Influences of intracellular pyridine nucleotide redox states on fatty acid synthesis in isolated rat hepatocytes

ARCHIVES

OF

Influences

BIOCHEMISTRY

AND

BIOPHYSICS

179,

310-321 (1977)

of Intracellular Pyridine Nucleotide Redox States on Fatty Acid Synthesis in Isolated Rat Hepatocytesl

GEORGE

A. COOK,

ANN

C. SULLIVAN,

AND JOSEPH

A. ONTKO

Cardiovascular Research Program, Oklahoma Medical Research Foundation and Department of Biochemistry and Molecular Biology, College of Medicine, University of Oklahoma, Oklahoma City, Oklahoma 73104, and Roche Research Center, Department of Biochemical Nutrition, Hoffmann-La Roche Inc., Nutley, New Jersey 07110 Received April 23, 1976 The regulation of fatty acid synthesis, measured by 3H,0 incorporation into fatty acids, was studied in hepatocytes from rata meal-fed a high carbohydrate diet. Ca2+ increased fatty acid synthesis, which became maximal at physiological concentrations of Ca*+. Ethanol markedly inhibited fatty acid synthesis. Maximum inhibition was reached at 4 rnM ethanol. However, ethanol did not decrease lipogenesis in the presence of pyruvate. Dr.-3-Hydroxybutyrate increased fatty acid synthesis. Acetoacetate decreased lipogenesis when used alone and reversed the effect of m-3-hydroxybutyrate when both were added. m-3-Hydroxybutyrate moderately decreased flux through the pyruvate dehydrogenase system and markedly inhibited citric acid cycle flux. By measurement of glycolytic intermediates, two ethanol-induced crossover points were observed: one between fructose 6-phosphate and fructose 1,6-diphosphate and the other between glyceraldehyde 3-phosphate and 1,3-diphosphoglycerat. The concentrations of pyruvate and citrate were decreased by ethanol and increased by m-3-hydroxybutyrate. Aminooxyacetate and L-cycloserine inhibited fatty acid synthesis and these effects were overcome by Dr.-3-hydroxybutyrate. Results indicate that in hepatocytes in a metabolic state favoring a high rate of lipogenesis, .production of reducing equivalents in the cytosol via ethanol metabolism inhibits fatty acid synthesis from glucose by inhibition of both phosphofructokinase and glyceraldehyde 3-phosphate dehydrogenase and by promoting reduction of pyruvate to lactate. Production of reducing equivalents in the mitochondria via m-3-hydroxybutyrate enhances fatty acid synthesis in liver cells by altering the partition of citrate between oxidation in the citric acid cycle and conversion to fatty acids in favor of the latter pathway. These interactions indicate the importance of the intracellular pyridine nucleotide redox states in the rate control of hepatic fatty acid synthesis.

The conversion of carbohydrate to fatty acids is a complex process and numerous factors have been implicated in the regulation of this multireaction sequence (l-3). In the present study, influences of the intracellular pyridine nucleotide redox states on fatty acid synthesis in rat hepatocytes were investigated. Ethanol was employed to generate reducing equivalents in

both the cytosol and mitochondria (4-6); m-3-hydroxybutyrate was used to produce reducing equivalents specifically in the mitochondria (7, 8). Ethanol increased the incorporation of [1,2-Wacetate into fatty acids in rat liver slices (91, although in other experiments with the same system ethanol suppressed incorporation of 14C-labeled acetate into fatty acids (10). Although fatty acid synthesis in the perfused liver from fed rats was reportedly inhibited by ethanol (111, other work has suggested that ethanol does not appreciably affect the rate of fatty acid synthesis in this tissue (12, 13).

1This investigation was supported by Research Grant HL 13302 from the U.S. Public Health Service. G. A. C. was supported during these studies by a Cardiovascular Research Traineeship (U.S. Public Health Service Grant No. 5-TOl-HL-05403) and the Oklahoma Medical Research Foundation. 310 Copyright 0 1977 by Academic press, Inc. All rights of reproduction in any form reserved.

ISSN 0003-9861

LIPOGENESIS

REGULATION

The activity of the citric acid cycle is of potential regulatory significance with respect to the supply of carbon for fatty acid synthesis, since intramitochondrial citrate is an intermediate in both processes. Citric acid cycle flux in hepatocytes is inhibited by ethanol (14) and by nL-&hydroxybutyrate (151, but the effects of these substrates on fatty acid synthesis have not been compared. A correlation between the intracellular pyridine nucleotide redox states and the conversion of lY4C]glucose into fatty acids has been projected (16) from data obtained with animals in different hormonal and nutritional states. In this comparison, the rates of lipogenesis in liver slices were directly proportional to the NAD+:NADH ratios in both the cytosol and mitochondria. In the current study it was observed that ethanol inhibits and m-3-hydroxybutyrate elevates the rate of fatty acid synthesis in isolated hepatocytes. The mechanisms operative in the metabolic expression of these effects are presented herein. MATERIALS

AND METHODS

regimen. Female Charles River CD strain rats weighing 180 to 220 g were housed individually in a light-controlled room (6 AM to 6 PM light) with free access to water. Rata were fed a commercial diet (Purina Laboratory Chow) ad Zibitum for 1 week before each experiment. Animals were fasted for 48 h, and then meal-fed (for 3 h daily, 8:30-11:30 AM) a synthetic diet for 5 to 8 days prior to cell isolation. The synthetic diet (17) consisted of 70% glucose, 23% vitamin-free casein, 5% Phillips and Hart salt mixture IV, 1% corn oil, 1% complete vitamin mixture, 0.5% butylated hydroxytoluene, and 40 g of cellulose/kg of diet. Isolation and incubation of hepatocytes. Rata were anesthetized with Nembutal (50 mg/kg ip) immediately following the 3-h meal and hepatocytes were isolated by a procedure modified from that of Berry and Friend (18). Each liver was perfused (nonrecirculating) with calcium-free Krebs-Henseleit (19) medium, pH 7.4, containing 16.5 mM glucose and equilibrated with 95% O,-5% CO, at 37°C. When the liver became blanched, it was excised and suspended in a perfusion chamber maintained at 37°C without interruption of the flow of perfusion medium. The perfusion rate was 25 to 30 ml/min. After the liver had been perfused for approximately 5 min, the flowthrough perfusion was changed to a recirculating perfusion (total volume, 70 ml). A high oxygen concentration was maintained (determined using a Animals

and

feeding

IN RAT HEPATOCYTES

311

Clark-type oxygen electrode) by passing 95% OX-51 CO* through a temperature-regulated disc oxygenator (20) which was included in the perfusion circuit. CaCl, was added to the perfusion medium as suggested by Seglen (21). The CaCl, (2 rn@ was added 5 min after adding 30 mg of collagenase. The liver was removed from the perfusion chamber after 15 to 20 min of exposure to collagenase and minced in cold Krebs-Henseleit medium containing no enzyme. Cells were filtered through silk and washed as previously described (22). The final cell suspensions in Krebs-Henseleit medium plus 16.5 mM glucose contained 5 to 21 mg dry wt of cells per milliliter. Greater than 90% of the cells excluded trypan blue. Hepatocytes were incubated at 37°C in 25-ml Erlenmeyer flasks in a Dubnoff shaker at 90 oscillations per minute. Each flask contained 2 ml of cell suspension unless otherwise indicated and was gassed for 20 s with 95% O,-5% CO, before stoppering for incubation. The total volume of gas used was >250 cm3 at STP.* All incubations were carried out in triplicate and each experiment was repeated at least once. Determination of fatty acid synthesis rates. Rates of lipogenesis were determined by the incorporation of tritium from “HZ0 into fatty acids. Each flask contained 0.5 mCi of 3H20. Rates were expressed as nanogram atoms of tritium incorporated into fatty acids per milligram dry weight of hepatocytes per time. Hepatocyte incubations were terminated by adding 2.1 ml of 5 N NaOH to each flask. Incubation mixtures were transferred to culture tubes with tefIon-lined screw-caps and combined with two 1.5-ml HZ0 rinses of the flask. The pooled mixture was saponified at 90°C overnight, and then extracted three times with 5 ml of petroleum ether (boiling point range, 30-60°C). This nonsaponified fraction was discarded. The aqueous, saponified phase was acidified with 2.5 ml of 5 N HCl. The pooled extracts were backwashed with 7.5 ml of water, evaporated, and counted (17). Assays of metubolites. At the end of incubation, a protein-free solution was obtained as previously described (22). Citric acid cycle intermediates and adenine nucleotides were measured enzymatically as described by Williamson and Corkey (23). Glucose (24) and glycerol 3-phosphate (251 were assayed enzymatically by spectrophotometer. Coupled enzymatic assays were used to measure glucose 6-phosphate and fructose 6-phosphate (26); fructose 1,6diphosphate, glyceraldehyde 3-phosphate, dihydroxyacemne phosphate, and 1,3diphosphoglycerat, (27); and 3-phosphoglycerate, 2-phosphoglycerate, and phosphoenolpyruvate (28) fluorometrically. Pyr2 Abbreviations used: STP, standard temperature and pressure; EGTA, [ethylenebis(oxyethylenenitrilo)]tetraacetic acid; CoA, coenzyme A.

312

COOK, SULLIVAN

AND %ONTKO

uvate (29), L-lactate (301, glutamate (31), acetoacetate (32), and n-3-hydroxybutyrate (33) were also assayed using enzymatic techniques. Measurement of ‘4C02production from ‘YXabeled citric acid cycle intermediates. Evolution of ‘%02 from hepatocytes incubated with [6J4Clcitrate, [1,5‘*Clcitrate, 2-oxo[l-‘4Clglutarate, or 11 4J4Clsuccinate was measured as previously described (15, 22). Materiuls.

Collagenase, CLS Type II, was purchased from Worthington Biochemicals. Enzymes for assays of metabolic intermediates and adenine nucleotides were from Boehringer-Mannheim. ATP, NAD+, and NADH were from P-L Biochemicals. 3H,0 and ‘%-labeled citric acid cycle intermediates were obtained from New England Nuclear. Nembutal was a product of Abbott Laboratories. Dietary components were from Nutritional Biochemicals. n-Cycloserine was obtained from Regis Chemical Company. RESULTS

In earlier studies on fatty acid oxidation (14, Xi), hepatocytes were incubated in the absence of added calcium. Since it was found in the present study that the addition of calcium chloride to the incubation medium at a physiological concentration of 2.5 mu enhanced fatty acid synthesis, the effect of varying concentrations of calcium chloride was investigated (Fig. 1). The hepatocytes were preincubated with EGTA to promote calcium depletion. Even at a concentration of 0.1 mM, calcium significantly elevated the rate of fatty acid synthesis (P < 0.01). Fatty acid synthesis was near maximum at 1.0 mM calcium chloride and was not increased by calcium chloride concentrations greater than 2.5 mu. In other experiments in which calcium was omitted from the isolation medium and the cells were not preincubated with EGTA, the addition of 2.5 mM CaCI, to the incubation medium increased the rate of fatty acid synthesis to the same maximum rate. However, the control rate of fatty acid synthesis (no calcium added) in hepatocytes preincubated with EGTA was much lower (15% of the maximum rate) than the control rate in hepatocytes not preincubated with EGTA (39-‘76% of maximum). Ethanol inhibited (P < 0.01) the incorporation of tritium from 3H,0 into fatty acids at all concentrations employed (0.5 to

0

0.5

1.0

1.5

2.0

:

[CaC12](mW

FIG. 1. The effect of calcium concentration on the rate of fatty acid synthesis in isolated rat hepatocytes. Hepatocytes were isolated in the absence of calcium and preincubated for 10 min at 25°C in 10 mM EGTA, then washed twice in calcium-free Krebs-Henseleit medium. Cells (26.8 mg dry wt) were incubated for 60 min at 37°C. Calcium chloride was added to yield total concentrations of 0.10, 0.25, 1.00, and 2.50 mM. Fatty acid synthesis was measured by incorporation of tritium from 3H,0 into fatty acids. Each point represents the mean of three determinations k SD.

20 mM> (Fig. 2). Maximum inhibition of fatty acid synthesis (62%) occurred at 4 mu ethanol. Half-maximum inhibition was estimated at 0.9 mu ethanol. When hepatocytes isolated from mealfed rats were incubated in the presence of increasing concentrations of nL-&hydroxybutyrate, rates of fatty acid synthesis were sequentially increased (Fig. 3). At the highest concentration used, 100 mM, the rate of lipogenesis was increased by 145% over the control rate. At concentrations of 10 mu or higher the rates of lipogenesis were significantly different (P < 0.01 by Duncan’s Multiple Range Test) from rates at all other concentrations tested. The addition of 50 mu acetoacetate caused a decrease of 38% (P < 0.01) in the lipogenic rate (Fig. 4). Acetoacetate also reversed the effect of 20 rnM m-&hydroxybutyrate. The degree of reversal was dependent upon the concentration of acetoacetate present; 20 mM acetoacetate signiii-

LIPOGENESIS

I

5

-REGULATION

15

10

20

0

10

0

20

40

60

[DL -3-HYDROXYBUTYRATE]

20 [Acetoacetate]

[ETHANOL] (mM)

FIG. 2. The effect of ethanol concentration on the rate of fatty acid synthesis in isolated rat hepatocytes. Cells (21.5 mg dry wt) were incubated for 60 min at 37°C. Rates were determined from 3H20. Each point represents the mean of three determinations k SD.

80

10

(mM)

FIG. 3. The effect of m-3-hydroxybutyrate concentration on the rate of fatty acid synthesis in isolated rat hepatocytes. Cells (37.1 mg dry wtl were incubated for 60 mm at 37°C. Rates were determined horn 3H,0. Each point represents the mean of three determinations f SD.

cantly (P < 0.01) decreased the effect of nL3-hydroxybutyrate and 50 mM acetoacetate decreased lipogenesis below that observed when neither acetoacetate nor DL-3-

313

IN RAT HEPATOCYTES

30

40

50

I

(mM)

of acetoacetate and nL-3-hydroxybutyrate on the rate of fatty acid synthesis in isolated rat hepatocytes. Cells (22.6 mg dry wtl were incubated for 60 min at 37°C. Acetoacetate was added to yield the concentration shown in the absence (0) or presence (A) of 20 mM m-3-hydroxybutyrate. Rates were determined from 3H,0. Each point represents the mean of three determinations k SD. FIG.

4. The

effects

hydroxybutyrate was added (P < 0.01). The production of 14C0, from [l14C]pyruvate and [l-14Cllactate was decreased by 31% (P < 0.005) in the presence of 50 mM m-3-hydroxybutyrate (Table I). Since the total lactate plus pyruvate was not significantly affected by m-3hydroxybutyrate, this indicates moderate inhibition of pyruvate dehydrogenase. Citric acid cycle flux was markedly inhibited by 40 mM nc&hydroxybutyrate (Table II). The greatest inhibition in the evolution of 14C0, was observed when [ 1,514C]citrate was the substrate. This intermediate must undergo the reactions of the citric acid cycle twice before both labeled carbons are converted to 14C02. The highest inhibition of 14C02 evolution from an intermediate with a single 14C label was observed using [6J4Clcitrate. The calculated value for the inhibition of 14C0, production from [1,5J4Clcitrate showed only a 6% difference from the observed value. This indicated that the inhibition values obtained with the various 14C-labeled intermediates were extremely consistent

314

COOK,SWLLIVAN

AND ONTKO

TABLE I OF DL-3-HYDBOXYBUTYRATE ON PRODUCTION OF “CO2 FROM [l-WILACTATE [I-14C]PY~~~~~ti WO, Production (PCi) Amount present at end of incubation

THE EFFECT

DL-~-HYdroxybutyrate added

Lactate (pmol)

(rnM) 0 50

0.274

c

0.012

0.190 r 0.007”

4.44

f

0.27

4.41 k 0.31

Pyruvate ( pmol)

Total (pmol)

0.49 + 0.02 0.40 2 0.036

4.93 -+ 0.26 4.81 + 0.31

AND

a The specific activity of the [1-Wllactate and [I-Wlpyruvate was 0.25 &i/pmol; 5.0 pmol of lactate and 1.0 pm01 of pyruvate were added in a total volume of 1.0 ml. Hepatocytes (10.5 mg dry wt) were incubated for 30 min at 37°C. Data represent the means 2 SD of three determinations. * Significantly different from control values (P < 0.005).

and closely correlated, even though the procedure used here would not yield an absolute value for the inhibition of citric acid cycle flux due to the possibility of changes in specific radioactivity during the incubation. Consistency of calculated and observed values also indicated that DL3-hydroxybutyrate had no effect on the specific radioactivity of the citric acid cycle intermediates. Calculated nL-3-hydroxybutyrate inhibition of the citric acid cycle reactions converting succinate to citrate was 25%. Fatty acid synthesis was measured at various intervals for 2 h with and without the addition of 50 mM Dr.,-Q-hydroxybutyrate (Fig. 5). There was a lag in the lipogenie rate which persisted for about 1 h. After 1 h the rates of fatty acid synthesis were constant in the presence and absence of r&-3-hydroxybutyrate; however, the rate of synthesis was 55% higher when DL3-hydroxybutyrate was present. The added nL-3-hydroxybutyrate was rapidly metabolized (Fig. 5, inset); the acetoacetate concentration rose rapidly and the ratio of the n-3-hydroxybutyrate concentration to the acetoacetate concentration decreased. After 2 h the ratio of n&hydroxybutyrate to acetoacetate was 1.81 in incubation mixtures to which r&3-hydroxybutyrate had been added and 0.58 in those with no such addition. Hepatocytes incubated without adding nc3-hydroxybutyrate had a constant D - 3 - hydroxybutyrate : acetoacetate ratio throughout the entire incubation period. Malate concentrations were measured at various intervals for 2 h in the presence and absence of 50 mM nL-3-hydroxybutyr-

ate (Fig. 6). The concentration of malate initially increased during incubation but became constant at 60 min. The malate concentration throughout the incubation period was higher in the presence of DL-3hydroxybutyrate. The steady-state concentration was equivalent to that reported in intact rat liver (34). Concentrations of various metabolites were measured after incubation of hepatocytes for 60 min in the absence or presence of 10 mu ethanol or 50 mM m-3-hydroxybutyrate (Table III). The final glucose concentration was greater in the presence of ethanol, indicating less utilization of glucose; m-Shydroxybutyrate did not affect the final concentration of glucose (P > 0.05). Ethanol increased the concentrations of glycolytic intermediates preceding the phosphofructokinase reaction and decreased the fructose 1,8diphosphate concentration; nc8hydroxybutyrate produced no such effects. The concentrations of glyceraldehyde 3-phosphate and dihydroxyacetone phosphate were increased and intermediates beyond the glyceraldehyde 3-phosphate dehydrogenase reaction were markedly decreased in the presence of ethanol but not m-3-hydroxybutyrate. These dual crossover results indicate inhibition of both phosphofructokinase and glyceraldehyde 3-phosphate dehydrogenase in hepatocytes incubated with 10 mM ethanol, accounting for the decrease in glucose utilization and the marked decrease in the concentrations of pyruvate and lactate (Table III). The increase in lactate and pyruvate concentrations in the presence of nc3-hydroxybutyrate provides further evidence for a moderate inhibition

LIPOGENESIS TABLE INHIBITION

315

Inhibitjf;;f

FLUX BY DL-3Method of determining inhibition

prod& tion” Pm Observed [6-W]Citrate 52 2-0x011-Wlglutarate 38 Observed 11 4-WlSuccinate 71 Observed 76 Observed 1115~WlCitrate [1,5-“C]Citrate 81 Calculatedd n Hepatocytes (13.9 mg dry wt) were incubated in a total volume of 1.0 ml f 40 mM nn-3-hydroxybutyrate for 15 min at 37°C. Data represent the means of three determinations. b Citric acid cycle intermediates were added in the following amounts: [6-14Clcitrate, 0.5 $i (4.5 &Zi/~mol); 2-oxo[lJ4C]glutarate, 0.13 &i (10.4 &i/pmol); [1,4J4C]succinate, 0.17 &i (6.28 $i/ pmol); and [l,5J4C]citrate, 0.5 @i (9.4 &i/pmol). c 14C0, production from controls, as a percentage of the amount added, were: [6-“Clcitrate, 58; 2oxo[lJ4C]glutarate, 52; [1,4-Wlsuccinate, 44; and [1,5-“Clcitrate 26. d Inhibition ‘of 14C02 evolution was calculated for [1,5J4C]citrate using values for 14C02 evolution observed for the other three labeled intermediates. First, the inhibition of the cycle between succinatc and citrate was calculated from the formulas: Percentage inhibition

IN RAT HEPATOCYTES

II

OF CITRIC ACID CYCLR HYDROXYBUTYRAT@

Citric acid cycle intermediateb

REGULATION

= 11 - (FJFJ]

TIME (mm)

5. Fatty acid synthesis in isolated rat hepatocytes as a function of time. Cells (24.8 mg dry wtl were incubated at 37’C in the absence %(O) or presence (A) of 50 mM nL-3-hydroxybutyrate. Reactions were terminated by adding NaOH for determination of the rate of fatty acid synthesis from 3Hz0 or by adding HCIO, for determining acetoacetate and n-3hydroxybutyrate concentrations. Each point represents the mean of three determinations f SD. (Standard deviations for all points in the inset were less that 10% of the mean.) FIG.

(100)

and where the rates of 14COi evolution from the various cycle intermediates were designated as follows: S = [l,4-14C]succinate, C = [6J4C]citrate, 0 = P-oxoll14C]glutarate, and F = citric acid cycle flux between succinate and citrate, Subscripts e and c designate experimental and control, i.e., the presence or absence of 40 mM m-3-hydroxybutyrate. (Therefore, 11 - (SJS,)] x 100% = percentage inhibition of 14C0, evolution from [1,4-“Clsuccinate.) Next, the inhibition of CO, evolution from [1,W4C]citrate was calculated from the formula: Percentage inhibition = [l - (X + Y + 211 (loo), where: X = [1,5-‘*C]citrate

converted to [1,4-‘*C]succinate + 14C0, = [(C e/c e) (O,/O,W2, Y = [1,4-“Clsuccinate converted to 2-ox~[l-*~C]glutarate + “CO, = X.{[(F,/FJ CC,/CJl/Z}, and Z = 2-oxo[l-Wlglutarate converted to succinate + wo, = Y. (0,/O,).

TIME (mm)

6. The effect of m-3-hyroxybutyrate on the concentration of malate in isolated rat hepatocytes. Cells (28.4 mg dry wt.) were incubated at 37°C in the absence (0) or presence (A) of 50 mM m-3-hydfoxybutyrate. Each point represents the mean of three determinations + SD. FIG.

316

COOK, SULLIVAN

AND ONTKO

TABLE EFFECTS

OF ETHANOL

III

AND DL-3-HYDROXYBUTYRATE

ON METABOLITE

(10 mM)

CONCENTRATION@

Metabolite

Control

Ethanol

m-B-Hydroxybutyrate (50 mM)

Glucose Glucose 6-phosphate Fructose 6-phosphate Fructose 1,6-diphosphate Glyceraldehyde 3-phosphate Dihydroxyacetone phosphate Glycerol 3-phosphate 1,3-Diphosphoglycerate 3-Phosphoglycerate 2-Phosphoglycerate Phosphoenolpyruvate Pyruvate Lactate Citrate 2-Oxoglutarate Malate Glutamate Acetoacetate n-3-Hydroxybutyrate ATP ADP AMP ATP + ADP + AMP ATPADP i/z I(2 ATP + ADP)/(AMP f ADP +

697 2 24 0.689 t 0.020 0.245 r 0.009 0.093 r 0.003 0.025 -c 0.004 0.445 ” 0.008 0.943 r 0.029 0.007 -c 0.001 0.832 + 0.024 0.088 + 0.005 0.349 f 0.022 13.73 + 0.51 71.86 + 0.95 1.88 t 0.24 0.87 + 0.16 0.88 + 0.01 10.33 + 0.31 4.69 k 0.39 3.80 5 0.47 5.97 -c 0.14 1.24 f 0.16 0.30 f 0.02 7.51 zt 0.27 4.87 k 0.63 0.88 k 0.02

744 0.847 0.324 0.026 0.049 0.611 5.240 0.002 0.031 0.017 0.063 0.31 22.05 1.30 0.22 1.14 9.80 9.67 14.97 4.95 0.81 1.09 6.85 6.11 0.78

2 9 k 0.041’ in 0.017c -t 0.007d k 0.001’ c 0.026 e 0.440” 2 0.001~ 2 0.004d t 0.008 r 0.004d -+ 0.19 2 1.23” + O.lT -c 0.05’ 2 0.13b f 0.60 + 0.451 + 0.8od 5 0.49 k 0.04* ? o.osd + 0.57 f. 0.37 k O.OY

704 -c 13 0.846 k 0.068’ 0.284 r?: 0.077 0.091 ” 0.015 0.014 ” 0.007 0.373 2 O.OOP 0.700 k 0.06W 0.007 2 0.001 0.846 ” 0.030 0.086 2 0.002 0.369 + 0.055 18.31 k 0.6Bd 80.69 + 3.20 2.67 + 0.19’ 1.14 + 0.040 1.75 k 0.16 14.91 k 0.6Y 328 + 29’ 615 -f. 59” 5.58 2 0.18’ 1.08 k 0.02 0.52 k 0.04 7.18 -c 0.18 4.77 ? 0.34 0.85 k 0.02

0.81 12.16 5.13 2.12

1.57 30.01 88.96 8.58

+ L 2 2

1.89 13.12 4.41 1.88

ATP)I n-3-Hydroxybutyrateacetoacetate Glutamate:2-oxoglutarate Lactate:pyruvate Glycerol 3-phosphakdihydroxyacetonephosphate a Hepatocytes (41.9 mg are reported in nanomoles determinations. b Significantly different ’ Significantly different d Significantly different

+ k k 2

0.08 2.36 0.16 0.10

0.08 2.11’ 4.6ob 0.95d

-c 0.36b f 0.65 rfr 0.19 t 0.01*

dry wt) were incubated for 60 min at 37°C in a total volume of 2 ml. Metabolites per milligram dry weight of cells. Data represent the means 5 SD of three from control (P i 0.05). from control (P < 0.01). from control (P < 0.001).

of pyruvate dehydrogenase (cf. Table I). Ethanol elevated the ratios of glycerol 3phosphate : dihydroxyacetone phosphate and lactate:pyruvate as a result of the conversion of cytosolic NAD+ to NADH via alcohol dehydrogenase. The elevation of the n-3-hydroxybutyrate:acetoacetate and glutamate:2-oxoglutarate ratios in the presence of ethanol indicated conversion of mitochondrial NAD+ to NADH by the oxidation of acetaldehyde. Reducing equivalents transported from the cytosol may also contribute to this effect. The concentrations of citrate and 2-oxoglutarate were

lower in the presence of ethanol but higher in the presence of m-3-hydroxybutyrate. Concentrations of malate and glutamate were also higher in the presence of DL-3hydroxybutyrate. Increases in concentrations of all citric acid cycle intermediates measured in the presence of m-3-hydroxybutyrate provide further evidence for an inhibition of citric acid cycle flux (cf. Table II). Even though ethanol also inhibits citric acid cycle flux in liver cells (14) accumulation of citrate and 2-oxoglutarate in the presence of ethanol is probably prevented by the marked decrease in glycoly-

LIPOGENESIS

REGULATION

sis. The ethanol-induced accumulation of malate may be due to the high NADH:NAD+ ratio and/or a decrease in malate-citrate exchange across the inner mitochondrial membrane. Adenine nucleotide concentrations were altered by ethanol. Concentrations of ATP and ADP were lower and the concentration of AMP was much higher in the presence of ethanol and energy charge W2[(2 ATP + ADP)/(AMP + ADP + ATP)]) was therefore reduced. In the presence of 50 mu m-3-hydroxybutyrate the concentration of ATP was slightly lower and the concentration of AMP slightly higher than the control, but there was no significant change in either the ratio of ATP:ADP or the calculated value for energy charge. When hepatocytes were incubated for 60 min in other experiments in the presence and absence of m-3-hydroxybutyrate at concentrations of 40 mM or lower, there was no significant difference (P > 0.05) in the concentrations of ATP, ADP, or AMP (results not shown). The results in Table III indicate that ethanol metabolism effectively depressed the pyruvate concentration via both decreased glycolysis and diversion of pyruvate to lactate. If ethanol inhibited fatty acid synthesis (Fig. 2) by decreasing the pyruvate concentration, addition of pyruvate should overcome the inhibition. This was examined experimentally and the following results were observed (mean nmol of 3H,0/mg dry wt/h + SD): control, 32.5 + 3.5; + 10 mu ethanol, 7.6 -C 0.8; + 10 mM pyruvate, 52.0 + 1.7; and + 10 mM pyruvate + 10 mM ethanol, 49.2 -+ 1.3. Lipogenesis was stimulated by pyruvate and was unaffected by ethanol when pyruvate was supplied. Aminooxyacetic acid, an inhibitor of the malate-aspartate shuttle, also inhibited fatty acid synthesis (Table IV), but its effect was decreased by the addition of 50 mM nL-3-hydroxybutyrate. Aminooxyacetate inhibited lipogenesis by 59%, but in the presence of m-3-hydroxybutyrate inhibition was only 15%. Another inhibitor of the malate-aspartate shuttle, L-cycloserine, also decreased lipogenesis (29%) but did not cause inhibition in the presence of nL-3-hydroxybutyrate.

317

IN RAT HEPATOCYTES TABLE INHIBITION

OF LIPOCENESIS AND

IV BY AMINOOXYACETATE

L-CYCLOGERINE”

Addition

Nanomoles 3H20 converted to fatt acids per rni.I.hgram dry wcii@$ per

Percent.“jy im bition

0

None 1 mM aminooxyacetate 1 mh4 L-cycloserine 50 mM m-3-hydroxybutyrate 50 mM m-3-hydroxybutyrate plus 1 mM aminooxyacetate 50 mM nn-3-hydroxybutyrate plus 1 mM Lcycloserine

25.20 10.26 17.89 31.76

2 2 2 2

0.79 0.27* 0.38 1.02

59 29 -

27.08 * 1.57’

15

30.40 k 0.47d

4

D Hepatocytes (18.5 mg dry wt) were incubated for 60 min at 37°C in a total volume of 2 ml. Data represent the means k SD of three determinations. * Significantly different from control (P < 0.0005). r Significantly different from on-3-hydroxybutyrate alone (P < 0.005). d Not significantly different from m-S-hydroxybutyrate alone (P > 0.05). DISCUSSION

Fatty acid synthesis in hepatocytes was decreased by preincubation of the liver cells with EGTA. However, EGTA did not damage the liver cells since the addition of calcium restored the rate of fatty acid synthesis to that observed when calcium was added to untreated cells. The results therefore indicate that EGTA treatment of the liver cells removes calcium and thereby provides a system more sensitive to calcium addition. These data indicate that calcium is required for high rates of conversion of glucose to fatty acids. Calcium was known to cause an increase in the mitochondrial NADH:NAD+ ratio (351, but it was not known whether this effect of calcium was the factor which caused the increase in fatty acid synthesis. Ethanol and m-3-hydroxybutyrate each increase the hepatic mitochondrial NADH:NAD+ ratio. Ethanol produces reducing equivalents in both the cytosolic and mitochondrial compartments (51 while m-3-hydroxybutyrate produces reducing equivalents only in the mitochondrial

318

COOK, SULLIVAN

compartment (36). These two substrates have therefore been employed to investigate the involvement of the pyridine nucleotide redox state in the regulation of fatty acid synthesis. Inhibition of fatty acid synthesis in isolated hepatocytes was produced at a low concentration (0.5 mM) of ethanol and the maximum inhibition (62%) was similar to the inhibition produced by ethanol (52%) in perfused rat liver (11). Since ethanol should increase both mitochondrial and cytosolic NADH: NAD+ ratios and m-3-hydroxybutyrate should increase primarily the mitochondrial ratio, it might have been expected that the addition of Dr.-3-hydroxybutyrate would either inhibit fatty acid synthesis or have no effect. However, the addition of m-3-hydroxybutyrate enhanced fatty acid synthesis (Fig. 3) and the effects of this compound were therefore investigated in more detail. Hepatocytes isolated from meal-fed rats rapidly converted n-3-hydroxybutyrate to acetoacetate (Fig. 51, resulting in a higher mitochondrial NADH:NAD+ ratio. This ratio may be the factor which was responsible for the stimulation of fatty acid synthesis because acetoacetate inhibited lipogenesis when added alone and reversed the effect of m-3-hydroxybutyrate when added at a concentration greater than that of n-3-hydroxybutyrate (Fig. 4). The rate of lipogenesis therefore increases with an increase of the mitochondrial NADH:NAD+ ratio and decreases when the mitochondrial pyridine nucleotides are in a more oxidized condition. Since there was no increase in ATP in these hepatocytes (Table III) the added nL-3-hydroxybutyrate was not simply making up an energy de& ciency in the cells. Neither the ATPADP ratio nor energy charge were expected to produce a change in the rate of lipogenesis since these ratios did not change when DL3-hydroxybutyrate was added (Table III). Brunengraber et al. (37) have reported high rates of lipogenesis in meal-fed rats perfused with glucose. It is of interest that the n-3-hydroxybutyrate:acetoacetate ratio was much higher in livers from fed rats perfused with 25 mM glucose than in livers from fed rats perfused with 4 mu glucose and these, in turn, had higher ratios than

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livers from rats fasted 1 or 2 days (the ratios were 1.9,0.58,0.16, and 0.18, respectively). These ratios correlated with rates of fatty acid synthesis in the four groups. The work of Lagunas et al. (38) also supports this hypothesis. These investigators fed nicotinamide to rata and observed a decrease in mitochondrial NADH:NAD+, a decrease in liver content of citrate and malate, an increase in citric acid cycle flux, and a decrease in fatty acid synthesis. The decrease in the production of 14C02 from [l-W]pyruvate and [l-Wllactate (Table I) indicated a decrease in production of acetyl-CoA from pyruvate. The change was not due to changes in specific radioactivity because of the very high activity of lactate dehydrogenase in the liver of fed rats compared to the activity of pyruvate kinase (39) and because there was no change in the lactate plus pyruvate concentrations in the whole cell in the absence or presence of m-3-hydroxybutyrate. These data indicate a moderate inhibition of pyruvate dehydrogenase by m-3hydroxybutyrate probably due to an increase in the mitochondrial NADH:NAD+ ratio (40). Therefore, the elevation in fatty acid synthesis caused by Dr.,-&hydroxybutyrate is not the result of increased carbon flow from glycolytic products to acetyl-CoA. The change in the mitochondrial NADH:NAD+ ratio caused an inhibition of citric acid cycle flux (Table II). Observed inhibition of WO, production from ‘4clabeled citric acid cycle intermediates indicated that there were two sites of inhibition (Table II). The major site was the isocitrate dehydrogenase reaction and a second site was 2-oxoglutarate dehydrogenase. NAD-linked isocitrate dehydrogenase is known to be competitively inhibited by NADH and the effect is potentiated by NADPH (41, 42). Therefore, changes in the NADH:NAD+ ratio should greatly affect the activity of this enzyme and substantially reduce oxidation of citrate by the citric acid cycle. Regulation at this point rather than at the citrate syntase reaction would allow citrate to be partitioned between oxidation to CO, or transport to the cytosol for support of the biosynthesis of fatty acids. Isocitrate dehydro-

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genase is activated by ADP (41, 42). DL-3Hydroxybutyrate did not change the ADP concentration in isolated hepatocytes (Table III). Therefore, the NADH:NAD+ ratio appears to be the major modifier of isocitrate dehydrogenase under these conditions. Citrate synthase has been proposed as a site for citric acid cycle regulation by the adenine nucleotide (43, 44) but there was no change in this ratio in isolated hepatocytes (Table III). The secondary site of inhibition of the citric acid cycle at the 2oxoglutarate dehydrogenase step has been reported previously in hepatocytes isolated from fasted rats (15) and in isolated rat liver mitochondria (45) to be influenced by the NADH:NAD+ ratio. The result of inhibition of the citric acid cycle by nL-Q-hydroxybutyrate should be a change in the partitioning of citrate so that less is oxidized and more is available for transport to the cytosol for the synthesis of fatty acids. The increase in cytosolic citrate could increase the rate of fatty acid synthesis in two ways. Besides increasing the substrate concentration for ATP-citrate lyase, the increase in citrate could positively modify the activity of acetyl-CoA carboxylase (46). The lag in the lipogenic rate observed during the first hour of-incubation (Fig. 5) was probably caused by a diminished concentration of intermediates due to repeated washing during the cell isolation procedure. The initial concentration of malate in the hepatocyte incubation mixtures was near zero (Fig. 6) but increased during the first hour of incubation. The concentration of malate did not change during the second hour of incubation but was significantly higher in incubation mixtures containing m-3-hydroxybutyrate. The concentration of malate and the rate of lipogenesis were therefore mutually related. These data (Figs. 5 and 6) also indicated that a metabolic steady state had been reached after 60 min of incubation at 37°C. Scholz et al. (11) suggested that ethanol inhibited fatty acid synthesis by diverting pyruvate to lactate, thereby causing a de& ciency of acetyl-CoA. Measurement of glycolytic intermediates in isolated hepatocytes confirmed the decrease in pyruvate (Table III); however, the present results

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indicate inhibition of phosphofructokinase and glyceraldehyde 3-phosphate dehydrogenase as the primary interactions responsible for the decreased pyruvate level, with conversion-of pyruvate to lactate as a contributing effect. Another factor was carbon diversion from dihydroxyacetone phosphate to glycerol S-phosphate (Table III). The combined result of these four effects of ethanol metabolism is severe inhibition of glucose conversion to fatty acids, which may be partially masked since ethanol itself provides carbon for fatty acid synthesis (12). The crossover at glyceraldehyde 3phosphate dehydrogenase is mediated by the elevated cytosolic NADH:NAD ratio. However, the molecular basis of the crossover at phosphofructokinase remains uncertain in the absence of information regarding intracellular compartmentation of adenine nucleotides. The stimulation of fatty acid synthesis by added pyruvate (text), as also observed by Clark et al. (471, indicates that the pyruvate concentration in the hepatocyte system was limiting, in agreement with the results of Harris (48). The marked decrease in the pyruvate level caused by ethanol is consonant with the concept that ethanol inhibits fatty acid synthesis by decreasing the pyruvate supply. Ethanol did not inhibit lipogenesis when pyruvate was added, presumably because of the direct supply of the limiting precursor and because pyruvate removes the reducing equivalents which mediate the action of ethanol. Lack of inhibition of hepatic lipogenesis by ethanol in uivo in meal-fed rats (13) may be related to the delivery of a multiplicity of precursors to the liver under these conditions. Increased concentrations of citric acid cycle intermediates and decreased 14C0, production from labeled cycle intermediates are both indicators of inhibition of the citric acid cycle. Concurrent n-S-hydroxybutyrate oxidation effectively substitutes as an energy source; the ATPADP ratio was maintained. The level of ATP was normal in the hepatocytes incubated with glucose alone as substrate and the ATP content was not increased by nL-3-hydroxybutyrate. Thus, the metabolism of D-3hydroxybutyrate did not elevate fatty acid

320

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synthesis simply by counteracting a highenergy phosphate deficiency. Therefore, the primary effect of n-3-hydroxybutyrate on fatty acid synthesis, mediated by the NADH produced, was alteration in the differential flow of citrate carbon into the competing pathways of oxidation and transport to the cytosol for fatty acid synthesis. Results are consonant with the concept that inhibition of isocitrate dehydrogenase increased the mitochondrial citrate concentration; thus, more citrate was available for transport to the cytosol. Aminooxyacetate and n-cycloserine inhibit transaminases which function in the malate-aspartate shuttle for transport of reducing equivalents into mitochondria (49). Aminooxyacetate is more effective since it inhibits both cytosolic and mitochondrial transaminases; L-cycloserine does not penetrate the mitochondrial membrane, and therefore, inhibits only the cytosolic transaminase (49). These agents therefore cause mitochondrial pyridine nucleotide oxidation (50). Fatty acid synthesis was inhibited by each of these agents (Table IV); m-3-hydroxybutyrate, which causes mitochondrial pyridine nucleotide reduction, relieved the inhibition of lipogenesis in each case. The results of these experiments support a ,hypothesis that the mitochondrial NADH:NAD+ ratio regulates fatty acid synthesis by partitioning citrate between transport from mitochondria to cytosol for fatty acid synthesis and oxidation within the mitochondria by reactions of the citric acid cycle. REFERENCES 1. PORTER, J. W., KUMAR, S., AND DUGAN, R. E. ~1971)Progr.Biochem.Pharmacol. 6,1-101. 2. GIBSON, D. M., LYONS, R. T., SCOTT, D. F., AND MUTO, Y. (1972) in Advances in Enzyme Regulation, Vol. 10, pp. 187-204, Pergamon Press, oxford. 3. NUMA, S., AND YAMASHITA, S. (1974) in Current Topics in Cellular Regulation (Horecker, B. L., and Stadtman, E. R., eds.), Vol. 8, pp. 197246, Academic Press, New York. 4. FOR~ANDER, 0. A., MAENPAA, P. H., AND SALASPURO, M. P. (1965) Acta Chem. &and. 19, 1770-1771. 5. RAWAT, A. K. (1968) Eur. J. Btichem. 6, 585592.

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