced tissue was homogenized in 2 vol medium H l2b of [ I]iodoinsulin at low speed in a Potter-Elvehjem Teflon glass homogenizer. The homogenate was filtered through Weight-matched mice were injected ip with 5 2 four layers of cheesecloth and diluted with medium jul/g BW 2 X 10~ M solution of either ZnSO4 or Na2SO4 in saline containing 800 U/ml sodium hep- H to a final volume of approximately 5 ml/g tissue. arin. One hour later, all animals were injected ip A 1.0-ml aliquot (homogenate; see Table 2) was with 1 X 106 cpm (0.6 pmol) [125I]iodoinsulin in 150 removed for analysis. The remaining homogenate was centrifuged at p\ 7 mM sodium barbital-acetate buffer, pH 7.4, 10,000 X gat 4 C for 10 min. The supernatant was containing 0.15 M sodium chloride and 1.5% normal guinea pig serum (RIB-GPS). At 2.5, 5, 10, 20, 40, decanted and centrifuged a second time. The two 80, and 160 min after the injection of [125I]iodoin- pellets containing subcellular organelles were dissulin, animals were sacrificed by exsanguination to carded. A 1.0-ml aliquot of the supernatant (Sup A; obtain a maximum amount of blood. The livers and see Table 2) was removed for analysis. The superkidneys were excised and immediately frozen on natant was centrifuged for 60 min at 75,000 X g at 4C to pellet the crude plasma membranes. The dry ice. The blood from each animal was centrifuged at crude membranes were then gently suspended in 1740 X g and the plasma was decanted. Duplicate 10% Ficoll-5 mM Tris, pH 7.4, at 4 C using a hand 100-jul samples of the plasma were diluted with 400 homogenizer (eight strokes). A 1.0-ml aliquot (peljul veronal buffered saline, pH 7.4 (10), containing 5 let B; see Table 2) was removed for analysis. The X 10"4 M MgCl2, 1 X 10"4 M CaCl2, and 0.15% suspended pellet B (20 ml) was layered on a disconcrystallized bovine plasma albumin (VBS-albumin) tinuous Ficoll gradient (12 ml 15%, 6 ml 26%) and centrifuged at 90,000 X g at 4 C for 90 min. A 1.0and assayed for intact and degraded insulin. Frozen liver and kidney samples were thawed, ml aliquot of the 10% Ficoll fraction was removed minced, and washed three times in VBS-albumin for analysis. Analysis of all samples included determination of protein concentration by the method (5 ml/g wet tissue). Samples were centrifuged at of Lowry (15) and the amounts of intact and de1250 X g at 4 C for 5 min and the supernatants were graded [125I]iodoinsulin. discarded. The tissue pellets were then resuspended in 3.0 ml (liver) or 1.5 ml (kidney) VBS-albumin and homogenized with a motorized pestle. OneAssay for intact and degraded [l2bI]iodoinsulin milliliter samples of homogenized tissue were used for assay of intact and degraded insulin. Each sample was treated with an equal volume of 10% trichloroacetic acid (TCA) for 10 min at 4 C, At 60, 65, and 85 min after the injection of Zn or Na into mice, plasma and liver samples were taken and centrifuged at 1740 X g for 10 min. The superand processed, as described above. The Zn content natant, which contained TCA-soluble [125I]iodoin-
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 08 July 2015. at 14:31 For personal use only. No other uses without permission. . All rights reserved.
ARQUILLA ET AL.
1442
sulin, designated as degraded insulin, was decanted. The pellet contained TCA-precipitable [125I]iodoinsulin and was designated as intact insulin. The quantity of degraded and intact [125I]iodoinsulin was determined by measuring the radioactivity in the TCA supernatant and TCA pellet, respectively, in a Beckman Biogamma Counter.
Endo • 1978 Vol 103 • No 4
ZnSO« 'No 2 SO 4
o
-2
c/> z en co
c c
In vitro effect of added Zn on binding and degradation of f1251] iodoinsulin by liver plasma membranes Purified liver plasma membranes for three in vitro studies were isolated from a crude liver membrane pellet by centrifugation on a discontinuous Ficoll gradient, as described above. The 10% Ficoll fractions were pooled and stored in 5-ml aliquots at -70 C. The characterization of this preparation has been well documented by House and Weidemann (11-13). Plasma membranes (250 /xg protein) were incubated for 30 min in RIB supplemented with 1.5% human serum albumin (RIB-HSA) at 30 C with 6.25 fmol [125I]iodoinsulin which has been preincubated with varying amounts of Zn in a final volume of 250 fA. The final concentrations of exogenous Zn in the reactions were 0, 6 X 10"6, 3 X 10"5, 6 X 10"5, 3 X 10"4, and 6 X 10~4 M. In competitive binding studies, 4.0 JUM monocomponent insulin was added to the reaction mixture. The reactions were stopped by the addition of 0.5 ml cold RIB-HSA. The liver plasma membranes were pelleted by centrifugation at 27,000 X g and the supernatant was decanted. The pellet was washed once with 0.5 ml RIB-HSA. Both the combined supernatants and the resuspended pellet were treated with an equal volume of 10% TCA. The [125I]iodoinsulin in the combined supernatants was separated into TCA-precipitable (free intact) [I25I]iodoinsulin and TCA-insoluble (free degraded) [125I]iodoinsulin. Similarly, the resuspended liver plasma membranes were treated with an equal volume of 10% TCA. The [125I]iodoinsulin recovered in the TCA precipitate was designated as bound intact [125I]iodoinsulin and that in the TCA supernatant as bound degraded [125I]iodoinsulin (see Fig. 4).
Results In vivo effect ofZn on binding and degradation of [mI]iodoinsulin with time The most pronounced effect observed was the binding and degradation of [125I]iodoinsulin by the livers of Zn-treated mice (Fig. 1A).
60
o5 g
Vol. 103, No. 4 Printed in U.S.A.
The Effect of Zinc on Insulin Metabolism* EDWARD R. ARQUILLA, STEVEN PACKER, WARREN TARMAS, AND SUZANNE MIYAMOTO Department of Pathology, University of California Irvine Medical Center, Orange, California 92668 ABSTRACT. Experiments were designed to study the effect of Zn on in vivo and in vitro insulin metabolism. The in vivo experiments involved pretreating mice with either Zn or Na, followed by ip [126I]iodoinsulin injection. Pretreatment of mice with Zn resulted in an accelerated and increased magnitude of binding of [125I]iodoinsulin to the liver compared to mice pretreated with Na. Results are submitted which support the probability that the changes in the amounts of intact and degraded insulin in circulation with time are related to the binding and degradation of insulin in the liver rather than in the kidney. In vivo ip injected insulin was demonstrated to
preferentially bind to the plasma membrane of the liver. Liver plasma membranes isolated from mice pretreated with Zn bound more [125I]iodoinsulin than plasma membranes of Na-pretreated mice. In vitro experiments employing isolated liver plasma membranes demonstrated that added Zn increased the binding and inhibited the degradation of insulin. Evidence is presented that supports the concept that two receptors exist, one at which degradation of [126I]iodoinsulin occurs and another at which degradation does not occur. (Endocrinology 103: 1440, 1978)
I
N MOST mammalian species, insulin is the presence of Zn might have upon insulin stored in the /?-cells of the pancreas as Zn binding and degradation in vivo and in vitro. The results obtained support the interprecrystals (1-3). Therefore, it is likely that insulin is secreted from the pancreas in a Zn tation that pretreatment with Zn causes an form and reaches the liver in a very short accelerated and increased magnitude in the time. The conformation of insulin as it circu- binding and simultaneously inhibits the deglates in the periphery and back to the liver is radation of insulin by liver plasma membranes. In the same experiments, pretreatless certain. The addition of Zn to insulin is known to ment with Zn did not affect the binding and cause conformational changes. These changes degradation of insulin by the kidney. Therehave been detected by near and far ultraviolet fore, it was concluded that ip injected insulin circular dichroic spectroscopy (4-7). Recently, is metabolized differently in mice pretreated Bently et al. have demonstrated a structural with Zn than in those pretreated with Na. The difference of three of the six insulin molecules events which affect this metabolism are cenin 4-Zn insulin crystals compared to 2-Zn crys- trally focused on the binding and degradation tals (8). We have recently demonstrated that of the insulin by receptors in the liver plasma the antigenic determinants of insulin in solu- membranes. The data presented in this report tion at monomeric concentrations are per- support earlier findings that there seems to be turbed by Zn (6, 7). In view of these Zn-in- two types of insulin receptors present on mice duced structural alterations of insulin, exper- liver plasma membranes, one concerned with iments were designed to determine the effect the degradation of insulin and another at which degradation does not take place (6). Received November 7,1977. Address requests for reprints to: Dr. Edward R. Arquilla, California College of Medicine, University of California Irvine Medical Center, Department of Pathology, 101 City Drive South, Orange, California 92668. * Presented in part at the Ninth Congress of the International Diabetes Federation, November, 1976, New Delhi, India (Excerpta Medica Proceedings, in press) and at the 59th Annual Meeting of The Endocrine Society, June 8,1977, Chicago, IL {Endocrinology 100:108,1977).
Materials and Methods Animals Swiss Webster outbred male albino mice (28-40 g; Simonsen Laboratories) were maintained on a standard diet of Purina mouse chow, starved overnight, and fed ad libitum 1 h before the experiments.
1440 The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 08 July 2015. at 14:31 For personal use only. No other uses without permission. . All rights reserved.
EFFECT OF Zn ON INSULIN METABOLISM Chemicals
1441
of the samples was measured (Table 1) by atomic absorbtion by Elek Microanalytical Laboratories (Torrance, CA).
Chemicals were purchased from the following suppliers: l25I (SA, 11-17 mCi/ju,g), AmershamSearle; ZnSO4, A. R. Baker; Na2SO4, A. R. Mallinckrodt Co.; sodium heparin, Riker Laboratories; in- Innh vivo effect ofZn on binding and degradation of sulin (Novo lot YZ0223 AMX), Elanco Products [ I]iodoinsulin by liver fractions Co.; sucrose, Schwarz Mann; Trizma base, Sigma; Weight-matched Swiss Webster male mice were and Ficoll, Pharmacia. injected ip with 5 /xl/g BW 2 X 10~2 M solution of either ZnSO4 or Na2SO4 in saline containing 800 Preparation of monocomponent insulin and car- U/ml sodium heparin. One hour later, all mice were rier-free fl25ljmonoiodotyrosylinsulin injected with 3 x 106 cpm (1.75 pmol) [125I]iodoinsulin in 150 jul RIB-GPS. Five minutes after this Both monocomponent insulin and [125I]monoioinjection, the animals were sacrificed and their 125 dotyrosylinsulin ([ I]iodoinsulin) were prepared livers were excised, weighed, and processed immeby a modification (9) of the method of Hamlin and diately. 125 Arquilla (5). The preparation of [ I]iodoinsulin Isolation of liver plasma membranes was percontained 1 atom iodine/insulin molecule, and had formed by a modification of the method of House a specific activity of approximately 250 mCi/mg and Weidemann (11-14). Livers from five animals (9). were minced and washed three times with medium H (0.25 M sucrose-5 mM Tris, pH 7.4, 4C). The In vivo effect ofZn on the binding and degradation minced tissue was homogenized in 2 vol medium H l2b of [ I]iodoinsulin at low speed in a Potter-Elvehjem Teflon glass homogenizer. The homogenate was filtered through Weight-matched mice were injected ip with 5 2 four layers of cheesecloth and diluted with medium jul/g BW 2 X 10~ M solution of either ZnSO4 or Na2SO4 in saline containing 800 U/ml sodium hep- H to a final volume of approximately 5 ml/g tissue. arin. One hour later, all animals were injected ip A 1.0-ml aliquot (homogenate; see Table 2) was with 1 X 106 cpm (0.6 pmol) [125I]iodoinsulin in 150 removed for analysis. The remaining homogenate was centrifuged at p\ 7 mM sodium barbital-acetate buffer, pH 7.4, 10,000 X gat 4 C for 10 min. The supernatant was containing 0.15 M sodium chloride and 1.5% normal guinea pig serum (RIB-GPS). At 2.5, 5, 10, 20, 40, decanted and centrifuged a second time. The two 80, and 160 min after the injection of [125I]iodoin- pellets containing subcellular organelles were dissulin, animals were sacrificed by exsanguination to carded. A 1.0-ml aliquot of the supernatant (Sup A; obtain a maximum amount of blood. The livers and see Table 2) was removed for analysis. The superkidneys were excised and immediately frozen on natant was centrifuged for 60 min at 75,000 X g at 4C to pellet the crude plasma membranes. The dry ice. The blood from each animal was centrifuged at crude membranes were then gently suspended in 1740 X g and the plasma was decanted. Duplicate 10% Ficoll-5 mM Tris, pH 7.4, at 4 C using a hand 100-jul samples of the plasma were diluted with 400 homogenizer (eight strokes). A 1.0-ml aliquot (peljul veronal buffered saline, pH 7.4 (10), containing 5 let B; see Table 2) was removed for analysis. The X 10"4 M MgCl2, 1 X 10"4 M CaCl2, and 0.15% suspended pellet B (20 ml) was layered on a disconcrystallized bovine plasma albumin (VBS-albumin) tinuous Ficoll gradient (12 ml 15%, 6 ml 26%) and centrifuged at 90,000 X g at 4 C for 90 min. A 1.0and assayed for intact and degraded insulin. Frozen liver and kidney samples were thawed, ml aliquot of the 10% Ficoll fraction was removed minced, and washed three times in VBS-albumin for analysis. Analysis of all samples included determination of protein concentration by the method (5 ml/g wet tissue). Samples were centrifuged at of Lowry (15) and the amounts of intact and de1250 X g at 4 C for 5 min and the supernatants were graded [125I]iodoinsulin. discarded. The tissue pellets were then resuspended in 3.0 ml (liver) or 1.5 ml (kidney) VBS-albumin and homogenized with a motorized pestle. OneAssay for intact and degraded [l2bI]iodoinsulin milliliter samples of homogenized tissue were used for assay of intact and degraded insulin. Each sample was treated with an equal volume of 10% trichloroacetic acid (TCA) for 10 min at 4 C, At 60, 65, and 85 min after the injection of Zn or Na into mice, plasma and liver samples were taken and centrifuged at 1740 X g for 10 min. The superand processed, as described above. The Zn content natant, which contained TCA-soluble [125I]iodoin-
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 08 July 2015. at 14:31 For personal use only. No other uses without permission. . All rights reserved.
ARQUILLA ET AL.
1442
sulin, designated as degraded insulin, was decanted. The pellet contained TCA-precipitable [125I]iodoinsulin and was designated as intact insulin. The quantity of degraded and intact [125I]iodoinsulin was determined by measuring the radioactivity in the TCA supernatant and TCA pellet, respectively, in a Beckman Biogamma Counter.
Endo • 1978 Vol 103 • No 4
ZnSO« 'No 2 SO 4
o
-2
c/> z en co
c c
In vitro effect of added Zn on binding and degradation of f1251] iodoinsulin by liver plasma membranes Purified liver plasma membranes for three in vitro studies were isolated from a crude liver membrane pellet by centrifugation on a discontinuous Ficoll gradient, as described above. The 10% Ficoll fractions were pooled and stored in 5-ml aliquots at -70 C. The characterization of this preparation has been well documented by House and Weidemann (11-13). Plasma membranes (250 /xg protein) were incubated for 30 min in RIB supplemented with 1.5% human serum albumin (RIB-HSA) at 30 C with 6.25 fmol [125I]iodoinsulin which has been preincubated with varying amounts of Zn in a final volume of 250 fA. The final concentrations of exogenous Zn in the reactions were 0, 6 X 10"6, 3 X 10"5, 6 X 10"5, 3 X 10"4, and 6 X 10~4 M. In competitive binding studies, 4.0 JUM monocomponent insulin was added to the reaction mixture. The reactions were stopped by the addition of 0.5 ml cold RIB-HSA. The liver plasma membranes were pelleted by centrifugation at 27,000 X g and the supernatant was decanted. The pellet was washed once with 0.5 ml RIB-HSA. Both the combined supernatants and the resuspended pellet were treated with an equal volume of 10% TCA. The [125I]iodoinsulin in the combined supernatants was separated into TCA-precipitable (free intact) [I25I]iodoinsulin and TCA-insoluble (free degraded) [125I]iodoinsulin. Similarly, the resuspended liver plasma membranes were treated with an equal volume of 10% TCA. The [125I]iodoinsulin recovered in the TCA precipitate was designated as bound intact [125I]iodoinsulin and that in the TCA supernatant as bound degraded [125I]iodoinsulin (see Fig. 4).
Results In vivo effect ofZn on binding and degradation of [mI]iodoinsulin with time The most pronounced effect observed was the binding and degradation of [125I]iodoinsulin by the livers of Zn-treated mice (Fig. 1A).
60
o5 g
