Vol. 126, No. 3. 1985 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

February 15, 1985 Pages 1146-1153

LOSS OF GLUCAGON CONTROL OF GLUCONEOGENESIS IN LIVER CELLS FROM RATS WITH BILE DUCT OBSPRUCTION*

Jiirgen Scholmerich** Ulrich Baumgartner'

Maria-Sybille Becher, and Wolfgang Gerok

Department of Internal Medicine, University of Freiburg Freihurg, West Germany

Received December 20, 1984

Bile acids induce membrane alterations including reduced

response to peptide hormones in vitro. Isolated liver cells from rats with bile duct obstruction were studied regarding

gluconeogenesis and its hormonal control. While cells from shamoperated animals showed an 63% increase of glucose re-

lease in the presence of glucagon (1 PM), cells from cholestatic livers did not response regardless of the dura- tion of obstruction. Cholestatic cells also showed other

signs of membrane alterations, such as an increased enzyme

leakage while redoxstatus and other metabolic responses

were unchanged. These results suggest that a loss of hormonal control in the liver could contribute to disturbations of

glucose homeostasis in cholestatic conditions. 0 1985 Academic Press, Inc.

Bile salts (BS) reduce the susceptibility of isolated

rat liver cells to peptide hormones in vitro. While dihy-

droxylated bile salts are effective only at concentrations

ahove 500 pM, taurolithocholate, a monohydroxy BS with

cholestatic properties, alters response at severalfold

*This work was supported by the Deutsche Forschungsgemeinschaft (SFB 154: Klinische und experimentelle Hepatologie, Freiburg).

**Address correspondence to: Dr. J. Schglmerich, Medizinische Universitstsklinik, D-7800 Freiburg, West Germany

+Present address: Department of Pathology, University of California at San Diego, La Jolla, CA 92093 USA

0006-291X/85 $1.50 Copyright 0 I985 by Academic Press. Inc. All rights qf reproduction in any form reserved. 1146

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lower concentrations (1). In addition, it has been shown

that BS reduce the concentration of insulin and glucagon

receptors in blood monocytes (2). This effect may be due

to alterations of membrane fluidity and destruction of sub-

memhraneous structures by BS which have been described in

several models (3,4,5,6,7). It is, however, unknown if this

loss of hormonal control has a role in cholestasis in vivo.

Recently, the isolation of viable cells from the liver of

cholestatic rats has been described (8). In order to elucidate

a possible alteration of hormone response in cholestasis, we

studied isolated cells from rats 1 - 6 days after ligation of

the common bile duct regarding their response to glucagon using

gluconeogenesis as parameter. In addition, several parameters

of cell function and membrane leakage were studied.

MATERIALS AND METHODS

Materials:

Female Sprague-Dawley rats (weight 239 2 35 g) from

Ivanovas (Kisslegg, Allgau, West Germany) fed a standard diet (AltrominR, Altrogge, Lage, West Germany) were used.

Collagenase was obtained from Roth KG (Karlsruhe, West Germany), biochemical substrates and enzymes were purchased

from Boehringer (Mannheim, West Germany). All chemicals used

for huffers and media were obtained in analytical purity from Merck (Darmstadt, West Germany). Glucagon was from

Serva (Heidelberg, West Germany).

Methods:

Cholestasis: After an overnight fast the common bile duct was ligated atraumatically after median laparotomy.

Wounds were cautiously closed and the animals held in single cages with free access to food and water. Weight was monitored

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until experimentation. Shamoperated controls were treated

identically with the exception that the ligature was not

tightened.

Isolation and incubation of cells: After a 24 h fast cells

were isolated according to Bojar et al. (9). Viability testing,

cell counting and incubation were essentially done as described previously (1). Three different groups of three incubations

each were done with cells from each animal in Krebs-Ringer- Bicarbonate buffer (pH 7.4, 37’ C, gassed with Carbogen) for

I hour: Either alanine (10 mM) alone; or alanine (10 mM), lac-

tate (5.5 m), and pyruvate (1.1 mM); or alanine (IO mM) and

glucagon (1 p) were added from the beginning to the incubation medium. Incubation was stopped after 1 hour by rapid centri-

fugation (30 x g, I min) at 4O C. Supernatants and pellets were collected and used for analysis.

Assays: The activities of lactate dehydrogenase (LDH), glutamate oxalacetate transferase (GOT) and 5-nucleotidase

(5-Nucl) were measured in untreated supernatants and homo-

genized pellets according to published methods (10). Concen- trations of urea, glucose, lactate and pyruvate were determined in deproteinized supernatants as described (10). Activities and

concentrations were calculated per g dry weight cells and per IO6 viable cells.

RESULTS

A similar weight decrease occured in both groups of rats.

The yield of viable cells isolated from the liver of chole-

static rats decreased with duration of cholestasis (C) (mean

4.0 + 2.5 x 106 cells) and was allways lower than that ob-

tained from shamoperated controls (S) (mean 22.7 + 9.1 x IO6

cells). The average lactate / pyruvate ratio in the incubation

medium was not different between both groups (C: 5.8 _+ 1.3;

S: 5.4 f 0.5). The release of GOT and LDH from the cells during

incubation increased with longer duration of cholestasis and

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was higher in the cholestatic cells (C: GOT: 4.8 t 3*1 U/IO6

cells, LDH: 28.8 ? 18.3 U/IO6 cells; S: GOT: 0.9 2 0.3, LDH:

13.9 ,+ 5.6). A'larger amount of 5-Nucl was released by the

cholestatic cells (C: 90 2 30 mu/IO6 cells; S: 20 2 10).

Lactate and pyruvate addition to the incubation medium resul-

ted in an increase of glucose release of 56% in cells from

cholestatic animals and of 22% in controls while basic glu-

cose release was lower in the cholestatic than in the sham-

operated group (C: 3.0 2 0.7 pmoles/106 cells/h; S: 4.1 f 1.5).

Glucagon addition did never result in an increase of glu-

cose release in cells from cholestatic livers regardless of

the duration of cholestasis (figure 1). In contrast, cells

from shamoperated rats showed a 63% increase (figure 2).

Only the animal studied 24 h after surgery presented with

an impairment of glucagon response.

Figure 1. Glucose release from rat liver cells isolated 1 - 6 days after bile duct ligatioll in the pre-

sence of alanine (IO mK) ( 1; alalline

(10 mM), lactate (5.5 mM), and pgruvate (1.1 mM)

(I ); and alanine (IO mM) and glucagon (1 PM)

(B>. Each bar represents mean 2 SD of three

incubations.

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LAC GLUC LAC GLUC

chdestosis shomoperated

Figure 2. Glucose release from cells isolated from chole- static and shamoperated rats in the presence of alanine (10 mM) (ALA); lactate (5.5 mM) and pyruvate (1.1 mM) (LAC); and glucagon (1 PM) (GLUC). Each bar represents mean 2 SD of 18

(cholestasis) or 6 (shamoperated) incubations.

DISCUSSION

Stimulation of gluconeogenesis is preserved in viable

isolated liver cells from fasted rats (11,12). Concentration

(2) or affinity (13) of membrane receptors can be modified

by alterations of membrane fluidity which, furthermore, is

changed by BS (6,7,14). This effect may be due to a changed

cholesterol/phospholipid ratio in the membrane (3,15,16,17).

Accordingly it has been shown that BS alter hormone-receptor

interaction (2), thus leading to a loss of hormonal control

in isolated liver cells from normal rats in the presence of

pathological BS concentrations (1). The data presented in

this study suggest that this loss occurs also in cells from

cholestatic livers, i.e. in vivo.

These cells are viable using the commonly accepted cri-

teria (18). However, the yield is much lower than in normal

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livers. This is probably due to an increased destruction of

predamaged cells during the isolation procedure which is

indicated by a large amount of cytoplasmic enzymes in the

washing solutions during isolation. In addition, the cells

show an increased enzyme loss compared to controls during

incubation which suggests that the remaining cells have also

some kind of sublethal membrane damage. Basic synthetic func-

tions such as glucose production from alanine are reduced.

This could be due to postoperative stress leading to glyco-

gen depletion. In contrast, redox state and synthetic activity

in response to substrate supply are well preserved.

Several possible explanations for the missing response

to glucagon in the cholestatic cells have to be considered.

A decreased food intake is unlikely since the weight loss

was identical in both groups. Glycogen depletion due to

stress induced alterations of metabolism is possible. How-

ever, it has been shown, that glucagon produces gluconeo-

genesis from amino acids rather than glycogenolysis in iso-

lated liver cells from fasted rats (19). A direct inter-

action of BS with the receptor is unlikely due to the

finding that affinity of hormones to the receptor is un-

changed by BS (2). Since the metabolic response to suh-

strate supply is also preserved we do not believe that

intrinsic metabolic impairments are the reason for the

missing hormonal response. Thus, the known membrane alterations

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with a change of lipid composition (3,16,17,21), fluidity

(6,7,15), transport ability (1,8,17) and enzyme activity

such as Na+- ,K+-ATP-ase (5,2l), may also include a reduced

concentration or an impaired function or internalisation

(14) of membrane receptors for peptide hormones. This seems

the most likely explanation at least for the alterations

found after 48 h obstruction, while those found in the

initial phase may indeed be due to postoperative stress as

indicated by a missing response in the controls in this early

period.

We conclude from our results that the impairment of

hormonal control found in isolated cells from normal rat

livers in the presence of cholestatic BS (I) occurs in vivo

as well and, thus, may have a role in the disturbance of the

metabolism of glucose and other substrates in cholestatic

conditions.

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