ARCHIVES

OF BIOCHEMISTRY

AND

Vol. 283, No. 1, November

BIOPHYSICS

15, pp. 90-95,199O

Simultaneous Stimulation of Fatty Acid Synthesis and Oxidation in Rat Hepatocytes by Vanadate Manuel

Guzm&

Department

and Jo& Castro’

of Biochemistry

and Molecular

Biology I, Faculty of Chemistry,

Complutense

University,

28040-Madrid,

Spain

Received March 15,1990, and in revised form May 31,199O

When added to the hepatocyte incubation medium, vanadate increased the rate of fatty acid synthesis de nouo as well as the activity of acetyl-CoA carboxylase, whereas it had no effect on the activity of fatty acid synthase. On the other hand, and despite elevating the intracellular levels of malonyl-CoA, vanadate diverted exogenous fatty acids into the oxidation pathway at the expense of the esterification route. This was concomitant to an increase in carnitine palmitoyltransferase I activity. All these effects were not significantly different between periportal and perivenous hepatocytes and were also evident in cells incubated in Ca2+-free medium. Nevertheless, Ca2+ ions enhanced carnitine palmitoyltransferase I activity in isolated liver mitochondria. In addition, the effects of vanadate on acetyl-CoA carboxylase and carnitine palmitoyltransferase I were only evident in a permeabilized-cell assay, disappearing upon cell disruption and isolation of the corresponding cell subfraction for enzyme assay. Results show that vanadate exerts specific insulin-like and non-insulinlike effects on hepatic fatty acid metabolism, and suggest that the intracellular concentration of malonylCoA is not the only factor responsible for the regulation of the fatty-acid-oxidative process in the liver. Q isso Academic

Press,

Inc.

A number of metabolic effects of vanadate have been described so far, many of which are insulin-like actions on intact cells (reviewed in (l-3)). Thus vanadate is able to mimic insulin action upon the stimulation of glucose transport and metabolism in adipocytes (4-7) and in skeletal muscle (6-8). Vanadate also increases both the glycolytic flux and the levels of fructose 2,6-bisphosphate in hepatocytes (9). Furthermore, vanadate enhances the phosphorylation of the insulin receptor (7). i To whom correspondence

90

should be addressed.

This effect could be accounted for by stimulation of the tyrosine kinase activity of the insulin receptor (7), although inhibition of a phosphotyrosine phosphatase activity may also be involved (10). In addition, vanadate exerts insulin-like effects in Go. For example, oral administration of vanadate to diabetic rats normalizes blood glucose levels and restores insulin responsiveness in several target tissues (11-14). Hence vanadate is considered an important probe to relate to insulin in studies of mechanisms of action. Nevertheless, the effects of vanadate on hepatic metabolism seem to be rather complex, since vanadate also exerts non-insulin-like effects on key enzymes of glycogen metabolism, activating glycogen phosphorylase and inactivating glycogen synthase in isolated hepatocytes (15). Although the effects of vanadate on carbohydrate metabolism have received considerable attention, fewer and less extensive studies have focused on the effects of vanadate on lipid metabolism. Insulin-like actions of vanadate have been reported in rat adipocytes, including inhibition of ACTH-2 or epinephrine-induced lipolysis (16,17) and stimulation of lipogenesis from glucose (17). Vanadate is also able to mimic insulin action in the stimulation of fatty acid synthesis (18,19) and in the inhibition of apolipoprotein B secretion (20) in rat hepatocytes. In the present study we examined in detail the effects of vanadate upon fatty acid metabolism in periportal and perivenous rat hepatocytes. Our results show that vanadate exerts insulin-like actions in the stimulation of fatty acid synthesis and acetyl-CoA carboxylase activity. However, vanadate also produced non-insulin-like effects by strongly depressing fatty acid esterification and stimulating fatty acid oxidation. The latter was con’ Abbreviations used: CPT, carnitine palmitoyltransferase (EC 2.3.1.21); CPT-I, overt form of mitochondrial CPT activity; CPT-II, latent form of mitochondrial CPT activity; HPG, 4-hydroxyphenylglyoxylate; ACTH, adrenocorticotropic hormone.

0003-9861/90 $3.00 Copyright 0 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.

EFFECTS

comitant to an increase ase I activity.

MATERIALS

OF VANADATE

of carnitine

ON HEPATIC

palmitoyltransfer-

AND METHODS

Materials. 3H20 (5 Ci/ml), [1-“Clpalmitic acid (58 Ci/mol), [U‘*C]palmitic acid (403 Ci/mol), L-[Me-3H]carnitine (71 Ci/mol) and [l-14C]acetyl-CoA (54.3 Ci/mol) were supplied by Amersham International (Amersham, Bucks, UK). Digitonin, sodium orthovanadate, and collagenase (type I) were purchased from Sigma Chemical Co. (St. Louis, MO). 4-Hydroxyphenylglyoxylate was purchased from Aldrich (Steinheim, Germany). Hepatocyte isolation. Male Wistar rats (225-275 g) were used all through this study. Hepatocytes from the whole liver were isolated according to the procedure of Seglen (21) with the modifications indicated by Beynen et al. (22), whereas periportal and perivenous hepatocytes were isolated by the method of Chen and Katz (23) and further characterized on the basis of the distribution pattern of several marker enzymes (24). Since lipogenesis is markedly depressed just after the isolation procedure, hepatocytes were incubated for 15 min at 37°C and then filtered through nylon mesh, as recommended by Holland et al. (25). Cell viability, as determined by Trypan blue exclusion, was always higher than 85% in the final hepatocyte suspension. Hepatocyte incubation. Hepatocytes were incubated in KrebsHenseleit bicarbonate buffer supplemented with 10 mM glucose and 2% (w/v) defatted and dialysed bovine serum albumin. Incubations (4-6 mg of cellular protein/ml) were carried out in a total volume of 2 ml at 37°C under an atmosphere of Op/COp (19:l). (i) The rates of fatty acid synthesis de nouo were determined in cell incubations containing 3HZ0 (0.4 mCi/ml). After 60 min, reactions were stopped and total fatty acids were extracted as in Ref. (26). (ii) The rates of fatty acid esterification were monitored by incubating hepatocytes with 0.4 mM [U-‘*C]palmitate (0.05 Ci/mol) bound to albumin. After 60 min, reactions were stopped and lipids were extracted (26). Neutral lipids were separated by TLC on silica-gel G plates with hexane/diethylether/formic acid (40/20/l, v/v/v) as developing system. Polar lipids were separated by TLC on silica-gel H plates (supplemented with 1% sodium carbonate and 0.4% ammonium sulfate) using chloroform/methanol/ acetic acid/water (50/25/8/l, v/v/v/v) as developing system. (iii) The rates of fatty acid oxidation were measured in cell incubations containing 0.4 mM [l-‘4C]palmitate (0.05 Ci/mol) bound to albumin. After 30 min, reactions were stopped and oxidation products were extracted exactly as described before (27). Part of the cells were incubated Measurement of enzyme activities. in radioisotope-free flasks and used for the determination of the activities of acetyl-CoA carboxylase, fatty acid synthase, and carnitine palmitoyltransferase I (CPT-I). (i) Acetyl-CoA carboxylase activity was measured by determining the incorporation of [l-‘4C]acetyl-CoA into fatty acids in a reaction coupled to the fatty acid synthase reaction (26, 28). For the determination of acetyl-CoA carboxylase activity in digitonin-permeabilized hepatocytes, 100 ~1 of hepatocyte suspension was added to 100 ~1 of isoosmotic assay medium supplemented with 64 pg of digitonin/mg of cellular protein, exactly as described in Ref. (28). After 4 min, reactions were stopped with 0.1 ml of 10 M NaOH and fatty acids were extracted with petroleum ether (bp 40-60°C) (28). Measurement of enzyme activity in the 12,000g supernatant was performed according to Tijburg et al. (26). (ii) Fatty acid synthase activity was determined in digitonin-permeabilized hepatocytes exactly as described by Bijleveld and Geelen (28). Digitonin concentration in the assay medium was the same as for the assay of acetyl-CoA carhoxylase. (iii) CPT-I activity was determined as the malonyl-CoA-sensitive incorporation of L-[Me-3H]carnitine into palmitoylcarnitine by previously described methods, both in isolated mitochondria (29) and in cells permeabilized with digitonin (30). In the second case, 100 ~1 of hepatocyte suspension was added to 100 ~1 of isoosmotic assay me-

FATTY

ACID

dium supplemented

METABOLISM with

91

40 pg of digitonin/mg

of cellular

protein.

Reactions were stopped by the addition of either 1.0 ml (isolated mitocbondria) or 0.3 ml (permeabilized cells) of 1 M HCl, and palmitoyl-carnitine product was extracted with butan-l-01 (29). MalonylCoA-insensitive CPT activity, representing the latent form of mitochondrial CPT activity (CPT-II) (31), was always discounted from the CPT activity experimentally determined. In both assays, CPT-II activity

never accounted

for more than 10% of the total CPT activity

experimentally determined (results not shown). Preparations of mitochondria were practically devoid of peroxisomes, as judged from experiments of recovery of catalase activity (results not shown). Other analytical

methods.

Intracellular

levels of malonyl-CoA

were

determined in neutralized perchloric acid cell extracts using the radioenzymatic method of Beynen et al. (22), with 70 mM potassium phosphate (pH 6.8) as assay buffer. Protein was determined by the method of Lowry et al. (32), with bovine serum albumin as a standard. Statistical analysis. Results represent the means + SD of the numbers of cell preparations indicated in every case. Cell incubations and/ or enzyme assays were always carried out in triplicate. Statistical analysis was performed by Student’s t test.

RESULTS Effects of Vanadate on Fatty Acid Synthesis de Novo The addition of vanadate to the hepatocyte incubation medium increased the rate of fatty acid synthesis de novo (Table I), which correlated well with the vanadate-mediated stimulation of acetyl-CoA carboxylase activity, as measured in the permeabilized-cell assay (Table I). In

addition, vanadate had no effect on fatty acid synthase activity (Table I). Acetyl-CoA carboxylase activity was also determined in the 12,000g supernatant obtained from cell suspensions which had been preincubated in the absence or in the presence of vanadate. When hepatocytes were preincubated with 2 mM vanadate and subsequently the 12,000g supernatant was isolated for enzyme assay, no effect of vanadate on acetyl-CoA carboxylase activity was apparent. This lack of effect of vanadate was still observed after increasing the vanadate concentration up to 5 mM and the preincubation time of the hepatocytes up to 30 min, even when 2 mM EDTA and 10 mM NaF were present all along the cell fractionation procedure as well as in the enzyme assay (results not shown). In addition,

a direct

effect of vanadate

on the enzyme

pro-

tein may be ruled out in view that acetyl-CoA carboxylase activity in the 12,000g supernatant was unaffected when 2 mM vanadate was added to the enzyme assay (results not shown). Effects of Vanadate on Fatty Acid Esterification The insulin-like effect of vanadate on fatty acid synthesis described above was, however, accompanied by a non-insulin-like effect on fatty acid esterification. Thus, vanadate decreased the rate of fatty acid esterification from

exogenous

substrate

into

the

major

glycerolipid

92

GUZMAN TABLE Effects

Metabolic

AND

I

of Vanadate on Fatty Acid Metabolism in Rat Hepatocytes

parameter

Fatty acid synthesis (n = 7) Fatty acid esterification (n = 9) Total lipids Triacylglycerols Phosphatidylcholines Phosphatidylethanolamines Cholesterol esters Fatty acid oxidation ( n = 12) Total product co* Acid-soluble product Enzyme activities Acetyl-CoA carboxylase (n = 5) Fatty acid synthase ( n = 4) CPT-I (n = 10)

No additions

+ Vanadate

2 mM

62.82 k 7.53

87.51 + 8.09”

16.11 7.47 4.15 2.03 0.58

11.75 4.80 3.07 1.42 0.53

+ f + f s

1.79 0.93 0.63 0.38 0.11

+_1.26b + 0.81b k 0.45” L 0.16” f 0.10

34.57 f 3.91 5.47 + 0.62 29.10 f 3.53

49.08 2 3.94b 13.81 f 2.73b 35.27 + 2.19”

0.51 k 0.08 3.94 + 0.45 5.89 + 0.76

0.67 2 0.09” 3.88 f 0.61 8.54 + 0.96”

CASTRO

mitochondria or (unlike malonyl-CoA) in intact hepatocytes (33). Since certain effects of vanadate are abolished when hepatocytes are incubated in Ca’+-free medium (15), we next studied whether the effects of vanadate on fatty acid oxidation and CPT-I activity were dependent on the presence of extracellular Ca’+. As shown in Fig. 1, basal rates of fatty acid oxidation, as well as basal CPTI activity, were lower in hepatocytes incubated in the absence of Ca2+ as compared with cells incubated in normal, Ca’+-containing medium. However, in both cases vanadate induced similar increases on these two parameters. Nevertheless, it is curious that Ca2+ was able to enhance CPT-I activity in a dose-dependent manner

Note. Hepatocytes were isolated from the whole liver and preincubated in the absence or in the presence of 2 mM vanadate. After 20 min, part of the cells were used for determination of the rates of fatty acid synthesis, esterification, and oxidation in intact hepatocytes. The rest of the cells were used for measurement of enzyme activities in digitonin-permeabilized hepatocytes. Rates of fatty acid synthesis are expressed as nmol of acetyl units/h per milligram of cellular protein. Rates of fatty acid esterification are expressed as nmol of [U“C]palmitate into lipid/h per milligram of cellular protein. “Total lipids” represents the sum of total phospholipids, cholesterol esters, and mono-, di-, and triacylglycerols. Rates of fatty acid oxidation are expressed as nmol of [1-Wlpalmitate into product/h per milligram of cellular protein. Enzyme activities are expressed as nmol/min per milligram of cellular protein. Results represent the means f SD of the number of cell preparations indicated in every case. Significantly different from incubations with no additions: “P < 0.05; bP -c 0.01.

x $

Y I

fractions, including triacylglycerols, lines, and phosphatidylethanolamines, acid incorporation into cholesterol affected (Table I).

phosphatidylchowhereas fatty esters was less

Effects of Vanadate on Fatty Acid Oxidation The rate of [ l-‘*C Jpalmitate oxidation was increased in a dose-dependent manner by vanadate (Fig. 1A). This stimulation affected both the CO, and the acid-soluble component of total oxidation product (Table I). Similarly, vanadate stimulated CPT-I activity in a dose-dependent manner (Fig. 1B). Moreover, these effects of vanadate were also evident in the presence of 4-hydroxyphenylglyoxylate (HPG) (Fig. 1A). HPG, a synthetic inhibitor of CPT-I activity, was chosen for these studies because it exhibits properties very similar to those of malonyl-CoA, the physiological inhibitor of CPT-I, so that it may be used to titrate CPT-I activity in isolated

1

5

2.5 [ Vanadate]

ImMl

FIG. 1. Vanadate-induced stimulation of fatty acid oxidation and CPT-I activity: dose-dependence and effects of Ca2+ depletion and inhibitors. Hepatocytes were isolated from the whole liver. Part of the cells were washed twice in Ca’+-free Krebs-Henseleit buffer and further incubated in the same buffer (closed symbols). The rest of the cells were similarly washed twice in normal Krebs-Henseleit buffer (containing 2.5 mM Ca’+) and further incubated in the same buffer (open symbols). Hepatocytes were preincubated for 20 min in the presence of different concentrations of vanadate and then the rates of both fatty acid oxidation and CPT-I activity were monitored. Results represent the means + SD of five different cell preparations. (A) Rate of [l14C]palmitate oxidation to total product. Hepatocytes were preincubated as follows: (0) +Ca’+, -HPG; (0) +Ca*‘, +5 mM HPG; (0) -Ca’+, -HPG; @) -Ca ‘* , +5 mM HPG. (B) CPT-I activity in permeabilized hepatocytes. Hepatocytes were preincubated as follows: (0) +Ca*+, -HPG; (0) +Ca’+, -HPG, enzyme assays in the presence of 20 PM malonyl-CoA; (Cl) +Ca’+, +5 mM HPG; (A) +Ca’+, +5 mM HPG, enzyme assays in the presence of 20 I.LM malonyl-CoA. (0) -Ca”, -HPG;

EFFECTS

OF VANADATE

ON HEPATIC

I “++‘7

6

-log

CCaCI,]

5

4

3

I Ml

FIG. 2. Effect of Ca2+ on CPT-I activity in isolated mitochondria. Mitochondria were isolated from liver homogenates and were preincubated for 4 min in the presence of different concentrations of CaCI,, as well as with (closed symbols) or wit,hout (open symbols) 0.5 mM HPG and with (squares) or without (circles) 10 pM malonyl-CoA. Reactions were then started by the addition of L-[Me-3H]carnitine and CPT-I activity was determined. Results represent the means _+SD of six different mitochondrial preparations.

when enzyme activity was determined in isolated liver mitochondria (Fig. 2). Inhibition of CPT-I activity by malonyl-CoA is a welldescribed property of the enzyme (34,35). Hence, we determined the effects of vanadate on hepatic malonylCoA levels. Surprisingly, vanadate induced a significant increase in the intracellular concentration of malonylCoA (Fig. 3). Nevertheless, this is in line with the vanadate-induced stimulation of acetyl-CoA carboxylase activity (Table I). Similarly as for acetyl-CoA carboxylase, the vanadate-mediated increase of CPT-I activity did not survive upon cell disruption and isolation of the corresponding cell subfraction (mitochondria in this case) for determination of enzyme activity. In addition, no direct effect of vanadate was observed on CPT-I activity, as measured in isolated mitochondria (results not shown). We finally studied whether all the above-mentioned effects of vanadate on hepatic fatty acid metabolism differed between the periportal and the perivenous zone of the liver. Nevertheless, no significant differences between the two hepatocyte subpopulations were found with regard to the effects of vanadate on the rates of fatty acid synthesis, esterification, and oxidation, on the activities of acetyl-CoA carboxylase and CPT-I, and on the intracellular levels of malonyl-CoA (results not shown). DISCUSSION

The effects of vanadate on fatty acid synthesis, esterification, and oxidation were studied in rat hepatocytes. In addition, digitonin-permeabilized cells were used for the study of the effects exerted by vanadate on the two

FATTY

ACID

METABOLISM

93

enzymes involved in fatty acid synthesis de novo, i.e., acetyl-CoA carboxylase and fatty acid synthase (36), as well as on CPT-I, the key enzyme of the fatty-acid-oxidative process (34, 35). Measurement of these enzyme activities in a permeabilized-cell system is essential to preserve the short-term alterations induced by different cellular modulators on the respective enzyme proteins, mainly when these effects are mediated by a putative mechanism of labile modification (28,30,37). In agreement with other reports (18; 19), the rates of hepatic fatty acid synthesis de novo were enhanced by vanadate. The vanadate-induced stimulation of acetylCoA carboxylase described herein is new, and supports the general view that acetyl-CoA carboxylase is a key regulatory point of the fatty-acid-synthesizing process (36, 38). Nevertheless, vanadate diverted exogenously added fatty acids into the oxidation pathway at the expense of the esterification route. Therefore, as for hepatic carbohydrate metabolism (9, 15), vanadate exerts insulin-like and non-insulin-like effects upon hepatic lipid metabolism, depending on the pathway studied. It is commonly agreed that malonyl-CoA, the physiological inhibitor of CPT-I, plays an essential role in the partition of fatty acids between esterification and oxidation in the liver (34). Surprisingly, vanadate enhanced the intracellular levels of malonyl-CoA as well as CPT-I activity and, concomitantly, the rate ofpalmitate oxidation. As far as we know, this is the first time in which a rapid stimulation of the rate of both long-chain fatty acid oxidation and CPT-I activity takes place despite the presence of parallel elevated levels of malonyl-CoA. In addition, this indicates that the intracellular concentration of malonyl-CoA may not be the only factor responsible for the regulation of hepatic fatty acid oxidation, at least in our experimental system. However, it has been recently reported that CPT activity in peroxisomes is sensitive to malonyl-CoA and accounts for up to 20% of total CPT activity in livers from fed rats (31). Just like mitochondria, peroxisomes remain inside the permeabilized cells (cf. Ref. (28)). Thus, we are aware that malonyl-CoA-sensitive CPT activity in permeabilized cells mostly (but not exactly) represents CPT-I activity. On the other hand, our preparations of mitochondria are practically devoid of peroxisomes, as judged from experiments on the recovery of catalase activity. Hence, it may be assumed that malonyl-CoA-sensitive CPT activity in isolated mitochondria represents CPT-I activity. The molecular bases of vanadate action on cellular metabolism are largely unknown (2,3,14). Nevertheless, vanadate has been suggested to act by different mechanisms of covalent and noncovalent modification of target enzymes (2,3,7,9,14). Although it is not possible to deduce from our results a detailed mechanism of vanadate action on hepatic fatty acid metabolism, several conclusions may be obtained with regard to mechanism:

94

GUZMAN

AND

CASTRO

ies are required to elucidate the molecular basis of these insulin-like and non-insulin-like effects of vanadate. ACKNOWLEDGMENTS This study was supported by a research grant (89/0220) from the Fondo de Investigaciones Sanitarias de la Seguridad Social, Spain. REFERENCES Time

(mm1

FIG. 3. Effect of vanadate on the cellular concentration of malonylCoA. Hepatocytes were isolated from the whole liver and incubated in the absence (0) or in the presence (0) of 2 mM vanadate. Aliquots were taken at different time points in order to determine the intracellular levels of malonyl-CoA. Results represent the means + SD of five different cell preparations.

(i) Vanadate does not seem to induce a very stable activation of acetyl-CoA carboxylase and CPT-I, since the modifications induced by vanadate on these two enzyme activities are observed in digitonin-permeabilized hepatocytes but do not survive upon cell disruption and isolation of the corresponding cell subfraction for enzyme assay. Therefore, it is tempting to speculate that vanadate causes a nonc’ovalent activation of these two enzymes. A similar conclusion was obtained by GGmez-Foix et al. for 6-phosphofructo-2-kinase (9). Nevertheless, the possibility that vanadate induces a labile covalent modification on these target enzymes may not be excluded. (ii) Although cells take up vanadate (4,39), the activation of acetyl-CoA carboxylase and CPT-I does not seem to be due to a direct effect of vanadate on the two enzyme proteins. However, the possible role of a metabolite of vanadate upon the modulation of hepatic lipid metabolism may not be ruied out. (iii) Although certain effects of vanadate disappear when cells are incubated in Ca’+-free medium (15), the vanadate-induced stimulation of fatty acid oxidation and CPT-I activity does not seem to be dependent on the presence of extracellular Ca’+. This is also evident for the vanadate-mediated activation of fructose 2,6-bisphosphate levels (9) as well as of fatty acid synthesis (18, 19), and results not shown). Vanadate is a well-known inhibitor of membrane ion-transporting ATPases, including the outwardly directed Ca*+-ATPase (l-3, 39). Thus, it has been suggested that vanadate increases the intracellular concentration of Ca*+ by blocking the efflux of cytosolic Ca *+ (39). In addition, vanadate has been shown to increase the efRux of Ca” from liver mitochondria (40). In this respect, it is interesting that Ca*+ activates CPT-I activity when added directly to the enzyme assay. In conclusion, the results reported herein show that vanadate simultaneously stimulates fatty acid synthesis and oxidation in rat hepatocytes. However, further stud-

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T., and Kurup,

Simultaneous stimulation of fatty acid synthesis and oxidation in rat hepatocytes by vanadate.

When added to the hepatocyte incubation medium, vanadate increased the rate of fatty acid synthesis de novo as well as the activity of acetyl-CoA carb...
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