- Email: [email protected]
Effect of insulin on aminoisobutyric acid uptake by human non-rheumatoid and rheumatoid synovial cells
Volume 214, number 2, 327-330
FEB 04616
April 1987
Effect of insulin on aminoisobutyric acid uptake by human non-rheumatoid and rheumatoid synovial cells Christian Aussel, D o m i n i q u e D e s m o u l i n s , J e a n A g n e r a y a n d O v h a n e s s e G. E k i n d j i a n Laboratoire de Biochimie Gbn4rale C N R S UA 622, Universitb Paris-Sud, Tour D4, 5, rue J.B. Clement, 92296 Chatenay-Malabry Cedex, France
Received 19 February 1987 The biological sensitivity of cultured non-rheumatoid human synovial cells (NRSCs) and rheumatoid synovial cells (RSCs) was examined in terms of the ability of insulin to stimulate the uptake of ct-aminoisobutyrate (AIB). NRSCs, like numerous fibroblastic lines, were sensitive to physiological concentrations of the hormone: half-maximal stimulation was obtained with (4 x 10 10 M) insulin, while maximum transport was found with a 60-90 min association time. On the contrary, although the basal transport was similar in RSCs, insulin was totally unable to accelerate AIB transport in these cells. Inflammatory processes lead to an insulin resistance which most likely involves a post-receptor step at the cellular level. Insulin; Amino acid transport; Insulin response; Rheumatoid arthritis; (Synovial cell)
1. I N T R O D U C T I O N Synoviocytes are fibroblastic cells which might undergo metabolic modifications during inflammatory processes such as rheumatoid arthritis, through a stimulation by numerous soluble factors or local hormones [1-3]. In a recent paper we described modifications in the initial step of rheumatoid cell culture, i.e. attachment and spreading [4]. On the other hand, the specific ligands which arrive at the synovial membrane via the blood stream have largely been ignored apart from the work of Goldring et al. [5] on the parathyroid hormone. Insulin was chosen with a view to investigating synoviocyte sensitivity to circulating hormones. Insulin is known to stimulate amino acid transport in fibroblasts [6,7] through system A [8]. The cr-aminoisobutyrate (AIB) uptake process was thus studied in synoviocytes and Correspondence address: C. Aussel, Laboratoire de Biochimie G6n6rale CNRS UA 622, Universit6 ParisSud, Tour D4, 5, rue J.B. C16ment, 92296 ChatenayMalabry Cedex, France
the influence of acute inflammation states on AIB transport with regard to insulin was investigated. 2. M A T E R I A L S A N D M E T H O D S 2.1. Materials
Dulbecco's modified Eagle's medium (DMEM), fetal calf serum (FCS), glutamine, bicarbonate, trypsin, phosphate buffered saline (PBS) were purchased from Eurobio (Paris, France). Clostridia collagenase (type II) and bovine pancreas insulin were from Sigma (St. Louis, USA). a,[1-14C]Aminoisobutyrate (spec. act. 57 m C i / mmol) was obtained from C E A (Saclay, France). 2.2. Cell cultures Synovial cells were isolated from samples of knee synovium obtained at synovectomy from patients with either osteoarthritic joint disease or clinically and biologically confirmed rheumatoid arthritis. Patients presented neither diabetes nor clinical insulin resistance. Synovial cell cultures were prepared using a proteolytic procedure as described [4]. When cellular confluence was attain-
Published by Elsevier Science Publishers B. V. (Biomedical Division)
00145793/87/$3.50 © 1987 Federation of European Biochemical Societies
327
Volume 214, number 2
FEBS LETTERS
ed, a passage was carried out using trypsin. 7 x 10 4 cells were s u s p e n d e d in 2 ml o f D M E M plus 4 . 8 % b i c a r b o n a t e a n d 2 % g l u t a m i n e s u p p l e m e n t e d with 10% F C S a n d p l a c e d in 9.6 cm 2 plastic dishes. S u b - c o n f l u e n t cultures were used for cell experiments. 2.3. A I B uptake T h e e x p e r i m e n t was c a r r i e d out as described [9]. Briefly, to reduce the i n t r a c e l l u l a r p o o l o f a m i n o acids a n d thus m i n i m i s e possible trans-effects [10], the cells were p l a c e d for 1 h in d e p l e t i o n m e d i u m , i.e. PBS a l o n e or with effector. Cells were then inc u b a t e d for 10 m i n in P B S c o n t a i n i n g 8/~M ( 0 . 5 / z C i / m l ) [14C]AIB. Cells were dissolved in 1 M N a O H , r a d i o a c t i v i t y was c o u n t e d , a n d p r o t e i n c o n t e n t m e a s u r e d b y the L o w r y m e t h o d [11]. T h e results were expressed as c p m / m g p r o t e i n per 10 min. 2.4. Statistics D a t a are given as m e a n s + SD o f triplicate m e a s u r e m e n t s . C o m p a r i s o n s were m a d e by use o f a n o n - p a r a m e t r i c o n e - w a y analysis o f v a r i a n c e acc o r d i n g to K r u s k a l l - W a l l i s test [12].
April 1987
lo~ cpm/rng protein / lornin
I
3-
Insulin[M]
l/
,5.~o_, 0
,i, ,o.9
,
,.,,o_8
i
1.~1o-'
I,
Fig.1. Dose responses of insulin stimulation of AIB transport in donor GE.M (e) and donor TE.J ( I ) of NRSCs. Each point is the mean _+ SD of triplicate determinations. * p < 0.05, ** p < 0.02, *** p < 0.01 and **** p < 0.001 compared to basal uptake. lo3,¢pm/rng protein/lomin
3. R E S U L T S T o e v a l u a t e the sensitivity a n d responsiveness o f human non-rheumatoid (NRSCs) and rheumatoid synovial cells (RSCs) to insulin, the biological response to the h o r m o n e was a n a l y z e d b y p e r f o r m ing d o s e - r e s p o n s e curves a n d time courses o f insulin action.
Insulin(M} ~/
1',7.1o-rO
117. lo-9
1:7.1o-8
117.1o-7
3.1. Dose-response curves for insulin S u b - c o n f l u e n t cultures o f h u m a n synovial cells were i n c u b a t e d at 37°C with v a r i o u s c o n c e n t r a tions o f b o v i n e insulin (1.7 x 10 -1° to 1.7 x 10 -7 M ) . The a s s o c i a t i o n time in the presence o f insulin was 1 h, since we have p r e v i o u s l y f o u n d t h a t this time allows the m a x i m a l s t i m u l a t i o n effect o f A I B u p t a k e in f i b r o b l a s t s [13]. T h e d a t a for A I B u p t a k e b y N R S C s are given in fig.1. The t r a n s p o r t rate was significantly higher t h a n in controis f r o m 1.7 x 10 -9 to 1.7 x 10 -7 M insulin a n d f r o m 1.7 x 10 -8 to 1.7 x 10 -7 M insulin in the two cell cultures, G E . M . a n d T E . J . , respectively. T h e m a x i m u m s t i m u l a t i o n was o b t a i n e d between 1.7 x 328
Fig.2. Dose responses of insulin stimulation of AIB transport in donor MO.J (e), donor MI.A (A) and donor VU.C ( I ) of RSCs. Each point is the mean _+ SD of triplicate determinations. 10 - 9 M a n d 1.7 x 10 -8 M insulin, c o r r e s p o n d i n g to a 6 0 % increase over basal u p t a k e . T h e N R S C s were sensitive to the a c t i o n o f insulin with halfm a x i m a l s t i m u l a t i o n at a h o r m o n e c o n c e n t r a t i o n o f a p p r o x . 4 x 10 -1° M. In the cultures f r o m three rheumatoid donors, AIB uptake was not s t i m u l a t e d w h a t e v e r the c o n c e n t r a t i o n o f insulin used (fig.2).
Volume 214, number 2
April 1987
FEBS LETTERS Table 1 Time course of insulin effect on AIB uptake in NRSCs and RSCs
Cell cultures
Time (min) 0
30
60
90
120
NRSC
5505 ± 455
6763 ± 343
8058 ± 576**
8488 ± 451"*
7940 ± 450*
RSC
3754 ± 390
4566 ± 620
3168 ± 502
3412 ± 416
3230 ± 426
Each point is the mean _+ SD of triplicate determination. * p < 0.01 and **p < 0.001 compared to control. NRSCs were from donor PA.C and RSC from donor RS.A
3.2. Time courses o f insulin effect For these experiments, a physiological stimulating insulin concentration (1,7 × 10 -9 M) was used. Results are given in table 1. In NRSCs, AIB uptake stimulation was obtained after 60 min, but the insulin effect gradually increased to a maximum at 90 min ( + 52°7o of basal uptake) when a steady state was reached. In RSCs, insulin treatment at various association times had no effect on AIB uptake. 4. D I S C U S S I O N The present study provides evidence that insulin at concentrations within physiological values stimulates the uptake of AIB in NRSCs. This observation is in agreement with previous reports on the insulin effect on AIB uptake in human fibroblasts [14,15]. The hormone-mediated stimulatory effect was detected after a 60 rain exposure of the synoviocytes to the hormone, but was only fully expressed 1 V2h later. It is well known that insulin enhances amino acid uptake through m R N A and protein synthesis-dependent processes [7]. It appears that the sensitivity and responsiveness of synoviocytes to the hormone (half-maximum response 4 x 10 -1° M, stimulation + 60°70) were quite similar to numerous reports in the literature [14-16]. The basal transport of AIB varied in the different experiments. However, the mean basal transport in NRSCs and RSCs was not significantly different. The AIB transport system can be considered as thus similar in NRSCs and RSCs. Our results are in disagreement with those of Byers et al. [17] who showed a decrease in amino acid
transport induced by inflammation in chondrocytes. However, their experiments were performed using rabbit articular cartilage tissue, not cell cultures after in vivo treatment with carrageenin. We have shown impaired insulin action in cultured synovial cells from patients with rheumatoid arthritis. These results cannot be .related to any clinical state of insulin resistance such as leprechaunism [15], diabetes [18] or pregnancy [19]. Moreover, the interference of drugs such as aspirin or glucocorticoids which induce insulin resistance [20,21] appears unlikely; in effect, it was removed during the various steps of the culture. In RSCs, we observed a combination of a lack of sensitivity and responsiveness to insulin, whatever the rheumatoid patient. Insulin resistance at the cellular level is due to an alteration at a pre-receptor, receptor or post-receptor level. As insulin resistance at the pre-receptor level would involve a factor reducing the free insulin concentration, we verified that RSC culture medium did not release an insulin degrading proteolytic enzyme (not shown). At the membrane level, alterations in receptor affinity or concentration could also result in a decreased sensitivity to the hormone. We have demonstrated (submitted) that the number and the affinity of insulin receptors were similar in NRSCs and RSCs. This implies that the anomaly exists at the post-receptor step, preventing the hormone from increasing either the velocity of translocation of amino acids across the membrane or the turnover of carriers [22]. In conclusion, these findings indicate a normal biological effect of insulin in NRSCs and a specific defect in RSCs involving a decreased sensitivity 329
Volume 214, number 2
FEBS LETTERS
and an unresponsiveness to the hormone. These data will be of use in the interpretation of future work on RSCs with regard to the biological effects of polypeptide hormones.
ACKNOWLEDGEMENTS The authors wish to thank the Department of Orthopedic Surgery of the Hospital Cochin (Professor Postel) and particularly Dr A. Kindermans and Mrs A. Dollo for providing tissue specimens. Many thanks to Mrs P. Jue for her secretarial assistance.
REFERENCES [1] Krane, S.M. and Amento, E.P. (1983) J. Rheumatol. Suppl. 11, 7-11. [2] Daireaux, M., Penfornis, H., Langris, M., Bocquet, J., Pujol, J.P., Beliard, R. and Loyau, G. (1981) FEBS Lett. 132, 93-97. [3] Hamerman, D. and Wood, D.D. (1984) Proc. Soc. Exp. Biol. Med. 177, 205-210. [4] Desmoulin, D., Aussel, C., Cals, M.J., Agneray, J. and Ekindjian, O.G. (1986) Cell Biol. Int. Rep. 10, 501-508. [5] Goldring, S.R., Dayer, J.M. and Krane, S.M. (1984) Inflammation 8, 107-121. [6] Cynober, L., Aussel, C., Vaubourdolle, M., Agneray, J. and Ekindjian, O.G. (1987) Diabetes 36, 27-32. [7] Hill, D.J., Crace, C.J., Strain, A.J. and Milner, R.D.G. (1986) J. Clin. Endocrinol. Metab. 62, 753-759.
330
April 1987
[8] Longo, N., Franchi-Gazzola, R., Bussolati, O., Dall'Asta, V., Foa, P.P., Guidotti, G.G. and Gazzola, G.C. (1985) Biochim. Biophys. Acta 844, 216-223. [9] Guermeur, C., Cynober, L., Auget, J.L., Aussel, C., Agneray, J. and Ekindjian, O.G. (1984) Cell, Mol. Biol. 30, 529-535. [10] Gazzola, G.C., Dall'Asta, V., Franchi-Gazzola, R., Bussolati, O., Longo, N. and Guidotti, G.G. (1984) Biochem. Biophys. Res. Commun. 120, 172-178. [11] Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J. (1951) J. Biol. Chem. 193,265-275. [12] Norheim, E.T. (1986) Comput. Method. Program. Biomed. 23, 57-62. [13] Cynober, L., Aussel, C., Chatelain, P., Vaubourdolle, M., Agneray, J. and Ekindjian, O.G. (1985) Biochimie 67, 1185-1190. [14] Hollenberg, M.D. (1976) Life Sci. 18, 521-528. [15] Craig, J.W., Larner, J., Locker, E.F., Widom, B. and Elders, M.J. (1984) Metabolism 33, 1084-1095. [16] Martin, M.S. and Pohl, S.L. (1979) J. Biol. Chem. 254, 9976-9978. [17] Byers, S., Handley, C.J., Lowther, D.A. and Sriratana, A. (1985) Ann. Rheum. Dis. 44, 477-484. [18] MacElduff, A., Hedo, J.A., Taylor, S.I., Roth, J. and Gorden, P. (1984) J. Clin. Invest. 74, 1366-1374. [19] Plehwe, W.E., Maitland, J.E., Williams, P.F., Shearman, R.P. and Turtle, J.R. (1985) J. Clin. Endocrinol. Metab. 61, 68-77. [20] Newman, W.P. and Brodows, R.G. (1983) J. Clin. Endocrinol. Metab. 57, 1102-1105. [21] Muggeo, M., Saviolakis, G.A., WachslichtRodbard, H. and Roth, J. (1983) J. Clin. Endocrinol. Metab. 56, 1169-1175. [22] Shotwell, M.A., Kilberg, M.S. and Oxender, D.L. (1983) Biochim. Biophys. Acta 737, 267-284.
FEB 04616
April 1987
Effect of insulin on aminoisobutyric acid uptake by human non-rheumatoid and rheumatoid synovial cells Christian Aussel, D o m i n i q u e D e s m o u l i n s , J e a n A g n e r a y a n d O v h a n e s s e G. E k i n d j i a n Laboratoire de Biochimie Gbn4rale C N R S UA 622, Universitb Paris-Sud, Tour D4, 5, rue J.B. Clement, 92296 Chatenay-Malabry Cedex, France
Received 19 February 1987 The biological sensitivity of cultured non-rheumatoid human synovial cells (NRSCs) and rheumatoid synovial cells (RSCs) was examined in terms of the ability of insulin to stimulate the uptake of ct-aminoisobutyrate (AIB). NRSCs, like numerous fibroblastic lines, were sensitive to physiological concentrations of the hormone: half-maximal stimulation was obtained with (4 x 10 10 M) insulin, while maximum transport was found with a 60-90 min association time. On the contrary, although the basal transport was similar in RSCs, insulin was totally unable to accelerate AIB transport in these cells. Inflammatory processes lead to an insulin resistance which most likely involves a post-receptor step at the cellular level. Insulin; Amino acid transport; Insulin response; Rheumatoid arthritis; (Synovial cell)
1. I N T R O D U C T I O N Synoviocytes are fibroblastic cells which might undergo metabolic modifications during inflammatory processes such as rheumatoid arthritis, through a stimulation by numerous soluble factors or local hormones [1-3]. In a recent paper we described modifications in the initial step of rheumatoid cell culture, i.e. attachment and spreading [4]. On the other hand, the specific ligands which arrive at the synovial membrane via the blood stream have largely been ignored apart from the work of Goldring et al. [5] on the parathyroid hormone. Insulin was chosen with a view to investigating synoviocyte sensitivity to circulating hormones. Insulin is known to stimulate amino acid transport in fibroblasts [6,7] through system A [8]. The cr-aminoisobutyrate (AIB) uptake process was thus studied in synoviocytes and Correspondence address: C. Aussel, Laboratoire de Biochimie G6n6rale CNRS UA 622, Universit6 ParisSud, Tour D4, 5, rue J.B. C16ment, 92296 ChatenayMalabry Cedex, France
the influence of acute inflammation states on AIB transport with regard to insulin was investigated. 2. M A T E R I A L S A N D M E T H O D S 2.1. Materials
Dulbecco's modified Eagle's medium (DMEM), fetal calf serum (FCS), glutamine, bicarbonate, trypsin, phosphate buffered saline (PBS) were purchased from Eurobio (Paris, France). Clostridia collagenase (type II) and bovine pancreas insulin were from Sigma (St. Louis, USA). a,[1-14C]Aminoisobutyrate (spec. act. 57 m C i / mmol) was obtained from C E A (Saclay, France). 2.2. Cell cultures Synovial cells were isolated from samples of knee synovium obtained at synovectomy from patients with either osteoarthritic joint disease or clinically and biologically confirmed rheumatoid arthritis. Patients presented neither diabetes nor clinical insulin resistance. Synovial cell cultures were prepared using a proteolytic procedure as described [4]. When cellular confluence was attain-
Published by Elsevier Science Publishers B. V. (Biomedical Division)
00145793/87/$3.50 © 1987 Federation of European Biochemical Societies
327
Volume 214, number 2
FEBS LETTERS
ed, a passage was carried out using trypsin. 7 x 10 4 cells were s u s p e n d e d in 2 ml o f D M E M plus 4 . 8 % b i c a r b o n a t e a n d 2 % g l u t a m i n e s u p p l e m e n t e d with 10% F C S a n d p l a c e d in 9.6 cm 2 plastic dishes. S u b - c o n f l u e n t cultures were used for cell experiments. 2.3. A I B uptake T h e e x p e r i m e n t was c a r r i e d out as described [9]. Briefly, to reduce the i n t r a c e l l u l a r p o o l o f a m i n o acids a n d thus m i n i m i s e possible trans-effects [10], the cells were p l a c e d for 1 h in d e p l e t i o n m e d i u m , i.e. PBS a l o n e or with effector. Cells were then inc u b a t e d for 10 m i n in P B S c o n t a i n i n g 8/~M ( 0 . 5 / z C i / m l ) [14C]AIB. Cells were dissolved in 1 M N a O H , r a d i o a c t i v i t y was c o u n t e d , a n d p r o t e i n c o n t e n t m e a s u r e d b y the L o w r y m e t h o d [11]. T h e results were expressed as c p m / m g p r o t e i n per 10 min. 2.4. Statistics D a t a are given as m e a n s + SD o f triplicate m e a s u r e m e n t s . C o m p a r i s o n s were m a d e by use o f a n o n - p a r a m e t r i c o n e - w a y analysis o f v a r i a n c e acc o r d i n g to K r u s k a l l - W a l l i s test [12].
April 1987
lo~ cpm/rng protein / lornin
I
3-
Insulin[M]
l/
,5.~o_, 0
,i, ,o.9
,
,.,,o_8
i
1.~1o-'
I,
Fig.1. Dose responses of insulin stimulation of AIB transport in donor GE.M (e) and donor TE.J ( I ) of NRSCs. Each point is the mean _+ SD of triplicate determinations. * p < 0.05, ** p < 0.02, *** p < 0.01 and **** p < 0.001 compared to basal uptake. lo3,¢pm/rng protein/lomin
3. R E S U L T S T o e v a l u a t e the sensitivity a n d responsiveness o f human non-rheumatoid (NRSCs) and rheumatoid synovial cells (RSCs) to insulin, the biological response to the h o r m o n e was a n a l y z e d b y p e r f o r m ing d o s e - r e s p o n s e curves a n d time courses o f insulin action.
Insulin(M} ~/
1',7.1o-rO
117. lo-9
1:7.1o-8
117.1o-7
3.1. Dose-response curves for insulin S u b - c o n f l u e n t cultures o f h u m a n synovial cells were i n c u b a t e d at 37°C with v a r i o u s c o n c e n t r a tions o f b o v i n e insulin (1.7 x 10 -1° to 1.7 x 10 -7 M ) . The a s s o c i a t i o n time in the presence o f insulin was 1 h, since we have p r e v i o u s l y f o u n d t h a t this time allows the m a x i m a l s t i m u l a t i o n effect o f A I B u p t a k e in f i b r o b l a s t s [13]. T h e d a t a for A I B u p t a k e b y N R S C s are given in fig.1. The t r a n s p o r t rate was significantly higher t h a n in controis f r o m 1.7 x 10 -9 to 1.7 x 10 -7 M insulin a n d f r o m 1.7 x 10 -8 to 1.7 x 10 -7 M insulin in the two cell cultures, G E . M . a n d T E . J . , respectively. T h e m a x i m u m s t i m u l a t i o n was o b t a i n e d between 1.7 x 328
Fig.2. Dose responses of insulin stimulation of AIB transport in donor MO.J (e), donor MI.A (A) and donor VU.C ( I ) of RSCs. Each point is the mean _+ SD of triplicate determinations. 10 - 9 M a n d 1.7 x 10 -8 M insulin, c o r r e s p o n d i n g to a 6 0 % increase over basal u p t a k e . T h e N R S C s were sensitive to the a c t i o n o f insulin with halfm a x i m a l s t i m u l a t i o n at a h o r m o n e c o n c e n t r a t i o n o f a p p r o x . 4 x 10 -1° M. In the cultures f r o m three rheumatoid donors, AIB uptake was not s t i m u l a t e d w h a t e v e r the c o n c e n t r a t i o n o f insulin used (fig.2).
Volume 214, number 2
April 1987
FEBS LETTERS Table 1 Time course of insulin effect on AIB uptake in NRSCs and RSCs
Cell cultures
Time (min) 0
30
60
90
120
NRSC
5505 ± 455
6763 ± 343
8058 ± 576**
8488 ± 451"*
7940 ± 450*
RSC
3754 ± 390
4566 ± 620
3168 ± 502
3412 ± 416
3230 ± 426
Each point is the mean _+ SD of triplicate determination. * p < 0.01 and **p < 0.001 compared to control. NRSCs were from donor PA.C and RSC from donor RS.A
3.2. Time courses o f insulin effect For these experiments, a physiological stimulating insulin concentration (1,7 × 10 -9 M) was used. Results are given in table 1. In NRSCs, AIB uptake stimulation was obtained after 60 min, but the insulin effect gradually increased to a maximum at 90 min ( + 52°7o of basal uptake) when a steady state was reached. In RSCs, insulin treatment at various association times had no effect on AIB uptake. 4. D I S C U S S I O N The present study provides evidence that insulin at concentrations within physiological values stimulates the uptake of AIB in NRSCs. This observation is in agreement with previous reports on the insulin effect on AIB uptake in human fibroblasts [14,15]. The hormone-mediated stimulatory effect was detected after a 60 rain exposure of the synoviocytes to the hormone, but was only fully expressed 1 V2h later. It is well known that insulin enhances amino acid uptake through m R N A and protein synthesis-dependent processes [7]. It appears that the sensitivity and responsiveness of synoviocytes to the hormone (half-maximum response 4 x 10 -1° M, stimulation + 60°70) were quite similar to numerous reports in the literature [14-16]. The basal transport of AIB varied in the different experiments. However, the mean basal transport in NRSCs and RSCs was not significantly different. The AIB transport system can be considered as thus similar in NRSCs and RSCs. Our results are in disagreement with those of Byers et al. [17] who showed a decrease in amino acid
transport induced by inflammation in chondrocytes. However, their experiments were performed using rabbit articular cartilage tissue, not cell cultures after in vivo treatment with carrageenin. We have shown impaired insulin action in cultured synovial cells from patients with rheumatoid arthritis. These results cannot be .related to any clinical state of insulin resistance such as leprechaunism [15], diabetes [18] or pregnancy [19]. Moreover, the interference of drugs such as aspirin or glucocorticoids which induce insulin resistance [20,21] appears unlikely; in effect, it was removed during the various steps of the culture. In RSCs, we observed a combination of a lack of sensitivity and responsiveness to insulin, whatever the rheumatoid patient. Insulin resistance at the cellular level is due to an alteration at a pre-receptor, receptor or post-receptor level. As insulin resistance at the pre-receptor level would involve a factor reducing the free insulin concentration, we verified that RSC culture medium did not release an insulin degrading proteolytic enzyme (not shown). At the membrane level, alterations in receptor affinity or concentration could also result in a decreased sensitivity to the hormone. We have demonstrated (submitted) that the number and the affinity of insulin receptors were similar in NRSCs and RSCs. This implies that the anomaly exists at the post-receptor step, preventing the hormone from increasing either the velocity of translocation of amino acids across the membrane or the turnover of carriers [22]. In conclusion, these findings indicate a normal biological effect of insulin in NRSCs and a specific defect in RSCs involving a decreased sensitivity 329
Volume 214, number 2
FEBS LETTERS
and an unresponsiveness to the hormone. These data will be of use in the interpretation of future work on RSCs with regard to the biological effects of polypeptide hormones.
ACKNOWLEDGEMENTS The authors wish to thank the Department of Orthopedic Surgery of the Hospital Cochin (Professor Postel) and particularly Dr A. Kindermans and Mrs A. Dollo for providing tissue specimens. Many thanks to Mrs P. Jue for her secretarial assistance.
REFERENCES [1] Krane, S.M. and Amento, E.P. (1983) J. Rheumatol. Suppl. 11, 7-11. [2] Daireaux, M., Penfornis, H., Langris, M., Bocquet, J., Pujol, J.P., Beliard, R. and Loyau, G. (1981) FEBS Lett. 132, 93-97. [3] Hamerman, D. and Wood, D.D. (1984) Proc. Soc. Exp. Biol. Med. 177, 205-210. [4] Desmoulin, D., Aussel, C., Cals, M.J., Agneray, J. and Ekindjian, O.G. (1986) Cell Biol. Int. Rep. 10, 501-508. [5] Goldring, S.R., Dayer, J.M. and Krane, S.M. (1984) Inflammation 8, 107-121. [6] Cynober, L., Aussel, C., Vaubourdolle, M., Agneray, J. and Ekindjian, O.G. (1987) Diabetes 36, 27-32. [7] Hill, D.J., Crace, C.J., Strain, A.J. and Milner, R.D.G. (1986) J. Clin. Endocrinol. Metab. 62, 753-759.
330
April 1987
[8] Longo, N., Franchi-Gazzola, R., Bussolati, O., Dall'Asta, V., Foa, P.P., Guidotti, G.G. and Gazzola, G.C. (1985) Biochim. Biophys. Acta 844, 216-223. [9] Guermeur, C., Cynober, L., Auget, J.L., Aussel, C., Agneray, J. and Ekindjian, O.G. (1984) Cell, Mol. Biol. 30, 529-535. [10] Gazzola, G.C., Dall'Asta, V., Franchi-Gazzola, R., Bussolati, O., Longo, N. and Guidotti, G.G. (1984) Biochem. Biophys. Res. Commun. 120, 172-178. [11] Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J. (1951) J. Biol. Chem. 193,265-275. [12] Norheim, E.T. (1986) Comput. Method. Program. Biomed. 23, 57-62. [13] Cynober, L., Aussel, C., Chatelain, P., Vaubourdolle, M., Agneray, J. and Ekindjian, O.G. (1985) Biochimie 67, 1185-1190. [14] Hollenberg, M.D. (1976) Life Sci. 18, 521-528. [15] Craig, J.W., Larner, J., Locker, E.F., Widom, B. and Elders, M.J. (1984) Metabolism 33, 1084-1095. [16] Martin, M.S. and Pohl, S.L. (1979) J. Biol. Chem. 254, 9976-9978. [17] Byers, S., Handley, C.J., Lowther, D.A. and Sriratana, A. (1985) Ann. Rheum. Dis. 44, 477-484. [18] MacElduff, A., Hedo, J.A., Taylor, S.I., Roth, J. and Gorden, P. (1984) J. Clin. Invest. 74, 1366-1374. [19] Plehwe, W.E., Maitland, J.E., Williams, P.F., Shearman, R.P. and Turtle, J.R. (1985) J. Clin. Endocrinol. Metab. 61, 68-77. [20] Newman, W.P. and Brodows, R.G. (1983) J. Clin. Endocrinol. Metab. 57, 1102-1105. [21] Muggeo, M., Saviolakis, G.A., WachslichtRodbard, H. and Roth, J. (1983) J. Clin. Endocrinol. Metab. 56, 1169-1175. [22] Shotwell, M.A., Kilberg, M.S. and Oxender, D.L. (1983) Biochim. Biophys. Acta 737, 267-284.