Acquisition of an Insulin-Sensitive Activity of Adenosine-3′,5′-Monophosphate Phosphodiesterase...

Eur. J. Biochem. 107, 217-224 (1980)

Acquisition of an Insulin-Sensitive Activity of Adenosine-3’,5’-Monophosphate Phosphodiesterase during Adipose Conversion of 3T3-L2 Cells Thomas MURRAY and Thomas R. RUSSELL

Department of Biochemistry and Medicine, University of Miami School of Medicine, Miami, Florida

(Received September 21, 1979/March 10, 1980)

Mouse 3T3-L2 fibroblasts differentiate when cultured and express a new phenotype which is characteristic of adipose cells. One aspect of this differentiation is the acquisition of hormone sensitivities typical of adipocytes. The sensitivity of adenosine-3’,5’-monophosphate (CAMP) phosphodiesterase to brief insulin treatment in 3T3-L2 cells at various stages of adipose conversion was examined. The capacity to increase the activity of a low-K,,, form of the enzyme in response to acute insulin exposure is a property which is acquired as the cells begin to express the adipose phenotype. Enzyme activity in preadipocytes is not affected by insulin, nor is there an effect in the non-differentiating 3T3-C2 line under any of the conditions tested. Since insulin receptors and cAMP phosphodiesterase are both present in the preadipocytes, one result of this cytodifferentiation is to effectively couple receptors to the enzyme activity.

Insulin-sensitive cAMP phosphodiesterase in 3T3-L2 adipocytes is characterized by a 40 - 50 :4 increase in enzyme activity during insulin treatment when determined at 0.1 yM CAMP. This activation results from an increase in the apparent I/ of the enzyme and does not involve a change in enzyme-substrate affinity. Maximal stimulation is seen within 2-5 min and is sustained for at least 45 min when high levels of insulin (850 nM) are used. Lower concentrations of insulin (1.7 nM) also bring about rapid activation, although the activation is not completely sustained during longer incubations. Stimulated activity falls off to about 60% of peak values by 30 min. Re-addition of insulin after 30 min raises enzyme activity back to the maximal level. Further, the insulin response is completely reversible in that the insulin-sensitive cAMP phosphodiesterase activity disappears within 10 min after removal of insulin from the cultures.

Several sublines of the established mouse 3T3 fibroblast line have been isolated through serial selec- tion on the basis of their greatly increased frequency to convert into adipose cells [l-41. This conversion is a type of cellular differentiation that can be promoted by short-term exposure of cultures to l-methyl-3-isobutylxanthine [5 ,6] , prostaglandin F z ~ [ 5 ] and dexamethasone [7]. Sublines such as 3T3-LI and 3T3-L2 differentiate with a high frequency and undergo numerous changes, both morphological and biochem- ical, as adipose conversion takes place. Expression of the differentiated phenotype has been shown to include large increases in the activities of at least twelve enzymes involved in fatty acid and triglyceride synthesis 18-15], The increase in activity of at least two of

Ahhrevzations. CAMP, adenosine 3’,5‘-monophosphate; S‘AMP, adenosine 5‘-monophosphate; QAE-Sephadex, quaternary diethyl- (2-hydroxypropy1)arninoethyl-Sephadex.

Enzyme. Adenosine-3’,S‘-monophosphate phosphodiesterase (EC 3.1.4.17).

the enzymes, acetyl-CoA carboxylase [8] and fatty acid synthetase [16], is known to result from new enzyme synthesis.

Adipocyte differentiation also includes the appearance of hormone sensitivity and responsiveness characteristic of mammalian fat cells. Adenylate cyclase in 3T3-LI cells first becomes responsive to adrenocor- ticotropic hormone during adipose conversion [I 71. Lipolytic agents, including epinephrine, isoproterenol, and dibutyryladenosine 3‘,5’-monophosphate, greatly reduce incorporation of palmitate into triglyceride [3]. Conversely, insulin, a lipogenic agent, causes increases in triglyceride accumulation [3]. Although preadipocytes have been shown to possess specific insulin receptors [7,17- 191, differentiation brings about a large increase in insulin receptor number [7,18]. Con- version also confers upon these cells an ability to demonstrate some acute metabolic effects of insulin in response to physiological concentrations of the hormone. Uptake of 2-deoxy-~-glucose and conversion

218 Development of Insulin-Sensitive Phosphodiesterase in vitro

of glucose into COz and lipid are greatly stimulated by short incubations with insulin [3,7]. Thus, in many respects these cells demonstrate metabolic responses which are qualitatively similar to those found with mammalian adipose tissue.

Adipose tissue from rats responds to intravenous injection of insulin incubations by increasing the activity of CAMP phosphodiesterase, the enzyme re- sponsible for degrading CAMP [20]. This is also true for cells isolated from epididymal fat pads where the activation is rapid, does not require protein synthesis, and alters the activity of the enzyme form having the lower Michaelis-constant for CAMP [21- 251. Studies were undertaken to determine if 3T3-L2 cells contain insulin-sensitive phosphodiesterase activity. Results indicate that phosphodiesterase activity is sensitive to insulin after adipose conversion takes place. Con- versely, undifferentiated preadipocytes do not show responsiveness to the hormone, even though specific insulin receptor and cyclic AMP phosphodiesterase are known to be present in these cells. Thus, differentiation results in the effective coupling of these two components.

EXPERIMENTAL PROCEDURES

Cell Culture

The 3T3-L2 and 3T3-C2 cell lines were provided by Dr Howard Green (Massachusetts Institute of Tech- nology). Cultures were seeded with 2 x 10“ cells/ 60-mm culture dish and fed three times weekly with 5 ml ofDulbecco’s modified Eagles’s medium (GIBCO) supplemented with 10% fetal calf serum (GIBCO), penicillin (SO units/ml), and streptomycin (50 pg/ml). Cells were maintained in a humidified 95 ‘%; air, 5 o/, COz atmosphere at 37 C.

Adipose Conversion of 3T3-L2 Cells

The cultured adipocytes were obtained by the standard procedure of Green and Kehinde [3] and by a modification of the method of Russell and Ho [5] which involves treatment of cultures with a mixture of I-methyl-3-isobutylxanthine and dexamethasone [7]. The first method involves chronic treatment of confluent cultures for 14- 17 days with 170 nM insulin [3]. Prior to the short-term studies on the effect of insulin on CAMP phosphodiesterase, cultures were changed to medium without insulin for three feedings. This feeding routine was established to minimize any effects of chronic insulin treatment on the acute effects of insulin on CAMP phosphodiesterase.

The second procedure involves treatment of confluent cultures for 48 to 72 h with a combination of 0.5 mM 1-methyl-3-isobutylxanthine plus 0.25 pM dexamethasone [7]. Cultures were then maintained

in the absence of added drugs for 4- 8 days. During the drug-free period the adipose phenotype develops to a greater extent and more uniformily than is seen with the first procedure [7]. The nature of the response was similar under both protocols, although the methylisobutylxanthine-dexamethasone treatment re- sulted in cultures in which insulin could stimulate phosphodiesterase to higher levels.

Insulin Incubations

Hormone incubations were initiated by adding insulin directly to the 5 ml of culture medium on each dish. Insulin was diluted in standard medium (Dul- becco’s modified Eagle’s medium containing 10 y ; fetal calf serum) to an appropriate concentration and in- troduced into the culture medium as a 25-kd aliquot (except where otherwise noted). Cultures were maintained in a humidified 5 % CO2 atmosphere at 37 C for the length of the incubation period.

Insulin incubations were stopped by pouring off culture medium and washing cells three times with warm (30 C) phosphate-buffered saline (pH 7.2). A fourth wash was performed with warm (30 C) ho- mogenization buffer which contained 0.25 M sucrose in 10 mM Tris-HCl pH 7.4 (unless otherwise indicated). Cells were harvested (4 ml of bufler/60-mm dish) with a Teflon scraper and disrupted in a Dounce homogenizer (15 strokes with a B pestle). Protein values were determined by the method of Bradford 1261 with ovalbumin as standard.

Pliosphodiesterase Assay

The assay for phosphodiesterase activity is a modification of the two-step procedure of Thompson and Appleman [27] which involves the measurement of adenosine formed from CAMP subsequent to hydro- lysis by phosphodiesterase and snake-venom nucleotidase. 3H-labeled CAMP is converted into 3H-labeled S‘AMP by cyclic nucleotide phosphodiesterase. The [jH]5’AMP is then converted into [’Hladenosine by a snake-venom nucleotidase (Crotalus atrox). The 0.2-ml reaction mixture for the first step was 20 niM in MgC12 and contained 0.05 ml of 0.3 pM unlabeled cAMPand 0.05ml of [3H]cAMP(5 pmol, 90000 counts/ min) in 40 mM Tris-HCI pH 7.4. The remainder of the reaction volume consisted of 0.1 ml of homogen- ization buffer (10 mM Tris in 0.25 M sucrose pH 7.4) containing an appropriate amount of enzyme. The reaction was started by the addition of enzyme solutions to the mixtures and samples were incubated for 3 min at 30°C. The mixtures were boiled for 1 min to halt the reaction and then chilled in an ice bath. All 5’AMP produced during the first reaction was converted into adenosine by incubation for 10 min at 30°C with 50 pg (0.1 ml) of snake venom. Samples were chilled and brought to a 1 ml volume by dilution

T. Murray and T. R. Russell

with 0.7 ml of H20. QAE-Sephadex A-25 column4 [28] equilibrated with 20 mM ammonium formatc pH 7.5 were employed to separate [3H]adenosine from t3H]cAMP. The 1 ml samples were poured onto QAE- Sephadex columns and eluted with 4ml of 20mM ammonium formate pH 7.5. The eluates containing [3H]adenosine were mixed with 15 ml of scintillation fluid. Radioactivity in the samples was determined with a Packard Tri-Carb liquid scintillation spec- trometer. A unit, U, of cAMP phosphodiesterase activity is defined as 1 pmol of cAMP hydrolyzed per min.

Inuctivution of Insulin

Dithiothreitol was used to reduce the disulfide bonds and inactivate insulin. The hormone was mixed with serum-medium containing 100 mM dithiothreitol and incubated for 4 h at 25°C. Hormone action was also blocked with anti-insulin serum. Guinea pig anti- (porcine insulin) serum was obtained from Dr P. H. Wright (Indianapolis, Indiana). An 11-fold excess of anti-insulin serum (as determined by its binding capacity) was incubated with insulin for 1 h at 25 'C prior to its use in the insulin stimulation experiments.

Muteriuls Dexamethasone, CAMP, Crotulus utrox snake ven-

om, ovalbumin and dithiothreitol were purchased from Sigma. 1 -Methyl-3-isobutylxanthine was obtained from Aldrich and purified by recrystallization from ethanol. [G-3H]cAMP (42.5 Ci/mmolj was from New England Nuclear and purified by chromatog- raphy on BioRad AG-50-XX. QAE-A25 Sephadex was purchased from Pharmacia.

RESULTS

Properties of Insulin-Sensitive cAMP Phosplzodiesteruse

Cultures of differentiated 3T3-L2 adipocytes dem- onslrate an ability to respond to brief incubation with insulin by increasing the activity of cAMP phosphodiesterase. The dose-response pattern of this effect for insulin concentrations ranging from 0.01 7 to 850 nM is shown in Fig. 1. Insulin was added directly to the culture medium for 3 min and phosphodiesterase activity was determined in the subsequently prepared homogenates. As little as 0.034 nM insulin produced a significant effect (P < 0.05).

Time courses of insulin action show very rapid activation of phosphodiesterase in cultures of 3T3-L2 adipocytes and vary somewhat with hormone concentration. Effects of the hormone can be detected when cells are exposed to insulin for as little as 1 rnin (Fig. 2A). Activation is near maximum by 5 min and remains

T

0 0.01 0.1 1.0 10 100 000 (Insulin] (nM)

Fig. 1. f ? j / i ~ t o/' insulin ( ~ ~ t ~ t ~ n f r i i t i o n on pl~ospliiidiesterusc. uctirirj in cultures of differenriafed 3T3-LZ cells. Insulin was diluted to the appropriate concentrations in standard medium and added as 25.~1 aliquots directly to the 5 ml of medium in each 60-mm culture dish. After incubation with the hormone for 3 min under standard conditions, culturcs were homogenized and phosphodiesterase activity was determined at 0.1 pM CAMP. Enzyme activity is expressed as a percentage of the activity in cultures receiving 25 pl of standard medium which does not contain insulin. Points represent the mean

S.E. of three separate incubations

0 K3 20 33 40 0 10 20 30 Time (min)

Fig. 2. 15flect of insulin incuhution time on pl~ospliodiesteruse ucfivitj in 373L2 udipocytes. Differentiated cultures were prepared as described under Experimental Procedure. Cells were then incubated with 1.7 pM (A) or 1.7 n M (B) insulin for the indicated times. cAMP phosphodiesterase activity in the subsequently prepared homogenates was measured using 0.1 pM cAMP as substrate. Points are averages of duplicate determinations. The patterns presented are typical of similar experiments, although the maximal stimulation varied from one experiment to another

at that level throughout a 45 min incubation with a relatively high concentration of insulin (1.7 pM).

The time course of the response was found to be somewhat different when lower concentrations of the hormone were used (Fig.2B). Initial aspects of the activation process appear to be similar regardless of the insulin concentration employed, in that the response is rapid and reaches peak levels with short incubations. Maximal activation is obtained within 2 to 3 min. However, unlike the pattern seen with high insulin, activation of insulin-sensitive phosphodiesterase activity is not sustained at peak level when

220 Development of Insulin-Sensitive Phosphodieateraae in vitro

- 200 e - c *---.

0 5 10 15 2 0 25 30 35 Time (rnin)

Fig. 3. Response of' phosphodiesteruse in insztlin-stirnuluted c,ells to addition of a secondaliquot of insulin. Cultures of 3T3-L2 adipocytes were prepared as described under Experimental Procedure. At t = 0, 0.05 pg (0-0) or 25 pg (.---a) of insulin was added to the medium of adipocyte cultures making them 1.7 n M or 850 nM with regard to the hormone. At t = 30 min, a second aliquot of insulin containing either 0.05 pg (e-0) or 25 pg (0 --.) was added to some cultures for 3 min. Phosphodiesterase activity at 0.1 pM CAMP was determined at the time indicated. Points represent the average of two determinations which differ from each other by less than 15%

Table 1. Reversihility of the insulin ej'ect on pkosplzodiesteruse Cultures received 0 or 1.7 n M insulin a t t = 0. Basal and insulin stimulated activity was determined 30 min later (A and B). Cul- tures which had been pretreated with 0 or 1.7 n M insulin for 30 min were rinsed with insulin-free medium, refed and incubated for an additional 10min in the absence of added insulin (C and D). Insulin was readded at t = 40 min for 3 rnin to cultures which had received insulin during the initial 30 min incubation prior to refeeding. Enzyme activity at 0.1 pM cyclic AMP is expressed as the mean value S.E. (n), and S.E. = 1/2 range when n = 2. Significance was assessed by Student's t test

Experimental protocol Phosphodiesterase P < activity

pmol x min-' % x mg-' control

30.6 f 0.7 (4) 100) 0.02 A. Insulin-free 30 min B. Insulin 30 min 39.3 f 2.3 (4) 129 C. Insulin-free 40 min 27.3 2.6(4) 89 D. Insulin 30 min, then

insulin-free 10 min E. Insulin 30 min, insulin-

free 10 min, then insulin 3 min 36.7 f 0.3 (2) 120

27.4 f l . X (4) 8 9 1 0.03

lower insulin concentrations (1.7 nM) are used. Rather, the stimulated phosphodiesterase activity begins to decrease at incubation times longer than 4 min. This fall-off continues such that by 30 min after addition of 1.7 nM insulin, the stimulated enzyme activity is about 40% lower than the maximal values seen at t = 2 min.

The difference in the pattern of response to low and high concentrations of insulin was of interest, as this phenomenon has not previously been reported in other systems. Experiments were conducted to deter-

mine the sensitivity of the system to additional insulin during the period of decreasing enzyme activity. Cul- tures of differentiated 3T3-L2 cells were treated for 3 or 30 min with both high (850 nM, 25 pg in 5 ml of medium) and low (1.7 nM, 0.05 pg in 5 ml of medium) insulin concentrations (Fig. 3). After 30 min of incubation a second aliquot of insulin was added to cultures which had been previously exposed to either 1.7 or 850 nM insulin. Addition of 25 pg of insulin to cultures which were pre-treated with 850 nM insulin for 30 rnin had no effect on CAMP phosphodiesterase activity over the next 3 min. In contrast, CAMP phosphodiesterase activity returned to peak levels in cultures which had received 1.7 nM insulin at t = 0 when either low (0.05 pg) or high (25 pg) insulin was used to restimulate at t = 30 min.

Just as the fall-off in phosphodiesterase levels from 3 to 30 min (Fig. 3) can be reversed by the re-addition of insulin, the enzyme activation caused by insulin can be reversed by removal of hormone from the culture medium (Table 1). Cultures of differentiated 3T3-L2 cells incubated for 30 rnin with 1.7 nM insulin (experiment B) contained 129% as much phosphodiesterase activity as controls (experiment A). Washing cells at t = 30 min, refeeding and incubating for an additional 10 min with insulin-free medium (experiment D) caused the enzyme activity to drop to slightly below control values. Thus, the effects of acute insulin incubations on phosphodiesterase activity rapidly decay upon removal of the hormone and completely disappear within 10 min. Further, cultures which were preincubated with insulin and then washed free of hormone are capable of responding to a second challenge with insulin. The readdition of 1.7 nM insulin brings about an increase in enzyme activity of 9.3 pmol x min-' x mg protein-', a difference which is equivalent to the change seen with the first insulin incubation (compare experiments D and E, and experiments A and B).

Kinetic analyses were performed to determine how insulin was affecting the K , and V parameters of the enzyme. Substrate concentrations were chosen for a range (0.1 to 1 .O pM) which would preferentially detect the activity of a low-K, form of cAMP phosphodiesterase [27,29]. Results indicate that brief incubations with insulin cause an increase in the apparent V of this form of the enzyme (Fig. 4). This is accom- plished without a change in the apparent Km value.

Evidence that it is the insulin molecule itself and not a contaminant in the hormone preparation which is causing the activation of phosphodiesterasc is given in Table 2. Adipocyte cultures respond to 1.7 nM insulin by increasing phosphodiesterase activity. In- sulin pretreated with an 11-fold excess of anti-insulin serum failed to stimulate the enzyme. Also insulin denatured by incubation with 100 mM dithiothreitol was ineffective in activating cAMP phosphodiesterase.

T. Murray and T. R. Russell

t

22 1

Table 3. Eflfltct o j insulin on pho.sppkodie.~teruse activity in 3T3-L.2 preadipocytes Cultures of 3T3-L2 cells were maintained 1-3 days pastconfluence in standard medium. Cells were incubated with or without 1.7 nM insulin for 3 min and phosphodiesterase activity was determined at 0.1 pM CAMP. Cultures in experiments 1 and 2 were homogenized in buffer containing 10 mM Tris-HC1 pH 7.4, while those in experiment 3 were homogenized in 40 mM Tris-HCI pH 7.4. Enzyme activities are expressed as the mean value f S.E. (n), where S.E. = 1/2 range for n = 2

l / [ cAMP] (piM')

Fig. 4. Kinetic analysis .f c,AMPpliospiiodiesleru.s~, activity in control (0) und insulin-treated (0) udiporytes. Cultures of 3T3-L2 adipocytes were incubated with 850 nM insulin for 5 min prior to harvesting. Homogenates were assayed for CAMP phosphodiesterase activity at substrate concentrations from 0.1 to 1.0 pM. K, and V values were calculated from points representing the average of duplicate incubations which were within 10% of each other. Velocity ( 1 ' ) is expressed as pmol CAMP hydrolyzed x min-' x mg protein-'

Table 2. E f f c t s of anti-insulin serum und ditliiotkreitol-treuted insulin on C A M P phosphodiesterase uctivity Cultures containing differentiated 3T3-L2 cells were incubated with the various solutions for 3 min at 37°C. Phosphodiesterase activity was assayed in the homogenates at 0.1 pM CAMP. Insulin treated with antiserum was prepared by incubating the hormone with antiserum for 1 h at 25°C prior to its usc in the incubations. Insulin was also denatured by incubating hormone in 100 mM dithiothreitol for 4 h at 25 "C. Cells werc treated with dithiothreitol- denatured insulin (0.5 nM dithiothreitol, 1.7 nM insulin) or an equivalent concentration of dithiothreitol. Enzymc activity is reported as the mean & S.E. of three incubations. Student's f test was used to determine significance

Additions Phosphodiesterase activity

pmol x min-' x mg-'

None 18.8 f 1.3 Insulin, 1.7 nM 31.1 f 3.2" Anti-insulin serum 18.2 f 0.8 Anti-insulin serum plus insulin 19.5 & 0.7 Dithiothreitol, 0.5 nM 16.9 f 0.9 Dithiothreitol-treated insulin 16.8 0.8

a Significant at P < 0.05. All others are not significantly different from the control.

Acquisition of un Insulin-Sensitive Plzo,splzodiesterase Activity during AdQose Conversion

Once it was established that insulin-sensitive phosphodiesterase activity is present in 3T3-L2 cells after adipose conversion, experiments were conducted to determine if this property is unique to the differentiated phenotype. Growing cells of the 3T3-L2 lines have similar morphological characteristics to those seen in the mouse 3T3 fibroblast cultures from which

~ ~~

Experi- Insulin Phosphodiesterase Stimulation ment activity

pmol x min-' x mg-' control

1 0 8.8 0.3 (3) + 10.4 f 0.5 (3) 119"

2 0 14.3 & 2.1 (2)

3 0 12.3 0.4 (2)

+ 14.5 f 0.1 (2) 102

+ 10.8 0.3 (2) 88

Specific activities are expressed as a percentage of activity in cultures not receiving added insulin.

the L2 line was derived [3]. The preadipocyte cultures remain predominantly fibroblast-like as they reach confluence and enter the non-growing state. Phospho- diesterase activity in preadipocytes 1 to 3 days past confluence is not activated by acute insulin treatment (Table 3) as it is in cells following the adipose conversion. As shown in Table 3, on some occasions slight increases in phosphodiesterase activity were seen in preadipocytes, but the increases were not repro- ducible nor were they statistically significant under the experimental conditions employed.

Development of insulin-sensitive phosphodiesterase was followed over a time course while adipose conversion was taking place (Fig. 5) . Cultures were treated at confluence for 48 h with 0.5 mM l-methyl- 3-isobutylxanthine plus 0.25 pM dexamethasone. The appearance of the response to insulin was followed after removal of the drugs. The stimulatory response to insulin appeared after day 4 in this experiment and increased in magnitude thereafter as the cells became more adipose-like. The pattern of the development depicted in Fig. 5 is typical of its appearance, although the time frame was found to vary between experiments. Batches of cells differed in the speed to which methylisobutylxanthine-dexamethasone treatment promoted the appearance of the insulin response. Similar inter- experimental variability at early times during and after drug treatment has been reported by Rubin et al. [7] with regards to the development of insulin-binding activity.

Evidence that insulin-sensitive phosphodiesterase activity was acquired as a result of the adipose conversion, and not due to prolonged maintenance of cells in the confluent state is given by experiments with

722 Development of Insulin-Sensitive Phosphodiestcrase in vitro

Table 4. Ef/icts uf insulin on pko.~pp/Iodiesterusr uctivity in 3T3-CZ cells Cultures of 3T3-C2 cells were grown to confluence and treated as indicated. In experiment A, cells were maintained 9 days past confluence on serum media alone. Cultures in experiment B received 0.5 mM methylisobutylxanthine plus 0.25 pM dexamethasone for 72 h followed by serum media alone for 6 days. Culture media in experiment C was supplemented with 170 nM insulin for 14 days beginning at confluence, followed by 6 days in the absence of added insulin. Cells were incubated with insulin for the times shown, homogenized, and phosphodiesterase activity was determined. Activities are expressed as the mean value k S.E. ( n )

___

Drugs added Insulin Incu- Phosphodiesterase after confluence bation activity

time

nM

A. No additions 0

B. Cells pre-treated 1.7

with methylisobutylxanthine + dexa- 0 methasone for 72 11 1.7

C. Cells pre-treated 0 with insulin 1.7 for 14 days 1.7

850

min pmoI x min-' x mg-'

38.4 1.6 ( 3 ) 3 37.1 F 1.4(3)

39.1 k 1.3 (3) 3 38.6 1.0 (3)

33.9 0.4 (4) 3 33.9 k 0.7 (4)

5 32.1 1.1 (4) 5 33.5 * 1.1 (4)

Yo control

100" 97

100 97

100 100 99 95

180

35

0

Time (days)

Fig. 5. Develo~miwr of' it~.rul,l~-sc~iz,sitivr i t l ios~l io~i~ , .~ /c ,~~tr . ,c . cictiiirj. in drffirentiuring 3T3-LZ c , r l l b . Confluent cultures received 0.5 m M I-methyl-3-isobutylxanthine and 0.25 pM dexamethasone for 2 days. Drugs were removed at t = 0 and CAMP phosphodiesterase activity at 0.1 pM substrate was determined at the times indicated. (A) The percentage increase in phosphodiesterase activity in cultures exposed to 1.7 nM insulin for 3 min. (B) The specific activities in insulin- treated (. ---.) and non-treated (-0) cultures. Points represent the mean * 1/2 range of two incubations

* Specific activities are expressed as a percentage of activity in cultures not receiving added insulin.

DISCUSSION

the 3T3-C2 cell line. These cells, like the 3T3-L2 cells, were derived from the original Swiss 3T3 fibroblast population [4,9]. However, the C2 cells were selected as a line which had a very low frequency of adipose conversion and remained fibroblast-like even when maintained in culture for long periods [9]. Cultures of the non-differentiating 3T3-C2 cells were tested for insulin-sensitive cAMP phosphodiesterase activity under a variety of conditions. These included procedures which were found to be effective for detecting stimulation of enzyme activity in 3T3-L2 adipocytes. As noted under experimental procedures, two methods were employed to promote expression of the adipose phenotype in 3T3-L2 cells. One involved treatment of confluent cultures with methylisobutylxanthine plus dexamethasone for 48 to 72 h. Expression of adipose conversion was also stimulated by chronic treatincnt of cultures with insulin (170 nM) for 14 to 17 days. When 3T3-C2 cultures were tested for insulin-scnsitivc cAMP phosphodiesterase activity after either of these treatments, no stimulatory effect could be found (Table 4). Thus, cultures of non-differentiating 3T3-C2 cells do not have the capacity to increase phosphodiesterase activity in response to insulin under conditions which were found to have a significant stimulatory effect on cultures of 3T3-L2 cells after adipose conversion.

The cytodifferentiation of 3T3-L2 fibroblasts into adipose cells involves the expression of a new cellular program in which much of the intracellular activity is redirected towards fatty acid metabolism. Not only is there an increase in the activities of the enzymes required for triglyceride production, but there also develops the sophisticated physiological mechanisms for regulating metabolism in fat cells. Among the hormone-dependent functions that develop is a cAMP phosphodiesterase activity which is sensitive to insulin.

3T3-L2 preadipocytes contain both specific insulin receptors [7,18,19] and low-K, CAMP phosphodiesterase activity. Prior to adipose conversion, insulin has no acute effects on the activity of the enzyme. Thus, part of the expression of the differentiated phenotype involves development of the mechanisms needed to eirectively couple the hormone aiid the response. This coupling capacity begins to appear as the cells differentiate and take on the morphological appearance of developing fat cells. Further, the coupling is a property specific to the differentiated phenotype, as insulin has no effect on phosphodiesterase in non- differentiating 3T3-C2 cells under any of the conditions employed.

Insulin-sensitive phosphodiesterase activity can first be detected 3-5 days after a 48-11 treatment of preadipocyte cultures with 1-methyl-3-isobutylxan-

T. Murray and T. R. Russell 223

thine and dexamethasone. Shortly after drug treatment there is also a large increase in the number of insulin-binding sites [7]. The proximity in time of appearance suggests that these two events might be related. However, the extent to which the increased hormone binding capacity facilitates the appearance of insulin-sensitive phosphodiesterase activity is yet to be determined.

Results reported here indicate that 3T3-L2 cells develop an insulin-sensitive phosphodiesterase activity during adipose conversion that is similar in many respects to the activity found in fat cells isolated from animals [21- 251. Cyclic AMP phosphodiesterase from adipose tissue is known to exist in more than one kinetic form, and it is the form having the lower Kn, (0.1 to 1.0 M) which responds to insulin treatment. Loten and Sneyd [21], and later others [23,25] reported that physiological concentrations of insulin (0.01 to 5 nM) were effective in rapidly elevating CAMP phosphodiesterase activity. Likewise, the low-k:, phosphodiesterase in cultured 3T3-L2 adipocytes also responds to physiological levels of the hormone. Concentrations as low as 0.034nM bring about a significant increase in phosphodiesterase under assay conditions designed to preferentially detect the activity of the low-K, form.

Phosphodiesterase activation in 3T3-L2 adipocytes as a function of insulin incubation times showed two distinct phases under the experimental conditions employed here when either high (850 nM) or low (1.7 nM) concentrations of insulin was used. There is an initial rapid increase in enzymatic activity when 850 nM insulin is used to stimulate phosphodiesterase. Within 2-5 min the system enters a steady state and activity remains constant at peak levels during longer incubations for up to 45 min. Other investigators who have looked at the effects of varying insulin incubation times on the enzyme from isolated adipocytes have reported activity patterns similar to the one obtained here with high (850 nM) insulin. Loten and Sneyd [21] using 5.5 nM insulin, and Manganiello and Vaughan [22], using 6.9 nM insulin, both found a rapid activation to maximal levels which were maintained during longer incubations with the hormone. Further, rat liver also contains insulin-sensitive phosphodiesterase activity [30,31] and the time course of enzyme activation in thal tissue follows the same pattern [31].

The time course of phosphodiesterase activity ob- served in 3T3-L2 adipocytes in response to more physiological levels of insulin (1.7 nM) differs from that seen with higher levels of insulin. Although initial aspects of the activation are similar, the second phase of the response is characterized by a slow decay in activity which begins soon after maximal levels are reached. The reason for this time-dependent decrease remains unknown at this time. However results of experiments in which a second aliquot of insulin was

added after an initial 30 min incubation with 1.7 nM insulin (10 ng/nd) indicates that the fall-off in stimulated activity can be completely reversed. Introduc- tion of as little as 10 ng/nil of insulin to treated cultures at t = 30 inin rapidly returns phosphodiesterase activity back to maximal levels, and demonstrates that the cells are still fully responsive to low levels of the hormone.

Acute effects of insulin on D-glucose and amino acid transport occur within minutes, are readily reversible, and depend upon concurrent binding of insulin to specific receptors [32]. Activation of phosphodiesterase by insulin fits into this category of responses to the hormone. Not only is there rapid activation of the enzyme, there is also a complete reversal of the stimulation when insulin is removed. Further, cells which have been stimulated with insulin and then washed free of the hormone possess the ability to respond to a second challenge with insulin by increasing phosphodiesterase activity. Thus, the results presented here are all consistent with a need for the cells to be in direct contact with the hormone ror insulin-sensitive phosphodiesterase activity to be detected.

In conclusion, i t has been shown that phosphodiesterase activity in 3T3-L2 adipocytes increases in response to brief exposures to insulin. The hormone has this effect on 3T3-L2 cells only after adipose conversion takes place, even though both phosphodiesterase activity and specific insulin receptors are present in preadipocytes. Mechanisms develop during cytodifferentiation which effectively join these two cellular components. The 3T3-L2 cell line, therefore, presents a unique model system for studying not only the nature of this hormonal response, but also its development.

This work was supported by the National Institutes of Health (USPHS-AM-21575). In partial fulfillment by T. Murray for the requireiiicnts for the degree of Doctor of Philosophy.

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T. Murray and T. R. Russell, Department of Biochemistry, University of Miami School of Medicine. P.O. Box 016129, Miami, Florida, U.S.A. 33101

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Acquisition of an Insulin-Sensitive Activity of Adenosine-3′,5′-Monophosphate Phosphodiesterase...

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