THE OF BIOLOGICAL CHEMISTRY Vol. No. 1, of IO. in U.S. of ... · THE JOURNAL OF BIOLOGICAL CHEMISTRY Printed in U.S.A. Vol. 258. No. 1, Issue of January IO. pp. 483-490, 1983 Effects

T H E JOURNAL OF BIOLOGICAL CHEMISTRY

Printed in U.S. A. Vol. 258. No. 1, Issue of January IO. pp. 483-490, 1983

Effects of Insulin on COz Fixation in Adipose Tissue EVIDENCE FOR REGULATION OF PYRUVATE TRANSPORT*

(Received for publication, May 25, 1982)

Rafiqur RahmanS, Flavia O’Rourke, and Robert L. Jungasg From the Department of Physiology, University of Connecticut Health Center, Farmington, Connecticut 06032

Insulin was found to double the rate of incorporation of HI4CO3- into protein by segments of rat epididymal adipose tissue provided the incubation medium contained a suitable energy substrate such as fructose. Overall protein synthesis was increased by insulin to a lesser extent, one-third as measured by tritiated water indicating that insulin also increased C 0 2 fixation into amino acids. The latter could be demonstrated only when the tissue amino acid pools were expanded by the addition of aspartate to the incubation medium. The pattern of labeling observed in the amino acids indicated that COz fixation occurred primarily at the pyruvate carboxylase step. Addition of pyruvate to the incubation medium also increased COz fixation and this effect was not additive with that of insulin, suggesting that insulin acted by increasing the availability of pyruvate to the carboxylase. No change in carboxylase activity could be measured. Mitochondria isolated from tissue exposed to insulin retained a higher capacity to fix COz into acid-soluble products provided they were not freeze-thawed or sonicated. Uptake of pyruvate by mitochondria incubated 1 min at 2 “C or 5 s at 15 “C was doubled by prior insulin treatment of the tissue. It is concluded that insulin increases the flux through pyruvate carboxylase in adipose tissue in part by increasing the transport of pyruvate through the inner mitochondrial membrane.

Very little study has been directed at the effects of insulin on CO, fixation. The studies of Manchester (Manchester and KrahI, 1959; Manchester and Young, 1959) established that insulin increases the incorporation of label from H14C03- into protein by rat diaphragms about 50%, with the label appearing mainly in aspartate and glutamate. Since insulin causes a similar increase in the incorporation of labeled amino acids and other precursors into protein, the increased bicarbonate incorporation was considered a reflection of insulin’s action on overall protein synthesis and was not pursued further.

Herrera and Renold (1965) reported that insulin would increase HI4CO3- incorporation into protein in rat adipose tissue segments 2-fold, provided glucose was present in the incubation medium. Incorporation of label from I4C-aminoacids was also unaffected by insulin unless glucose was added

* This work was supported by Grant AM 22142 from the National Institute of Arthritis, Metabolism, and Digestive Diseases. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduer- tisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

!$ Present address, Department of Biochemistry, University of Dacca, Dacca, Bangladesh.

5 To whom reprint requests should be addressed.

(Herrera and Renold, 1960; Carruthers and Winegrad, 1962) and, perhaps for this reason, the observation with bicarbonate was considered unremarkable. Further work was limited to establishing that the label appeared largely in aspartate and glutamate with traces in alanine, serine, and glycine. Since under the conditions of these experiments the addition of glucose or pyruvate was sufficient to increase amino acid incorporation into protein (Carruthers and Winegrad, 1962; Chistophe and Wodon, 1964; KraN, 1964; Herrera and Ren- old, 1965), the effects of insulin on bicarbonate or amino acid incorporation into protein could be readily interprebed as secondary to its effects on carbohydrate metabolism.

We report here our studies related to the effects of insulin on COY fixation which have led us to a reinterpretation of the pioneering studies just summarized. Preliminary reports of these studies have appeared (Rahman and Jungas, 1977; 1979).

EXPERIMENTAL PROCEDURES

Source of Tissue and Materials Male CD rats were obtained from Charles River Breeding Labo-

ratories, Wilmington, MA, and were maintained on Purina Laboratory Chow ad libitum for 1 to 3 weeks before use. Animals in the weight range 130 to 250 g were selected for all experiments. The rats were killed by decapitation, and the epididymal fat bodies were removed and floated on 0.15 M NaCl at room temperature. After discarding thick portions, each fat body was halved longitudinally. One such half from each of two rats was placed in each incubation flask containing 2 ml of Krebs-Henseleit bicarbonate medium as described previously (Taylor et al., 1973). In general, tissue segments were incubated at 37 “C in control medium for 30 min to establish basal conditions and then transferred to fresh media for the experimental incubation period of 15 to 60 min. When fasted refed rats were employed, the animals were fasted 3 days and refed breadsticks for 2 days prior to use. Statistical analyses of significance used Student’s t test for paired observations on the logarithms of the data.

Beef insulin obtained from Sigma was utilized as previously described (Ball et al., 1959). All isotopic compounds were from New England Nuclear. a-Cyano-4-OH-cinnamic acid and a-cyanocinnamic acid were obtained from AIdrich. Bovine serum albumin (Miles Lab- oratories, fatty acid-free fraction V) was dialyzed against 2 rnM EDTA 24 h, against distilled water 24 h, and lyophilized. Ficoll (Pharmacia) was dialyzed against distilled water for 24 h prior to lyophilization.

Isolation of Acid-soluble and Acid-insoluble Products Reactions were terminated by adding 0.5 ml of 100% (w/v) trichlo-

roacetic acid to the incubation flasks. The flask contents were immediately transferred to an all glass TenBroeck tissue grinder (Arthur H. Thomas) and homogenized. The homogenate was transferred to a centrifuge tube, and the grinder was rinsed with carrier bovine serum albumin, 20 mg in 2 ml of water. The combined homogenate and rinse were heated for 30 rnin at 85 “C and after cooling, lipids were extracted with diethyl ether (three times with 3 ml) and discarded. To the aqueous residue was added 0.5 ml of 100% (w/v) trichloroacetic acid followed by centrifugation. After recovering the supernatant, the precipitate was washed with 2 ml of ice-cold 10% (w/v) trichloroacetic acid and the washing combined with the initial supernatant to form the fraction termed “acid-soluble products.” After gassing with 95%

483

484 C 0 2 Fixation in Adipose Tissue

air, 5% COn for I min to remove traces of 14c02, an aliquot was transferred to 5 ml of Aquasol (New England Nuclear) for radioactivity measurements. The precipitate from the above procedure was dissolved in 1 ml (88%) (w/v) of formic acid, left a t room temperature for 30 min, and reprecipitated with 5 ml of ice-cold 10% (w/v) trichloroacetic acid. The pellet collected by centrifugation was suspended in 2 ml of 10% trichloroacetic acid and recollected on a glass fiber filter (Whatman). The residue, termed acid-insoluble products, along with the fiiter was transferred to a scintillation vial, 0.5 ml of water and 0.5 ml of Protosol (New England Nuclear) were added, and the vial was heated a t 55 "C for 2 h with occasional shaking. After the further addition of 10 ml of Aquasol, the vial was shaken vigorously and allowed to stand until the next day before making radioactivity measurements.

In experiments wherein tritiated water was utilized to measure protein synthesis rates, after the incubation was terminated by the addition of trichloroacetic acid, the tissue segments were blotted on absorbent tissue and shaken with 10 mi of 0.025 M NaCl for 20 min. This washing process was repeated six times or until the radioactivity removed by a 10-min wash was less than 2000 cpm. The tissue segments were then homogenized in 10% (w/v) trichloroacetic acid and processed as just described.

Resolution of Acid-soluble Products

The solution containing the acid-soluble products was extracted three times with equal volumes of diethyl ether to remove trichloroacetic acid and lyophilized. The residue was dissolved in 0.5 ml of water and passed over a column (0.8 X 14 cm) of AG2-X8 resin (Bio- Rad) in the hydroxide form. The column was eluted with I M acetic acid, and the first 7 ml emerging after the acid front were collected. This fraction, containing about 80% of the radioactivity in the acid- soluble fraction, was lyophilized and taken up in a small volume of water containing 500 pg each of carrier alanine, aspartate, glutamate, serine, and proline. This mixture was spread as a narrow band on a silica gel-coated plastic sheet (EM Laboratories, type 60, 0.2 mm), and the thin layer chromatogram was developed twice with metha- nol:chloroform:ammonium hydroxide:acetic acid (20:20:10:1). Amino acid bands were located with ninhydrin and scraped off into scintillation vials. Two ml of water were added and the vials were warmed to 60 "C for 15 min to extract the acids from the gel. Finally, 0.2 ml of 30% (w/v) H202 was added, the vials warmed a second time to decolorize, and 12 ml of Aquasol added for radioactivity measurements.

Preparation of Mitochondria

Three methods were employed depending on the use of the mitochondria. In Method I, used for ['4C]bicarbonate fixation studies, fat cells were isolated from 0.3 to 0.5 g of tissue according to Rodbell (1964) using 2 mg/ml of collagenase (Sigma) and a 50-min digestion period. The cells were washed and suspended in 5 ml of an ice-cold medium containing sucrose (0.3 M), EDTA (1 m), and Tris-C1 (5 mM) with the pH adjusted to 7.30 measured at room temperature. The suspension was vortexed vigorously for 30 s (Arthur H. Thomas Super Mixer) and centrifuged for 30 s a t 800 X g,,,. After collecting the milky infranatant fluid, the residual supernatant fatty layer was washed with 5 ml of the same medium, mixing and centrifuging as just described. The combined infranatant fractions were then centrifuged 10 min a t 10,ooO X g,,, and the mitochondrial pellet resuspended in the same medium for immediate use.

Method I1 was used when mitochondria were utilized for studies of pyruvate uptake in the presence of inhibitors and is essentially that described by Martin and Denton (1970). Cells from 3 to 3.5 g of tissue were suspended in 10 ml of ice-cold buffered sucrose (sucrose (0.5 M ) , Tris-CI (20 mM), ethylene glycol bis(P-aminoethyl ether)- N,N,N',N'-tetraacetic acid (2 mM), reduced glutathione (10 mM), and 2% (w/v) defatted (Chen, 1967) and dialyzed bovine serum albumin (Miles Laboratories, fraction V), pH 7.30, at room temperature) and vortexed in the Super Mixer for 1.5 min. After centrifugation for 2 min at 200 X g,,,, the mitochondria were collected from the infranatant fluid by centrifuging a t 20,000 X gave for 5 min. The pellet was washed once in the buffered sucrose medium and then resuspended in 5 ml of cold KC1-Tris buffer (KC1 (125 mM), Tris-C1 (20 mM), pH 6.80, at room temperature). The mitochondria were repelleted as above and the KC1 wash was repeated. Finally, the mitochondria were suspended in 1.0 ml of the KC1-Tris buffer for use. Mitochondrial protein was measured according to Lowry et al. (1951) after heating samples 30 min at 90 "C with 1 M NaOH and 4 mg/ml of deoxycholate.

Method I11 was developed to provide larger quantities of highly purified mitochondria used in studies of pyruvate uptake without inhibitors present. Fat bodies from 20 130 to 140-g rats were minced with scissors and apportioned into a control and an experimental flask each containing 10 ml of Krebs-Henseleit medium supplemented with 2% bovine serum albumin, 11 mM glucose, and 3 mg/ml of collagenase (Sigma). After 30 min, cells were washed three times, resuspended in 10 ml of fresh medium, and incubated 30 min a t 37 "C to &ow recovery of surface insulin receptors. Insulin (14 nM) was added to the experimental flask and incubation continued for 25 min. Cells were then homogenized in 7 ml of a hypertonic sucrose medium (0.5 M sucrose, 20 mM Tris-C1, 1 m~ ethylene glycol bis(P-aminoethyl ether)-N,N,N',N'-tetraacetic acid, 5 m~ sodium succinate, 3% albumin, pH 7.4, at 4 "C) using a Dounce grinder with a loose pestle (9 to 12 strokes). The homogenate was centrifuged 25 min at 800 X g,,,, and the aqueous layer underlying the fat cake was transferred carefully to a second tube preloaded with 1 ml of the homogenizing medwm adjusted to a density of 1.08 ( 2 4 "C) with Ficoll and 2 ml of unsupplemented homogenizing medium (density, 1.06). Centrifuga- tion for 15 min a t 29,000 X gave drove the mitochondria into a pellet, whereas plasma membrane fragments collected at the density inter- face and could be discarded. The mitochondrial pellet was resuspended in cold medium containing I25 I" KCI, 20 mM Tris-C1, 1.6 I" KH2P04, 2 mM MgC12, 0.1 I" ethylene glycol bis(P-aminoethyl ether)-N,N,N',N'-tetraacetic acid, and 1 m~ sodium succinate, pH 7.4, at 4 "C using a Dounce grinder and loose pestle. Such mitochondria exhibited a respiratory control ratio of 4 with pyruvate and a matrix volume of 2.1 2 0.3 (4) pl/mg of protein.

Measurement of Mitochondrial Spaces a n d of Pyruvate Uptake

Method I: Use of Inhibitors-The reaction was initiated by adding an aliquot of the mitochondrial suspension (Method 11) to plastic centrifuge tubes (1.5 ml) previously loaded with the following materials at 2 "C: 20 pl of 70% (w/v) perchloric acid, 200 p1 of silicone oil obtained by mixing Harwick F 50 and S F 96100 (7:3), and 800 pl of a solution containing KC1 (125 m ~ ) , Tris-C1 (20 m ~ ) , rotenone (20 p ~ ) , antimycin A (20 p ~ ) , [3-I4C]pyruvate (50 pM and 0.25 pCi/ml), and with or without a-cyanocinnamate (0.5 m ~ ) , all at pH 6.80 as measured at room temperature. After 200 pl of the chilled mitochondrial suspension (150 to 300 pg of protein) were added, the tubes were left on ice for 1 min and then centrifuged for 90 s at 15,000 X g,,,. Then 200 pl of the upper aqueous phase were transferred to a second tube previously charged with 20 p1 of 70% (w/v) perchloric acid. After mixing and centrifuging, an aliquot of the supernatant fluid was added to 5 ml of Aquasol for radioactivity measurements. The remainder of the original upper aqueous phase and the silicone oil were aspirated off so that 200 pl of water could be added to the perchloric acid containing the mitochondrial pellet. After mixing and centrifuging down the debris, an aliquot of this phase was taken for radioactivity measurements. To determine intra- and extramitochondrial volumes, identical incubations were carried out using [U-'4C]sucrose and tritiated water. This allowed the pyruvate uptake to be corrected for pyruvate remaining outside the sucrose-impermeable barrier.

Method II: Without Inhibitors-The reaction vessel consisted of 3/32-inch (inside diameter) Tygon tubing attached to the male Luer fitting of a 5-ml syringe. The syringe was fiist filled with 5 ml of wash medium (same as used to resuspend mitochondria in Method 111 except omitting succinate) and immersed in an ice bath with the Luer fitting exposed. A 5-cm length of Tygon tubing was then attached to the syringe and 200 pl of assay medium (same as wash medium with 0.78 mM [3-'4C]pyruvate, 1 pCi/tube, added) were injected midway to the tubing. The assay medium was at room temperature (20-22 "C). The reaction was initiated by adding rapidly 100 pl of cold mitochondrial suspension (250 to 300 pg of protein) to the assay medium via a second syringe fitted with a blunt 19-gauge needle. The mixture, now at 15 "C, was allowed to incubate for 5 s, after which it was ejected onto a polycarbonate filter (Nucleopore, 2.5 cm, 0.6-pm pore) in a vacuum well and washed immediately with the 5 ml of cold wash medium in the same syringe. The washing procedure was complete within 4 s. The filter was immersed immediately in 100 p1 of 5% trichloroacetic acid. The filtrate was collected in tubes containing 100 pl of 5% trichloroacetic acid for later analysis. Blanks lacking mitochondria were run in parallel and amounted to 15% or less of experimental values.

Labeled metabolites either collected on the filters or in the fitrate were separated by high voltage electrophoresis essentially as described by Lardy et al. (1966) using a Savant flat plate apparatus. Samples must be processed as soon as possible for highest recovery

COa Fixation in Adipose Tissue

TABLE I Effect of insulin on [I4CJbicarbonate incorporation into trichloroacetic acid-insoluble material by adipose

tissue segments Mean f S.E. of number of paired observations in parentheses. All incubation media contained 4.7 pci of

H14C03-/ml with or without insulin (7 nM). Bicarbonate concentration either 25 m~ (bicarbonate medium) or 0.5 mM (phosphate medium). Incubation period, 60 min. Tissue segments were paired in rows but not columns; vertical comparisons are, therefore, approximate only. Conversion of counts/min to nanomoles is based solely on the specific activity of the bicarbonate in the starting medium and is intended only to give an estimate of the magnitude

485

of the rates involved. Conditions Control Insulin Effect

cprn.g".h" nrnol.g".h" cprn.g".h-l nrnol.g".h-' B

Krebs-Ringer bicarbonate No substrate 3,800 f 700 (14) Fructose, 11 mM 5,690 f 330 (59) Fructose and cycloheximide, 100 200 f 70 (4)

Krebs-Ringer phosphate Fructose, 11 mM 103,000 f 6,000 (19) Fructose and cycloheximide, 100 6,700 f 1,000 (4)

M/ml

of labeled pyruvate. Separation was accomplished in 90 min on an 8 "C plate using 4000 volts and a current of 5 mA/strip of Whatman 3" paper (4 X 57 cm). Standards of pyruvate and oxalacetate were located with a 2,4-dinitrophenylhydrazine spray (Waldi, 1965) and other acids with an indicator spray containing glucose and aniline in butanol-ethanol (Schweppe reagent) (Michl, 1975). The strips were cut into I-cm sections and radioactivity determined using a scintillant containing 10 g of 2,5-diphenyloxazole and 0.126 g of 1,4-bis[2-(5- phenyloxazolyl)]benzene/liter of toluene.

The total pyruvate content of mitochondria prepared by Method 111 was determined after filtration by fluorimetric assay (Passonneau and Lowry, 1974).

RESULTS

When segments of rat epididymal adipose tissue were incubated with ['4C]bicarbonate in a Krebs-Henseleit bicarbonate medium, label was incorporated into trichloroacetic acid- insoluble material. In conf i ia t ion of Herrera and Renold (1965), the rate of this process could be accelerated %fold by the addition of insulin provided that fructose was also present (Table I). In the absence of added substrate, insulin was totally without effect. Since the incorporation was nearly completely inhibited by cycloheximide addition, it is pre- sumed that the measured incorporation is largely into protein and that it occurs only during de novo protein synthesis. When the bicarbonate medium was replaced by Krebs-Ringer phosphate, the insulin effect was not as pronounced. The lower absolute rate of incorporation in the phosphate medium is most likely the result of the far lower bicarbonate concentration in this buffer (as noted in the legend).

The substrate requirement for the insulin effect was pursued further as summarized in Table 11. Glucose served equally as well as fructose but not better, even though glucose uptake is increased far more by insulin than is fructose uptake (Ball and Cooper, 1960; Froesch and Ginsberg, 1962; Pozza and Ghidoni, 1962). Pyruvate or aspartate also supported the hormonal effect but less completely than the sugars. When glycogen-rich tissue from fasted-refed rats (Frerichs and Ball, 1962) was employed, the insulin stimulation could be seen without added substrate, indicating that intracellular glycogen could also meet the substrate requirement. Indeed, it served fully as well as medium fructose in the refed tissue.

Since it is widely accepted that insulin increases protein synthesis in adipose tissue (Krahl, 1959; Minemura et al., 1970), it seemed possible that insulin's effect on bicarbonate incorporation into protein could be a simple reflection of this fact. To investigate this point, we wished to compare the magnitude of insulin's effect on bicarbonate incorporation into

12.3 3,800 f 650 12.3 0 18.4 11,400 f 790 36.8 100 0.6 240 & 40 0.8

5.5 150,OoO f 10,000 8.0 46 0.4 9,400 f 2,200 0.5

TABLE I1 Ability of various carbon sources to support the action of insulin

on [I4C]bicarbonate incorporation into trichloroacetic acid- insoluble material

pCi of HI4COy-/ml with or without insulin (7 m). Glycogen-rich Incubation medium was Krebs-Ringer bicarbonate containing 4.7

tissue segments were obtained from rats fasted 3 days and then refed 2 days on a high carbohydrate diet. Values given are relative to normal tissue segmentsincubated 60 min without substrate (2800 cpm. g".h-').

Conditions Control Insulin Change 4

Normal tissue No substrate (8) 100 f 10 100 f 17 0 Glucose, 11 mM (2) 207 f 24 423 f 67 104 Pyruvate, 5 mM (4) 274 -t 5 374 k 3 36 Aspartate, 1 mM (4) 89 f 6 132 -C 1 48

Glycogen-rich tissue No substrate (2) 765 f 294 I100 f 235 44 Fructose, 11 mM (2) 903 & 286 1309 f 288 45

protein with its effect on overall protein synthesis. Measure- ment of the latter parameter is not straightforward, owing to the difficulty of determining the specific activity of the immediate precursors utilized for protein assembly. We sus- pected that simply measuring insulin's effect on label incorporation into protein from radioactive amino acids added to the incubation medium might not serve this purpose reliably as insulin is known to decrease proteolysis in adipose tissue (Christophe and Wodon, 1964; Minemura et al., 1970), an effect which might well alter precursor pool specific activities. We therefore turned to a rarely used procedure and utilized tritiated water to measure protein synthesis (Humphrey and Davies, 1976). The method rests on the assumption that the a-hydrogens of many amino acids in the cellular pool will rapidly exchange with the hydrogens of water through the action of aminotransferases and that this exchange will cease once the amino acids are incorporated into protein. For some amino acids, additional hydrogen atoms will also exchange rapidly with water, owing to metabolic reactions. If these exchange reactions are rapid relative to the rate of protein synthesis, the incorporation of label from tritiated water into protein might serve as a simple reliable method for measuring the total rate of protein synthesis even as it serves as a useful measure of the total rate of fatty acid synthesis (Jungas, 1968). Tissue segments were incubated in medium containing ["C] bicarbonate and :'H20, and the incorporation of both labels

486 Con Fixation in Adipose Tissue

into trichloroacetic acid-insoluble material was measured. As summarized in Table 111, insulin addition increased label incorporation from bicarbonate somewhat over P-fold, while it increased tritium incorporation by only one-third. Note that there were no exogenous amino acids present in this experiment. Evidence that the tritium incorporation serves at least as a rough measure of protein synthesis is provided from two sources. The incorporation is largely inhibited by cycloheximide (Table 111), and the magnitude of the insulin effect on tritium incorporation is similar to that seen with labeled amino acids. Thus, in five experiments where 1 mM L-[U-'~C] leucine was added to the medium, insulin increased label incorporation into acid-insoluble material by 34%, while with 1 mM ~-[U-'~C]alanine, the average increment was 35% in 11 tissue pairs. When 1 mM ~-[U-'~C]aspartate was used as the labeled precursor, it was necessary to omit medium fructose to avoid undue label dilution within the cell; the insulin effect was then 31% averaged over 27 pairs (fructose itself had little effect on label incorporation into protein from other precursors). We conclude from these data that insulin increases overall protein synthesis in adipose tissue under these conditions only about one-third. This effect is, therefore, too small to account for the greater part of the insulin stimulation of bicarbonate incorporation into protein.

It follows from the above results that insulin would also be expected to increase the fixation of CO, into the cellular amino acid pool, although to a somewhat lesser extent than it increases fixation into protein. This was examined by looking at bicarbonate incorporation into the total trichloroacetic acid- soluble pool of metabolites of the tissue. Contrary to expec- tation, it was found that insulin had no significant effect on this parameter (Table IV) whether the tissue segments were incubated with or without fructose. Since there was the sug- gestion of an effect in the presence of fructose, we repeated the experiments with 1 mM aspartate added to the medium. It was thought that this maneuver might expand the pool of acid-soluble metabolites and thus serve to trap label entering the pool more effectively. It can be seen from the final line of Table IV that this procedure did allow the demonstration of a 58% increase in bicarbonate incorporation into acid-soluble metabolites in the presence of insulin and fructose. This is very close to the magnitude of effect anticipated from the data on C02 fixation into protein.

I t was thought likely that additional insight into the bio- chemical processes involved in the COz fixation phenomenon might result if we looked at incorporation into individual components of the free amino acid pool. Data are presented in Table V both with and without pool expansion with aspartate. The majority of label was recovered in just the four amino acids shown in the table. The data make clear the

TABLE I11 Comparison of incorporation of HI4C03- and " 2 0 into

trichloroacetic acid-insoluble material Tissue segments were incubated 60 min in Krebs-Ringer bicarbon-

ate containing fructose (11 nM) with or without insulin (7 nM). Tritiated water (1 mCi/ml) and H14COs-, (4.7 pCi/ml) were present throughout. When present, cycloheximide concentration was 100 pg/ ml. Values are mean k S.E. of the number of replicates given in parentheses.

No cycloheximide Plus cycloheximide Conditions

H~~co.; ~ H ~ O ~ 1 4 ~ 0 : ~ - , 'H~o cpm.g".h"

Control 2600 -+ 200 (18) 4720 k 200 289 (2) 824 Insulin 5510 -+ 420 (18) 6480 k 270 297 (2) 941 '% Effect +113 -+ 23 +37 f 8 Ratio of effects 3.05 f 1.10 (p < 0.05)

TABLE IV Effect of insulin on ['4C]bicarbonate incorporation into acid-

solubleproducts by rat adipose tissue segments Mean k S.E. of number of paired observations indicated in paren-

theses. Tissue was incubated I h in Krebs-Ringer bicarbonate medium containing 4.7 pCi/ml of Hi4CO:r- with or without insulin (7 nM). Tissues were paired in rows but not columns; vertical comparisons are, therefore, approximate only. Substrate added I Control Change Insulin

~~

cpm.g".h" B

mM

aspartate

NS, not significant.

reason for the difficulty in demonstrating an insulin effect on bicarbonate incorporation into acid-soluble metabolites in the absence of added aspartate. While label incorporation into aspartate itself was doubled by insulin under these conditions, the labeling of three other amino acids was actually dimin- ished by about 25%. This unanticipated result was not evident in the presence of added aspartate when labeling of all the amino acids was at least doubled, save that of serine. The same four amino acids also contained nearly all of the label incorporated into tissue protein from [I4C]bicarbonate as determined by acid hydrolysis of the trichloroacetic acid-insoluble fraction and chromatographic resolution of the resultant amino acids (Table VI). In particular, there was negligible label incorporated into arginine, demonstrating the absence of urea cycle activity in adipose tissue. We conclude from this pattern of labeling that the major site of bicarbonate fixation is the reaction catalyzed by pyruvate carboxylase within the mitochondria of the tissue and that in some manner insulin promotes the flow of carbon through this pathway.

It remains to consider possible mechanisms for this action of insulin. Contrary to the situation found earlier in studying flow-through pyruvate dehydrogenase (Jungas, 1970), it has not been possible to demonstrate any increase in the activity of pyruvate carboxylase by assays conducted in homogenates of tissues previously exposed to insulin (data not presented; Coore et al., 1971). There was also no change in phospho- enolpyruvate carboxykinase activity in tissue extracts. It seemed possible that insulin might act by increasing the supply of pyruvate available to pyruvate carboxylase, and we therefore examined the effect of adding exogenous pyruvate to the incubation medium. The results are shown in Table VII. The addition of as little as 1 r r m pyruvate increased bicarbonate incorporation into acid-insoluble products as effectively as insulin even though 11 rn fructose was also present. Moreover, in the presence of excess pyruvate, the further addition of insulin increased incorporation only about 40% instead of the usual doubling. In other words, in the presence of excess pyruvate, the insulin effect on bicarbonate incorporation into protein could be accounted for simply by the increase in overall protein synthesis caused by the hormone. There was no longer any need to postulate an additional action on bicarbonate fixation at the level of pyruvate carboxylase. This interpretation is supported by the data on incorporation into acid-soluble products. Even though the soluble pool was being expanded by the addition of pyruvate as shown by the increase in incorporation in the control column, there was no significant effect of insulin. Thus, carbon flow through the carboxylase can be increased either by increasing the availability of pyruvate or by adding insulin and the effects

CO2 Fixation in Adipose Tissue 487

TABLE V

Effect of insulin on [‘4C/bicarbonate incorporation into adipose tissue free amino acid pools

Tissue was incubated 1 h in Krebs-Ringer bicarbonate medium containing 100 &i/ml of H’4C0z- and 11 mM fructose with or without insulin (7 nM). In one of the two experiments, alanine and proline were not adequately resolved to be measured individually.

No aspartate added 1 rn~ aspartate added Amino acid

Control Insulin Effect Control Insulin

cpm,g-‘.h-’ % cpm.g-‘.h-’ Aspartate 40,000 f 1,200 (2) 82,100 f 15,000 +103 251,000 r+ 10,200 (2) 705,000 * 99,000 Glutamate 206,000 f 39,000 (2) 145,000 f 22,500 -30 236,000 + 18,700 (2) 472,000 + 107,000 Serine 98,400 + 3,500 (2) 73,400 -t 2,000 -25 43.700 f 1,300 (2) 49,400 + 14,000 Alanine 11,800 (1) 8,700 -26 36,000 (1) 81,300

Effect 7

+I81 +100

+13 +126

TABLE VI Effect of insulin on [‘4C/bicarbonate incorporation into individual

amino acids of the acid-soluble pool Tissue segments were incubated in Krebs-Ringer bicarbonate me-

dium containing 47 @i/ml of HL4CO:~- and 11 mu fructose with or without insulin (7 no). Acid-insoluble products were isolated as described in the text and hydrolyzed in 6 N HCl in a sealed tube at 100 “C for 48 h. Amino acids were separated on an automatic analyzer equipped with a stream splitter to allow samples of each peak to be sampled for radioactivity measurements. Data are from a single experiment.

Amino acid Control Insulin Ratio Control Insulin Effect

pmol/sample dpm.pmol-’ 90 Aspartate 6.39 7.62 1.19 1180 2830 140 Glutamate 6.69 8.25 1.23 2230 4080 83 Serine 3.12 3.75 1.20 550 590 7 Alanine 5.16 6.48 1.25 490 1040 112 Arginine 3.24 3.95 1.22 None None

TABLE VII Effect ofpyruvate on the stimulation by insulin of [‘4C]bicarbonate

incorporation into acid-soluble and insolubleproducts Adipose tissue segments were incubated 60 min in Krebs-Ringer

bicarbonate medium containing 11 mbr fructose and 4.7 @i/ml of H14C03- with or without insulin (7 nM). Data at different pyruvate concentrations were obtained from different rats and are expressed relative to control tissue without pymvate (7,900 cpm. g-’ . h-’ acid- insoluble and 61,700 cpm. g-’ . h-’ acid-soluble). There was no significant effect of insulin on acid-soluble products.

Pymvate Concen- tration

Acid-insoluble I

Control Insulin Effect

Average ,% effect, pyruvate present

% +102

+35 +48 +49 +41

+43 -+ 3

Acid-soluble

Control

relatwe 100 -t 8 (7)

450 (2) 515 (2) 843 (2)

1122 (2)

Insulin

dues

152 -c 29 359 510

1036 1064

are not additive. While these data favor the view that insulin acts by increas-

ing the availability of pyruvate, previous workers had shown that insulin probably does not increase whole tissue levels of pyruvate when the only added substrate is fructose (Coore et al., 1971). Thus, it seemed likely to us that insulin might make more pyruvate available to pyruvate carboxylase by increasing the rate of entry of pyruvate into the mitochondrial matrix space. This possibility would be open for investigation if mitochondria prepared from insulin-treated tissue retained their altered metabolic rates after isolation. In the case of adipose tissue, it is difficult to isolate intact mitochondria from homogenates of whole tissue; much better results are obtained if isolated fat cells are first prepared and then gently ruptured to release the mitochondria (Halperin et al, 1969;

Martin and Denton, 1970; Pate1 and Hanson, 1970). We have had difficulty in obtaining consistent effects of insulin on bicarbonate incorporation into acid-soluble products by isolated fat cells. Therefore, we found it useful to expose intact tissue segments to insulin, digest the segments with collagenase also in the presence of insulin, and then rupture the cells and recover the mitochondria by centrifugation. Using this protocol, it was possible to demonstrate that mitochondria prepared from tissue exposed to insulin retained an increased ability to incorporate [‘*C]bicarbonate into acid-soluble products (Table VIII, first line). This was true even though the mitochondria were incubated in medium containing 5 mM pyruvate. Thus, the action of insulin on bicarbonate incorporation into acid-soluble products must involve events occur- ring in the mitochondrial fraction and cannot be the result simply of an increase in cytosolic pyruvate levels.

If the action of insulin were on the transport of pyruvate across the impermeable inner mitochondrial membrane, it should be apparent only in intact mitochondria. Mitochondria were therefore isolated as before, but were subjected to freeze- thawing prior to incubation with labeled bicarbonate. The results are shown in Table VIII. Note that the intact mitochondria exposed to insulin exhibited a 56% greater rate of fixation of CO* as seen in the previous experiments, but this effect was lost after freeze-thawing. Thus, the insulin stimulation could be mimicked by destroying the permeability barrier. To demonstrate that the permeability barrier had been lost after freezing and that there was no change induced by insulin treatment in the activity of the carboxylase itself, we made further measurements after adding excess exogenous acetyl-CoA and ATP. Addition of these cofactors greatly increased the rate of bicarbonate fixation into acid-stable acid- soluble products (Table VIII), and this fixation was dependent on the presence of pyruvate (data not shown). However, no insulin effect was now apparent. Similar results were obtained after sonication of the mitochondrial suspension (data not shown).

While these results are consistent with an effect of insulin on pyruvate transport, were the insulin effect on bicarbonate fixation secondary to an enhanced supply of intramitochondrial acetyl-CoA (an allosteric activator of the carboxylase) generated via an activated pyruvate dehydrogenase, the effect might also disappear when the membranes were made perme- able or excess acetyl-CoA was added. We therefore undertook to measure directly the uptake of pyruvate by mitochondria under conditions where little metabolism occurred. For this purpose, mitochondria were incubated with low levels of [3- ‘*C]pyruvate (50 FM) in an ice bath (2 “C) for only 1 min with added rotenone and antimycin A. Under these conditions, we could measure essentially no flux through pyruvate dehydrogenase (using [ l-‘4C]pyruvate conversion to 14COz). Uptake of pyruvate was measured by centrifuging the mitochondria through silicone oil into perchloric acid. The results are given in Table IX. Pyruvate uptake in the mitochondria isolated

488 COZ Fixation in Adipose Tissue

TABLE VI11 Effect of insulin treatment on ['4CJbicarbonate incorporation into acid-soluble products by isolated

mitochondria Mitochondria were isolated from tissue which had been incubated 15 min a t 37 "C in Krebs-Ringer bicarbonate

medium containing 11 nm fructose and 4% (w/v) defatted albumin with or without insulin (7 m). Insulin was also present during the collagenase digestion period (Method I), as described in the text. Mitochondria were incubated 20 min at 30 "C in medium containing pyruvate, 5 mM; KHI4CO:,, 12 mM (10 pCi/ml); KeHPO,, 6 mM; MgCln, 6 mM; sucrose, 0.3 M; Tris-C1, 1.2 mM; and EDTA, 0.2 mM. The reaction was stopped by adding trichloroacetic acid to 10% (w/v) and label incorporated into acid-soluble acid-stable products measured. In Experiment 2, freeze-thawing was done twice utilizing dry ice-alcohol and tap water. Each assay was performed in triplicate on the number of mitochondrial preparations indicated in parentheses.

. ,

TABLE IX Effect of insulin onpyruvate uptake by isolated mitochondria Mitochondria were isolated (Method 11) from tissue previously

incubated 15 min at 37 "C in Krebs-Ringer bicarbonate medium containing 11 mM fructose and 3% (w/v) bovine serum albumin with or without insulin (7 nM). Incubation was for 1 min a t 2 "C as described in the text. Measurement was of total intramitochondrial label assumed for calculation purposes to be entirely pyruvate.

Control p Value Effect Insulin Q

Pyruvate uptake (nmol .mg protein-min")

No additions

NS" 0.27 f 0.05 0.22 f 0.06 Plus a-Cyanocinnamic acid,

~ 0 . 0 1 +83 0.42 f 0.05 0.23 k 0.03 (6)

0.5 mM (6) '' NS, not significant.

from insulin-treated tissue was some 80% greater than in the controls. This increment in pyruvate uptake was virtually abolished by the specific inhibitor of the pyruvate transport system, a-cyanocinnamic acid (Halestrap, 1975), although the basal uptake rate was not affected. Similar results were observed when the incubation was terminated after 30 s or 2 min (data not shown).

In the experiment just described, we could not be certain that the increased label found in the mitochondria from insulin-treated tissue was in the form of pyruvate even though inhibitors were present. Moreover, the presence of the inhibitors undoubtedly drastically alters the physiological state of the mitochondria, as does the lowered temperature, and study of the hormonal effect under more normal conditions is clearly desirable. We therefore performed an additional series of experiments using mitochondria prepared as carefully as possible, consistent with reasonable speed (Method 111), and incubated them with labeled pyruvate but without inhibitors and at a higher temperature for only 5 s. Following the rapid termination of the incubation by filtration (Method 11), the mitochondrial content of labeled pyruvate was measured. As shown in Table X, mitochondria prepared from tissue previously exposed to insulin consistently showed a higher content of labeled pyruvate. Uptake of label by mitochondria is linear for more than 10 s under these conditions, and the mitochondria prepared from insulin-treated cells did not contain a higher level of pyruvate when isolated than did the controls (control, 0.18 & 0.05 and insulin, 0.21 k 0.07 nmol/mg of protein, respectively) as determined by fluorometric assay. We conclude that in some manner insulin increases the rate of entry of pyruvate into adipose tissue mitochondria.

Preparation Control Insulin "-

Effect ."

cpm (mgprotein) (20 mzn) ' 't -

1 Intact mitochondria 39,400 f 7,200 (8) 60,800 f 8,300 +54 (p < 0.05) 2 Intact mitochondria 26,500 f 200 (2)

Frozen-thawed mitochondria 41,500 -C 2,600 +56

No additions 40,200 f 1,500 (2) 39,100 f 17,100 Acetyl-coA, 500 PM 773,000 f 47,000 (2) 667,000 f 144,000 Acetyl-coA plus ATP. 6 mM 1.560.000 f 30.000 (2) 1,300,000 f 106,000

TABLE X Pyruvate transport by mitochondria prepared from adipocytes

incubated with and without insulin Mitochondria were isolated (Method 111) from tissue previously

incubated 25 min at 37 "C in Krebs-Henseleit medium containing 11 mM glucose and 2% (w/v) bovine serum albumin with or without insulin (14 nM). Mitochondria, 250 pg of protein, were incubated for 5 S a t 15 "c with 0.78 mM [3-'4C]pyruvate (1 pCi) as described in the text. Mitochondrial constituents were separated by high voltage electrophoresis.

Mitochondrial labeled pyruvate

Control Insulin Experiment Increase

nmol/mgprotein I

1 0.521 1.270 144 2 0.267 0.707 165 3 0.238 0.789 232 4 0.429 0.546 27 5 0.253 0.396 57 6 0.131 0.323 147

Average p = 0.003

0.307 k 0.142 0.672 f 0.342 129 k 31

Finally, in c o n f i a t i o n of the involvement of the pyruvate transporter in the action of insulin on bicarbonate fixation into acid-soluble products, we show in Table XI that another inhibitor of that carrier system, a-cyano-4-hydroxycinnamate (Halestrap and Denton, 1974a), totally abolished the insulin stimulation seen in intact fat cells. This particular preparation of cells was unusually responsive to the hormone. Note that again the inhibitor had little effect on basal rates. Halestrap and Denton (1975) observed earlier that the effect of insulin on glucose conversion to fatty acids by fat cells was reduced about 40% in the presence of 0.1 mM a-cyano-4-hydroxycinnamate.

DISCUSSION

Carbon flow through pyruvate carboxylase plays an important role in lipogenesis in rat adipose tissue (Pate1 and Hanson, 1970). The oxalacetate so produced is utilized for citrate synthesis which ultimately provides virtually all the carbon and about one-half the NADPH used in fatty acid synthesis (Flatt and Ball, 1964). It is thus widely appreciated that insulin addition increases the flux of carbon through the carboxylase anytime it promotes the conversion of carbohydrate to fat. The mechanisms responsible for this alteration of flux remain incompletely understood. In the face of repeated failures, including our own, to observe an activation by insulin of the carboxylase as assayed in tissue extracts, it has often

COZ Fixation in Adipose Tissue 489

TABLE XI Effect of an inhibition ofpyruvate transport on insulin's action on

['4C]bicarbonate incorporation into acid-solubleproducts by adipocytes

Isolated fat cells were incubated 60 min at 37 "C in Krebs-Ringer bicarbonate medium containing 11 mM fructose, 3% defatted dialyzed bovine serum albumin, 4.7 pCi/ml of HL4C03- with or without 7 nM insulin. Values are means & S.E. of eight replicate determinations all from a single preparation of cells.

Conditions Control Insulin Effect p Value cpm.g" fat cel1s.h" 7c

No additions 22,400 f 1,300 61,900 f 5,900 +176 t0.003 a-Cyano-4-OH- 18,000 f 1,900 15,700 f 700 NS"

cinnamic acid, 1 rnM

" NS, not significant.

been suggested (e.g. Randle and Denton, 1973) that the increased flux is caused by an increased mitochondrial concentration of the enzyme's positive effector, acetyl-coA, resulting from insulin's activation of pyruvate dehydrogenase. Direct evidence for this view is sparce owing to the difficulty of ascertaining the actual effect of insulin on mitochondrial levels of metabolites. Yamazaki and Haynes (1975) ruled out this explanation in the case of the activation of pyruvate carbox- ylation by glucagon in liver.

The evidence presented here suggests that an additional previously unrecognized action of insulin may be involved, namely, an activation of the transport of pyruvate across the mitochondrial membrane. While all of the data obtained in intact cells are consistent with this interpretation, the most direct evidence comes from the pyruvate uptake measurements with isolated mitochondria (Tables IX and X). Under the conditions of those measurements, uptake is doubled in mitochondria prepared from insulin-treated tissue. This effect could be accomplished rather indirectly, for example, by insulin causing a change in the intramitochondrial concentration of a metabolite which persists throughout the isolation procedure and causes accelerated pyruvate entry. Alternatively, it could be a more direct manifestation of the same insulin second messenger substance which is thought to be responsible for the activation of pyruvate dehydrogenase in these cells (Larner et al., 1979; Seals and Jarett, 1979, 1980; Seals and Czech, 1981). Further studies of the nature of this action of insulin are in progress. It may be noted here, however, that the concentration of pyruvate inside the mitochondria after a 1-min incubation with 50 ~ L M external pyruvate was about 200 ~ L M in both control and insulin-treated samples (data not presented). The 4-fold concentration gradient is similar to that reported in liver mitochondria by Paradies and Papa (1973) and by Halestrap and Denton (1974a) and presumably reflects the fact that a proton enters the mitochondria with the pyruvate anion (Francavilla et al., 1971; Papa and Para- dies, 1974; Halestrap, 1975). The pH gradient across the inner mitochondrial membrane thus becomes another important factor to consider. Titheradge and Coore (1976a) and Hales- trap (1977) have suggested that glucagon increases pyruvate uptake into mitochondria from liver via an indirect mecha- nism, ultimately increasing the driving force for pyruvate entry. Indeed, pyruvate transport into mitochondria may be a rate-controlling step in liver gluconeogenesis (Thomas and Halestrap, 1981). I t is curious that insulin should have an action on adipose tissue mitochondria pyvuate uptake resem- bling that of glucagon in liver, while in liver, insulin seems to have no effect at all (Titheradge and Coore, 1976b). Data to be presented separately indicate that glucagon and epineph- rine have effects opposite to those of insulin on adipose tissue pyruvate metabolism.

The measurements of the effect of insulin on bicarbonate incorporation into tissue free amino acids revealed a complex pattern. The measurements were conducted over a rather long time period of 1 h so that these data could be compared with the incorporation into protein. Thus, changes in both pool sue and specific radioactivity must be considered in interpreting the insulin effect. Incorporation into amino acids in protein linkage when corrected for the one-third increase in o v e r d protein synthesis should reflect, however, just the specific activity of the free amino acid pools and not their size. The data in Table VI yield estimates of an increase in specific activity for aspartate of about 8055, about 40% for glutamate, and about 60% for alanine, while serine shows a 20% decline. Comparing these estimates with the effects of insulin on labeling of free amino acid pools in Table V, it would appear that in the absence of added aspartate, insulin has no effect on the serine pool size, that it increases somewhat the aspartate pool, but that it decreases the tissue pools of glutamate and alanine to about one-third the basal level. The increase in the specific activity of aspartate must result from an increased rate of label incorporation into the pool, but these data do not indicate whether the specific radioactivity increases of glutamate and alanine result from this cause or from a decreased input of cold amino acids into these pools. If the latter were true, expanding the pools by adding aspartate to the medium should tend to overwhelm the effect and reduce the magnitude of the insulin effect. The opposite was seen. We conclude that insulin probably acts by increasing the flow of label from ["C] bicarbonate into all three amino acids, aspartate, glutamate, and alanine, but has no such effect on flow to serine.

By what means might insulin lower the pool sizes of glutamate and alanine? If insulin increases the activity of acetyl- CoA carboxylase (Halestrap and Denton, 1974133, this would remove one of the products of the citrate cleavage reaction. This should favor the generation of cytosolic oxalacetate while draining citrate. Transamination of cytosolic oxalacetate is the probable source of most labeled aspartate, while citrate would be the source of 2-oxoglutarate and thus of labeled glutamate. Flatt and Ball (1964) found that insulin reduced the flow of citrate into the citric acid cycle some 60% and a proportionate decrease in the glutamate pool size would not be unreasonable. A similar argument can be applied to the alanine pool since insulin is known to activate pyruvate utili- zation. What remains most unclear to us is the process by which label is incorporated into serine in amounts as great or greater than recovered in alanine.

While we have presented here only limited data bearing on the issue, we believe the use of tritiated water as a method for measuring overall protein synthesis has considerable promise. Further work is needed to document the stoichiometry of the tritium incorporation under a variety of conditions. If the method proves sufficiently exact, it would eliminate the ne- cessity for determining the specific activity of amino acyl- tRNA pools as required for accurate interpretation of I4C- aminoacid incorporation data. Rapid degradation of newly synthesized protein would interfere with either method. Dou- ble label studies utilizing tritiated water and 'T-aminoacids may allow simultaneous assessment of the effects of hormones or other agents on protein synthesis and degradation.

Acknowledgment-We thank Professor E. Khairallah of the Storrs campus of the University of Connecticut for performing the analyses of specific radioactivities of amino acids in protein hydrolysates.

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