Exp. Eye Res. (1991) 52, 51-57
Requirement of Insulin or IGF-I for the Maintenance Retinyl Ester Synthetase Activity by Cultured Retinal Epithelial Cells ROSS Boston
University
B. EDWARDS*, School
(Received
ALICE
of Medicine,
74 December
J. ADLER”AND
ROBERT
Department of Ophthalmology, Boston, MA, U.S.A. 1989 and accepted
of Pigment
C. CLAYCOMB and “Eye Research
Institute,
in revised form 76 May 1990)
Previous work from these laboratories showed that the retention of retinyl ester synthetase activity by cultured human retinal pigment epithelium is up to tenfold greater with PM medium (Medium 199 plus insulin, other added defined components, 1% serum and 1 y0 retina extract) than with conventional culture media. The present work shows that insulin is the component of PM medium required for maintenance of ester synthetase activity and that insulin-like growth factor type 1 (IGF-1) also is effective at maintaining ester synthesis. In addition, insulin can maintain ester synthetase activity in cultured rat RPE. Preliminary dose-response measurements provide additional support for these findings and strongly suggest that both insulin and IGF-1 are maximally effective at physiological concentrations (l-10 ng ml-‘). Key words: retinyl ester synthesis ; retinal pigment epithelium : insulin : insulin-like growth factor type 1; vitamin A; cell culture: human RPE.
1. Introduction The retinal pigment epithelium (RPE) plays a central role in the uptake and metabolsm of vitamin A (retinol) by the vertebrate eye. Receptors on the RPE basal surface bind serum retinol-binding protein and facilitate the uptake of retinol from the blood (Heller, 1975; Bok and Heller, 1976; Pfeffer et al., 1986). The RPE also appears to be the principal site of metabolism of vitamin A to the form required for vision, since this tissue contains the highest known specific activities of retinyl ester synthetase, retinyl ester hydrolase, vitamin A isomerase, and 1 1-cis retinol oxidoreductase. These activities, respectively, convert all-trans retinol to retinyl esters of fatty acids (Krinsky, 19 5 8 ; Andrews and Futterman, 1964; Berman et al., 1980; Saari and Bredberg, 19 8 8, 19 8 9), hydrolyze retinyl esters to free retinol (Blaner et al., 1987), isomerlze all-trans retinyl esters to 11-cis retinol (Bernstein, Law and Rando, 1987; Deigner et al., 1989; Trehan, Cannada and Rando, 1990), and oxidize 11-cis retinol to the aldehyde (Lion et al., 1975). Retinyl ester synthetase (RES) activity is particularly high in the RPE (Berman et al., 1980; Saari and Bredberg, 1988). A likely reason for this is that retinol is toxic, being highly membranoIyytic, while the retinyl esters of long-chain fatty acids are non-toxic (Goodall, Fisher and Lucy, 1980) : thus, during an intense bleach when the photoreceptors release large amounts of retinol, RES in effect detoxifies retinol by converting it to fatty acid * For reprint requests at: Boston University School of Medicine. Department of Ophthalmology G907, 80 East Concord Street, Boston,
MA 02118.
U.S.A.
00144835/91/010051+07
$03.00/0
esters. In addition, retinyl esters of long-chain fatty acids are more soluble in membranes than is retinol (Rando and Bangerter, 1982; Ho. Pownall and HollyfieId, 1989). which facilitates storage of vitamin A in RPE oil droplets and intracellular membranes, the principal ocular storage sites of vitamin A (Bridges, 1976). It has also been recently shown that retinyl esters are the most likely substrate for the isomerization of vitamin A (Deigner et al., 1989: Trehan et al., 1990). RES, because of its high activity in the RPE, also serves as an easily quantifiable biochemical marker for the RPE. RES activity is rapidly lost in RPE cultured in conventional media (e.g. commercially available media supplemented with 20 % fetal bovine serum) but is retained at levels up to 40% of fresh tissue levels in human RPE grown in PM medium (Edwards, Adler and Southwick, 1987). Therefore, human RPE cultured in PM medium should be a useful system with which to explore the nutritional, hormonal and growth-factor requirements for the expression of this differentiated property of RPE. The object of the present work was to determine which of the components of PM medium are required for this maintenance of RES activity in cultured human RPE. A preliminary account of this work has been reported elsewhere (Edwards and Adler, 1989). 2. Materials
and Methods
RPE cells were isolated, as described previously. from human autopsy eyes obtained from the National Disease Research Interchange, Philadelphia, PA (Edwards, 1982), or from S- to 6-day-old, Long-Evans, 0 199 1 Academic Press Limited 4-2
52
pigmented rat eyes (Charles River, Wilmington, MA: Edwards, 1981) and seeded as primary cultures at 1040 x lo3 cells per cm* culture surface in 24- or 12cluster plastic plates (Costar, Cambridge, MA). In each experiment cells from a different set of donors were used. An experiment is defined as a comparison of different media during a given 14-day culture period. First-passagehuman RPE cultures that were obtained from Dr B. A. Pfeffer were used in one experiment. In most cases an aliquot of freshly isolated cells was washed twice in isotonic saline and stored at - 75°C for subsequent comparison with cultured cells derived from the samedonor. Cellswere initially seededin PM medium, which consistsof Medium 19 9 supplemented with seven additional defined components (10 pg ml-l insulin, 5 ,ug ml transferrin-‘, 20 nM hydrocortisone, 1 nM triiodothyronine, 0.3 pugml-’ putrescine, 0.3 ,uM linoleic acid, and 10 pg ml-’ bovine serum albumin) and two undefined components (1 y0 newborn calf serum and 1% bovine retina extract; Pfeffer et al.. 1986 ; Edwards et al., 198 7). Insulin and IGF-1 were obtained from Collaborative Research, Bedford, MA: all other media components were from sources stated previously (Edwards et al., 198 7). One to 3 days after the cells were seeded,the medium was replaced with variations of PM medium to be tested (i.e. PM medium with one or more components omitted, or with the 10 ,ug ml-’ insulin replaced with different concentrations of insulin or IGF-l), and was subsequently changed ever 3 or 4 days. For dose-responsemeasurements, PM medium was used with the 10 pg ml-’ insulin replaced with different concentrations of insulin or IGF-1, and the media were changed daily to minimize the in vitro degradation of labile medium components (Straus, 1984). Ten to 14 days after the initial medium change, the cells were rinsed in 0.145 M NaCl:O.OS M phosphate, pH 7.2, sonicated, and centrifuged at 50000 g for 2 hr at 3°C. The resulting pellets were resuspended by sonication in 0.05 M sodium phosphate, pH 7.2, assayedfor protein (Lowry et al., 1951), and stored at -75°C. Aliquots of the pellet (15 pg protein) were combined with 2 ,&i [l 1 ,123H]all-truns retinol (New England Nuclear, Boston, MA, specific activity 53 Ci mmol-I), 1 IIIM dithiothreitol, and Tris buffer (0.1 M, pH 8.2) (Saari and Bredberg, 1988) in a final volume of 02 ml, and incubated in the dark under nitrogen for 15 min at 37°C. Under these conditions no more than 2 5 % of the substrate was esterified. The mixtures were then extracted with hexane and analyzed by HPLC for radioactive retinoi and retinyl esters, as previously described (Edwards et al.. 1987) except that radioactivity of the HPLC effluent was continuously monitored with a flow-through liquid scintillation counter (Flow-l Beta, Radiomatic Instruments, Inc.. Meriden, CT). Results are expressedas the percent of radioactive retinol converted to retinyl esters(% retinyl esters). In some cases, to pool data from different experiments, RES activity was normalized to 100%
R. B. EDWARDS
ET AL.
PM medun
PM w/o undefined
PM w/o defined Medium +20%
199 serum 0 Retlnyl
FIG.
1. Effects
I
I
1
I
25
50
75
100
ester
synthesis
(% of PM medium)
on retinyl estersynthesisof omitting classes
of PM medium components.Two
sets of human
RPE
cultures, each from a different donor, were used in this experiment.For each set. singlecultures were maintained with completePM medium,PM withoutundefined components (i.e. without 1y0 serumor 1y0 retina extract). PM without defined components (i.e. without the seven added defined components, see Materials and Methods), or Medium 199+20% serum [Medium 199 supplemented with 10% (v/v) fetal bovine serum]. After 14 days the cultures were processed and assayed for RES activity as described in Methods. The data are normalized to 100 % for complete PM medium : for one set of cultures this was equivalent to 7.2 % conversion of retinol to retinyl esters, and for the other set this value was 13.5%. Bars indicate the normalized means for each pair of cultures: lines show the higher normalized value for each pair. These data are from one of three experiments that gave similar results.
for cellsgrown in complete PM medium ; this was done only for Figs 1 and 2. 3. Results When human RPE cells were maintained on PM medium from which two undefined components (1 “/o serum and 1 Y0retina extract) were omitted, significant levels of RES activity were still retained by cultured human RPE cells. In one experiment the mean RES activity (per 15 pg protein of cell pellet fraction) was 40% of the synthesis observed with the cell pellet fraction from cells grown with complete PM medium (Fig. 1) ; in some experiments with this medium, RES activity was as high as in cells grown with complete PM medium (data not shown). In contrast, when cells were maintained on PM medium from which the seven added defined components were omitted, virtually no RESactivity was detected (Fig. 1). As previously reported (Edwards et al., 1987), comparable low levels of ester synthesis also were observed with pellet fractions of cells maintained with a conventional culture medium. i.e. Medium 199 supplemented with 20% fetal bovine serum (Fig. 1). From the results shown in Fig. 1 it appeared that one or more of the seven added defined components was required for the maximum maintenance of RES activity. Therefore, media were tested that consisted of PM medium with individual, defined,’ added components omitted. (In the caseof linoleic acid and BSA, which were obtained as a combined preparation, both were omitted together.) Only the omission of insulin
RETINYL
ESTER
SYNTHETASE
IN CULTURED
TABLE
RPE
I
Effect on retinyl ester synthesis of omitting insulin from PM medium oARetinyl esters
Donor 1
2 3 4
PM with
PM without
insulin*
insulin
7.2 13.5 6.5 23.8
Ratio? without/with insulin
1.2 0.6 1.2 4.4
0.17 0.05 0.18 0.18
* Insulin concentration = 10 ,/hgml-‘. t Mean ratio k S.D. = 0.14 f 0.07. P < @OOOl, compared to ratio of 1.0. based on two-tailed t-test.
resulted in a marked lossof RES activity from cultured human RPE (Fig. 2). Additional experiments confirmed that the omission of insulin from PM medium resulted in a marked reduction in the maintenance of RES activity in cultured human RPE. As illustrated by the results from Fig. 2 and from two other experiments, RES activity in cultures grown in PM medium without insulin was lessthan 0.2 that of cultures grown with 10 begml-’ insulin (Table I). This difference was statistically significant (P < O.OOOl), based on a two-tailed t-test analysis of the ratios of Table I. In addition, rat RPE cultured for 2 weeks with PM medium containing insulin retained higher levels of RES activity (11% conversion of all-trans retinol to esters) than replicate cultures grown in PM medium without insulin (2 % conversion to esters; Fig. 3). Since insulin at 1 L&gml-’ or higher has been shown to stimulate the receptors for insulin-like growth factor type 1 (IGF-1: see Straus. 1984; Rechler and Nissley, 1985 ; for reviews), IGF-1 was also tested for its capacity to maintain RES activity of cultured human RPE. When insulin was replaced with IGF-1 at
0
25 Retlnyl
FIG.
50
ester
synthesis
75
100
(% of PM medium)
2. EXectson retinyl estersynthesisof omitting single,
added, defined components
of PM medium.
Details are as
describedfor Fig. 1: i.e. human RPEcultures were from the sametwo setsas usedfor Fig. 1. with singlecultures from each set maintainedwith each of the indicated test media. Results are from one experiment.
53 100 Dr 300 ng ml-l and the media changed every 34 days, the retained levels of RES activity were comparable to, or exceeded, RES activity retained in the presence of 10 pg ml-’ insulin (Fig. 4). Preliminary dose-responsemeasurements indicated that both insulin and IGF-1 are effective in the physiological concentration range. With insulin, retention of RES activity increased sharply as the concentration was increased above l-3 ng ml-’ (Fig. 5). For comparison, the physiological range for insulin is 14 ng ml-’ (equivalent to 25-100 PU ml-l : Larner, 1985). Retention of RES activity by primary cultures (m in Fig. 5) increased further for insulin concentrations of 80 ng ml-’ or higher; such an increase was not seenin the experiment with the firstpassagecultures (0 in Fig. 5). With IGF-1. retention of RES activity increased sharply above 3-10 ng ml-l {Fig. 6), which compareswith a physiological range of l-20 ngml-’ or more (Zapf and Froesch, 1986). Although a complete set of IGF-1 concentrations was tested only for the first-passage cells, the increased retention of RES activity for IGF-1 concentrations between 3 and 100 ng ml-’ was statistically significant. To demonstrate this, the experimental values within each of three concentration ranges were pooled and compared, using a two-tailed t-test. In Fig. 6, horizontal lines indicate the concentration ranges over which IGF-1 was effective (3-100 ng ml-‘) or not effective (O-3 ng ml-‘, and greater than 300 ng mlml). The mean ( &SD.) of the six high RES values at 3-100 ng ml-’ (% ester = 20.8 f 3.5 %) is significantly greater than the mean of the five low values at O-3 ng ml-’ (% ester = 11.3 & 2.8 %, P = 00009) and the mean of the three low values at 300 ng ml-’ (% ester = 9.6 f 0.5 %, P = 00011). For both insulin and IGF-1 the upward breaks in the dose-response curves occurred within a narrowly defined concentration range for cultures from different donors. IGF-1 at the highest concentration tested (300 ng ml-‘) did not maintain RES activity when the daily medium change schedule was used for doseresponsemeasurements (Fig. 6). This result contrasted markedly with the maintenance of high RES activity by 300 ng ml-’ IGF-1 when the medium was changed every 34 days (Fig. 4). Also noteworthy are the higher basal levels of RESactivity seen in the absence of added insulin or IGF-1 when the media were changed daily (Figs 5 and 6) compared with medium changes every 34 days (Fig. 4). For possible explanations of these differences, see Discussion. For some sets of replicate cultures a subtle morphological difference was seen between cells maintained with insulin and those maintained without insulin. Cultures of human or rat RPE grown for 2 weeks with PM medium that contained 10 pg ml-’ insulin appeared to contain cells that were somewhat more refractile and more cobblestone-like in shape (i.e. thought to more nearly resemble RPE in vivo) compared to cells grown in PM medium without
54
R. B. EDWARDS
i
ET AL.
30
5 5 .c P .$ 200 L
jl~I 0
2 Retinyl
4 ester
6
8
synthesis
IO (% retlnyl
I2
lnSulln
or IGF-,
b
0
,
,
2
4
Retlnyl
ester
,
6 synthesis
/
14
esters)
FIG. 3. Effects of insulin on retinyl ester synthesis by cultured rat RPE. Cultures from 6-day-old pigmented rats were maintained for 14 days in duplicate with complete PM medium (PM) or with PM medium without insulin and assayed for RFS activity. Details are otherwise as described for Fig. 2 except that results are expressed as the percent retinol esterified during a 15-min incubation. Results are from one experiment.
?M WI0
$p
,
,
8
IO
(% retlnyl
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0
\I
100
I
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IO2
IO3
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I IO5
(ng ml-‘)
FIG. 5. RES activity as a function of insulin concentration. Human RPE cultures were maintained for 14 days in PM medium with the indicated concentrations of insulin. Media were changed daily to minimize the in vitro degradation of insulin. (m) Primary cultures from one donor, (0) firstpassage cultures from a seconddonor.Eachpoint represents one culture with each curve being from a separate experiment. Results are expressed as percent retinol esterified during a 15-min incubation. For comparison, the equivalent RES activity for fresh tissue from the primary-culture donor was 87% esterification of retinol.
j
12 esters)
FIG. 4. Effects of substituting IGF-1 for insulin in PM medium. Single cultures of human RPE from a replicate set were maintained for 14 days with the following media: PM medium that contained the normal concentration of insulin (10000 ng ml-‘), PM medium with the insulin replaced with 100 or 300 ng ml-’ IGF-1, or PM medium without insulin or IGF-1. Media were changed ever 34 days. RES activity is expressed as the percent retinol esterified during a 15-min incubation. Results are from one experiment.
insulin (data not shown). These differences were not perceived in all experiments, nor were these observations made as part of a blind experimental design, so that further work is required to determine the reproducibility of this subtle difference.
iol
,\\I 0
I IO0 IGF-I
IO’ (ng
I IO2
IO3
ml-‘)
FIG. 6. KES activity as a function of IGF- 1 concentration. Human cultures were maintained for 14 days in PM medium without insulin and with the indicated concentrations of IGF-1. Media were changed dally. (0, n ) The same sets of cultures as the corresponding symbols in Fig. 5. Each point represents one culture. The three horizontal lines indicate the mean values of RES activity for the data points of the corresponding ranges of IGF-1 concentration (see Results). Other details are as in Fig. 5.
4. Discussion These results show that insulin is a component of PM medium that is required by cultured human RPE for the maintenance of RESactivity. Of the components tested, only the omission of insulin from PM medium resulted in a marked loss of RES activity compared to cells grown with complete PM medium or PM medium with other components omitted (Figs 1 and 2, Table I). Preliminary dose-response measurements (Fig. 5)
further support this finding and strongly suggest that insulin is effective in the physiological dose range (l-4 ng ml-’ ; Larner, 198 5). Insulin also maintains RES activity in cultured rat RPE (Fig. 3). Note, however, that these results do not rule out the possibility that other components of PM medium are required for maintenance of RES activity. Synergistic relationships between insulin and other medium
RETINYL
ESTER
SYNTHETASE
IN CULTURED
RPE
components have not been examined, nor have we tested the removal of standard components of Medium 199 such as specific amino acids or vitamins. In addition, IGF-1 was effective in maintaining RES levels in cultured human RPE. (IGF-1 has not yet been tested with cultured rat RPE.) As with insulin, dose-response measurements (Fig. 6) suggest that IGF-1 is effective at physiological concentrations (l-20 ng ml-‘: Zapf and Froesch, 1986). It must be emphasized that the dose-response curves for both insulin and IGF-1 are preliminary ; additional measurements are required to confirm some of the details of the relationships shown in Figs 5 and 6. For insulin (Fig. 5) it will be of interest to confirm the increase in the retention of RES activity above 80 ng ml--’ observed in the primary cultures. This increase was not seen in the first-passage cells. Although it is speculative at this time to suggest a reason for this increase, it may be due to the stimulation of IGF-1 receptors at the higher insulin concentrations (Straus, 1984; Rechler and Nissley, 19 8 5). Since the first-passage cells were in culture for a longer period and presumably underwent more cell division than the primary cultures, the surface density of IGF-1 receptors may have been diluted in the firstpassage cells compared to the primary cells. Such a reduced density might explain the absence of the rise in RES activity in the first-passage cells at insulin concentrations above 80 ng ml-l. This might also explain why a slightly higher concentration of IGF-1 was required to achieve maximum RES activity in the first-passage cells (10 ng ml-l) than in the primary cells (3 ng ml-‘) (Fig. 6). On the other hand, the difference between 3 and 10 ng ml-’ IGF-1 is small compared to the lOOO-fold difference between insulin concentrations that elicited the additional increases in RES activity in primary cultures (above 80 ng ml-l) and concentrations that failed to do so in first-passage cultures (up to 100000 ng ml-’ ; Fig. 5). It should also be noted that the apparently modest differences in RES activity in the presence compared to the absence of added insulin or IGF-1 in the doseresponse curves (Figs 5 and 6) are due largely to the high basal levels of RES activity without added insulin or IGF-1. These high basal levels resulted from the daily changes of the culture medium (compare Fig. 4 with Figs 5 and 6). We speculate that these high basal levels may have been due to trace amounts of insulin, IGF-1, or as yet unidentified factors in the serum or retina extract that maintain RBS activity; these factors may become degraded in 34 days in vitro, but not in 1 day. IGF-1 at the highest concentration tested (300 ng ml-‘) was not effective when the medium was changed daily (Fig. 6). This was observed in two experiments with three cultures. This observation is consistent with the idea that IGF-1 receptors may have been reduced in number (downregulated) after exposure to sufficiently high IGF-1 concentrations, as
55
has been reported for a number of other cell types (Rechler and Nissley, 1985). Confirmation of this point will require measuring the number of IGF-1 receptors on RPE cells before and after incubation with a variety of IGF-1 concentrations in the range of 100-10 000 ng ml-‘. The ineffectiveness of IGF-1 at 300 ng ml-’ in the experiments of Fig. 6 may seem to contradict the results of Fig. 4, in which 300 ng ml-’ IGF-1 was very effective in maintaining RES activity. However, the data of Fig. 6 were obtained with cultures that received daily medium changes, while the data of Fig. 4 were from cultures that had media changed every 34 days. Our present interpretation is that, over the 34-day interval between changes of medium, the 300 ng ml-’ IGF-1 became partially degraded in vitro, resulting in reduced concentrations of IGF-1 that were effective in maintaining RES activity ; daily changes with 300 ng ml-’ IGF-1 apparently preserved a sufilciently high concentration of IGF-1 to prohibit the maintenance of RES activity, possibly by downregulation of IGF-1 receptors. It has been reported that RES activity in bovine RPE decreased to about 5% of fresh tissue levels after 3 days in culture, regardless of whether the cells were grown in a defined medium with 105 ,ug ml-l insulin or a conventional medium supplemented with 20% serum (Bridges et al., 1986). The initial cell density in those experiments was much lower (35 cells cme2) than in the present work (1040 x lo3 cells cme2), and the bovine cultures were confluent by 7 days, suggesting that the cells had divided rapidly and extensively. The depletion of RES activity could have resulted from a combination of dilution from mitosis and insufficient time for the newly divided cells to resynthesize RES. Differences between PM medium and the media used by Bridges et al. (1986) may also be involved. The mechanisms by which insulin and IGF-1 inIluence the levels of RES activity are not known. The effectiveness of insulin and IGF-1 at physiological concentrations suggests that cultured RPE cells possess surface receptors for each of these factors. Although 600-1000 ng ml-’ insulin can stimulate IGF-1 receptors, and IGF-1 above 350 ng ml-’ can stimulate insulin receptors (Straus, 1984; Rechler and Nissley, 1985) the concentrations at which these factors are maximally effective in maintaining RES activity (l-10 ng ml-l) are much lower; this fact is consistent with insulin and IGF-1 each acting at its own respective receptor to exert its effect on maintaining RES activity. IGF-1 receptors have been directly demonstrated on cultured human RPE cells (Haskell et al., 1989). Other evidence of the response of cultured human RPE to insulin and IGF-1 is the stimulation of DNA synthesis by insulin and IGF-1 (Leschey et al., 1990) and the stimulation of N-ras oncogene mRNA synthesis by IGF-1 (Wilcox, Blochberger and Sossi, 1989). Insulin and IGF-1 both elicit the phos-
56
phorylation of their respective membrane receptors in a number of other tissues, and stimulate the phosphorylation or dephosphorylation of a variety of other cellular proteins (Straus, 1984 ; Rechler and Nissley, 1985; Rosen, 1987). Insulin alters the transcription of the genes for phosphoenolpyruvate carboxykinase and other enzymes (Rosen, 198 7), and IGF-1 stimulates hydroxysteroid dehydrogenase activity (Lin et al., 1987) and synthesis of the mRNA for aromatase (Steinkampf, Mendelson and Simpson, 1988) in other cell types, establishing the precedent for the possible induction or maintenance of enzyme activity by insulin and IGF-1 in the RPE. IGF-1 also appears to play a supportive role in differentiation; according to the dual effector model (Zezulak and Green, 1986), growth hormone acts on a predifferentiated cell to stimulate differentiation, to stimulate synthesis of IGF-1, and to sensitize the cell to divide in response to IGF-1. In this way the newly differentiated cell proliferates in response to its own newly synthesized IGF-1. This clonal expansion model appears to be applicable to adipocyte development (Zezulak and Green, 1986) and developing bone (Zapf and Froesch, 1986 ; Isaksson et al., 198 7). IGF-1 also stimulates the fusion of myoblasts to myotubes (Zapf and Froesch, 1986 ; Isaksson et al., 1987) and neurite formation (Recio-Pinto and Ishii, 1988). Whether any of these observations apply to maintenance of RES activity in RPE requires further study. Insulin does not influence glucose uptake by frog RPE (DiMattio and Streitman, 1986), suggesting that insulin-induced glucose uptake is not involved in the maintenance of RES activity in RPE. A number of questions remain to be answered regarding the significance of these findings. First, whether or not insulin or IGF-1 actually maintains RES activity in the RPE in vivo will require experiments with animals that are pancreatectomized to reduce insulin levels, or hypophysectomized to remove the source of growth hormone, the principal stimulant of IGF-1 synthesis in many tissues (Isaksson et al., 198 7). Second, the role of insulin and IGF-1 in the induction of RES in early development of the RPE is not known ; determination of the time of onset of RE% activity in the RPE of fetal or neonatal animals and the correlation of RES activity with insulin and IGF-1 levels will help answer this question. It will also be necessary to determine if insulin or IGF-1 can induce RES activity in RPE cultured before RES is normally detectable. Third, we must clearly identify the surface receptors through which these growth factors influence RES activity, and determine whether the effects of low doses of insulin and IGF-1 are additive. Fourth, the effects of insulin and IGF-1 on the levels of other
enzymes and binding proteins involved in retinoid metabolism should be examined in cultured RPE. Finally, it will be of interest to determine if these findings imply a role for RES in the etiology of diabetic retinopathy.
R. B. EDWARDS
ET AL.
Acknowledgments We thank Dr B. A. Pfeffer for a sample of cultured human RPE and the National Disease Research Interchange, Philadelphia, PA, for providing human autopsy eyes. This research was supported by NIH grants EY02028 (RBE) and EY04368 (AJA), the Retinitis Pigmentosa Foundation Fighting Blindness, Baltimore, MD, and by grants to the Department of Ophthalmology, Boston University School of Medicine, from the Massachusetts Lions Eye Research Fund. Inc., and Research to Prevent Blindness, Inc., New York, NY. References Andrews, J. S. and Futterman. S. (1964). Metabolism of the retina. V. The role of microsomes in vitamin A esterification in the visual cycle. J. BioZ. Chem. 239, 4073-6. Berman, E. R., Horowitz, J.. Segal, N.. Fisher, S. and FeeneyBurns, L. (1980). Enzymatic esterification of vitamin A in the pigment epithelium of bovine retina. Biochim. Biophys. Acta 630, 3646.
Bernstein, P. S., Law, W. C. and Rando, R. R. (1987). Isomerizationof all-trans-retinoidsto 1 1-cis-retinoidsin vitro. Proc. Natl. Acad. Sci. U.S.A. 84, 1849-53.
Blaner.W. S.,Das,S.R., Gouras,P. andFlood,M. T. (198 7). Hydrolysisof 1l-cis- and all-trans-retinyl palmitateby homogenatesof human retinal epithelial cells.J. BioZ. Chem.262, 53-8. Bok, D. and Heller,J. (1976). Transport of retinol from the blood to the retina: an autoradiographicstudy of the pigment epithelial cell surface receptor for plasma retinol-bindingprotein. Exp. Eye Res.22, 395402. Bridges,C. D. B. (1976). Vitamin A and the role of the pigmentepitheliumduring bleachingand regeneration of rhodopsin in the frog eye. Exp. Eye Res. 22, 435-55. Bridges,C. D. B., Oka, M. S., Fong, S.-L. , Liou, G. I. and Alvarez, R. A. (1986). Retinoid-bindingproteins and retinol esterification in cultured retinal pigment epithelium cells.Neurochem Int. 8. 527-34. Deigner.P. S.. Law, W. C., Cannada,F.J. and Rando,R. R. (1989). Membranes as the energy source in the endergonic transformation of vitamin A to 1l-cisretinol. Science 244, 968-71. DiMattio, J. and Streitman. J. (1986). Facilitated glucose transport acrossthe retinal pigment epitheliumof the bullfrog (Ram catesbeiann). Exp. Eye Res. 43, 15-28. Edwards,R. B. (1981). The isolationand culturing of retinal epitheliumof the rat. VisionRes. 21, 147-50. Edwards, R. B. (1982). Culture of mammalian retinal pigment epithelium and neural retina. Methods Enzymol. 81, 147-50. Edwards,R. B., Adler, A. J. and Southwick, R. E. (1987). Maintenance of retinyl ester synthetase levels in cultured human retinal pigment epithelial cells. Exp. Eye Res. 45, 187-90.
Edwards,R. B. and Adler, A. J. (1989). Insulin and IGF-1 promote the retention of retinyl ester synthesisby cultured human retinal pigment epithelium (RPE). 1. Cell BioZ. 109, 333a. Goodall,A. H., Fisher,D. and Lucy, J. A. (1980). Cellfusion, haemolysisand mitochondrial swelling induced by retinal and derivatives. Biochim. Biophys. Acta 595, 9-14. Haskell,J. F., Haws, L. E., Davis, A. and Hunt, R. (1989). Comparisonof insulin-like growth factor receptorsin human retinal cells.In Molecular and Cellular Biologyof insulin-Like Growth Factors and Their Receptors (Fds. Le Roith. D. and Raizada,M. K. ). Pp. 297-308. Plenum Press:New York.
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ESTER
SYNTHETASE
IN
CULTURED
RPE
Heller, J. (1975). Interactions of plasma retinol binding protein with its receptor. Specific binding of bovine and human retinol binding protein to pigment epithelium cells from bovine eyes. J. Biol. Chem. 250, 3613-9. Ho, M. I. P., Pownall, H. J. and Hollylleld, J, G. (1989). The spontaneous transferof retinal. retinoic acid,andretinyl esters between single unilamellar vesicles. Invest. Ophthahnol.Vis. Sci. 30 (Suppl.). 332. Isaksson.0. G. P., Lindahl, A., Nilsson,A. and Isgaard,J. (1987). Mechanismof the stimulatory effectof growth hormoneon longitudinal bone growth. Endocr. Rev. 8, 426-38. Krinsky. N. I. (1958). The enzymatic esterification of vitamin A. I. Biol. Chem. 232, 881-94. Larner, J. (198 5). Insulin and oral hypoglycemic drugs; glucagon.In Goodman and Gilman’s The Pharmacological Basis of Therapeutics, 7th edn (Eds Gilman, A. G., Goodman, L. S., Rall. T. A. and Murad, F.). Pp. 1490-516. Macmillan: New York. Leschey,K. H., Hackett, S.F., Singer,J. H. and Campochiaro, P. A. (1990). Growth factor responsiveness of human retinal pigment epithelialcells. Invest. Ophthalmol. Vis. Sci. 31, 83946. Lin. T., Vinson, N.. Haskell,J. and Murono, E. P. (1987). Induction of 38-hydroxysteroid dehydrogenaseactivity by insulin-like growth factor-I in primary culture of purified Leydig cells.Adv. Exp. Med. Biol. 219, 603-7. Lion, F., Rotmans,J. P., Daemen,F. J. M. and Bonting, S.L. (1975). Biochemical aspectsof the visual process. XXVII. Stereospecificityof ocularretinol dehydrogenases and the visual cycle. Biochem. Biophys. Acta 384. 283-92.
Lowry, 0. H., Rosebrough,N. J., Farr, A. L. and Randall, R. J. (i 95 1). Proteinmeasurement with the Folinphenol reagent.J. Biol. Chem. 193, 265-75. Pfeffer, B. A., Clark, V. M., Flannery, J. G. and Bok, D. (1986). Membranereceptor for retinol-binding protein in cultured human retinal pigment epithelium. Invest. Ophthalmol. Vis. Sci. 27, 103140.
57
Rando, R. R. and Bangerter, F. W. (1982). The rapid intennembraneoustransfer of retinoids. Biochem.Biophys. Res. Commun. 104, 430-6. Rechler,M. M. and Nissley,S. P. (1985). The nature and regulation of the receptors for insulin-like growth factors. Annu. Rev. Physiol. 47, 425-42. Recio-Pinto,E. and Ishii, D. N. (1988). Insulin and insulinlike growth factor receptorsregulating neurite formation in cultured humanneuroblastomacells.1, Neurosci. Res. 19, 312-20. Rosen, 0. M. (1987). After insulin binds. Science237, 1452-8. Saari,J. C. and Bredberg,D. L. (1988). CoA- and non-CoAdependent retinol esterification in retinal pigment epithelium.J. Biol. Chem. 263, 8084-90. Saari, J. C. and Bredberg, D. L. (1989). Lecithin: retinol acyltransferasein retinal pigment epithelial microsomes.I. Biol. Chem. 264, 863640. Steinkampf, M P., Mendelson, C. R. and Simpson, E.R. (1988). Effectsof epidermalgrowth factor and insulinlike growth factor I on the levels of mRNA encoding aromatase cytochrome P-450 on human ovarian granulosacells.Mol. CelI. Endocrinol. 59, 93-9. Straus, D. S. (1984). Growth-stimulatory actionsof insulin in vitro and in vivo. Endocr. Rev. 5, 356-69. Trehan,A., Cannada,F. J.andRando,R. R. (1990). Inhibitors of retinyl esterformation alsoprevent the biosynthesis of ll-cis-retinal. Biochemistry 29, 309-12. Wilcox, D. K., Blochberger,T. and Sossi,N. (1989). IGF-1 Induces abnormal N-RAS expressionin retinal pigmented epithelium (RPE). Invest. Ophthalmol. Vis. Sci. 30 (Suppl.), 117. Zapf, J. and Froesch, E. R. (1986). Insulin-like growth factors/somatomedins : structure, secretion,biological actions and physiological role. Hormone Res. 24. 121-30. Zezulak, K. M. and Green, H. (1986). The generation of insulin-like growth factor-l-sensitive cells by growth hormoneaction. Science 233, 551-3.
Requirement of Insulin or IGF-I for the Maintenance Retinyl Ester Synthetase Activity by Cultured Retinal Epithelial Cells ROSS Boston
University
B. EDWARDS*, School
(Received
ALICE
of Medicine,
74 December
J. ADLER”AND
ROBERT
Department of Ophthalmology, Boston, MA, U.S.A. 1989 and accepted
of Pigment
C. CLAYCOMB and “Eye Research
Institute,
in revised form 76 May 1990)
Previous work from these laboratories showed that the retention of retinyl ester synthetase activity by cultured human retinal pigment epithelium is up to tenfold greater with PM medium (Medium 199 plus insulin, other added defined components, 1% serum and 1 y0 retina extract) than with conventional culture media. The present work shows that insulin is the component of PM medium required for maintenance of ester synthetase activity and that insulin-like growth factor type 1 (IGF-1) also is effective at maintaining ester synthesis. In addition, insulin can maintain ester synthetase activity in cultured rat RPE. Preliminary dose-response measurements provide additional support for these findings and strongly suggest that both insulin and IGF-1 are maximally effective at physiological concentrations (l-10 ng ml-‘). Key words: retinyl ester synthesis ; retinal pigment epithelium : insulin : insulin-like growth factor type 1; vitamin A; cell culture: human RPE.
1. Introduction The retinal pigment epithelium (RPE) plays a central role in the uptake and metabolsm of vitamin A (retinol) by the vertebrate eye. Receptors on the RPE basal surface bind serum retinol-binding protein and facilitate the uptake of retinol from the blood (Heller, 1975; Bok and Heller, 1976; Pfeffer et al., 1986). The RPE also appears to be the principal site of metabolism of vitamin A to the form required for vision, since this tissue contains the highest known specific activities of retinyl ester synthetase, retinyl ester hydrolase, vitamin A isomerase, and 1 1-cis retinol oxidoreductase. These activities, respectively, convert all-trans retinol to retinyl esters of fatty acids (Krinsky, 19 5 8 ; Andrews and Futterman, 1964; Berman et al., 1980; Saari and Bredberg, 19 8 8, 19 8 9), hydrolyze retinyl esters to free retinol (Blaner et al., 1987), isomerlze all-trans retinyl esters to 11-cis retinol (Bernstein, Law and Rando, 1987; Deigner et al., 1989; Trehan, Cannada and Rando, 1990), and oxidize 11-cis retinol to the aldehyde (Lion et al., 1975). Retinyl ester synthetase (RES) activity is particularly high in the RPE (Berman et al., 1980; Saari and Bredberg, 1988). A likely reason for this is that retinol is toxic, being highly membranoIyytic, while the retinyl esters of long-chain fatty acids are non-toxic (Goodall, Fisher and Lucy, 1980) : thus, during an intense bleach when the photoreceptors release large amounts of retinol, RES in effect detoxifies retinol by converting it to fatty acid * For reprint requests at: Boston University School of Medicine. Department of Ophthalmology G907, 80 East Concord Street, Boston,
MA 02118.
U.S.A.
00144835/91/010051+07
$03.00/0
esters. In addition, retinyl esters of long-chain fatty acids are more soluble in membranes than is retinol (Rando and Bangerter, 1982; Ho. Pownall and HollyfieId, 1989). which facilitates storage of vitamin A in RPE oil droplets and intracellular membranes, the principal ocular storage sites of vitamin A (Bridges, 1976). It has also been recently shown that retinyl esters are the most likely substrate for the isomerization of vitamin A (Deigner et al., 1989: Trehan et al., 1990). RES, because of its high activity in the RPE, also serves as an easily quantifiable biochemical marker for the RPE. RES activity is rapidly lost in RPE cultured in conventional media (e.g. commercially available media supplemented with 20 % fetal bovine serum) but is retained at levels up to 40% of fresh tissue levels in human RPE grown in PM medium (Edwards, Adler and Southwick, 1987). Therefore, human RPE cultured in PM medium should be a useful system with which to explore the nutritional, hormonal and growth-factor requirements for the expression of this differentiated property of RPE. The object of the present work was to determine which of the components of PM medium are required for this maintenance of RES activity in cultured human RPE. A preliminary account of this work has been reported elsewhere (Edwards and Adler, 1989). 2. Materials
and Methods
RPE cells were isolated, as described previously. from human autopsy eyes obtained from the National Disease Research Interchange, Philadelphia, PA (Edwards, 1982), or from S- to 6-day-old, Long-Evans, 0 199 1 Academic Press Limited 4-2
52
pigmented rat eyes (Charles River, Wilmington, MA: Edwards, 1981) and seeded as primary cultures at 1040 x lo3 cells per cm* culture surface in 24- or 12cluster plastic plates (Costar, Cambridge, MA). In each experiment cells from a different set of donors were used. An experiment is defined as a comparison of different media during a given 14-day culture period. First-passagehuman RPE cultures that were obtained from Dr B. A. Pfeffer were used in one experiment. In most cases an aliquot of freshly isolated cells was washed twice in isotonic saline and stored at - 75°C for subsequent comparison with cultured cells derived from the samedonor. Cellswere initially seededin PM medium, which consistsof Medium 19 9 supplemented with seven additional defined components (10 pg ml-l insulin, 5 ,ug ml transferrin-‘, 20 nM hydrocortisone, 1 nM triiodothyronine, 0.3 pugml-’ putrescine, 0.3 ,uM linoleic acid, and 10 pg ml-’ bovine serum albumin) and two undefined components (1 y0 newborn calf serum and 1% bovine retina extract; Pfeffer et al.. 1986 ; Edwards et al., 198 7). Insulin and IGF-1 were obtained from Collaborative Research, Bedford, MA: all other media components were from sources stated previously (Edwards et al., 198 7). One to 3 days after the cells were seeded,the medium was replaced with variations of PM medium to be tested (i.e. PM medium with one or more components omitted, or with the 10 ,ug ml-’ insulin replaced with different concentrations of insulin or IGF-l), and was subsequently changed ever 3 or 4 days. For dose-responsemeasurements, PM medium was used with the 10 pg ml-’ insulin replaced with different concentrations of insulin or IGF-1, and the media were changed daily to minimize the in vitro degradation of labile medium components (Straus, 1984). Ten to 14 days after the initial medium change, the cells were rinsed in 0.145 M NaCl:O.OS M phosphate, pH 7.2, sonicated, and centrifuged at 50000 g for 2 hr at 3°C. The resulting pellets were resuspended by sonication in 0.05 M sodium phosphate, pH 7.2, assayedfor protein (Lowry et al., 1951), and stored at -75°C. Aliquots of the pellet (15 pg protein) were combined with 2 ,&i [l 1 ,123H]all-truns retinol (New England Nuclear, Boston, MA, specific activity 53 Ci mmol-I), 1 IIIM dithiothreitol, and Tris buffer (0.1 M, pH 8.2) (Saari and Bredberg, 1988) in a final volume of 02 ml, and incubated in the dark under nitrogen for 15 min at 37°C. Under these conditions no more than 2 5 % of the substrate was esterified. The mixtures were then extracted with hexane and analyzed by HPLC for radioactive retinoi and retinyl esters, as previously described (Edwards et al.. 1987) except that radioactivity of the HPLC effluent was continuously monitored with a flow-through liquid scintillation counter (Flow-l Beta, Radiomatic Instruments, Inc.. Meriden, CT). Results are expressedas the percent of radioactive retinol converted to retinyl esters(% retinyl esters). In some cases, to pool data from different experiments, RES activity was normalized to 100%
R. B. EDWARDS
ET AL.
PM medun
PM w/o undefined
PM w/o defined Medium +20%
199 serum 0 Retlnyl
FIG.
1. Effects
I
I
1
I
25
50
75
100
ester
synthesis
(% of PM medium)
on retinyl estersynthesisof omitting classes
of PM medium components.Two
sets of human
RPE
cultures, each from a different donor, were used in this experiment.For each set. singlecultures were maintained with completePM medium,PM withoutundefined components (i.e. without 1y0 serumor 1y0 retina extract). PM without defined components (i.e. without the seven added defined components, see Materials and Methods), or Medium 199+20% serum [Medium 199 supplemented with 10% (v/v) fetal bovine serum]. After 14 days the cultures were processed and assayed for RES activity as described in Methods. The data are normalized to 100 % for complete PM medium : for one set of cultures this was equivalent to 7.2 % conversion of retinol to retinyl esters, and for the other set this value was 13.5%. Bars indicate the normalized means for each pair of cultures: lines show the higher normalized value for each pair. These data are from one of three experiments that gave similar results.
for cellsgrown in complete PM medium ; this was done only for Figs 1 and 2. 3. Results When human RPE cells were maintained on PM medium from which two undefined components (1 “/o serum and 1 Y0retina extract) were omitted, significant levels of RES activity were still retained by cultured human RPE cells. In one experiment the mean RES activity (per 15 pg protein of cell pellet fraction) was 40% of the synthesis observed with the cell pellet fraction from cells grown with complete PM medium (Fig. 1) ; in some experiments with this medium, RES activity was as high as in cells grown with complete PM medium (data not shown). In contrast, when cells were maintained on PM medium from which the seven added defined components were omitted, virtually no RESactivity was detected (Fig. 1). As previously reported (Edwards et al., 1987), comparable low levels of ester synthesis also were observed with pellet fractions of cells maintained with a conventional culture medium. i.e. Medium 199 supplemented with 20% fetal bovine serum (Fig. 1). From the results shown in Fig. 1 it appeared that one or more of the seven added defined components was required for the maximum maintenance of RES activity. Therefore, media were tested that consisted of PM medium with individual, defined,’ added components omitted. (In the caseof linoleic acid and BSA, which were obtained as a combined preparation, both were omitted together.) Only the omission of insulin
RETINYL
ESTER
SYNTHETASE
IN CULTURED
TABLE
RPE
I
Effect on retinyl ester synthesis of omitting insulin from PM medium oARetinyl esters
Donor 1
2 3 4
PM with
PM without
insulin*
insulin
7.2 13.5 6.5 23.8
Ratio? without/with insulin
1.2 0.6 1.2 4.4
0.17 0.05 0.18 0.18
* Insulin concentration = 10 ,/hgml-‘. t Mean ratio k S.D. = 0.14 f 0.07. P < @OOOl, compared to ratio of 1.0. based on two-tailed t-test.
resulted in a marked lossof RES activity from cultured human RPE (Fig. 2). Additional experiments confirmed that the omission of insulin from PM medium resulted in a marked reduction in the maintenance of RES activity in cultured human RPE. As illustrated by the results from Fig. 2 and from two other experiments, RES activity in cultures grown in PM medium without insulin was lessthan 0.2 that of cultures grown with 10 begml-’ insulin (Table I). This difference was statistically significant (P < O.OOOl), based on a two-tailed t-test analysis of the ratios of Table I. In addition, rat RPE cultured for 2 weeks with PM medium containing insulin retained higher levels of RES activity (11% conversion of all-trans retinol to esters) than replicate cultures grown in PM medium without insulin (2 % conversion to esters; Fig. 3). Since insulin at 1 L&gml-’ or higher has been shown to stimulate the receptors for insulin-like growth factor type 1 (IGF-1: see Straus. 1984; Rechler and Nissley, 1985 ; for reviews), IGF-1 was also tested for its capacity to maintain RES activity of cultured human RPE. When insulin was replaced with IGF-1 at
0
25 Retlnyl
FIG.
50
ester
synthesis
75
100
(% of PM medium)
2. EXectson retinyl estersynthesisof omitting single,
added, defined components
of PM medium.
Details are as
describedfor Fig. 1: i.e. human RPEcultures were from the sametwo setsas usedfor Fig. 1. with singlecultures from each set maintainedwith each of the indicated test media. Results are from one experiment.
53 100 Dr 300 ng ml-l and the media changed every 34 days, the retained levels of RES activity were comparable to, or exceeded, RES activity retained in the presence of 10 pg ml-’ insulin (Fig. 4). Preliminary dose-responsemeasurements indicated that both insulin and IGF-1 are effective in the physiological concentration range. With insulin, retention of RES activity increased sharply as the concentration was increased above l-3 ng ml-’ (Fig. 5). For comparison, the physiological range for insulin is 14 ng ml-’ (equivalent to 25-100 PU ml-l : Larner, 1985). Retention of RES activity by primary cultures (m in Fig. 5) increased further for insulin concentrations of 80 ng ml-’ or higher; such an increase was not seenin the experiment with the firstpassagecultures (0 in Fig. 5). With IGF-1. retention of RES activity increased sharply above 3-10 ng ml-l {Fig. 6), which compareswith a physiological range of l-20 ngml-’ or more (Zapf and Froesch, 1986). Although a complete set of IGF-1 concentrations was tested only for the first-passage cells, the increased retention of RES activity for IGF-1 concentrations between 3 and 100 ng ml-’ was statistically significant. To demonstrate this, the experimental values within each of three concentration ranges were pooled and compared, using a two-tailed t-test. In Fig. 6, horizontal lines indicate the concentration ranges over which IGF-1 was effective (3-100 ng ml-‘) or not effective (O-3 ng ml-‘, and greater than 300 ng mlml). The mean ( &SD.) of the six high RES values at 3-100 ng ml-’ (% ester = 20.8 f 3.5 %) is significantly greater than the mean of the five low values at O-3 ng ml-’ (% ester = 11.3 & 2.8 %, P = 00009) and the mean of the three low values at 300 ng ml-’ (% ester = 9.6 f 0.5 %, P = 00011). For both insulin and IGF-1 the upward breaks in the dose-response curves occurred within a narrowly defined concentration range for cultures from different donors. IGF-1 at the highest concentration tested (300 ng ml-‘) did not maintain RES activity when the daily medium change schedule was used for doseresponsemeasurements (Fig. 6). This result contrasted markedly with the maintenance of high RES activity by 300 ng ml-’ IGF-1 when the medium was changed every 34 days (Fig. 4). Also noteworthy are the higher basal levels of RESactivity seen in the absence of added insulin or IGF-1 when the media were changed daily (Figs 5 and 6) compared with medium changes every 34 days (Fig. 4). For possible explanations of these differences, see Discussion. For some sets of replicate cultures a subtle morphological difference was seen between cells maintained with insulin and those maintained without insulin. Cultures of human or rat RPE grown for 2 weeks with PM medium that contained 10 pg ml-’ insulin appeared to contain cells that were somewhat more refractile and more cobblestone-like in shape (i.e. thought to more nearly resemble RPE in vivo) compared to cells grown in PM medium without
54
R. B. EDWARDS
i
ET AL.
30
5 5 .c P .$ 200 L
jl~I 0
2 Retinyl
4 ester
6
8
synthesis
IO (% retlnyl
I2
lnSulln
or IGF-,
b
0
,
,
2
4
Retlnyl
ester
,
6 synthesis
/
14
esters)
FIG. 3. Effects of insulin on retinyl ester synthesis by cultured rat RPE. Cultures from 6-day-old pigmented rats were maintained for 14 days in duplicate with complete PM medium (PM) or with PM medium without insulin and assayed for RFS activity. Details are otherwise as described for Fig. 2 except that results are expressed as the percent retinol esterified during a 15-min incubation. Results are from one experiment.
?M WI0
$p
,
,
8
IO
(% retlnyl
,
0
\I
100
I
I
I
I
IO’
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IO3
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I IO5
(ng ml-‘)
FIG. 5. RES activity as a function of insulin concentration. Human RPE cultures were maintained for 14 days in PM medium with the indicated concentrations of insulin. Media were changed daily to minimize the in vitro degradation of insulin. (m) Primary cultures from one donor, (0) firstpassage cultures from a seconddonor.Eachpoint represents one culture with each curve being from a separate experiment. Results are expressed as percent retinol esterified during a 15-min incubation. For comparison, the equivalent RES activity for fresh tissue from the primary-culture donor was 87% esterification of retinol.
j
12 esters)
FIG. 4. Effects of substituting IGF-1 for insulin in PM medium. Single cultures of human RPE from a replicate set were maintained for 14 days with the following media: PM medium that contained the normal concentration of insulin (10000 ng ml-‘), PM medium with the insulin replaced with 100 or 300 ng ml-’ IGF-1, or PM medium without insulin or IGF-1. Media were changed ever 34 days. RES activity is expressed as the percent retinol esterified during a 15-min incubation. Results are from one experiment.
insulin (data not shown). These differences were not perceived in all experiments, nor were these observations made as part of a blind experimental design, so that further work is required to determine the reproducibility of this subtle difference.
iol
,\\I 0
I IO0 IGF-I
IO’ (ng
I IO2
IO3
ml-‘)
FIG. 6. KES activity as a function of IGF- 1 concentration. Human cultures were maintained for 14 days in PM medium without insulin and with the indicated concentrations of IGF-1. Media were changed dally. (0, n ) The same sets of cultures as the corresponding symbols in Fig. 5. Each point represents one culture. The three horizontal lines indicate the mean values of RES activity for the data points of the corresponding ranges of IGF-1 concentration (see Results). Other details are as in Fig. 5.
4. Discussion These results show that insulin is a component of PM medium that is required by cultured human RPE for the maintenance of RESactivity. Of the components tested, only the omission of insulin from PM medium resulted in a marked loss of RES activity compared to cells grown with complete PM medium or PM medium with other components omitted (Figs 1 and 2, Table I). Preliminary dose-response measurements (Fig. 5)
further support this finding and strongly suggest that insulin is effective in the physiological dose range (l-4 ng ml-’ ; Larner, 198 5). Insulin also maintains RES activity in cultured rat RPE (Fig. 3). Note, however, that these results do not rule out the possibility that other components of PM medium are required for maintenance of RES activity. Synergistic relationships between insulin and other medium
RETINYL
ESTER
SYNTHETASE
IN CULTURED
RPE
components have not been examined, nor have we tested the removal of standard components of Medium 199 such as specific amino acids or vitamins. In addition, IGF-1 was effective in maintaining RES levels in cultured human RPE. (IGF-1 has not yet been tested with cultured rat RPE.) As with insulin, dose-response measurements (Fig. 6) suggest that IGF-1 is effective at physiological concentrations (l-20 ng ml-‘: Zapf and Froesch, 1986). It must be emphasized that the dose-response curves for both insulin and IGF-1 are preliminary ; additional measurements are required to confirm some of the details of the relationships shown in Figs 5 and 6. For insulin (Fig. 5) it will be of interest to confirm the increase in the retention of RES activity above 80 ng ml--’ observed in the primary cultures. This increase was not seen in the first-passage cells. Although it is speculative at this time to suggest a reason for this increase, it may be due to the stimulation of IGF-1 receptors at the higher insulin concentrations (Straus, 1984; Rechler and Nissley, 19 8 5). Since the first-passage cells were in culture for a longer period and presumably underwent more cell division than the primary cultures, the surface density of IGF-1 receptors may have been diluted in the firstpassage cells compared to the primary cells. Such a reduced density might explain the absence of the rise in RES activity in the first-passage cells at insulin concentrations above 80 ng ml-l. This might also explain why a slightly higher concentration of IGF-1 was required to achieve maximum RES activity in the first-passage cells (10 ng ml-l) than in the primary cells (3 ng ml-‘) (Fig. 6). On the other hand, the difference between 3 and 10 ng ml-’ IGF-1 is small compared to the lOOO-fold difference between insulin concentrations that elicited the additional increases in RES activity in primary cultures (above 80 ng ml-l) and concentrations that failed to do so in first-passage cultures (up to 100000 ng ml-’ ; Fig. 5). It should also be noted that the apparently modest differences in RES activity in the presence compared to the absence of added insulin or IGF-1 in the doseresponse curves (Figs 5 and 6) are due largely to the high basal levels of RES activity without added insulin or IGF-1. These high basal levels resulted from the daily changes of the culture medium (compare Fig. 4 with Figs 5 and 6). We speculate that these high basal levels may have been due to trace amounts of insulin, IGF-1, or as yet unidentified factors in the serum or retina extract that maintain RBS activity; these factors may become degraded in 34 days in vitro, but not in 1 day. IGF-1 at the highest concentration tested (300 ng ml-‘) was not effective when the medium was changed daily (Fig. 6). This was observed in two experiments with three cultures. This observation is consistent with the idea that IGF-1 receptors may have been reduced in number (downregulated) after exposure to sufficiently high IGF-1 concentrations, as
55
has been reported for a number of other cell types (Rechler and Nissley, 1985). Confirmation of this point will require measuring the number of IGF-1 receptors on RPE cells before and after incubation with a variety of IGF-1 concentrations in the range of 100-10 000 ng ml-‘. The ineffectiveness of IGF-1 at 300 ng ml-’ in the experiments of Fig. 6 may seem to contradict the results of Fig. 4, in which 300 ng ml-’ IGF-1 was very effective in maintaining RES activity. However, the data of Fig. 6 were obtained with cultures that received daily medium changes, while the data of Fig. 4 were from cultures that had media changed every 34 days. Our present interpretation is that, over the 34-day interval between changes of medium, the 300 ng ml-’ IGF-1 became partially degraded in vitro, resulting in reduced concentrations of IGF-1 that were effective in maintaining RES activity ; daily changes with 300 ng ml-’ IGF-1 apparently preserved a sufilciently high concentration of IGF-1 to prohibit the maintenance of RES activity, possibly by downregulation of IGF-1 receptors. It has been reported that RES activity in bovine RPE decreased to about 5% of fresh tissue levels after 3 days in culture, regardless of whether the cells were grown in a defined medium with 105 ,ug ml-l insulin or a conventional medium supplemented with 20% serum (Bridges et al., 1986). The initial cell density in those experiments was much lower (35 cells cme2) than in the present work (1040 x lo3 cells cme2), and the bovine cultures were confluent by 7 days, suggesting that the cells had divided rapidly and extensively. The depletion of RES activity could have resulted from a combination of dilution from mitosis and insufficient time for the newly divided cells to resynthesize RES. Differences between PM medium and the media used by Bridges et al. (1986) may also be involved. The mechanisms by which insulin and IGF-1 inIluence the levels of RES activity are not known. The effectiveness of insulin and IGF-1 at physiological concentrations suggests that cultured RPE cells possess surface receptors for each of these factors. Although 600-1000 ng ml-’ insulin can stimulate IGF-1 receptors, and IGF-1 above 350 ng ml-’ can stimulate insulin receptors (Straus, 1984; Rechler and Nissley, 1985) the concentrations at which these factors are maximally effective in maintaining RES activity (l-10 ng ml-l) are much lower; this fact is consistent with insulin and IGF-1 each acting at its own respective receptor to exert its effect on maintaining RES activity. IGF-1 receptors have been directly demonstrated on cultured human RPE cells (Haskell et al., 1989). Other evidence of the response of cultured human RPE to insulin and IGF-1 is the stimulation of DNA synthesis by insulin and IGF-1 (Leschey et al., 1990) and the stimulation of N-ras oncogene mRNA synthesis by IGF-1 (Wilcox, Blochberger and Sossi, 1989). Insulin and IGF-1 both elicit the phos-
56
phorylation of their respective membrane receptors in a number of other tissues, and stimulate the phosphorylation or dephosphorylation of a variety of other cellular proteins (Straus, 1984 ; Rechler and Nissley, 1985; Rosen, 1987). Insulin alters the transcription of the genes for phosphoenolpyruvate carboxykinase and other enzymes (Rosen, 198 7), and IGF-1 stimulates hydroxysteroid dehydrogenase activity (Lin et al., 1987) and synthesis of the mRNA for aromatase (Steinkampf, Mendelson and Simpson, 1988) in other cell types, establishing the precedent for the possible induction or maintenance of enzyme activity by insulin and IGF-1 in the RPE. IGF-1 also appears to play a supportive role in differentiation; according to the dual effector model (Zezulak and Green, 1986), growth hormone acts on a predifferentiated cell to stimulate differentiation, to stimulate synthesis of IGF-1, and to sensitize the cell to divide in response to IGF-1. In this way the newly differentiated cell proliferates in response to its own newly synthesized IGF-1. This clonal expansion model appears to be applicable to adipocyte development (Zezulak and Green, 1986) and developing bone (Zapf and Froesch, 1986 ; Isaksson et al., 198 7). IGF-1 also stimulates the fusion of myoblasts to myotubes (Zapf and Froesch, 1986 ; Isaksson et al., 1987) and neurite formation (Recio-Pinto and Ishii, 1988). Whether any of these observations apply to maintenance of RES activity in RPE requires further study. Insulin does not influence glucose uptake by frog RPE (DiMattio and Streitman, 1986), suggesting that insulin-induced glucose uptake is not involved in the maintenance of RES activity in RPE. A number of questions remain to be answered regarding the significance of these findings. First, whether or not insulin or IGF-1 actually maintains RES activity in the RPE in vivo will require experiments with animals that are pancreatectomized to reduce insulin levels, or hypophysectomized to remove the source of growth hormone, the principal stimulant of IGF-1 synthesis in many tissues (Isaksson et al., 198 7). Second, the role of insulin and IGF-1 in the induction of RES in early development of the RPE is not known ; determination of the time of onset of RE% activity in the RPE of fetal or neonatal animals and the correlation of RES activity with insulin and IGF-1 levels will help answer this question. It will also be necessary to determine if insulin or IGF-1 can induce RES activity in RPE cultured before RES is normally detectable. Third, we must clearly identify the surface receptors through which these growth factors influence RES activity, and determine whether the effects of low doses of insulin and IGF-1 are additive. Fourth, the effects of insulin and IGF-1 on the levels of other
enzymes and binding proteins involved in retinoid metabolism should be examined in cultured RPE. Finally, it will be of interest to determine if these findings imply a role for RES in the etiology of diabetic retinopathy.
R. B. EDWARDS
ET AL.
Acknowledgments We thank Dr B. A. Pfeffer for a sample of cultured human RPE and the National Disease Research Interchange, Philadelphia, PA, for providing human autopsy eyes. This research was supported by NIH grants EY02028 (RBE) and EY04368 (AJA), the Retinitis Pigmentosa Foundation Fighting Blindness, Baltimore, MD, and by grants to the Department of Ophthalmology, Boston University School of Medicine, from the Massachusetts Lions Eye Research Fund. Inc., and Research to Prevent Blindness, Inc., New York, NY. References Andrews, J. S. and Futterman. S. (1964). Metabolism of the retina. V. The role of microsomes in vitamin A esterification in the visual cycle. J. BioZ. Chem. 239, 4073-6. Berman, E. R., Horowitz, J.. Segal, N.. Fisher, S. and FeeneyBurns, L. (1980). Enzymatic esterification of vitamin A in the pigment epithelium of bovine retina. Biochim. Biophys. Acta 630, 3646.
Bernstein, P. S., Law, W. C. and Rando, R. R. (1987). Isomerizationof all-trans-retinoidsto 1 1-cis-retinoidsin vitro. Proc. Natl. Acad. Sci. U.S.A. 84, 1849-53.
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