The Metabolism of Isolated Fat Cells

THE JOURNALS OF BIOLOGICAL CHEMISTRY Vol. 241, No. 17, Issue of September 10, pp. 39093917, 1966 Printed in U.S.A. The Metabolism of Isolated Fat Cells IV. REGULATION OF RELEASE OF PROTEIN BY LIPOLYTIC HORMONES AND INSULIN MARTIN RODBELL (Received for publication, March 10,1966) From the Section on Endocrinology, Laboratory of Nutrition and Endocrinology, National Institute of Arthritis and Metabolic Diseases, National Institutes of Health, Bethesda, Maryland ZOO14 SUMMARY Proteins of fat cells, isolated from rat epididymal fat pads, were labeled with 14Chistidine or 14Cleucine. The fate of the labeled protein was examined after subsequent treatment of the cells with hormones. Release of labeled proteins was stimulated by lipolytic hormones (adrenocorticotropic hormone, glucagon, and epinephrine) and by theophylline. Proteolysis, as measured by the appearance in the medium of trichloracetic acidsoluble radioactivity, occurred to a small extent but was not influenced by lipolytic hormones. Release of such nonparticulate enzymes as malic dehydrogenase, glucose 6phosphate dehydrogenase, lactic dehydrogenase, and hexokinase was also enhanced by lipolytic hormones. In intact adipose tissue, lipolytic hormones had the same effect on release of enzymes as observed with isolated fat cells. Release of protein was secondary to the rise in intracellular concentration of fatty acids in response to lipolytic hormones. Fatty acids, not their peroxides, were responsible for the release of protein. Isolated fat cells released protein after albumin in the medium became saturated with fatty acids. Intact adipose tissue released enzymes in response to lipolytic hormones when the concentration of fatty acids in the tissue reached approximately 16 peq per g. With isolated fat cells, accumulation of intracellular fatty acids and protein release were prevented by either providing an excess of albumin in the medium or supplying glucose to the cells, especially in the presence of insulin, which accelerated reesterification of intracellular fatty acids. It is suggested that the metabolic capacity of adipose tissue, in Vitro and in Go, is regulated in part by the release of cellular protein mediated through the intracellular concentration of fatty acids which is the product of the opposing actions of insulin and lipolytic hormones. The metabolism of adipose tissue depends, to an extraordinary degree, upon alterations in the nutritional and hormonal state of the animals. Particularly noticeable is the fall in metabolism associated with fat mobilization that occurs during either fasting or diabetes (l4). Although the relationship between hormones that stimulate fat mobilization and the fall in metabolic activity has not been clearly established in t&o, several studies in vitro indicate that lipolytic hormones, such as adrenocorticotropic hormone and epinephrine, accelerate protein breakdown (5) and decrease incorporation of labeled amino acids into proteins in adipose tissue (57). Since proteins comprise a small but highly active portion of the fat cell, it is likely that enzymes represent a large percentage of the protein in the cell. Accordingly, decreased synthesis and accelerated breakdown of enzymes in response to effects of lipolytic hormones could be major factors that regulate the metabolism of adipose tissue. Lipolytic hormones have a greater effect on isolated fat cells than on intact adipose tissue (8, 9). Accordingly, the reported stimulation of proteolysis by lipolytic hormones was reexamined with isolated fat cells. Proteins in the cells were prelabeled in vitro, and the release of labeled amino acids to the medium was measured in response to hormones. During the course of this study, it was found unexpectedly that, rather than stimulating proteolysis, lipolytic hormones stimulated the release of intact proteins from the cells to the incubation medium. Some of these proteins have enzymatic activity. This finding and its implications in the hormonal regulation of the metabolism of adipose tissue are presented in this report. EXPERIMENTAL PROCEDURES Male SpragueDawley rats (140 to 160 g) were used in this study; they were fed Purina laboratory chow ad lib&urn. The procedures used for isolating, dispensing, and incubating isolated fat cells have been described previously (10). Glucose was omitted during treatment of tissue with bacterial collagenase (Worthington) except in those experiments in which radioactive amino acids were incorporated into protein of fat cells (see below). Unless stated otherwise, fat cells or intact adipose tissue was incubated at 37, with shaking, in a final volume of 2 ml of 0.5% albumin in bicarbonate buffer, ph 7.4. The composition of the bicarbonate buffer has been described elsewhere (10). Triplicate samples were incubated in each experiment. Each experiment was carried out at least three times. Data were statistically evaluated by Student s t test. Analytical ProceduresProcedures for determining free fatty acid and triglycerides content have been reported elsewhere (10). Triglyceride content was used to determine the quantity of cells

present in each flask: 1 g of fat1 cells was set equivalent to 1 mmole of triglyceride. Fatty acid peroxides were determined by the thiobarbituric acid method (11). Radioactivity determinations were made in a liquid scintillation spectrometer with the use of a naphthalenedioxane solvent system (12). Combination of Fatty Acids with A&urn&Triglycerides obtained from extracts of fat cells were saponified. The free acids were isolated in heptane. The heptane solution was evaporated to dryness, and the fatty acids were neutralized with an equivalent amount of NaOH. Solution of the fatty acid soaps was obtained by heating at 60 for approximately 10 min. The solution was added dropwise to a constantly stirred solution of albumin in bicarbonate buffer. After standing for 15 min at room temperature, the solution was passed through a 0.22p Millipore filter. Molar ratios of fatty acids to albumin were determined from the fatty acid and protein content (13), assuming the molecular weight of bovine albumin to be 68,000. Fresh solutions of albumin combined with fatty acids were prepared for each experiment. Enzyme AssaysAll assays were based on measurements of the fluorescence of NADH or NADPH. Fluorescence was measured with an Aminco fluoromicrophotometer equipped with a compensating voltage control. The 360 rnp activation light was provided by a General Electric Blacklite (No. F4T4) and a primary filter (Aminco No. 47113). The secondary filter holder contained a Wratten 2A filter (Aminco No. 47116) which passed light above 415 rnp; a 30% neutral density filter was interposed to disperse light from particlecontaining suspensions. An attached recorder (Varicord, model 43) was operated at a chart speed of 2 inches per min. The sensitivity of the apparatus was adjusted so that 5 pm NADH would give full scale (loinch) deflection with a noise level of cl%. Enzyme activities were determined in a lml volume at room temperature (22 rt 2 ) and rates as slow as 1 x 10m3 pmole of NADH changed per min were measurable with an error of less than 10%. Lactic or malic dehydrogenase activities were determined in media containing 50 mm glycine, 240 mm hydrasine, 2 mm EDTA, 3 InM NAD, and either 10 mm c&lactate or Zmalate. Final ph was 9.4. Metabolism of Isohted Fat Cells. IV Vol. 241, No. 17 The incubation medium for glucose 6phosphate dehydrogenase activity contained 0.12 mm NADP, 2.5 mm MgS04, 50 mm Tris (ph 7.4), and 5 mm glucose 6phosphate. Hexokinase (glucoseatp transferase) was assayed RESULTS The initial phase of this study involved the question of whether containing 0.12 mm NADP, 2.5 mm MgS04,2.5 mm ATP, 50 mm or not ACTH had any effect on protein breakdown in isolated fat Tris (ph 7.4), 0.1 unit of glucose 6phosphate dehydrogenase cells. This was investigated by preincubating adipose tissue (Sigma, type IV), and 10 InM glucose. It was found later that with leucinell% or with uniformly labeled histidinel*c in the glucose 6phosphate dehydrogenase could be omitted from the reaction mixture; sufficient enzyme was present in extracts of fat cells. All reactions were initiated by the addition of substrate. Rates were determined from the initial, linear portion of the rate presence of glucose in order to label the protein of fat cells. After isolated fat cell preparations had been washed, an average of 2.4% of the radioactivity of added leucine or histidine was recovered per g of fat cells. Approximately 80% of the radioactivity recovered in the isolated fat cells was in protein (TCAcurves. One unit of enzyme is defined as the amount necessary insoluble). The results of incubation studies, recorded below, to cause reduction of 1 pmole of NAD or NADP per min. It was were the same with cells labeled with either histidine or leucine. assumed that 2 moles of NADPH were formed per mole of substrate consumed in the determinations of hexokinase and glucose 6phosphate dehydrogenase. In most experiments, enzyme activities found in incubation media and in fat cells after incubation were calculated as the percentage of enzyme activity found in homogenates of cells be fore incubation. Homogenization was carried out at room temperature with a motordriven Teflon plunger and a Pyrex test tube. Fat was removed from homogenates by centrifuging briefly and withdrawing the infranatant fluid with a Pasteur pipette. Labeling of Protein in Isolated Fat CellsEpididymal adipose tissue (500 to 800 mg) was incubated in 3 ml of 4% albumin bicarbonate medium containing 10 RIM glucose, 10 mg of collagenase, and 1 to 10 PC of either leucine114c or uniformly labeled histidine1% (New England Nuclear; specific activity, 1 mc per mmole). After incubation at 37 for la hours, fat cells were separated from stromalvascular cells by centrifugation. The fat cells were washed twice with 4a/, albuminbicarbonate medium and then three times in 0.5 y0 albuminmedium. Fat cells were dispensed into 40~ plastic vials (Nalgene) that contained 0.5% albuminmedium to give a final volume of 2.0 ml. Radioactivity was determined in both medium and cells after their separation by a brief centrifugation. One portion of the medium was added directly to scintillation mixture for counting; an equal portion was precipitated with 5% trichloracetic acid. The precipitate was washed once with 5% TCAI and, after excess TCA had been drained off, was dissolved in 0.5 ml of Hyamine 10X (Packard) and counted. TCAsoluble material was counted after the medium had been neutralized with Hyamine. Fat cells were washed once with 0.5% albuminmedium and extracted with hexaneisopropyl alcohol and dilute sulfuric acid followed by addition of hexane and water (14). Aliquots of the hexane phase were taken for triglyceride and radioactivity determinations. The lower phase was heated at 60 for 15 min. The precipitate was either dissolved directly in Hyamine and counted or dissolved with performic acid and reprecipitated with 5% TCA according to the procedure described by Fain (15). The precipitated protein was resuspended in 5 % TCA and heated for 15 min at 90 ; this was followed by centrifugation and collection of the protein. Protein was dissolved in 88% formic acid and precipitated with 5% TCA. The precipitate was dissolved in Hyamine and counted. ChemicalsThe sources of hormones were the same as previously described (10). Nucleotides and glucose Bphosphate were purchased from Sigma. Quercitin and theophylline were obtained from Mann. Albumin (bovine, Fraction V) was obtained from Armour. During the 1st hour of incubation of fat cells with ACTH, labeled protein disappeared from the cells at a linear rate which was reflected by an equal rate of appearance of radioactivity in the medium (Fig. 1). No further change occurred after 1 hour 1 The abbreviations used are: TCA, trichloracetic acid; ACTH, adrenocorticotropic hormone; FFA, free fatty acid.

Issue of September 10, 1966 M. Rodbell 3911 of incubation. Surprisingly, after incubation, 85% of the radioactivity in the medium was TCAinsoluble material. Radioactive proteins in the medium were relatively large since fractionation of the medium on Sephadex G75 columns revealed that 20% of the radioactive protein appeared in the void volume of the column and the remainder was eluted coincident with albumin. These experiments demonstrated that ACTH stimulated the release of intact proteins from fat cells. In the absence of ACTH, some labeled protein disappeared from the cells during incubation; the amount was about onethird less than that observed with ACTHtreated cells (Fig. 1). The amount lost was accounted for in the medium as both TCAsoluble and TCAinsoluble radioactivity. The amount of labeled protein released to the medium was less than 20 y0 of that released by ACTHtreated cells. ACTH had no effect on the rate or amount of labeled TCAsoluble material appearing in the medium from labeled protein in fat cells. ACTH, therefore, did not increase proteolysis of cellular protein. Comparative Effects of Adrenocorticotropic Hormone, Epinephrine, Theophylline, and Glucagon on Protein Release from Fat CellsEpinephrine, glucagon, and theophylline also stimulate lipolysis in isolated fat cells (16). As shown in Table I, they also stimulated the release of protein from fat cells to the same extent as ACTH. Theophylline was 50% as effective at 0.1 mm as at 1 mm. Concentrations of hormones less than 0.1 pg per ml had no effect on protein release in 2 hours of incubation. The finding that agents that stimulate lipolysis also stimulate prot.ein release implicated fatty acids in the release of protein from fat cells. Release of Labeled Protein, Enzymes, and Membranes from Fat Cells in Response to Adrenocorticotropic HormoneIt was expected that some of the proteins released from fat cells in response to ACTH would have enzymatic activity. One of the most active and easily assayed enzymes in fat cells is malic dehydrogenase, which is found primarily in the soluble (100,000 x g) fraction of homogenates. As shown in Fig. 2, the rate of appearance of malic dehydrogenase in the medium was proportionate to that of labeled protein; both rates were accelerated by ACTH. After 1 hour of incubation, all of the malic dehydrogenase of the fat cells was released to the medium. Similar results were obtained with glucose 6phosphate dehydrogenase, lactic dehydrogenase, and hexokinase (Table II). Hexokinase could not be measured in the medium since it was inactivated when incubated in the medium for 1 hour at 37. However, its disappearance from fat cells treated with ACTH coincided with the disappearance of the other three enzymes. Incomplete recovery of glucose 6phosphate dehydrogenase in fat cells and medium was also observed and may reflect instability of this enzyme in the medium; this was not investigated further. In response to ACTH, opalescent material appeared in the medium in increasing amounts but at a slower rate than the appearance of labeled protein or malic dehydrogenase. Change in opalescence of the medium was arbitrarily measured at 600 rnp in a spectrophotometer. The material causing opalescence was completely sedimented in 15 min at 10,000 X g. Electron microscopic examination of osmiumfixed sediment revealed the presence of undefined membranes, some of which appeared to be relatively large saclike structures. Because the prolonged incubation of the membranes in album might have changed + 8000 +ACJH 0 +6000 +4000 +2000 F E 0 u I z 2000 % 5 6 4000 6000 8000 t \ t ACTH 0 i@ MEDIUM I I I I I 0 30 60 90 120 MINUTES FIG. 1. Effects of ACTH and time of incubation on release of I%protein from fat cells. Fat cells (62 mg per flask) prelabeled with uniformly labeled histidine *C were incubated in 2 ml of 0.5% albuminmedium. At indicated times, fat cells were separated from medium. 14CProtein in fat cells was precipitated with hot TCA as described under Experimental Procedures. Radioactivity in the medium was separated into TCAinsoluble (protein) () and soluble ( ) radioactivity with 5% TCA. Radioactivity and cells at zero time incubation was subtracted from that found in incubated cells and medium. ACTH concentration was 1 rg per ml. Results are the averages for three replicate flasks. TABLE I Effects of lipolytic agents on loss of Wprotein from fat cells Fat cells (average of 60 mg per flask) prelabeled with uniformly labeled histidinej4c were incubated for 2 hours containing 0.5% albumin. WProtein represents hot TCAinsoluble material (see Experimental Procedures ) extracted from fat cells after incubation. Results are the averages for three experiments f the standard error. Lipolytic agent WProtein in cells after incubation ACTH (1 rg/ml).... 52 f 4 Epinephrine (1 pg/ml).... 51 f 2 Glucagon (1 &g/ml).... 48 4; 2 Theophylline (0.1 mm)... 22 rt: 4 Theophylline (1.0 mm).... 59 f 3 a Percentage change from control; control = 100. the original structures, the origin and nature of the membranes could not be determined. The nature and origin of this material in the fat cell is still under investigation. In the experiments described in Fig. 2, only 60% of the radioactive protein was released to the medium in response to ACTH, as compared to 100% of the malic dehydrogenase activity. The remaining 40% of the labeled protein was found in the layer of fat cells. After homogenization of the fat cell layer and centrif

3912 Metabolism of Isolated Fat Cells. IV Vol. 241, No. 17 the concentration of albumin. At the end of 2 hours of incubation with ACTH, albumin became saturated with FFA, as indicated by the molar ratios of FFA to albumin of 7.0, which is the maximum binding capacity of albumin with FFA (17). However, the time at which the saturation point was attained was inversely related to the albumin concentration; namely, 15 min for 0.5% albumin, 1 hour for 2%, and 2 hours for 4% albumin. Thus, there was a direct relationship between the time at which albumin became saturated with FFA and the increase in enzyme released to the medium. It will also be noted in Table III that basal release (no ACTH) of malic dehydrogenase increased as the albumin concentration was lowered. This protective effect of albumin cannot be as 0 MDH 30 60 90 I20 MINUTES FIG. 2. Effect of ACTH on release from fat cells of Wprotein, malic dehydrogenase (MDH), and membranes to the medium. Fat cells (average of 49 mg per flask) prelabeled with Whistidine were incubated at indicated times containing 0.5% albumin. WProtein (TCAinsoluble material) and malic dehydrogenase activity are expressed as the percentage of that found in fat cells before incubation. Turbidity in the medium due to membranes was measured by determining the optical density (600 rnp) of 1 ml of medium in a Beckman spectrophotometer with the use of cuvettes with a lcm light path. Concentration of ACTH was 1 pg per ml. Results are the averages for three experiments f the standard error (vertical lines). ugation at 10,000 X g for 15 min, half of the radioactivity was found in the sedimented material, suggesting that this labeled protein was present in cells as particulate material. Relationship between Concentration of Albumin and Release of Malic Dehydrogenase in Response to Adrenocorticotropic Hormone Since the dehydrogenase activity in the medium was a reliable and sensitive index of protein release from fat cells, its measurement was used in all subsequent studies. In the previous studies the incubation medium contained 0.5% albumin. It has been shown that the degree of lipolysis in fat cells increases in proportion to the concentration of albumin and the ability of ACTH to stimulate release of protein was examined. As shown in Table III, there was an inverse relationship between the amount of malic dehydrogenase released to the mea urn and TABLE II E$ect of ACTH on release from fat cells of malic dehydrogenase, lactic dehydrogenase, glucosesp dehydrogenase, and hexokinase Isolated fat cells (40 mg per flask) were incubated for 2 hours at 37 in 2 ml of 0.5Q/, albuminbicarbonate medium. After incubation period, cells were separated from medium by centrifugation, washed once with medium, and homogenized in 1 ml of medium. Aliquots of fatfree homogenates and incubation medium were assayed for the indicated enzymes as described under Experimental Procedures. The values are the percentage of enzyme content of homogenates of an equal amount of unincubated fat cells homogenized in the same incubation medium. Results are the averages for three experiments f the standard error. Enzymes assayed ACTH ~~ Recovery of enzymes and fat cells after incubation Medium +ACTH % % Malic dehydrogenase. 15 f 7 93 f 4 Lactic dehydrogenase.. 13 f 5 84 f 3 Glucose6P dehydrogenase. 6 f 2 34 f 5 Hexokinase. 0 0 TABLE III Cells ACTH 1 +ACTH % % 85f5 7f2 83 f 5 6 f 0.9 76&S 11&2 75f5 llf4 Effect of albumin concentration on release of malic dehydrogenase and FFA by fat cells in response to ACTH Fat cells (average of 55 mg per flask) were incubated in 2 ml of media containing the indicated concentration of albumin. Molar ratios of FFA to albumin were calculated from FFA and protein content of medium after 2 hours of incubation. Results are averages for four experiments f the standard error. Concentration of albumin % 4.0 2.0 0.5 a Concentration, ACTH + + + 1 pg per ml. Malic Molar ratio of FFA to ehydrogenase albumin after incubation u?zits/g 8f2 15 f 5 13 f 3 27 f 2 20 f 5 88*7 0.8 7.0 0.9 7.0 1.5 7.0

Issue of September 10, 1966 M. Rodbell 3913 Albumin Concentration % FFA Albumin Zero 2 Time Hours ACTH plg/ml 4.0 I:1 1.5:1 4.0 5:1 5:1 4.0 7:1 7:1 4.0 I:1 7:1 + I I I I 0 25 50 75 100 MALIC DEHYDROGENASE IN MEDIUM (% of cell homogenate) FIG. 3. Comparison of effects of ACTH and FFA on release of malic dehydrogenase to medium by fat cells incubated in 0.5 and 4% albumin. Fat cells (average of 52 mg per flask) were incubated for 2 hours in incubation medium containing FFA with which was combined albumin described under Experimental Procedures. Numbers of experiments are indicated in parentheses. Standard errors of the means are indicated by horizontal lines. 0.5 0.5 0.5 II 5:1 7:1 II 1.3:1 5:1 7:1!:I + scribed to its ability to bind FFA. It has been previously shown Thus, fatty acids that were not bound to albumin mimicked the that lipolysis does not occur in the absence of hormone treatment in isolated fat cells obtained from normal, fed animals (8). effects of lipolytic hormones on protein release. However, maximal release was not observed even after 2 hours of incubation. Effects of Varying Concentrations of Added Free Fatty Acid in Addition of ACTH to this medium accelerated release of the Medium on Malic Dehydrogenase ReleaseTo determine the eff ects dehydrogenase, which was complete within 2 hours of incubation. of fatty acids on malic dehydrogenase release, fat cells were incu This is in contrast to control experiments (shown in Fig. 3), in bated in either 0.5 or 4% albumin, with which were combined which ACTH, in the presence of 4y0 albumin (molar ratio of FFA acids derived from saponification of fat cell triglycerides. Molar ratios of FFA to albumin ranging from 1.0 to 7.0 were utilized. As shown in Fig. 3, no significant effects of added fatty acids were observed even when albumin was saturated with fatty acids, to albumin = 1) stimulated release of only 40% of the malic dehydrogenase of the cells in 2 hours. It has been shown previously (9) that glycerol release (a measure of total hydrolysis of triglycerides) from fat cells does not occur in response to lipolytic whereas ACTH stimulated complete release of the dehydrogenase hormones when the medium albumin is saturated with FFA. when cells were incubated in 0.5% albumin. In the experiments The slower release of malic dehydrogenase by fat cells in the with 4% albumin plus ACTH, considerable variation was encountered in the release of the dehydrogenase; the effects were not significantly different from those for the controls. presence of albumin saturated with FFA may reflect a very slow rate of production of FFA which, because the medium albumin is already saturated with FFA, is confined within the cells. Addition of fatty acids to 4 y0 albumin slightly in excess (molar Comparison of Effects of Theophylline and ACTH on Release of ratio, 7.2) of the binding capacity of albumin (molar ratio, 7.0) Ma& Dehydrogenase by Fat Cells Incubated in 4% Albuminstimulated release of malic dehydrogenase as described in Fig. 4. With fat cells incubated in 4$& albumin, ACTH had a variable

3914 Metabolism of Isolated Fat Cells. IV Vol. 241, No. 17 20 IO 0 30 60 90 I20 MINUTES FIN. 4. Comparison of etfects of FFA in excess of binding capacity of 4% albumin on release of malic dehydrogenase (MDH) by fat cells incubated with and without ACTH. Fat cells (average of 59 mg per flask) were incubated in 2 ml of medium containing 4% albumin to which fatty acids had been added to give a final concentration of 8.2 Meq per ml (molar ratio of FFA to albumin = 7.2). ACTH concentration was 1 rg per ml. Results are the averages for three experiments f the standard error. and relatively small effect on release of malic dehydrogenase, whereas theophylline, a more potent lipolytic agent than the hormones (16), invariably stimulated maximal release of the dehydrogenase. One such experiment comparing the effect of theophylline and ACTH on fat cells incubated in 4% albumin is shown in Fig. 5. The release of FFA for the first 15 min was the same from cells treated with theophylline or ACTH. However, theophylline sustained the rate of release of FFA which reached, within 1 hour, levels sufficient to saturate albumin. In the presence of 1 pg of ACTH per ml, the maximal effective concentration (9), fatty acids did not saturate albumin until 2 hours of incubation. Malic dehydrogenase was not released until after albumin had been saturated with FFA. Release of the dehydrogenase in the presence of theophylline was evident 1 hour sooner than observed with ACTH. Thus, the time required for saturation of albumin with FFA was related to the time at which maximal release of malic dehydrogenase occurred. Fatty Acid Peroxidation and Release of Malic Dehydrogenase Formation of fatty acid peroxides accompanies the release of fatty acids stimulated by lipolytic hormones in adipose tissue (18). Ethylenediaminetetraacetic acid or quercitin (an antioxidant), each of which has been found to prevent peroxide formation in adipose tissue (US), had the same preventative effects with isolated fat cells but did not prevent the release of malic deyhdrogenase (Table IV). These results indicate that fatty 0 I I I I I 1 30 60 90 120 I50 I80 MINUTES FIG. 5. Comparison of efiects of theophylline and ACTH on release of FFH and malic dehydrogenase (MDH) by fat cells incubated in 4% albumin. Fat cells (55 mg per flask) were incubated containing 4% albumin. Concentrations of theophylline and ACTH were 1.0 rnrd and 1.0 pg per ml, respectively. TABLE Effects of ACTH, quercitin, and EDTA on release by fat cells of malic dehydrogenase and FFA and formation of lipid peroxides Fat cells (average of 63 mg per flask) were incubated for 2 hours containing 0.597, albumin. When added, ACTH concentration was 1 pg per ml; EDTA, 1 mm; and quercitin, 1 mm. Results are averages for three experiments f the standard error. None... ACTH... EDTA.... ACTH + EDTA.... Quercitin... ACTH + quercitin.... Malic dehydrogenase units/g 10 f 2 102 f 4 15 f 4 107 * 9 10 f 3 99 f 2 IV FFA Lipid peroxides P@ ml Ad2 ml 0.1 f 0.01 0.04 f 0.005 1.08 i 0.08 0.13 z!z 0.02 0.09 f 0.01 0.04 f 0.005 1.07 f 0.06 0.03 * 0.005 0.80 zk 0.04 0.04 f 0.005 1.04 f 0.06 0.04 f 0.005

Issue of September 10, 1966 M. Rodbell 3915 acids per se and not their peroxides were responsible for the release of protein by fat cells. Quercitin, in the absence of ACTH, stimulated release of FFA (Table IV). This effect of quercitin has also been observed with intact adipose tissue (19). During 2 hours of incubation, quercitin did not stimulate sufficient release of FFA to saturate albumin in the medium. Effects of Insulin and Glucose on Release of Ma& Dehydrogenase and Free Fatty Acid in Response to Adrenocorticotropic Hormone The previous studies were carried out in the absence of glucose in the incubation medium. Under these conditions, maximal release of malic dehydrogenase occurred within 1 hour of incubation of fat cells containing 0.5% albumin. As shown intablev, insulin (1 milliunit per ml) did not inhibit the ACTHinduced release of dehydrogenase unless glucose (3 mm) was included in the medium. As an inhibitor of dehydrogenase release, glucose alone was less effective than insulin plus glucose. Insulin plus glucose or glucose alone reduced the release of FFA in response to ACTH to the same degree. Malic dehydrogenase release has been shown to be dependent upon the accumulation of of intracellular FFA (Fig. 4). Reesterification of intracellular FFA would reduce, therefore, the release of malic dehydrogenase. Insulin, by stimulating glucose transport (20, Zl), enhances reesterification of FFA and thereby inhibits the release of the dehydrogenase in the presence of ACTH (Table V). There was no relationship between the content of FFA in the medium and the release of malic dehydrogenase in the absence of ACTH. As stated before, basal release of the dehydrogenase or proteins in general probably reflects instability of isolated fat cells during incubation, particularly in 0.5% albumin. Release of Malic Dehydrogenase by Intact Adipose Tissue in Response to Adrenocorticotropic HormonePieces of epididymal fat pad (approximately 50 mg) incubated containing 0.5 or 4% albumin released malic dehydrogenase to the medium in response to ACTH, as shown in Fig. 6. In contrast to isolated fat cells, which released essentially all of their dehydrogenase activity in 1 hour in the presence of 0.5% albumin, intact adipose tissue released a maximum of 30% of the tissue content of the dehydrogenase under the same conditions. Maximal release was observed within 90 min of incubation in 0.5% albumin. In TABLE V and glucose on release of malic dehydrogenase and Effects of insulin FFA by fat cells in response to ACTH Fat cells (average 45 mg per flask) were incubated for 2 hours in medium containing 0.5% albumin. When added, ACTH concentration was 1 pg per ml; insulin, 1 milliunit per ml; and glucose, 3 mm. Results are averages for four experiments i the standard error. Additions to incubation medium None... Glucose.... Glucose + insulin.... ACTH... ACTH + insulin.... ACTH + glucose.... ACTH + glucose + insulin. Malic dehydrogenase units/g 20 zk 5 18 * 8 24 I 6 91 It 5 92 f 4 61 zk 5 46 z!z 3 FFA wq/z ml 0.29 f 0.02 0.11 z!z 0.03 0.12 f 0.02 1.06 f 0.09 1.05 * 0.07 0.71 f 0.03 0.77 f 0.05 2 40 t FFA MDH IN TISSUE $ 4.0% Albumin 3 0.5% % Albumin IN MEDIUM MINUTES FIG. 6. Effects of ACTH on release of malic dehydrogenase (MDH) to medium and FFA release by intact adipose tissue incubated in 0.5 and 4y0 albumin. Approximately 50 mg of adipose tissue per flask were incubated in 2 ml of medium containing ACTH (1 pg per ml) and either 0.5 or 4% albumin. Paired tissues were used as control (no ACTH) and were incubated for the same time periods. FFA content of tissues and media in the control flasks was subtracted from values obtained with ACTHtreated tissues. No malic dehydrogenase activity was found of tissues not incubated with ACTH. The percentage of tissue malic dehydrogenase released to the medium was calculated from the average malic dehydrogenase content of 10 pieces of adipose tissue. This was found to be 95 f 5 units per g as compared with 98 f 8 units per g of isolated fat cells. Results are the averages for three experiments (three paired flasks per time point) f the standard error. 4 To albumin tissues did not release the dehydrogenase in response to ACTH until after 90 min of incubation, whereupon release rose sharply to the value observed in 0.5% albumin. Comparison,of FFA production (in tissue versus medium) in 0.5 or 4% albumin is also shown in Fig. 6. Production of FFA for the first 30 min was the same in both media. However, when 0.5% albumin was in the medium, 65% of the FFA remained in the tissue from 30 min up to 2 hours of incubation. Significant release of malic dehydrogenase had occurred in 30 min. In contrast, when 4% albumin was present, 70 to 80% of FFA formed in response to ACTH appeared in the medium during the 1st hour of incubation; there was no release of the dehydrogenase during this period. Thereafter, the concentration of FFA rose in the tissue, until at 2 hours, half of the total FFA produced was presec ; in the tissue; only at this time was the dehydrogenase ob

3916 Metabolism of Isolated Fat Cells. IV Vol. 241, No. 17 served in the medium. Thus, release of malic dehydrogenase by intact tissue was related to the concentration of FFA in the tissue and not in the medium. Release of the dehydrogenase became apparent when t,he concentration of FFA in the tissue was approximately 16 peq per g of tissue, whether incubated in 0.5 or 4% albumin. DISCUSSION The present findings indicate that lipolytic hormones stimulate the release of protein both from isolated fat cells and from intact adipose tissue. This phenomenon appears to be directly related to the lipolytic action of the hormones. The finding that fatty acids added in excess of the binding capacity of albumin mimic the effects of lipolytic hormones is strong evidence that fatty acids are responsible for the release of protein. This is also suggested from the finding that release of protein from isolated fat cells occurred after albumin in the medium had become saturated with fatty acids produced during lipolysis. Since fatty acids tend to accumulate in isolated fat cells as the medium albumin becomes saturated (9), it is likely that fatty acids, directly or indirectly, caused release of proteins. The observation that ACTH had a greater effect on protein release than did externally added fatty acids (see Fig. 5) suggests that internally produced fatty acids have a more selective and localized effect on the sites responsible for the release of protein. The preferential action of internally produced fatty acids can also be inferred from the inhibition of the proteinreleasing action of lipolytic hormones when glucose was available in the cells for reesterification of intracellular fatty acids. Finally, the direct correspondence between the rise in fatty acids in intact tissue and the release of enzymes is further evidence that the release of protein from fat cells is a consequence of an accumulation of fatty acids in the tissue. Unlike isolated fat cells, in which release of enzyme was correlated with saturation of albumin with FFA, release of enzyme by fat pads was related to the concentration of fatty acids in the tissue. Albumin in the medium did not become saturated with fatty acids during incubation of adipose tissue with lipolytic hormones under the conditions employed. Thus, the difference in the requirements for protein release between isolated fat cells and fat pad may reflect the slow diffusion of FFA from tissue compared to the rapid flow of FFA from isolated fat cells to the medium which is in intimate contact with isolated cells. The more rapid egress of FFA from isolated fat cells than from tissue cells may also be due to the absence of the usual basement membrane that surrounds fat cells in intact adipose tissue (10). The basement membrane may constrain diffusion of fatty acids from cells in the tissue and thereby cause sufficient accumulation of fatty acids within the tissue cells to elicit release of cellular protein. To the extent that intact tissue fat cells can accumulate FFA irrespective of the presence of albumin in the medium, tissue cells are more responsive than isolated fat cells to the effects of lipolytic hormones on release of proteins. Measurements of the uptake of labeled fatty acids by isolated fat cells indicate that the cells can accumulate a maximum of about 5 peq of FFA per g (9). With the methods available, it has not been possible thus far to measure accurately the concentration of fatty acids within the small amount of isolated fat cells used in this study. Based on the studies with intact tissue, a maximum of 16 peq of FFA per g was required before release of malic dehydrogenase occurred. This concentration may be a composite of both extracellular (basement membrane?) and intracellular concentrat ions of FFA. Although a relationship was found between fatty acid accumulation and the release of protein from fat cells, the present study does not reveal either the primary cause of the release of protein or, if fatty acids are directly responsible, at what site in the cell the fatty acids exert their effect. Membrane was released to the medium after the release of soluble proteins or enzymes was initiated. The source of the membranes reieased from fat cells is still under investigation as are the structural features of the fat cell after it has been depleted of soluble protein. As stated in the introduction, two observations prompted this study: (a) that epinephrine or ACTH stimulates proteolysis in adipose tissue (5) and (b) that these hormones inhibit incorporation of amino acids into proteins of adipose tissue (57). The finding that these hormones stimulate the release of proteins by fat cells does not directly explain the first observation. However, it is possible that with intact adipose tissue, in which 500/, of the cells are not fat cells (22), hydrolysis of protein released from fat cells might have been affected by other cells, e.g. macrophages, mast cells, etc., in adipose tissue. This possibility warrants further study, but it could explain why only 30% of the malic dehydrogenase activity of intact adipose tissue was released to the medium whereas in the isolated fat cells all of this enzyme was found in the medium after treatment with lipolytic hormones. Inhibition of labeled amino acid incorporation into fat cells by ACTH has been confirmed with isolated fat cells2 provided that only the labeled protein of the cells was examined, However, when the radioactive protein found in the medium was combined with the cellular radioactive protein, no significant effects of ACTH were obtained on protein synthesis in fat cells. These findings indicate that lipolytic hormones do not initially affect protein metabolism per se in the fat cell, but rather the loss of protein and enzymes from the cell. The observation that enzymes leave the fat cells under the influence of lipolytic hormones is consistent with the gradual alteration in protein content and in metabolic activity that accompanies hormonal imbalance in viva. For example, the absolute amount of protein as well as fat in adipose tissue falls during fasting (23). The enzymes that have been reported to change when rats either are fasted (relative insulin lack) or are made diabetic include such soluble, cytoplasmic enzymes as hexokinase (24), glycolytic enzymes (24), pentose shunt enzymes (24, 25), malic enzyme (25), citrate cleavage (26), lipoprotein lipase (27), and stearate oxygenase (28). Studies now in progress in this laboratory indicate that the list can be extended to include malic and lactic dehydrogenases, which fall to the same extent as the abovementioned enzymes in adipose tissue from rats that have been fasted. Refeeding fasting rats with carbohydraterich diets (relative excess of insulin) causes an increase in all of the abovementioned enzymes, presumably by the stimulatory effect of insulin on protein synthesis. These studies, which reflect hormonal changes in vivo, are compatible with the evidence presented here that lipolytic hormones and insulin regulate the metabolic capacity of adipose tissue. Based on the present study, insulin, in the presence of glucose, conserves proteins in fat cells by inhibiting the effects of lipolytic hormones on release of fatty acids and the related loss of proteins * Unpublished observations.

Issue of September 10, 1966 M. Rodbell 3917 or enzymes. Parentheticallv. insulin also has a notent antili ological Society, Washington D. C., 1965, Chapter 47, p. polytic eff ect in the absence of glucose (29) if the concentration of lipolytic hormones is less than that used in the present study (16). Insulin also causes a general increase in protein or enzyme synthesis through its effects on transport processes in fat cells (20, 21). These actions of insulin, counteracting the lipolytic and proteinreleasing action of lipolytic hormones and stimulating amino acid and carbohydrate transport, may be attributed to a primary action of the hormone at the plasma membrane (16,21). A direct action of insulin on protein synthesis at the level of DNAdependent RNA synthesis has been suggested in liver (30), muscle (31, 32), and adipose tissue (33). It remains to be determined whether the dual actions of insulin presented here can provide an explanation for the suggested direct action of insulin on protein synthesis. AcknowledgmentsThe author thanks Mrs. Ann B. Jones for her skilled technical assistance and Dr. Sidney S. Chernick for his advice and discussion during the course of the investigation. 1. 2. 3. 4. 5. 6. 7. 8. REFERENCES ROSE, G., AND SHAPIRO, B., Biochim. Biophys. Acta, 18, 504 (1955). HAUSBERGER: F. X., AND MILSTEIN, S. W., J. Biol. Chem., 214, 483 (1955). ENGEL, F. L., AND WHITE, J. E., Am. J. Clin. Nuf~.~ 8, 691 (1960). MOORE, R. O., Am. J. Physiol., 206, 222 (1963). CHRISTOPHE, J., AND WODON, C., Arch. Intern. Physiol. Biochim., 72, 100 (1964). HERRERA, M. G., AND RENOLD, L%. E., Biochim. Biophys. Acta., 44, 165 (1960). HERRERA. M. G.. AND RENOLD. A. E.. in A. E. RENOLD AND G. F. CAHILL, JR. (Editors); Handdook of physiology, Section V, American Physiological Society, Washington, D. C., 1965, p. 375. RODBELL, M., in A. E. RENOLD AND G. F. CAHILL, JR. (Editors), Handbook of physiology, Section V, American Physi 471. 9. RODBELL, M., Ann. N. Y. Acad. Sci., 131, 302 (1965). 10. RODBELL, M., J. Biol. Chem., 239, 375 (1964). 11. WILBUR, K. M., BERNHEIM, F.,.~ND SHAPIRO, W. O., ilrch. Riochem. Biophys., 24, 305 (1949). 12. BRAY, 0. A., Anal. Biochem., 1, 279 (1960). 13. LOWRY, 0. II., ROSEBROVOH, N. J., FARR, A. L., AND R.IN DALI,, R. J., J. Biol. Chem., 193, 265 (1951). 14. DOLE, V. P., AND MEINERTZ, H., J. Biol. Chem., 235, 2595 (1960). 15. FAIN, J. N., Biochim. Biophys. Acta, 84, 636 (1964). 16. RODBELL, M., AND JONES, A. B., J. Biol. Chem., 241, 140 (1966). 17. GOODMAN, D. S., J. Am. Chem. Sot., 80, 3892 (1958). 18. ENGEL. F. L.. BALL. M. F.. AND BLACKARD. W. G.. J. Liuid Res., 6, 21 (1965). 19. LYNN, W. S., in A. E. RENOLD AND G. F. CAHILL, JR. (Editors), Handbook of physiology, Section V, American Physiological Society, Washington, D. C., 1965, p. 349. 20. CROFFORD, 0. B., AND RENOLD, A. E., J. Biol. Chem., 240, 3237 (1965). 21. RODBELL, M., J. Biol. Chem., 241, 130 (1966). 22. RODBELL, M., J. Biol. Chem., 239, 753 (1964). 23. HAIJSBERGER, F. X., in A. E. RENOLD AND G. F. CAHILL, Jn. (Editors), Handbook oj physiology, Section V, American Physiological Society, Washington, D. C. 1965, p. 519. 24. BALL, E. 6.. AND JUNG.&, R. L.1 Bi&hemistry, a,586 (1963). 25. YOUNG. J. W.. SHRAGO. E.. AI\ D LARDY. H. A.. Biochemistru. 3, 1657 (1964). 26. KORNACKER, M. S., AND B.~LL, E. G., Proc. Natl. Acad. Sci. U. S., 64, 899 (1965). 27. HOLLENBERG, C. H.? Am. J. Physiol., 197, 667 (1959). 23. BENJAMIN, W., AND GELLHORN, A., J. Biol. Chem., 239, 64 (1964). 29. JUNGAS, R. L., AND BILL, E. G., Biochemistry, 2, 383 (19G3). 30. WEBER, G., SINGHAL, R. L., AND SRIVASTAVA, S. K., Proc. Natl. Acad. Sci. U. S., 63, 96 (1965). 31. MANCHESTER, K. L., AND YOUNG, F. G., Vitamins Hormones, 19, 95 (1961). 32. WOOL, I. G., in G. LITW~CH AND D. KRITCHEVSKY (Editors), Actions of hormones on molecular processes, John Wiley and Sons, Inc., New York, 1964, p. 422. 33. GELLHORN, A., AND BENJAMIN, W., Science, 146, 1166 (1964)..

The Metabolism of Isolated Fat Cells: IV. REGULATION OF RELEASE OF PROTEIN BY LIPOLYTIC HORMONES AND INSULIN Martin Rodbell J. Biol. Chem. 1966, 241:39093917. Access the most updated version of this article at http://www.jbc.org/content/241/17/3909 Alerts: When this article is cited When a correction for this article is posted Click here to choose from all of JBC's email alerts This article cites 0 references, 0 of which can be accessed free at http://www.jbc.org/content/241/17/3909.full.html#reflist1