Biochimica et Biophysica Acta, 1052 (1990) 229-234 229 Elsevier

BBAMCR 12661

Effects of glucagon and a- and fl-agonists on glycogenolysis and gluconeogenesis in isolated ovine hepatocytes

Anne Faulkner and Helen T. Pollock

Hannah Research Institute, Ayr (U.K.)

(Received 2 August 1989) (Revised manuscript received 22 November 1989)

Key words: Gluconoegenesis; Glycogenolysis; Glucagon; Isoproterenol; Phenylephrine; Adrenergic agonist; (Ovine hepatocyte)

(1) The effects of glucagon, dibutyryl cyclic AMP, vasopressin, phenylelphrine, and isoproterenol on glycogenolysis and gluconeogenesis were investigated using isolated ovine hepatocytes. (2) Glycogenolysis was stimulated by all effectors except vasopressin. The response to a-agonists was greater than that of/~-agonists in older animals. Stimulation by jO-agonists increased after 30 h primary culture. (3) Glucoeneogenesis from propionate or L-lactate plus pyruvate was stimulated to a small extent by dibutyryl cyclic AMP, glucagon and isoproterenol but not by vasopressin or phenylephrine. (4) No effects of lactation were observed. (5) Data are compared to results obtained in other species and the physiological significance of the results in relation to the ruminant is discussed.

Introduction

Glucagon and catecholamines can stimulate the re- lease of glucose from the mammalian liver by increasing rates of both gluconeogenesis and glycogenolysis in rats [1-7], guinea pigs [8] and rabbits [9-11]. In rats and other species studied, this stimulation of glycogenolysis and gluconeogenesis effectively occurs independently. Gluconeogenesis is low but stores of glycogen are high in fed animals; hence the effects of glucagon and cate- cholamines in stimulating hepatic glucose release by increasing the rates of glycogenolysis are quantitatively more important in the fed state: in starved animals, rates of gluconeogenesis are high but glycogen reserves are reduced so that the hormones exert their effect mainly on gluconeogenesis in the starved state.

In ruminants which absorb little glucose from the diet and rely on gluconeogenesis for their glucose re- quirements [12,13] gluconeogenesis is highest in the fed state when reserves of glycogen are also high. It is of interest, therefore, to investigate the effects of glucagon and catecholamines on gluconeogenesis and glycogeno- lysis in fed ruminants where both processes can operate at high rates simultaneously. Both glucagon and cate- cholamines are thought to be important in energy mobilisation in ruminants in times of stress [14]. Cate-

cholamines influence plasma glucose concentrations in the exercising ruminant [15,16] and account, in part, for the metabolic and hormonal adaptations to hypo- glycaemia [17]. Glucagon concentrations also rise dur- ing exercise in the sheep [16] but decline during fasting and increase on refeeding, and glucagon release can be stimulated by intravenous administration of propionate and butyrate [18].

During lactation the requirement for glucose in- creases dramatically to accomodate lactose synthesis, and rates of gluconeogenesis in ruminants rise 2-3-fold [19,20]. In rats during lactation some tissues demon- strate a reduced response to adrenergic agents [21]. We have, therefore, investigated the effects of glucagon and a- and fl-adrenergic agonists (phenylephrine and iso- proterenol) on gluconeogenesis and glucogenolysis in hepatocytes isolated from livers of both lactating and non-lactating sheep to compare the response of rumi- nants to that previously described in rats and also to investigate any modifications which may have been imposed by the lactating state. In addition, modifica- tions to the a- and fl-adrenergic system in response to development and culture have also been studied, as changes in other species have been observed under these conditions [11,22,23].

Materials and Methods

Correspondence: A. Faullmer, Hannah Research Institute, AYR, KA6 5HL, U.K.

Animals . Sheep were 2-5-year-old Finn-Dorset Horn cross-breeds. Lactating animals were 18-22 days post-

0167-4889/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)

230

partum. Sheep were fed and killed as described previ- ously [24].

Preparation and incubation of hepatocytes. Im- mediately after killing the sheep, the caudate lobe of the liver was removed and perfused with Ca’+-free Krebs- Henseleit bicarbonate buffer for 5 mins. The perfusion was then changed to a recirculating system containing 150 ml Ca2+-free Krebs-Henseleit bicarbonate buffer plus 50 mg collagenase which was gassed continuously with 95% 0,/S% CO,. After 30-40 min the lobe was removed from the perfusion apparatus and minced with scissors, suspended in complete Krebs-Henseleit bi- carbonate buffer and filtered through cheese-cloth. Hepatocytes were harvested by centrifuging at 60 x g

for 3 min and washed twice with buffer [25]. After resuspension in Krebs-Henseleit bicarbonate buffer cells were incubated with shaking in 25 ml plastic conical flasks containing 3 ml bicarbonate buffer, 1% dialysed fatty acid free bovine serum albumim, 0.5 FCi NaHi4C0, and substrates and hormones as indicated. Unless otherwise stated incubations were for 60 min after which 0.5 mm01 HClO, was added. Precipitated protein was removed by centrifuging and the super- natant neutralised with KOH, before being used for the determination of glucose [26], glucose specific activity [27], L-lactate [28] and pyruvate [29].

A study of the time-course of glucose production showed that release of glucose was linear over the 60 min period in the absence of added substrate (Fig. 1A). Rates of gluconeogenesis were also linear over the 60 min incubation period, irrespective of whether data were expressed as net glucose production (i.e., total glucose production minus that produced in the absence of substrate) or as radioactive incorporation (Fig. 1B).

The techniques of isolating hepatocytes from pre- dominantly periportal or perivenous regions of the rat liver [30,31] has demonstrated that gluconeogenesis and glycolysis are zonally distributed in this species [30-321. Our technique of isolating ovine hepatocytes should yield a mixture of periportal and perivenous cells, but the interpretation of the data might be affected if one form predominanted. However, this seems unlikely as, between individual hepatocyte preparations, having quite large variations in rates of gluconeogenesis and lactate production, there was no significant inverse rela- tionship between glycolysis and gluconeogenesis, nor were there significant differences in data obtained from hepatocytes prepared by perfusing the caudate lobe via the portal or hepatic vein.

Culture of hepatocytes. Isolated hepatocytes were cul- tured for up to 30 h as described previously [33]. Cul- tured hepatocytes were incubated for 2 h with 20 mM glucose to increase their glycogen content prior to wash- ing and incubating for 30 min with the hormones and additions indicated.

Expression of data and statistical analysis. Data are

25or A

__--.

200 - ,.__---

/ /

/ A’

0

.z o-

5 5 2 200-

P a

% 8 : 150-

loo-

50-

O-

, I I I

0

I I I I I 1

0 30 60 90 120 150

Time (min)

Fig. 1. Effect of varying incubation time on glucose production in the

absence (A) or presence (B) of propionate. In (A) hepatocytes were

incubated with no addition (O), with 10e7 M glucagon (0) or with

10m5 phenylephrine (v). In (B) glucose production was determined as

net formation (0) or incorporation of radioactivity (0). Data are

means of four non-lactating sheep.

expressed as means + SE. Statistically significant ef- fects of hormones within groups was determined using Student’s t-test for paired observations. Statistically sig- nificant effects of hormones between groups of animals (i.e., lactating vs. non-lactating) was determined by analysis of variance.

Chemicals. Glucagon, phenylephrine, yohimbine, prazosin, dibutyryl cyclic AMP, vasopressin, albumin and isoproterenol were obtained from Sigma Chemical Co., Poole, U.K. Collagenase, enzymes and co-enzymes were obtained from Boehringer, Lewes, U.K. NaH’4C0, was obtained from Amersham International, Amersham, U.K. All other reagents were from British Drug Houses, Poole, U.K.

Determination of gluconeogenesis using NaH”C0,. Hepatocytes from fed sheep had high rates of endoge- nous glucose production due to the presence of glyco- gen. Thus estimates of gluconeogenesis based on net glucose production in the presence of precursor were variable. To obtain a more accurate value, rates of incorporation of NaH14C0, into glucose were followed. Using propionate as precursor radioactivity is incorpo-

rated into methyl malonyl CoA and equilibrates com- pletely between C1 and C4 of fumarate and hence oxaloacetate. On synthesis of PEP, radioactivity from C1 is lost as 14CO2. Thus for every molecule of glucose synthesised one molecule of NaH14CO3 is incorporated. With L-lactate or pyruvate as precursor, radioactivity is incorporated into oxaloacetate and equilibration be- tween C1 and C4 can occur through the fumarase reaction. But equilibration may not be complete and ratios of NaHa4CO3 incorporated to glucose synthesised may be less than 1.

This method assumes that no exchange reactions are occurring to enrich or dilute either C1 or C4 of oxaloacetate (as, for example, dilution by amino acids entering the citric acid cycle). In these simple in vitro experiments, where only single glucose precursors were supplied and any such dilution could only come from endogenous sources, the error introduced is probably small. The linearity of incorporation of radioactivity into glucose (Fig. 1B) indicates that this method gives reliable data under these conditions.

Results

Influence of effectors on glycogenolysis in hepatocytes High rates of glycogen breakdown were observed in

the absence of added substrates or effectors, but these were increased significantly by glucagon, phenylephrine, isoproterenol and dibutyryl cyclic AMP (Table I), due entirely to the stimulation of glucose production. The highest stimulation was observed with dibutyryl cyclic AMP and the a-agonist, phenylephrine, with smaller effects being observed with the fl-agonists, isoproterenol

TABLE I

Effects of a- and fl-agonists and glucagon, dibutyryl cyclic A M P and vasopressin on glycogenolysis in isolated ovine hepatocytes

Hepatocytes were prepared and incubated as described in Materials and Methods. Glydogenolysis was calculated as the sum of glucose, L-lactate and pyruvate production (C6 units). Complete oxidation of glucose to CO 2 was not determined but is believed to be low in ruminants [441. Results are means + S.E. for 12 animals. A statistically significant effect of added hormones or agopnists is shown by * * * P < 0.001, * * P < 0.01.

Addition Glycogenolysis ( n m o l / h per mg dry wt. cells)

None Prazosin (10 -6 M) Yohimbine (10 -6 M) Isoproterenol (10 -6 M) Phenylephrine (10- s M) Glucagon (10 -7 M) Vasopressin (10- s M) Dibutyryl cyclic AMP (10 - a M) Phenylephrine + yohimbine Phenylephrine + prazosin

141 + 38.2 138 ± 36.8 139 + 42.6 190±40.3 * * * 227±40.1 * * * 197+39.7 ** 162 + 35.2 213+40.2 * * * 203+39.9 *** 137±35.9

231

TABLE II

Effects of a- and fl-agonists and glucagon on glucose production in cultured ovine hepatocytes

Hepatocytes were cultured for 30 h as described in Materials and Methods. During the last 2 h of culture glucose was added to give a final concentration of 20 raM. Plates were then washed and incubated with Krebs-Henseleit bicarbonate buffer and additions as given be- low. Results are means±S .E , for four animals. Plates contained approx. 4 rag dry wt ceils. A statistically significant effect of the addition is given by * * P < 0.01, * P < 0.05.

Addition Glucose production ( n m o l / h per plate)

None 238 + 53 Isoproterenol (10 - 6 M) 489 + 112 * * Phenylephrine (10 -5 M) 305± 68 * Dibutyryl cyclic AMP (10 -4 M) 458 ± 61 * Glucagon (10 -7 M) 340+ 68 *

and glucagon. Vasopressin was without effect. Prazosin but not yohimbine prevented the stimulation due to phenylephrine.

Hepatocytes which had been cultured showed a dif- ferent pattern of stimulation by a- and fl-agonists. After 30 h in culture a greater stimulation of glucose release was observed by t - than by a-agonists (Table II). In young animals (3-4 months) the stimulation by a- and fl-agonists was about the same (results not shown).

Influence of effectors on gluconeogenesis in hepatocytes Rates of glucose synthesis were measured both by the

net increase in glucose output in the presence of added substrate and by the incorporation of radioactivity from NaH14CO3 . Using both methods, statistically significant stimulation of gluconeogenesis was observed during treatment of hepatocytes with dibutyryl cyclic AMP, glucagon and the/3-agonist, isoproterenol, but the ex- tent of the stimulation was small being only 25% maxi- mally. Vasopressin and phenylephrine had no signifi- cant stimulatory effect (Table III).

Effects of lactation on the response of hepatocytes to effectors

No statistically significant differences were found in the response of gluconeogenesis or glycogenolysis to any of the effectors tested in hepatocytes prepared from livers of lactating and non-lactating ewes. However, as modifications during lactation may take the form of altered sensitivity rather than response, the effects of varying concentrations of glucagon, isoproterenol and phenylephrine on glycogenolysis were determined (Fig. 2). No statistically significant differences were observed in the concentration of effector required to give half- maximal stimuation. The concentrations of glucagon, isoproterenol and phenylephrine which gave half-maxi- mal response w e r e 10 - 9 , 10 - 7 and 10 - 6 M , respectively.

232

TABLE III

Effects of glucagon, dibutytyl cyclic AMP and a- and B-agonists on gluconeogenesis in ovine hepatocytes

Hepatocytes were prepared and incubated as described in Materials and Methods. Results are means* S.E. of 12 animals. A statistically significant

effect of hormone or agonist compared to corresponding result with no addition is shown by * P < 0.05. Net ghrcose production was calculated as the difference in glucose production in the presence and absence of substrate.

Addition Substrate

None propionate (3 mM)

Isoproterenol (1O-6 M) propionate (3 mM)

Phenylephrine (10m5 M) propionate (3 mM)

Vasopressin (lo-’ M) propionate (3 mM)

Glucagon (lo-’ M) propionate (3 mM)

Dibutyryl cyclic AMP (10m4 M) propionate (3 mM)

None L-lactate + pyruvate (3 mM)

Isoproterenol (lOme M) L-lactate + pyruvate (3 mM)

Phenylephrine (lo-’ M) L-lactate + pyruvate (3 mM)

Vasopressin (lo-* M) L-lactate + pyruvate (3 mM)

Glucagon (10F7 M) L-lactate + pyruvate (3 mM)

Gluconeogenesis (nmol/h per mg dry wt. cells)

net glucose production t4C incorporation

61.8!c 8.2 7O.Ok4.8 76.0* 7.5 * 73.6 + 6.3 *

59.8& 7.9 62.0 * 5.4 *

53.4* 7.1 * 71.0 f 9.1

71.6* 6.8 * 75.0 f 5.9 *

78.55 6.9 * 76.0 f 5.9 *

43.2+ 5.5 26.7+2.6

48.1 !c 11.8 * 28.5+3.7 *

30.8k 8.1 27.2 + 5.4

43.5 f 6.2 27.0 f 2.9

51.6k 5.8 * 31.0*3.8 *

L-Lactate and pyruvate output from hepatocytes L-Lactate and pyruvate output increased in the pres-

ence of propionate, but there were no statistically sig- nificant effects of the various effecters, either in the presence or absence of propionate (Table IV).

Discussion

The principal finding reported here is that, in the isolated hepatocyte from the fed sheep, glucagon and (Y- and /3-agonists have a far greater effect on glycogen breakdown than on gluconeogenesis. This contrasts with the situation in the rat, where both processes are stimu- lated under these conditions [1,7]. In addition, hepatic

TABLE IV

Effects of glucagon, vasopressin and a- and B-agonists on L-lactate and pyruvate output from ovine hepatocytes

Hepatocytes were prepared and incubated as described in the Meth-

ods. Results are means * SE. of 12 animals.

Addition Substrate L-Lactate and

pyruvate output

(nmol/h per mg

dry wt. cells)

None none 46.lf 9.2

Isoproterenol (10m6 M) none 45.5 f 10.5

Phenylephrine (low5 M) none 40.8* 7.2

Vasopressin (lo-* M) none 52.2kl3.6

Glucagon (lo-’ M) none 45.2 f 12.8

None propionate (3 mM) 87.4* 8.8

Isoproterenol (10K6 M) propionate (3 mM) 89.6* 9.5

Phenylephrine (low5 M) propionate (3 mM) 103.2 f 15.0

Vasopressin (10e5 M) propionate (3 mM) 90.4* 8.1

Glucagon (lo-’ M) propionate (3 mM) 92.5 f 15.2

catabolism of released glucose appeared to be unaf- fected (as indicated by unchanged rates of L-lactate and pyruvate production). This is again different from the rat, where glycolysis is inhibited by glucagon through decreased pyruvate kinase activity, the reduction in futile cycling at this reaction contributing to the stimu- lation of gluconeogenesis by glucagon [34,35]. Adren- ergic agonists did not significantly affect glycolysis in hepatocytes from adult rats, although P-agonists in- hibited in young rats in one study [35], while others have found a stimulation of glycolysis by cY-agonists in perfused rat livers [36].

The larger responses of glycogenolysis to IY- than to /3-agonists in ovine hepatocytes was similar to that observed in the rat [3-71 although smaller in magnitude, but unlike that described in rabbits [9,11] or guinea-pig [8], where the /3-stimulated response predominates. Greater stimulation of glycogen breakdown was evident with the cu-agonist, phenylephrine, than with the /3- agonist, isoproterenol, and inhibition by prazosin was indicative of action through the ru,-receptor [37]. After maintaining hepatocytes overnight as a primary mono- layer culture, the stimulation of glycogenolysis by the P-agonist was more pronounced than that of the (Y- agonist and in hepatocytes isolated from younger animals the response to both was about equal. This, too, has been observed in rats [l&19,38]. Dibutyryl cyclic AMP stimulated glycogen breakdown to the same ex- tent as phenylephrine and glucagon was also effective, although the stimulation was less than that of the (Y- agonist. This contrasts with the observations in the rat where glucagon and cyclic AMP produce a higher re- sponse than phenylephrine [6]. In isolated hepatocytes from Smonth-old sheep, phenylephrine, isoproterenol and glucagon activate phosphorylase

233

.c

F

o

@

(.9

180

160

140

12C

100

8O

140

120

100

8C

140

120

100

80

0

0

OlD

i i I I I I i i ! I I

0 2 4 6 8 10

i i

0 I I

"C

oe

#-

I I I 1 ~ o 02 4

I ! i i

0.6 0.8 i i

1 . 0

O o

e O

O O

I I I I I 0 0.025 0.05 0.075 0.10

Hormone conc ( .uM)

Fig. 2. Effect of varying the concentrations of (a) phenylephrine, (b) isoproterenol and (c) glucagon on glucose output from bovine hepatocytes. Hepatocytes from lactating (o) and non-lactating (o) sheep were isolated and incubated as described in the text. Data are

means of eight lactating and five non-lactating sheep.

is similar to that reported for isolated rabbit hepato-

cytes [10]. The smaller response of ~uconeogenesis in sheep

liver compared to the rat may be a consequence of the different nutritional status of the animal. In the sheep, glucose has to be synthesised within the body (mainly in the liver), as very little glucose is obtained from the diet [12,13]. To compensate for this, .the ruminant animal has evolved mechanisms for sparing glucose and its precursors, such as the utilisation of acetate, not glu- cose, for fatty acid synthesis [42,43] and low rates of glucose oxidation [44]. The reduced response of sheep hepatocytes to hormones stimulating gl, ucogenesis may be a consequence of such a glucose-sparing mechanism. In the rat liver it has been proposed that hormones, especially those acting via cyclic AMP, stimulate gluco- neogenesis in the short term by decreasing futile cycling which occurs at the pyruvate kinase and phosphofruc- tokinase catalysed reaction [1,2,34,35]. If, as a method of preserving glucose, there is normally much lower rates of futile cycling in the livers of ruminants, hormortes acting to reduce futile cycling could not be expected to be as effective in these animals as in rats. The lack of effect of any of the agents studied on rates of L-lactate and pyruvate output argues against any major changes in recycling rates at the pyruvate kinase catalysed reaction.

Whatever the mechanisms involved in the reduced sensitivity of gluconeogenesis to short-term hormonal regulation in ruminants, it appears that arty transient deficit in the supply of glucose to the animal is com- pensated for more by mobilisation of glycogen than by increased rates of glucose synthesis. Even in lactation when demands for glucose are greatly increased, no significant modifications in the response of hepatocytes to the hormones and agonists were detected.

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