449

Joutrnal of Physiology (1991). 436. pp. 449-468 With 3 figures Printed in Great Britain

RELEASE OF GLUCOSE FROM THE LIVER OF FETAL AND POSTNATAL SHEEP BY PORTAL VEIN INFUSION OF CATECHOLAMINES OR GLUCAGON

BY RICHARD S. K. APATU AND RICHARD J. BARNES From the Physiological Laboratory, Downing Street, Cambridge, CB2 3EG

(Received 23rd November 1989) SUMMARY

1. The blood flow to the liver in fetuses near term, newborn and adult sheep was measured by the Fick principle, using radionuclide-labelled plastic microspheres, before and during infusion of adrenaline, noradrenaline or glucagon. 2. Glucose output and lactate consumption by the liver in sheep of each age group were calculated by application of the Fick principle using the concentration gradients of these metabolites measured in blood samples obtained, simultaneously with blood flow measurements, from catheters chronically implanted in the inflow and outflow vessels of the liver. 3. Catecholamines were infused into the portal vein of fetuses near term at a rate comparable with that at which they are known to be secreted in the sheep fetus during moderate to severe hypoxia. The cardiovascular and metabolic responses to these infusions were found to be comparable with those that occur in the fetus during hypoxia. 4. Catecholamines increased glucose output from the liver in all except the immediate post-partum animals. Catecholamines were less effective than glucagon in promoting glucose release. The mean increments in glucose output during adrenaline infusion were 0055+0-015 mmol min- (100 g liver)-1 in the fetus, 0-122+ 0024 mmol min- (100 g)-1 in the 2-week-old lambs, 0078+0019 mmol min-' (100 g)-' in young lambs and 0-049+0-012 mmol min-' (100 g)-' in the adult sheep. During glucagon infusion the mean glucose output increments were 0-146 + 0023 mmol min-1 (100 g)- in the fetus, 0-274+0-085 mmol min- (100 g)-1 in the 2-week-old and young lambs and 0 180+0-054 mmol min-' (100 g)-1 in the adult. Adrenaline was more potent than noradrenaline, suggesting that the major glycogenolytic response might be f-receptor mediated. 5. In the immediate newborn period the output of glucose from the liver was high (0-20 + 0-05 mmol min-' (100 g liver)-' and was not statistically significantly increased by infusion either of glucagon or of catecholamines which resulted in similar increments of glucose output of about 04128 +0-133 mmol min-' (100 g)-1. It is probable that the high output of glucose reflected the high endogenous circulating levels of catecholamines and glucagon in these animals at birth and that further infusions failed to add significantly to the already near-maximal glucose release. 6. The relative stability of fetal liver glycogen stores may reflect not only an M S 8096 15

PH Y 436

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R. S. K. APATU AND R. J. BARNES

absence of high levels of circulating catecholamines and glucagon in the resting fetus but also may indicate a deficiency in either the receptor mechanism for catecholamines or of the intracellular second messenger system for glycogenolysis. The greater effectiveness of glucagon (than adrenaline) as a mediator of glucose release in these animals tends to support the idea of a fl-receptor rather than a second messenger deficiency. It is postulated that a deficiency of hepatic ,-adrenergic receptors, provided that it can be corrected in time for birth, would help to protect glycogen stores in the liver in utero. INTRODUCTION

Many in vitro studies have shown that catecholamines stimulate release of glucose from the liver (for review see Hems & Whitton, 1980). In vivo administration of catecholamines increases blood glucose concentration in fetal and postnatal sheep (McClymont & Setchell, 1956; Jones & Ritchie, 1978). In near-term fetal sheep ritodrine, a fl2-adrenergic agonist decreased hepatic glycogen by 50 % (Warburton, Parton, Buckley, Cosico & Saluna, 1988). It has always, therefore, been assumed that the major cause of the hyperglycaemia resulting from catecholamine infusions in vivo is an increase in the release of glucose from the liver, although it has also been thought possible that reduced peripheral utilization of glucose might play a part. The immature fetal adrenal synthesizes very little adrenaline but, as the gland matures, the induction of phenyl-ethanolamine-N-methyl transferase (PNMT) under the influence of cortisol enables the medulla to methylate noradrenaline into adrenaline and, although noradrenaline remains the predominant amine stored, the sheep fetus near term is able to release both adrenaline and noradrenaline, e.g. during fetal hypoxia (Comline & Silver, 1961; Comline, Silver & Silver, 1965; Silver & Edwards, 1980; Cohen, Piasecki, Cohn, Young & Jackson, 1984). This response is mediated by the splanchnic nerve, direct stimulation of the adrenal medulla occurs only in extreme hypoxia which is incompatible with fetal survival (Silver & Edwards, 1980). There is an increase in sympathetic activity in the fetus during maternal labour and at birth, shown particularly by large increases in concentrations of circulating plasma catecholamines at that time (Eliot, Klein, Glatz, Nathanielsz & Fisher, 1981). It has been suggested that circulating catecholamines might stimulate glucose release from the liver at birth (Dawes & Shelley, 1968; Jones & Ritchie, 1978; Apatu & Barnes, 1991). The role of glucagon in vivo in fetal life appears to be less important. There is very poor stimulation of glucagon secretion by the fetal pancreas by L-alanine and hypoglycaemia which are potent secretagogues in adult animals (Chez, Mintz, Epstein, Oakes & Hutchison, 1974). Although glucagon is a more effective glycogenolytic agent in adult liver than catecholamines (Sokal, Sarcione & Henderson, 1964), pharmacological doses of glucagon are needed to cause hyperglyeaemia and endogenous glucose output in the fetus (Devaskar, Ganguli, Syer, Devaskar & Sperling, 1984) and this glucagon resistance is known to persist until after birth (Ganguli, Sinha, Sterman, Harris & Sperling, 1983). There have, however, been no in vivo investigations to test whether circulating catecholamines can stimulate glucose release from the liver of the adult sheep and

PER INA TAL HEPA TIC GL YCOGEN OL YSIS IN VIIVO

451

certainly no such studies have been made of the newborn. Accordingly the effects of catecholamines on in vivo hepatic glucose output and lactate consumptions were investigated in chronically catheterized fetal, newborn and adult sheep. METHODS

Animals Wtelsh Mountain ewes of known gestational age, lambs and adults from the same flock were used in these studies. The housing, feeding and pre-operative treatment have been described elsewhere (Comline & Silver, 1972; Apatu, 1985; Apatu & Barnes. 1991).

Surgical procedures and anaesthesia Full details of the methods for insertion of intra-vascular catheters and Caesarean delivery have already been published (Apatu & Barnes, 1991). Insertion of catheters and sampling techniques In fetuses, newborn lambs, older lambs and adult sheep catheters were inserted into the inferior vena cava, aorta, one or more tributaries of the portal vein and one or more of the hepatic veins. In addition, catheters were introduced from a peripheral artery into the left ventricle in animals studied postnatally and into the umbilical vein in fetuses studied in utero. The techniques for obtaining blood samples under aseptic conditions have been fully described elsewhere (Comline & Silver, 1972).

Experimental protocols Five groups of animals were studied. They were: (a) mature fetuses in utero (age 135+2 days, n = 7) studied at least 3 days after surgery, (b) newborn animals delivered by Caesarean section (age 137 + 2 days, n = 5) following infusion of adrenocorticotrophic hormone (ACTH; Synacthen, Ciba-Geigy; 0-5 mg for 48 h) to ensure maturation of the lungs, studied within 6 h of birth, (c) lambs, 1-2 weeks old (n = 7), studied at least 3 days after surgery, (d) older lambs, at least 6 weeks old (n = 5), studied at least 3 days after surgery and, (e) adult sheep (n = 7) studied at least 3 days after surgery. In each group in each animal the study consisted of up to six measurements of blood flow and metabolism using radioactive microspheres and the Fick principle (Apatu & Barnes, 1991). Measurements of blood flow and metabolism were made serially (a) before any infusion, (b) after infusion of either adrenaline or noradrenaline for 10 min. (c) 30 min after stopping the infusion, (d) after 10 min of an infusion of the catecholamine not infused at (b), (e) 30 min after stopping the second infusion, (f) after 10 min of an infusion of glucagon.

Catecholamine infusions in fetuses Either adrenaline (1/1000 adrenaline BP, MacCarthy's Ltd, London) or noradrenaline (1/1000 noradrenaline BP (Levophed, Winthrop, London) was diluted with saline, protected from light, and infused into the portal vein at a rate of 0-388 ml min-' so that each fetus received 0 5 ,ug adrenaline kg-' min-' for 10 min. This was to mimic the known rate of maximum secretion from the adrenal medullae during moderate to severe hypoxia in fetal animals (Comline et al. 1965). An initial rate of infusion of 10 ml min-' for approximately 30 s was used to clear the dead space of the portal vein catheters (0 5 ml on average). Bradycardia, usually within 30 s of the start of the rapid infusion, signalled entry of catecholamines into the general circulation and immediately the infusion rate was reduced in order to deliver 05 yum min-' kg-' to the animal. Catecholamine infusions in postnatal sheep Catecholamine infusions and blood flow measurements in newborn lambs delivered by Caesarean section and studied within 6 h ('immediate newborns') and older postnatal sheep were performed under a protocol which was similar to that used in fetal experiments. The dose for catecholamine infusion in postnatal animals was 05 jtm min-' kg-', which was identical to the dose in the fetus, but was chosen, following preliminary dose-response experiments, 15-2

452

R. S. K. APATU AND R. J. BARNES

because at that dose there were significant changes in the plasma glucose concentration with only moderate cardiovascular effects of the infused catecholamines (see Figs 1 and 2). In a few newborn animals a higher infusion rate of 10 jug min-' kg-' was used.

Glucagon infusions Glucagon (Novo, Copenhagen) was used as a final test infusion (to see if the liver was still able to release glucose) in all animals. It was infused at the same rate as the catecholamines (0 5 ,tg min-' kg-') under an identical protocol of sampling and blood flow measurement. Although the concentration of plasma glucagon was not determined it was recognized that this dose would result in concentrations of plasma glucagon much higher than would occur under any physiological conditions in the fetus. Measurements during infusion regimes Abdominal aortic blood pressure and, in some fetuses and postnatal sheep, hepatic vein pressures were measured during the experiments. Umbilical vein pressures were measured in some fetuses. Pressure transducers (Elcomatic, EM 750) were calibrated to read zero when at the estimated level of the right atrium in postnatal animals and the pressure in the amniotic space was used as the reference zero in fetuses. The heart rate was monitored continuously. Samples of blood from umbilical, portal and hepatic veins and abdominal aorta were taken immediately before a blood flow measurement and were handled as previously described (Apatu & Barnes, 1991). After the measurement of the blood flow a volume of blood equivalent to that removed (approximately 10-12 ml in fetuses and immediate newborns, 25-30 ml in older lambs and adults) was infused into the animal using donor blood from another fetus or adult, as appropriate.

Biochemical analyses, determination of hepatic blood flow and glucose and lactate consumption Sample preparation for hepatic blood flow estimation, glucose output and lactate consumption by the liver in fetal and postnatal sheep have been described elsewhere (Apatu & Barnes, 1991). Measurements of plasma concentrations of catecholamines The catecholamine assay, modified from that of Peuler & Johnson (1977), has been described in detail by Silver, Ousey, Dudan, Fowden, Knox, Cash & Rossdale (1984). The coefficient of variation between assay batches was less than 12% and within assay batches was about 10%.

Statistical analyses Statistical analyses were after the methods of Snedecor & Cochran (1967). Student's t test for paired data was used to compare the effects of infusion in animals of the same age group, and Student's t test for unpaired data was used when animals of different age groups were compared. Where appropriate analysis of variance was used to test statistical significance observed. Differences with P < 0 05 were considered to be statistically significant. All values are given as means + S.E.M. RESULTS

Dose-response experiments The changes in blood pressure, heart rate, plasma lactate and plasma glucose concentrations during dose-response experiments were similar in lambs between 1 and 2 weeks old and those between 3 and 9 weeks old were therefore analysed together. Figure 1 shows the mean heart rate, blood pressure (pulsatile and mean) and portal and hepatic vein pressures in lambs between 1 and 9 weeks old during dose-response experiments.

PERINA TAL HEPA TIC GL YCOGENOL YSIS IN VIVO

453

Figure 2 shows the changes in plasma lactate and plasma glucose concentrations in arterial, portal and hepatic vessels in young lambs during infusion of catecholamines at different rates. Either catecholamine increased systolic blood pressure significantly only when infused at 25 ,ug min-' kg-'. However, diastolic and mean blood pressures were 200 _ 0-0 Systolic 180 v-v Mean 160- *-* Diastolic I 140EE 120_ < 100_ v-7- -V-sQ 3 80 -

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increased significantly at a 05 jug min-1 kg-' dose of noradrenaline but only at 25 ,ug min-' kg-' for adrenaline. At the doses used adrenaline did not have a significant effect on heart rate. By contrast, noradrenaline infused at 2-5 ,tg min-' kg-' decreased heart rate significantly (Fig. 1). There was an increase in heart rate within 30 s when the infusion of either of the catecholamines was stopped. The heart rate then fell to the control value within 30 min after adrenaline infusion. The immediate increase with noradrenaline, which had significantly lowered heart rate, was greater (from 123+12 to 207+13 beats min-') than that with adrenaline (from 176+ 16 to 222 + 15 beats min-') and did not return to the basal level even after 30 min. By contrast adrenaline was more effective than noradrenaline in raising circulating plasma glucose concentrations and also in increasing plasma lactate.

R. S. K. APATU AND R. J. BARNES

454

The inset in Fig. 2 shows the linear relation between mean change in plasma glucose concentration and the logarithm of adrenaline dose (log-dose) infused over 0 5 ,tg min-' kg-1 to 1 0 jug min-' kg-' (y = 91x + 65-7; r = 0708; P < 0-002; n = 26 arterial values). A loss of linearity is observed when the infusion dose was increased to 2-5 4ug min-' kg-'. 14

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As a result of these experiments a dose of 0 5 ,ug min-1 kg-l for both catecholamines was chosen for all except a few of the immediate newborns since it produces metabolic effects with little or no cardiovascular change.

Plasmna catecholamine concentrations Fetuses and newborn lambs immediately after delivery Resting plasma concentrations of adrenaline were 0-61+±0-73 ng ml-l and of noradrenaline were 0~54 + 066ng ml-. During infusion of the appropriate catecholamine the concentrations obtained in fetal plasma were 132 +074 ng mP1 (adrenaline) and 217 +0-85 ng ml (noradrenaline). The concentrations of adrenaline and noradrenaline in newborn lambs immediately after delivery were 2e9+1p1 ng ml (n = 5) and 5923M + 8 ng ml-l (n = 4). These values were significantly higher than the basal catecholamine concentrations in the

PERINA TAL HEPA TIC GL YCOGEANOL YSIS IN VIVO

455

mature fetus (P < 0-05, unpaired t test) and than those observed at rest in any of the other animal groups studied. During infusion of adrenaline or noradrenaline plasma concentrations increased to 9-7 + 2-3 ng ml-' and 11 6 + 3 3 ng ml-' respectively. Adults In two animals the average concentration of adrenaline rose from 0Q20 ng ml-1 at rest to 144 ng ml-' and that of noradrenaline from 098 to 231 ng ml-1 during infusion.

The effects of catecholamines and glucagon on blood pressure and heart rate Adrenaline and noradrenaline The infusion of either catecholamine led to varying degrees of increase in blood pressure and bradycardia in the fetal and postnatal animals. These were similar to the changes observed during the dose-response experiments in lambs described above. In fetuses either catecholamine increased systolic and diastolic blood pressure significantly. The mean blood pressure rose from about 50 to 60 mmHg with either infusion and an attendant fall in heart rate from about 170 to 140 beats min-' was also significant. Changes in blood pressure and heart rate were observed within 30 s of the start of the rapid infusion of catecholamines used to clear the dead space of the catheters (see Methods). In newborn lambs the increase in blood pressure during the infusion of either catecholamine became significant only when the rate of infusion was increased to 0 jtg min-' kg-'. Attendant falls in heart rate with each catecholamine infusion were not statistically significant. In adult sheep while the infusion of adrenaline did not have any significant effects on blood pressure and heart rate noradrenaline infusion increased systolic, diastolic and mean blood pressure. The increase in blood pressure was associated with a significant fall in heart rate.

Glucagon In fetal sheep the mean blood pressure was unchanged during the infusion of glucagon. Although a fall in systolic blood pressure from (57 4 + 2-3 to 54-6 + 2-2 mmHg) was not statistically significant (P = 0 085) there was a significant fall in diastolic blood pressure which was associated with a significant increase in heart rate from 174 to 194 beats min-m. Glucagon infusion decreased systolic, diastolic and mean blood pressure but had no effect on the heart rate in the immediate newborn. In adult sheep the significant falls in systolic, diastolic and mean blood pressures observed during glucagon infusion were associated with a significant increase in heart rate.

The effects of infusions of catecholamines on blood pH and blood gas tensions In the fetuses arterial pH remained stable at between 7-31 and 7-45. Arterial oxygen tension varied between 19 and 30 mmHg but showed no consistent trends

456

R. S. K. APATU AND R. J. BARNES

and no correlation was noted between changes in blood gas tensions or pH with the different infusions. Similarly, in postnatal sheep there were no significant changes in blood gas tensions or pH during infusions. The effect of infusion of catecholamines or glucagon on blood flow to the liver Fetuses Adrenaline. Mean umbilical venous return was 227 + 30 ml min-1 (kg fetal weight)-' (n = 7) in the control period and 50 % of it bypassed the liver through the ductus venosus. The placental resistance calculated for four fetuses in which umbilical venous pressures were measured was 1-66 + 036 mmHg min ml-' (100 g fetus)-1. During adrenaline infusion the umbilical blood flow was 165+17 ml min-1 kg-' (n = 7) and the placental resistance was 272+030 mmHg min ml-' (100 g fetus)-' (n = 4). The differences in umbilical venous return and placental resistance were not statistically significant. Blood flow to the whole liver was 4401 +41V7 ml min-' (100 g liver)-'. Umbilical venous return contributed 75 % of this total flow. Left and right lobes of the liver were perfused by nearly equal volumes of umbilical blood (52-7 % to the left and the rest to the right). The percentage of umbilical venous return distributed to the liver fell by 14 % (to 366 + 6-6 %) during adrenaline infusion. Although the change was not statistically significant (P = 0 09) the reduction in flow of umbilical blood to the right liver (from 305-5+38-0 ml min-1 (100 g)-1 to 166-5+4341 ml min-1 (100 g)-1) was significant. The reduction in umbilical blood flow to the left lobe was more variable and was not statistically significant. Although the reduction in portal flow to the right lobe of the liver (from 240 7+53-8 ml min-1 (100 g)-1 to 130-2+20-1 ml min-' (100 g)-') was not statistically significant (P = 0 08), together with the fall in umbilical blood flow it led to a significant fall in total blood flow (from 546 + 80-2 ml min-1 (100 g)-1 to 296-7 +56-6 ml min-' (100 g)-f) to the right liver. Noradrenaline. Umbilical venous return to the fetus was unchanged during noradrenaline infusion and the placental resistance was not increased significantly. The umbilical blood flow to the whole liver was maintained and changes in umbilical flow to each lobe were not statistically significant or consistent. Portal blood flow was significantly decreased by noradrenaline infusion. Although the umbilical blood flow to the right lobe was maintained, the fall in portal flow (from 281V8+39-3 ml min-' (100 g)-1 to 1600+ 30-4 ml min-1 (100 g)-1) was enough to cause a significant reduction in total blood flow to the right lobe. Blood flow to the whole liver however, was unchanged by noradrenaline infusion. Glucagon. Glucagon infusion resulted in a significant increase in portal blood flow. Although umbilical blood perfusion of the right lobe was significantly reduced a large increase in portal flow (from 2834 +44-2 ml min-' (100 g)-' to 408-5+55-8 ml min-' (100 g)-') maintained total blood flow to the right lobe. An apparent decrease in umbilical perfusion to the left lobe was not statistically significant. Blood flow to the whole liver was also apparently maintained.

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457

Newborns immediately after delivery There were no significant changes in total blood flow to the liver with adrenaline, noradrenaline or glucagon infusions into the portal vein in newborn animals immediately after delivery. Total blood flow to the liver at birth in four newborns was 216+42 ml min-' (100 g)-1 of which arterial flow was 33X5+ 13-9 ml min-' TABLE 1. Plasma lactate concentrations and uptake of lactate, glucose and oxygen from the umbilical circulation by the fetus during catecholamine or glucagon infusions. Values are means, +S.E.M. in parentheses, n = 7

Fetal uptake (mmol min-' kg-')

Plasma lactate (mmol I`)

UV Adrenaline Control

Infusion

Noradrenaline Control Infusion

Glucagon Control

PV

HV

Art

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Glucose

Oxygen

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1-57 (0 04) 2-44**

0-042 (0-015) 0-012

0-022 (0-004) 0-023

(0-17)

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(0-1I9)

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(0-010)

0-348 (0 039) 0-180** (0 028)

2-46 (0-27) 2-77 (0-41)

2-37 (0-29) 2-51 (0-41)

2-22 (0 28) 2-60 (0 42)

2-30 (0-29) 2-64 (0 40)

0-029 (0 009) 0-023 (0-010)

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0-230 (0 042) 0-227 (0-035)

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0-310 0-020 0-008 (0-060) (0033) (0-005) 0 200 0 -0 005 (0-039) (0-013) (0-004) Art = artery. **P < 005; value

(100 g)-' thus forming 14-0 + 3-5 % of the total flow. During adrenaline infusion total blood flow to the liver was 185 + 23 ml min-' (100 g)-1 and arterial flow (24-6 + 5-4 ml min-' (100 g)-') was 15-5 + 5% of the total flow. Arterial and total blood flow to the liver during noradrenaline infusion were 44-1 + 12-0 ml min-' (100 g)-1 and 247-7 + 73-4 ml min-' (100 g)-'. Apparent decreases in both arterial and portal flows that occurred when adrenaline was infused were insignificant. Although noradrenaline, like adrenaline, had no effect on total liver blood flow it appeared to increase the fraction of arterial blood flow to total blood flow. Thus adrenaline did not alter the fraction of the total liver blood flow contributed by the hepatic artery (from 14-0 + 3-5 % to 15-5 + 5-5 %, P > 0 50) while noradrenaline caused a marginal increase in the arterial contribution to total liver blood flow (from 13-2 + 3-5 to 19-5 + 5-5 %, P = 0111). This is similar to the effect on liver blood flow seen following surgical stress (Greenway & Stark, 1971) and raises the possibility that autonomic rather than autoregulatory factors are influencing the flow in the surgically stressed animal. Changes in portal blood flow during glucagon infusion were small and insignificant.

458

R. S. K. APATU AND R. J. BARNES

Young lambs and adult sheep There were no significant changes in blood flow (which remained at about 200 ml min-1 (100 g)-1) to the liver in the older postnatal sheep during either adrenaline or noradrenaline infusion. Small increases in blood flow with glucagon 2.5 0--O Hepatic v ein

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infusions in the adult were statistically insignificant. Arterial contribution to liver blood flow always remained at less than 10%. The effect of infusion of catecholamines or glucagon on plasma glucose and lactate concentrations, liver glucose output and lactate consumption Fetus Figure 3 shows the mean (+S.E.M.) plasma glucose concentrations and glucose output from the right liver in the fetal lamb during catecholamine or glucagon infusions. Table 1 shows the plasma lactate concentrations and amounts of glucose, lactate and oxygen taken up by the fetus from the umbilical circulation during catecholamine or glucagon infusions. Glucose. All three infused substances produced significant increases in plasma glucose concentrations in arterial, umbilical, portal and hepatic venous samples. Adrenaline and glucagon increased arterial plasma glucose concentration by 100 and 70 % respectively. The increase during noradrenaline infusion, although

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461

Noradrenaline similarly increased glucose output from 0X146 + 0099 to 0X346 + 0148 mmol min-1 (100 g liver)-'. The wide range in values again made the change statistically insignificant. Lactate consumption was 0 109 + 04128 mmol min-1 (100 g)-1 during noradrenaline infusion and was therefore similar to the control value Glucagon-induced increases in glucose output were also non-uniform and insignificant statistically. No change was observed in lactate consumption.

Newborns (2 weeks old) and young lambs (between 3 and 9 weeks old) The changes in plasma glucose and lactate concentrations in lambs in these groups during either adrenaline infusion or noradrenaline infusion during blood flow experiments are shown in Table 2. Hepatic glucose output. Adrenaline and noradrenaline: in lambs about 2 weeks old resting hepatic glucose output was 0 114 + 0-025 mmol min-1 (100 g)-' and rose significantly (P < 0 05) to 0-236+0-035 mmol min-1 (100 g)-1 when adrenaline was infused. However, the increase (to 0-130 mmol min-' (100 g)-1) during noradrenaline infusion was not statistically significant (P > 0-05). A similar pattern of a significant increase in hepatic glucose output during adrenaline infusion (from 0-072+0-025 mmol min-1 (100 g)-1 to 0-150+0-016 mmol min-1 (100 g)-1, P < 0-05) but not during noradrenaline infusion (to 0-103 + 0-03 mmol min-1 (100 g)-', p > 0-05) was observed in five lambs between 3 and 9 weeks old. Glucagon: in four lambs (two between 1 and 2 weeks old and two between 3 and weeks 9 old), glucagon was infused as a first infusion after control measurements. Liver glucose output increases were large (P < 0-02). From basal outputs of 0-056 + 0-013 mmol min-' (100 g)-1 hepatic glucose output rose to 0-330 + 0-086 mmol min-' (100 g)-'. Hepatic lactate consumption. The liver in lambs between 2 and 9 weeks old consumed 0-061 + 0-008 mmol min-' (100 g)-1 of lactate during the control period and 0-108+0-023 mmol min- (100 g)-1 and 0 101 +0-022 mmol min- (100 g)-l during adrenaline and noradrenaline infusions respectively. There were thus no significant differences in lactate uptake during catecholamine infusions. In four lambs infused with glucagon, lactate consumption by the liver remained at 0-089 + 0-027 mmol min-' (100 g)-'. Adults The changes in plasma glucose and lactate concentrations in adult sheep during either adrenaline infusion or noradrenaline infusion during blood flow experiments are also shown in Table 2. Plasma glucose concentrations. Adrenaline infusion increased plasma glucose concentration significantly in all vessels sampled. Noradrenaline infusion also apparently increased plasma glucose concentration in all vessels. However, the increases were smaller and achieved statistical significance only in hepatic venous blood samples. Glucagon infusion gave the biggest increases in plasma glucose concentrations and its effects were significant in all vessels.

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Heptic glucose output. Adrenaline increased glucose output by the adult liver significantly (from 0-038+0013 mmol min- (100 g)-1 to 0090+0019 mmol min-' (100 g)-1) but, while noradrenaline infusion appeared to further increase glucose output above the relatively high level at which it had remained after the end of adrenaline infusion this second increase (from 0-067 + 0032 mmol min-' (100 g)-1 to 0098 + 0028 mmol min-' (100 g)-1) was not statistically significant. Noradrenaline was used as a first catecholamine infusion in only two adult sheep. The increase in glucose output from the liver was about half that following adrenaline. Glucagon infusion led to a significant increase (P < 0 05) in glucose output (from 0028+0X018 mmol min- (100 g)-l to 0X222+0X042 mmol min- (100 g)-') by the liver in adult sheep. Plasma lactate concentration and hepatic lactate uptake. There was very little variation in plasma lactate concentrations and hepatic lactate uptake during infusions of catecholamines or glucagon, although plasma lactate changes in some vessels were significant (Table 2). DISCUSSION

The response of the sheep (a ruminant) to infusions of catecholamines is similar to that of many other species (including non-ruminants) studied previously (McClymont & Setchell, 1956; Himms-Hagen, 1972) and includes varying degrees of hyperglycaemia and cardiovascular changes. In the present study adrenaline was more potent in inducing hyperglycaemia than noradrenaline but less potent in its cardiovascular effects. For both catecholamines the cardiovascular changes were minimal at doses below 1 0 jtg min-' kg-', higher doses were therefore not used because of their generalized cardiovascular effects. The effects of catecholamines on blood flow, heart rate and blood pressure in the fetus The cardiovascular changes observed during the infusion of catecholamines into the portal vein of sheep fetuses were similar to those which have been seen during fetal hypoxia (Jones & Robinson, 1975; Bristow, Rudolph, Itskovitz & Barnes, 1983). The present results show that some of the catecholamine infused into the portal vein escaped destruction by the fetal liver. It is not known if activities of monoamine oxidase (MAO) and catecholamine 0-methyl transferase (COMT) fall towards term and increase to adult levels only after birth in the sheep fetus in a similar way to that found in the rat and rabbit fetus (Parvez & Parvez, 1980). The fall in portal blood flow and increase in mean blood pressure, seen with either catecholamine, is consistent with splanchnic and peripheral vasoconstriction. In addition there was an increased shunting of umbilical blood through the ductus venosus. These changes which have also been observed in hypoxic fetuses support the hypothesis that increased sympathetic nerve activity, partly directly and partly through release of circulating catecholamines, increases the amount of umbilical blood shunted through the ductus venosus and redistributes fetal cardiac output to the upper body and the brain (Heymann & Rudolph, 1967; Edelstone & Rudolph, 1979). This would protect the central nervous system by providing it with welloxygenated umbilical blood (Edelstone & Rudolph, 1979; Bristow et al. 1983).

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Although a direct stimulation of chemoreceptors in the carotid sinus by low arterial oxygen tension (Lewis, Donovan & Platzker, 1980) has been suggested to be responsible for the bradyeardia during fetal hypoxia, the bradyeardia in these fetuses (which were not hypoxic) closely followed increases in blood pressure and was probably through a vagal reflex via the stimulation of baroceptors (Jones & Robinson, 1975; Jones & Ritchie, 1978). The maintenance of total umbilical venous return suggests that the rise in arterial blood pressure is greater than the increase in placental resistance during catecholamine infusion and this view is consistent with the limited number of measurements of placental resistance that it was possible to make in this study. The finding of a significant fall in umbilical venous blood flow to the right liver but not the left liver is contrary to that of Zink & Petten (1980) who found a reduced flow of umbilical venous blood to the left liver when they infused catecholamines into fetuses. However, their infusions were made into the umbilical venous blood (which is evenly distributed to the right and left lobes of the liver) and would deliver a higher concentration of catecholamine to the left lobe, undiluted by portal venous blood. In the present studies a higher concentration of catecholamines will reach the right liver which receives all the portal drainage (Bristol et al. 1983). In adult animals glucagon is known to cause vasodilation in the splanchnic bed (for review see Farah, 1983). It seems that glucagon infusion in the fetus also induces a decrease in the vascular resistance of the splanchnic bed. There was a fall in blood pressure (with a tachyeardia which was probably in response to this fall) and blood flow to the gut was significantly increased.

Changes in plasma glucose concentration during infusions The changes in plasma glucose concentrations during portal vein infusion of catecholamines and glucagon agree with previous studies in which catecholamines were infused into a peripheral vein. Noradrenaline, when compared with either adrenaline or glucagon, has been shown to be a poor hyperglyeaemic agent in many species, for example in fetal sheep (Jones & Robinson, 1975) and the young calf (Comline & Edwards, 1968; Edwards, 1971). Adrenaline, however, increased fetal blood glucose concentrations significantly when infused at rates of 1 0 ,ug min-1 kg-1 (Comline & Silver, 1972; Jones & Robinson, 1975). The differential effects of adrenaline and noradrenaline have not been explained in the sheep but preliminary studies (Apatu, Barnes & Martin, 1986) indicate that hepatocytes in the sheep lack oc-receptors and the effect of catecholamines on glycogenolysis is probably via fl-receptors. Adrenaline has a greater affinity for fl-receptors. Prolonged (24 h) administration of ritodrine, a fl2adrenergic agonist, to near-term fetal sheep decreased hepatic glycogen by 50 % (Warburton et al. 1988). Glucose concentrations were higher in portal vein plasma than in arterial plasma in newborn lambs which had previously suckled, and in young lambs, because these animals were absorbing glucose from ingested milk. In immediate newborn lambs, which had not suckled and therefore had no glucose to absorb from the gastrointestinal tract, and in the adult, the glucose concentrations in portal vein plasma were lower than in arterial samples. In adult ruminants also no glucose is

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available from the gut because it is fermented by the symbiotic bacteria in the reticulo-rumen (Ballard, Hanson & Kronsfeld, 1969). Thus, in both the immediate newborn and in the adult the liver, by glycogenolysis or gluconeogenesis, is primarily responsible for the provision of blood glucose. The effect of catecholamines and glucagon on liver glucose output in fetal and postnatal sheep The liver of the fetal sheep at rest is in zero glucose balance. Both adrenaline and noradrenaline stimulate glucose output from the fetal liver, and both reduce liver blood flow but adrenaline causes a bigger decrease in liver blood flow than noradrenaline. This suggests that their metabolic effects on the liver are direct and not secondary to variations in blood flow. Glucagon infusion resulted in a dramatic output of glucose from the liver in the fetus. The dose of glucagon used was clearly pharmacological and the direct infusion into portal blood would deliver enough hormone to the liver to overcome the relative glucagon resistance found in the fetus by Devaskar et al. (1984). However, in the isolated perfused liver of the adult rat glucagon has been found to be much more effective than catecholamines in releasing glucose (Sokal, Sarcione & Henderson, 1964). It would appear from these studies that the same is true for the sheep fetal liver in vivo. In contrast to the fetal liver, newborn and adult animal livers release glucose at rest. The output in the newborn was particularly high. There was no significant increase from this high level of output in the newborn with any of the three infusions. The apparent failure of catecholamines and glucagon to increase the output of glucose from the liver in the newborn group must be interpreted in context. In man and many other species there is a large increase in circulating catecholamine concentrations at birth (Lagercrantz, 1984; Silver, Ousey, Dudan, Fowden, Knox, Cash & Rossdale, 1984). It is known that glucagon concentrations also rise at birth (Girard & Ferre, 1982). Indeed the catecholamine concentrations determined in some of the sheep fetuses delivered by Caesarean section were high. In vitro studies in the rat indicate that stimulation by catecholamines of glucose output from hepatocytes from newborn liver is mediated by an increase in cyclic AMP (Blair, James & Forster, 1979). It is most likely that high plasma catecholamines and glucagon in the newborn, within the first few hours of birth, will maximally stimulate cyclic AMP production in the liver. The rapid glucose release measured in the newborn without any infusions may be a reflection of this situation. It is likely that receptors to catecholamines in the liver are saturated at these high concentrations and infusions of catecholamines will have no further effect. Although glucagon is known to be important for glycogenolysis at birth (see Girard & Ferre, 1982), the newborn rat liver has been found to be refractory to stimulation by glucagon. Prolonged exposure of liver cells in vitro to either adrenaline or glucagon has been shown to diminish the ability of either hormone to stimulate adenosine 3'5'-monophosphate (cyclic AMP) formation (Gurr & Ruh, 1980). It is not unlikely that a similar 'down-regulation' of sensitivity to glucagon and adrenaline occurs at birth. Adrenaline significantly increased glucose output from adult liver. Noradrenaline

PERINA TAL HEPA TIC GL YCOGENOL YSIS IN VIVO 465 was without any further effect. In adult animals noradrenaline, if infused first also increased glucose output from the liver, although less markedly than adrenaline. In adult sheep, cat, dog and the newborn calf direct stimulation of sympathetic nerves mobilizes glucose from glycogen (Edwards, 1971). Although the physiological role of catecholamines in mobilizing glucose from the liver has been questioned (Sokal, Sarcione & Henderson, 1964), it is generally accepted that the hyperglycaemic effects of catecholamines include glucose mobilization from the liver and many in vitro studies have shown catecholamines to stimulate liver glycogen break-down and glucose release (for review see Hems & Whitton, 1980). Both catecholamines exert their action in mobilizing glucose through the same kind of adrenergic receptor in sheep liver (Apatu et al. 1986). However, the hyperglycaemic effect of noradrenaline is less than that of adrenaline. The greater efficacy of adrenaline in increasing plasma free fatty acid concentration via ,adrenergic receptors (Jones & Ritchie, 1978) could contribute to its enhanced hyperglycaemic effect. Glucose utilization by peripheral tissue is inhibited by high plasma free fatty acid concentration (Newsholme, Randle & Manchester, 1962). Both adrenaline and noradrenaline would also reduce plasma insulin concentration by an a-adrenergic effect (Jones & Ritchie, 1978) and so further reduce the uptake of glucose by insulin-dependent peripheral tissue (Young & Landsberg, 1977). In the sheep fetus the poor mobilization of glucose by infused catecholamines compared to glucagon might be explained either by extracellular or by intracellular factors. Failure of catecholamines per se to mobilize liver glycogen might be due to inadequate amounts of catecholamines reaching the liver cells or inhibition of the action of the catecholamines by antagonistic hormones. However, in these studies catecholamines were infused into the portal vein (which drains directly into the liver) at the rates comparable to endogenous release by the fetal adrenal medulla during spontaneous or experimentally induced hypoxic episodes. By contrast the adrenal medulla releases its output into the general circulation. The placenta clears at least 50 % of the catecholamines reaching it from the fetus (Jones & Robinson, 1975) and thus the umbilical vein blood catecholamine concentration is low. Since the hepatic artery contributes negligible amounts of the total blood flow to the fetal liver, catecholamine delivery to the liver via the hepatic arteries must be very small. It was, therefore, likely that amounts of catecholamines reaching the liver during the experiments might well have exceeded those even during times of maximum adrenal release of about 1 jtg min-' (kg fetal weight)-' (Comline & Silver, 1961). The local concentrations of noradrenaline released at the sympathetic nerve endings within the fetal liver would, however, be high and under circumstances of severe stress might be high enough to result in glycogen mobilization. The importance of hepatic innervation in relation to liver function in the fetus in unknown. The role of insulin, the most likely effective antagonist of catecholamine action on the fetal liver, in the control of glucose homoeostasis in the fetus is still very much undetermined. The near-term fetal pancreas can be stimulated by hyperglycaemia, hyperaminoacidaemia and hyperlipidaemia; conditions similar to those which provoke insulin release from the adult pancreas (Fowden, 1980a). Hyperglycaemia, caused by adrenaline in particular, may well induce an increase in insulin levels

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which would antagonize subsequent effects of adrenaline on the fetal liver. Unfortunately the amounts of blood required for accurate determination of insulin in the fetal plasma are quite large (about 200 ,l fetal plasma) and, in this study, insufficient sample remained for insulin assay after other essential measurements had been made. However, the depression of the release of insulin from the pancreas by catecholamines through a-receptors (for review see Malaisse, 1972) has also been observed in fetal sheep (Fowden, 1980b). It is therefore unlikely that insulin levels were much increased during the catecholamine infusion experiments. However, the influence of insulin on the glucose-mobilizing effects of catecholamines on the liver cannot be determined from these experiments. Failure of liver cells to release glucose may be due to failure of the fl-adrenergic receptor-mediated cyclic AMP-protein kinase, the ac-adrenergic receptor-mediated cyclic AMP-independent Ca2+ enzyme activation system, lack of enzyme systems for glycogenolysis, low or absent glycogen stores or lack of interaction by liver cells with catecholamines because of the absence or low levels of catecholamine receptors. Although /l-adrenergic agonists and glucagon have different receptor sites on the liver cell membrane (Tomasi, Koretz, Ray, Dunnick & Marinnete, 1970) their intracellular glycogenolytic effects are mainly through adenylate cyclase-stimulated cyclic AMP formation. Indeed, their pathways of action almost certainly converge. The fact that glucagon caused significantly large increases in the fetal liver glucose output suggests that events distal to the cyclic AMP-stimulated protein kinase activity occur normally in the sheep fetal liver and are similar in magnitude to the comparable events in adult sheep or rat livers. It is also known that glycogen is stored in large amounts in the liver of the sheep fetus in late gestation (Shelley, 1961; Barnes, Fowden, Silver & Comline, 1977; Barnes, Comline & Silver, 1978). It seems unlikely that the infused catecholamines were ineffective due to glycogen lack in the liver of the experimental fetuses and, indeed, the effectiveness of glucagon, given at the end of the experimental protocol, suggests that glycogen stores were more than adequate in these animals. The most likely explanation of the reduced responsiveness to catecholamines in the fetus thus seems to be that there is a deficiency at the receptor level. Experiments designed to test this hypothesis are in progress (Apatu et al. 1986, 1991). We are grateful to P. Hughes, T. Grimes, D. Clarke, J. Knox and R. Tindall for skilled technical assistance. R. S. K. A. was a holder of a British Council (Technical Co-operation Training) grant. REFERENCES

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