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Sensitivity of cultured fibroblasts to human, bovine and porcine insulins
Cell Biology
International
Reports,
Vol. 11, No. 8, August
1987
591
SENSITIVITY OF CULTUREDPIBROBLASTSTO IlUHAN, BOVINEAND PORCINEINSULINS. Alain
Aussel, Luc Cynober, Christian Hernvannl, Jean Agneray and Ohvanesse Ekindjian. CNRS UA 622 - Universite 92296 Chatenay-Malabry
Paris XI cedex
ABSTRACT We have studied the effects of human, bovine and by cultured chicken embryo porcine insulin on sugar transport monolayers. For a 30 min. associa_gion time, human fibroblast and bovine insulin at a concentration of 5.10 M stimulated 2-deoxy-D-glucose uptake. (respectively by an average 58 p.cent and 55 p.cent over basal). Porcine insulin was less potent since a concentration similar stimulation. of5.10m7M was necessary to obtain occur only Moreover, the maximal effect of porcine insulin time instead of 30 min. for the after 60 min. association other peptides. The differences between the effects of insulin from different sources is related to species-dependent differences in their structure. INTRODUCTION Studies concerning the action of insulin show that fibroblasts are sensitive to insulin for glucose uptake. However, from one study to another, there is considerable heterogeneity in the results (Cynober et al., 1986 for a Among the factors which could explain these review). the source of insulin might be involved since discrepancies, both porcine or bovine insulin are generally used. However, this aspect remains largely unexplored. The problem is now compounded by the emergence of commercially-available human insulin . Indeed, studies performed in vivo in humans (Schluter et al., 1983) and rabbits (Pingel et al., 1985) suggest that human insulin is more biologically active than porcine insulin. There is a lack of data concerning in vitro studies, . For these reasons, it appeared of interest to test the potency of porcine, bovine and human insulin on the uptake of 2-deoxy-D-glucose (2- DOG), a non-metabolizable analogue of D-glucose (Smith and Gorski, 1968), in chick embryo fibroblasts (CEF), a model commonly used for studies of insulin action on the sugar uptake process. 1 To whom reprint
requests
0309-1651/87~080591-08/$03.00/o
should be directed. @ 1987 Academic
Press Ltd.
592
Cell Biology
International
Reports,
Vol. 7 1, No. 8, August
MATERIALS ANDMETHODS e&s Bovine, porcine and biosynthetic human insulins were a gift from Dr.W.R FIELDS (ELI LILLY Research Laboratories, Indianapolis, Indiana-U.S.A.) 2-deoxy-D-glucose
was from
SIGMA (St
Louis-MO,
U.S.A.) Ci/mmol)
2-deoxy-D was supplied
1-3H - glucose (spec.act. 15-25 by Amersham International (Bucks, UK).
Culture medium (Eagle's minimum essential medium by fetal calf serum) and Dulbecco's (MEW, supplemented phosphate buffered saline (PBS) were obtained from Eurobio (Paris, France). cells Chick embryo fibroblast cultures were prepared from 10 day-old embryos as previously described (Cals et al., 1981) and cultured until confluency in MEM containing 5 p. cent fetal calf serum. Since it has been found (Fujimoto and Williams, 1974) that the presence of serum conceals any potential effect of insulin on glucose uptake, serum was removed from the culture medium 4 hours prior to the experiments. Cultures were incubated either for 30 min. with different concentrations of insulin or for various association times with one concentration of insulin. The medium was removed and cultures were washed 3 times with 2 ml of PBS (pH 7,5) at 37" C. Control cultures were submitted to the same experimental protocol but without exposure to insulin. 2-DoGuptake -The experiment was carried out as described all cultures were elsewhere (Guermeur et al., 1984). Briefly, incubated 5 min. with PBS containing 500 uM 2-DOG (2 uCi/ml). At the end of this period, the incubation medium was aspirated and the dishes were rinsed three times with ice-cold PBS (2 ml each time). The cell monolayers were dissolved in 1 ml NaOH 1M. Radioactivity was counted after dilution in Aqualuma solvent and protein content per dish was measured by Lowry's method (Lowry et al, 1951). Results are expressed in 10' cpm/5 min/mg protein Statistics Values were exnressed as mean + S.D. from trinlicate measurements and comparisons were made using the Mann and Whitney U-test. Differences with p values of less than 0,05 were considered significant.
1987
Cell Biology
international
Reports,
Vol. 11, No. 8, August
1987
593
RESULTS * Dose response -curves of various iusulius --on 2-DOG transport Confluent cultures were incubated at 37°C in serumfree culture medium with various_6 concentrations of bovine, human or porcine insulin (0 to 10 M). The association time in the presence of insulin was 30 min. As shown in figure 1, 2-DOG-transport was significantly increased8 (p < 0,05) with human insulin at concentrations above lo- M. Maximal action wa28 obtained for human peptide at a concentration of 5.10 M. (+ 58 p. cent). Bovine insulin gave similar results (maximal stimulation + 55 p. cent). The stimulation was _ 7si$yl&E~;ll~ with a concentration of porcine insulin of 10 maximal effect 46-7 p.cent) was obtained with higher concentrations (5.10 M).
g;,
c 0
10-a
10-6
10-1
insulin concentration CM) Fioure
Insulin
1 :
Cultures
action on Z-DOG uptake. treated 30 min. with human (
n
1. bovine
(A 1
or porcine ( l 1 insulin at various concentrations ( 0 to 10e6 MLUptake assays were then performed. Results are given as the mean + S.D. of 3 determinations l
P
and expressed <0.05
compared
in lo3
to basal
cpm/mg protein. values.
594
Cell Biology
International
Reports,
Vol. 11, No. 8, August
* Time-tours of insulin action --on 2-DOG transport. lO%iconcentrations of insulin were used in order to evaluate the action of various insulin on 2-DOG uptake with different association times. Results are given in figure 2 : 2-DOG uptake stimulation was obtained after 15 min. in the case of human and bovine insulins. Longer times led only to a slight increase in the biological effect. A 60 min.period was required to observe a significant action with porcine insulin.
L 0
15
36
60
120
time ( min. 1 FIaure 2 : Time course of insulin action on cultures were treated with 10-'M human ( n 1, or porcine (0 ) insulin for the times Indicated. assay5 were then performed. Results ( 10' cpm/mg protein) are given as mean * p <0.05 comparedto basal values
2-DOG uptake: bovine ( A 1 Uptake +
S.D.
DISCUSSION induce modifications in the Various factors which by insulin in cultured stimulation of glucose uptake have been extensively studied : glucose fibroblasts time of of fetal calf serum, presence starvation, preincubation in serum-free medium, time of exposure to data are few 1986). However, insulin (Cynober et al., available on the influence of the source of the insulin.
1987
Cell Biology
International
Reports,
Vol. 11, No. 8, August
1987
The data reported here show a relationship between relative to sugar uptake. The human and bovine insulin sensitivity and responsiveness of CEF to these hormones are similar and 2-m uptake stimulation appears rapidly. Cells are less sensitive to porcine insulin and stimulation requires longer association times. In a previous study, Gavin (1972) showed that porcine, bovine and human insulin compete equally for binding to human skin fibroblasts. However, he used very high concentrations of insulin (10 -4to lo-7M) and there was thus no convicting evidence that these 3 insulins bound to cells with equal affinity at physiological concentrations. More recently, Baizada (1976) demonstrated in CEF a maximal effect with 5.10-6 M porcine insulin, whereas stimulating Cynober (1985) found 5.10w7 M to be an effective concentration with using bovine insulin. These results appear to conflict our present data since we found a higher sensitivity of the cells to different insulins (factor 10). These discrepancies would appear to be related to a longer preincubation time in serum-free medium before the study of the hormone action on sugar uptake (4 hours instead of the 2 hours in previous studies). The amino acid sequences associated with the threedimensional structures might aid the comprehension of the biological activities. The insulins differ as shown in the table below. Insulin
;gF;;
Source
Aminoacid and position
on the A or B chain.
'Tj--yqy
The molecular heterogeneity is greater between human and bovine insulins than between human and porcine insulins. With regard to these differences in structure and to our results, the amino acid in position B 30 could be a major factor in the action of the hormone. However, although the same difference is observed between human and bovine insulins, the activity of these two peptides on sugar uptake was not found to differ. In bovine insulin, both the presence of an alanine in A8 (instead of threonine) and of valine in A 10 (instead of an isoleucine) could compensate for the possible (t negative effect N of the B 30 alanine. Indeed, the threedimensional configuration of the insulin monomer could explain the inter-relations between these amino acids. Even if these amino acids do not seem to be involved directly in
596
Cell Biology
International
Reports,
Vol. 11, No. 8, August
the spatial structure (Blundell et al., 1972) A8 lies just outside the putative receptor binding region of the molecule (Pullen et al., 1976). Thus, A8, A 10 and B 30 amino acids being on the same side of the molecule could form further stabilizing interactions with receptor residues. This hypothesis is strengthened by the work of Marki (1979), who demonstrated that the replacement, in human insulin, of one amino acid in various positions modified the biological activity. Among the seventeen analogues studied modifications on A8 led to large quantitative differences in the effect of insulin on glucose oxydation by rat adipocytes. The analogues with the greatest potency in vitro are those containing HisA which is present in chicken insulin. This was confirmed by Simon (1977) who found that chicken insulin is 1.5- to 1.9- fold as potent as porcine insulin in stimulation of glucose oxydation in isolated rat adipocytes. The in vivo potencies of human and porcine insulins still remain under discussion : Schluter (1983) demonstrated that the effect of exogenous human insulin in man was significantly higher than that of porcine insulin. On the contrary, Arias (1984)reported that the peptides did not differ in their action. It is noteworthy that former authors used biosynthetic human insulin, the latter semi-synthetic hormone from porcine insulin by a chemical modification on B 30 amino acid. the action of insulin on 2-DOG uptake In conclusion, in chick embryo fibroblasts varies according to the source of insulin. It would be interesting to investigate whether these differences correlate with variations in affinity for the insulin receptor or are due to differences in post-receptor processing. REFERENCES Arias, P., Kerner, W., Navascues, I., Schafauer, G. and human insulin and Pfeiffer, E.F. (1984) Semi-synthetic purified pork insulin do not differ in their biological potency. Klinische Wochenschrift 62, 1145-1150 Blundell, T.L., Dodson, C;, Hodgkin, D.C. and Mercola, D.A. (1972) The structure and biology of insulin. Advances in Protein Chemistry 26, 279 -402. Cals, M.J., Adolphe, M., Jardillier, J.C., Miocque, M., O.G.(1981) Fibroblast cultures in Agneray, J. and Ekindjian, conditions. Effects of antiexperimental disturbed metabolic inflammatory drugs. International Journal on Tissue Reactions 2, 113 - 119.
1987
Cell Biology
International
Reports,
Vol. 11, No. 8, August
1987
597
Cynober, L., Aussel, C., Chatelain, P., Vaubourdolle, M., Agneray, J. and Ekindjian, O.G.(1985) Insulin-like growth factor I/somatomedin-C action on 2-deoxy-D-glucose and alphaamino isobutyrate uptake in chick embryo fibroblasts. Biochimie 67, 1185 - 1190 Cynober, L., Capeau, J. and Ekindjian, O.G. (1986) Cultured fibroblasts as a model for studying insulin action on glucose uptake. Diabete et Metabolisme 12, 331 - 337 Fujimoto, W.Y. and Williams, R.H. (1974) Insulin action on the cultured human fibroblast : glucose uptake, protein synthesis, RNA synthesis. Diabetes 23, 443 - 448 Gavin, JR., Roth, J., Jen P. and Freychet, P. (1972) Insulin receptors in human circulating cells and fibroblasts. Proceedings of the National Academy of Sciences 69, 747-751 Guermeur, C., Cynober, L., Auget, J.L., Aussel, C., Agneray J and Ekindjian O.G. (1984) Effect of enzyme treatment on nytrient uptake by chick embryo fibroblasts. Cellular and r Molecular Biology 30, 529-535 Lowry, O.H., Rosebrough, N.J., Farra, L. and Randall, (1951) Protein measurement with folin phenol reagent. Journal of Biological Chemistry 132, 265 - 275.
R.J. The
Marki, F., Gasparo, M., Eisler, K., Kamber, B., Riniker, B., Rittel, W.and Sieber, P.(1979) Synthesis and biological activity of seventeen analogues of human insulin. HoppeSeyler's Zeitschrift Physiologische Chemie 360, 1619-1632. Pingel, M., Volund, A., Sorensen, E., Collins, J.E.and Dieter, C. (1985) Biological potency of porcine, bovine and human insulins in the rabbit bioassay system. Diabetologia 28, 862 869. Pullen, R.A., Lindsay, D.G., Wood, S.P., Tickle, I.J., Blundell, T.L., Wollmer, k, &ail, G., Brandeburg, D., Zahn, H., Gliemann, J. and Gammeltoft, S. (1976) Receptor-binding region of insulin.Nature 259, 369 - 373. Raizada, M.K. and Perdue, J.F. (1976) Mitogen receptors in chick embryo fibrqpsts. Kinetics, specificity, unmasking and synthesis of I Insulin binding site. The Journal of Biological Chemistry 251, 6445 - 6455.
598
Cell Biology
International
Reports
Vol. I?, No. 8, August
Schluter, K.J., Enzmann, F. and Kerp, L. (1983) Different potencies of biosynthetic human and purified porcine insulin. Hormone and Metabolic Research 15, 271 - 274. Simon, J., Freychet, insulin binding sites 13, 219-228.
P. and Rosselin, G. (1977) A study of in the chicken tissues. Diabetologia,
Smith, D.F. and Gorski, J. (1968) Oestrogen control of uterine glucose metabolism. Analysisbased on the transport and phosphorylation of 2-deoxy-Dglucose. The Journal of Biological Chemistry 243, 4169 - 4174.
Received:
29141 a7
Accepted:
3016187
1987
International
Reports,
Vol. 11, No. 8, August
1987
591
SENSITIVITY OF CULTUREDPIBROBLASTSTO IlUHAN, BOVINEAND PORCINEINSULINS. Alain
Aussel, Luc Cynober, Christian Hernvannl, Jean Agneray and Ohvanesse Ekindjian. CNRS UA 622 - Universite 92296 Chatenay-Malabry
Paris XI cedex
ABSTRACT We have studied the effects of human, bovine and by cultured chicken embryo porcine insulin on sugar transport monolayers. For a 30 min. associa_gion time, human fibroblast and bovine insulin at a concentration of 5.10 M stimulated 2-deoxy-D-glucose uptake. (respectively by an average 58 p.cent and 55 p.cent over basal). Porcine insulin was less potent since a concentration similar stimulation. of5.10m7M was necessary to obtain occur only Moreover, the maximal effect of porcine insulin time instead of 30 min. for the after 60 min. association other peptides. The differences between the effects of insulin from different sources is related to species-dependent differences in their structure. INTRODUCTION Studies concerning the action of insulin show that fibroblasts are sensitive to insulin for glucose uptake. However, from one study to another, there is considerable heterogeneity in the results (Cynober et al., 1986 for a Among the factors which could explain these review). the source of insulin might be involved since discrepancies, both porcine or bovine insulin are generally used. However, this aspect remains largely unexplored. The problem is now compounded by the emergence of commercially-available human insulin . Indeed, studies performed in vivo in humans (Schluter et al., 1983) and rabbits (Pingel et al., 1985) suggest that human insulin is more biologically active than porcine insulin. There is a lack of data concerning in vitro studies, . For these reasons, it appeared of interest to test the potency of porcine, bovine and human insulin on the uptake of 2-deoxy-D-glucose (2- DOG), a non-metabolizable analogue of D-glucose (Smith and Gorski, 1968), in chick embryo fibroblasts (CEF), a model commonly used for studies of insulin action on the sugar uptake process. 1 To whom reprint
requests
0309-1651/87~080591-08/$03.00/o
should be directed. @ 1987 Academic
Press Ltd.
592
Cell Biology
International
Reports,
Vol. 7 1, No. 8, August
MATERIALS ANDMETHODS e&s Bovine, porcine and biosynthetic human insulins were a gift from Dr.W.R FIELDS (ELI LILLY Research Laboratories, Indianapolis, Indiana-U.S.A.) 2-deoxy-D-glucose
was from
SIGMA (St
Louis-MO,
U.S.A.) Ci/mmol)
2-deoxy-D was supplied
1-3H - glucose (spec.act. 15-25 by Amersham International (Bucks, UK).
Culture medium (Eagle's minimum essential medium by fetal calf serum) and Dulbecco's (MEW, supplemented phosphate buffered saline (PBS) were obtained from Eurobio (Paris, France). cells Chick embryo fibroblast cultures were prepared from 10 day-old embryos as previously described (Cals et al., 1981) and cultured until confluency in MEM containing 5 p. cent fetal calf serum. Since it has been found (Fujimoto and Williams, 1974) that the presence of serum conceals any potential effect of insulin on glucose uptake, serum was removed from the culture medium 4 hours prior to the experiments. Cultures were incubated either for 30 min. with different concentrations of insulin or for various association times with one concentration of insulin. The medium was removed and cultures were washed 3 times with 2 ml of PBS (pH 7,5) at 37" C. Control cultures were submitted to the same experimental protocol but without exposure to insulin. 2-DoGuptake -The experiment was carried out as described all cultures were elsewhere (Guermeur et al., 1984). Briefly, incubated 5 min. with PBS containing 500 uM 2-DOG (2 uCi/ml). At the end of this period, the incubation medium was aspirated and the dishes were rinsed three times with ice-cold PBS (2 ml each time). The cell monolayers were dissolved in 1 ml NaOH 1M. Radioactivity was counted after dilution in Aqualuma solvent and protein content per dish was measured by Lowry's method (Lowry et al, 1951). Results are expressed in 10' cpm/5 min/mg protein Statistics Values were exnressed as mean + S.D. from trinlicate measurements and comparisons were made using the Mann and Whitney U-test. Differences with p values of less than 0,05 were considered significant.
1987
Cell Biology
international
Reports,
Vol. 11, No. 8, August
1987
593
RESULTS * Dose response -curves of various iusulius --on 2-DOG transport Confluent cultures were incubated at 37°C in serumfree culture medium with various_6 concentrations of bovine, human or porcine insulin (0 to 10 M). The association time in the presence of insulin was 30 min. As shown in figure 1, 2-DOG-transport was significantly increased8 (p < 0,05) with human insulin at concentrations above lo- M. Maximal action wa28 obtained for human peptide at a concentration of 5.10 M. (+ 58 p. cent). Bovine insulin gave similar results (maximal stimulation + 55 p. cent). The stimulation was _ 7si$yl&E~;ll~ with a concentration of porcine insulin of 10 maximal effect 46-7 p.cent) was obtained with higher concentrations (5.10 M).
g;,
c 0
10-a
10-6
10-1
insulin concentration CM) Fioure
Insulin
1 :
Cultures
action on Z-DOG uptake. treated 30 min. with human (
n
1. bovine
(A 1
or porcine ( l 1 insulin at various concentrations ( 0 to 10e6 MLUptake assays were then performed. Results are given as the mean + S.D. of 3 determinations l
P
and expressed <0.05
compared
in lo3
to basal
cpm/mg protein. values.
594
Cell Biology
International
Reports,
Vol. 11, No. 8, August
* Time-tours of insulin action --on 2-DOG transport. lO%iconcentrations of insulin were used in order to evaluate the action of various insulin on 2-DOG uptake with different association times. Results are given in figure 2 : 2-DOG uptake stimulation was obtained after 15 min. in the case of human and bovine insulins. Longer times led only to a slight increase in the biological effect. A 60 min.period was required to observe a significant action with porcine insulin.
L 0
15
36
60
120
time ( min. 1 FIaure 2 : Time course of insulin action on cultures were treated with 10-'M human ( n 1, or porcine (0 ) insulin for the times Indicated. assay5 were then performed. Results ( 10' cpm/mg protein) are given as mean * p <0.05 comparedto basal values
2-DOG uptake: bovine ( A 1 Uptake +
S.D.
DISCUSSION induce modifications in the Various factors which by insulin in cultured stimulation of glucose uptake have been extensively studied : glucose fibroblasts time of of fetal calf serum, presence starvation, preincubation in serum-free medium, time of exposure to data are few 1986). However, insulin (Cynober et al., available on the influence of the source of the insulin.
1987
Cell Biology
International
Reports,
Vol. 11, No. 8, August
1987
The data reported here show a relationship between relative to sugar uptake. The human and bovine insulin sensitivity and responsiveness of CEF to these hormones are similar and 2-m uptake stimulation appears rapidly. Cells are less sensitive to porcine insulin and stimulation requires longer association times. In a previous study, Gavin (1972) showed that porcine, bovine and human insulin compete equally for binding to human skin fibroblasts. However, he used very high concentrations of insulin (10 -4to lo-7M) and there was thus no convicting evidence that these 3 insulins bound to cells with equal affinity at physiological concentrations. More recently, Baizada (1976) demonstrated in CEF a maximal effect with 5.10-6 M porcine insulin, whereas stimulating Cynober (1985) found 5.10w7 M to be an effective concentration with using bovine insulin. These results appear to conflict our present data since we found a higher sensitivity of the cells to different insulins (factor 10). These discrepancies would appear to be related to a longer preincubation time in serum-free medium before the study of the hormone action on sugar uptake (4 hours instead of the 2 hours in previous studies). The amino acid sequences associated with the threedimensional structures might aid the comprehension of the biological activities. The insulins differ as shown in the table below. Insulin
;gF;;
Source
Aminoacid and position
on the A or B chain.
'Tj--yqy
The molecular heterogeneity is greater between human and bovine insulins than between human and porcine insulins. With regard to these differences in structure and to our results, the amino acid in position B 30 could be a major factor in the action of the hormone. However, although the same difference is observed between human and bovine insulins, the activity of these two peptides on sugar uptake was not found to differ. In bovine insulin, both the presence of an alanine in A8 (instead of threonine) and of valine in A 10 (instead of an isoleucine) could compensate for the possible (t negative effect N of the B 30 alanine. Indeed, the threedimensional configuration of the insulin monomer could explain the inter-relations between these amino acids. Even if these amino acids do not seem to be involved directly in
596
Cell Biology
International
Reports,
Vol. 11, No. 8, August
the spatial structure (Blundell et al., 1972) A8 lies just outside the putative receptor binding region of the molecule (Pullen et al., 1976). Thus, A8, A 10 and B 30 amino acids being on the same side of the molecule could form further stabilizing interactions with receptor residues. This hypothesis is strengthened by the work of Marki (1979), who demonstrated that the replacement, in human insulin, of one amino acid in various positions modified the biological activity. Among the seventeen analogues studied modifications on A8 led to large quantitative differences in the effect of insulin on glucose oxydation by rat adipocytes. The analogues with the greatest potency in vitro are those containing HisA which is present in chicken insulin. This was confirmed by Simon (1977) who found that chicken insulin is 1.5- to 1.9- fold as potent as porcine insulin in stimulation of glucose oxydation in isolated rat adipocytes. The in vivo potencies of human and porcine insulins still remain under discussion : Schluter (1983) demonstrated that the effect of exogenous human insulin in man was significantly higher than that of porcine insulin. On the contrary, Arias (1984)reported that the peptides did not differ in their action. It is noteworthy that former authors used biosynthetic human insulin, the latter semi-synthetic hormone from porcine insulin by a chemical modification on B 30 amino acid. the action of insulin on 2-DOG uptake In conclusion, in chick embryo fibroblasts varies according to the source of insulin. It would be interesting to investigate whether these differences correlate with variations in affinity for the insulin receptor or are due to differences in post-receptor processing. REFERENCES Arias, P., Kerner, W., Navascues, I., Schafauer, G. and human insulin and Pfeiffer, E.F. (1984) Semi-synthetic purified pork insulin do not differ in their biological potency. Klinische Wochenschrift 62, 1145-1150 Blundell, T.L., Dodson, C;, Hodgkin, D.C. and Mercola, D.A. (1972) The structure and biology of insulin. Advances in Protein Chemistry 26, 279 -402. Cals, M.J., Adolphe, M., Jardillier, J.C., Miocque, M., O.G.(1981) Fibroblast cultures in Agneray, J. and Ekindjian, conditions. Effects of antiexperimental disturbed metabolic inflammatory drugs. International Journal on Tissue Reactions 2, 113 - 119.
1987
Cell Biology
International
Reports,
Vol. 11, No. 8, August
1987
597
Cynober, L., Aussel, C., Chatelain, P., Vaubourdolle, M., Agneray, J. and Ekindjian, O.G.(1985) Insulin-like growth factor I/somatomedin-C action on 2-deoxy-D-glucose and alphaamino isobutyrate uptake in chick embryo fibroblasts. Biochimie 67, 1185 - 1190 Cynober, L., Capeau, J. and Ekindjian, O.G. (1986) Cultured fibroblasts as a model for studying insulin action on glucose uptake. Diabete et Metabolisme 12, 331 - 337 Fujimoto, W.Y. and Williams, R.H. (1974) Insulin action on the cultured human fibroblast : glucose uptake, protein synthesis, RNA synthesis. Diabetes 23, 443 - 448 Gavin, JR., Roth, J., Jen P. and Freychet, P. (1972) Insulin receptors in human circulating cells and fibroblasts. Proceedings of the National Academy of Sciences 69, 747-751 Guermeur, C., Cynober, L., Auget, J.L., Aussel, C., Agneray J and Ekindjian O.G. (1984) Effect of enzyme treatment on nytrient uptake by chick embryo fibroblasts. Cellular and r Molecular Biology 30, 529-535 Lowry, O.H., Rosebrough, N.J., Farra, L. and Randall, (1951) Protein measurement with folin phenol reagent. Journal of Biological Chemistry 132, 265 - 275.
R.J. The
Marki, F., Gasparo, M., Eisler, K., Kamber, B., Riniker, B., Rittel, W.and Sieber, P.(1979) Synthesis and biological activity of seventeen analogues of human insulin. HoppeSeyler's Zeitschrift Physiologische Chemie 360, 1619-1632. Pingel, M., Volund, A., Sorensen, E., Collins, J.E.and Dieter, C. (1985) Biological potency of porcine, bovine and human insulins in the rabbit bioassay system. Diabetologia 28, 862 869. Pullen, R.A., Lindsay, D.G., Wood, S.P., Tickle, I.J., Blundell, T.L., Wollmer, k, &ail, G., Brandeburg, D., Zahn, H., Gliemann, J. and Gammeltoft, S. (1976) Receptor-binding region of insulin.Nature 259, 369 - 373. Raizada, M.K. and Perdue, J.F. (1976) Mitogen receptors in chick embryo fibrqpsts. Kinetics, specificity, unmasking and synthesis of I Insulin binding site. The Journal of Biological Chemistry 251, 6445 - 6455.
598
Cell Biology
International
Reports
Vol. I?, No. 8, August
Schluter, K.J., Enzmann, F. and Kerp, L. (1983) Different potencies of biosynthetic human and purified porcine insulin. Hormone and Metabolic Research 15, 271 - 274. Simon, J., Freychet, insulin binding sites 13, 219-228.
P. and Rosselin, G. (1977) A study of in the chicken tissues. Diabetologia,
Smith, D.F. and Gorski, J. (1968) Oestrogen control of uterine glucose metabolism. Analysisbased on the transport and phosphorylation of 2-deoxy-Dglucose. The Journal of Biological Chemistry 243, 4169 - 4174.
Received:
29141 a7
Accepted:
3016187
1987