Quantitative studies on fatty acid metabolism in isolated parenchymal cells from normal and cirrhotic livers in rats

Jourrral of Hepatoiogy, 1992; 15: 10-16 @ 1992 Elsevier Science Publishers B.V. All rights reserved. 016%8278/92/$05.00

10 HEPAT 01044

Quantitative studies on fatty acid metabolism in isolated parenchymal cells from normal and cirrhotic livers in rats Thomas

Zimmermann,

Hans Franke,

Mathias

Peuker

and Rolf Dargel

lttstituteof Pathologicalfliochetnistry, Friedrich-Schiller-Urzik*ersity, Jeua, Federal Republic of Gertnatzy (Received 15 November

1988)

Parenchymal cells isolated from normal and thioacetamide-induced micronodular cirrhotic rat livers were used to evaluate changes in hepatocellular fatty acid (FA) metabolism in cirrhosis. Exogenous free FA (FFA) are rapidly taken up by hepatocytes obtained either from normal or cirrhotic livers. They are predominantly esterified to triglycerides and accumulate intracytoplasmatically as lipid droplets with increasing cellular FFA uptake. In the parenchymal cells of cirrhotic livers, however, the following changes were observed when compared with controls: (i) decreased cellular output of esterified FA derived both from exogenous sources and from de novo FA synthesis; (ii) increased total ketone body production, mainly as /3-hydroxybutyrate; (iii) decreased cholesterol synthesis: and (iv) enhanced incorporation of newly synthesized FA into phospholipids in spite of an unaffected rate of FA synthesis. In summary, the data provide evidence for intrinsic alterations in the lipid metabolism in the parenchymal cells of cirrhotic livers which are preserved in the isolated hepatocytes under optimum incubation conditions for oxygen and substrate supply.

The iiver plays a key role in lipid and lipoprotein metabolism (l-3). Thus, one could expect chronic liver diseases to be accompanied by striking abnormalities in hepatic lipid and lipoprotein metabolism. However, the current knowledge about lipid metabolism in hepatic failure (4-8) is, in many respects, considerably poorer than about amino acids for instance. Therefore, it was the aim of the studies presented here, to quantitatively investigate the hepatocellular processes of fatty acid uptake and utilization in cirrhosis. These studies were carried out with isolated parenchymal cells prepared from normal and micronodular cirrhotic rat livers according to a procedure described recently (9) in order to exclude extrahepatic factors and the effects of disturbed microcirculation. Experimental cirrhosis was induced by chronic administration of the hepatotoxic agent thioacetamide (lo,1 1). This animal model has been shown to resemble the major features of the human disease (10-16). Correspondetlce: Dr. Thomas Jena. F.R.G.

Material and Methods Animals

Female virgin rats from the Uje: WIST strain (average weight 230 g) were kept under constant conditions and fed a stock-pelleted laboratory diet ad libitum. To induce micronodular cirrhosis, animals of the experimental group received drinking water containing 300 mg TAA/I from the fourth to the sixth month of life (10,11,16). After two additional weeks under the same conditions, but without TAA administration, the animals were used in experiments. Age-matched control animals received tap water for a corresponding time period. The concentrations of serum and liver lipids as well as the Triton WR 1339-induced increase in serum TG concentration were determined in vivo as described (10). The Triton-induced increase in serum TG is commonly held to represent hepatic VLDL-TG output (17). Ketone bodies were measured enzymatically.

Zimmermann.Institute of Pathological Biochemistry,

Abbrevialions:FA = fatty acids:FFA = iree fatty acids; TAA = thioacetamide; TG = density lipoproteins.

Friedrich-Schiller-University, triglycerides; PL

Lijbderstrape

= phospholipids; VLDL

3, O-6900 = very low

FATTY ACID METABOLlSM

IN LIVER

CIRRHOSIS

Isolation and incubation of liver parenchymal cells

Liver parenchymal cells were isolated from fed animals by perfusion with a calcium-free solution followed by 0.03% collagenase essentially as described (9). After repeated washings a batch of packed hepatocytes was obtained, which was suspended in approx. 200 ml of Eagle’s MEM-medium (SIFIN, Berlin) and used for incubation experiments. Parenchymal cells represent more than 95% of all cells in the purified suspensions prepared both from normal and cirrhotic rat livers. In each case the viability was higher than 85% as judged by the Trypan blue exclusion test. The loss of cells during subsequent incubation was negligible. Ultrastructurally, the cells were identical with those characterized previously (9). After isolation and purification 45.10’ hepatocytes were incubated in 30 ml of Eagle’s MEM-medium under a gas phase of 0, and CO, (95:5, v/v) at 37°C in Erlenmeyer flasks in a rotary shaker at about 100 rpm. After a 15-min preincubation period (given as t = 0 on the abscissa, in Fig. 2A,B) albumin-bound palmitate was added at a final concentration of 0.50 mM (BSA, FA free, Serva), if not stated otherwise (see below). At that time point the lipid concentratiom of the cells and the medium were measured in preparations of both normal and TAA-injured cells. The insignificant differences of the medium lipid concentrations at t = 0 mainly reflected the different secretory activities of cells from normal and cirrhotic rat livers after a short preincubation which became apparent during the long-incubation period. Since the number of cells remained constant during the incubation period. as assessed by means of the Trypan blue exclusion test, their loss might not essentially contribute to the slight difference at t = 0. In a separate set of experiments the influence ot increasing initial medium FFA concentrations on the cellular processes of FA metabolism was tested. In these incubations the molar palmitate/albumin ratio was kept constant, since otherwise different binding sites with different affinities would be involved. Principally, a molar ratio palmitate/albumin = 2.0 was used. At the end of incubation, the cells were immediately separated from the medium by centrifugation and the two fractions were processed as described below. All reagents were of analytical grade. Rates of lipogenesis

Rates of FA de nova synthesis were estimated by measuring the incorporation of 3H from “Hz0 into FA. The experiments were started by the addition of 5.53.10’ dpm 3H,0 per flask and carried out essentially as described (9). The rates of FA synthesis were calculated

11 according en Iv *%heassumption that the incorporation of 4.8 molecules 3 @ into FA reflects the synthesis of one molecule palmitate (18). The rates of de novo synthesis of ch$llesteroi were also determined from experiments wi:> tr?iated water assuming that the incorporation of 9.7 molecules of ‘H,O represents the Lynthesis of 1 molecule cholesterol (19). Statistical methods

The numbers of observations given in the tables and figures refer to results obtained from separate cell preparations. All values are given as means f S.E. Statistical comparisons were performed by Student’s t test.

200

J 0.25

0 05

1.0

Inttlol

15

medwm

palmltate

[mM]

Fig. I. Changes of lipid concentrations in liver parenchymal cell suspensions prepared from normal and TAA-treated rats during incubation with increasing initial medium palmitate concentration. Data are means +- S.E. from three to five separate experiments. Negative values indicate disappearance from the medium (i.e.. cellular uptake) or cellular loss. whereas positive values indicate accumulation in medium or cells, respectively. The initial lipid concentrations expressed in absolute terms and given as nmoi FAIlOh cells or an equivalent volume of the medium have been determined as follows:Cells: FFA 22 f 3 (control) and 21 _+4 (TAA): TG-FA 180 + 27 and 100 + 33: PL-FA 370 ?I 51 and 290 2 37. Medium (concentrations in /tM are given in brackets): TG-FA 1.1 ?I 0.30 (1.7 f 0.5) (control) and 0.44 k 0.22 (0.66 = 0.33) (TAA): PL-FA 0.89 + 0.27 (1.34 f 0.41) and 0.44 + 0.33 (0.66 ? 0.5). The medium FFA concentrations were adjusted to values ranging from 0.25 to 1.5 mM by adding albumin-bound palmitate at a constant molar palmitatelalbumin ratio (for details see Materials and Methods). *p < 0.05 vs. control. Stripped bars. control; open bars. TAA. FFA. free fatty acids: TG-FA. triglyceride FA: PL-FA. phospolipid FA.

T. ZIMMERMANN

12

et al.

TABLE 1 Characterization

of control and TAA-treated

-~

rats

___

Body weight (g) Liver weight (g) Food intake (g/animal per day) Water consumption (ml/animal per day) Serum enzymes (U/1) ASAT ALAT Alkaline phosphatase Choline esterase Serum protein (g/l) Serum glucose (mM)

230 9.0 20 24

+ + + r

6.8 0.32 0.43 1.1

(15 ) (18 ) (15 ) (15)

22@+ 11 f 21 -t 24 +

5.5 0.21” 0.94 0.77

(13 (21 (15 (15

110 49 1100 2000

r r + r

9.4 4.4 78 190

(23 ) (23 ) (8) (23 )

110 + 55 + 1100 & 980 +

6.0 5.1 130 80’”

(18 ) (18) (5 ) (9 )

70 + 2.8

(15 )

5.3 + 0.51 (10 )

4.2 + 0.22

(10 )

0.22 0.55 0.96 0.33 0.62

Liver tissue lipids @moVg wet weight) Free fatty acids Triglycerides Phospholipids Cholesterol Cholesterol ester

3.1 6.6 33 3.3 0.42

Data are means + SE. (number of experiments).

) ) ) )

76 + 3.5 (5 )

Serum lipids (mM) Free fatty acids Triglycerides Phospholipids Cholesterol Cholesterol ester

Triton WR 1339-induced increase in serum TG concentration (jcmol/min per I)

TAA

Control

(11) * 0.012 f 0.037 + 0.10 2 0.027 f 0.072

0.17 0.38 1.2 0.45 0.74

(11) + rt + + r

(11)

0.25 0.48 2.4 0.29 0.033

35 + 4

(11) + 0.026 + 0.040’” * 0.19 -+ 0.082 rf: 0.11

4.0 3.8 29 4.8 0.58

(6 )

+ + ? + t

0.73 0.46* 2.7 0.15 0.059

18 + 3

(6 )*

*p < 0.05 vs. control.

Results Characterization of control and TAA-treated animals

Several parameters were measured to characterize control and TAA-treated animals (see Table 1). The most important findings are the significant reduction of serum and hepatic TG concentrations and the lowered rate of hepatic TG secretion in cirrhotic rats when compared with the controls.

conditions, the latter effect being more pronounced in cells of cirrhotic livers. The rates of TG-FA and PL-FA secretion did not change significantly in spite of the 6-fold rise of initial medium FFA concentration and the increased cellular FFA uptake. It should be noted, however, that the output of TG-FA and PL-FA from parenchymal cells of cirrhotic livers into the medium was reduced by about 50% in comparison with the controls.

Palmitate utilisation

TABLE 2

Fig. 1 shows that within the concentration range of medium FFA applied the cellular FFA uptake increased almost linearly and equally into cells isolated both from normal and cirrhotic livers during a 90-min incubation. In spite of the enhanced FFA uptake, the cellular FFA content was never increased. At higher initial concentrations even reduced cellular concentrations of FFA were observed at the end of the incubation. However, increasing initial concentrations of medium FFA augmented the accumulation of TG-FA within the cells, whereas the intracellular content of PL-FA decreased under the same

Ketone body production normal and TAA-treated

B-Hydroxybutyrate Acetoacetate Total ketone bodies /3-Hydroxybutyrate iacetoacetate

in parenchymal rats

liver cells prepared

from

Control

TAA

42.2 + 7.3 37.7 f 7.2 80.1 + 7.6

95.5 If: 14.6* 35.5 i 3.7 131.1 rf: 10.2*

1.1 2.7* ___Cells (45.10b) were incubated for 1 h in 30 .nl of Eagle’s h4EM-medium supplemented with 0.5 mM palmitate. Data represent means + S.E. from eight separate experiments. Values are given as nmol/106 cells per h. *p < 0.05 vs. control.

FAnY

ACID METABOLISM

IN LIVER

0

60 Time

CIRRI-IOSIS

120

180

13

240

0

60

of mcubatlon [min]

Time

120

180

of lncubatlon

240

[min]

Fig. 2. Time-course of lipid concentrations in liver parenchymal cells isolated from normal and TAA-treated rats (A) and in the incubation medium (B) during 4 h of incubation with 0.5 mM initial palmitate concentration. Data are means + S.E. from three to six separate experiments. *p < 0.05 vs. control. control: 0. TAA. Chol, cholesterol; CholE, cholesterol ester; other abbreviations, see Fig. 1.

In separate experiments it could be shown that at least 90% of esterified FA accumulated in the medium floated in the VLDL density range (data not #own). The FFA uptake, intracellular esterification and lipid secretion during a 4-h incubation period in the presence of an initial palmitate concentration of 0.5 mM are given in Fig. 2. Parenchymal cells isolated from cirrhotic rat livers had a reduced cellular TG content when compared with cells isolated from normal livers (Fig. 2 and Table 1). It should be mentioned that during the 4-h incubation period the TG content of cells isolated from cirrhotic livers was approximately doubled and nearly reached the concentration measured in the controls. Under the same conditions the cellular PL content declined in both groups. The cellular contents of FFA, cholesterol and cholesterol esters were identical in both preparations and did not change throughout the four-hour incubation period. The uptake of FFA from the incubation medium did not differ significantly between cells obtained either from normal or cirrhotic livers (Fig. 2B). However, the output of TG-FA and PL-FA from cells of cirrhotic livers into the incubation medium was significantly lower in comparison with the controls, whereas there was no difference with respect to cholesterol and cholesterol esters.

Uetone body producrion

In the presence of 0.5 mM palmitate the total ketone body production of cells prepared from cirrhotic livers increased to about 105% when compared with the controls. In detail, the data given in Table 2 show that the production of /S-hydroxybutyrate was elevated to about 230% in cells isolated from cirrhotic livers, whereas no difference was found with respect to acetoacetate formation. T~Js, after 1 h of incubation, the ratio /3-hydroxybutyrate to acetoacetate formation was significantiy in-

Cell% +

e

45

0 Time

135

of !ncubatron [min]

Fig. 3. Incorporation of ‘H from -‘Hz0 into total fatty acids in suspensions of liver parenchymal cells prepared from normal and TAA-treated rats. Data are means + S.E. from three to six sepaand 0, cells plus merate exoeriments. *p < 0.05 vs. control. and L1. medium. Filled symbols, controls: and q, cells; dium: open symbols. TAA.

14 creased (2.7 vs. 1.1) in suspensions containing cells isolated from cirrhotic livers in comparison with those obtained from controls. De novo FA synthesis Cells both from normal and cirrhotic rat livers incor-

porated 3H from 3H,0 into the total FA fraction of cell pellet plus medium at nearly constant rates, and the absolute values did not differ significantly between the controls and the experimental group (Fig. 3). The incorporation of 3H into the intracellular TG-FA, however, was significantly reduced from 18 + 1.6 nmol/106 cells per h in the controls (n = 6) to 9.4 f 0.78 nmoY106 cells per h in the experimental group (n = 6; p < 0.05). whereas incorporation into PL-FA was significantly elevated from 5.1 & 0.45 (n = 6) to 11.0 +_ 0.96 nmol/106 cells per h (n = 6; p < 0.05), respectively. Furthermore, the total amount of 3H-labeled newly synthesized FA delivered into the incubation medium as esterified FA (mainly TG-FA and PL-FA) was significantly reduced to 73% in cell suspensions prepared from cirrhotic livers. While the secretion of TG esterified with newly synthesized FA was lowered by 50%, PL-FA was increased to 150%. De novo synthesis of cholesterol

Under our experimental conditions the incorporation of 3H from 3H,0 into total cholesterol backbone amounted to 29.2 k 5.1 (n = 6) and 17.6 f 1.8 dpm/ incubation per h (n = 6; p < 0.05) in cells from normal and cirrhotic livers, respectively. This is equivalent to rates of cholesterol de nova synthesis of about 140 2 17 and 70 k 7.0 nmol total cholesterol/g cells per h in normal and TAA-treated animals, respectively.

Discussioa It is gen,erally accepted that the hepatocellular uptake of long-chain FFA is directly related to its concentration in serum. Until now, however, it was not clear whether the cellular processes of FFA uptake were impaired in cirrhosis. Our experimental data show that the uptake of FFA per cell is directly correlated with the palmitate concentration in the incubation medium up to unphysiologically high values in parenchymal cells of both normal and cirrhotic livers. Furthermore, the rates of FFA uptake are similar to or higher than those obtained in vivo in normal perfused rat livers (20) and in isolated normal rat liver parenchymal cells (21). Experiments with increasing amounts of medium FFA could be performed by keeping either the concentration

T. ZIMMERMANN

et

a;.

of albumin or the molar palmitate/albumin ratio constant. We decided to use the latter approach, since only identical FFA binding sites of albumin were involved in spite of increasing FFA concentrations. The protein concentration of serum from rats treated with T.4A for 3 months did not differ significantly froin controls. When 1.5 mM palmitate was applied, the albumin concentration of the medium nearly reached serum values. The decreased albumin and increased FFA serum concentrations found in more advanced stages of human cirrhosis lead to higher palmitatefalbumin ratios, thus causing occupation of additional FFA binding sites having lower affinity. Further experiments are needed to test whether involvement of these sites favours cellular FFA uptake. Our data show that in experimental cirrhosis parenchymal cells exhibited a great capacity for FFA utilization since, in spite of greatly enhanced rates of FFA uptake, no intracellular accumulation of FFA occurred. However, neither cells from normal or cirrhotic livers could deliver increasing amounts of esterified FA into the medium when the medium concentration of FFA was elevated to 1.5 mM. Metabolic studies in normal livers (22,23) yielded similar results. It was concluded that with increasing rates of TG synthesis following enhanced FFA uptake, the liver had an upper limit for TG secretion. Obviously, under our experimental conditions this limit was reached at 0.5 mM palmitate as seen in the high rates of TG secretion observed even at low FFA concentrations. It should be added that in cells of cirrhotic rats the rates of TG-FA and PL-FA output into the medium were reduced by about 50% at all medium FFA concentrations used. Therefore, the increasing amount of FFA taken up must be channelled into cellular FA accumulation or into oxidation. Indeed, the bulk of FFA additionally taken up by the cells at higher palmitate concentrations accumulated as TG-FA. Several years ago, it was shown that the esterification of long-chain FA into liver TG was not saturable in normal parenchymal cells (24). Thus, a great reserve capacity for FA esterification exists both in cells from normal and cirrhotic livers. On the other hand, the cellular content of PL-FA tended to decrease. This effect was slightly but not always significantly higher in cells of cirrhotic animals. Our data obtained with isolated parenchymal cells from cirrhotic rat livers confirm and extend studies of patients suffering from cirrhosis (7,25-27). These studies used catheter techniques for measuring splanchnic arteriovenous differences and concluded that liver TG but not ketone body output was decreased in cirrhotic patients. The shift of FFA towards ketone body formation rather than TG secretion was attributed to underinsulin-

FATTY ACID METABOLISM

IN LIVER

CIRRHOSIS

isation related to reduced effective hepatic blood flow (28). Our data clearly show that in cells isolated from cirrhotic livers ketone body production was increased, whereas the hepatoccllular secretion of esterified FA wzs markedly reduced. Thus, the reciprocal relationship between the rates of hepatic ketogenesis and TG secretion which can be found

under

various

conditions

(29) was

in vitro conditions. Until now, de novo synthesis of FA in human cirrhosis has not been investigated. Our data provide evidence that in experimental rat cirrhosis hepatocellular synthesis of FA is not changed. However, the secretion of TG esterified from newly synthesized FA is significantly diminished. This finding corresponds to the general reduction of TG secretion from the parenchymal liver cells of cirrhotic animals. On the other hand, the PL-FA turnover of newly synthesized and added FA is markedly enhanced. It is worth mentioning that the FA patterns of also observed

in experimental

cirrhosis

under

eferences 1 McIntyre N, Harry DS, Owen JS. Lipoprotein disorders in liver disease. In: Calandra S, Carulli N, Salvioli G, eds. Liver and Lipid Metabolism. Amsterdam: Elsevicr, 1984: 51-9. 2 Cooper AD. Ro:; of ibe liver in the degradation of lipoprotein. Gastroenterology 1985: 88: 192-206. 3 Harry DS, Owen JS, McIntyre N. Plasma lipoproteins and the liver. In: Wright R, Millward-Sadler GH, Alberti KGMM, Karran S, eds. Liver and Bili;ir-y Disease - Pathophysiology. Diagnosis, Management Lond.Lyn: Bailliere Tindall, 1985; 6.5-86. 4 Holm E, Tschepe A, Steadt U. Leweling H, Gladisch R. Bassler KH. Fatty acid and ketone body metabolism in hepatic failure. In: Advances in Hepatic Encephalopathy and Urea Cycle Diseases. Base]: Karger. 1984; 645-66. 5 Sandhofer F. Sailer F, Braunsteiner H. Fettslure- und Triglyzeridumsltze bei Patienten mit Leberzirrhose. Wiener Klin Wochenschr 1966: 78: 731-3. 6 Ducasse MCR, Bahnsen M. McCullogh AJ, James OFM. Is ketogenesis impaired in patients with hep?tic cirrhosis? Clin Sci 1983: 64: 68-72. 7 Nosadini R, Avogaro A, Mollo F. et al. Carbohydrate and lipid metabolism in cirrhosis. Evidence that hepatic uptake of gluconeogenic precursors and of free fatty acids depends on effective hepatic flow. J Clin Endocrinol Metabol 1984; 58: 1125-32. 8 Riggio 0, Merli M. Cantefora A, et al. Total and individual free fatty acid concentrations in liver cirrhosis. Metabolism 1984: 33: 646-51. 9 Zimmermann T. Franke H. Dargel R. Isolation and characterization of parenchymal cells from normal and cirrhotic rat liver. Cell Biochem Function 1987; 5: 47-54. 1C Zimmermann T, Franke H. Dargel R. Studies on lipid and lipoprotein metabolism in rat liver cirrhosis induced by different regimens of thioacemmide administration. Exp Pathol 1986; 30: 109-17. 11 Zimmermann T, Miiller A, Machnik G. Franke H. Schubert H. Dargel R. Biochemical and morphological studies on production and regression of experimental liver cirrhosis induced by thioacetamide in Uje: WIST rats. Z Versuchstierkd 1987; 30: 16% 80. 12 Brodehl J. Thioacetamid in der experimentellen Leberforschung. Kiin Wochenschr 1961: 39: 956-62.

1.5

mitochondrial and microsomal PL from liver cells of TAA-pretreated animals showed a marked change (30,31). In summary, this report presents evidence that parenchymal cells isolated from experimental micronodular cirrhotic rat livers are a useful tool for studying metabolic processes. The data reveal differences in the FA and cholesterol metabolism between parenchymal cells of normal and cirrhotic livers, thus supporting our view that cirrhosis leads to alterations cf intrinsic hepatocellular properties. These changes can be observed under experimental conditions where microcirculation and shortages of either substrates or oxygen do not play a role.

The authors wish to thank Marita Wunder, Christa Fritzlar and Marlies Kahn for expert technical assistance.

13 Wohlgemuth B. Experiment
16 Invest 1983; 72: 1821-32. 27 Owen OE. Mozzoli MA, Reichle FA, et al. Hepatic and renal metabolism before and after portasystemic shunts in patients with cirrhosis. J Clin Invest 1985; 76: 1209-17. 28 McGarry JD, Wright PH, Foster DW. Hormonal control of ketogenesis. J Clin Invest 1975; 55: 1202-9. 29 Ide T. Ontko JA. Increased secretion of very low density lipoprotein triglyceride following inhibition of long chain fatty

T. ZIMMERMANN

et al.

acid oxidation in isolated rat liver. J Biol Chem 1981; 256: 10247-55. 30 Mijller B. Dargel R. Structural and functional impairment of mitochondria from rat livers chronically injured by thioacetamide. Acta Pharmacoi Toxicol 1984: 55: 126-32. 31 Moller D. Sommer M. Kretzschmar M. et al. Lipid peroxidation in thioacetamide-induced maronodular liver cirrhosis. Arch Toxicol 1991; 65: 199-204.