Hormonal control of glucose production by Amphiuma means liver in organ culture

GENERAL AND COMPARATIVE ENDOCRINOLOGY 27, 380-388 (1975)

Hormonal Control of Glucose Production by Amphiuma means Liver in Organ Culture

DENNIS BROWN,~ NORMAN FLEMING,~ AND MICHAEL BALLS

School of Biological Sciences, University of East Anglia, Norwich NR4 7TJ, England

Accepted July 7, 1975

The effects of glucagon, insulin, and adrenalin on glucose production by Amphiuma means liver organ cultures maintained at 25” were studied. Glucagon stimulated gluconeogenesis from pyruvate and ah-mine, and adrenalin treatment resulted in a more rapid glycogenolysis than glucagon treatment. Glucagon increased the tissue levels of glutamate- oxaloacetate transaminase (GOT), glutamate-pyruvate transaminase (GPT), and fructose- l&diphosphatase after 40 hr, while insulin decreased GOT and GPT levels after 72 hr. The presence of pyruvate in the medium significantly reduced nitrogenous excretion by the liver fragments. We conclude that cultured A. means liver responds to glucagon, insulin, and adrenalin in ways that are compatible with the known effects of these hormones in mammals.

Since adult mammalian and avian tissues survive for only short periods in organ culture (Trowell, 1959; MacDougall and Coup- land, 1%7), there has been little long- term in vitro work on the responses of such organs as liver, kidney, and pancreas to hormones or other factors. In contrast, we have recently found that fragments of a number of organs from adults of several amphibian species survive particularly well in long-term organ culture, retaining many aspects of normal structure and function (Balls et al., 1975; Fleming et al., 1975). Monnickendam et al. (1974) found that the addition of insulin to cultured liver fragments from Amphiuma means (the Congo eel) caused glycogen synthesis, while adrenalin resulted in glycogenolysis. In the experiments described in this paper, we in- vestigated the effects of insulin and glucagon on gluconeogenesis. Fructose-

1,6-diphosphatase, glutamate-oxalacetate transaminase (GOT), glutamate-pyruvate transaminase (GPT), and glycogen levels in cultured liver fragments were assayed, and the effects of alanine and pyruvate on glucose production and nitrogenous excretion were measured. The relative rates of adrenalin- and glucagon-induced glycogenolysis were compared.

MATERIALS AND METHODS

1. Animals Adult A. means were obtained from Carolina Bio-

logical Supply Company, Burlington, North Carolina, USA, kept at about 20”, and fed on chopped beef liver and heart.

2. Culture Technique and Media Liver fragments were maintained in submerged

organ culture, as previously described by Monnick- endam and Balls (1972). The basic medium was 50%

i Present address: Institut d’aistologie et d’Embryologie, Bcole de Medicine, Universite de Geneve, 1211 Genbve 4, Switzerland.

* Present address: Anatomisches Institut, Universitiit Ziirich, Ziirich, Switzerland. 3 Present address: Department of Human Morphology, The Medical School, University of Not-

tingham, Nottingham NG7 2UH, England.

380 Copyright @ 1975 by Academic Press, Inc. AU rights of reproduction in any form reserved.

HORMONAL CONTROL OF GLUCOSE PRODUCTION 381

Minimum Essential Medium (MEM-Auto-Pow, Flow Laboratories, Irvine, Ayrshire), 5 or 10% foetal bo- vine serum (Flow Laboratories), and 4045% double distilled deionised water, with 100 IU/rnl Benzylpeni- cillin (Glaxo, Greenford, Middlesex), 100 &ml Streptomycin sulphate (Glaxo), and 2 cLg/ml Fungi- zone (Squibb, New York). Tissues were incubated at 25” in all the experiments described below.

3. Glycogen Determination Glycogen levels were measured as described pre-

viously (Monnickendam et al., 1974), amylo al,4-ol,6 glucosidase being used to hydrolyse the glycogen to glucose, which was then determined by the hexokinaseiglucose-6-phosphate dehydrogenase method. Glycogen levels are given as a percentage of the wet weight of the tissue.

4. Glucose Uptake or Release

The glucose content of 0.1 ml samples of medium was measured by the glucose oxidase/perioxidase method (Sigma Chemical Company Ltd., London). Results are expressed as milligrams of glucose taken up or released per gram of wet weight of tissue.

5. Transaminase Activity

Fragments of fresh or cultured liver were blotted, weighed, and homogenised in 0.1% sodium cholate in the ratio 1 g of liver to 50 ml of solution. The ho- mogenate was centrifuged for 10 min at 20,OOOg at 0”. Part of the supematant fluid was diluted by half and its GOT activity determined by a method based on that of Karmen (1955). Oxalacetate produced by incubation of the crude extract with aspartate and oxoglutarate is reduced to malate by malate dehydrogenase at the expense of NADH. The resulting change in OD at 366 nm was used to calculate GOT activity, which was expressed as units per gram of wet weight of liver. One unit is the amount of enzyme which con- verts 1 pmole of substrate per minute at 25”.

GPT activity in a similar dilution of the supernatant fluid was determined by the method of Wroblewski and LaDue (1956). In this case, pyruvate resulting from incubation of the crude extract with alanine and cu-oxoglutarate is reduced by lactate dehydrogenase to lactate at the expense of NADH, and, as for GOT, the change in OD at 366 nm was used to calculate GPT activity. GPT activity was expressed as units per gram of wet weight of tissue.

6. Fructose-1,6-Diphosphatase

The enzyme activity in a crude extract prepared by homogenisation of liver in 0.03 M HCl (approx 30 mg tissue in 2 ml acid) was assayed as described by Pogell and McGilvery (1952). The amount of inorganic phosphate released during incubation of the

extract with fructose diphosphate (purified cyclohex- ammonium salt, Sigma, London) at 37” being measured by the method of Lowry and Lopez (1946). One unit of FDPase is defined as the amount of enzyme which will liberate 1 pmole of inorganic phosphate per hour under the given assay conditions.

7. Nitrogenous Excretion The total nitrogen in the culture medium was as-

sayed as follows: 0.1 ml samples were incubated with 0.1 ml urease suspension (2 mg/ml, Boehringer London Ltd.) and 1 ml distilled water to facilitate mixing. The ammonium ions produced by the hydrol- ysis of urea, together with preformed ammonia (A. lltean~ excrete both ammonia and urea) were then assayed by the method of Fawcett and Scott (1960). Re- sults are given as micromoles NH, released per gram of wet weight of tissue.

8. Hormones The hormones used were adrenalin solution B.P.

(British Pharmacopoeia drug standard) (equivalent to 0.1% w/v adrenalin-5 x 1O-3 M), insulin B.P. (A.B. Insulin Ltd., London: 80 units/ml), and glucagon hydrochloride (Eli Lilly, Basingstoke, England: 1 mglml-2.8 x lOelM).

9. Statistics

When three or more replicates were involved, results are given as the arithmetic mean *‘standard error of the mean. The mean values of groups were compared by using Student’s t test, with a modifica- tion for small sample analysis (Bailey, 1959).

10. Histology

In all experiments on cultured tissue, routine histo- logical examination of the pieces of liver was carried out as described by Monnickendam and Balls (1972). Examples of the appearance of cultured liver are shown in papers by Monmckendam and Balls (1972) and Fleming, Brown, and Balls (1975).

RESULTS

The livers from four Amphiumae were used in the following work, one per experiment .

1. Induction of Glycogenolysis by Glucagon and Adrenalin

The liver used in this experiment had been in culture for 1 wk. The initial glycogen level was 7.7%, and it was hoped that

382 BROWN. FLEMING AND BALLS

Glucose content of mediumlmg/mll

Hours aftw hommm t-t FIG. 1. Effect of glucagon (A - 1.4 X lo-’ M; 0.5

&n-J: A - 1.4 x 1O-8 M; 0.05 &ml) and adrenalin (8 - 5.5 x lo-& M; 1 &ml: q - 5.5 x lo-’ M; 0.1 /.&ml) on glucose release by A. means liver cultures. Each curve represents the response of a single flask of tissue to a single hormone dose, applied at time 0. The results of typical rates of release from individual flasks are presented because when the increase in medium glucose content is measured, the magnitude of the response depends upon the weight of the tissue in any particular flask (O- control rate).

this would lead to a large increase in medium glucose level following hormone- induced glycogenolysis. The rate of glucose release was followed in single flasks after the addition of glucagon or adrenalin. Each flask contained about 40 mg of tissue, and the hormone doses ranged from 10m6 M to lo-l2 M. The results (only doses which produced a marked effect are in- cluded) are shown in Fig. 1.

Adrenalin had a much more rapid effect than glucagon. Sufficient glucose was released in the 24 hr following treatment with 5.5 x lO-‘j M adrenalin to account for the loss of 65% of the tissue glycogen. The lower dose, 5.5 x lo-’ M, caused gly-

cogenolysis at a similar rate for 6 hr, then the rate declined, possibly because the hormone was more rapidly degraded to an ineffective level.

Glucagon (1.4 x lo-’ M and 1.4 X

10ms M) caused a steady release of glucose for up to 120 hr-enough to account for 25% of the initial glycogen. Some effect was observed at concentrations down to 1.4 x lo-lo M, but at such a low rate of glucose release it is not possible to rule out an increase in gluconeogenesis as the cause. At this dose, the medium glucose content rose to 1.80 mg/lO ml, after 120 hr, as opposed to 1.59 mg/lO ml in the controls.

The slower glycogenolytic action of glucagon was, however, directly measured in another experiment. After 14 days in culture, 32 liver cultures were changed into fresh MEM, and 1.4 x lo+ M glucagon was added to 16 of them. Four treated and four untreated flasks were sampled each day for 4 days, the tissue being removed, blotted, weighed, and frozen over dry ice. The results of subsequent glycogen determination are given in Table 1. Control cultures retained their glycogen, whereas those treated with glucagon showed a

TABLE 1 EFFECTS OF GLUCAGON ON GLYCOGEN CONTENT

OF CULTURED A. means LIVER=

Glycogen content (% wet weight,

mean f standard error)

Hours after Glucagon- glucagon Control treated

added cultures cultures P

0 1.9 + 0.3 - - 24 1.9 +- 0.1 1.6 f 0.1 NS 48 2.0 + 0.2 1.5 + 0.2 NS 72 1.8 + 0.05 1.2 f 0.05 <O.OOl 96 2.2 2 0.1 1.4 ? 0.1 co.002

a Experimental period = cultural days 12-16; P = glucagon-treated culture values compared with control culture values; NS = not significant (P > 0.05). Glucagon dose = 1.4 X lo-@ M (5 pghd).


steady decline in glycogen level. After 72 hr, the equivalent of 40% of the initial glycogen had been broken down.

2. The Effects of Glucagon on Gluconeogenesis

Three groups of eight flasks of liver, which had been cultured in normal MEM (10% serum) for 7 days, were changed into glucose-free MEM (5% serum). Sodium pyruvate or alanine (final concentration 2.8 mM) were added to two groups, the third

Days in cultura

Ghcose uptaketl or rdeasel+l hg/g wet weight) T

1 I

FIG. 2. A. Glucose production by cultured A. means liver over a 7-day period in glucose-free MEM. Effect of glucagon (1.4 x 10-r M; 0.5 pg/ml) on glucose release in the presence of alanine, pyruvate or glucose-free MEM alone. Each point represents the mean + SE of four cultures. n - 2.8 mM pyruvate (0 - glucagon), A - 2.8 mM alanine (A + ducagonh 0 - glucose-free MEM alone (0 + glucagon). B. Glucose uptake (-) or release (+) over the next 7 days, following a medium change and second hormone treatment on day 7. Symbols as for Fig. 2A.

acting as controls. Four cultures from each group were given 1.4 X lo-’ M glucagon at the beginning of each week, when the medium was changed. The glucose and total ammonia content of the used medium was measured over a 4-wk period, after which the tissues were weighed and assayed for glycogen. The results are given in Figs. 2-4.

Initially, glucagon caused an increase in medium glucose in all groups, presumably as a result of glycogenolysis. After 4 days, the glucose levels in the alanine and pyruvate groups treated with glucagon were significantly higher than in all the others (P < 0.02 andP < 0.01, respectively). This suggests that alanine and pyruvate were being used as gluconeogenic substrates under the influence of the hormone. In the

Total glucose uptake-) Or releaM+l

(mg/g wet weight) +30-l

FIG. 3. Total glucose uptake (-) or release (+) by cultured A. means liver over a 28-day period. Each point is the sum of all previous weekly values and is the mean -C SE of four cultures. Effect of glucagon, alanine, and pyruvate. Symbols are as for Fig. 2A. Final glycogen contents of the tissues are shown next to the 28-day points. (% wet weight of tissue).

384 BROWN, FLEMING AND BALLS

Total nitrogenous excretion

(pmoles Nl-+/g wet weight)

Days in culture

FIG. 4. Total nitrogenous excretion (urea + free-NH,) by cultured A. means liver over a 2% day period. Each. point is the sum of all previous weekly values, and represents the mean f SE of four cultures. Effect of glucagon, alanine, and pyruvate-symbols as for Fig. 2A.

absence of glucagon, little change in medium glucose was observed, although alanine seemed to cause an increase in glucose equivalent to that caused by glucagon, but more slowly.

Over the 4-wk period, glucagon had more effect with pyruvate as a substrate, less effect with alanine, and no effect (apart from the initial glycogenolysis) when used alone. The group treated with alanine alone followed a very similar pat- tern of glucose release to that of cultures treated with glucagon alone. The final glycogen level of the tissue was lowest when ahmine and glucagon were administered together.

Added pyruvate caused a significant reduction (P < 0.001) in nitrogenous excre-

tion from the control level. Alanine also caused a reduction (P < O.OOl), but the reduction was less than with pyruvate. Glu- cagon had no significant effect on nitrogenous excretion in either control cultures or those where pyruvate or alanine were present.

3. Effects of Insulin and Glucagon on Fuctose-1 ,bDiphosphatase Levels

Sets of 15 flasks which were set up 14 days previously, were changed into fresh MEM and treated with either insulin (10 mu/ml) or glucagon (1.4 x lo-’ M). A third set of flasks were left untreated, and three flasks were sampled at time 0 for determination of the initial level of the enzyme. Every 8 hr up to 40 hr, the tissue from three flasks of each type was weighed and assayed for fructose- 1,6-diphosphatase activity. The results are given in Table 2, and show that insulin had no effect on the activity of the enzyme during the 40-hr period, but that by this time glucagon had caused a significant increase.

The level of the enzyme in freshly isolated liver was 4.3 U/g wet weight, so the

TABLE 2 EFFECTS OF INSULIN AND GLUCAG~N ON

FRUCTOSE-1,6-DIPHosPHATASE LEVELS IN CULTURED A. means LIVERY

Fructose-1,6diphosphatase level (units per gram wet weight,

Hours mean f standard error) after

hormone Control Cultures with, Cultures with added cultures added insulin added glucagon

0 2.8 * 0.3 - - 8 3.6 + 0.1 4.1 k 0.4 3.4 f 0.1

16 2.7 k 0.3 3.7 2 0.1 2.4 + 0.3 24 3.6 k 0.2 3.5 2 0.2 3.9 f 0.2 32 4.1 +- 0.2 3.6 IL 0.3 3.6 ” 0.1 40 3.2 -+ 0.3 3.8 + 0.2 8.0 + 0.9*

a Experimental period = culture days 14-16; * = significantly higher than all other levels (P < 0.001). Glucagon dose = 1.4 x 10-r it4 (0.5 &ml). Insulin dose = 10 mu/ml.


normal level of the enzyme is maintained in vitro.

4. Effects of Insulin and Glucagon on Tissue GOT and GPT Levels

Three sets of 16 flasks of liver which had been in culture for 16 days, were treated with insulin (10 mu/ml) or glucagon (1.4 x lo-’ M) or were left as untreated controls. At 24-hr intervals, four flasks from each group were removed and tissue GOT and GPT levels determined. At each sample time, the hormone treatment was repeated in the remaining flasks. The results are shown in Fig. 5. In the insulin- treated tissues, the activities of both trans-

24 1 lb %

Hours after initial treatment

T 2i 4b l!! % Hours after initial treatment

FIG. 5. Effect of glucagon (0 - 1.4 x lo-’ M; 0.5 &ml) and insulin @ - 10 mu/ml) on glutamate- oxaloacetate transaminase (GOT) and glutamate- pyruvate transaminase (GPT) activity of cultured A. ntean~ liver (0 - level in MEM alone). Each point is the mean k SE of four cultures. *-significantly diierent (P < 0.05) from untreated controls. Activities of the enzymes in freshly isolated liver were 5.3 f 0.3 units/g wet weight (GPT) and 17.3 units/g wet weight (GOT). Each value is the mean + SE of four samples from different animals.

aminases dropped to a minimum after 72 hr (P < 0.05), after which they increased again to give levels higher than those in control cultures. Glucagon stimulated both GOT and GPT activity to a maximum at 48 hr (P < 0.05), following which the levels fell back to the control value by 96 hr.

DISCUSSION

The experiments described show that glucagon has marked effects on glucose production both from glycogenolysis and gluconeogenesis in A. means liver fragments in organ culture. A comparative study showed that although glycogenolysis is stimulated by both glucagon and adrenalin, the response to glucagon is slower. In contrast, Monnickendam et al. (1974) found that adrenalin induced a fall in glycogen level from 3.5 to 1.4% in 24 hr, when added to A. means liver cultures. However, whereas glucagon had an effect at doses as low as lo-lo M, glycogenolysis is not stimulated by adrenalin below lo-’ M (Monnickendam et al., 1974).

In designing experiments such as these, it is difficult to determine how often the hormone dosage should be repeated. In vivo, both glucagon and adrenalin have very short half-lives (Goodman and Gilman, 1970), but their stability in our culture medium is not known. In the experiments concerning glucose production in the presence of pyruvate and alanine, glucagon was administered once weekly. Although it is probable that most of the hormone would have been degraded during the subsequent 7-day period, glucagon increased glucose production from pyruvate and, to a lesser extent, from alanine. Since cultures treated with glucagon alone produced less glucose over the 4-wk period, despite breaking down most of their glycogen, the extra glucose must have come from glucagon-induced gluconeogenesis. Pyruvate caused no increased glucose production in the absence of glucagon. Alanine, however, seemed to


stimulate an initial glycogenolysis, and the final glycogen level was lower than in control cultures.

Pyruvate had a marked inhibitory effect on total nitrogenous excretion, as was observed by Metz et al. (1968) in rat liver slices in vitro. The addition of glucagon did not change this response. Alanine (2.8 mM) addition resulted in a smaller de- crease in nitrogenous excretion, but subsequent work (Fleming and Balls, in prepara- tion) has shown that a higher concentration of alanine (10 mM) had a stimulatory effect.

This action of pyruvate may be a result of the ornithine-pyruvate transaminase (OPT) reaction, decreasing the availability of ornithine to the urea cycle. A similar inhibition by alanine would be possible, if pyruvate, produced by transamination of alanine by GPT, then took part in the OPT reaction. One product of the OPT reaction is alanine itself, so the presence of 2.8 mM alanine in the medium may partially inhibit OPT, resulting in a lower rate of ornithine loss than with pyruvate added to the medium. However, the higher level of alanine (10 m&f) may completely inhibit OPT, so that no ornithine is lost in this way and it is all available to the urea cycle enzyme ornithine transcarbamylase (OTC). This would result in the observed increase in nitrogenous excretion with 10 mM alanine.

The effects of insulin and glucagon on enzyme activity are consistent with the ob- servation that gluconeogenesis is inhibited by insulin and stimulated by glucagon (Exton et al., 1966). The significant fall in transaminase activity caused by insulin after 72 hr would be expected as protein synthesis increased and fewer amino acids were transaminated to glutamate. The subsequent rise in activity after 96 hr may be a result of the stimulation of transamination in the reverse direction, i.e., glutamate to other amino acids as protein synthesis pro- ceeded at a higher rate. An alternative

explanation is that, as protein synthesis was stimulated by insulin, the higher level of transaminase activity after 96 hr simply reflected this increased synthetic activity.

Glucagon produced its maximum stimulatory effect on transaminase activity by 48 hr. The redirection of amino acids to glucose via glutamate and a-ketoglutarate would require such an increase in transamination. The gradual drop in activity after 48 hr may be a result of a build-up of glucose both inside and outside the hepato- cytes, which would inhibit the process, leading to a reduction in transamination. Glucagon inhibits protein synthesis, so it is also possible that the fall in transaminase activity is a reflection of the general reduction in protein synthesis level in the cultured liver.

The level of fructose- 1,6-diphosphatase, a key enzyme in gluconeogenesis, was also increased by glucagon after 40 hr. This enzyme controls the reversal of the phosphofructokinase reaction of glycolysis, and, together with glucose-6-phosphatase, is one of two nonglycolytic enzymes utilised in gluconeogenesis from pyruvate. Although glucagon probably stimulates a rate-limiting step in gluconeogenesis between pyruvate and phosphoenolpyru- vate in rat liver (Exton et al., 1971), it is still possible that specific induction of fructose-1,6-diphosphatase takes place. This would enhance the rate of glucose forma- tion. Indeed, a recent report by Kneer et al. (1974) provides evidence for a hormone- mediated site of regulation of gluconeogenesis at phosphofructokinase-fructose diphosphatase, controlled by glucagon via cyclic-AMP.

The delayed action of both glucagon and insulin may be a reflection of the generally low metabolic rate of the amphibian used in this work which was carried out at 25”, whereas mammalian tissues require a culture temperature of 37”, and reaction rates will be greater (Miller, 1960; Mon- nickendam and Balls, 1973). This lag period

is also apparent in Fig. 2, where the rate Long-term amphibian organ culture. Merh.

of glucose production in liver cultures with Cell Bid. 13, in press.

added pyruvate plus glucagon did not rise Epple, A. (1966). Islet cytology in urodele amphib-

above the rate in control cultures until ians. Gen. Camp. Endocrinol. 7, 207-214.

48 hr after hormone treatment. On the Exton, J. H., Jefferson, L. S., Butcher, R. W., and

Park, C. R. (1966). Gluconeogenesis in the per- other hand, glycogenolysis in response to fused liver. Amer. J. Med. 40, 709-715.

adrenalin is a comparatively rapid process. Exton, J. H., Ui, M., Lewis, S. B., and Park, C. R.

Anuran amphibians have more islet (1971). Mechanism of glucagon activation of glu-

tissue than urodeles, where the endo- coneogenesis. In “Regulation of Gluconeogene-

crine pancreatic tissue is scattered sis” (H-D. Soling and C. R. Park, eds.), pp. X0-178. Academic Press, New York.

throughout the acinar tissue in small Fawcett, J. K., and Scott, J. E. (1960). A rapid and

clusters of cells. Wurster and Miller precise method for the determining of urea.

(1960) and Miller (1961) reported that J. Clin. Pathol. 13, 156-159.

the islet tissue of three urodele species Fleming, N., Brown, D., and Balls, M. (1975). Hepa-

(Taricha torosa, A. means, and Ensatina tocyte function in long-term organ culture of Amphiuma means liver. J. Cell. Sci. 18, 533-544.

eschscholtzi) was composed almost en- Goodman, L. S., and Gilman, A. (1970). “The

tirely of /3 cells (insulin-producing) and Pharmacological Basis of Therapeutics.” Col-

that they could find no cells with a-cell lier-Macmillan, London.

(glucagon-producing) characteristics. How- Grossner, D. (1%7). Uber das Inselorgan des Axolotl

(Siredon mexicanum). Z. Zellforsch. 82, 82-91. ever, Epple (1%6), using electron micro- Karmen, A. (1955). A note on the spectrophotometric scopy and differential staining techniques, assay of glutamic-oxalacetic transaminase in

found (Y and /3 cells in various urodele human blood serum. J. Clin. Invest. 34, 131-

species, including A. means. Grossner 133.

(1%7) and Trandaburo (1970) also found Kneer, N. M., Bosch, A. L., Clark, M. G., and

Lardy, H. A. (1974). Glucose inhibition of epi- cells with a-cells characteristics in the nephrine stimulation of hepatic gluconeogenesis pancreas of various urodeles, and Sato et by blockade of the o-receptor function. Proc.

al. (1%6) observed a! cells in the A. means Nat. Acad. Sci., U.S.A. 71,4523-4527.

pancreas. Nate et al. (1965) found that Lowry, 0. H., and Lopez, J. A. (1946). The deter-

alloxan, insulin, and glucagon had effects mination of inorganic phosphate in the presence

on blood sugar level and islet function in A. of labile phosphate esters. J. Biol. Chem. 162, 421-428.

means and Necturus maculosus. Thus, the MacDougall, J. D. B., and Coupland, R. E. (1967). balance of opinion now favours the exis- Organ culture under hyperbaric oxygen. EXP.

tence of (Y cells in the urodele pancreas, and Cell Res. 45, 385-398.

our experiments clearly demonstrate that Metz, R., Salter, J. M., and Brunet, G. (1968). Effect

the urodele liver in vitro responds to of pyruvate and other substrates on urea synthesis in rat liver slices. Metabolism 17, 158-

glucagon in ways which areconsistent with 167. the known effects of the hormone in Miller, M. R. (1960). Pancreatic islet histology and

mammals. carbohydrate metabolism in amphibians and reptiles. Diabetes 9, 318-323.

ACKNOWLEDGMENTS Miller, M. R. (l%l). Carbohydrate metabolism in amphibians and reptiles. In “Comparative

We acknowledge the support of postgraduate re- Physiology of Carbohydrate Metabolism of search studentships from the Science Research Heterothermic Animals” (A. W. Martin, ed.), Council (D.B.) and The Wellcome Trust (N.F.). pp. 125-144. University of Washington Press,

Seattle.

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Balls, M., Brown, D., and Fleming, N. (1975). MoMickendam, M. A., and Balls, M. (1973). The



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Monnickendam, M. A., Brown, D., and Balls, M. (1974). Organ culture of Amphiuma means liver: control of glycogen content. Camp. Bio- them. Physiol. 47A, 569-572.

Nate, P. F., Blair, M., and Dass, B. (1%5). Blood sugar and islet function in salamanders; Am- phiuma and Necturus. Anat. Rec. 151, 391-392.

Pogell, B. M., and McGilvery, R. W. (1952). The proteolytic activation of fructose-l$- diphosphatase. .I. Biol. Chem. 197, 292-302.

Sato, T., Herman, L., and Fitzgerald, P. J. (1966). The comparative ultrastructure of the pan-

creatic islet of Langerhans. Gen. Camp. Endo- crinol. 7, 132-157.

Trandaburo, T. (1970). Light and electron micro- scopic investigations on the endocrine pancreas of the newt [Triturus Y. vulgaris (L)]. Z. Mcrosk. Anat. Forsch. 82, 322-340.

Trowell, 0. A. (1959). The culture of mature organs in a synthetic medium. Exp. Cell Res. 16, 118-147.

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Wurster, D. H., and Miller, M. R. (1960). Studies on the blood glucose and pancreatic islets of the salamander, Taricha torosa. Camp. Bio- them. Physiol. 1, 101-109.

Hormonal control of glucose production by Amphiuma means liver in organ culture

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