Regulation of insulin-stimulated glucose transport in the isolated rat ...

Vol. 262, No. 1, Issue of January 5, p p . 245-253,1987 Printed in U.S.A.

THEJOURNAL OF BIOLOGICAL CHEMISTRY

Regulation of Insulin-stimulated Glucose Transportin the Isolated Rat Adipocyte CAMP-INDEPENDENTEFFECTS OF LIPOLYTIC AND ANTILIPOLYTIC AGENTS* (Received for publication, April 3, 1986)

Masao Kuroda$$, Rupert C. HonnorllII, Samuel W.CushmanS, Constantine Londosll, and Ian A. Simpson$** From the $.Experimental Diabetes, Metabolism, and Nutrition Section, Molecular, Cellular, and Nutritional Endocrinology Branch, the llMembrane Regulation Section, Laboratory of Cellular and Developmental Biology and the National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892

This paper examines the modulation of insulin-stimulated glucose transport activityin rat adipose cells by ligands for receptors (R)that mediate stimulation (Rs; lipolytic) or inhibition (Ri; antilipolytic) of adenylate cyclase. The changesin glucose transport activityand CAMP, as assessed by 3-0-methylglucose uptake and (-/+) CAMP-dependent protein kinase (A-kinase) activity ratios, respectively, weremonitored under conditions that maintain steady-state A-kinase activity ratios (Honnor, R. C., Dhillon, G . S., and Londos, C. (1985) J. Biol. Chern. 260, 15122-15129). Removal of endogenous adenosine with adenosine deaminase decreased insulin-stimulated glucose transport activity by -30%, which was prevented or restored with Ri agonists such as phenylisopropyladenosine, nicotinic acid, and prostaglandinEl. These changes in transport activity werenot accompanied by changes in A-kinase activity ratios, indicating that Ri-mediated effects on transport areindependent of cAMP changes. Addition of an R, ligand, isoproterenol, in the presence of adenosine increased kinase activity but did not change glucose transport activity. Conversely, upon removal of adenosine, addition ofR. ligands such as isoproterenol, adrenocorticotropic hormone, or glucagon strongly inhibited transport (-50%) and stimulated kinase activity. However, subsequent addition of phenylisopropyladenosine nearlyrestored transport activity without alterationof A-kinase activity. These data and additional kinetic experiments suggest that R.-mediatedglucose transport modulations are also independent of CAMP. The interchangeability of ligands for both R. and Ri receptors inmodulating transport activity suggests that these CAMP-independent effects are mediated by the stimulatory(N,) and inhibitory (Ni) guanyl nucleotide-binding regulatory proteins of adenylate cyclase. All R.- and Ri-induced changes in transport activity occurred without a change in glucose transporter distribution, as assessed by D-glucose-inhibitable cytochalasin B binding, suggesting that R. and Ri ligands modulate the intrinsic activity of the glucose trans* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisemnt” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Present address: Fermentation Research Laboratories, Sankyo Co., Ltd., 1-2-58 Hiromachi, Shinagawa, Tokyo, 140 Japan. 11 Present address: Dept. of Biochemistry, The University, Newcastle upon Tyne, NE1 7RU, United Kingdom. ** To whom correspondence should be addressed Bldg. 10,Room 5N102, NIDDK/NIH, Bethesda, MD 20892.

porter present in the plasma membrane. Studies from this (1-3) and Kono’s (4-6) laboratories have led to the formulation of the translocationhypothesis to explain insulinstimulation of glucose transport in ratadipose cells. Essentially, it isproposed that insulin induces the translocation of glucose transporters from a large intracellular pool to the plasmamembrane by arapid, reversible, and energy-dependent process. Accordingly, increased glucose transporter concentration in the plasma membrane, rather than a change in transporter intrinsic activity, is thought to be the basis of the increased VmaX for glucose transport activity in response to insulin. This concept has been extended to explain certain pathophysiological conditions in which insulin resistance or a diminished response to insulin is associated with a diminished intracellular pool of glucose transporters in thebasal state and,consequently, fewer transporters available fortranslocation to the plasma membrane in response to the hormone (7-9). Lipolytic agents which stimulate adenylate cyclase, such as catecholamines, ACTH,’ and glucagon, inthe absence of endogenous adenosine, acutely inhibit theV,,, for both basal and insulin-stimulated glucose transport activity. This inhibition may be reversed by antilipolytic agents which act via receptors toinhibit adenylate cyclase, such as adenosine, nicotinic acid, and prostaglandins (10-16). It has been suggested, by implication, that thesemodulations in glucose transport activity result from changes in cellular cAMP concentration and the resultant changes in CAMP-dependent protein kinase activity. Hereafter, we shall refer to CAMPdependent protein kinase as A-kinase and use this activity to reflect intracellular cAMP concentrations (17-19). To assess the putative relationships between changes in cAMP concentration, glucose transport activity, and glucose transporter subcellular distribution, we have applied the stringently defined protocol for adipose cell incubations reported by Honnor et al. (20, 21). By eliminating the transient“peaking” phenomenon in kinase activity in response to lipolytic agents, this procedure permits the establishment and maintenance of relatively invariant A-kinase activity levels for up The abbreviations used are: ACTH, adrenocorticotropic hormone; PGE1, prostaglandin El;A-kinase, CAMP-dependent protein kinase; Bt,cAMP, dibutyryl CAMP; PIA, N6-[R-(-)-l-methyl-2-phenethyl] adenosine; Hepes, 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid; Ro-20-1724, dl-4-(3-butoxy-4-methoxybenzyl)-2-imidazolidinone; R. and 9, receptors that mediate stimulation or inhibition, respectively of adenylate cyclase; N. and Ni, the stimulatory and inhibitory guanyl nucleotide-binding proteins, respectively, of adenylate cyclase.

245

246

Regulation of Adipocyte Glucose Transport Activity

t o 30 min following application of a lipolytic stimulus. Thus, the relationship between steady-state A-kinase activity and glucose transport activity can be assessed, allowing one to determine whether or not changes in transport by adenylate cyclasestimulators and inhibitorsresultfromchangesin cellular CAMP concentration. For increased sensitivity, we have examined these effects in the insulin-stimulated state although a previous report describes comparable findings in the basal state (16). The outcome of such studies reported here strongly suggests that 1) the alterations in the V,, for both lipolyticand antilipolytic hormonesoccur predominantly through a CAMP-independent process, and 2) the changes in glucose transport activity observed in the intact cell are t h e result of alteration in the intrinsic activity of the glucose transporters residing in the plasma membranes and not in the translocation of glucose transporter.

in a solution of20mM Tris-HC1, pH 7.4, 1 mM EDTA, and 255 mM sucrose, and immediately homogenized. Three subcellular membrane fractions were then prepared by differential ultracentrifugation as previously described (22):a plasma membrane fraction, a high-density and a low-density microsomal membrane fraction. Incubation of cells with either lipolytic or antilipolytic hormones and agents had no effect on the subcellular fractionation of membranes, as assessed by the distribution of marker enzyme activities (22). The concentration of glucose transporters in each membrane fraction was determined using the equilibrium D-glucose-inhibitable [3H]cytochalasinB-bindingprocedure described by Wardzala et al. (I), asmodified by Simpson et al. (22). Protein content was assayed by the Coomassie Brilliant Blue method (Bio-Rad protein assay), as described by Simpson and Sonne (25), with crystalline bovine serum albumin (Sigma) as the standard. Data shown under “Fksults” are from representative experiments. Each experiment was performed at least three and, in most cases, five times. RESULTS

EXPERIMENTALPROCEDURES

Materials-(-)-Isoproterenol, BhcAMP, PGE1, Hepes, adenosine deaminase, and the A-kinase inhibitor were purchased from Sigma. PIA was from Boehringer Mannheim, nicotinic acid from Eastman Kodak, ACTH”“ from Behring Diagnostics, and glucagon from Lilly. Sources for all other reagents were as reported previously for the glucose transport (3, 22) and A-kinase (20) procedures. Rats of the CD strain were supplied by the Charles River Breeding Laboratories. Insulin was a generous gift of Dr. Ronald E. Chance, Lilly. The cyclic nucleotide phosphodiesterase inhibitor, Ro-20-1724, wasfrom Dr. H. Shepperd, Hoffmann-La Roche. Bovine serum albumin, fraction V, was from Reheis Chemical Co. and collagenase was from Cooper Biomedical. Cell Preparation and Measurement of Glucose Transport ActivityAdipose cells were isolated from the epididymal fat pads of 180-200g rats fed ad libitum with standard National Institutes of Health chow. The fat pads were digested with collagenase according to the method of Rodbell (23) as modified by Cushman et al. (24). All incubations were carried out in Krebs-Ringer buffer, pH 7.4, supplemented with 30 mM Hepes, 10 mM HCO,, 5% (w/v) bovine serum albumin, and 2.5 mM glucose. Following their isolation, the cells were distributed to plastic vials in a final volume of 400 pl; cell concentrations did not exceed 106/ml. Incubations were conducted a t 37 “C in a Precision Scientific Model 50 reciprocating water bath with rapid shaking (110 strokes/min, stroke length = 5 cm). Unless otherwise stated, cells were preincubated for 15 min, after which they were exposed to 7 nM insulin for 15 min. Temporal sequences of further additions were as indicated in the legends to theFigures and Tables. Where applicable, the final ethanol concentration in the incubation mixture did not exceed 0.015% (w/v). Glucose transport activity was determined by the 3-0-methylglucose uptake technique, as described by Karnieli et al. (3). The small inhibition of 3-0-methylglucose transport caused by the presence of glucose in the medium was not accounted for in the calculations. Determination of A-kinuse Activity Ratios-A-kinase activity was assayed according to Honnoret at. (20). Briefly, the 400-plcell incubations were terminated by the addition of 600 pl of an extraction medium containing EDTA and Ro-20-1724.The final concentrations of the chelator and Ro-20-1724 were, respectively, 10 mM and 500 p ~ homogenates ; also contained 1%ethanol carried over from the Ro-20-1724 stock solution. This mixture was rapidly transferred to a glass homogenizing tubeand homogenized with 10 strokes of a ground-glass pestle and the homogenate placed in a 1.5-ml conical centrifuge tube pre-cooled to 4 “C. After completion of all incubations in a given experiment, the homogenates were centrifuged at 10,000 X g for 15 min. The infranatants were withdrawn for protein kinase assays, which wereperformed either immediately or on samples stored at -80 “C, as previously described (20). A-kinase activities are expressed as the-cAMP/+cAMP activity ratios, with corrections made for non-A-kinase activities (20). Preparation of Subcellular Membrane Fractions and Determination of the Concentration of Glucose Transporters-Isolated cells prepared from 12 g of adipose tissue (12-15 rats) were diluted to a final volume of 72 ml with the Krebs-Ringer incubation medium described above, and incubated in 950-ml polypropylene containers at 37 “C with moderate shaking (70 strokes/min) in the above described shaking water bath. Following incubation, the cells were washed twice at 17 ‘C

Effects of Cell Mixing Speed-Under the protocol established by Honnor et al. (ZO), rapid agitation of lipolytically of released fatty acids stimulated adipocytes to ensure removal by the medium albumin is essential for eliminating thetransient peaking of A-kinase activity ratios. Fig. 1 presents a comparison of the time courses of kinase activity ratios in response to isoproterenol under both rapid and slow mixing conditions. With the slower, conventional shaking speed (40 strokes/min), kinase activities reached a peak within a few minutes and declined dramatically over 30 min, whereas with the higher mixingspeed (110 strokes/min), elevated A-kinase activities were maintained throughout the incubation period. Table I depicts representative experiments comparing the effects of cell mixing speed on both glucosetransport activities and the A-kinase activity ratios. Comparable inhibitions of insulin-stimulated glucose transport activities were observed irrespective of either the rate of cell mixing or the kinase activityratios. Thus, despiteconsiderabledecreasesin Akinase activity at the slower shaking speed, the inhibition of transport activity persisted, suggesting that the maintenance of elevatedkinaseactivity is not necessaryfor transport inhibition. Effects of Removal of Endogenous Adenosine-Adenosine, a potent inhibitor of both adenylate cyclase and lipolysis, is inevitably present in isolated adipose cell suspensions (26, 27). The above experiments were performed in the presence order to convert endogenous adenof adenosine deaminase in osine to inosine, which is not an agonist of adenosine receptors. Fig. 2 shows the effectsofadenosineremovalwith adenosinedeaminaseon both insulin-stimulatedglucose A Low Shaking Speed

0

B High Shaking Speed

0.6

a

0.4

q

0.2

w

4

TIME (rnin)

TIME (rnin)

FIG. 1. Effects of cell incubation shaking speed on A-kinase activity ratios in isolated adipose cells. Isolated adipocytes were incubated with 7 nM insulin at 37 “C for 15 min. Subsequently, adenosine deaminase (1unit/ml) and isoproterenol were added and cells were subjected to either low (A) (40 cycles/min) or high ( B ) (110 cycles/min) shaking speed. At the indicated times, cells were homogenized and A-kinase activity ratios determined. The data are derived from several different experiments, each denoted by a separate line in the figure; isoproterenol concentrations varied between 50 and 200 nM.


247

TABLEI The effects of shaking speed on glucose transport activity and A-

1 .o

kinase activity ratios in isolated rat adipose cells Isolated adipocytes were incubated for 15 min in the presence of 7 nM insulin. Subsequently, cells were incubated in the absence (“Control”) or presence (“IS0 + ADA”) of 1 unit/ml adenosine deaminase plus isoproterenol at either low or high shaking speed, 40 or 110 cpm, respectively. The isoproterenol concentration was 100 nM for Experiment A and 200 nM for Experiments B, C, and D. At the indicated times, glucose transport activities were measured and parallel samples were taken for A-kinase activity determinations.Transport activities represent the mean values of triplicate samples and A-kinase activity ratios are themeans of triplicate determinationsof duplicate samples. For Experiments B and C, data are shown for 30 min, a time at which the A-kinase activity ratios had declined from the high values seen a t 5 min (see Fig, 1).The Table shows only one example of results at the higher shaking speed, Experiment D, since all other experiments presented in this paper were performed at this speed (see Figs. 4-8). Incubation condition

Shaking activity transport Time speed min

Experiment A Control

A-kinase

activity

ratio

Px

1.0

i

=

8

:

R

0.0

3

1.0

0.5

0.0 -15

fmol/cell/min

0

5

10

15

TIME (rnin)

0.075 30 5 30

2.83 3.03 1.55 1.54

30 30

3.15 1.15

30 30

3.05 1.10

15 0.14 30 1.07 15 30

2.60 2.72 0.65

0.11

0.80

1.03

0.07 0.84 0.19

Low 0.09

0.26

Low 0.05 0.41

High

IS0 + ADA

0.0

0.5 5

Low

ISO” + ADA* Experiment B Control IS0 + ADA Experiment C Control IS0 + ADA Experiment D Control

Glucose

0.5

FIG. 2. Effects of adenosine removal by adenosine deaminase on glucose transport activity and on the A-kinase activity ratio in rat adipose cells. A , isolated cells were incubated with 7 nM insulin ( I N S ) a t 37 ‘C in the presence or absence of 1 unit/ml adenosine deaminase ( A D A ) .B, isolated cells were preincubated with 7 nM insulin for 15 min and thenincubated in the presence or absence of adenosine deaminase. C, isolated cells were preincubated for 1min with or without adenosine deaminase prior to the addition of 7 nM insulin. At the indicated times cells were sampled for measurements of 3-0-methylglucose uptake (0,0 ) and A-kinase activity ratios (0, H). Open and closed symbols denote activities determined in the absence and presence of adenosine deaminase, respectively. Transport rates are the mean values obtained from triplicate samples. The results of A-kinase ratios are the means of triplicate determinations of duplicate samples.

ISO, isoproterenol.

’ADA, adenosine deaminase. transport activity and the A-kinase activity ratio. Adenosine removal led to a 30% reduction in the maximum rate of 3-0methylglucose transport irrespective of whether deaminase was added before, together with, or after the addition of insulin. Adenosine deaminase addition either prior to or simultaneous with insulin did not alter the time course of insulin stimulation of transport activity (Panels A and C), and transport activity declined with a tlh of 60-90 s when adenosine was removed subsequent to establishment of the transport response with insulin (Panel B ) . In all cases, the lower transport rate resulting from adenosine depletion occurred without achange in theA-kinase activity ratio. Kinase activity did rise slightly upon addition of deaminase prior to insulin, but rapidly returned to control levels after insulin addition (Panel C). All of the actions of adenosine deaminase seen in Fig. 2 were prevented or reversed by 100 nM PIA, a potent adenosine receptor agonist that is not metabolized by deaminase. Taking the adenosine-depleted condition as the reference point, Table I1 and Fig. 2 show that adenosine or PIA increased insulin-stimulated glucose transport by 40%. Similarly, in an adenosine-free medium, other antilipolytic agents that inhibit adenylate cyclase, such as PGEl and nicotinic acid, prevented the decrease in glucose transport activity upon adenosine removal with adenosine deaminase (Table 11). Moreover, the enhancement of transport activity by these agents was not accompanied by a change in the A-kinase activity ratio (data not shown). Thus, both the reduction in insulin-stimulated glucose transport activity by metabolism

TABLEI1 Effects of antilipolytic agents on insulin-stimulated glucose transport activity in isolated rat adipose cells Isolated adipocytes were incubated for 15 min in the presence of 7 nM insulin and then for an additional 15 min in the further presence of the indicated agents. Adenosine deaminase was added at 1 unit/ ml, PIA at 1pM, nicotinic acid a t 1 pM, and PGE, at 10 nM. Glucose transport activity is expressed as the percent of control activity, which wasthe rateobtained in the presence of insulin plus adenosine deaminase. For the experiment shown, the control glucose transport activity was 2.7 2 0.1 fmol/cell/min. Values represent the means k S.E. of triplicate determinations. Additions incubations to Insulin

ADA“

Others

+ +

-

None None PIA PGE, Nicotinic acid

+ + +

Glucose transport activity % of control

+ + + +

143 k 6 100 141 k 4 137 k 7 140 k 7

ADA, adenosine deaminase.

of endogenous adenosine with adenosine deaminase and the enhancement of transport by ligands for adenylate cyclase inhibitory receptors occur without changes in A-kinase activity ratios. In all subsequent discussions we will refer to that condition in which endogenous adenosine has been removed by adenosine deaminase as the“ligand-free’’ state. Effects of Isoproterenol-The effects of isoproterenol in the presence of endogenous adenosine on both glucose transport activity and A-kinase activity are illustrated in Fig. 3. Applying a protocol similar to that seen inFig. 2B, cells were

248


incubation. Isoproterenolalso produced atime-dependent ( t H 2 min) -70% inhibition of glucose transport activity (Fig. 4). The isoproterenol concentrationdependency for both the inhibition of glucose transport and the stimulation of Akinase is illustrated in Fig. 5. Note that the controlvalue for transport activity depicted in Fig. 5 represents the insulin stimulated level of the ligand-free state. Under these conditions, the isoproterenol concentration-dependent increase in A-kinase activitywas essentially paralleled by the decrease in H glucose transport activity. Half-maximally effective concen5 4- - - - - - - I - - - - - - - - 0 f I I f 0.0 trations for modulation of both responseswere approximately 15 30 30-40 nM, suggesting that both systems are mediated through TIME (mid the same P-adrenergic receptor. However, the data in Figs. 4 FIG. 3. Insulin-stimulated glucose transport activity and AFig. 3, in which kinase activityratios in isolated rat adipose cells: time course and 5 are to be contrasted with those in isoproterenol, in the presence of adenosine, leads to an inof effects of isoproterenol (ISO).Isolated cells were preincubated crease in kinase activity without any decrease in transport with 7 nM insulin for 15 min a t 37 "C and then incubated in the presence (0,A) or absence (0,A) of 3 y M isoproterenol. Cells were activity. sampled at indicated times for the measurements of 3-0-methylgluEffects of Combinations of Isoproterenol and PIA-The excose uptake and A-kinase activity ratios. Solid lines denote glucose periments depicted in Figs. 6-8, involving manipulations with 0 ) and dushed lines represent A-kinase activity transport rates (0, ratios (A, A).Results of transport rates are the means obtained from combinations of ligands for the adenosine and @-adrenergic triplicate samples. The results of A-kinase ratio are the means of receptors, demonstrate a dissociation between the effects of triplicate determinations of duplicate samples. these ligands on glucose transport activity and A-kinase activity ratios. Fig. 6 shows the effect of PIA addition to cells that had been challenged first by isoproterenol in a ligandINSULIN IS0 +ADA free environment. In the initial phase,isoproterenol inhibited k t t transport activityby -50% and elevated the A-kinase activity 2 4 ratio to 1.0. Subsequent addition of PIA rapidly (tab = 2 min) v) z restored the glucose transport to -90% of the initial insulinU lx stimulated rate but,surprisingly, had littleeffect on A-kinase I-2 activity. The data in Fig. 7 show that the temporalsequence % Z 0% u u 2 of ligand addition determined whether or not PIA lowered isoproterenol-stimulated A-kinase activity. PIA, when added together with isoproterenol, prevented the rise in kinase ac> I tivity induced by a moderately high concentration of the @kl """_" -&-" - -"A adrenergic agonist. However, addition of PIA after exposure 0 15 30 to isoproterenolfailed to reduce kinase. Despite the temporal TIME (mid dependency for achieving PIA effects on A-kinase, the adenFIG. 4. Insulin-stimulated glucose transport activity and A- osine receptor agonist consistently maintained or restored kinase activity ratiosin isolated rat adipose cells: time course glucose transport activity under all conditions tested. of effects of isoproterenol (ISO) plus adenosine deaminase Fig. 8 shows the effects of varying concentrations of both (ADA). Isolated cells were preincubated with 7 nM insulin for 15 min at 37 "C then incubated either with no further additions (0,A) isoproterenol and PIA on glucose transport activity and Aor with 200 nM isoproterenol plus 1unit/ml adenosine deaminase (0, kinase activity ratios. Two features of the data are to be

-

~

4 o l d

A). Cells were sampled at indicated times for the measurements of 30-methylglucose uptake and A-kinase activity. Solid lines denote glucose transport rates (0,0 ) and dashed lines represent A-kinase activity ratios (A, A). Results of transport ratesare the means obtained from triplicate samples. The results of A-kinase ratio are the means of triplicate determinations of duplicate samples.

stimulatedwithinsulinfor 15 min, at which point 1 I.~M isoproterenol was added. The addition of isoproterenol induced a rapid increase in the A-kinase activity ratio to 0.45 within 1 min which remained essentially constant over the E -."+~ remainder of the incubation period. Despite this substantial 1 , 0.0 O 0 ' 1 2 5 10 20 M 100 200 5w low increase in the A-kinase activity, no inhibition of glucose ISOPROTERENOL inMl transport activitywas observed over the correspondingperiod. FIG. 5. Isoproterenol concentration dependency for the inThe data depicted in Fig. 3 represent a typical experiment. hibition of insulin-stimulated glucose transport activity and However,occasionally, presumablywhenthe endogenous stimulation of A-kinase activity ratios in isolated rat adipose adenosine concentrationwas less than saturating (see below), cells inthe absence of adenosine. Isolated cells were preincubated inhibitions of glucose transport of up to 20% were observed with 7 nM insulin for 15 min a t 37 "C and then exposed to increasing of this relatively high concentration of isopro- concentrations of isoproterenol in combination with 1 unit/ml of in the presence adenosine deaminase for a further 15 min. Cells were sampled for the terenol. The effects of 200 nM isoproterenol in a ligand-free envi- measurements of 3-0-methylglucose uptake (0)and A-kinase activity (A).Transport rates are expressed as percentages of the value obronment are shown inFig. 4. Two minutes after the simulta- tained in the presence of adenosine deaminase with no added isoproneousaddition of the@-adrenergicagonistandadenosine terenol, which was 2.2 fmol/cell/min. Results of transport rates are deaminase, the A-kinase activity ratio reached the maximumthe means of triplicate samples. Results of A-kinase ratio are the value, 1.0, which was maintained for the remainder of the means of triplicate determinations of duplicate samples.

kt"""' " "

INSULIN

ISO+ADA

PIA

c

/

Regulation of Adipocyte Glucose Transport Activity + a

c

249

0

i

TIME (minl

FIG. 6. Insulin-stimulated glucose transport activity and Akinase activity ratios in rat adipose cells: time course of effects of PIA added subsequent to exposure to isoproterenol (ISO)and adenosine deaminase (ADA).Isolated cells were preincubated with 7 nM insulin for 15 min at 37 "C and then incubated with 200 nM isoproterenol plus 1 unit/ml adenosine deaminase for a further 15 min. PIA (100 nM) was then added and, at the indicated times, cells were sampled for the measurement of 3-0-methylglucose uptake (solid line) and A-kinase activity (dashed line). 0, A, cells incubated with insulin alone; 0, A, cells in the presence of insulin, isoproterenol, and adenosine deaminase; O, A, cells treated with insulin, isoproterenol, adenosine deaminase, and PIA. Transport are themeans of triplicate samples. A-kinase activity rates (0,0,O) ratios (A,A, A) are the means of triplicate determinationsof duplicate samples. A

lOOr

R 0

7

1.0

- 0.8

D

fz

- 0.6 % z

-

9s 0.4

2 D 3

- 0.2 6

-

A$?

0 ADA(lU/ml)

lSO(nM)

- i- i0

0

+

i-

50 2001000

+ + + + + +

-

i-

0

0

0.0

15 30 50 100 2001000

FIG. 7. Insulin-stimulated glucose transport activity and Akinase activity ratios in rat adipose cells: comparison of the effects of PIA when added simultaneously with or subsequently to exposure to isoproterenol (ISO) plus adenosine deaminase (ADA).A , isolated cells were preincubated with 7 nM insulin for 15 min a t 37 "C and then the indicated concentrations of isoproterenol in combination with 1 unit/ml adenosine deaminase were added in the presence (0,A) or absence (0,A) of 100 nM PIA for an additional 30 min. B, isolated cells were preincubated with 7 nM insulin for 15 min a t 37 "C and incubated for a further 15 min with the indicated isoproterenol concentrations in the presence of 1 unit/ml adenosine deaminase. Subsequently, the cells were incubated for a further 15 min in the presence (0,A) or absence (0,A) of 100 nM PIA. After a total incubation time of 45 min, cells were taken for measurements of 3-0-methylglucose uptake (0, 0 ) and A-kinase activity (A, A). Transport rates are expressed as percentages of the value obtained in the presence of insulin in the absence of isoproterenol and deaminase (3.1 fmol/cell/min). The results are the means obtained from triplicate samples. The A-kinase ratio data are the means of triplicate assays from duplicate samples.

noted. First, the PIA concentration requirementreversing for isoproterenol-induced transport inhibition was independent of the isoproterenol concentration (see "Discussion"). Second, as in Fig. 7 B , all changes in transport activity elicited by PIA Fig. 8, occurred without a change in the kinase activity ratio.

PIA (nM)

FIG. 8. Insulin-stimulated glucose transport activity and Akinase activity ratios in rat adipose cells: concentration-dependent effects of PIA addedsubsequent to exposure to adenosine deaminase plus varying concentrations of isoproterenol. Isolated cells were preincubated with 7 nM insulin for 15 min at 37 "C and then exposed for 15 min to 1 unit/ml adenosine deaminase plus isoproterenol at the following nanomolar concentrations: 0 (O),50 (A),200 (B), and 1000 (VI.Subsequently, cells were exposed to the indicated PIA concentrations for a further 15 min, resulting in a total incubation time of 45 min. Glucose transport rates are expressed as percentages of the value obtained in the presence of insulin (2.9 fmol/ cell/min) and theabsence of adenosine deaminase and isoproterenol. Results are the means obtained from triplicate samples. The toppanel shows rates of 3-0-methylglucose transport and the bottom panel depicts the corresponding A-kinase activity ratios.

top, also shows that the final level to which PIA restored transport activity was dependent upon the concentration of isoproterenol in the incubation, but seemingly independent of the kinase activity, which was maximal for all isoproterenol concentrations tested. Other Lipolytic and Antilipolytic Agents-In Table 111 the actions of isoproterenol and PIA are compared with two other lipolytic hormones, ACTH and glucagon, and two other antilipolytic agents, PGE, andnicotinic acid. As with isoproterenol (Fig. 3), ACTH and glucagon did not inhibit glucose transport activity in the presence of adenosine (Table 111). However, in aligand-free environment,allthree lipolytic hormones at maximally effective concentrations induceda 50-60% inhibition of insulin-stimulated glucose transport activity. Their effects were additive at submaximal, but not a t maximal concentrations. Furthermore, the three antilipolytic agents equally restored, to within 80-90% of the uninhibited control, glucose transport activity which had been inhibitedby isoproterenol.At submaximalconcentrations, combinations of these antilipolytic agents were additive, but not at maximal concentrations. Thus, all thehormones, both lipolytic and antilipolytic, appear t o exert their respective actions on insulin-stimulatedglucose transport activity interchangeably,suggesting mediation by common mechanisms distal to the initial hormone receptor interactions. Effects of BtSAMP-The data in Table IV compare the effects of BtzcAMP and isoproterenol on glucose transport activity. In the ligand-free state, Bt2cAMP inducedonlya 20% inhibition of transport compared to the 63% inhibition by isoproterenol. However, inthe presence of adenosine,


250

TABLE III Effects of various lipolytic and antilipolytic hormones and agents on insulin-stimulated glucose transport activity in isolated adipocytes Adipocyteswere incubated for 15 min in the presence of 7 nM insulin, and then for an additional 15 min in the further presence of the ligands indicated. Values represent the means f S.E. of triplicate determinations, expressed as a percentage of the transport rate of insulin-stimulated cells measured in the presence of adenosine deaminase but without other additions. The control values for glucose transport activity were 2.3 and 2.7 fmol/cell/min, respectively, for ExDeriments A and B. Incubation condition

Adenosine deaminase

Isoproterenol

1 unit/ml

200 nM

Other additions

Glucose transport activity % ' of control

Experiment A

+

+ + + + +

None None Isoproterenol, 1000 nM ACTH, 100 nM Glucagon, 1000 nM Isoproterenol, 15 nM Isoproterenol, 1000 nM ACTH, 1.5 nM ACTH, 100 nM Glucagon, 15 nM Glucagon, 1000 nM Isoproterenol, 15 nM

+

ACTH, 1.5 nM Isoproterenol, 15 nM

-

-

+

+

glucagon, 75 nM Isoproterenol, 1000 nM + ACTH, 1000 nM

+

100 143 +- 6 136 f 6 133 & 9 139 f 3 79 f 9 44 f 6 76 rt 7 39 f 3 73 f 6 51 5 6 47 rt 7 56 f 6 36 k 3

Experiment B

+

-

+ + + + + + + +

+

+ " "

a

-

None None + None + PIA, lo00 nM + + PIA, 1 nM + + NA," 1000 nM + + NA, 100 nM + + PGE1,lO nM + + PGE,, 0.5 nM + + PIA, 1 nM NA, 100 nM + + PIA, 1nM + PGE,, 0.5 nM + + PIA, 1000 nM + NA,"'iOOO nM __-_

-

+

+

100 143 k 7 46 -t 7 125 -t 9 64 -C 11 124 k 10 80 -t 9 94 f 7 57 -C 3 102 f 4 93 f 14 131 f 4

NA, nicotinic acid.

Bt,cAMP inhibited transport activity by 47% whereas isoproterenol induced only a 16% inhibition. Thus, the effects of Bt,cAMP and isoproterenol on glucose transport activity are modulated differently by adenosine. Subcellular Distribution of Glucose Transporters-A previous report from this laboratory suggested that isoproterenol in combination with adenosine deaminase induced botha reduction in the intrinsic activity of the glucose transporters in the plasma membrane and an impairment of the insulininduced translocation of glucose transporters from the intracellular pool to theplasma membrane (16). Applying the new incubation protocols as described in Fig. 5, Fig. 9 shows that these conclusions require re-evaluation. As previously reported, insulin's stimulatory effect on glucose transport activity in the presence of adenosine was accompanied by an

TABLE IV Comparison of the effectsof isoproterenol and Bt,-cAMP on insulinstimulated glucose transport activity in isolated rat adipose cells Isolated adipocytes were incubated for 15 min with 7 nM insulin followed by a further 15-min incubation in the presence or absence of 1 unit/ml adenosine deaminase and the additions as indicated in the Table. Transport activities are expressed as the percent of control, which is the activity measured in the presence of adenosine deaminase plus insulin. For this experiment the control glucose transport rate was 3.2 & 0.21 fmol/cell/min. Values shown represent the means ? S.E. of triplicate determinations. Incubation condition

Control Isoproterenol, 1 h~ Bg-CAMP, 1 mM

Adenosine deaminase

-

+-

+ +

Glucose transport activitv % of control

154 f 7 100 130 & 4 37 -t 3 82 f 7 80 f 5

approximately 2.5-fold increase in the concentration of glucose transporters in the plasma membranes, as assessed by cytochalasin B binding. However, incubation with insulin in a ligand-free environment didnot alter the plasma membrane glucose transporter concentration despite a 30% decrease in glucose transport activity observed in the intact cell. Moreover, in contrast to previous observations, the addition of isoproterenol in the absence of adenosine did not induce a significant decrease in the number of transporters in the plasma membranes, despite eliciting a 70% reduction in insulin-stimulated 3-0-methylglucose transport activity in intact cells. Finally, addition of PIA subsequent to isoproterenol stimulation restored transport activity to 80% of the initial activity seen with insulin plus adenosine, again without changing the concentration of glucose transporters in the plasma membranes. Correspondingly, the insulin-induced 60% reduction in transporters residing in the low density microsomal fraction was not modified by either adenosine or isoproterenol. Also, there were no significant changes in the low concentrations of glucose transporters found in the highdensity microsomal fraction inthe presence or absence of any of the above agents (data notshown). DISCUSSION

Rat adipocyte adenylate cyclase is regulated by two opposing circuits, each containing acomplement of stimulatory (R.) and inhibitory (RJ receptors and their associated GTP-binding regulatory proteins, N, and Ni, respectively (28-30). The R, and Ri receptors have been shown to regulate the CAMPindependent antilipolytic insulin effect in adipose cells, leading to theproposal that theR . N circuits impinge on processes other than adenylate cyclase (31). In this report we have attempted to answer two questions. First, with the use of Akinase activity ratios to monitor cellular CAMP concentrations, can oneattribute R and Ri effects on glucose transport activity to changes in CAMP?Second, we asked whether these R, and Ri effects resulted from changes in the subcellular distribution of the glucose transporters or from changes in intrinsic activity of the transporter. In both cases, we employed maximally insulin-stimulated cells to optimize our ability to measure small changes in transport activity. However, in our previous study (16),qualitatively similar effects of F& and Ri ligands were observed in both basal and insulinstimulated cells. The evidence appears to suggest that R, and Ri modulate the intrinsic activity of the plasma membrane glucose transporter througha CAMP-independent mechanism.

251


2.5

?Lrn 2

4

2.0 15

8; 3 % 1.0 25 ?

50

50

g;a

4o

40

;g

30

30

20

20

10

10

rnE 4-

0.5

7

4

60

t rn

0 zE C

+F

:j

(0

W

a

LOW DENSITY MICROSOMES

PLASMA MEMBRANES

GLUCOSE TRANSPORT ACTIVITY

> 0

0.0 BASAL INS

0

0

ADA ADA ADA IS0 I S 0

BASAL INS INS INS INS ADA ADA ADA IS0 I S 0

PIA

PIA

INS

INS

INS

BASAL INS

INS INS INS ADA ADA ADA IS0 I S 0 PIA

FIG. 9. Subcellular distribution of glucose transporters in plasma membranes and low density microsomal membranes from adipose cells exposed to various incubation conditions. Isolated cells from 12-15 rats were suspended in 72 ml of incubation buffer (see “Experimental Procedures”). Basal cells were incubated in the absence of any addition for 30 min. All insulin-stimulated cells ( I N S ) were incubated for an initial 15 min with 7 nM insulin, followed by a further 15 min with the indicated additions: 1 unit/ml adenosine deaminase (ADA);200 nM isoproterenol (ZSO). Where indicated 100 nM PIA was added for a further 15 min. Immediately prior to homogenization, triplicate aliquots of cells were sampled for determination of 3-0-methylglucose transport activities. Plasma membranes and low density microsomal membranes were prepared and the concentrations of glucose transporters determined by specific D-glucose-inhibitablecytochalasin B binding. Values for cytochalasin B binding represent the means S.E. of the single values obtained by Scatchard analysis in at least three separate experiments. Transport activities are the means & S.E. of three separate experiments.

Although not as clear-cut as the data with the Ri effectors, Consider first theeffects of adenosine on glucose transport rates.Insulin-stimulatedtransportactivityis significantly the data presented herein argue against cAMP involvement in glucose transport inhibition by R. ligands, such as isoprodecreased upon removal of theadenosinethatinevitably appears as a contaminant of isolated adipocyte suspensions, terenol, the limitationbeing the inevitable increase in cAMP especially at the relatively high cell concentrations used rou- and A-kinase activity upon application of an R, stimulant. tinely in glucose transport studies (Figs. 2 , 7, and 8; Table 11) Nevertheless, the following disparities may be noted. First, (26). When added tocells in the ligand-free state (adenosine stimulation of kinase activity ratios to nearly 0.5 by isopromediumdoes notinhibit removed), the adenosine receptor agonist PIA enhancesglu- terenolinanadenosine-replete cose transport activity by 40%. Despite these large fluctua- transport activity (Fig. 3). However, with increasing isoproterenol concentrations in an adenosine-freemedium, signifitions in glucose transport activity, A-kinase activity ratios remain low (10.05) and unchanged,which is explainedby the cant inhibitionof glucose transport is seen as the kinase ratio presence of supramaximal insulin concentrations (See Fig. approaches 0.5 (Fig. 5). Similarly, the inhibitionof transport 2 C ) . Another apparent A-kinase-independent effect of the by isoproterenol in an adenosine-freemedium persists when, adenosinereceptoragonistis observed upon fixing kinase under slow cell mixing conditions, the A-kinase activity ratio ratios at varyinglevels by adding a n R, ligand (isoproterenol) declines to 0.19-0.41 (Table I). Thus, one may activate Ato with isoproterenol and,depending to cells in an otherwise ligand-free environment (Figs. 6-8). kinase activity ratios 0.5 That is, if one first establishes inhibited transport rates and on the incubation conditions, see either a marked inhibition high kinase levels with isoproterenol, subsequent addition of or no effect on glucose transport activity. PIA nearly restores transport activity but the kinase activity Second, dependent on the incubation condition (compare remains elevated and unchanged (see below). Finally, with Figs. 5 and 8), progressive glucose transport inhibition may be seen with increasing isoproterenol concentrations beyond lipolysis, an R,- and Ri-mediated process linked tightly to cAMP metabolism, increasing concentrations of Ri ligands those required to achieve kinase ratios of 1.0. Finally, the are required to reverse the actions of increasing concentra- observation that R, reversal of the effects of isoproterenol apparently occurs through a CAMP-independentprocess tions of R. ligands(21). However, thePIAconcentration required t o reverse transport inhibition by increasing isopro- tends toraise doubts about the role of cAMP in the mediation terenol concentrations does not vary (Fig. 8). Therefore, inof transport inhibition by isoproterenol in the first instance. sofar as A-kinase activity ratiosreflect cellular cAMP concen- Taken together, these results stronglysuggest a CAMP-indetrations,adenosinereceptor-mediatedchangesin glucose pendent inhibition of glucose transport by P-adrenergic effectransport in insulin-stimulated adipocytes are not mediated tors. Analogous with the Ri system discussed above, the findby changes in CAMP. ing that otherR, ligands, such as corticotropin and glucagon, Ligands for other adipocyteRi receptors, such as nicotinic mimic isoproterenol and exhibit additive effects on glucose acid and prostaglandins have been shownmimic to the effects transport a t submaximal concentrations (Table 111) suggests of adenosine and PIA on transport activity both in the absence that theR, receptors modify transport activityvia their GTP (Table 11) or presence of isoproterenol (Table 111). Further- regulatory protein, N,. a t submaximal more, the effects of these agents are all additive In our earlier report on the actions of lipolytic and antilipconcentrations (Table 11). Thus, it is likely that the CAMP- olytic agents on both basal and insulin-stimulated glucose independent effectsof the Ri receptors are mediated by factors transport, we reported thatisoproterenol inhibition of glucose separate from but common to all of the R, receptors, presum- transport was associated, in part, with an impairmentof the ably the Ni regulatory proteins. insulin-induced translocationof glucose transporters from the

252


intracellular pool to the plasma membrane(16). By contrast, sequence of ligand addition is critical for eliciting resistance in this paper we show that glucose transport regulation by to the adenosinereceptor agonist. This phenomenon did not isoproterenol and adenosine occurs without a change in the result from an apparent overshoot of CAMP to concentrations subcellular distribution of the transporter, indicating thatR, beyond those required to activate kinase, since resistance to and Ri receptors modulate intrinsic transporter activity within PIA was observed at kinase activity ratioswell below 1.0 (Fig. the plasma membrane. The present study employed lower cell 7). Tests on adenylate cyclase activities in purified plasma concentrations, shorter incubation times, and more vigorous membranes from suchcells revealed that they were normally cell mixingconditions than earlier studies. Another important responsive to inhibition by GTP, PIA, and other inhibitory difference is thesequence of addition of the various hormones ligands.' Whatever the mechanism, resistance R, to ligands is and agents. In this paper, we report data on cells that were elicited only in insulin-stimulatedcells, since in many experstimulated firstby insulin, following which R, and Ri ligands iments we found that cells first exposed to isoproterenol in were tested, whereas previously the R. ligands and insulin the absence of insulin were highly susceptible to subsequent were added simultaneously. Whether the different methodol- inhibition of kinase with PIA (21).3It is of interest that this ogies account for the different results remains to be deterpeculiar lack of response to PIA occurs despite an adenosine mined. receptor-mediated effect on the restoration of glucose transThe hypothesis proposed here that R, and R, ligands alter port activity, indicating that cells may differentially regulate the intrinsic activity or turnover number of those glucose different responses to R,or Ri.Ni signals. Apotentially related transporters residing in the plasma membrane is in marked anomaly is the relationship between the inability of PIA to contrast with previous studies of insulin resistance, such as seen in the high-fat fed rat (8) and thediabetic rat (9), where fully restore isoproterenol-inhibitedglucose transport and the the observed 40-50% inhibition of insulin-stimulated glucose initial degree of inhibition induced by the increasing isoproterenol concentrations (Fig. 8). Only the extentof restoration transport activity directly correlates with the concentration is of plasma membrane glucose transporters. The converse has of glucose transport activity and not the sensitivity to PIA correlated to the degree of inhibition. This absolute loss of alsobeenobserved in the youngZucker rat (33) and the hyperinsulinemic rat (34) in which insulin hyper-responsive- glucose transport activity may well be related to the, albeit ness is associated with an increase in the concentration of unphysiological, effect of BtzcAMP (Table IV). This agent also induces an absolute decrease in insulin-stimulated glutransporters in the plasma membranes. The ability readily to by the detect these changes in transporter concentration using the cose transport which isinsensitivetorestoration cytochalasin B binding assay clearly demonstrates that the antilipolytic hormones. These effects potentially reflecta assay is of sufficient sensitivity to have detected changes in temporal response of the adipose cells to elevated cAMP/Akinase activity leading to a general secondary, slowly reverstransporter distribution induced by R, and Ri ligands had they occurred. ible desensitization of the initial mechanism regulating gluMore recently, Joost et al. (35, 36) have demonstrated that cose transport. the inhibitionof insulin-stimulated glucose transport activity In summary, our data indicate that Ri, and probably R,, in the intact adipose cell induced by isoproterenol in the ligands modify glucose transport activity in isolated adipose absence of adenosine can be retained in preparations of iso- cells by a CAMP-independent mechanism(s) involving regulated plasma membranes by homogenizing the cells in the lation of the intrinsic activity of the glucose transporter in presence of KCN. Moreover, the preservationof this inhibited the plasma membrane.Of perhaps broader significance than glucose transport activity occurs without alteration of the transport regulation is the demonstration of involvement of concentration of glucose transporters in the plasma memadipocyte receptor complexes known primarily for their linkbrane, which remains unchanged with isoproterenol treatage to adenylate cyclase in events apparently unrelated to ment. These results appear therefore todirectly demonstrate CAMP, a findingreminiscent of themodulation by these the inhibitory actionof isoproterenol on the intrinsic activityreceptors of the CAMP-independent inhibition of lipolysis by of the plasma membraneglucose transporter. insulin (31). Based on arguments presentedabove and previCoupled with the realization that recent technical improve- ously (31), we speculate that in exerting their CAMP-indements in manipulating isolated adipocytes permit establishpendent actions, R, and Ri receptors act through their respecment and maintenance of steady-state A-kinase activity ratios tive G T P regulatory complexes, N,and Ni. Thus, regulation (20), an impetus for re-examining the actions of R, and Ri of the glucose transporter would join the family of other R.effectors on insulin-stimulatedglucose transport activity was and Ri-mediated, CAMP-independentprocesses, such as pthe finding that these ligands modify inhibition of lipolysis adrenergic receptor-mediatedinhibition ofM$+ transport by insulin (31). Lipolytic activity ina ligand-free environment acid is significantly less sensitive to insulin thanlipolytic activity (37) and N-formyl peptide receptor-mediated arachidonic release (38). Examples in which other insulin responsive seen in the presenceof both R, and Ri ligands (31), and such regulation applies to both CAMP-related and CAMP-inde- systems appear t o be modulated by R. N complexes include pendent insulin effects on lipolysis. Similarly, in studies pre- phosphodiesterase activation in adipose (39) and liver (40) viously reported (12, 32),we and othershave found that both cells and phosphatidylinositol turnover (41). Thus, there is a growing body of evidence suggesting that the concertedreguisoproterenol and adenosine modulate the sensitivity to insulin in initiating the glucose transport response, which might lation of lipogenesis and lipolysis in rat adipocytes involves the interactionof R -N complexes with insulin a t many levels. reflect an action of these ligands on the insulin signaling process and subsequent transporter translocation. It should Acknowledgments-We wish to thank Mary Jane Zarnowski, Dena be noted that to eliminate such effects in this study, a satuR. Yver, and Douglas L. Johnson for expert technical assistance. We rating concentration of insulin was employed in all experialso thank Drs. Soraya Naghshineh and Hans Joost for making data ments. available prior to publication. An enigma presented by the results is the inabilityof PIA to reduce A-kinase activity ratios in cells previously exposed * S. Naghshineh, unpublished observations. R. C. Honnor and C. Londos, unpublished observations. firsttoinsulinandthentoisoproterenol.Notethatthis

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