CAMP-dependent Protein Kinase and Lipolysis in Rat Adipocytes

Transcription

1 THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 260, No. 28, Issue of December 5, pp ,1965 Printed in U.S.A. CAMP-dependent Protein Kinase and Lipolysis in Rat Adipocytes 111. MULTIPLE MODES OF INSULIN REGULATION OF LIPOLYSIS AND REGULATION OF INSULIN RESPONSES BY ADENYLATE CYCLASE REGULATORS* (Received for publication, May 8, 1984) Constantine Londosg, Rupert C. Honnor, and Gurpreet S. Dhillon With the technical assistance of Douglas L. Johnson From the Section on Membrane Regulation, Laboratory of Cellular and Developmental Biology, National Institute of Arthritis, Diabetes, and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland The relationship between CAMP-dependent protein kinase (A-kinase) activity ratios and lipolysis in the presence of insulin was compared to the standard relationship between these two parameters established with a variety of adenylate cyclase modulators (Honnor, R. C., Dhillon, G., and Londos, C. (1985) J. Biol. Chem. 260, ). Three phases of insulin action were observed. First, when tested in control cells exhibiting A-kinase activity ratios up to approximately 0.25, insulin inhibition of lipolysis could be accounted for by the decrease in A-kinase activity. Second, in cells exhibiting A-kinase activity ratios >0.3, the decrease in kinase activity by insulin did not account for the decrease in lipolysis. Finally, as the A- kinase activity ratio approached 0.6 the insulin effect on lipolysis was lost. The data suggest that protein phosphatase activation accounts for the CAMP-independent insulin action. Moreover, the insulin effect not accounted for by a decrease in A-kinase activity appears to be elicited only upon elevation of A-kinase activity. The method by which cells were stimulated determined the IC5,, for insulin inhibition of: 1) A-kinase activity ratios, 2) lipolysis explained by the decrease in A-kinase activity ratios, and 3) lipolysis not explained by a decrease in A-kinase activity ratios. For all three parameters, cells stimulated by lipolytic hormones were approximately 5 times more sensitive to insulin than cells stimulated by incubation in a ligandfree environment achieved with adenosine deaminase; insulin ICso values were approximately 120 and 600 PM, respectively. Such data establish a link between insulin actions in modifying camp concentrations and in modifying events apparently independent of changes in CAMP. It is proposed that the receptors and regulatory components associated with adipocyte adenylate cyclase are associated also with components of the insulin response system separate from cyclase. Since the discovery that insulin lowers camp in fat pads (1) and in isolated adipocytes (2, 3), numerous investigators have attempted to determine whether or not the effect of insulin on cyclic nucleotide concentration accounts for the * The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. $To whom reprint requests should be sent: NIADDK, LCDB, NIH, Building 6, Room B1-16, Bethesda, MD antilipolytic actions of the hormone (for review, see Refs. 4-9). The realization that only small changes in camp concentrations are necessary for the response to lipolytic hormones has led to the use of CAMP-dependent protein kinase as a means of measuring indirectly that CAMP which might be relevant to physiological responses. With the use of A-kinasel activity ratios as an index of cellular camp concentrations, it has been reported that the antilipolytic actions of insulin in antagonizing lipolytic stimulation by corticotropin (10) receptors may be accounted for by insulin s ability to decrease cellular camp concentrations. In no case was it observed that insulin altered the relationship between A-kinase activity and glycerol production. In the preceding papers, methods were described for preparing adipocytes that behave in a highly predictable manner (12) and the steady-state relationship between A-kinase ac- tivity ratios and lipolysis was defined with a number of ligands, both inhibitory and stimulatory, thought to act on the adenylate cyclase system (13). This paper describes attempts to determine whether or not one may relate the antilipolytic actions of insulin to its ability to lower A-kinase activity. EXPERIMENTAL PROCEDURES Materials-Insulin was purchased from the Eli Lilly. PIA was from Boehringer Mannheim, ACTH 24 from Ciba-Geigy, and nicotinic acid from Eastman Kodak. Sources for other materials are described in the preceding papers (12,13). Methods-Detailed methodologies for all procedures employed in these studies may be found in the preceding papers (12,13). Briefly, epididymal adipocytes from g Sprague-Dawley rats fasted for h were prepared and suspended in Krebs-Ringer media bufferred with 25 mm Hepes, ph 7.4, containing 200 nm Ado, 2.5 mm CaC12, and 2 mm glucose. Cells were prepared in 1% BSA, but subsequent incubations were in 4% defatted BSA solutions. Typically, the final cell suspension prior to delivery to incubation vials contained 1 ml of packed fat cells/lo to 15 ml of medium. Incubations were initiated by introducing 100 pl of the adipocyte suspension into prewarmed, 37 C, incubation vials containing 1 unit/ml adenosine deaminase plus the various lipolytic stimulators and inhibitors. Con- centrations of specific ligands are indicated in the legends to the figures. Unless otherwise indicated, incubations were carried out for 25 min. Cells were homogenized and cell extracts were prepared as described previously (12). A-kinase was assayed and corrections were made for non-a-kinase activities, and lipolysis was determined by measuring the glycerol content in 100-pl aliquots of the cell extracts (12). The abbreviations used are: A-kinase, CAMP-dependent protein kinase; PIA, N6-[R-(-)-l-methyl-2-phenethyl]adenosine; adrenocorticotropin 1-24; Hepes, 4-(2-hydroxethyl)-l-piperazineethanesulfonic acid; BSA, bovine serum albumin.

2 15140 Insulin Effects on Metabolism Adipocyte RESULTS The general scheme for experiments presented in this paper is as described in the preceding paper (13). For reasons discussed previously (12), cells were prepared and suspended in media containing 200 nm Ado. The ability of insulin to lower both glycerol production and A-kinase activity ratios was tested under two basic conditions. First, both A-kinase activity ratios and lipolysis were stimulated by Ado removal (ligand-free condition) and a range of responses was produced by varying concentrations of adenylate cyclase inhibitors. Second, both kinase activity and lipolysis were stimulated with increasing concentrations of adenylate cyclase stimulators. It should be noted that since Ado removal evokes in- creases in both glycerol production and A-kinase activity ratios, it was necessary to examine the actions of lipolytic hormones (isoproterenol and ACTH"24) in the presence of adenylate cyclase inhibitors, such as nicotinic acid or the deaminase-resistant Ado receptor analog, PIA. The actions of insulin were tested on the spectrum of responses produced by these two basic manipulations, both of which have been shown to elicit an identical relationship between glycerol production and A-kinase activity ratios (13). Insulin Effects on Isoproterenol- and ACTH"24-stimulated Adipocytes-Fig. 1 depicts insulin effects on both glycerol production and A-kinase activity ratios upon stimulation of both processes by a range of isoproterenol and ACTH1-24 concentrations. As demonstrated in the preceding paper (13), lipolysis and A-kinase activity ratios are closely related, and increased glycerol production is associated with a rise in ratio from approximately 0.05 to 0.3. Upon stimulation by either hormone with concentrations that led to A-kinase activity ratios of 0.25 or less, addition of insulin reduced both kinase and lipolysis, and the reductions in kinase activity ratios were sufficient to account for the drop in glycerol production. On the other hand, with lipolytic hormone concentrations that resulted in A-kinase activity ratios of greater than 0.25, the effects of insulin on kinase activity were insufficient to account for the reduction in lipolysis. Thus, in contrast to numerous other antilipolytic agents tested, all of which inhibited lipolysis in an A-kinase-related fashion (13), insulin appears to inhibit lipolysis both by mechanisms related to and unrelated to the decrease in A-kinase activity. Insulin Effects on Adenosine Deaminase-stimulated Adipo- cytes-seasonal differences among adipocyte preparations dictate the extent to which cells are stimulated upon Ado removal; cells isolated in the spring exhibit a higher response than those isolated later in the year (12). Thus, in order to compare insulin effects on both lipolysis and A-kinase activity over a broad response range, one may test either strongly responding cells whose responses are progressively muted with submaximal PIA concentrations (see Ref. 13) or cells isolated at different times of the year, in which case inherent properties of the adipocytes determine the level of response to Ado removal (12). The experiments shown in Fig. 2 depict the effects of insulin on cells isolated at different times of the year, although both methods produced essentially identical data. The A-kinase activity ratios rose to only 0.18 with the cells tested in panel A, producing approximately 50% of maximal lipolysis, whereas those tested in panel B showed a relatively strong kinase response, 0.46, and maximal lipolysis 0 / I I I I I, B i A-Kinase activity ratio FIG. 1. Antilipolytic effects of insulin in hormone-stimu- A-Kinase activity ratio FIG. 2. Antilipolytic effects of insulin in ligand-free-stimlated cells are both related to and unrelated to the decrease in ulated cells are both related to and unrelated to the decrease A-kinase activity ratios. Adipocytes, 7.0 and 8.8 p1/800 pl of in A-kinase activity ratios. Adipocytes, 9.8 and 7.8 pl of cells/800 incubation medium in panels A and B, respectively, were incubated pl of medium in panels A and B, respectively, were incubated for 25 for 25 min in the presence of 1 unit/ml adenosine deaminase and 100 min with 1 unit/ml adenosine deaminase. The formats for panels A nm PIA. Control incubations are indicated by solid circles, and open and B are identical. The uppermost solid circles (C) represent the circles show results with insulin. Panel A, both solid and open circles, control values, indicating the absence of either PIA or insulin. Readreading from left to right, indicate increasing concentrations of iso- ing from right to left, solid circles represent increasing concentrations proterenol in the following order: 0, 10, 20, 40, 80, 160, 320, and 640 of PIA; the order for panel A was 0, 0.25,0.5, 1, 4, and 100 nm and nm. Dashed lines lead from control data to insulin-replete data for for panel B, 0, 1,2,4,8, and 100 nm. Again, reading from right to left, each concentration of isoproterenol tested. The insulin concentration open circles represent increasing concentrations of insulin; nanomolar was 2 nm. Panel B, the protocol and presentation are similar to panel insulin concentrations are shown in the figure. Panel A shows an A, but here 0 and 0 indicate increasing concentrations, experiment performed in September in which incubation with adenreading from left to right, in the following order: 0, 1, 2, 4, 8, 16, 32, osine deaminase resulted in an A-kinase activity ratio of 0.18 and 64,128, and 256 nm. The insulin concentration was 0.67 nm. Lipolysis lipolysis was only 47% of the maximal glycerol production as deteris expressed as the per cent of maximal glycerol release, which was mined by incubation with adenosine deaminase plus 10 pm isoproterdetermined with the highest concentration of either lipolytic hormone enol. Panel B shows an experiment conducted in May in which in the presence of adenosine deaminase but without added PIA. incubation with adenosine deaminase stimulated A-kinase activity Maximal glycerol release rates were 281 and 290 nmol of glycerol/25 ratios to 0.44 and lipolysis to 98% of maximum. See Ref. 12 for a min in panels A and B, respectively. discussion of seasonal variations in adipocyte behavior.

3 under Ado-free conditions. It is seen in panel A that the relationship between lipolysis and A-kinase activity ratios did not differ in cells inhibited by either PIA or insulin. Thus, as with cells stimulated to a limited degree by lipolytic hormones (Fig. l), cells so-stimulated by Ado removal were inhibited by insulin, and the effects of insulin on lipolysis do not appear to be dissociated from those on A-kinase activity. Fig. 2, Panel B, on the other hand, shows that in more strongly stimulated cells one may dissociate the insulin effects on lipolysis from those on A-kinase activity ratios. It is evident that, compared to the relationship between A-kinase activity ratios and lipolysis described by varying PIA concentrations, the decreases in A-kinase activity ratios by increasing insulin concentrations were insufficient to account for the inhibition of glycerol production. Thus, as with cells stimulated by lipolytic hormones (Fig. l), cells stimulated by Ado removal exhibit both A-kinase related and unrelated antilipolytic actions of insulin, depending on the extent to which cells were stimulated. In the above discussion of Figs. 1 and 2 we used the kinase activity ratios in control (minus insulin) incubations as a reference point for discriminating between A-kinase-related and -independent insulin actions on lipolysis. Alternatively, this discrimination may be made based on the resulting kinase activity ratio of insulin-replete incubations, in which case the A-kinase-independent antilipolytic effect becomes evident if insulin is unable to lower the activity ratio below approximately 0.15 (Figs. 1 and 2). A-kinase-related Loss of Insulin Effects-It is known that the antilipolytic action of insulin is lost upon increasing stimulation of fat cells by agents that increase CAMP production (3, 6, 14-16). If this loss of insulin action were due to overproduction of CAMP, and if A-kinase were the only target enzyme for CAMP, one should be able to relate the loss of insulin action to the A-kinase activity ratio. Fig. 3 shows that this is the case. Under conditions that permitted the A-kinase activity ratio to vary from approximately 0.15 to nearly 0.95, " Insulin Effects on Metabolism Adipocyte I the effects of two insulin concentrations, 0.67 and 6.7nM, were examined (Fig. 3). Lipolytic inhibition by the supramaxirnal insulin concentration was reversed as the kinase activity ratio reached 0.6. Several other experiments revealed that the antilipolytic effects of maximal insulin concentrations were consistently lost as the A-kinase activity ratio rose from 0.5 to 0.6. Thus, the loss of insulin action does appear to be related to the A-kinase activity ratio, and it is the CAMPindependent effect of insulin that is reversed by increased A- kinase activity. Relationship between Insulin Concentration Dependency on Lipolysis and Method of Adipocyte Stimulation-A series of studies on the concentration dependency for insulin inhibition of lipolysis revealed the surprising finding that sensitivity to insulin was dependent on the manner in which the lipolytic response was evoked. As noted previously, a common relationship obtains between A-kinase activity ratios and lipolysis when a range of responses is produced either by stimulation upon addition of lipolytic hormones or stimulation upon incubation in a ligand-free environment, i.e. addition of adenosine deaminase (13). However, as shown in Fig. 4, it is evident that lower insulin concentrations were required to inhibit lipolysis stimulated by isoproterenol than were required to inhibit lipolysis stimulated by adenosine deaminase. It is also apparent from the data in Fig. 4 that the concentration dependency for insulin inhibition of lipolysis was independent of the magnitude of stimulation; data are in marked contrast to those for other antilipolytic agents (13). Fig. 5 shows the combined data from a number of experiments similar to that depicted in Fig. 4. The insulin ICso for inhibiting isoproterenol-stimulated lipolysis derived as described in the appendix to the preceding paper (13) was 123 PM, whereas inhibition of adenosine deaminase-stimulated lipolysis exhibited an ICso of 616 PM for insulin. Thus, inhibition of cells stimulated by incubation in a ligand-free environment (Ado-free) required 5 times higher insulin concentrations than did cells stimulated by a lipolytic hormone. ' A ) I ' ' r Solid symbols t ' -Isoproterenol Open symbols -Ligand free T l E3 l I I I I = e -80 c c i n - I I I I I o A-Kinase activity ratio FIG. 3. The relationship between increasing A-kinase activity ratios and the loss of insulin antilipolytic actions. Adipocytes, 8.4 pl of cells/800 pl of incubation medium, were incubated for 25 min with 1 unit/ml adenosine deaminase and 1 nm PIA. Reading from left to right, solid circles show results with increasing isoproterenol concentrations in the following order: 0,1, 2, 4, 8, and 100 nm. Each isoproterenol concentration was incubated with either 670 (0) or 6700 (0) p~ insulin. Insulin-replete incubations may be matched with corresponding control incubations by matching symbols from left to right. In other words, the data are presented as in Fig. 1, but the dashed lines and arrows have been omitted. Maximal lipolysis, determined as described in the previous figures, was 294 nmol of glycerol produced per 25 min ' Insulin concentration. pm FIG. 4. Method of cell stimulation determines insulin sensitivity for inhibition of lipolysis. Adipocytes, 6.4 pl of cells/800 pl of medium were incubated for 25 min with 1 unit/ml adenosine deaminase. The antilipolytic effects of varying insulin concentrations were tested in cells in which the lipolytic response was varied as follows. A range of increasing lipolytic activities was produced with 100 nm PIA plus 30 (A), 100 (m), and 300 (0) nm isoproterenol. A range of decreasing lipolytic responses was produced with no PIA (O), 0.2 (U) and 0.4 (A) nm PIA. In paneel A the results are expressed as the per cent of the maximum lipolytic response, which was 211 nmol of glycerol produced per 6.4 pl of cells/25 min. Panel B shows the data from punel A with all control (minus insulin) values normalized to 100%.

4 15142 Insulin Effects on Adipocyte Metabolism m +Adenosine deaminase (Ligand-free) I 1 1 I I I I Insulin concentration, pm FIG. 5. Sensitivity to insulin is altered by dietary status and method of cell stimulation. Adipocytes were prepared from either fed (0) rats or from rats that had been starved overnight (0 and m). A range of lipolytic responses against which insulin was test was produced as follows. Cells from starved animals were incubated with 1 unit/ml adenosine deaminase alone or with deaminase plus varying PIA concentrations (m), following the general protocol shown in the legend to Fig. 4. Cells from both starved (0) and fed (0) animals were incubated also with 100 nm PIA plus a range of isoproterenol concentrations of from 30 to 100 nm. The range of lipolytic responses with each of these two basic incubation conditions was tested against varying insulin concentrations for 25 min. The figure shows all control (minus insulin) normalized to loo%, and bars indicate standard deviations. The curves were fitted and the insulin concentrations that produced 50% inhibition of lipolysis were computed according to the appendix to Ref. 12. Insulin ICs0 values (mean f S.D., number of experiments) were as follows: starved rats, isoproterenol-stimulated, 122 f 13 p ~ n, = 9; starved rats, adenosine deaminase-stimulated, 616 k 129 pm, n = 5; fed rats, isoproterenol-stimulated, 360 f 42 pm, n = 5. The different sensitivity to insulin in isoproterenol- and adenosine deaminase-stimulated cells from starved animals (p < 0,001) was maintained over a broad response range. That is, insulin concentration dependency for inhibition of lipolysis was independent of whether or not the insulin effect was accounted for or apparently unrelated to the ability of insulin to decrease A-kinase activity ratios as determined in Figs The increased insulin sensitivity in cells stimulated by a lipolytic hormone was not limited to the condition of isoproterenol in combination with PIA. Identical results were produced upon substitution of ACTH1-24 for isoproterenol when tested against a background of PIA (data not shown). Similarly, upon substitution of nicotinic acid for PIA, stimulation with either isoproterenol or ACTH1-24 resulted in increased glycerol release that was more sensitive to insulin inhibition than that produced by Ado removal. Moreover, the ICs0 for insulin was PM in inhibiting lipolysis produced by all ligand combinations (isoproterenol or ACTH1-24 plus PIA or nicotinic acid) used to stimulate lipolysis, whereas the ICs0 Insulin concentration, pm for insulin inhibition of adenosine deaminase-stimulated li- FIG. 6. Sensitivity to insulin of A-kinase activity ratio inpolysis was consistently p ~ Thus,. increased sensi- hibition altered by method of cell stimulation. Adipocytes were tivity to insulin was independent of the specific hormones or incubated with 1 unit/ml adenosine deaminase alone (0 and 0) or agents employed to elicit stimulation of cells, and all ligand with 100 nm PIA plus either isoproterenol at 100 (0) and 1000 (m) nm, or 10 nm ACTH"24 (A). The elevated A-kinase activity ratios combinations were easily distinguishable from the Ado-free produced in this way were tested against varying insulin concentra- (ligand-free) condition. tions for 25 min. The data have been normalized so that all control Relationship between Insulin Concentration Dependency on (minus insulin) conditions are 100% and the A-kinase activity ratios A-kinase Activity Ratios and Method of Adipocyte Stimula- tested with the maximum insulin concentration are zero. The maxima tion-based on the evidence that a common relationship (control) and minima (plus insulin) for each experiment are as follow: adenosine deaminase-stimulated, 0, 0.18 and 0.08; 0, 0.44 and 0.28; obtains between A-kinase activity ratios and glycerol producisoproterenol-stimulated, 0, 0.19 and 0.10; a, 0.41 and 0.22; and tion upon stimulation of adipocytes by either addition of lipolytic hormone or by removal of inhibitory ligands, it was concluded that these two modes of lipolytic stimulation pro- ceed via a common pathway, i.e. increased cellular CAMP concentration. Also, as shown above, cells stimulated by either method exhibit antilipolytic effects of insulin that appear both related and unrelated to the insulin-induced decrease in A-kinase activity (Figs. 1 and 2). However, it is clear that lower insulin concentrations are required to inhibit lipolysis stimulated by lipolytic hormones than are needed to inhibit lipolysis stimulated by Ado removal (Figs. 4 and 5). Consideration of these phenomena brings forth the following questions: 1) are the different concentration dependencies for insulin action on lipolysis reflected in the insulin requirements for depression of A-kinase activity ratios, and 2) are the insulin requirements for decreasing lipolysis governed by whether or not the decrease in lipolysis is related to decreased kinase activity? Fig. 6 reveals that lower insulin concentrations were required to inhibit A-kinase activity ratios stimulated by lipolytic hormones than were required to inhibit kinase activities elevated as a result of Ado removal. The calculated ICso value for insulin inhibition of hormone-stimulated A-kinase activity ratios was approximately 105 PM, whereas that for deaminasestimulated kinase activities was 560 PM. Note that these values for insulin inhibition of A-kinase activity ratios are rather similar to the ICso values for inhibiting lipolysis under the two different conditions of stimulation (Figs. 4 and 5). Also, either isoproterenol or ACTH"'* reduced the insulin concentration requirement for inhibition of A-kinase activity ratios. Moreover, the initial control value for the kinase activity ratio (see legend to Fig. 5) had no bearing on the insulin concentration required to lower kinase activities. That is, whether tested against cells exhibiting activity ratios of less than 0.2 or greater than 0.4 in the absence of insulin, all hormone-stimulated cells required lower insulin concentra- I I I I I I Adenosine deaminase stimulated.free) ACTH1-24-stimulated, A, 0.30 and Different responses to adenosine deaminase alone were obtained in experiments performed at different times of the year: November (0) and April (0); see Ref. 12.

5 tions for kinase inhibition than did deaminase-stimulated cells. It may be concluded, therefore, that it is the method, but not the extent, of stimulation that dictates insulin sensitivity for decreasing A-kinase activity ratios in adipocytes. Increased Insulin Sensitivity of Hormone-stimulated Lipolysis Is Independent of the Extent to Which Cells Are Stimulated-The second question posed above deals with the relationship between insulin requirements for suppression of glycerol release and the extent which to cells are stimulated. That is, are the insulin requirements a function of whether or not insulin inhibits lipolysis in an A-kinase-related or A-kinaseunrelated manner? This question has been answered in part, by &ta already presented. The data on lipolytic inhibition of isoproterenol-stimulated cells shown in Fig. 5 represent the combined results of experiments on cells that exhibited control A-kinase activity ratios of from less than 0.15 to greater than 0.4. Under conditions wherein the insulin effects on depression of A-kinase activity and lipolysis were apparently related, the insulin IC,, for lipolytic inhibition was 115 f 16 (S.D., n = 5), whereas under conditions where insulin effects on kinase could not account for the decrease in glycerol production, the ICso for insulin was 136 f 26 pm (S.D., n = 4). Similar results were found upon substitution of ACTH 24 for isoproterenol as the lipolytic stimulant. In ACTH1-24- stimulated cells, the insulin IC, was 117 k 22 (S.D., n = 3) and 157 f 25 (S.D., n = 4) over A-kinase-related and -unrelated, respectively, phases of insulin inhibition of lipolysis. Thus, with hormone-stimulated cells, the concentration dependency for insulin inhibition of lipolysis is independent of the relationship between insulin effects on lipolysis and A- kinase activity ratios. Decreased Insulin Sensitivity of Adenosine Deaminase-stimulated Lipolysis Is Independent of the Extent to Which Cells Are Stimulated-As with cells stimulated by lipolytic hormones, the insulin requirement for inhibition of lipolysis in deaminase-stimulated cells was independent of the extent to which cells were stimulated. A typical example is shown in Fig. 2, where it may be seen that relatively high insulin concentrations, approximately 700 PM, were required to in- hibit lipolysis by 50% both in the case where the insulin effects on A-kinase and lipolysis were apparently related (Fig. 2, panel A) and in the case where the two effects of insulin were apparently unrelated (Fig. 2, panel B). Other experiments established that the relatively high insulin IC,, value for inhibition of deaminase-stimulated cells was applicable regardless of the extent to which cells were stimulated. Thus, the data establish a link between events apparently related and unrelated to camp in that the method for stimulating cells determines the insulin sensitivity of the following phenomena: 1) depression of A-kinase activity ratios by insulin 2) A-kinase-related inhibition of lipolysis by insulin, and 3) A-kinase-unrelated inhibition of lipolysis by insulin. Dietary Considerations-The experiments in this and the preceding papers (12, 13) were conducted with adipocytes from starved rats. The primary reason for fasting the animals was to obtain a consistently strong lipolytic response upon removal of Ado from the incubation medium (12). Starving of animals did not modify lipolysis stimulated by hormones (12). On the other hand, as seen in Fig. 5, adipocytes from fed animals exhibited a lower sensitivity to insulin than did cells from starved animals. Upon stimulation with isoproterenol, the insulin ICs, (123 k 13, S.D., n = 9) for inhibition of lipolysis in cells from starved rats was significantly lower 0, < 0.001) than the insulin IC,, with cells from fed animals (360 f 42, S.D., n = 5). Unfortunately, the erratic responses in cells from animals to Ado removal (12) precluded testing Insulin Effects on Metabolism Adipocyte of insulin sensitivity of fed animals upon Ado removal. Thus, although it is clear that starvation results in altered insulin sensitivity, one may not state whether or not starvation contributes to the differences between insulin sensitivity under hormone-stimulated and deaminase-stimulated conditions. Temporal Considerations-For all experiments discussed above, adipocytes were exposed simultaneously to insulin and lipolytic stimulants. Although all of the data presented were from incubations that were terminated after 25 min, preliminary experiments established that both insulin inhibition of A-kinase activity ratios and lipolysis were established within a few minutes and maintained over the full time course. Also, maintenance of insulin effects occurred both at submaximal and supramaximal insulin concentrations. DISCUSSION The studies presented in this paper were rendered feasible by the demonstration that one may define, with the use of a variety of adenylate cyclase stimulators and inhibitors, the steady-state relationship between A-kinase activity ratios and glycerol release. A common relationship was found between these two parameters upon their modulation over a wide response range with a variety of ligands other than insulin (13). Insulin, on the other hand, exhibits both convergence with and divergence from the standard relationship between A-kinase activity ratios and lipolysis, and three distinct phases of insulin action may be defined. First, when added to cells exhibiting A-kinase activity ratios of up to approximately 0.25, a condition associated with maximal or nearly maximal lipolysis, it is not possible to dissociate insulin effects on glycerol release and kinase activity. Thus, over the stimulation range up to that just required for maximal lipolysis, one need not invoke any mechanism other than a lowering of A-kinase activity to account for lipolytic inhibition by insulin. Such data seem to establish camp depression as a key factor in insulin-mediated lipolytic inhibition. However, application of this conclusion to the physiological condition, as opposed to isolated adipocytes, must await a description of the relationship between kinase activity and lipolysis in situ. A second phase of insulin action is evident when the hormone is added to cells exhibiting A-kinase activity ratios from approximately 0.3 to 0.6, in which case resulting kinase ratios remain in the 0.15 to 0.5 range. Under such conditions, insulin inhibits both lipolysis and kinase activity, but the decrease in kinase activity is insufficient to account for the decrease in fat metabolism. From such data it may be concluded that insulin exerts antilipolytic effects independent of its ability to lower cellular camp concentrations. The findings of Nilsson et al. (17) suggest that the phosphorylation state of the hormone-sensitive lipase correlates well with the lipolytic rate under all conditions. Thus, if one excludes the possibility of an endogenous inhibitor of the lipase that may be regulated by insulin, increased dephosphorylation of the lipase might account for the A-kinase-independent inhibition of lipolysis by insulin. The final phase of insulin interaction with respect to kinase activity occurs as the A-kinase activity ratio rises to values beyond 0.5 in the presence of maximal insulin concentrations. Within a well-defined and reproducible range of kinase activity ratios, 0.5 to 0.6, insulin effects on lipolysis disappear. The ability to define quantitatively these three phases of insulin action not only answers a long-standing question on the relationship between insulin effects on cellular camp concentrations and fat metabolism, but invites the following speculation on possible underlying mechanisms of CAMP-independent insulin action.

6 15144 Insulin Effects on Metabolism Adipocyte If, in the presence of insulin, the kinase activity ratio remains at or above 0.15, insulin and the elevated kinase act in concert to activate a protein phosphatase, resulting in dephosphorylation and inactivation of the hormone-sensitive lipase. At yet higher kinase activities, phosphorylation of lipase by the kinase proceeds at a rate sufficient to overcome the increased phosphatase activity. The unique feature in such a formulation is the notion that insulin-dependent activation of enzymes that dephosphorylate lipase is facilitated by increased A-kinase activity. Such a proposal is based on: 1) the failure to detect greater lipolytic inhibition by insulin than that predicted by the reduction of A-kinase activity upon challenging cells whose kinase ratios were less than 0.3; and 2) the kinetic features, under insulin inhibition, of the relationship between A-kinase activity ratios and lipolysis as the kinase activity is varied. On this latter point, it should be noted that, if insulin were to increase a lipase phosphatase independent of the A-kinase activity state, one would have observed a mere rightward shift in the A-kinase activity ratio associated with lipolysis. However, as is evident in Figs. 1 and 3, insulin promotes a divergence from the standard A-kinase to lipolysis relationship only as the kinase activity ratio approaches and exceeds Thus, it may be argued that the mechanisms by which insulin inhibits lipolysis in a manner apparently unexplained by the decrease in kinase activity may be, nevertheless, dependent on, or facilitated by, A-kinase. Although the above arguments attempt to link elevated A- kinase activity to the phenomenon whereby insulin decreases lipolysis in a manner not explained by a decrease in kinase activity, we shall, for purposes of discussion, continue to refer to this phase of insulin action as A-kinase independent or A- kinase-unrelated. An unexpected outcome of this study were the data suggesting that adenylate cyclase-linked receptors may be associated also with processes other than cyclase in adipocytes. As proposed earlier by this laboratory (18-20) and confirmed by recent biochemical data (21, 22), receptors for cyclase stimulatory (RJ and inhibitory (Ri) hormones are associated with their respective GTP-binding regulatory components, N, and Ni, providing for dual regulation of cyclase activity. The conditions used herein for lipolytic stimulation dictated either that both circuits were occupied by hormones or that neither circuit was occupied. These conditions were termed, respectively, hormone-stimulated and ligand-free-stimulated. For all parameters of insulin action investigated, we found hormone-stimulated cells 5 times more sensitive to insulin than ligand-free-stimulated cells; insulin IC5o values were 120 and 600 PM, respectively. The key observation was that the shift in insulin sensitivity applied not only to CAMP-related processes but to insulin antilipolysis not explained by a decrease in A-kinase activity ratios. Moreover, the insulin shift was independent of the specific hormone combinations employed, suggesting involvement of factors common to but separate from receptors, presumably the N components. Thus, the R,N, circuit, the RiNi circuit, or possibly both circuits seem to be involved in mediating insulin actions independent of their actions on adenylate cyclase. Unfortunately, the requirement for having an Ri ligand present in order to elicit stimulation by R, ligands precluded testing if either one or the other R. N circuit had a greater effect in producing the insulinsensitivity shift. Two general mechanisms may be envisioned to explain the insulin-sensitivity shift. One, the %N,/RiNi circuitry may be linked to either the insulin receptor or to a signalling process promoted by the receptor. Alternatively, the R. N circuitry may be linked to different effector systems that respond to signals from the insulin receptors. Although the data do not permit discrimination between these two possibilities, the finding that both CAMP-related and CAMP-independent insulin actions were similarly modified tends to suggest a role for R. N components at or near the point of insulin receptor signal generation. Support for the above proposals is provided by recent evidence on the effects of a Bordatella pertussis toxin that modifies selectively the Ni regulatory component associated with adenylate cyclase (21, 22). This toxin reduces or elimi- nates insulin stimulation of a phosphodiesterase (23) and enhances insulin stimulation of phosphatidylinositol turnover (24) in adipocytes, two effects not thought to be the result of insulin depression of cellular camp concentrations. Also, it has been reported that both insulin activation of phosphodiesterase (25) and inhibition of adenylate cyclase (26) in isolated hepatic membranes is regulated by GTP. Thus, it is not unreasonable to suggest that the regulatory components associated with adenylate cyclases may be associated also with the insulin receptor or its associated effector systems. Maguire and colleagues (27) reached a similar conclusion upon finding that 8-adrenergic-mediated inhibition of magnesium ion transport requires a functional N component, but is observed in A-kinase-deficient mutants. Finally, the recent findings of Bokoch and Gilman (28), showing that pertussis toxin inhibition of n-formyl peptide actions on neutrophils is not ex- plained by changes in CAMP, support a general, cyclaseindependent role for the GTP regulatory proteins. It should be noted that any direct effects of insulin on the adenylate cyclase system would be mediated by circuits other than the Ni circuit described above, since adipocytes were highly responsive to insulin in these studies even in the presence of maximal or supramaximal concentrations of PIA or nicotinic acid, ligands thought to operate via the Ni mechanism. This conclusion is reinforced by recent data showing that elimination of a functional Ni circuit by pertussis toxin treatment enhances adipocyte sensitivity towards insulin (29). 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