117
Biochimica et Biophysica Acta, 585 ( 1 9 7 9 ) 1 1 7 - - 1 2 7 © E l s e v i e r / N o r t h - H o l l a n d B i o m e d i c a l Press
BBA 28918
INSULIN ACTION IN ISOLATED FAT CELLS II. EFFECTS OF DIVALENT CATIONS ON STIMULATION BY INSULIN OF PROTEIN SYNTHESIS, ON INHIBITION OF LIPOLYSIS BY INSULIN, AND ON THE BINDING OF 12SI-LABELLED INSULIN TO ISOLATED FAT CELLS
R.A. A K H T A R * a n d M.C. P E R R Y
Department of Biochemistry, Basic Medical Sciences Group, Chelsea College, University of London, Manresa Road, London SW3 6LX (U.K.) ( R e c e i v e d O c t o b e r 4 t h , 1978) (Revised m a n u s c r i p t received J a n u a r y 2 n d , 1 9 7 9 )
Key words: Ca2+;Mg2+; Protein synthesis; Antilipolysis; Insulin binding; (Fat cell)
Summary The effects of omission of Ca ~÷ and Mg2+ from the incubation medium on three aspects of insulin action in isolated fat cells have been investigated. In the (Ca 2+ + Mg2+)-free incubation medium incorporation of L-[14C]leucine into fat cell protein was reduced in the absence of insulin. Insulin stimulated L-[14C]leucine incorporation only in the presence of added CaC12 or MgC12. Incubation of the cells in the (Ca 2÷ + Mg2+)-free medium reduced but did n o t abolish the ability of adrenaline to stimulate lipolysis or the ability of insulin to inhibit the adrenaline-stimulated lipolysis. Specific binding of 12SI-labelled insulin to the fat cells was reduced in the absence of Ca 2+ and Mg2÷ but was not abolished, even in the presence of EDTA. Ca 2÷ was routinely the most effective divalent cation in supporting these aspects of insulin action, but similar responses were obtained with M g 2+, Sr 2+ and Ba 2+. Since insulin still binds to the cells under conditions in which some of the cellular effects of the h o r m o n e are abolished, it is suggested that divalent cations m a y have a role, either direct or indirect, in the processes linking the insulin-insulin receptor complex to certain effector systems in the cells. It is tentatively suggested that this action occurs at the level of the fat cell plasma membrane.
* Present address: D e p a r t m e n t of Cell and Molecular Biology, School of Medicine, Medical College of Georgia, Augusta, GA 30901, U.S.A.
118
Introduction In the preceding paper [1], the effects of divalent cations on the stimulation b y insulin of glucose uptake by isolated fat cells were reported. L o w levels o f Ca :÷ or Mg 2÷ in the incubation medium of the cells were found to be necessary for insulin to be able to stimulate glucose uptake b y the cells. It was suggested that the omission of these cations from the incubation medium may have led to a structural change in the fat cell membrane which in turn m a y result in failure o f insulin to exert its normal action. We have also investigated the effects of divalent cations, in particular Ca 2÷ and Mg :÷, on other actions of insulin in the cells. These additional actions are (1) the ability of insulin to stimulate the incorporation of L-[~4C]leucine into protein; (2) the ability of insulin to inhibit hormone-stimulated lipolysis, and (3) the specific binding of ~:SI-labelled insulin to the fat cells. The results from these studies are reported in this paper. Materials and Methods
Chemicals. Adenosine triphosphate (trisodium salt), bovine serum albumin (fraction V), crystalline zinc insulin from bovine pancreas, phosphoenolpyruvate (tricyclohexylammonium salt), reduced nicotinamide adenine dinucleotide (sodium salt), triethanolamine hydrochloride, glycerol kinase and pyruvate kinase were obtained from Sigma (London) Chemical Company Ltd., Surrey, U.K. Adrenaline and talc were purchased from British Drug Houses Ltd., Poole, U.K. L-['4C]Leucine (specific activity 62 Ci/mol) and Nal~SI (carrier free; specific activity 14 mCi/#g iodine) were from the Radiochemical Centre, Amersham, Bucks., U.K. Cellulose acetate strips were obtained from Oxoid Ltd., Basingstoke, Hampshire, U.K. Collagenase was obtained from the Worthington Biochemical Corporation, Freehold, NJ, U.S.A. Incubation medium. Treatment of the bovine serum albumin used with EDTA, and preparation of the (Ca 2÷ + Mg2÷)-free incubation medium were as described in the previous paper [ 1 ]. Insulin and adrenaline. Insulin was dissolved in 0.003 N HC1 and stored at --20°C in small aliquots. It was n o t refrozen after thawing for use. Adrenaline was dissolved and stored in 0.1 M ascorbic acid. Preparation of isolated fat cells. Isolated fat cells were prepared from the epididymal adipose tissue of male albino Wistar rats weighing 140--180 g as described previously [2]. The isolated fat cells were washed 4 times with 10 ml (Ca2*+ Mg2+)-free incubation medium containing 3% (w/v) EDTA-treated bovine serum albumin and suspended in a fresh aliquot o f this medium. Incorporation of L-[14C]leucine into isolated fat cell protein. Aliquots (0.5 ml) of the isolated fat cell suspension were incubated in (Ca 2÷ + Mg2*) free incubation medium containing 3% (w/v) EDTA-treated bovine serum albumin under an atmosphere of 95% O2/5% CO:. L-[14C]Leucine was added to a final concentration of 0.1/~Ci/ml. Ca 2÷ and Mg 2÷ were added, as appropriate, as concentrated solutions of their chlorides. At the end of the incubation (usually after 90 rain), 0.1 ml of the fat cell suspension was spotted on to a 30 mm diameter filter disc cut from Whatman No. 3 filter paper. The discs
119 were d r o p p e d gently onto the surface of ice-cold 10% (w/v) trichloroacetic acid to precipitate the protein of the sample in the filter paper. After 10 min the filter discs were washed twice with fresh trichloroacetic acid. The filter discs were then extracted for 15 min with ethanol and for 10 min with diethyl ether to remove lipids and finally dried in air. The discs were cut into strips and counted in 10 ml scintillation fluid using an Intertechnique Liquid Scintillation Counter at an efficiency for 14C o f approximately 60%. Controls were run in parallel to determine the nonspecific adsorption of radioactivity to the filter discs. This was always less than 0.1% o f the total radioactivity added to each incubation. The count rate was corrected for this nonspecific adsorption and for the dry weight of the cells determined as described previously [3]. Measurement of lipolysis. The production o f glycerol b y isolated fat cells incubated in the presence and absence of hormones was used as an index of lipolysis. Glucose was omitted from the media used for the preparation and incubation of the cells in these experiments. At the end of the incubation with the hormones and divalent cations, 1 ml 10% (w/v) trichloroacetic acid was added to each incubation vial. The contents of the vial were immediately mixed and centrifuged at 1200 × g for 5 min. 1 ml of the supernatant was taken and the trichloroacetic acid removed b y shaking three times with 5 ml diethyl ether. After centrifugation the ether phase was discarded and aliquots o f the deproteinized aqueous phase assayed for glycerol using the method described by Garland and Randle [4]. Preparation of 12SI-labelled insulin. Insulin was iodinated by the m e t h o d of Cuatrecasas [5]. The iodine content of the preparations of l:SI-labelled insulin used was always less than one iodine/molecule of insulin, and the specific activity ranged between 0.81 and 0.96 Ci/pmol insulin. Degradation of the 125Ilabelled insulin during the iodination was assessed b y the method of Freychet et al. [6]. The 12SI-labelled insulin was more than 96% precipitable with 10% (w/v) trichloroacetic acid, and more than 94% of the ~2SI-labelled insulin adsorbed to talc. Binding of 12SI-labelled insulin to isolated fat cells. Studies of the binding of ~2SI-labelled insulin to isolated fat cells in the presence and absence of divalent cations were carried out as described b y Cuatrecasas [5]. Aliquots (0.5 ml) of the fat cell suspension were incubated at 37°C for 30 min in small uncapped polythene vials with ~2SI-labelled insulin ( 1 . 5 . 1 0 -9 M) in the presence and absence of 20 ttg unlabelled insulin and divalent cations. The incubation was terminated b y the addition of 3 ml ice-cold (Ca 2÷ + Mg2÷)-free Krebs-Ringerbicarbonate, pH 7.4, containing 0.1% (w/v) bovine serum albumin. The suspension was filtered immediately through cellulose acetate filters under reduced pressure. The filtered cells were washed with 10 ml ice-cold medium containing 0.1% (w/v) bovine serum albumin. The total filtration time was 5--10 s. The filter papers were removed, dried and placed in 10 ml scintillation fluid and counted (Intertechnique Liquid Scintillation Counter) at an efficiency of approximately 45%. The specific binding of ~2SI-labelled insulin to the fat cells was calculated b y subtracting the radioactivity b o u n d to the fat cells in the presence of 20 ttg unlabelled insulin from the radioactivity b o u n d in the absence of unlabelled hormone. The nonspecific binding of ~2SI-labelled insulin averaged 10% o f the total binding.
120
Statistics. The significance of any differences observed was tested using Student's 't' test. Res~
Incorporation of L-[14C]leucine into fat cell protein Omission of b o t h Ca 2÷ and Mg 2+ from the incubation medium resulted in a significant depression of incorporation in the absence of insulin (Table I). Addition of 2 mM CaC12 to the incubation medium increased incorporation to an extent that was n o t significantly different from the incorporation observed in the presence of 2 mM CaC12 + 2 mM MgC12. At the same concentration MgC12 was approximately 50% as effective as CaC12. A similar pattern of behavior was observed when the cells were incubated in the presence, of insulin. In the absence o f added CaC12 and MgC12 insulin failed to stimulate the incorporation of L-[14C]leucine into protein (Table I). In the presence of 2 mM CaC12 insulin stimulated incorporation as effectively as it did in the presence o f 2 mM CaC12 + 2 mM MgC12. The same concentration of MgC12, while supporting a significant stimulation o f incorporation b y insulin, was again approximately 50% as effective as CaC12. The ability of the cells to incorporate L-[14C]leucine into protein and the ability of insulin to stimulate this process are restored over the concentration range 0--0.5 mM of divalent cation added to the incubation medium (Figs. 1 and 2). Increasing the concentration of either divalent cation above 0.5 mM resulted in no further enhancement in the presence or absence of insulin. The residual low incorporation of L-[~4C]leucine into fat cell protein in the absence of MgC12 and CaC12 was further reduced by 50% when 1 mM EDTA was added to the incubation medium. The residual incorporation observed was therefore probably the result of the traces of Mg 2÷ and Ca 2÷ known to be present in the (Ca2+ + Mg2÷)-free incubation medium even though the levels
TABLE I E F F E C T S O F Ca 2+ A N D Mg 2+ ON T H E I N C O R P O R A T I O N O F L - [ 1 4 C ] L E U C I N E I N T O F A T C E L L PROTEIN Fat cells w e r e i s o l a t e d in c o m p l e t e K r e b s - R i n g e r b i c a r b o n a t e c o n t a i n i n g 3% ( w / v ) a l b u m i n ; w a s h e d f o u r t i m e s w i t h (Ca 2+ + Mg2+)-free i n c u b a t i o n m e d i u m c o n t a i n i n g 3% ( w / v ) E D T A - t r e a t e d a l b u m i n a n d susp e n d e d in this (Ca2+ + Mg2+)-free i n c u b a t i o n m e d i u m . L - [ 1 4 C ] L e u c i n e i n c o r p o r a t i o n w a s d e t e r m i n e d as d e s c r i b e d u n d e r Materials and M e t h o d s . T h e results are the m e a n s ± S.E. o f t h r e e d e t e r m i n a t i o n s . A d d i t i o n t o (Ca 2+ + Mg2+)-free m e d i u m (raM)
I n c o r p o r a t i o n o f L-[ 14 C ] l e u c i n e i n t o p r o t e i n ( n m o l / 1 0 0 m g cells p e r 90 rain)
CaCI2
MgCl 2
Control
+ I n s u l i n (1 m U / m l )
--2.0 2.0
-2.0 -2.0
0.15±0.01 0.39±0.03** 0.65±0.06** 0.64±0.02**
0.13±0.02 0.76±0.01" 1.28±0.02. 1.35±0.05'
* S i g n i f i c a n t (P ~ 0 . 0 0 5 ) d i f f e r e n c e f r o m c o n t r o l in t h e a b s e n c e o f insulin. ** S i g n i f i c a n t (P ~ 0 . 0 0 5 ) d i f f e r e n c e f r o m c o n t r o l in a b s e n c e o f MgCI 2 and CaCI 2.
121 .c_ E 0
E
0
F
'I
o
3" J $ "6 E c
0 I 0
,
50
- - ~
I00
~
L
500
I000
2000
/u.M CoCI 2 added as final concentration in the incubation medium
Fig. 1. T h e d e p e n d e n c e of L - [ 1 4 C ] l e u c i n e i n c o r p o r a t i o n o n CaCl 2 c o n c e n t r a t i o n . E x p e r i m e n t a l details w e r e as d e s c r i b e d in T a b l e I. Cells w e r e i n c u b a t e d in t h e p r e s e n c e (A A) a n d a b s e n c e (~ ~) of insulin (1 m U / m l ) . E a c h p o i n t is t h e m e a n of t h r e e d e t e r m i n a t i o n s w i t h t h e b a r i n d i c a t i n g -+1 S.E. T h e v e r t i c a l c o l u m n s t o t h e r i g h t s h o w t h e l e u c i n e i n c o r p o r a t i o n w h e n 2 m M CaC12 + 2 m M MgC12 w e r e a d d e d in t h e p r e s e n c e ( s h a d e d c o l u m n ) a n d a b s e n c e ( o p e n c o l u m n ) o f insulin.
present in this medium were insufficient to support a stimulation of incorporation by insulin. Basal leucine incorporation was increased in the presence of either 2 mM SrC12 or 2 mM BaC12, although neither cation could restore incorporation to the level seen in the presence of 2 mM CaC12 + 2 mM MgC12. From all the experiments we have performed, the relative effectiveness of the divalent cations at a concentration of 2 mM in supporting the incorporation of leucine
._= E O
2
8
i I .
"6 E
c
0 0
.
.
.
50
,/
I00
"
,
,
500
I000
2000
~ M MgCI2 added as final concentration in the incubation medium
Fig. 2. T h e d e p e n d e n c e of L - [ 1 4 C ] l e u c i n e i n c o r p o r a t i o n o n MgC12 c o n c e n t r a t i o n . E x p e r i m e n t a l details w e r e as d e s c r i b e d in Fig. 1 e x c e p t MgCI 2 was u s e d in Place of CaC12.
122 in the absence of insulin was Ca 2÷ > Mg 2÷ > Sr 2÷ > Ba 2÷. The same rank order holds in the presence of insulin, although all of the cations supported a significant stimulation of incorporation by insulin.
The antilipolytic effect of insulin One of the major physiological functions of insulin is to suppress lipolysis in adipose tissue and so regulate the rate at which non-esterified fatty acids are released into the blood. This effect can be demonstrated also in vitro with isolated fat cells, and we have investigated the effects of changes in the divalent cation composition of the incubation medium on this process. The concentrations of adrenaline (0.1 pg/ml) and insulin (10 pU/ml) used in these experiments were those that had been shown previously to give the maximum degree o f inhibition by insulin o f hormone-stimulated lipolysis [ 7 ]. In the (Ca 2÷ + Mg2+)-free medium, adrenaline produced a significant stimulation of glycerol production when used at a submaximal concentration of 0.1 #g/ml (Table II). Unlike the action of insulin to promote glucose uptake and to stimulate leucine incorporation into protein, in no experiment using the (Ca2÷ + Mg2÷)-free medium, with or without the addition o f EDTA, was it possible to completely abolish the stimulation of lipolysis by adrenaline. The addition of either 2 mM CaCI~ or 2 mM MgC12 caused an increase in the glycerol production by the cells in response to adrenaline. Routinely the addition of CaC12 caused a greater degree of enhancement than did the addition of the same concentration o f MgC12. Insulin, at a concentration of 10 #U/ml, always produced a significant inhibition of the adrenaline-stimulated glycerol production by the fat cells even when Ca 2÷ and Mg 2÷ were omitted from the incubation medium (Table II). Thus, this effect o f the hormone differs markedly from the two effects described earlier,
TABLE
II
EFFECTS O F C a 2+, M g 2+ A N D BITION BY INSULIN
EDTA
ON ADRENALINE-STIMULATED
LIPOLYSIS
AND
ITS INHI-
Fat cells w e r e i s o l a t e d in c o m p l e t e K r e b s - R i n g e r b i c a r b o n a t e c o n t a i n i n g 3% b o v i n e s e r u m a l b u m i n ; t h e n w a s h e d ( 4 t i m e s 1 0 m l ) and s u s p e n d e d in ( C a 2+ + M g 2 + ) - f r e e K r e b s - R i n g e r b i c a r b o n a t e c o n t a i n i n g 3 % E D T A - t r e a t e d b o v i n e s e r u m a l b u m i n . A l l q u o t s ( 1 m l ) o f t h e fat cell s u s p e n s i o n in t h e ( C a 2+ + M g 2 + ) - f r e e i n c u b a t i o n m e d i u m w e r e i n c u b a t e d at 3 7 ° C f o r 1 h w i t h t h e a d d i t i o n s s h o w n . G l y c e r o l w a s d e t e r m i n e d at t h e e n d o f t h e i n c u b a t i o n as d e s c r i b e d in Materials and M e t h o d s . T h e results are the m e a n s ± S . E . o f t h r e e determinations. A d d i t i o n t o ( C a 2+ + M g 2 + ) - f r e e m e d i u m (raM) CaC12
MgC12
EDTA
-2.0 -2.0
--2.0 2.0
-----
Glycerol production (~mol/100
m g cells per h )
Control
+Adrenaline (0.1 ~g/ml)
+Adrenaline (0.1 ~g/n~) +insulin (10 # U / m l )
0.21±0.01 0.22±0.01 0.25±0.02 0.22±0.02
0.76±0.03* 1.07±0.04" 0.82±0.06* 1.22±0.02"
0.33 0.65 0.55 0.62
± 0.05 ± 0.01 ± 0.05 -+ 0 . 0 7
* Significant (P (0.001) s t i m u l a t i o n b y a d r e n a l i n e as c o m p a r e d t o t h e a p p r o p r i a t e c o n t r o l . * * S i g n i f i c a n t ( P ~ 0 . 0 0 1 ) i n h i b i t i o n b y insulin o f t h e a d r e n a l i n e - s t i m u l a t e d g l y c e r o l p r o d u c t i o n .
** ** ** **
123
i.e. the insulin stimulation of glucose uptake [1] and the insulin stimulation of the incorporation of leucine into fat cell protein (Table I), in that it is clearly not abolished by the omission of Ca 2÷ and Mg 2÷ from, or addition of EDTA to, the incubation medium. The addition of CaCI: and MgC12, either singly or together, produced little change in the percentage inhibition observed when insulin was added, although the overall stimulation of lipolysis by adrenaline increased when the divalent cations were added.
Binding of 12SI-labelled insulin to isolated fat cells In the absence of added CaC12 and MgC12 the specific binding of 12SI-labelled insulin to isolated fat cells was reduced (Table III). This was a consistent and reproducible observation although the extent to which the binding was reduced varied from experiment to experiment with different preparations of isolated fat cells. The average reduction in specific binding from all experiments was approximately 50%. In no case, however, when Ca 2÷ and Mg 2÷ were omitted from the incubation medium or when EDTA was added to the (Ca 2÷ + Mg2+) free medium (Table III), was the specific binding of 12SI-labelled insulin abolished. Indeed when EDTA was added to the (Ca 2÷ + Mg2÷)-free medium a small, but usually significant, increase in insulin binding was observed. This increase was not seen when EDTA was added in the presence of excess CaC12: and MgC12. Addition of 2 mM CaC12 to the incubation medium increased the binding of 12SI-labelled insulin to levels approaching those seen with 2 mM CaC12 + 2 mM MgC12 present in the medium (Table III). MgC12, at the same concentration, was less effective than CaC12. Other divalent cations, Sr 2÷ and Ba 2÷, also increased
TABLE
III
EFFECTS O F C a 2+ A N D M g 2+ A N D E D T A LIN TO ISOLATED FAT CELLS
ON THE
SPECIFIC
BINDING
OF 125I-LABELLED
INSU-
F a t cells w e r e i s o l a t e d in I~cebs-Ringer b i c a r b o n a t e c o n t a i n i n g C a 2+, M g 2+, 3 % b o v i n e s e r u m a l b u m i n and 3 m M g l u c o s e ; t h e n w a s h e d ( 4 t i m e s 1 0 m l ) and s u s p e n d e d in ( C a 2 + + M g 2 + ) - f r e e K r e b s - R i n g e r b i c a r b o n a t e c o n t a i n i n g 3% E D T A - t r e a t e d b o v i n e s e r u m a l b u m i n and 3 m M g l u c o s e . A l i q u o t s ( 0 . 5 m l ) o f the fat cell s u s p e n s i o n in t h e ( C a 2+ + M g 2 + ) - f r e e i n c u b a t i o n m e d i u m w e r e i n c u b a t e d at 3 7 ° C for 3 0 m i n w i t h 1 2 5 1 - l a b e l l e d i n s u l i n ( 1 . 5 • 1 0 - 9 M ) in t h e p r e s e n c e and a b s e n c e o f 2 0 p g u n l a b e l l e d insulin. The 1 2 S I l a b e l l e d insulin b o u n d s p e c i f i c a l l y t o t h e fat cells w a s d e t e r m i n e d as d e s c r i b e d in Materials and M e t h o d s b y s u b t r a c t i n g the c p m b o u n d in the p r e s e n c e o f 2 0 ~ g u n l a b e l l e d insulin f r o m the c p m b o u n d in its a b s e n c e . T h e results are t h e m e a n s ± S . E . o f 3 - - 6 d e t e r m i n a t i o n s . A d d i t i o n t o t h e ( C a 2+ + M g 2 + ) - f r e e medium (mM) CaCl 2
MgC12
EDTA
-2.0 -2.0 -2.0
--2.0 2.0 -2.0
----1.0 1.0
1 2 5 i . l a b e l l e d insulin s p e c i f i c a l l y b o u n d ( c p m b o u n d / 1 0 0 rag cells)
5 700 12000+ 9900± 13 250 7 800 14 800
* S i g n i f i c a n t ( P ~ 0 . 0 1 ) increase in *,he 1 2 5 I - l a b e l l e d b i n d i n g o b s e r v e d in t h e a b s e n c e o f any a d d i t i o n .
±
556 500" 600" ± 420 * ± 1200 * ± 400 *
insulin s p e c i f i c a l l y b o u n d t o t h e fat ceils o v e r t h e
124
TABLE
IV
EFFECTS OF INCREASING CONCENTRATIONS OF CaCl 2 OR MgCl2 ON THE SPECIFIC OF 125I-LABELLED INSULIN TO ISOLATED FAT CELLS
BINDING
T h e e x p e r i m e n t a l d e t a i l s w e r e as d e s c r i b e d i n T a b l e I I I . T h e r e s u l t s a r e t h e m e a n s + S . E . o f t h r e e d e t e r m i nations in each case. Separate preparations of isolated fat cells were used for the two different experiments. CaC12 a d d e d t o t h e ( C a 2+ + M g 2 + ) - f r e e medium (raM) -0.05 0.10 0.50 1.00 2.00 2.0 mM CaCl 2 + 2 . 0 m M MgC12
125 I - l a b e l l e d i n s u l i n specifically bound (cpm bound/100 mg cells)
5 6 6 7 7 9
600 100 500 300 400 200
+ 300 + 300 + 300 + 400 _+ 2 0 0 + 300
10 900 + 200
MgC12 a d d e d t o t h e ( C a 2+ + M g 2 + ) - f r e e medium (mM)
125 I - l a b e l l e d i n s u l i n specifically bound (cpm bound/100 mg cells)
-0.05 0.10 0.50 1.00 2.00 2 raM CaCI 2 + 2 raM MgCl 2
6200 6900 6100 6500 6100 7600
+ 100 + 1200 + 1300 + 600 + 100 + 800
8900 +
300
insulin binding when they were added, b u t at a concentration of 2.0 mM neither o f these ions could completely restore binding to the levels seen when both Mg 2+ and Ca 2+ were added (data not shown). The relationship between increasing concentrations of either CaC12 or MgC12 in the incubation medium and insulin binding to isolated fat cells is shown in Table IV. With increasing concentrations of CaC12 the binding of 12SI-labelled insulin increased steadily. With MgC12, there was no increase in insulin binding until the concentration of MgC12 added exceeded 1.0 mM. Over the concentration range of 0--0.1 mM added divalent cation, which almost completely restored the ability of insulin to stimulate the uptake of glucose [1] and the incorporation of L-[14C]leucine into fat cell protein (Figs. 1 and 2), the increase in insulin binding was small in the case of CaC12 and non-existent in the case of MgC12. Discussion
The omission o f Ca 2+ and Mg 2+ from the incubation medium has been shown to modify various aspects of insulin action in isolated fat cells. For instance, L-[14C]leucine incorporation into protein was suppressed in the (Ca 2÷ + Mg 2+)free medium and insulin failed to stimulate this process (Table I and Ref. 8). A comparison of these results and those obtained in the glucose u p t a k e experiments [1] show several similarities. Thus, in both cases there was total abolition of the insulin stimulation in the (Ca ~+ + Mg2+)-free medium. Further, the addition of 25--50 pM CaC12 or MgC12 supported a significant effect o f the h o r m o n e on both glucose uptake [1] and leucine incorporation (Fig. 1). In both cases under saturating conditions the presence of Ca 2÷ allowed a greater insulin stimulation than the presence of the same concentration of Mg 2÷. The one marked difference between the t w o processes was the inhibition of leucine incorporation in the absence of insulin in the (Ca 2+ + Mg2+)-free medium. Basal
125
glucose uptake wm not affected by the absence of the two cations. Although glucose uptake (wh! ch includes transport of glucose into the cell and its subsequent metabolism and the incorporation of leucine into protein involve m a n y different, discrete m~flecular events, the one obvious feature that t h e y have in c o m m o n is a facilitated transport step across the fat cell membrane. Under the present experimental conditions, glucose transport is probably the rate-limiting step for glucose met~ bolism in the cells [9,10] and therefore changes in glucose uptake may closely :eflect changes in the rate of glucose transport. In view of these considerations it is possible that the loss of the insulin effects under these conditions could be a reflection of a requirement of these transport systems for a divalent cation ~d their consequent inhibition in the (Ca2++ Mg2÷)-free medium. However, little appears to be known of any specific requirement of these systems in t~e fat cell membrane for divalent cations, although it has been suggested t h ~ Ca 2+ m a y control glucose transport across the fat cell membrane [11]. It ~ also possible, of course, that the suppression of leucine incorporation in/to protein in the absence of extracellular Ca 2÷ and Mg 2÷ and in the absence o~/insulin may indicate a requirement for a divalent cation in amino acid ~2ansport. A n y requirement of these transport systems for a divalent cat/i/on under the present experimental conditions would, however, have to bg/a relatively nonspecific one, since Sr 2÷ and Ba 2÷, in addition to Mg2÷ and Ca 2÷', could support an insulin stimulation of glucose uptake [1] and leucine incorporation. Two other sites that could explain the inhibition of these effects of insulin in the absence of Ca 2÷ and Mg2÷ are, (1) the binding of the h o r m o n e to its receptor, and (2) the system or systems, as yet unknown, which functionally link the hormone-receptor complex to the various effector systems, including the transport processes. It is clear that inhibition of insulin binding to its receptor cannot fully explain the effects observed for, in the (Ca 2÷ + Mg2+)-free m e d i u m under conditions in which insulin failed to stimulate glucose uptake or leucine incorporation, the binding of ~2SI-labelled insulin to the cells, although reduced, was never completely inhibited. It has been reported previously that binding of ~2SI-labelled insulin to liver and fat cell membranes [12] and to insulin receptors solubilized from t h e m [13] does not require the presence of divalent cations and is not inhibited by chelating agents. The increase in specific ~2SI-labelled insulin binding observed when CaC12 or MgC12 were added to the incubation medium could then have been due to a change in membrane structure produced by the ions which in turn caused the exposure of more specific insulin binding sites. The observation that the h o r m o n e still binds to the cells under conditions in which it is unable to m o d i f y glucose metabolism and protein synthesis implies that the divalent cations are acting subsequently to the formation of the hormone-receptor complex, perhaps in the process by which this complex modulates these metabolic processes. In support of this is the finding that increasing the concentration of CaC12 or MgC12 added to the m e d i u m over the range 0--100/aM permitted almost full expression of the action of the h o r m o n e on glucose uptake [1] and protein synthesis (Fig. 1), but caused little (Ca 2+) or no (Mg 2÷) change in the a m o u n t of 12SI-labelled insulin specifically bound to the cells. Thus, divalent cations are viewed as having a 'permissive' effect in allowing certain, if not all, of the consequences of insulin-
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insulin receptor interaction to be expressed in the cells. It has, however, to be noted that insulin still inhibited adrenaline-stimulated lipolysis in the (Ca2÷ + Mg2÷)-free medium and in the presence of EDTA, demonstrating that this effect o f the h o r m o n e is less sensitive to the divalent cation concentration in the medium than were glucose uptake and the incorporation of leucine into protein. So a possible alternative explanation for these findings, but one for which there is little supporting evidence, could be the existence of t w o populations of insulin receptors, one population to which the binding of the h o r m o n e is in some way divalent cation dependent and which is functionally linked to glucose transport and protein synthesis, and a second population of insulin receptors in which the binding is insensitive to changes in the divalent cation concentration o f the incubation medium and which is functionally linked to the lipolytic system. While it is clear from these results that the actions of insulin on isolated fat cells are sensitive to changes in the divalent cation concentration of the incubation medium, the cellular site or sites at which these cations act to permit expression of the effects of the h o r m o n e have not been identified. Although Ca 2÷ was in general the most effective of the cations in supporting insulin action, the responses observed were by no means specific for Ca 2÷ and were also obtained with Mg 2÷, Sr 2÷ and Ba :÷. This relative nonspecificity makes it difficult t o interpret these results in relation to suggestions that Ca :+ [14,15] or Mg 2÷ [16] might act as a specific intracellular second messenger for insulin. Recently it has also been reported that depletion of intracellular Ca 2÷ b y EGTA significantly inhibited the insulin-stimulated cyclic GMP level, protein synthesis and lipoprotein lipase activity in adipose tissue [17]. Since no a t t e m p t was made in these experiments to deplete the cells of intracellular Ca 2÷ and Mg 2*, nothing is known of any possible changes in or redistribution of intracellular Ca 2÷ and Mg 2÷. In view of these considerations it seems more probable, at least as a working hypothesis, that the effects observed may relate to changes occurring on or within the fat cell plasma membrane when the cells were placed in the (Ca 2+ + Mg2+)-free medium. In this medium Ca 2+ and Mg ~+ b o u n d to the plasma membrane might be expected to dissociate from the membrane and this m a y lead to changes in membrane structure and/or charge distribution within the membrane. For instance, Ca 2+ and Mg 2+ are known to influence lipid phase transitions in membranes [19,20]. These changes m a y in turn cause inhibition of insulin action perhaps by blocking the processes involved in linking the hormone-hormone receptor complex to certain of the effector systems, e.g. glucose transport. Ca 2+ has been shown to bind to plasma membranes prepared from isolated fat cells [20,21]. There is evidence for at least two classes of binding sites [20], the binding capacity of which is increased by insulin [ 21]. However, the chemical identity of these sites, i.e. whether they represent negative charges on phospholipids, sialic acid, proteins, etc., has n o t been established. This information would appear to be necessary in order to a t t e m p t a more detailed analysis of the way in which these ions may act to permit insulin to act on certain cellular processes. In particular i t would be valuable to identify those binding sites from which divalent cations readily dissociate in the (Ca 2+ + Mg2+)-free medium. It can, however, be predicted that these sites will be relatively nonspecific and will bind a variety of divalent cations.
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Acknowledgement The financial support of the Wellcome Trust during the course of this work is gratefully acknowledged. References 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21
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