Organ culture of Amphiuma means liver: Control of glycogen content

Camp. Biochem. Physic&, 1974, Vol. 47A, pp. 567 to 572. Pergamon Press. Printed in Great Bn’tain

ORGAN CULTURE OF AMPHIUMA MEANS LIVER: CONTROL OF GLYCOGEN CONTENT MARJORIE

A. ~ONNICKENDA~,

D. BROWN

and M. BALLS

School of Biological Sciences, University of East Anglia, Norwich NOR 88C (Received 14 March 1973)

Abstract-l.

Amphiuma means liver fragments retained their glycogen for at least 35 days in organ culture. 2. Glycogen levels fell when adrenalin was added, then increased when insulin and/or extra glucose were added to the culture medium.

INTRODUCTION

LIVERfragments from Amphiuma means (Congo eel) survive particularly well in organ culture. We have already shown that fragments retain their normal histology for several weeks in a range of media, and that hepatocyte proliferation rates are low (Monnickendam & Balls, 1972). Electron microscope studies show large numbers of glycogen granules in hepatocytes after 35 days in vitro (Monnickendam & Balls, 1973a). In the experiments described here, we measured the glycogen content of liver fragments in organ cultures, compared different media and looked at the effects of insulin and adrenalin on glycogen levels. MATERIALS

AND

METHODS

1. Animals Adult A. means were supplied by Carolina Biological Supply Company, Burlington, North Carolina, U.S.A., were kept at 18-23°C and fed on chopped beef liver and heart. 2. Culture technipue and media Liver fragments were cultured as described previously (Monnickendam & Balls, 1972); each flask contained ten fragments with a mean total weight of 60 mg. The media used were : (a) MEM--50% Minimal Essential Medium with Earle’s salts (Auto-Pow, Flow Laboratories, Irvine, Ayrshire), 10 mM L-glutamine (Flow Laboratories), 15 mM HEPES (Hopkin & Williams, Chadwell Heath, Essex), 10% foetal bovine serum (Flow Laboratories), 40% double distilled deionized water, 100 i.u./ml Benzylpenicillin (Glaxo, Greenford, Middlesex), 100 pg/ml Streptomycin sulphate (Glaxo), 2 &ml Fungizone (Squibb, New York). The pH was adjusted by the addition of 1 N NaOH; (b) MEM + G-as (a) with l-5 g glucose per 1. extra, giving a final concentration of 2 g glucose per 1.; (c) LlS--50% Leibovitz L15 medium without NaHCO, (Flow Laboratories), 10% foetal bovine serum, 40% double distilled deionized water, plus antibiotics as in (a). The culture medium was changed once a week. The culture temperature was 25°C. 567

568

~ARJORI~A.~ONN~CK~DAM,

D.BROWN

and M. BALLS

3. Hor?noTles The hormones used were adrenalin solution B.P. (equivalent to 0.1% w/v adrenalin5 x 10V3M) and insulin B.P. (A. B. Insulin Ltd., 80 units/ml). 4. Assays The optimal adrenalin concentration was determined using fresh A. means liver fragments with a total weight of 60 mg, which were incubated for 30 min at 25°C in 10 ml of 50% Dulbecco’s phosphate buffered saline before the addition of adrenalin. Samples (O-2 ml) of saline were taken immediately before the addition of adrenalin and 1, 2, 3 and 4 hr after addition. These were assayed for glucose using glucose oxidase/peroxidase [PGO enzymes, Sigma (London) Chemical Co. Ltd., Kingston-upon-Thames, Surrey]. Results are given as the rate of glucose release (mg glucose/hr per g tissue) relative to the control rate. The glycogen content was determined as follows : liver fragments were blotted, weighed and frozen at - 2O”C, homogenized in 0.03 N HCl and heated for 10 min at 100°C to disaggregate the glycogen. The pH was adjusted to 4.5 with 0.5 N NaOH and the glucose content of a 0*5-ml sample measured (initial glucose level). A further sample was treated with 5 mg/ml amylo a1,4-n1,6 glucosidase [Boehringer (London) Ltd., Ealing, London] for 30 min to hydrolyse the glycogen to glucose. The glucose content of the hydrolysate was also measured (final glucose level) using the hexokinase/glucose-6-phosphate dehydrogenase method (Slein, 1965), with final concentrations of 5 mgirnl hexokinase (Boehringer) and 0.7 mg/ml glucose-6-phosp~te dehydrogenase (Sigma). The glycogen content was obtained from the difference between initial and final glucose levels, using a calibration curve of rabbit liver glycogen (O-4 mg/ml, Sigma). Results are given as percentage of the wet weight of tissue. 5. Statistics Results are given as the arithmetic mean+ standard error of the mean. The mean values of different groups were compared using the Student t-test with a modification for comparing small samples (Bailey, 1959). EXPERIME~S

AND RESULTS

Liver was cultured in L15 or MEM and assayed for glycogen after 1, 3 and 5 weeks. The mean values of duplicate samples were 1.34, 0.54 and 0.66 per cent respectively for L15, and l-41, 1.85 and l-60 per cent for MEM. Since a higher levelof glycogenwas maintained in MEM, it was used in all subsequent experiments.

2. Efiect

ofadrenalin

on fresh liwer

This experiment was carried out to determine the adrenalin concentration giving the most rapid rate of glucose release. The results are given in Fig. 1. It can be seen that glucose was most rapidly released at 5 x low5 M and 5 x 10G M. Adrenalin has a smaller stimulator-y effect at 5 x 10~” M; below thii concentration it had no effect. One hr after the addition of 10-b M adrenalin or more, there was no detectable glucose in the saline solution, although glucose was present at the time of adrenalin addition. After this, glucose was again released at the same rate as in controls. We do not know whether this inhibition is due to adrenalin or to the other components of adrenalin solution B.P.

ORGAN CULTURE OF AMPHIUMA

MEANS

569

LIVER

.

4-

\

.

33 I

B t

l

% 8 2 0

2-

I-

l

I :-•

\./*\*/*

I ,()-‘I

I ,()*I0

I to-9

I lo+

I

I

I

I

I

10-T 10-S 10-S 164 10-S

Adrenalin concentration,

M

FIG. 1. Effect of adrenalin on rate of glucose release from fresh A. meu~tsliver. Abcissa : adrenalin concentration; ordinate : experimental rate,kontrol raie.

3. Preliminary experiments using cultured Iiver

When day 7 liver fragments were transferred into saline containing 3 x IO-“iM adrenalin, there was a threefold increase in the rate of glucose release compared with controls in saline. Thus the cultured liver fragments responded in the same way as fresh tissue. Adrenalin (5 x 10M6M) was also added to day 19 fragments in MEM and the glycogen content measured after 6 and 24 hr, to show that glycogen was being degraded. Since MEM contains glucose (O-5 g/l.), the increase in glucose content of the medium was too small to measure. The mean glycogen content of the pair of control cultures was 2.7 per cent, the 6-hr cultures 2.3 per cent and the 24hr cultures 1.1 per cent. To find out whether adrenalin-treated cultures could recover and synthesize more glycogen, and to compare different insulin levels, adrenalin (5 x 10-s M) was added to six cultures on day 21. Twenty-four hr later, two cultures were changed into fresh MEM, two into MEM containing 0.1 units insulin per ml and two into MEM containing O-01 units insulin/ml. The cultures were frozen 28 hr later. The mean glycogen content was 1.1 per cent for the control pair of cultures, 1.3 per cent for those given 0~1 units/ml and l-8 per cent for those given O-01 units/ml. Thus, adren~in-treated cultures did not synthesize any glycogen when transferred to MEM, but when insulin was added there was a significant increase in glycogen levels, the increase being greater at the lower insulin concentration. 4. The effects

of adrenalin,

insulin and glucose on glycogen levels in vitro

In this experiment, fifty cultures were used. On culture day 26, 5 x 10~~ M adrenalin was added to half the cultures. Twenty-four hr later, five adrenalintreated and five control cultures were weighed and frozen. All the other cultures were transferred to clean flasks containing fresh media, five from each group into

570

MARJORIEA. MONNICKENDAM, D. BROWN AND M. BALLS

1’

’

1

I

A

-

I

AG

1

AI

I

AGI 29

2

I

I

I

GI Treatmrnt

Wtun

day

FIG. 2. Effects of adrenalin, insulin and extra glucose on glycogen content of cultured A. means liver (mean& standard error). C, Control cultures; A, plus adrenalin (5 x lo-& M); I, plus insulin (0.01 units/ml); G, plus added glucose (final concentration, 2 mg/ml).

MEN, MEM + G, MEM + insulin (@Ol units/ml) and MEM + G + insulin (0.01 units/ml). These cultures were incubated for a further 48 hr before being weighed and frozen. Five cultures were assayed in each treatment group. The results are given in Fig. 2. It can be seen that 24 hr adrenalin treatment caused a significant decrease in glycogen content (O-01
The experiments described here show that A. mans liver organ cultures maintain their functional normality for far longer than has been observed in mammalian liver organ cultures (Campbell & Hales, 1971), probably because

ORGAN

CULTURE

OF

AMPHIUMA

MEANS

LIVER

571

Amphiuma tissues have lower respiration rates, oxygen is more soluble at the lower culture temperature and amphibians can tolerate wider variations in their internal environment (Monnickendam & Balls, 1973b). Glycogen is still present in day 35 cultures, and levels are higher in MEM than in L15. Glycogen is degraded when adrenalin is added, and is synthesized when insulin or extra glucose are added. This shows that the enzyme and membrane systems controlling glycogen levels are retained irz vitro and can still respond in a normal integrated manner to hormones. Frieden & Lipner (1971) have said that in vitro hormone effects have not been successfully related to in viva hormone effects, because the hormone concentrations needed were usually much greater (up to lo5 times) than those found in z&o. This criticism does not apply to our experiments. Assuming that the concentrations of insulin and adrenalin are the same in tissue and medium, an insulin concentration of 0.01 units/ml in vitro gives a tissue level equivalent to 0.6 units/kg, and an adrenalin level of 5 x 10e6 M gives a tissue level equivalent to 6 pg/kg. No in wivo dose levels for A. means have been published, but Wurster & Miller (1960) found that 10 units/kg insulin induced hypoglycaemia in Taricha torosa (Urodela) and adrenalin caused hyperglycaemia (dose not given). Frieden & Lipner (1971) indicated that an injection of O-2 mg/kg adrenalin induced a rise in blood glucose and a fall in liver glycogen in rats. Amphibian liver organ cultures have mainly been used for looking at problems in amphibian biology, such as the role of thyroxine in inducing urea cycle enzymes, and oestrogen and gonadotrophin in inducing vitellogenin synthesis (see Monnickendam & Balls, 1973a). Liver cultures from Discoglossus picta and Pleurodeles waltlii tadpoles could accumulate glycogen prematurely, when glucose was present in the medium (Beaumont, 1956; Bellec, 1957). There are few reported measurements of glycogen content in mammalian liver organ cultures. Campbell & Hales (1971) found that the glycogen content of liver fragments from 5- to 6-week-old rats fell rapidly. After 24 hr in vitro there was no glycogen, even when the fragments were incubated in 3 atmospheres of oxygen with 5% carbon dioxide. Monder & Coufalik (1972), comparing liver from 20 _+1 day rat foetuses and adults, found that adult liver lost more than 50 per cent of its glycogen within 1 hr. Foetal liver, with an initial glycogen content of 5 per cent of wet weight, lost 90 per cent of its glycogen within 22 hr, and at 44 hr the glycogen content was 0.02 per cent. When cortisol was present in the medium, the glycogen content at 44 hr was O-3 per cent. Cortisol stimulated the synthesis of glycogen added to 42-hr liver cultures, but the increase was very small compared with the amount which had been lost. There was a bigger increase in glucose-supplemented media, but only when cortisol was present. In our experiments, the glycogen level in adrenalin-treated cultures increased significantly in glucose-supplemented medium without added insulin. These experiments also confirm the prediction (Monnickendam & Balls, 1973a) that HEPES-buffered media should be as suitable as L15 for long-term amphibian organ culture. We are currently comparing other functions in liver fragments cultured in the two media.

MARJORIEA. MONNICKENDAM, D. BROWNANDM. BALLS

572

SUMMARY

Fragments of A. meuns liver were cultured at 25°C in MEM or L15 for up to 35 days. Glycogen levels were higher in MEM, which was used in all subsequent experiments. The addition of adrenalin (5 x 10” M) to liver cultures caused glucose release and a reduction in glycogen content. The addition of insulin (O-01 units/ml) and/or glucose (increasing the medium concentration from 05 to 2 mg/l.) induced further glycogen synthesis. Achnowledgements-This work was supported by the Medical Research Council (M. A. M. and M. B.) and the Science Research Council (D. B.) of the United Kingdom. REFERENCES BAILEY N. T. J. (1959) Statistical Methods in Biology. English Universities Press, London. BEAUMONTA. (1956) Sur la culture in oitro de foie larvaire d’amphibien: apparition du glycogene en milieu synthetique. C. Y. hebd. Skanc. Acad. Sci., Paris 243, 676-677. BELLEC A. (1957) Apparition du glycogene en culture in witro de foie larvaire de Pleurodeles. C. Y. S&anc. Sot. Biol., Paris 151, 1353-1355. CAMPBELLA. K. & HALEY C. N. (1971) Maintenance of viable cells in organ culture of mature rat liver. Expl Cell Res. 68, 33-42. FRIEDENE. & LIPNER H. (1971) Biochemical Endocrinology of the Vertebrates. Prentice-Hall Englewood Cliffs, New Jersey. MONDERC. & COUFALIKA. (1972) Influence of cortisol on glycogen synthesis and gluconeogenesis in fetal rat liver in organ culture. J. biol. Chem. 247, 3608-3617. MONNICKENDAM M. A. & BALLS M. (1972) The long-term organ culture of tissues from adult Amphiuma, the Congo eel. J. Cell Sci. 11, 799413. MONNICKENDAM M. A. & BALLS M. (1973a) Amphibian organ culture. Experientia 29, l-17. MONNICKENDAM M. A. & BALLS M. (1973b) The relationship between cell sizes, respiration rates and survival of amphibian tissues in long-term organ cultures. Camp. Biochem. Physiol. 44A, 871-880. SLEIN M. W. (1965) D-Glucose determination with hexokinase and glucose-6-phosphate dehydrogenase. In Methods of Enzymatic Analysis (Edited by BERGMEYERH.-U.), pp. 117-123. Academic Press, New York. WURSTERD. H. 8z MILLER M. R. (1960) Studies on the blood glucose and pancreatic islets of the salamander, Taricha torosu. Camp. Biochem. Physiol. 1, 101-109. Key

Word Index-Amphiuma

; organ culture ; liver; glycogen.