The effects of lactate and acetate on fatty acid and cholesterol biosynthesis by isolated rat hepatocytes

The effects of lactate and acetate on fatty acid and cholesterol biosynthesis by isolated rat hepatocytes

0020.71 lX/g2/030165-OSSO3.00/0 Copyright 0 1982 Pergamon Press Ltd Int J. B~o...

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0020.71 lX/g2/030165-OSSO3.00/0 Copyright 0 1982 Pergamon Press Ltd

Int J. B~o
THE EFFECTS OF LACTATE AND ACETATE ON FATTY ACID AND CHOLESTEROL BIOSYNTHESIS BY ISOLATED RAT HEPATOCYTES ANTON C. BEYNEN’*, KENNETH F. BUECHLER’, ATE J. VAN DER MOLEN’ and MATH J. H. GEELEN’? *Laboratory of Veterinary Biochemistry, State University of Utrecht, Biltstraat 172, 3572 BP Utrecht, The Netherlands and ZDepartment of Biochemistry, Indiana University School of Medicine, 1100 West Michigan St, Indianapolis, IN 46223, U.S.A. (Received 28 July 1981) Abstract-l. The present study demonstrates that lactate and acetate stimulate fatty acid synthesis and inhibit cholesterogenesis by isolated rat hepatocytes. 2. Exposure of the intact cells to lactate increases the activity of acetyl-CoA carboxylase, as can be measured in homogenates of these cells. A similar effect by acetate was not observed. 3. Both acetate and lactate drastically increase the cellular level of citrate. 4. Possible mechanisms underlying the difference in response of fatty acid and cholesterol synthesis to an increase in substrate availability are discussed. Furthermore, a mechanism is proposed for the lactate effect on acetyl-CoA carboxylase.

Isolation and incubation of hepatocytes

INTRODUCTION Several studies have documented that lactate markedly increases the rate of de nouo fatty acid synthesis by isolated hepatocytes (Clark et al., 1974; Salmon er al., 1974; Geelen, 1974; Harris, 1975). This stimulatory effect of lactate on fatty acid synthesis cannot be explained completely by its role as a carbon precursor for fatty acids (Clark et al., 1974; Salmon et al., 1974; Geelen, 1974; Harris, 1975; Newton & Freedland, 1980). In a recent review we indicated that lactate has a “hormone-like” effect .on hepatic fatty acid biosynthesis (Geelen et al., 1980). The present investigation was triggered by the observation that added lactate stimulates the rate of fatty acid synthesis by isolated rat hepatocytes, whereas the concomitant synthesis of non-saponifiable lipids is unaffected (Beynen & Geelen, unpublished observations). These non-saponifiable lipids constitute a mixture of ill-defined composition. We therefore decided to measure the effect of lactate specifically on the synthesis of cholesterol. Furthermore, these effects of lactate on lipogenesis by isolated hepatocytes were compared with the effects of acetate, another effective substrate for fatty acid synthesis by hepatocytes (Harris, 1975).

Hepatocytes were isolated from meal-fed, male Wistar rats by the method of Seglen (1976) with modifications described previously (Beynen et al., 1979). Isolated hepatocytes were suspended in Krebs-Henseleit bicarbonate buffer (pH 7.4) supplemented with 3.5% bovine serum albumin (defatted and dialysed), 10 mM glucose and other additions as indicated. Incubations were carried out at 37°C in a metabolic shaker (90 strokes/min) in Z-ml Erlenmeyer flasks that contained about 120mg wet wt of cells (equivalent to about 18mg of cellular protein) in a volume of 3 ml. At the indicated times these vessels were sampled. During incubation, flasks were continuously gassed with 95% oxygen, 5% carbon dioxide. Lipogenesis by hepatocytes Rates of fatty acid synthesis from [“H]H,O or from [1-‘4C]acetate were estimated as described by Harris (1975). In experiments on cholesterogenesis, the nonsaponifiable fraction of total lipids was subjected to thinlayer chromatography after the addition of carrier cholesterol. Chromatography was performed on silica G with petroleum ether (b.p. 4O-6O”C)diethyl ether-acetic acid (80:20:2, by vol) as developing agent. The silica, containing the cholesterol, was scraped from the plate, mixed with scintillation liquid and counted for radioactivity. Acetyl-CoA carboxylase activity in hepatocytes

Acetyl-CoA carboxylase activity was measured in homogenates of cells previously incubated with various additions. Aliquots (2 ml) of the cell suspension were centrifuged at room temperature for 1 min at 1OOg. The pellet (intact cells) was resuspended in 0.3ml buffer containing 50 mM potassium phosphate (pH 7.0), 3 mM B-mercap toethanol and 1% (v/v) Triton X-100. In a 25-ul aliauot of this mixture carboxylase activity was measured as the fixation of [‘%Z]bicarbonate into acid-stable material (malonyl-CoA) during 1 min. The assay mixture contained in a final volume of 0.25 ml:50 mM potassium phosphate (pH 7.0). 3 mM p-mercaptoethanol, 6 mM MgCIZ, 3 mM ATP, 1 mM acetyl-CoA, 0.1 mg bovine serum albumin

MATERIALS AND METHODS Chemicals

All radioactive chemicals were purchased from the Radiochemical Centre, Amersham; acetyl-CoA, bovine serum albumin (fraction V), collagenase (type I) and nucleotides from Sigma; silica G from Merck; L-lactate from Serva; other chemicals from Baker. * Present address: Department of Human Nutrition, Agricultural University De Dreijen 12, 6703 BC Wageningen, The Netherlands. t To whom all correspondence should be addressed. 165

166

ANTONC. BEYNENet

al.

(fatty-acid free) and 20mM NaHr4COJ (1 Ci/mol). The assays were terminated by addition of 50~1 6 N HCI. Samples of the reaction mixture were transferred to glass scintillation vials and dried. The residue was resuspended in 50”/, (v/v) ethanol and counted for radioactivity. All values for acetyl-CoA carboxylase activity were corrected for acetyl-CoA-independent CO2 fixation.

biosynthesis, thereby indicating that the rate of substrate supply is not limiting the rate of fatty acid synthesis. As demonstrated previously (Beynen et al., 1979), in the presence of only 10mM glucose (Fig. 1, control incubations) fatty acid synthesis is not linear for the 60-min incubation. This lag phase in fatty acid synthesis is probably related to the time required for hepatocytes to accumulate lipogenic intermediates, Determination of the citrate content of hepatocytes such as lactate and pyruvate (Harris, 1975). In accordSince extensively dialysed bovine serum albumin still contains some citrate, samples intended for citrate determiance with this thesis is the fact that addition of lactate nation were quenched by transferring 1.0 ml to an Eppenplus pyruvate largely abolish the lag phase (Fig. 1). dorf microtube containing 0.2 ml ice-cold 1.2 M HC104 at In contrast to their effects on fatty acid synthesis, the bottom and about 0.3 ml silicone oil (Wacker 200lactate and acetate markedly inhibit the incorporation Wacker 20 = 5:1, v/v) above the bottom layer. The tube of C3H]H,0 into cholesterol (Fig. 2). It appears that was spun for 15 set at full speed using a modified Eppenan inverse relationship exists between the rates of indorf 3200 centrifuge with swing-out rotor. Neutralized corporation of [‘H]H20 into fatty acids and cholessamples of the acid layer were analysed for citrate (Czok & terol, at least under the conditions studied (cf. Figs 1 Lamprecht, 1974) using a double-beam Aminco spectro& 2). No satisfactory explanation can as yet be offered photometer. In other series of hepatocyte incubations bovine serum albumin was omitted. Samples from these for the fact that the increase in fatty acid synthesis incubations were quenched with 1.2 M HCl04 (final condoes not match the decrease in cholesterol synthesis. centration), neutralized and analysed for citrate (Czok KL The effects of lactate and acetate on C3HJH20 inLamprecht. 1974). corporation into cholesterol may not represent true effects on the rate of cholesterogenesis since the number of jH-atoms incorporated per atom of newly RESULTS synthesized sterol carbon may differ according to the The synthesis of fatty acids and cholesterol by the nature of the sterol precursor (Gibbons & Pullinger, hepatocytes was monitored by the incorporation of 1977) or the source of the necessary reducing equivalents (Lakshmanan & Veech, 1977). The data of ‘H from C3H]H,0 into these lipids. The [‘H]H20 Table 1 show that lactate also inhibits cholesterol method is considered to be the most reliable method when [l-i4C]acetate is used as a radioactive precuravailable to assess rates of lipogenesis (Brunengraber sor for sterol synthesis. This would have been anticiet al., 1972). Figure 1 confirms that lactate significantly stimulates fatty acid synthesis by the cells. pated since the addition of lactate will decrease the specific radioactivity of acetyl-CoA, the direct precurLikewise, acetate enhances the flux of 3H into the sor for cholesterol formation. On the other hand, fatty fatty acid biosynthetic route. The addition of both acid synthesis from [1-14C]acetate is not influenced lactate and acetate does not further enhance fatty acid

120-

.

. A 0 90 -

60 -

0

0

15

30 Tome

Fig. acid tate, (V).

45

60

(min)

1. Effects of lactate and acetate on the rate of fatty synthesis by isolated rat hepatocytes. Control (0); lac1OmM (A); acetate, 1OmM (0); lactate plus acetate All incubations contained 10mM glucose and C3H]Hz0 (1 mCi/ml).

30

15 Tme

65

60

(min)

Fig. 2. ERects of lactate and acetate on the rate of cholesterol synthesis by isolated rat hepatocytes. Control (0); lactate, 1OmM (A); acetate. 10mM (Cl); lactate plus acetate (V). All incubations contained 10mM glucose and [“H]H,O (1 mCi/ml).

Hepatic lipogenesis

167

Table 1. Effect of lactate on the incorporation of [1-‘4C]acetate into fatty acids and cholesterol by isolated rat hepatocytes (nmol acetate Fatty acids

Additions [I-‘%Z]acetate [l-“CJacetate

(1OmM) + lactate

(10

mM)

incorp./mg protein per hr) Cholesterol

29.4 + 1.1

2.16 + 0.04

32.4 + 0.7

1.61 f 0.04*

Hepatocytes were incubated for 1 hr with 10 mM glucose and the indicated additions. Specific radioactivity of acetate: 0.05 Ci/mol. Results expressed as means + SE for 3 different incubations. Versus control (Student’s r-test):* P < 0.01.

by lactate (Table 1). Since fatty acid and cholesterol synthesis draw upon the same cytoplasmic acetylCoA pool (Decker & Barth, 1973; Clinkenbeard et al., 1975) these observations indicate that lactate-inhibited cholesterogenesis, as measured by [‘H]H,O incorporation, is not an artifact. This may also hold for the inhibition of cholesterol synthesis observed in the presence of acetate. This notion is derived from the work of Gibbons & Pullinger (1979). These authors demonstrated a decreased accumulation of desmosterol when hepatocytes from 24hr donor rats were incubated with acetate and triparanol, suggesting an inhibition of cholesterogenesis by acetate. Decker & Barth (1973) have shown with supernatant fractions of rat-liver homogenates that activation of acetyl-CoA carboxylase by (-)hydroxycitrate is paralleled by an increase in the incorporation of [l-14C]acetate into fatty acids. [1-14C]Acetate incorporation into cholesterol is depressed by (-)hydroxycitrate. In the presence of avidin, on the other hand, fatty acid synthesis is inhibited, as is carboxylase activity, whereas cholesterogenesis is enhanced (Decker & Barth, 1973). It appears that an activated acetyl-CoA carboxylase drains off acetyl-CoA from cholesterol synthesis. Conversely, an inhibited carboxylase allows other acetyl-CoA requiring reactions in the cytosol, like cholesterogenesis, to utilize this mutual substrate at a higher rate. We therefore decided to investigate whether acetate and lactate modulate the activity of acetyl-CoA carboxylase in isolated hepatocytes. As shown in Table 2, lactate sigcarboxylase, as nificantly activates acetyl-CoA measured in homogenates of hepatocytes previously incubated with lactate. Exposure of the cells to acetate, on the other hand, does not influence the activity Table

2, Effects of lactate and acetate on acetyl-CoA boxylase activity in isolated rat hepatocytes

Additions None Lactate Acetate Lactate

(10 mM) (10 mM) + acetate

car-

Acetyl-CoA carboxylase activity (mU/mg protein) 2.10 2.93 2.34 2.99

+ + + +

0.03 0.05* 0.10 0.06*

Hepatocytes were incubated for 30 min with 10 mM glucose and the indicated additions. Afterwards. enzyme activity was measured in cellular homogenates. A mU of acetyl-CoA carboxylase activity is equivalent to l.Onmol H’%ZO; fixed per min at 37-C under the conditions of the assay. Each figure represents the mean _t SE of 3 different incubations. Versus control (Student’s r-test): *P -z 0.01.

of acetyl-CoA carboxylase (Table 2). Acetate also does not affect the lactate-induced stimulation of carboxylase activity (Table 2). The stimulatory effect of lactate on acetyl-CoA carboxylase, and hence on fatty acid synthesis, may be related to the lactate-stimulated generation of citrate in the cytosolic space (Siess et al., 1976; Watkins et al., 1977). Citrate is a well-known allosteric activator of acetyl-CoA carboxylase. Consistent with observations in other laboratories (Siess et al., 1976; Watkins et al., 1977; McGarry et al., 1978) lactate signiticantly elevates whole-cell citrate concentrations (Table 3). Acetate even has a more pronounced effect on the cellular level of citrate as compared to lactate. In the presence of 10 mM acetate a more than 2-fold increase in whole-cell citrate is observed (Table 3). The effects of acetate and lactate on the cellular concentration of citrate are approximately additive (Table 3). Results obtained by the two methods employed in this study for measuring cellular citrate levels do not differ significantly. DISCUSSION

In agreement with other investigations (Clark et al., 1974; Salmon et al., 1974; Geelen, 1974; Harris, 1975; Newton & Freedland, 1980) we have found that lactate markedly accelerates fatty acid biosynthesis by isolated hepatocytes. Likewise, incubation of hepatocytes with acetate results in enhanced rates of fatty acid synthesis (cf. Harris, 1975). In contrast to the stimulatory effects of lactate and acetate on fatty acid synthesis, these compounds inhibit cholesterogenesis. The inhibitory effect of acetate on cholesterol synthesis by isolated hepatocytes has also been observed by Gibbons & Pullinger (1979). We are not aware of any study demonstrating inhibition of cholesterogenesis by lactate. It is not clear at present why an increased substrate availability, effected by addition of lactate or acetate or both, stimulates fatty acid synthesis but inhibits cholesterogenesis. It is attractive to suppose that the lactate-activated acetyl-CoA carboxylase (Table 2) channels acetyl-CoA preferentially into the fatty acid biosynthetic pathway at the expense of cholesterol formation. This would be analogous to observations by Decker & Barth (1973) who demonstrated with supernatant fractions of rat-liver homogenates that activation of acetyl-CoA carboxylase by (- )hydroxycitrate inhibits cholesterol synthesis. However, a corresponding line of reasoning does not hold for acetate. In our studies acetate stimulates fatty acid synthesis and inhibits cholesterogenesis, but does not

ANTON C. BEYNEN et al.

168 Table 3. Effects of lactate

and acetate on cellular rat hepatocytes

citrate

levels in isolated

Citrate level (nmol/mg protein)

Additions None Lactate

(10 mM)

Acetate

(10 mM)

Lactate

+ acetate

Cells incubated with bovine serum albumin

Cells incubated without bovine serum albumin

3.09 (2.94. 3.24) 4.48 (4.50, 4.46) 8.10 (7.08. 9.12) 10.38 (10.00, 10.76)

3.78 (3.50, 4.05) 5.78 (5.93, 5.63) 8.99 (9.08, 8.90) 10.14 (10.30, 9.98)

Hepatocytes were incubated for 30min with 1OmM glucose and the indicated additions. Citrate was determined in cells only (cells incubated with bovine serum albumin) or in the total hepatocyte suspension (cells incubated without bovine serum albumin). Results are expressed as the

mean (and range) of 2 different incubations. At the start of the incubations, cells contained 1.63 (1.60, 1.66) and 1.55 (1.40, 1.70) nmol citrate/ mg protein in the presence and the absence of bovine serum albumin, respectively.

affect the measured acetyl-CoA carboxylase activity in hepatocyte homogenates. This suggests either that acetate does not affect acetyl-CoA carboxylase within the intact hepatocyte or that a possible effect is not preserved after cell disruption. As to the mechanism of the stimulatory effect of lactate on acetyl-CoA carboxylase (Table 2), we can only speculate at present. Lactate itself does not affect the activity of partially purified acetyl-CoA carboxylase (Carlson & Kim, 1974). Since citrate is a very potent allosteric activator of acetyl-CoA carboxylase, it could be argued that lactate exerts its stimulatory effect on the carboxylase through an elevation of the cellular content of citrate (Table 3). Because of compartmentation of citrate in the cell the results of Table 3 should be carefully interpreted with respect to regulation of acetyl-CoA carboxylase which is localized in the cytosol. However, lactate not only increases whole-cell citrate, but has also been shown to stimulate generation of citrate in the cytosolic space (Siess et al., 1976; Watkins et al., 1977). Inconsistent with the thesis that lactate exerts its stimulation of acetyl-CoA carboxylase through generation of citrate is the observation that acetate also drastically elevates the cellular content of citrate (Table 3) without affecting the activity of the carboxylase (Table 2). Activation of acetyl-CoA carboxylase through an increased concentration of citrate within the hepatocyte would be persistent upon cell disruption. This notion is derived from the fact that the stimulatory effect of citrate on partially purified acetyl-CoA carboxylase is still observed when the enzyme and effector are separated on a Sephadex G-25 column (data not shown). Furthermore, upon incubation of intact hepatocytes with lactate, the polymeric state of acetyl-CoA carboxylase is not changed as compared to the lactatefree control (Buechler, Beynen & Geelen, in preparation). In both conditions the carboxylase migrates to a 1617% sucrose band of a 5-200/, sucrose gradient. Activation of purified carboxylase prep-

arations with citrate is accompanied by polymerization of the enzyme (Lane et al., 1974). Therefore, we conclude that citrate-induced protomer-polymer transitions are not involved in the short-term control of acetyl-CoA carboxylase by lactate. Acetyl-CoA carboxylase activity is also controlled by phosphorylation-dephosphorylation cycles (for review, see Geelen & Beynen, 1981). Purified rabbit mammary gland acetyl-CoA carboxylase can be inactivated through phosphorylation by a cyclic AMPdependent protein kinase from rabbit skeletal muscle (Hardie & Cohen, 1978). The rate of phosphorylation of partially purified acetyl-CoA carboxylase from rat liver is increased (as is the rate of inactivation) in the presence of cyclic AMP (Lent et al., 1978). Possibly within the hepatocyte the activity of acetyl-CoA carboxylase is regulated by the level of cyclic AMP, which in turn dictates the activity of cyclic AMPdependent protein kinase. A high level of cyclic AMP would be associated with an inactivated carboxylase, whereas low concentrations of cyclic AMP would favor the dephosphorylated, active form of acetylCoA carboxylase. This concept is consistent with the observation that glucagon inactivates acetyl-CoA carboxylase in isolated hepatocytes (Geelen et al., 1978) and increases cyclic AMP levels (Geelen et al., 1977). Lactate has been shown to lower basal cyclic AMP levels in isolated rat hepatocytes (Zederman et al., 1977). Therefore, the observation that lactate activates acetyl-CoA carboxylase (Table 2) suggests that the mechanism underlying the control of acetyl-CoA carboxylase by lactate may be opposite to that of glucagon. Unfortunately, at present no data are available concerning a possible effect of acetate on cyclic AMP levels in hepatocytes. In summary, both lactate and acetate stimulate fatty acid synthesis by isolated hepatocytes and inhibit cholesterol synthesis. The inhibition of cholesterogenesis imposed by lactate cannot be unequivocally explained by the observed lactate-induced activation

Hepatic lipogenesis of acetyl-CoA carboxylase, diverting acetyl-CoA from cholesterol synthesis. Acetate does not affect acetylCoA carboxylase activity. The lactate-mediated activation of acetyl-CoA carboxylase does not appear to be a consequence of the increased citrate levels seen upon treatment of the cells with lactate. Acetate even more drastically elevates citrate levels but does not affect the measured acetyl-CoA carboxylase activity. Possibly, lactate modulates the activity of acetyl-CoA carboxylase through changes in the cellular level of cyclic AMP.

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Acknowledgemems-These investigations were supported