Fatty Acid Synthesis from Glucose and Acetate and the Control of Lipogenesis in Adipose Tissue

Transcription

1 European J. Biochem. I1 (1969) Fatty Acid Synthesis from Glucose and Acetate and the Control of Lipogenesis in Adipose Tissue J. DEL BOCA and J. P. FLATT Institut de Biochimie, Universitk de Lausanne (Received April 5/July 11, 1969) Epididymal fat pads of fed and 48 h fasted rats were incubated in vitro with I0 mm glucose and 0, 60, 120, or lo4 punits of insulin/ml. A comparison was made of the metabolism of glucose in the absence or presence of 15 mm acetate in the incubation medium. At physiological concentrations of insulin, the addition of acetate increases glucose uptake, glucose oxidation to CO,, and incorporation of glucose-c into fatty acids; concomitantly the rates of glycerol release and lactate production are decreased, and the lactate/pyruvate ratios are lowered. Fatty acid synthesis in the prcsence of glucose plus acetate was in all cases significantly higher than with glucose alone. It is concluded that, at a given insulin eoncentration, the conversion of carbohydrate to fat in intact adipose tissue is limited by the tissue s ability to supply acetyl-coa for fatty acid synthesis, and not by the enzymes directly engaged in the synthesis of fatty acid from acetyl- CoA. The effects of acetate were of greater importance in adipose tissue from rats fasted for 48 h than from fed rats. The impairment in lipogenesis caused by fasting appears to be due primarily to a decrease in the tissue s ability to produce acetyl-coa from glucose. In mammals, about one-third of the ingested carbohydrate is converted to fat before being utilized for energy production in the form of free fatty acids [I]. Adipose tissue is a primary site of lipogenesis from glucose, the major physiological precursor for de novo synthesis of fat [2,3]. Under normal conditions this conversion is controlled mostly by insulin, the regulator of the entry rate of glucose into adipose tissue [4-61. An important metabolic disturbance during fasting, fat feeding, or diabetes is a marked depression of lipogenesis [4,7,8], which can be only partially relieved by insulin [9]. Several hypotheses have been advanced to explain the reason for this metabolic defect, observed in whole cell preparation or extracts of liver and adipose tissue [lo-121. In liver homogenates, the enzyme acetyl-coa carboxylase, which catalyzes the first reaction in the cytoplasmic pathway of fatty acid synthesis [13], has been found to be less Enzymes. Glycerol-phosphate dehydrogenase (EC ); lactate dehydrogenase (EC ) ; malate dehydrogenase (EC ); malic enzyme or L-ma1ate:NAD oxidoreductase (decarboxylating) (EC ) ; glucose-6-phosphate dehydrogenase (EC ); glucose oxidase (EC ); glyceraldehyde-phosphate dehydrogenase (EC ); pyruvate dehydrogenase (EC ); peroxidase (EC ); hexokinase (EC ); phosphofructokinase (EC ); glycerol kinase (EC ); pyruvate kinase (EC ); lipase (EC ); citrate synthase (EC ); ATP citrate lyase or citrate cleavage enzyme (EC ); pyruvate carboxylase (EC ); acetyl-coa carboxylase (EC ); fatty acid synthetase. active than the fatty acid synthesizing enzyme complex [14]. The carboxylase is stimulated by citrate [I51 which acts also as a carrier for acetyl-coa across the mitochondria1 membrane [16,17] and is inhibited by long-chain fatty acyl-coa derivatives, the end products of fatty acid synthesis [18]. Acetyl-CoA carboxylase has provoked special interest as a possible pacemaker for fatty-acid synthesis [12,19]. Certain restrictions are imposed in an intact tissue because a balance between production and utilization rates of reducing-equivalents must be maintained. Detailed data on these rates, obtained during quantitative studies of the glucose catabolism during fat synthesis, led Flatt and Ball [20] to suggest that the rate-limiting reactions for glucose conversion to fat in intact adipose tissue may not be the same as the rate-limiting reaction in fatty acid synthesis from acetate or acetyl-coa in isolated enzyme systems [ZI]. In the presence of high doses of insulin, more fatty acids were synthesized in intact adipose tissue when the medium was supplemented with acetate in addition to glucose [22,23]. This confirms the notion that the pathway, by which acetyl-coa is formed from glucose, reaches its maximal rate before the total capacity for the conversion of acetyl-coa to fatty acid is attained [22]. Our present study extends these observations to the full range of insulin concentration. Furthermore, acetate is shown to have a greater relative effect in

2 128 Fatty Acid Synthesis from Glucose and Acetate in Adipose Tissue European J. Biochem. facilitating fat synthesis in epididymal adipose tissue from 48 h fasted rats than from fed animals. It is concluded that the impairment in lipogenesis caused by fasting is due primarily to a decrease in the tissue's ability to generate acetyl-coa from glucose, and not to inhibition, or to loss in the amounts of the enzymes directly involved in the synthesis of fatty acid from acetyl-coa. Preliminary reports have been presented [24,25]. MATERIALS AND METHODS Male albino Wistar rats were purchased from La Garenne, Station d'elevage (1261 Le Vaud, Switzerland). The fed rats were maintained on a standardized pellet chow (Altromin R) ; their drinking water was supplemented with glucose, the concentration of which was increased over a period of 4 days from 5O//, to 1O0/, and to 15O/,, where it was maintained for 5 to 10 days. When rats were fasted, drinking water without glucose was supplied. The rats which were fed had an average weight of 167 g, while the weight of those fasted was 147 g. The animals were decapitated with a miniature guillotine and their epididymal fat pads removed with minimal handling [4,26]. The fat pads of 2 fed or 2 fasted rats were randomly distributed over 6 incubation flasks [27]. The average amount of tissue per flask (determined to L0.5 mg) was 125 nig from fed and 110 mg from 48 h fasted rats. The nitrogen content of epididymal adipose tissue (determined by a micro-kjeldahl procedure) of fed and fasted rats treated as described, was (a,verage of 12 determinations 2- standard error) and C mg per 100 mg of wet weight, respectively. Unless otherwise indicated, pieces of adipose tissue from the same rats were incubated simultaneously in 3 flasks. In each of these groups, the first flask contained 10 mm [U-l*C]glucose, the second 10 mm [U-14C]glucose and 15 mm acetate (Na salt), and the third 10mM glucose and 15mM [UJ4C]- acetate; approximately 0.5 pc of I4C, added in 50 pl of radioactive stock solutions, was present in each flask. A few experiments were performed to determine the incorporation of 3H of tritiated water into fatty acids under conditions described in the legend to Table 4. 14C-labeled substrates and tritiated water were purchased from New England Nuclear Corporation. [U-14C]acetate was prepared by mixing cqual amounts of [lj4c]acetate and [2-14C]acetate. The incubations were performed in 25 ml Erlenmeyer flasks equipped with a sleeved serum rubber cap fitted with a stainless steel wire [28] supporting a small glass well. Each flask Contained a total volume of 2.5 ml of bicarbonate buffer [29] modified to contain only half of the prescribed (:a++, and supplemented with 300 mg/100 ml of gelatine (Merck) to prevent the adsorption of insulin to the glass walls [30]. Bovine insulin (Novo, 10 x recrystallized, 25 unitslmg. Lot no ) was present in concentrations of 0, 60, 120, or I0000 punits/ml. The flasks were flushed with a gas mixture of 95O/, 0, + 5OI0 CO,, previously saturated with water vapor. The incubations were carried out for 150 min at 37" in a Gallenkamp incubator shaking at a rate of 100 cycles/ min. At the end of this period the flasks were placed in an ice bath. Using syringes, 0.3 ml of 1 M hyamine in methanol (or I M NaOH) was introduced through the rubber cap into the small cup, followed by the addition of 0.6 ml of 6 N H,SO, to the medium. Complete absorption of the CO, by the alkali was achieved after 2.5 h of slow shaking at 37". The vessels were opened and their various contents processed. Blank values for the different detcrniinations were obtained from control vessels carried through the entire procedure without added tissue. Due to its moderate volatility in acid solutions, significant amounts of [Wlacetate accompanied the 14C0, absorbed by the alkali in the experiments with [U-14C]acetate. For this reason 1 M NaOH instead of hyamine was added to absorb the CO, in these incubation flasks. After transfer to new flasks, the CO, was liberated a second time with H,SO,, but this time in the presence of approximately 60 mg of unlabeled acetate. The vessels were left slowly shaking overnight at room temperature ; during this period the CO, was being absorbed with hyamine. Having a low specific activity, the acetate absorbed with the CO, contained only minor amounts of 14C. This procedure reduced the blank value for the 14C02 from approximately 0.4 to less than 0.02 pg atoms of acetate-c per flask. A 2 ml sample of the acidified incubation medium, left overnight in the cold, was neutralized with NaOH (using phenol red as an indicator), made up to 10 ml, and centrifuged. Enzymatic analyses were performed on aliquots of this solution using enzymes and coenzymes obtained from C. F. Boehringer & Soehne (Mannheim). Glucose utilization was determined using either glucose oxidase or hexokinase with glucose-6-phosphate dehydrogenase. The amounts of lactate and pyruvate released were measured with lactate dehydrogenase, using procedures given by C. F. Roehringer & Soehne (Mannheim), with minor modifications. Glycerol was measured with glycerokinase and either glycero-phosphate dehydrogenase [31] or pyruvate kinase plus lactate dehydrogenase The pieces of adipose tissue were gently transferred to alcoholic KOH [20]. After saponification, followed by acidification, the fatty acids were extracted with petroleum ether and washed free of water soluble radioactivity [20]. On the average, mg (standard deviation) and mg were recovered per 100 mg of wet weight tissue from fed or 48 h fasted rats. If the recovery was less than the average value diminished by 2 times the standard

3 ~ ~ Vol.11, No.1, 1969 J. DEL BOCA and J. P. FLATT 129 deviation, a correction was applied to the observed incorporation of 14C into fatty acid on the basis of the weight of the fatty acids recovered and the average recovery. Radioactive measurements were made by liquid scintillation counting in a Nuclear Chicago Model 725 three channel counter. The samples were dissolved in 2 ml of ethanol + 8 ml of toluene containing 4 g/l 2,5-diphenyloxazole and 50 mg/l 1,4-bis-(5-phenyloxazolyl-2)-benzene. Counting efficiencies for 14C were established for each sample using the channels ratio method [33]; they ranged from 0.67 to Incorporation of glucose or acetate-c into fatty acid and CO, was calculated on the basis of the determined dis./min. Several samples of the added radioactive stock solutions were counted in each experiment; 0.1 ml of I M hyamine was added to the vials with labeled acetate to prevent losses of free acetic acid, The corrections for quenching were checked by the addition of radioactive toluene to the different types of samples encountered ; the recoveries agreed within 301,. Incorporation of 3H into fatty acid was calculated from the observed counts/min since only this type of sample was to be counted. Analytical reagents and glass redistilledwater were used throughout the experiments. RESULTS The experimental results obtained for the adipose tissue from fed and 48 h fasted rats are presented in Tables 1 and 2 respectively; the data obtained in the three types of incubation are presented together. The difference between the results obtained with labeled glucose alone or in the presence of unlabeled acetate directly reflects the modifications in the metabolism of glucose resulting from the availability of acetate as additional substrate. In adipose tissue, acetate is expected to be metabolized chiefly to fatty acid and CO,, a fact which has been verified by comparing radioactive and manometric data [22]. During the Table 1. Measuremenis on the metabolism of glucose and acetate by adipose tissue of fed rats The results are averages of N groups of 3 incubations. All values are expressed in pg atoms of substrate-c & standard errors of the mean, per 100 mg of wet weight of adipose tissue, for an incubation period of 150 min, in the presence of 10 mm glucose with or without 15 mm acetate. The 14C02 values for the experiments with labeled acetate were obtained after elimination of contaminating counts from this precursor. This technique was perfected only during the second part of the study and the values given (without standard error) are based only on 1-4 experiments; in order to respect the constancy of the 14C0,/[14C]- fatty acid ratio (which we also observed in other experiments), the results were divided by the 14C-labeled fatty acid results obtained in these experiments, and multiplied by the 14C-labeled fatty acid results obtained in the N experiments. The amount of glucose-c recovered in the experiments with [U-14C]glucose was calculated by adding the pg atoms of 1% found in the CO, and in the fatty acids to the pg atoms of lactate, pyruvate, and glycerol released. It has been shown that in adipose tissue the specific activity of lactate is essentially the same as that of the glucose in the medium [34]. The incorporation of glucose-c into glyceride-glycerol is just slightly higher than the glycerol release [20]. The percentage of glucose-c recovered was obtained by dividing this sum by the amount of glucose taken up from the medium and multiplying the result by 100 The P values for the significance of the difference in the metabolism of glucose between paired flasks containing [U-14C]glucose with or without unlabeled acetate were calculated by the Student's t test Insulin N Substrate "co, ["OIFatty ~cid Gl;l;;;l Lactate Pyruvate Glc-C Glc-C release release recovered recovery dj/ml wg atoms substrate-c/100 nig wet tissue "0 0 8 [U-14C]Glc 9.1h k & k &0.09 (P< 0.05) [U-14C]Glc k j % & Glc $ & j j [U-14C]Ac 60 6 [U-14C]Glc 20.0h k j (P< 0.01) (P< 0.05) [U-14C]Glc 24.4h k k AC Glc A & [UJ4C]Ac [U-14C]Glc 29.6k k k f * (P<0.01) (P<O.01) (Pc0.02) [U-14C]Gl~ k % k Ac GlC k & [U- W]Ac lo4 6 [U-14C]Glc 39.2% & [U-14C]Gl~ k & j Glc % % k [U - W] Ac 9 European J. Biochem., Vol.11

4 130 Fatty Acid Synthesis from Glucose and Acetate in Adipose Tissue liuropc:in J. Hi~ri~~ni. Table 2. Measurements on the metabolism of glucose and acetate by adipose tissue of 48 h tasted rats The results are the averages of N groups of 3 incubations, expressed in pg atoms of substrate-c standard errors of the mean, per 100 mg of wet weight of adipose tissue, for an insubation period of 150 min. For details see Table 1 Glucose co, [,401Fstty arid Insulin N Substrate uptal;e GleY; ;l Lactate Pyruvatc Glc-C Glc-C release release recovered recovery [ U-14C]Glc [U-*4C]Glc Glc + [U-I4C]Ac [U- 4C]Glc [U- 4C]Glc Glcf [U-14C]Ac [U- 4C]Glc [U- 4C]Gl~ Glc + [U-14C]Ac [U-14C]Glc [U-14C]Gl~ Glcf [U-14C]A~ (P<0.01) 7.0h (P=0.01) h & Wg atoms snhstrate-c/100 mg wet tissue 0.3l,lt h k h1.6 (P<0.01) (P<0.01) (P=0.05) & & h & h & &0.7 (P<0.05) (P<0.01) 1.65:t & & & h h h k (P<0.05) 7.45h k incubations performed with unlabeled glucose and labeled acetate, the tissue encountered the same metabolic situations as in the incubations with labeled glucose and unlabeled acetate. These experiments provide the data necessary to determine the amounts of acetate converted to fatty acid and CO,; the sum of these two figures represents the total amount of acetate utilized. The data which are not based on radioactive measurements are similar in the two incubations with glucose and acetate; the greatest discrepancy is in the glucose uptake values with fasted tissue in the presence of 60 punits/ml of insulin ; the value obtained in the experiments with [U-14C]glucose plus acetate is too low as confirmed by the excessive recovery of glucose-c (123,/,). The uptake in the paired experiments with glucose and [U-14C]acctate would give a recovery of 82 O/,, which is consistent with the recoveries generally encountered. Insulin has a stimulating effect on the utilization and metabolism of glucose by adipose tissue [3,4]. This is reflected by the data on glucose uptake, and in those showing the amounts of glucose-c oxidized to CO, and incorporated into fatty acid. With tissue from the fed rats, the presence of 60 punits/ml of insulin elicits about half of the response seen with lo4 punits/ml, a dose about 10 times higher than that required to provoke a maximal stimulation [30]. It is interesting to observe that tissue taken from fasted animals requires a dose of 120 punits/ml to provoke a stimulation amounting to half of that obtained with the highest dose of the hormone. In addition, the tissue s maximal ability to synthesize fat from glucose is reduced to 46,/, by 48 h of fasting, even though the uptake of glucose is reduced only to 6501,. This is due to an increased rate of lactate production and to a greater demand for glycero-phosphate for re-esterification of free fatty acid produced by Iipolysis, which proceeds at a higher rate in fasting tissue as shown by the elevated rates of glycerol release [35,26]. It can be notsd that the glycerol releases are slightly depressed in the presence of acetate. When glucose entry into the cell is restricted, i.e. in the absence of insulin and with fasting tissue even in the presence of 60 punits/ml of insulin, an important portion of the acetate is channeled into the citric acid cycle, thus contributing to the formation of reduced coenzymes for the production of energy. The fate of acetate is markedly different when, under the influence of insulin, sizable uptakes of glucose can take place. Under these conditions acetate is converted primarily to fatty acid, a process requiring ATP and reducing equivalents in the cytoplasm in the form of NADPH, which must be generated by degradation of glucose. In studying the effects of acetate on the utilization ofglucose, one should consider the data on the uptake of glucose and those showing the amounts of glucose-c converted into the various measured end products ; the latter appear to be more reliable since they have

5 Vo:. 11, No J. DEL Boca and J. P. FLATT 131 Table 3. Effect of acetate on glucose uptake by adipose tissue of rats fed ad libitum or fasted for 48 k The results are the averages of N pairs of incubations standard errors of the mean, expressed in pg atoms of glucose-c per 100 mg of wet weight of adipose tissue. After 150 min of incubation, the uptake of glucose was determined with hexokinase and glucose-6-phosphate dehydrogenase. Aliquots of incubation medium incubated without adipose tissue were used as standards. The P values were calculated by the Student's t test, using the differences between paired incubations Substrates - Nutritional state of rats N Insulin Glucose (10 mm) Acetate effect P (lo ma') f Acetate (15 mm) wu/ml pg atoms Glc-C/100 mg wet tissue Fed ad libitum 9 GO k0.9G =0.02 with 15 o/o glucose in drinking water 9 lo4 30.3& & * 1.90 (<0.2) not significant Fasted 48 h & k &0.54 (0.05 (given above diet before fasting) 12 lo4 19.4kO.G 20.0& h0.52 (<0.3) not significant smaller standard errors. It is particularly interesting to observe that, in the presence of insulin, the tissue appears to take up more glucose from the medium when acetate is available. Since it is important to know whether or not the utilization of glucose is controlled exclusively by the insulin concentration, additional incubations were performed to study the effect of acetate on the glucose uptake (Table 3). In these experiments, the concentration of glucose in the medium at the end of the incubation was determined with hexokinase and glucose-6-phosphate dehydrogenase, after deproteinization with HC10,. With this method an improved precision of the measurements is indicated by decreases in the standard errors. At a concentration of lo4 punitslml of insulin, the average uptake of glucose is again slightly, but not significantly, higher in the presence of acetate. However, at the concentrations of insulin which elicit approximately half of the maximal response, 60 punits/ml with tissue from fed and 120 punits/ml with tissue from fasted rats, acetate increases the utilization of glucose significantly. In evaluating these data, it should be realized that, while the glucose uptakes are variable due to the individual variations among the experimental animals, the effects of acetate are relatively constant (cf. third column of Table 3). Flatt and Ball [22] have reported a similar significant stimulation with 1000 punits/ml, and in the data of Rognstad and Katz [23] utilization of glucose was also higher in the presence of acetate. Since acetate, in the absence of insulin, has no stimulatory effect on glucose uptake, it must be concluded that the increased glucose uptake is not due to acetate per se, but it is the consequence of a modification in the tissue's metabolic state, related to the availability of acetate as an additional source of acetyl-coa. This observation is surprising in view of the studies of Crofford and Renold [5,6] which showed that, under physiological conditions, glucose transport limits the uptake of glucose by adipose tissue. It may be that glucose phosphorylation and not glucose transport is limiting in some cells because of the inhibition of hexokinase by glucose-6-phosphate. This type of control appears to be of major importance for the regulation of glucose utilization in heart muscle [37]. Availability of acetate, by facilitating the catabolism of glucose-6-phosphate, as discussed later, could possibly relieve this inhibition. The rate of fatty acid synthesis is given by the amounts of 14C incorporated into fatty acid. In the case of the incubation carried out in the presence of both glucose and acetate, the sum of the incorporations observed with [U-14C]glucose in the presence of unlabeled acetate, and from [U-14C]acetate in the presence of unlabeled glucose, reflects the total rate of fatty acid synthesis when both glucose and acetate are available. This is shown in Fig. 1, where the total fatty acid synthesis is obtained by superposing the columns showing the incorporations measured in the incubations with glucose plus acetate. The rates of fatty acid synthesis from glucose and acetate are significantly higher than those obtained in the presence of glucose alone for all concentrations of insulin used in our study. The synthesis of fat in adipose tissue when glucose is the sole substrate available is therefore not limited by the amounts present in the tissue of acetyl-coa carboxylase and of fatty acid synthetase, which are the two enzymes that carry out the conversion of acetyl-coa to fatty acid. The results also show that the tissue is capable of producing more ATP, for the carboxylation of acetyl-coa to malonyl-coa, and more NADPH, for the reductive steps in the synthesis of long-chain fatty acid, than are required during the conversion of glucose to fat. These conclusions are valid over the whole range of insulin concentration for adipose tissue from fed as well as from 48 h fasted rats, where the acetate effect is much more pronounced. Following the evidence first presented by Spencer and Lowenstein [16], it is now generally believed that citrate mediates the transport of acetyl-coa through the mitochondria1 membrane. Accordingly, the acetyl- CoA formed by pyruvate dehydrogenase in the mito- 9*

6 132 Fatty Acid Synthesis from Glucose and Acetate in Adipose Tissue European J. Biochem. c.- E P 0.01 T Insulin (pmtshl) Fig. 1. Incorporation of glucose-c and acetate4 into fatty acid by adipose tissue of rats fed ad libitum or fasted for 48 h. The vertical columns indicate by their heights the numbers of pg atoms of I4C found in the fatty acids (Tables 1 and 2) in the presence of various concentrations of insulin. In each pair of columns, open areas show the incorporation of glucose-c measured after 150 min of incubation with 10 mm [U-14C]glucose (left column), or with [U-14C]glucose and unlabeled 15 mm acetate (right column). The third type of incubation, with unlabeled glucose and [U-14C]acetate in the medium, gave the incorporations shown by hatched areas. These are represented on the top of the right hand columns to show the total rate of fatty acid synthesis in the presence of the two substrates. The brackets show the size of the standard errors of the mean. The P values were obtained by the Student s t test, using the differences between fatty acid synthesis from glucose plus acetate, and from glucose alone; P values apply to the difference between glucose4 incorporation into fatty acid, with and without acetate in the incubation medium chondria is converted to citrate by the citratecondensing enzyme ; citrate then diffuses into the cytoplasmic compartment. Under the influence of the citrate cleavage enzyme, acetyl-coa is regenerated and oxaloacetate is formed. The latter can be reduced to malate and then decarboxylated to pyruvate by malic enzyme ; these two reactions provide for the transfer of reducing equivalents from NADH to NADP+ [17,38]. This transfer must contribute almost half of the NADPH required for fatty acid synthesis in tissue from fed rats incubated with glucose and a high concentration of insulin 120,231. The reoxidation of NADH accomplished by this transfer may be indeed more important in the conversion of glucose to fat than the generation of NADPH [39]. The possibility that the transport of acetyl-coa from the mitochondria to the cytoplasm may become limiting in the synthesis of fat from glucose must also be recognized. In our experiments with tissue from fasting rats, the conversion of glucose-c to fatty acid (Fig. I) was increased by acetate, providing evidence that acetyl-coa transport does not play a rate-limiting role. Acetate did not significantly augment the incorporation of glucose-c into fatty acid in tissue from fed rats, although the Gotal rate of fatty acid synthesis was greater in the presence of acetate. Table 4. Incorporation of 3H of 3Hz0 into fatty acid during lipogenesis in adipose tissue The results are averages of 5 experiments f standard errors of the mean, expressed in pg atoms of 3H of 3H,0 incorporated into fatty acid, per 100 mg of wet weight tissue, after 150 min of incubation. In each experiment, adipose tissue of fed rats was randomly distributed into 3 incubation flasks containing 2.5 ml of bicarbonate buffer, fortified with 10 mm glucose, insulin (lo4 punitslml) and tritiated water (0.8 mc/ flask). The media in the second and third flasks were also supplemented with sodium pyruvate (15 mm), or sodium oxaloacetate (15 mm); the latter was prepared extemporaneously by mixing oxaloacetic acid andnahc0,. Thevalues of P were calculated by the Student s t test, using the differences between paired incubations Substrate ah in fatty acid Increase F gg atoms/ 100 mg wet tissue Glucose Glucose + pyruvate <0.01 Glucose + oxaloacetate 14.2 & <0.01 However, since acetate may be activated directly in the cytoplasm, the experiments reported in Table 4 were performed. The results show that fatty acid synthesis, measured by the incorporation of 3H from tritiated water [40], is greater when pyruvate or oxaloacetate are provided in addition to glucose, than with glucose alone. As in the case of glucose, acetyl-coa from these substrates is formed by pyruvate dehydrogenase. Hence, it becomes evident that the mechanism for the transfer of acetyl-coa to the cytoplasm is not operating at its full capacity during fat synthesis from glucose. Thus, the reactions catalyzed by the citrate-condensing enzyme and the citrate cleavage enzyme are not rate-limiting in the conversion of glucose to fat in intact adipose tissue of fed or fasting rats. The striking similarily of the results from pyruvate and oxaloacetate can be observed also in the absence of glucose (unpublished observation). This may be due to a decarboxylation of oxaloacetate to pyruvate prior to or during its uptake by the cells. DISCUSSION Our findings coincide with other experimental observations which support the view that the formation of acetyl-coa from glucose limits de novo fat synthesis in tissue from fed animals [22]. When lipolysis is stimulated by small amounts of epinephrine in the presence of glucose, the free fatty acids which are formed are rapidly re-esterified. In this process glycero-phosphate and ATP are required ; both lead to the reoxidation of some NADH. Using 0.2 pg/ml of epinephrine, Flatt and Ball observed a small increase in the rate of glucose conversion to fat [21]. Felber et az. [41] found that the addition of

7 134 DEL Boca and FL~TT : Fatty Acid Synthesis from (ihcose and Acetate in Adipose Tissue European J. Biochem. technical assistance. This work constitutes a part of a doctoral thesis submitted by J. Del Boca to the Faculty of Sciences of the Univcrsiti: de Lausanne. REFERENCES 1. Wertheimer, H. E., In Handbook of Physiology, Section 5, Adipose Tissue [edited by A. E. Renold and G. F. Cahill, Jr.), Williams and Wilkins, Co., Baltimore, $Id. 1965, p Favarger, P., In Handbook of Physiology, Section 5, Adipose Tissue (edited by A. E. Renold and G. F. Cahill, Jr.), Williams and Wilkins, Co., Baltimore, Md. 1965, p Jeanrenaud, B., Ergebn. Physiol. Biol. Chem. Exptl. Pharmakol. 60 (1968) Winegrad, A. I., and Renold, A. E., J. Biol. Chem. 233 (1958) Crofford, 0. B., and Renold, A. E., J. Biol. Chem. 240 (1965) Crofford, 0. B., and Renold, A. E., J. Biol. Chem. 240 (1965) Wieland, O., Eeufeldt, I., Numa, S., and Lynen, F., Biochem (1963) Wieland, O., and Eger-Neufeldt, I., Biochem (1963) Herrera, M. G., Philipps, G. R., and Renold, A. E., Biochim. Biophys. Acta, 106 (1965) Masoro, E. J., J. Lipid Res. 3 (1962) Korchak, H. &I., and Masoro, E. J., Biochim. Biophys. Acta, 58 (1962) Wieland, O., Symp. Deut. Ges. Endokrinol. 12 (1967) Wakil, S. J., J. Am. Chem. SOC. 80 (1958) Numa, S., Matsuhashi, M., and Lynen, F., Biochem (1961) Vagelos, P. R., Alberts, A. W., and Martin, D. B., J. Biol. Chem. 238 (1963) Spencer, A. F., and Lowenstein, J. M., J. Biol. Chem. 237 (1962) Kornadker, M. S., and Ball, E. G., Proc. Natl. Acad. Sci. U. S. 54 (1965) Bortz, W. M., and Lynen, F., Biochem (1963) Numa, S., Bortz, W. M., and Lynen, F., Ad,van. Enzyme Reg. 3 (1965) Flatt, J. P., and Ball, E. G., J. Biol. Chem. 239 (1964) Flatt, J. P., and Ball, E. G., In The Control of Lipid Metabolism (edited by J. K. Grant), Academic Press, London 1963, p Flatt, J. P., and Ball, E. G., J. Biol. Chem. 241 (1966) Rognstad, R., and Katz, J., Proc. ATatl. Acad. Sci. U. S. 55 (1966) Del Boca, J., and Flatt, J. P., Helv. Physiol. Pharmacol. Acta, 25 (1967) CR Flatt, J. P., and Del Boca, J., Federation Proc. 28 (1969) Ball, E. G., and Merrill, M. A., Endocrinology, 69 (1961) Flatt, J. P., and Ball, E. Q., Biochem (1963) Cuppy, D., and Crevasse, L., Anal. Biochem. 5 (1963) Krebs. H. A.. and Hensrleit. K.. 2. Phwsiol. u Chem. 210 (1932) Ball, E. G., Martin, D. B., and Cooaer. 0.. J. Biol. Chem. 234 (1959) Wieland, O., Biochem (1957) Garland, P. B., and Randle, P. J., Nature, 196 (1962) Bush, E. T., Anal. Chem. 36 (1964) Denton, R. M., and Randle, P. J., Biochem. J. 104 (1967) Steinberg, D., In The Control of Lipid Metabolism (edited by J. K. Grant), Academic Press, London 1963, p Jungas, R. L., and Ball, E. G., Proc. Natl. Acad. Sci. U. S. 47 (1961) England, P. J., and Randle, P. J., Biochem. J. 105 (1967) Wise, E. M., Jr., and Ball, E. G., Proc. Natl. Acad. Sci. U. S. 52 (1964) Ball, E. G., Advan. Enzyme Reg. 4 (1966) Jungas, R. L., Biochemistry, 7 (1968) Felber, J. P., Zaragoza, N., Grassi, L., Moody, A. J., and Vannotti, A., Schweiz. Med. Wochschr. 96 (1966) Krebs, H. A., Advan. Enzyme Reg. 5 (1967) Hohorst, H. J., Kreutz, F. H., and Biicher, Th., Biochem. Z. 332 (1959) Utter, M. F., and Keech, D. B., J. Biol. Chem. 235 (1960) PC Atkinson, D. E., Ann. Rev. Biochem. 35 (1966) 85. J. Del Boca Institut de Biochimie de I Universit6 21 Rue de Bugnon, CH-1005 Lausanne, Switzerland J. P. Flatt s present address: Department, of Nutrition and Food Science Massachusetts Institute of Terhnology Cambridge, Massachusetts 02139, U.S.A.