Biochimica et Biophysica Acta, 1134(1992)53-60
53
© 1992ElsevierScience PublishersB.V. All rightsreserved0167-4889/92/$05.00
BBAMCR 13097
Correlation of insulin receptor level with both insulin action and breakdown of a potential insulin mediator precursor; studies in CHO cell-lines transfeeted with insulin receptor eDNA S. L a n c e M a c a u l a y a,c, Steila C l a r k a,b a n d R i c h a r d G . L a r k i n s ~ a Melbourne University Department of Medicine and b Busnet Clinical Research Unit, Walter and Eliza Hull Institute, Royal Melbourne Hospital. Melbourne (Australia) and c Commonwealth Scientific and Industrial Research O~,anization, Di~'ision of Biomolecular Engineering, Parkville (Australia)
(Received 23 May 1991) (Revised manuscriptreceived21 August1991)
Key words: Insulinreceptor;InsulinmediatorprecursorPl-glycan;Pyrovatedehydregenase;Glucosetransport A phosphatidylinositol-glycan(Pl-glycan) has been described previously that may set~e as the precursor for a mediator of some of insulins actions. The present study further addresses the potential relevance of this compound by correlating its breakdown with other insulin actions in Chinese hamster ovary (CLIO) cells which express different levels of insulin receptor. Comparisons were drawn between parent ClIO cells expressing 3-103 receptors/cell and two cell-lines tranxfected with human insulin receptor eDNA, that expressed 600 (CHO.TH) and 300 (CHO.T) times the parent receptor level. A Pl-glycan was isolated from all cells that incorporated [3H]glucosemine,[3Hlaalactuse and [3H]inositol and was rapidly turned over upon insulin stimulation. Maximal turnover by insulin of approx. 20% was achieved in each cell line consistent with the fibroblastic nature of these cells. The effect of increased insulin receptor expression was to increase the sensitivity of the Pl-glycun response to insulin. Increasing receptor number from 3.103 to 0.88.106 also increased the sensitivity of response of other insulin actions measured in this study, namely activation of pyruvate dehydrogenase (PDH), glucose utilization and transport. Thus turnover of the Pl-glycan is linked closely to both metabo~,ic actions of insulin and to cell surface insulin receptor expression further supporting its potential role in insulin action.
Introduction Insulin has been shown previously to stimulate the cleavage of a PI-glycan in a variety of cell types [1-9]. The head group generated by phospholipase mediated cleavage of this molecule has been shown to mimic several, but not all, actions of the hormone when added to in vitro enzyme systems or to intact cells [1-3,5,6,8,9-12]. The diacylglycerol generated with the head group has been postulated to contribute to the mechanism of insulin-stimulated glucose transport [13].
Abbreviations: PI, phusphatidylinositol; CHO, Chinese hamster ovate; PDH, pyravate dehydrogenase; PLC, phospholipaseC; TIC, thin-layer chromatography. Correspondence: S.L. Macaulay, CommonwealthScientific and Industrial Research Organization, Divisionof BiomolecularEngineering, 343 Royal Parade, Parkville, Victoria, Australia3052.
The ability of this head group and diacylglycerol to mimic some of the actions of the hormone has led to the proposal that the Pl-gl~an is the precursor of a mediator for some of the hormone's intracellular actions (see Ref. 14 for review), although this is not uniformly accepted [15,16]. in a previous study [17], it was established that the tyrosine kinase activity of the insulin receptor was necessary for insulin-stimulated breakdown of the PIglyean consistent with most, if not all, other actions of the hormone. The present study further addresses the potential contribution of this molecule to insulin action by correlating its breakdown foliowing insulin treatment with the level of cell surface insulin receptor expression and with other actions of insulin. Although effects of insulin on some insulin actions have been previously described in celis expressing different levels of insulin receptor, the present study is the first to correlate PI-glycan breakdown with these actions and to describe effects of increased insulin receptor expres-
54 sion on PDH activation and glucose utilization. These studies were performed in CHO cells which although not highly insulin responsive, because of their fihroblastic nature, are one of the few cell lines in which it has been possible to stably express different levels of insulin receptors. Materials and Methods
Materials D-[6-3H]Glucosamine hydrochloride, D-[G-~H]galactose, D-[5-3H]glucose and [1-J4C]pyruvic acid were obtained through DuPont from New England Nuclear, Boston, MA. myo-[2-3H]lnositol, [9,10(n)-3H]myristic acid, [9,10(n)-3H]palmitie acid and 2-deoxy-D-[U14C]glucose were from Amersham International, U.K. Purified phosphatidylinositol-specific phospholipase C (PI-PLC) from B. thuringiensis was a generous gift from Dr. M. Low, Columbia University, New York and was used at an activity dilution of 2.5-5 p.mol/min per ml estimated using [3H]phosphatidylinositol as substrate at pH 7 in the presence of 0.1% deoxTcholate. Preparations tested for proteinase activity were below detectable limits ( < 2.5 ng/25 U PI-PLC, M. Low, personal communication). Materials for cell culture were obtained from Flow Laboratories, McLean, VA, except for alpha modified minimum essential medium which was obtained from the Commonwealth Serum Laboratories, Parkville, Australia. Silica gel G plates for thin-layer chromatography (TLC) were from Whatman, Clifton NJ. Porcine insulin and most other chemicals were from Sigma, St. Louis, MO. Chinese hamster ovary (CHO) cell lines The CHO cell lines used in the present study were derived from ceils obtained from Dr. Leland Ellis (Howard Hughes Medical Inst., Dallas, TX). The parent CHO cell line expressed approx. 3.103 insulin receptors/cell. Two other stably transfected cell lines were used. The first, Ellis and co-workers termed CHO.T [18], was derived from cells transfected with human insulin receptor eDNA. These cells have been well characterized previously [18] and in our hands expressed approx. 0.88.10 ° insulin receptors/cell. The third cell line was derived from the CHO.T cell line in our studies by indirect immunolabelling and fluorescence-activated cell sorting (FACS). CHO.T cells were labelled with an anti-insulin receptor antibody 83-14 (obtained from Dr. Ken Siddle, Cambridge University, U.K. [19]) directed towards the a-subunit of the insulin receptor at a 1:100 dilution, followed by a second F1TC labelled goat anti-mouse antibody at a 1:50 dilution. Two sorts were performed to enrich those cells expressing human insulin receptors. In the first sort the top 10% of insulin receptor expressing cells were collected and expanded. These cells were re-
sorted, the top 10% of insulin receptor expressing cells collected and a pure cell line obtained by limiting dilution of these FACS sorted cells. This cell line, designated CHO.TH, expressed approx. 1.6" 106 insulin receptors/cell. Each of the three cell lines were grown to confluence from a 1:10 dilution in a-modified minimum essential medium (a-MEM), 5% fetal calf serum, 100 U / m i penicillin, 100 i,t g / m l streptomycin and 1/~g/ml fungizone (Squibb) and 10 /.tCi/ml tracers (D-[63H]glucosamine hydrochloride, D-[G-3H]galactose, and myo-[2-3H]inositol or [9,10(n)-3H]myristic acid), as required if precursor Pl-glycan was to be examined, in 6 well dishes (Nunc). When the cells reached approx. 80% confluence (48 h), the medium was removed and replaced with a-MEM, 1% bovine serum albumin (BSA) and tracer (at the same specific activity) as appropriate for the final 16 h by which time the cells were confluent. The medium was then removed and replaced with 1 ml fresh medium (a-MEM, 1% BSA, or Krebs-Ringer bicarbonate Hepes buffer (pH 7.4) containing half the normal Ca 2+ concentration (1.15 mM) (for glucose transport determination)). Cell stimulations with insulin were then carried out after a 20 min equilibration period. Insulin mediator precursor Pl-glycan oreakdown Cells labelled to equilibrium with [3H]glucosamine, or the other tracers as indicated, were extracted for glucosyl-lipids using a similar scheme to that employed previously for H35 cells, splenic T cells and adipocytes [3,6,8,20]. After the 20 rain equilibration period, the cells were stimulated with insulin for the appropriate time (1 min unless otherwise stated) and reaction stopped by the addition of 1 mi of methanol at 0°C. Control incubations were performed for the same period of time. The cells were then scraped, transferred into extraction tubes and the wells washed with a further 0.5 ml of methanol. 3 ml of chloroform was added to the extraction tubes followed by 1.5 ml of chloroform/methanol/HCI (200:100: 6). Phase separation was then induced by the addition of 1.5 ml of 0.1 M KCI. The organic phase was collected and the aqueous phase washed with a further 1 ml of chloroform. The Pl-glycan was then purified from the pooled organic phase by thin-layer chromatography (TLC) in two solvent systems [3,6,8]. It remained at or near the origin following TLC in chloroform/acetone/methanol/acetic acid/water (50:20:10:10:5) (solvent system I), in which it was separated from the major phospholipid classes, and following elution with methanol and TLC in c h l o r o f o r m / m e t h a n o l / ammonia/water (45 : 45 : 3.5 : 10) (solvent system !I) on oxalate impregnated plates, ran with an R F of approx. 0.5. This procedure, in our hands, yielded preparations of PI-glycan free of major contaminants although con-
55 taminants have been reported using this procedure in a previous study in liver microsomes [16]. Radiolabelled lipid and standard bands were located by autoradiography of TLC plates sprayed with Enhance R (New England Nuclear), and were quantified, following scraping of the bands, by liquid scintillation counting. Parallel wells were processed for measurement of cell protein and all results were corrected for differences between cell-lines. Several criteria were used to establish the band as the Pl-glycan we and others have described for other cell types [1-8]. First it was confirmed that the majority of the added tracer in the labelled band remained as the native sugars. In contrast to our earlier fat cell studies in which there was significant turnover of the added tracers during labelling [8], the majority of the added tracers remained as the native sugars in the CHO cell lines. After acid hydrolysis of the Pl-glycan band in the CHO cells used in the present study and TLC of the component sugars as in Refs. 3,8, we found that 65 + 3% of the added [3H]glucosamine tracer remained as the native sugar. Even less inositol and galactose were metabolized, with 82% and 71% remaining as the native sugars, respectively (data not shown). Phosphatidylinositol-specific phospholipase C digestion of the band extracted from [3H]glncosamine labelled cells, as in Refs. 3,8, resulted in a loss of 45% of the label associated with the band (data not shown), consistent with previous studies [3,6,8]. Furthermore, phosphatidylinositol-specific phospholipase C digestion of the band as in Ref. 8 generated a material that had a stimulatory effect on PDH activity of adipocyte mitochondria (Basal PDH activity = 0.694 + 0.072 n m o l / m g per rain, control (extract prepared in the absence of Pl-glycan band) --- 0.744 + 0.072 n m o l / l / m g per rain, Pl-glycan= 1.06 4-0.054 n m o l / m g per min (n = 3)). These data taken together all support the contention that the band extracted was a Pl-glycan similar to that described in previous studies [1-8]. Data given in Results support its insulin sensitivity.
Other assays of insulin action Glucose utilization was determined as the conversion of [5-~H]glucose to 3H20 as we have described previously for adipocytes [8,20]. Each well in 1 ml of a-MEM, 1% BSA (pH 7.4) was incubated in the presence or absence of insulin for 10 rain followed by 4 ~Ci [5-3H]glucose for an additional 30 min. 3H20 in the medium was then measured after absorption into CaCI 2. l~jruvate dehydrogenase (PDH) activity was measured in the same cells after removal of the medium washing cells in phosphate-buffered saline and extraction of the cells with 0.2% Triton X-100, 2 mM dithiothreitol, 2 mM EDTA, 2 mM EGTA, 50 mM potassium phosphate buffer (pH 7.4) (PC as described in Refs. 20-22. Each well was scraped and cellular sus-
pensions vortexed three times 10 s, if'C, and centrifuged 5 s, 4°C, at 10000 x g. PDH activity was determined in the supematant fractions by the 14COz release from [ 1-14C]pyruvate assay [20-22]. "Active" PDH was determined in the presence of 50/zM MgCI 2 and 5 0 / z M CaCI 2. 'Total' PDH was determined following preincubation of aliquots with 20 mM MgCI 2 and 0.5 mM CaCI 2 for 30 rain. Parallel wells were processed to enable results to be corrected for protein. 2-Deoxy-glucose was used as the index of glucose transport and was measured as in Refs. 23,24. Briefly, wells were washed twice in Krebs-Ringer bicarbonate Hepes buffer, 1% bovine serum albumin, 0.1 mM glucose, 2 mM pyruvate (pH 7.4) and the cells allowed to equilibrate in the incubator for 20 rain. Cells were then incubated in the presence or absence of insulin for 30 min and assay then initiated by the addition of 0.1 mM 2-deoxy-[U-*4C]glucose (0.25 p.Ci tube) for a further 5 min. Assay was terminated by washing the cells three times in phosphate-buffered saline at if'C, with the final addition of I ml of 1 M NaOH. 0.6 ml was taken for determination of 2-deo~glncose uptake and the remainder used for protein determination.
Statistical analyses Statistical analyses were performed using the Student's t-test. Results are expressed as the mean + S.E. Statistical significance was determined at the 0.05 level and is indicated in the results. Results
Effect of insulin on Pi-glycan breakdown Initially, the kinetics of insulin effects on the level of Pl-glycan, determined as [3H]glucosamine label associated with the Pl-glycan band after TLC of lipid extracts of insulin-treated cells was examined (Fig. 1). Cells were labelled to equilibrium with [3H]glucosamine to achieve steady state labelling of Pl-glycan, and then incubated in fresh medium for the next 20 rain prior to insulin stimulation. Insulin, at 0.7 nM, caused a rapid 10ss (17%) of [3H]glucosamine associated with the Pl-glycan band in the CHO.T cell line (Fig. 1) that was maximal at 1 rain and transient in nature. These studies establish that insulin stimulates breakdown of the Pl-glycan in CHO.T cells. In additional experiments (data not shown) in which other time-points were examined, it was apparent that there was variability between experiments over the point at which maximal effects were obtained (30 s - i rain). The reversibility of the response was also variable as has been described for other cell types. In some experiments labelling of the Pl-glycan returned towards basal at 5 rain whilst in others the response was still maximal at this time. The standard error bars for the 5 rain point in Fig. I reflect this fact. In contrast to the
56
CHO.T cells, no significant loss of [3H]glucosamine label associated with the Pl-glycan band was found in CHO cells following stimulation with this low concentration of insulin (0.7 nM) over the same time-course. This was despite the similar labelling of the Pl-glycan band in both cell types under basal conditions. In order to strengthen the contention that an insulin-sensitive Pl-glycan was extracted from within the PI-glycan band in CHO.T cells after TLC and that the observed effects were not a result of potential contaminants in the preparations, other potential sugars associated with this band were examined. Insulin caused a loss of 22.3 ± 5.5% (n = 3), 37.3 + 4.9% (n = 3) and 18.2 + 2.0% (n = 21) in the amount of galactose, inositol and glucosamine respectively associated with the Pl-glycan band implicating these sugars in Pl-glycan structure in the CHO cell lines, similar to previous studies in other cell types [2,3,6,8]. The time-course study (Fig. 1) established that an insulin-sensitive PI-glycan could be extracted from CHO cell lines and that the insulin effect was rapid. Further studies sought to establish the effect of increased cell surface insulin receptor expression on insulin sensitivity/responsiveness to PI-glycan breakdown (Fig. 2). The three cell lines expressing different levels of insulin receptor were incubated with concentrations of insulin over the range 0.007-7.0 nM. Insulin stimulated a loss of label associated with the PI-glycan band in each cell line. Similar maximal effects of ¢=
1
1
o
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0.007 0.07 07 Inlulln nononntmtlon (nM)
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Fig. 2. Concentration.response to insulin of PI-glycan breakdown. CHO (e), CHO.T ( • ) and CHO.TH ( A ) cells prelabelled to equilibrium with ['~Hlglucosamine were incubated with insulin at the concentratlons indicated, as desrlbed in the legend to Fig. I, except that incubations were carried out for ! rain. Results expressed as a percent of the respective basals are given as the means + S.E. of four or more experiments, in which the values for each point were determined in three independent cell incubations. The basal level of Pl-glycan did not differ in the three cell lines within any single experiment from cell line to cell line although its labelling in the three lines varied from experiment to experiment similar to that described in the legend to Fig. I (Range 24697-66244 dpm/mg protein). Significant differences compared with Pl-glycan extracted from cells incubated in the absence of insulin are indicated *. Stimulation of Pl-glycan breakdown in CHO.TH cells did not differ significantly from that of CHO.T cells at 0.007 nM insulin (O.l < P > 0.05) nor at higher insulin concentrations. Pl-glycan breakdown was significantly greater (P < 0.01) in CHO.TH cells than CHO cells at 0.007 nM and 0.07 nM insulin (confirmed by Bonfcrroni analysis). The CHO.T cell response was greater than the C l i O cell response at 0.07 nM and 0.7 nM insulin ( P < 0 . 0 0 I and P < 0.05, respectively).
~
100
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100
~eu
2
3
4
5
6
TIME (rain) Fig. I. Time-course of the effect of insulin on Pl-glycan turnover. CHO (e) and CHO.T (11) cells were prelabelled with ['~H]81ucosamine as described in Materials and Methods and taken into fresh serum free medium containing 1% bovine serum albumin for the final 30 min. Cell stimulations with 0.7 nM ;nsulin were carried out for the time course indicated. Incubations w e r e staggered so that each cell incubation was carried out for a similar length of time. Results expressed as a percent of basal represent the mean+S.E, of three experiments within which each point was determined in three independent cell incubations. The basal level of [3HJglucosamine incorporation into the PI-glycan band was similar for both cell lines but differed from experiment to experiment (range 48-171 dpm/nmol total lipid phosphorus, 2~0t9-49353 dpm/mg protein) due probably to differences in labelling times and to differences in the specific activity of the labelling medium. Significance at the 0.01 level compared with culls not stimulated with insulin is indicated **.
insulin were observed in each cell line, with breakdown of 14-21% of the initial label associated with the precursor band being achieved. As shown in Fig. 2, significant stimulation of loss of label associated with the Pl-glycan band occurred at 0.007 nM with the CHO.TH cells ( P < 0.05 after Bonferroni correction for multiple comparisons compared to the parent cell line) and significant and maximal breakdown occurred with both transfected cell lines (CHO.T a~td CHO.TH) at 0.07 nM insulin, a concentration that was ineffective in causing loss of label in the parent cell line (values for both CHO.T and CHO.TH were significantly different from CHO cells at this concentration, P < 0.05 after Bonferro~i correction). Significant loss of label from the parent cell line was not observed until 100 fold higher concentration (7 nM) of insulin was used (Fig. 2). There was no difference in the basal level of labelling of the Pl-glycan measured in each of the three cell lines confirming the data obtained in Fig. 1 and suggesting that the level of insulin receptor expression has little or no effect on the cellular level of Pl-glycan in these CHO cell lines. The insulin concentration response curve of Pl-glycan breakdown in CHO.T and CHO cells labelled to equilibrium with
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Insulin concentration(nM) Fig. 3. Concentration-response to insulin of glucose transport. CHO (o), CHO:I" (11) and CHO.TH ( • ) cells incubated as described in Materials and Methods were deprived of serum overnight and then incubated in Krebs-Ringer bicarbonate Hepes buffer, 1% bovine serum albumin, 0.1 mM glucose (pH 7.4) for 20 rain prior to the addition of insulin at the concentzations indicated for 30 rain. 2-Deoxyglucose uptake was then determined over the next 5 mio. Results are expressed as the percent of the respective basal transport rates and are the mean±S.E, of three or more experiments in which the values for each point were determined in three independent cell incubations. Basal glucose transport levels in the three cell lines did not differ and were 486±27 pmol/mg protein per rain (CHO), 590+39 pmol/mg protein per rain (CHO.T), 434±20 pmol/mg protein per rain (CHO.TH). Significant differences compared to cells incubated in the absence of insulin are indicated *. The stimulation by 0.007 nM and 7 nM insulin was statistically greater in CHO.TH cells than in CHO.T celts ( P < 0.05). After Bonferruni correction for multiple comparison the statistical significance at 7 nM insulin was eliminated.
0.007 0.07 mul~ ~
0.7 (n~
7.0
Fig. 4. Concentration-response to insulin of glucose utilization. CHO (o), CHO.T (11) and CHO.TH ( - ) cells were pre-ineubated as described in the legend to Fig. 2 except that they were incubated in the absence of [3H]glacosamioe. Following transfer into fresh serum frec medium containing I% bovine serum albumin, the cells were incubated in the presence or absence of insulin at the indicat:d concentrations for 10 rain. [5-3H]glucose was added for the next 30 rain, after which the medium was removed for determination of glucose utiih.,,ltion as described in Materials and Methods. Results, exFrc~cd as the percent of basal glucose utilization rate. are given as the mean± S.E. of six or more experiments in which three independent cell incubations were performed within each experiment. Basal glucose utilization rates did not differ between the three cell lines and were 3112:L 183 pmol/mg per mio (CHO), 3296± 183 pmol/mg .')er rain (CHO.T) 2918±203 pmoi/mg per min (CHO.TH). Significance from the appropriate basal for each point in each cell line is indicated *.
[3H]inositol w a s s i m i l a r t o t h a t s h o w n in F i g . 2 f o r [ 3 H ] g l u c o s a m i n e cells ( d a t a n o t s h o w n ) .
Effect of insulin on glucose transport The effect of insulin on glucose transport was studied using 2-deoxyglucose uptake to assess transport r a t e in t h e t h r e e cell l i n e s ( F i g . 3). I n s u l i n s t i m u l a t e d g l u c o s e t r a n s p o r t in e a c h o f t h e t h r e e cell lines, w i t h e f f e c t s o n t h e C H O . T H cell line b e i n g a p p a r e n t a t t h e l o w e s t c o n c e n t r a t i o n o f i n s u l i n t e s t e d (0.007 riM), e f f e c t s o n t h e C H O . T line e v i d e n t a t 0.07 n M , a n d e f f e c t s o n t h e C H O cell line a t 0.7 n M . B a s a l g l u c o s e t r a n s p o r t activity in t h e t h r e e cell l i n e s d i d n o t d i f f e r s i g n i f i c a n t l y ( s e e t h e l e g e n d t o F i g . 3). A l t h o u g h t h e C H O . T H cells s h o w e d a s i g n i f i c a n t l y d i f f e r e n t m a x i m u m r e s p o n s e ( c o m p a r e d t o t h e p a r e n t C l I O cell l i n e ) at the highest insulin concentration tested, the res p o n s e o f t h e C H O . T cell line w a s i n t e r m e d i a t e b e t w e e n t h e t w o o t h e r cell l i n e s a n d d i d n o t d i f f e r significantly from either.
Effects of insulin on glucose utilization T h e e f f e c t o f i n s u l i n o n g l u c o s e utilization, m e a sured as 3H20 production from [5-3H]glucose, was d e t e r m i n e d f o r t h e t h r e e cell l i n e s o v e r t h e s a m e concentration range as the PI-glycan and glucose trans-
0~
0.007
0.07
0.7
7.0
ImmlM ,~,,-,¢a,-,,'-ati~ nil Fig. 5. Concentration-response to insulin of pyrovate debydrogenase activation. The relationship between insulin concentration and 'aclive" PDH activity in the CHO, CHO.T and CHO.TH cell lines was determined in the same cells in which effects on 81acose utilization were measured (Fig. 4). Following removal of the medium for determination of glucose utilization, a 'cocktail" for determination of PDH activity was added to the cells. "Active" and "total" PDH activities were determined as described in Materials and Methods, "Total" PDH activity did not differ over the range of insulin concentrations tested, or from cell line to cell line and was 1079±269 pmol/mg per rain (CHO), 1376± 223 pmol/n,,8 per rain (CHO.T) and 1074+ 118 pmol/mg per rain (CHO.TH). Basal 'active' PDH also did not differ from cell line to cell line and was 125:t: 17 pmol/mg per rain (CHO) 97± 16 pmol/mg per rain (CHO.T) and 121 + 13 pmol/mg per rain (CHO.TH). Results are expressed as the percent of basal 'active" PDH for each cell line and are the meand:S.F-., of six or more experiments within which three independent cell incubations were performed and the PDH activity of each incubation determined in duplicate. Significance from the appropriate basal for each point in each cell line is indicated *.
58 port responses (Fig. 4). Significant stimulation of glucose utilization was obtained in both the CHO.T and CHO.TH cell lines at all concentrations of insuli,i tested with no statistical difference in the response of the two insulin receptor transfected lines. Insulin stimulation of glucose utilization in the CHO cell line was achieved at a !.00-fold higher concentration of insulin (0.7 nM), similar to the concentration at which significant effects on glucose transport were achieved (Fig. 3). Consistent with the previous data there was also no difference in the basal level of glucose utilization in each of the three cell lines (see legend to Fig. 4).
Effects' of insulin on pyruvate dehydrogenase The effect of insulin on PDH activity was examined in the three cell lines using the same cells in which the glucose utilization data were obtained (Fig. 5). Consistent with the glucose utilization data there was no significant difference between the C H O . T and CHO.TH cell lines with respect to the PDH response to insulin. Significant stimulation of PDH in both the CHO.T and CHO.TH cell lines was achieved at 0.07 nM insulin, whereas a significant response to insulin in the parent CHO cell line was only achieved at a 100-fold higher concentration of insulin (7 nM). Consistent with each of the other responses of the ceils to insulin, there was no difference between the three cell lines in either basal or total PDH activities (Fig. 5). Discussion
The present study correlates, for the first time in the one investigation, the effect of different levels of insulin receptor expression in a single cell type with insulin-induced breakdown of Pl-glycan, PDH activation and stimulation of glucose transport and utilization. In doing so, it establishes that the predominant effect of altered cell surface insulin receptor expression is to alter the sensitivity to insulin of breakdown of the Pl-glycan, PDH activation and stimulation of glucose transport and utilization. To our knowledge this is the first study attempting to correlate the effect of increasing insulin receptor expression with insulin activation of glucose utilization. Insulin receptor expression has been correlated with PDH activation in one other study [2.~] and the breakdown of the PI-glycan likewise in only one other study [17]. This study [17] established that a normal insulin receptor, containing a functional tyrosine kinase, is required for insulin-stimulated breakdown of the molecule. Comparison of insulinstimulated Pl-glycan breakdown was drawn between cells that either expressed high or low levels of insulin receptors or kinase deficient receptors, but no comparison was drawn to other actions of insulin. In the presem study, two cell lines expressing different receptor levels, derived from the same parent line, were
used to compare insulin actions. The parent cell line, expressing approx. 3.103 insulin receptors/cell, was compared to two stable cell lines transfeeted with human insulin receptor eDNA. One cell line designated CHO.T, expressed approx. 300-fold more insulin receptors than the parent fine. The other cell line designated CHO.TH, was generated by FACS sorting the CHO.T cell line and expressed approx. 2-fold more receptors than the CHO.T cell line. Initial studies established the presence of an insulin-sensitive PI-glycan in the CHO cell lines with characteristics similar to those we and others have described previously in other cell types [1-8]. The PI-glycan extracted from CHO cells in the present study separated in a similar manner to that extracted in a past CHO cell study [15] as well as that extracted from other ceil types, including adipocytes [8,20], H35 hepatoma cells [3,5] and T ceils t6]. As in other cell types, the PI-glycan extracted from CHO cells in the present study could be labelled with glucosamine, inositol and galactose, supporting the presence of these sugars within the Pl-glycan structure in these ceils. Most importantly the Pl-glycan was sensitive to insulin, which stimulated loss of approx. 20% of the label associated with the molecule in each of the three cell lines, indicating that insulin stimulated its breakdown. This maximum breakdown of PI-glycan of 20% in our study is consistent with the level of responsiveness of the C H O cells (a fibroblastic cell) to insulin (i.e., 2.3fold stimulation of PDH, Fig. 5), which although significant, is lower than the responsiveness of classical insulin target tissues such as fat (30% breakdown of Pl-glycan and 3-4-fold or greater effects on PDH, see for example Refs. 8,20,24). Our data therefore suggest that it is not purely the insulin receptor level that determines responsiveness of cells to insulin but that the breakdown of Pl-glycan is also a function of the cell line in which these receptors are expressed. The effects of insulin concentration on Pl-glycan breakdown and other actions of insulin, including PDH activation, glucose transport and utilization were compared in order to assess the potential effects of the level of cell surface insulin receptors on post-receptor insulin action in the CHO cell lines. Significant response in the parent CHO cell line (expressing 300-fold fewer insulin receptors than the CHO.T cell line) was obtained only at the higher concentrations of insulin tested, 0.7 and 7 nM. Significant response of the CHO.TH cells (expressing twice the number of insulin receptors as CHO.T cells) was obtained at insulin concentrations either not different to CHO.T cells (PDH activation (0.07 nM insulin) and stimulation of glucose utilization (0.007 nM insulin)) or the next lowest concentration tested (Pl-glycan breakdown and glucose transport (0.007 nM insulin)) although the sensitivity of the PI-glycan response obtained with the
CHO.TH cell line were not statistically different from those obtained with the CHO.T cell line after Bonferroni correction for multiple comparisons. The effects of insulin on the CHO.TH cell line, however, reinforce the results obtained with the CHO.T cell line. The data taken together clearly indicate that the major effect of altered insulin receptor expression was to alter the sensitivity of the cells to insulin. There was no difference in any of the measured parameters between the cell lines under ba~al or noninsulin stimulated conditions. In addition to the increased sensitivity of the cellular responses to insulin with increased insulin receptor expression, the glucose transport and glucose utilization measurements indicated a tendency for increased responsiveness of the cells to insulin. As the glucose transport rate is a major factor in determining the rate of glucose utilization, and in many cases limits glucose utilization, the findings for glucose transport effects support the likelihood that altered insulin receptor expression modifies slightly the responsiveness of glucose transport to imu!in in addition to affecting the sensitivity of responst, in contrast to the observed effects on PDH a.-.d Pl-glycan breakdown. The present report supports previous studies [6,9-528] in both Rat-I cells and ClIO cells that suggest that insulin sensitivity is proportional to the number of active iusuli~,~ receptors. These other investigations demonstrate~! increased sensitivity of response to insulin on $6 kinase activation, glycogen synthase activation, thymidine uptake and 2-deoxyglncose uptake in insulin receptor transfected cells. The present study extetio's these insulin effects to include glucose utilization, pyruvate dehydrogenase activation and breakdown of Pl-glycan. In the one previous study that examined the Pl-giycan response in a different population of CHO cells transfected with insulin receptors [17] it was found that the responsiveness, rather than the sensitivity, to insulin was increased following transfection of cells with insulin receptors in contrast to the sensitivity change observed in the present study. The reason for this difference in the manifestation of the effect of increased cell surface insulin receptor expression is unclear. However, a positive effect of increased expression of insulin receptors on insulin responsiveness would be predicted if there were no spare functional insulin receptors [29]. In the present study comparison of receptor occupancy with each biological response for the transfected cell lines indicated the presence of functional spare receptors, thus maximal biological activation was achieved when 15-20% of receptors were occupied. The parental CHO cell line reported in the previous study expressed fewer insulin receptors than those used in the present study. This difference may explain the differences observed in the manifestation of effect on Pl-glycan breakdown al-
though in the previous study one might also have expected sensitivity effects in addition to the observed effects on responsiveness. The present studies showing predominant effects on insulin sensitivity, rather than responsiveness, suggest that even in the non-trausfected CHO cells the number of insulin receptors are sufficient to fully stimulate Pl-glycan breakdown and subsequent steps that may be dependent on this process (such as PDH activation). The present studies showing predominant effects of insulin receptor level on the sensitivity of Pl-glycan breakdown are consistent with the other effects of insulin receptor expression on :nsulin action we observed in the present study and those that have been previously reported in the literature [6,26-28]. In conclusion, the present study provides further evidence to support the potential relevance of the Pl-glycan in insulin action by correlating the expression of cell surface insulin receptors with cellular sensitivity to insulin of its breakdown and of other classical actions of insulin, namely PDH activation and stimulation of glucose utilization and transport. The exact contribution of the breakdown of the Pl-glycan to overall cellular seusitivtiy and responsiveness to insulin remains to determined and will require full characterization of the molecule. .Acknowledgements This work was supported by grants from the National Health and Medical Research Council, Eli Lilly, Australia, and a grant-in-aid from the Diabetes Australia Research Trust. The authors are grateful to Dr. M. Low for the gift of B. thuringienesis phosphatidylinositol specific phuspholipase C, to Dr. Leland Ellis for the CHO.T cell line used in this study and to Dr. Ken Siddle for insulin receptor antibody 83-14. The excellent technical assistance of Mr. Warwick Atkinson was greatly appreciated.
References I Saltiel, A. and Cuatrecasas,P. (1~6) Proc. Natl. Acad. Sci. USA 83, 5793-5795. 2 Sahiel,A.R., Sherline,P. and Fox,J.A. (1987)J. Biol.Chem.262, 1116-1121. 3 Mato, J.M., Kelly, K.I.., Abler, A. and Jarett. L (1987) J. Biol. Chem. 262, 2131-2137. 4 Farese, R.V., Cooper, D.R., Konda, T.S., Hair, G., Standaert, M.L. and Pullet. R.J. (1988)giochem.J. 256,185-188. 5 Witter~ L.A. and Watts, "I.D. (1988) J. Biol. Chem. 263, 80278036. 6 Gaulton.G.N., Kelly, K.L., Pawiowski.J., Mato, J.M. and Jaren, L (1988) Cell 53, 963-970. 7 Romero, G., Lunrell, L, Rol;ol, A., Seller, K.. Hewlen, E. and Lamer, J. (1988)Science. 240,509-511. 8 Macaulay, S.L. and Latkins, R.G. (1990) Biochem. J. 271, 427435.
9 Standaert, M.L., Farese, R.V., Cooper, D.R. and Pollet, RJ. (1988) J. Biol. Chem. 263, 8696-8705. 10 Salt/el, A.R. (1987) Endocrinology 120, 967-972. 11 Kelly, K.[, Mato, J.M. and Jarett, L. (1986) FEBS Lett. 209, 238-242. 12 Alemany, S., Mato, J.M. and Stralfors, P. (1987) Nature 330, 77-79. 13 Stralfors, P. (1988) Nature 335, 554-556. 14 Low, M.G. and Saltiel, A.R. (1988) Science 239, 268-275. 15 Low, M.G. (1989) Biochim. Biophys. Acta 988, 427-454. 16 Thakkar, J.K., Raju, M.S., Kennington, A.S.~ Foil, B. and Caro, J.F. (1990) J. Biol. Chem. 265, 5475-5481. 17 Villalba, M., Alvarez, J.F., Russell, D.S., Mato, J.M. and Rosen, O.M. (1990) Growth Factors 2, 91-97. 18 Ellis, L., Clauser, E., Morgan, D.O., Edew, M., Roth, R.A. and Rutter, WJ. (1986) Cell 45, 721-732.
19 Soos, M.A., Siddle, K., Baron. M.D., Heward, J.M., Luzio, J.P., Bcllatin, J. and Lennox, E.S. (1986) Biochem. J. 235,199-208. 20 Macaulay, S.L. and Larkins, R.G. (1990) Cell Sig. 2, 9-19. 21 Macaulay, S.L. and Jarctt, L. (1985) Arch. Biochem. Biophys. 237, 142-150. 22 Jarett, L. (1974) in Methods in Enzymol. 31, 60-71. 23 Olefsky, J.M. (1978) Biochem. J. 172,137-145. 24 Macaulay, S.L. and Larkins, R.G. (1988) Metabolism 37, 958-965. 25 Gottschatk, W.K. (1991) J. Biol. Chem. 266, 8814-88119. 26 Chou, C.K., Dull, T.J., Rusell, D.S., Cherzi, R., Lebwohl, D., Ullrich, A. and Rosen, O.R. (1987) L Biol. Chem. 262,1842-1847. 27 McClain, D.A., Maegawa, H., Ice, J., Dull, T.J., Ullrich, A. and Olefsky, J.M. (1987) J. Biol. Chem. 262,14663-14672. 28 McClain, D.A. (1990) Diabetes Care 13, 302-316.
53
© 1992ElsevierScience PublishersB.V. All rightsreserved0167-4889/92/$05.00
BBAMCR 13097
Correlation of insulin receptor level with both insulin action and breakdown of a potential insulin mediator precursor; studies in CHO cell-lines transfeeted with insulin receptor eDNA S. L a n c e M a c a u l a y a,c, Steila C l a r k a,b a n d R i c h a r d G . L a r k i n s ~ a Melbourne University Department of Medicine and b Busnet Clinical Research Unit, Walter and Eliza Hull Institute, Royal Melbourne Hospital. Melbourne (Australia) and c Commonwealth Scientific and Industrial Research O~,anization, Di~'ision of Biomolecular Engineering, Parkville (Australia)
(Received 23 May 1991) (Revised manuscriptreceived21 August1991)
Key words: Insulinreceptor;InsulinmediatorprecursorPl-glycan;Pyrovatedehydregenase;Glucosetransport A phosphatidylinositol-glycan(Pl-glycan) has been described previously that may set~e as the precursor for a mediator of some of insulins actions. The present study further addresses the potential relevance of this compound by correlating its breakdown with other insulin actions in Chinese hamster ovary (CLIO) cells which express different levels of insulin receptor. Comparisons were drawn between parent ClIO cells expressing 3-103 receptors/cell and two cell-lines tranxfected with human insulin receptor eDNA, that expressed 600 (CHO.TH) and 300 (CHO.T) times the parent receptor level. A Pl-glycan was isolated from all cells that incorporated [3H]glucosemine,[3Hlaalactuse and [3H]inositol and was rapidly turned over upon insulin stimulation. Maximal turnover by insulin of approx. 20% was achieved in each cell line consistent with the fibroblastic nature of these cells. The effect of increased insulin receptor expression was to increase the sensitivity of the Pl-glycun response to insulin. Increasing receptor number from 3.103 to 0.88.106 also increased the sensitivity of response of other insulin actions measured in this study, namely activation of pyruvate dehydrogenase (PDH), glucose utilization and transport. Thus turnover of the Pl-glycan is linked closely to both metabo~,ic actions of insulin and to cell surface insulin receptor expression further supporting its potential role in insulin action.
Introduction Insulin has been shown previously to stimulate the cleavage of a PI-glycan in a variety of cell types [1-9]. The head group generated by phospholipase mediated cleavage of this molecule has been shown to mimic several, but not all, actions of the hormone when added to in vitro enzyme systems or to intact cells [1-3,5,6,8,9-12]. The diacylglycerol generated with the head group has been postulated to contribute to the mechanism of insulin-stimulated glucose transport [13].
Abbreviations: PI, phusphatidylinositol; CHO, Chinese hamster ovate; PDH, pyravate dehydrogenase; PLC, phospholipaseC; TIC, thin-layer chromatography. Correspondence: S.L. Macaulay, CommonwealthScientific and Industrial Research Organization, Divisionof BiomolecularEngineering, 343 Royal Parade, Parkville, Victoria, Australia3052.
The ability of this head group and diacylglycerol to mimic some of the actions of the hormone has led to the proposal that the Pl-gl~an is the precursor of a mediator for some of the hormone's intracellular actions (see Ref. 14 for review), although this is not uniformly accepted [15,16]. in a previous study [17], it was established that the tyrosine kinase activity of the insulin receptor was necessary for insulin-stimulated breakdown of the PIglyean consistent with most, if not all, other actions of the hormone. The present study further addresses the potential contribution of this molecule to insulin action by correlating its breakdown foliowing insulin treatment with the level of cell surface insulin receptor expression and with other actions of insulin. Although effects of insulin on some insulin actions have been previously described in celis expressing different levels of insulin receptor, the present study is the first to correlate PI-glycan breakdown with these actions and to describe effects of increased insulin receptor expres-
54 sion on PDH activation and glucose utilization. These studies were performed in CHO cells which although not highly insulin responsive, because of their fihroblastic nature, are one of the few cell lines in which it has been possible to stably express different levels of insulin receptors. Materials and Methods
Materials D-[6-3H]Glucosamine hydrochloride, D-[G-~H]galactose, D-[5-3H]glucose and [1-J4C]pyruvic acid were obtained through DuPont from New England Nuclear, Boston, MA. myo-[2-3H]lnositol, [9,10(n)-3H]myristic acid, [9,10(n)-3H]palmitie acid and 2-deoxy-D-[U14C]glucose were from Amersham International, U.K. Purified phosphatidylinositol-specific phospholipase C (PI-PLC) from B. thuringiensis was a generous gift from Dr. M. Low, Columbia University, New York and was used at an activity dilution of 2.5-5 p.mol/min per ml estimated using [3H]phosphatidylinositol as substrate at pH 7 in the presence of 0.1% deoxTcholate. Preparations tested for proteinase activity were below detectable limits ( < 2.5 ng/25 U PI-PLC, M. Low, personal communication). Materials for cell culture were obtained from Flow Laboratories, McLean, VA, except for alpha modified minimum essential medium which was obtained from the Commonwealth Serum Laboratories, Parkville, Australia. Silica gel G plates for thin-layer chromatography (TLC) were from Whatman, Clifton NJ. Porcine insulin and most other chemicals were from Sigma, St. Louis, MO. Chinese hamster ovary (CHO) cell lines The CHO cell lines used in the present study were derived from ceils obtained from Dr. Leland Ellis (Howard Hughes Medical Inst., Dallas, TX). The parent CHO cell line expressed approx. 3.103 insulin receptors/cell. Two other stably transfected cell lines were used. The first, Ellis and co-workers termed CHO.T [18], was derived from cells transfected with human insulin receptor eDNA. These cells have been well characterized previously [18] and in our hands expressed approx. 0.88.10 ° insulin receptors/cell. The third cell line was derived from the CHO.T cell line in our studies by indirect immunolabelling and fluorescence-activated cell sorting (FACS). CHO.T cells were labelled with an anti-insulin receptor antibody 83-14 (obtained from Dr. Ken Siddle, Cambridge University, U.K. [19]) directed towards the a-subunit of the insulin receptor at a 1:100 dilution, followed by a second F1TC labelled goat anti-mouse antibody at a 1:50 dilution. Two sorts were performed to enrich those cells expressing human insulin receptors. In the first sort the top 10% of insulin receptor expressing cells were collected and expanded. These cells were re-
sorted, the top 10% of insulin receptor expressing cells collected and a pure cell line obtained by limiting dilution of these FACS sorted cells. This cell line, designated CHO.TH, expressed approx. 1.6" 106 insulin receptors/cell. Each of the three cell lines were grown to confluence from a 1:10 dilution in a-modified minimum essential medium (a-MEM), 5% fetal calf serum, 100 U / m i penicillin, 100 i,t g / m l streptomycin and 1/~g/ml fungizone (Squibb) and 10 /.tCi/ml tracers (D-[63H]glucosamine hydrochloride, D-[G-3H]galactose, and myo-[2-3H]inositol or [9,10(n)-3H]myristic acid), as required if precursor Pl-glycan was to be examined, in 6 well dishes (Nunc). When the cells reached approx. 80% confluence (48 h), the medium was removed and replaced with a-MEM, 1% bovine serum albumin (BSA) and tracer (at the same specific activity) as appropriate for the final 16 h by which time the cells were confluent. The medium was then removed and replaced with 1 ml fresh medium (a-MEM, 1% BSA, or Krebs-Ringer bicarbonate Hepes buffer (pH 7.4) containing half the normal Ca 2+ concentration (1.15 mM) (for glucose transport determination)). Cell stimulations with insulin were then carried out after a 20 min equilibration period. Insulin mediator precursor Pl-glycan oreakdown Cells labelled to equilibrium with [3H]glucosamine, or the other tracers as indicated, were extracted for glucosyl-lipids using a similar scheme to that employed previously for H35 cells, splenic T cells and adipocytes [3,6,8,20]. After the 20 rain equilibration period, the cells were stimulated with insulin for the appropriate time (1 min unless otherwise stated) and reaction stopped by the addition of 1 mi of methanol at 0°C. Control incubations were performed for the same period of time. The cells were then scraped, transferred into extraction tubes and the wells washed with a further 0.5 ml of methanol. 3 ml of chloroform was added to the extraction tubes followed by 1.5 ml of chloroform/methanol/HCI (200:100: 6). Phase separation was then induced by the addition of 1.5 ml of 0.1 M KCI. The organic phase was collected and the aqueous phase washed with a further 1 ml of chloroform. The Pl-glycan was then purified from the pooled organic phase by thin-layer chromatography (TLC) in two solvent systems [3,6,8]. It remained at or near the origin following TLC in chloroform/acetone/methanol/acetic acid/water (50:20:10:10:5) (solvent system I), in which it was separated from the major phospholipid classes, and following elution with methanol and TLC in c h l o r o f o r m / m e t h a n o l / ammonia/water (45 : 45 : 3.5 : 10) (solvent system !I) on oxalate impregnated plates, ran with an R F of approx. 0.5. This procedure, in our hands, yielded preparations of PI-glycan free of major contaminants although con-
55 taminants have been reported using this procedure in a previous study in liver microsomes [16]. Radiolabelled lipid and standard bands were located by autoradiography of TLC plates sprayed with Enhance R (New England Nuclear), and were quantified, following scraping of the bands, by liquid scintillation counting. Parallel wells were processed for measurement of cell protein and all results were corrected for differences between cell-lines. Several criteria were used to establish the band as the Pl-glycan we and others have described for other cell types [1-8]. First it was confirmed that the majority of the added tracer in the labelled band remained as the native sugars. In contrast to our earlier fat cell studies in which there was significant turnover of the added tracers during labelling [8], the majority of the added tracers remained as the native sugars in the CHO cell lines. After acid hydrolysis of the Pl-glycan band in the CHO cells used in the present study and TLC of the component sugars as in Refs. 3,8, we found that 65 + 3% of the added [3H]glucosamine tracer remained as the native sugar. Even less inositol and galactose were metabolized, with 82% and 71% remaining as the native sugars, respectively (data not shown). Phosphatidylinositol-specific phospholipase C digestion of the band extracted from [3H]glncosamine labelled cells, as in Refs. 3,8, resulted in a loss of 45% of the label associated with the band (data not shown), consistent with previous studies [3,6,8]. Furthermore, phosphatidylinositol-specific phospholipase C digestion of the band as in Ref. 8 generated a material that had a stimulatory effect on PDH activity of adipocyte mitochondria (Basal PDH activity = 0.694 + 0.072 n m o l / m g per rain, control (extract prepared in the absence of Pl-glycan band) --- 0.744 + 0.072 n m o l / l / m g per rain, Pl-glycan= 1.06 4-0.054 n m o l / m g per min (n = 3)). These data taken together all support the contention that the band extracted was a Pl-glycan similar to that described in previous studies [1-8]. Data given in Results support its insulin sensitivity.
Other assays of insulin action Glucose utilization was determined as the conversion of [5-~H]glucose to 3H20 as we have described previously for adipocytes [8,20]. Each well in 1 ml of a-MEM, 1% BSA (pH 7.4) was incubated in the presence or absence of insulin for 10 rain followed by 4 ~Ci [5-3H]glucose for an additional 30 min. 3H20 in the medium was then measured after absorption into CaCI 2. l~jruvate dehydrogenase (PDH) activity was measured in the same cells after removal of the medium washing cells in phosphate-buffered saline and extraction of the cells with 0.2% Triton X-100, 2 mM dithiothreitol, 2 mM EDTA, 2 mM EGTA, 50 mM potassium phosphate buffer (pH 7.4) (PC as described in Refs. 20-22. Each well was scraped and cellular sus-
pensions vortexed three times 10 s, if'C, and centrifuged 5 s, 4°C, at 10000 x g. PDH activity was determined in the supematant fractions by the 14COz release from [ 1-14C]pyruvate assay [20-22]. "Active" PDH was determined in the presence of 50/zM MgCI 2 and 5 0 / z M CaCI 2. 'Total' PDH was determined following preincubation of aliquots with 20 mM MgCI 2 and 0.5 mM CaCI 2 for 30 rain. Parallel wells were processed to enable results to be corrected for protein. 2-Deoxy-glucose was used as the index of glucose transport and was measured as in Refs. 23,24. Briefly, wells were washed twice in Krebs-Ringer bicarbonate Hepes buffer, 1% bovine serum albumin, 0.1 mM glucose, 2 mM pyruvate (pH 7.4) and the cells allowed to equilibrate in the incubator for 20 rain. Cells were then incubated in the presence or absence of insulin for 30 min and assay then initiated by the addition of 0.1 mM 2-deoxy-[U-*4C]glucose (0.25 p.Ci tube) for a further 5 min. Assay was terminated by washing the cells three times in phosphate-buffered saline at if'C, with the final addition of I ml of 1 M NaOH. 0.6 ml was taken for determination of 2-deo~glncose uptake and the remainder used for protein determination.
Statistical analyses Statistical analyses were performed using the Student's t-test. Results are expressed as the mean + S.E. Statistical significance was determined at the 0.05 level and is indicated in the results. Results
Effect of insulin on Pi-glycan breakdown Initially, the kinetics of insulin effects on the level of Pl-glycan, determined as [3H]glucosamine label associated with the Pl-glycan band after TLC of lipid extracts of insulin-treated cells was examined (Fig. 1). Cells were labelled to equilibrium with [3H]glucosamine to achieve steady state labelling of Pl-glycan, and then incubated in fresh medium for the next 20 rain prior to insulin stimulation. Insulin, at 0.7 nM, caused a rapid 10ss (17%) of [3H]glucosamine associated with the Pl-glycan band in the CHO.T cell line (Fig. 1) that was maximal at 1 rain and transient in nature. These studies establish that insulin stimulates breakdown of the Pl-glycan in CHO.T cells. In additional experiments (data not shown) in which other time-points were examined, it was apparent that there was variability between experiments over the point at which maximal effects were obtained (30 s - i rain). The reversibility of the response was also variable as has been described for other cell types. In some experiments labelling of the Pl-glycan returned towards basal at 5 rain whilst in others the response was still maximal at this time. The standard error bars for the 5 rain point in Fig. I reflect this fact. In contrast to the
56
CHO.T cells, no significant loss of [3H]glucosamine label associated with the Pl-glycan band was found in CHO cells following stimulation with this low concentration of insulin (0.7 nM) over the same time-course. This was despite the similar labelling of the Pl-glycan band in both cell types under basal conditions. In order to strengthen the contention that an insulin-sensitive Pl-glycan was extracted from within the PI-glycan band in CHO.T cells after TLC and that the observed effects were not a result of potential contaminants in the preparations, other potential sugars associated with this band were examined. Insulin caused a loss of 22.3 ± 5.5% (n = 3), 37.3 + 4.9% (n = 3) and 18.2 + 2.0% (n = 21) in the amount of galactose, inositol and glucosamine respectively associated with the Pl-glycan band implicating these sugars in Pl-glycan structure in the CHO cell lines, similar to previous studies in other cell types [2,3,6,8]. The time-course study (Fig. 1) established that an insulin-sensitive PI-glycan could be extracted from CHO cell lines and that the insulin effect was rapid. Further studies sought to establish the effect of increased cell surface insulin receptor expression on insulin sensitivity/responsiveness to PI-glycan breakdown (Fig. 2). The three cell lines expressing different levels of insulin receptor were incubated with concentrations of insulin over the range 0.007-7.0 nM. Insulin stimulated a loss of label associated with the PI-glycan band in each cell line. Similar maximal effects of ¢=
1
1
o
1
9o
i~so °0¢'~
0.007 0.07 07 Inlulln nononntmtlon (nM)
7.0
Fig. 2. Concentration.response to insulin of PI-glycan breakdown. CHO (e), CHO.T ( • ) and CHO.TH ( A ) cells prelabelled to equilibrium with ['~Hlglucosamine were incubated with insulin at the concentratlons indicated, as desrlbed in the legend to Fig. I, except that incubations were carried out for ! rain. Results expressed as a percent of the respective basals are given as the means + S.E. of four or more experiments, in which the values for each point were determined in three independent cell incubations. The basal level of Pl-glycan did not differ in the three cell lines within any single experiment from cell line to cell line although its labelling in the three lines varied from experiment to experiment similar to that described in the legend to Fig. I (Range 24697-66244 dpm/mg protein). Significant differences compared with Pl-glycan extracted from cells incubated in the absence of insulin are indicated *. Stimulation of Pl-glycan breakdown in CHO.TH cells did not differ significantly from that of CHO.T cells at 0.007 nM insulin (O.l < P > 0.05) nor at higher insulin concentrations. Pl-glycan breakdown was significantly greater (P < 0.01) in CHO.TH cells than CHO cells at 0.007 nM and 0.07 nM insulin (confirmed by Bonfcrroni analysis). The CHO.T cell response was greater than the C l i O cell response at 0.07 nM and 0.7 nM insulin ( P < 0 . 0 0 I and P < 0.05, respectively).
~
100
°0[
100
~eu
2
3
4
5
6
TIME (rain) Fig. I. Time-course of the effect of insulin on Pl-glycan turnover. CHO (e) and CHO.T (11) cells were prelabelled with ['~H]81ucosamine as described in Materials and Methods and taken into fresh serum free medium containing 1% bovine serum albumin for the final 30 min. Cell stimulations with 0.7 nM ;nsulin were carried out for the time course indicated. Incubations w e r e staggered so that each cell incubation was carried out for a similar length of time. Results expressed as a percent of basal represent the mean+S.E, of three experiments within which each point was determined in three independent cell incubations. The basal level of [3HJglucosamine incorporation into the PI-glycan band was similar for both cell lines but differed from experiment to experiment (range 48-171 dpm/nmol total lipid phosphorus, 2~0t9-49353 dpm/mg protein) due probably to differences in labelling times and to differences in the specific activity of the labelling medium. Significance at the 0.01 level compared with culls not stimulated with insulin is indicated **.
insulin were observed in each cell line, with breakdown of 14-21% of the initial label associated with the precursor band being achieved. As shown in Fig. 2, significant stimulation of loss of label associated with the Pl-glycan band occurred at 0.007 nM with the CHO.TH cells ( P < 0.05 after Bonferroni correction for multiple comparisons compared to the parent cell line) and significant and maximal breakdown occurred with both transfected cell lines (CHO.T a~td CHO.TH) at 0.07 nM insulin, a concentration that was ineffective in causing loss of label in the parent cell line (values for both CHO.T and CHO.TH were significantly different from CHO cells at this concentration, P < 0.05 after Bonferro~i correction). Significant loss of label from the parent cell line was not observed until 100 fold higher concentration (7 nM) of insulin was used (Fig. 2). There was no difference in the basal level of labelling of the Pl-glycan measured in each of the three cell lines confirming the data obtained in Fig. 1 and suggesting that the level of insulin receptor expression has little or no effect on the cellular level of Pl-glycan in these CHO cell lines. The insulin concentration response curve of Pl-glycan breakdown in CHO.T and CHO cells labelled to equilibrium with
180 ~.
",,g o~
°oe'~m °wca 140~120
0~-'--
2iS
i~. 20o
160
o.oo7
o.o7
o.7
7.o
1.50
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Insulin concentration(nM) Fig. 3. Concentration-response to insulin of glucose transport. CHO (o), CHO:I" (11) and CHO.TH ( • ) cells incubated as described in Materials and Methods were deprived of serum overnight and then incubated in Krebs-Ringer bicarbonate Hepes buffer, 1% bovine serum albumin, 0.1 mM glucose (pH 7.4) for 20 rain prior to the addition of insulin at the concentzations indicated for 30 rain. 2-Deoxyglucose uptake was then determined over the next 5 mio. Results are expressed as the percent of the respective basal transport rates and are the mean±S.E, of three or more experiments in which the values for each point were determined in three independent cell incubations. Basal glucose transport levels in the three cell lines did not differ and were 486±27 pmol/mg protein per rain (CHO), 590+39 pmol/mg protein per rain (CHO.T), 434±20 pmol/mg protein per rain (CHO.TH). Significant differences compared to cells incubated in the absence of insulin are indicated *. The stimulation by 0.007 nM and 7 nM insulin was statistically greater in CHO.TH cells than in CHO.T celts ( P < 0.05). After Bonferruni correction for multiple comparison the statistical significance at 7 nM insulin was eliminated.
0.007 0.07 mul~ ~
0.7 (n~
7.0
Fig. 4. Concentration-response to insulin of glucose utilization. CHO (o), CHO.T (11) and CHO.TH ( - ) cells were pre-ineubated as described in the legend to Fig. 2 except that they were incubated in the absence of [3H]glacosamioe. Following transfer into fresh serum frec medium containing I% bovine serum albumin, the cells were incubated in the presence or absence of insulin at the indicat:d concentrations for 10 rain. [5-3H]glucose was added for the next 30 rain, after which the medium was removed for determination of glucose utiih.,,ltion as described in Materials and Methods. Results, exFrc~cd as the percent of basal glucose utilization rate. are given as the mean± S.E. of six or more experiments in which three independent cell incubations were performed within each experiment. Basal glucose utilization rates did not differ between the three cell lines and were 3112:L 183 pmol/mg per mio (CHO), 3296± 183 pmol/mg .')er rain (CHO.T) 2918±203 pmoi/mg per min (CHO.TH). Significance from the appropriate basal for each point in each cell line is indicated *.
[3H]inositol w a s s i m i l a r t o t h a t s h o w n in F i g . 2 f o r [ 3 H ] g l u c o s a m i n e cells ( d a t a n o t s h o w n ) .
Effect of insulin on glucose transport The effect of insulin on glucose transport was studied using 2-deoxyglucose uptake to assess transport r a t e in t h e t h r e e cell l i n e s ( F i g . 3). I n s u l i n s t i m u l a t e d g l u c o s e t r a n s p o r t in e a c h o f t h e t h r e e cell lines, w i t h e f f e c t s o n t h e C H O . T H cell line b e i n g a p p a r e n t a t t h e l o w e s t c o n c e n t r a t i o n o f i n s u l i n t e s t e d (0.007 riM), e f f e c t s o n t h e C H O . T line e v i d e n t a t 0.07 n M , a n d e f f e c t s o n t h e C H O cell line a t 0.7 n M . B a s a l g l u c o s e t r a n s p o r t activity in t h e t h r e e cell l i n e s d i d n o t d i f f e r s i g n i f i c a n t l y ( s e e t h e l e g e n d t o F i g . 3). A l t h o u g h t h e C H O . T H cells s h o w e d a s i g n i f i c a n t l y d i f f e r e n t m a x i m u m r e s p o n s e ( c o m p a r e d t o t h e p a r e n t C l I O cell l i n e ) at the highest insulin concentration tested, the res p o n s e o f t h e C H O . T cell line w a s i n t e r m e d i a t e b e t w e e n t h e t w o o t h e r cell l i n e s a n d d i d n o t d i f f e r significantly from either.
Effects of insulin on glucose utilization T h e e f f e c t o f i n s u l i n o n g l u c o s e utilization, m e a sured as 3H20 production from [5-3H]glucose, was d e t e r m i n e d f o r t h e t h r e e cell l i n e s o v e r t h e s a m e concentration range as the PI-glycan and glucose trans-
0~
0.007
0.07
0.7
7.0
ImmlM ,~,,-,¢a,-,,'-ati~ nil Fig. 5. Concentration-response to insulin of pyrovate debydrogenase activation. The relationship between insulin concentration and 'aclive" PDH activity in the CHO, CHO.T and CHO.TH cell lines was determined in the same cells in which effects on 81acose utilization were measured (Fig. 4). Following removal of the medium for determination of glucose utilization, a 'cocktail" for determination of PDH activity was added to the cells. "Active" and "total" PDH activities were determined as described in Materials and Methods, "Total" PDH activity did not differ over the range of insulin concentrations tested, or from cell line to cell line and was 1079±269 pmol/mg per rain (CHO), 1376± 223 pmol/n,,8 per rain (CHO.T) and 1074+ 118 pmol/mg per rain (CHO.TH). Basal 'active' PDH also did not differ from cell line to cell line and was 125:t: 17 pmol/mg per rain (CHO) 97± 16 pmol/mg per rain (CHO.T) and 121 + 13 pmol/mg per rain (CHO.TH). Results are expressed as the percent of basal 'active" PDH for each cell line and are the meand:S.F-., of six or more experiments within which three independent cell incubations were performed and the PDH activity of each incubation determined in duplicate. Significance from the appropriate basal for each point in each cell line is indicated *.
58 port responses (Fig. 4). Significant stimulation of glucose utilization was obtained in both the CHO.T and CHO.TH cell lines at all concentrations of insuli,i tested with no statistical difference in the response of the two insulin receptor transfected lines. Insulin stimulation of glucose utilization in the CHO cell line was achieved at a !.00-fold higher concentration of insulin (0.7 nM), similar to the concentration at which significant effects on glucose transport were achieved (Fig. 3). Consistent with the previous data there was also no difference in the basal level of glucose utilization in each of the three cell lines (see legend to Fig. 4).
Effects' of insulin on pyruvate dehydrogenase The effect of insulin on PDH activity was examined in the three cell lines using the same cells in which the glucose utilization data were obtained (Fig. 5). Consistent with the glucose utilization data there was no significant difference between the C H O . T and CHO.TH cell lines with respect to the PDH response to insulin. Significant stimulation of PDH in both the CHO.T and CHO.TH cell lines was achieved at 0.07 nM insulin, whereas a significant response to insulin in the parent CHO cell line was only achieved at a 100-fold higher concentration of insulin (7 nM). Consistent with each of the other responses of the ceils to insulin, there was no difference between the three cell lines in either basal or total PDH activities (Fig. 5). Discussion
The present study correlates, for the first time in the one investigation, the effect of different levels of insulin receptor expression in a single cell type with insulin-induced breakdown of Pl-glycan, PDH activation and stimulation of glucose transport and utilization. In doing so, it establishes that the predominant effect of altered cell surface insulin receptor expression is to alter the sensitivity to insulin of breakdown of the Pl-glycan, PDH activation and stimulation of glucose transport and utilization. To our knowledge this is the first study attempting to correlate the effect of increasing insulin receptor expression with insulin activation of glucose utilization. Insulin receptor expression has been correlated with PDH activation in one other study [2.~] and the breakdown of the PI-glycan likewise in only one other study [17]. This study [17] established that a normal insulin receptor, containing a functional tyrosine kinase, is required for insulin-stimulated breakdown of the molecule. Comparison of insulinstimulated Pl-glycan breakdown was drawn between cells that either expressed high or low levels of insulin receptors or kinase deficient receptors, but no comparison was drawn to other actions of insulin. In the presem study, two cell lines expressing different receptor levels, derived from the same parent line, were
used to compare insulin actions. The parent cell line, expressing approx. 3.103 insulin receptors/cell, was compared to two stable cell lines transfeeted with human insulin receptor eDNA. One cell line designated CHO.T, expressed approx. 300-fold more insulin receptors than the parent fine. The other cell line designated CHO.TH, was generated by FACS sorting the CHO.T cell line and expressed approx. 2-fold more receptors than the CHO.T cell line. Initial studies established the presence of an insulin-sensitive PI-glycan in the CHO cell lines with characteristics similar to those we and others have described previously in other cell types [1-8]. The PI-glycan extracted from CHO cells in the present study separated in a similar manner to that extracted in a past CHO cell study [15] as well as that extracted from other ceil types, including adipocytes [8,20], H35 hepatoma cells [3,5] and T ceils t6]. As in other cell types, the PI-glycan extracted from CHO cells in the present study could be labelled with glucosamine, inositol and galactose, supporting the presence of these sugars within the Pl-glycan structure in these ceils. Most importantly the Pl-glycan was sensitive to insulin, which stimulated loss of approx. 20% of the label associated with the molecule in each of the three cell lines, indicating that insulin stimulated its breakdown. This maximum breakdown of PI-glycan of 20% in our study is consistent with the level of responsiveness of the C H O cells (a fibroblastic cell) to insulin (i.e., 2.3fold stimulation of PDH, Fig. 5), which although significant, is lower than the responsiveness of classical insulin target tissues such as fat (30% breakdown of Pl-glycan and 3-4-fold or greater effects on PDH, see for example Refs. 8,20,24). Our data therefore suggest that it is not purely the insulin receptor level that determines responsiveness of cells to insulin but that the breakdown of Pl-glycan is also a function of the cell line in which these receptors are expressed. The effects of insulin concentration on Pl-glycan breakdown and other actions of insulin, including PDH activation, glucose transport and utilization were compared in order to assess the potential effects of the level of cell surface insulin receptors on post-receptor insulin action in the CHO cell lines. Significant response in the parent CHO cell line (expressing 300-fold fewer insulin receptors than the CHO.T cell line) was obtained only at the higher concentrations of insulin tested, 0.7 and 7 nM. Significant response of the CHO.TH cells (expressing twice the number of insulin receptors as CHO.T cells) was obtained at insulin concentrations either not different to CHO.T cells (PDH activation (0.07 nM insulin) and stimulation of glucose utilization (0.007 nM insulin)) or the next lowest concentration tested (Pl-glycan breakdown and glucose transport (0.007 nM insulin)) although the sensitivity of the PI-glycan response obtained with the
CHO.TH cell line were not statistically different from those obtained with the CHO.T cell line after Bonferroni correction for multiple comparisons. The effects of insulin on the CHO.TH cell line, however, reinforce the results obtained with the CHO.T cell line. The data taken together clearly indicate that the major effect of altered insulin receptor expression was to alter the sensitivity of the cells to insulin. There was no difference in any of the measured parameters between the cell lines under ba~al or noninsulin stimulated conditions. In addition to the increased sensitivity of the cellular responses to insulin with increased insulin receptor expression, the glucose transport and glucose utilization measurements indicated a tendency for increased responsiveness of the cells to insulin. As the glucose transport rate is a major factor in determining the rate of glucose utilization, and in many cases limits glucose utilization, the findings for glucose transport effects support the likelihood that altered insulin receptor expression modifies slightly the responsiveness of glucose transport to imu!in in addition to affecting the sensitivity of responst, in contrast to the observed effects on PDH a.-.d Pl-glycan breakdown. The present report supports previous studies [6,9-528] in both Rat-I cells and ClIO cells that suggest that insulin sensitivity is proportional to the number of active iusuli~,~ receptors. These other investigations demonstrate~! increased sensitivity of response to insulin on $6 kinase activation, glycogen synthase activation, thymidine uptake and 2-deoxyglncose uptake in insulin receptor transfected cells. The present study extetio's these insulin effects to include glucose utilization, pyruvate dehydrogenase activation and breakdown of Pl-glycan. In the one previous study that examined the Pl-giycan response in a different population of CHO cells transfected with insulin receptors [17] it was found that the responsiveness, rather than the sensitivity, to insulin was increased following transfection of cells with insulin receptors in contrast to the sensitivity change observed in the present study. The reason for this difference in the manifestation of the effect of increased cell surface insulin receptor expression is unclear. However, a positive effect of increased expression of insulin receptors on insulin responsiveness would be predicted if there were no spare functional insulin receptors [29]. In the present study comparison of receptor occupancy with each biological response for the transfected cell lines indicated the presence of functional spare receptors, thus maximal biological activation was achieved when 15-20% of receptors were occupied. The parental CHO cell line reported in the previous study expressed fewer insulin receptors than those used in the present study. This difference may explain the differences observed in the manifestation of effect on Pl-glycan breakdown al-
though in the previous study one might also have expected sensitivity effects in addition to the observed effects on responsiveness. The present studies showing predominant effects on insulin sensitivity, rather than responsiveness, suggest that even in the non-trausfected CHO cells the number of insulin receptors are sufficient to fully stimulate Pl-glycan breakdown and subsequent steps that may be dependent on this process (such as PDH activation). The present studies showing predominant effects of insulin receptor level on the sensitivity of Pl-glycan breakdown are consistent with the other effects of insulin receptor expression on :nsulin action we observed in the present study and those that have been previously reported in the literature [6,26-28]. In conclusion, the present study provides further evidence to support the potential relevance of the Pl-glycan in insulin action by correlating the expression of cell surface insulin receptors with cellular sensitivity to insulin of its breakdown and of other classical actions of insulin, namely PDH activation and stimulation of glucose utilization and transport. The exact contribution of the breakdown of the Pl-glycan to overall cellular seusitivtiy and responsiveness to insulin remains to determined and will require full characterization of the molecule. .Acknowledgements This work was supported by grants from the National Health and Medical Research Council, Eli Lilly, Australia, and a grant-in-aid from the Diabetes Australia Research Trust. The authors are grateful to Dr. M. Low for the gift of B. thuringienesis phosphatidylinositol specific phuspholipase C, to Dr. Leland Ellis for the CHO.T cell line used in this study and to Dr. Ken Siddle for insulin receptor antibody 83-14. The excellent technical assistance of Mr. Warwick Atkinson was greatly appreciated.
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