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

229

Elsevier BBAMCR12661

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.)

(Received2 August1989) (Revised manuscriptreceived22 November1989)

Keywords: Gluconoegenesis;Glycogenolysis;Glucagon;Isoproterenol;Phenylephrine;Adrenergicagonist;(Ovinehepatocyte)

(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 release 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 catecholamines 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 requirements [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 glycogenolysis in fed ruminants where both processes can operate at high rates simultaneously. Both glucagon and catecholamines 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 hypoglycaemia [17]. Glucagon concentrations also rise during 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 increases dramatically to accomodate lactose synthesis, and rates of gluconeogenesis in ruminants rise 2-3-fold [19,20]. In rats during lactation some tissues demonstrate a reduced response to adrenergic agents [21]. We have, therefore, investigated the effects of glucagon and a- and fl-adrenergic agonists (phenylephrine and isoproterenol) on gluconeogenesis and glucogenolysis in hepatocytes isolated from livers of both lactating and non-lactating sheep to compare the response of ruminants to that previously described in rats and also to investigate any modifications which may have been imposed by the lactating state. In addition, modifications 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,HannahResearchInstitute,AYR, KA6 5HL, U.K.

A n i m a l s . Sheep were 2-5-year-old Finn-Dorset Horn cross-breeds. Lactating animals were 18-22 days post-

0167-4889/90/$03.50 © 1990 ElsevierSciencePublishers B.V. (BiomedicalDivision)

230 partum. Sheep were fed and killed as described previously [24]. Preparation and incubation of hepatocytes. Immediately after killing the sheep, the caudate lobe of the liver was removed and perfused with Ca’+-free KrebsHenseleit 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 bicarbonate 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 supernatant 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 predominantly 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 relationship 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 cultured for up to 30 h as described previously [33]. Cultured hepatocytes were incubated for 2 h with 20 mM glucose to increase their glycogen content prior to washing and incubating for 30 min with the hormones and additions indicated. Expression of data and statistical analysis. Data are

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60

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120

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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 effects of hormones within groups was determined using Student’s t-test for paired observations. Statistically significant 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 endogenous glucose production due to the presence of glycogen. 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-

231 rated into methyl malonyl CoA and equilibrates completely 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 between 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 s u m 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 m e a n s + S.E. for 12 animals. A statistically

significanteffect of added hormonesor agopnists is shown by * * * P < 0.001, * * P < 0.01. Addition

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

None Prazosin (10 - 6 M) Yohimbine (10 - 6 M) Isoproterenol (10 - 6 M) Phenylephrine ( 1 0 - s M) Glucagon (10 - 7 M) Vasopressin ( 1 0 - s M) Dibutyryl cyclic A M P (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

T A B L E 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 below. Results are m e a n s ± 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 Isoproterenol (10 - 6 M)

238 + 53 489 + 112 * *

Phenylephrine(10 -5 M) DibutyrylcyclicAMP (10 -4 M)

305± 68 * 458 ± 61 * 340+ 68 *

Glucagon (10 -7 M)

and glucagon. Vasopressin was without effect. Prazosin but not yohimbine prevented the stimulation due to phenylephrine. Hepatocytes which had been cultured showed a different 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 extent of the stimulation was small being only 25% maximally. Vasopressin and phenylephrine had no significant 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 halfmaximal stimuation. The concentrations of glucagon, isoproterenol and phenylephrine which gave half-maximal response w e r e 1 0 - 9 , 1 0 - 7 and 1 0 - 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 Isoproterenol (1O-6 M) Phenylephrine (10m5 M) Vasopressin (lo-’ M) Glucagon (lo-’ M) Dibutyryl cyclic AMP (10m4 M)

propionate propionate propionate propionate propionate propionate

None Isoproterenol (lOme M) Phenylephrine (lo-’ M) Vasopressin (lo-* M) Glucagon (10F7 M)

L-lactate L-lactate L-lactate L-lactate L-lactate

Gluconeogenesis

(3 (3 (3 (3 (3 (3 + + + + +

mM) mM) mM) mM) mM) mM)

pyruvate pyruvate pyruvate pyruvate pyruvate

(3 (3 (3 (3 (3

mM) mM) mM) mM) mM)

L-Lactate and pyruvate output from hepatocytes

L-Lactate and pyruvate output increased in the presence of propionate, but there were no statistically significant 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 (Yand /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 stimulated under these conditions [1,7]. In addition, hepatic

TABLE

IV

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

on L-lactate and

Hepatocytes were prepared and incubated as described ods. Results are means * SE. of 12 animals. Addition

Substrate

None Isoproterenol (10m6 M) Phenylephrine (low5 M) Vasopressin (lo-* M) Glucagon (lo-’ M)

none none none none none

None Isoproterenol (10K6 M) Phenylephrine (low5 M) Vasopressin (10e5 M) Glucagon (lo-’ M)

propionate propionate propionate propionate propionate

in the Meth-

L-Lactate and pyruvate output (nmol/h per mg dry wt. cells) 46.lf 9.2 45.5 f 10.5 40.8* 7.2 52.2kl3.6 45.2 f 12.8 (3 (3 (3 (3 (3

mM) mM) mM) mM) mM)

87.4* 8.8 89.6* 9.5 103.2 f 15.0 90.4* 8.1 92.5 f 15.2

(nmol/h

per mg dry wt. cells)

net glucose production

t4C incorporation

61.8!c 76.0* 59.8& 53.4* 71.6* 78.55

8.2 7.5 7.9 7.1 6.8 6.9

7O.Ok4.8 73.6 + 6.3 62.0 * 5.4 71.0 f 9.1 75.0 f 5.9 76.0 f 5.9

43.2+ 48.1 !c 30.8k 43.5 f 51.6k

5.5 11.8 * 8.1 6.2 5.8 *

* * * *

* * * *

26.7+2.6 28.5+3.7 * 27.2 + 5.4 27.0 f 2.9 31.0*3.8 *

catabolism of released glucose appeared to be unaffected (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 stimulation of gluconeogenesis by glucagon [34,35]. Adrenergic agonists did not significantly affect glycolysis in hepatocytes from adult rats, although P-agonists inhibited 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 /3agonist, isoproterenol, and inhibition by prazosin was indicative of action through the ru,-receptor [37]. After maintaining hepatocytes overnight as a primary monolayer culture, the stimulation of glycogenolysis by the P-agonist was more pronounced than that of the (Yagonist 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 extent as phenylephrine and glucagon was also effective, although the stimulation was less than that of the (Yagonist. This contrasts with the observations in the rat where glucagon and cyclic AMP produce a higher response than phenylephrine [6]. In isolated hepatocytes from Smonth-old sheep, phenylephrine, isoproterenol and glucagon activate phosphorylase

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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.

breakdown in the rat liver [40,41] was without effect in sheep hepatocytes. The response of gluconeogenesis to the various effectors in the ovine hepatocyte appeared different from that of the rat. A statistically significant stimulation was observed only with agents which are believed to act through a cyclic-AMP-dependent pathway (i.e., dibutyryl cyclic AMP, glucagon and isoproterenol). Even with these, the response was small, stimulation of gluconeogenesis being only about 25% of basal. In the rat, stimulation by glucagon can be 200-300% and that of phenylephrine and vasopressin about 30-60% [1,5,6,41]. N o significant stimulation by vasopressin or phenylephrine was detected in the sheep hepatocytes. The overall smaller response of gluconeogenesis to effectors

is similar to that reported for isolated rabbit hepatocytes [10]. The smaller response of ~uconeogenesis in sheep liver compared to the rat m a y 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 glucose, 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 gluconeogenesis in the short term by decreasing futile cycling which occurs at the pyruvate kinase and phosphofructokinase 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 compensated 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. References

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