Aust. J. BioI. Sci., 1983,36,271-84
Glucose Turnover and Hepatocyte Glucose Production
of Starved and Toxaemic Pregnant Sheep
M. E. Wastney,A,B J. E. WOlffA,C and R. BickerstaffeB
A Ruakura Animal Research Station, Ministry of Agriculture and Fisheries,
Private Bag, Hamilton, New Zealand.
B Department of Biochemistry, Lincoln College, Canterbury, New Zealand.
C To whom correspondence should be addressed.
Abstract
Ewes bearing twins were starved for 10 days during the last month of gestation to induce ovine
pregnancy toxaemia (OPT). Glucose turnover was measl,lred by a primed continuous infusion of
[U_ '4 C]_ and [6- 3 HJglucose at the end of 10 days of starvation (non-susceptible), or earlier when
ewes became recumbent with OPT (susceptible). All ewes were slaughtered at the end of the infusion
and hepatocytes were prepared in order to measure glucose production from different substrates.
Many of the ewes had dead foetuses when slaughtered. Glucose production rates by hepatocytes
with the substrates propionate, lactate or alanine were significantly less from the susceptible ewes
than were those from non-susceptible ewes. These low rates were not stimulated by incubation with
glucagon (10- 8 M), glutamine or glycerol. Rates of glucose turnover and of hepatic glucose production
from all substrates were higher for ewes with dead than with live foetuses. The data support the
hypothesis that pathogenesis of OPT is related to an impairment of hepatic gluconeogenesis, and further
suggest that, in starved pregnant ewes, maternal glucose production may be restrained in the presence
of a live foetus.
Introduction
Ovine pregnancy toxaemia (OPT) is a metabolic disease of polytocous sheep. It
occurs in late gestation following undernutrition and stress, and is characterized by
hypoglycaemia, hyperketonaemia and elevated plasma concentrations of free fatty
acids. A fatty liver is generally found at autopsy. Neurological signs of the disease
include cerebral depression, loss of eye and ear reflexes, ataxia, adoption of unusual
posture, and loss of appetite. As the disease progresses, convulsions may occur but
the ewe generally becomes comatose and recumbent and dies quietly.
The pathogenesis of the disease has been studied by subjecting pregnant sheep to
10 days of starvation during the last month of gestation. Some animals appear capable
of withstanding the challenge, while other succumb with neurological signs similar to
those observed in field cases of OPT (Wolff et al. 1974; O'Hara et al. 1975). The
hypoglycaemia, prevalent during early phases of this disease (McClymont and
Setchell 1955; Reid 1968), is thought to arise from an inadequate rate of gluconeogenesis and is usually considered to be the main metabolic lesion responsible for
pathogenesis (Kronfeld 1970).
0004-9417/83/030271 $02.00
M. E. Wastney et al.
272
The object of our study was to examine the ability of isolated hepatocytes from
starved pregnant ewes bearing twins to synthesize glucose from various precursors.
Hepatocytes were chosen since the liver is the predominant site of gluconeogenesis in
ruminants (Bergman et al. 1970; Kaufman and Bergman 1974) and the effects of
substrates and hormones could be examined in the absence of complex in vivo feedback
controls. There have been several reports on the use of ovine hepatocytes for studying
gluconeogenesis (Clark et al. 1976a, 1976b; Ash and Pogson 1977; Morton et al.
1977; Richardson and Livesey 1979; Weekes et al. 1979) but none pertain to hepatocytes obtained from pregnant sheep. It was anticipated that the hepatocytes should
indicate a gluconeogenic potential of the liver in the intact animal, and glucose
production by the hepatocytes in some animals was compared with glucose turnover
rates measured by a primed continuous infusion of radioactive glucose.
Materials and Methods
Materials
Laboratory chemicals were of analytical grade. Enzymes, substrates and other chemicals were
obtained from Sigma Chemical Co., St Louis, MO, U.S.A., except for collagenase (EC 3.4.24.3
fype CLS, activity 210 units/mg) which was from Worthington Biochemical Corp., Freehold, NJ
U.S.A. Ovine glucagon was a gift from Eli Lilly, Indianapolis, IN, U.S.A. D_[U-14C]- and [6- 3 H]glucose were obtained from Amersham International, and the scintillation counting cocktail
Aquasol was from New England Nuclear, Boston, MA, U.S.A.
Animals and the Experimental Protocol
Experiments were performed on mature Perendale-Romney crossbred ewes which had been
mated on known dates. They were X-rayed at 80 days of gestation to determine the number of
foetuses, and ewes bearing twins were moved indoors into individual pens. They were fed daily on
40 g dry matter/kgo. 7s liveweight of a pelleted ration containing, on an air-dry basis, lucerne (600
g/kg), barley (300 g/kg), linseed meal (50 g/kg) and molasses (50 g/kg) with no less than 17 % crude
protein.
The experiments were undertaken over 2 years. In the first year ewes bearing twins (n = 23)
were starved at about 130 days of gestation, but had free access to water. The animals were slaughtered
for hepatocyte studies either when they became recumbent with signs characteristic of OPT (designated susceptible, S) or, if they failed to show signs of the disease, after 10, 11 or 12 days (nonsusceptible, NS). The longer periods were needed to schedule the observations. Some ewes were
found to have dead foetuses at the time of slaughter. In the second year, ewes bearing twins (n = 18)
were also starved, with free access to water, at about 130 days of gestation, to induce OPT. However,
prior to being slaughtered for preparation of hepatocytes they were infused for 3 h with radioactive
glucose to measure glucose turnover. In both years, throughout the starvation period, jugular blood
samples were analysed for circulating constituents and packed cell volumes as previously described
(Wastney et al. 1982).
Tracer glucose infusions
The infusions were undertaken during the starvation period, either when the sheep became
recumbent with OPT, or at the end of 10 days of starvation. Both jugular veins were catheterized on
the morning of the experiment with polyvinyl plastic tubing [Ld. 0·97 mm; o.d. 1·27 mm (Dural
Plastics, Dural, N.S.W.)]. A priming dose consisting of 20/lCi (0·74 MBq) [U_ 14C]_ and 40 /lCi
(1'48 MBq) [6- 3 H]glucose was injected through one of the catheters followed by an infusion of
13/lCi (0'48 MBq) [U_ 14C]_ and 25/lCi (0·93 MBq) [6- 3 H]glucose in 40 ml of 0·15 M saline per
hour, using a Harvard Apparatus Infusion/Withdrawal Pump (Millis, MA, U.S.A.). Blood samples
(10 ml) were withdrawn from the other catheter at 30, 60, 90, 120, 150, 165 and 180 min after the
priming dose. They were stored on ice in tubes containing 25 mg sodium fluoride and 20 mg potassium oxalate and the plasma separated by centrifuging for 15 min at 2700g and 4°C.
Glucose Production in Starved Pregnant Sheep
273
Hepatocytes
Isolation
Liver cells from ewes which were moribund or had survived 10 days of starvation were isolated
by the method of Clark et al. (1976b), with some modifications. The sheep were given an intravenous
injection of 500 units heparin/kg liveweight and 2 min later killed by stunning with a captive bolt
pistol. The liver was immediately exposed, the caudate lobe removed and rinsed with 10 ml KrebsRinger 1 buffer (without CaB) (Dawson et al. 1969) adjusted to pH 7·6. The lobe was perfused
with 250 ml of this buffer under the conditions described by Clark et al. (1976b), except that 75 mg
collagenase was added to the perfusate. Perfusions were carried out with a Harvard Apparatus 1215
Variable Speed Peristaltic Pump (Millis, MA, U.S.A.), and a constant-pressure device. After 30 min
the lobe was disconnected from the apparatus, the liver capsule was removed with forceps, and the
cells separated from each other in Krebs-Henseleit buffer (Dawson et al. 1969), containing 15 gil
gelatin and adjusted to pH 7· 6. This eliminated the second incubation procedure of Clark et aI,
(1976b), and reduced the isolation time. Since calcium is required for maintenance of cell junctions,
it was omitted from the perfusion medium (Berry 1976), but was reintroduced immediately after
the perfusion to maintain cellular respiration (Howard and Pesch 1968).
The suspension of cells was filtered through coarse nylon mesh and centrifuged at 200 g for 30 s
at ambient temperature. The cell pellet was gently washed in Krebs-Henseleit buffer, filtered through
a fine filter (100 tim pore size) and, after a second wash, made up to 50 ml in Krebs-Henseleit buffer.
The final suspension contained about 5 mg dry weight cells/ml. The overall preparation time was
generally 60 min, and the cells were used immediately for incubation studies.
Incubations
Incubation conditions with the substrates sodium propionate, lithium lactate, alanine and
glutamine or glycerol, each at a final concentration of 10 mM in Krebs-Henseleit buffer, were
the same as those described by Clark et al. (1976b). Glucagon at a concentration of 10- 8 M was
made up according to the method of Faloona and Unger (1974). All incubations were performed
in triplicate and lasted 30 min. The reactions were stopped by the addition of O· 5 ml of 60 gil
perchloric acid and the denatured protein removed by centrifugation. The supernatants (1. 5 ml)
were neutralized with o· 1 ml of 4·5 M potassium hydroxide and after· a second centrifugation,
analysed for glucose.
The viability of each cell preparation was assessed by the integrity of the cell membranes, as
judged by the following two methods:
(i) Exclusion of trypan blue. Cell suspensions (0'1 ml) were diluted with 0·3 ml saline containing 6 gil trypan blue (Seglen 1976) and after 5 min in a Burker chamber, 200 cells were
counted. Viability was expressed as the percentage of cells with unstained nuclei.
(ii) Stimulation of oxygen uptake by succinate. This is a sensitive test for membrane integrity
as only damaged membranes allow succinate permeation at a rate sufficient to stimulate
respiration (Baur et al. 1975). Oxygen uptake by 1 ml of cells in Krebs-Henseleit buffer was
measured polarographically in a Clarke apparatus (YSI Model 53 Biological Oxygen Monitor,
Yellow Springs Instrument Co, Yellow Springs, OH, U.S.A.). The uptake was measured before
and after the addition of succinate (final concentration, 1 mM), and data from suspensions in
which the oxygen uptake was stimulated by a factor of more than 1 . 30 were excluded.
The dry weight of cells was determined as the difference between 2 ml cells and 2 ml buffer dried
at 80°C for 24 h.
Analyses
Plasma samples from the tracer experiments were analysed for glucose as already described
(Wastney et al. 1982) and a portion deprotonized by the method of Somogyi (1945). Glucose in the
protein-free filtrate (and a dilution of the infusion solution) was converted to gluconate as described
by Blair and Segal (1960), with 100 mg of glucose as carrier. The potassium gluconate crystals were
isolated, weighed into glass scintillation vials, dissolved in 3 ml water and counted with 12 ml of
Aquasol in a Beckman Liquid Scintillation Counter. Counting efficiency of each isotope 'was corrected to 100 %from a quench correction curve and the external standard ratio.
M. E. Wastney et al.
274
Glucose in the protein-free supernatant from the hepatocyte incubations was measured on the
Technicon Autoanalyzer using the glucose oxidase method previously described (Wastney et al.
1982), but with sensitivity enhanced by removing the dialyser and introducing the sample directly
into the buffered reaction mixture.
Liver
A sample of liver homogenate (c. 10 g) was weighed, freeze-dried, weighed again to give the dry
weight of tissue, and refluxed in weighed Soxhlet thimbles with light petroleum (b.p. 60-80°C) for
6 h. The sample plus thimble was dried and weighed. The weight loss was taken to represent fat
content. Glycogen was extracted from portions of frozen liver (c. 2 g) by the procedure of Laskov
and MargoIiash (1963) and analysed by the iodine-iodide method of Krisman (1962).
Calculations
Glucose specific activities were calculated as dpm/mmol, graphed against time, and averaged over
the period when specific activity appeared to reach a plateau. Then
.
glucose turnover rate (GTR) (mmoI/mm) =
infusion rate (dpm/min)
.
mean specific activity (dpm/mmol)
The proportion of glucose recycled (expressed as a percentage) was estimated as
recycled glucose (%) = l00x ([6- 3 H1GTR- [U- '4 C]GTR)/[6- 3 H1GTR.
For the hepatocytes, glucose production rates of hepatocytes were expressed as ,umol/g cell dry
weight per min.
Statistical Analyses
One-way analyses of variance between classification groups were conducted for the following
variables: concentration of plasma or serum constituents prior to slaughter, ewe liveweights, combined weights of foetuses, ewe liver weights and liver composition. If the F-test was significant,
least significant differences were calculated for the between-group comparisons. Hepatocyte glucose
production rates were compared by analyses of variance using the GENSTAT package and glucose
turnover rates were compared using the t-test. For many of the variates (glucose, urea, creatinine
and inorganic phosphate concentrations, hepatocyte glucose production rates and glucose turnover
rates), variances were stabilized by logarithmic transformations of the data.
Results
The results are presented separately for the susceptible (S) and non-susceptible
(NS) ewes and further classified according to whether the foetuses were alive (L) or
dead (D) at slaughter.
Clinical Assessment and Classification of the Ewes
Table 1 shows the concentration of plasma constituents and packed cell volumes
on the last date that the ewes were sampled before slaughter, and these can be compared with the average value for all ewes before starvation began. For the NS-L
group, there was the hypoglycaemia and hyperketonaemia that is normally observed
during starvation. Slight increases in packed cell volumes (indicative of dehydration)
and urea concentrations occurred, but there was no elevation of creatinine to levels
of 265 J1M (3 mg/dl), which we have found to be indicative of severe renal failure.
Inorganic phosphate concentrations were slightly elevated and in one ewe (No. 193)
the rise coincided with a substantial drop in the serum CO 2 content. With the possible
exception of ewe No. 390, where the serum CO 2 content had fallen to a low level, all
of these ewes could, with confidence, be expected to survive had they been allowed
access to a palatable food.
275
Glucose Production in Starved Pregnant Sheep
Table 1.
Circulating metabolite concentrations and packed cell volumes of ewes before starvation and
prior to slaughter
Ewes were susceptible (S) or non-susceptible (NS) with live (L) or dead (D) lambs in utero at slaughter.
Ketones, acetoacetate plus 3-hydroxybutyrate; PCV, packed cell volume; Creat., creatinine; Pi,
inorganic phosphate; CO 2 , serum CO 2 content. Means with different superscripts are significantly
different ("b, P < 0'05; AD, P < 0'01)
Group
and year
All ewes
Mean
s.d.
NS-L
1st year
2nd year
Mean
s.d.
S-L
1st year
2nd year
Mean
s.d.
NS-D and S-D
1st year
2nd year
Mean
s.d.
Animal Days Glucose Ketones
No.
starved (mM)
(mM)
114
313
330
335
357
390
193
0
2·88
0·35
10
10
10
0·77
1·12
2·24
1·58
1·75
2·20
2'55
II
10
10
11
0·67
0·26
5·7
10·7
3·3
7·9
3·7
7·7
5·5
1'75aA
0·64
288
294
322
349
356
363
371
26
33
38
178
7
5
8
5
7
7
7
10
5
9
10
2·83
4·29
1·09
1·33
1·12
1·13
3·43
1·78
2·14
2·04
2·75
9·1
7·7
2·7
10·3
9·7
17·6
6·6
7·0
9·0
5·6
2·9
2·18 aA
1·05
4
300
368
6
57
61
84
93
107
132
155
164
172
174
182
II
8
II
12
10
4
4
9
10
10
8
10
7
10
11
3·85
3·82
5 ·17
2·68
4·65
2·94
12·29
1l·47
5·71
14·68
2·48
6·00
16·59
6·71
2·67
6·78
4·66
6·4"
2·6
8 '0"
4·0
1·3
7·0
5·0
0·6
3·7
10·5
4·0
4·2
0·6
0·9
2·4
1·2
2·1
bD
PCV
Pi
(mM)
(%)
Urea
(mM)
Creat.
(pM)
34
3
5·7
1·6
851
12
2·59
0·52
23·1
3·7
34
40
39
40
39
41
25
8·3
1l·5
18·8
19·9
12·8
19·2
12·7
77
99
106
206
111
190
145
3·20
4·87
2·45
6'14
5·36
4·94
6·42
22·0
23·7
21·8
23'5
16·0
10·6
13·7
37a
6
14·7"
4·5
133 a
49
4·77"
1·46
18·8a
5·3
37
46
32
34
46
33
41
35
40
35
9·9
18·9
9·6
7·3
18·7
13·5
9·3
59·4
27·6
41·1
76·0
104
247
67
126
200
204
110
419
89
378
697
4·54
3·27
2·87
3·88
5·84
5·52
6·62
6·20
6·37
6·24
7·03
15 ·1
14·8
26·2
23·5
12·3
10·5
4·8
7·2
6·2
13·3
4·1
38"
5
27·4 a
24'5
240"
191
5·31"
1·43
12·5 b
7·2
14·0
59·6
42·3
127·2
67'5
14·6
31·1
33·8
96·9
2·6
25·0
65·5
25·0
79·1
28·4
93
592
540
996
622
153
175
285
2080
96
355
945
841
472
256
8·22
10'53
9·77
12·06
10·05
5'78
1l·88
2·80
2·67
8·55
7·30
18·81
6·00
3·67
15 ·0
6·3
11·5
7·9
10·7
14·0
15·6
10·8
3·9
12·9
12·6
5·3
3·3
5·9
18·5
8·44b
4·33
1O·3 b
4·6
1·3
40
45
44
38
31
32
31
48
38
48
43
42
42
45
43
3·2b
2·8
41"
6
47·S"
34·6
567 b
514
CO 2
(mM)
276
M. E. Wastney et al.
By comparison, many of the ewes in the S-L group had circulating levels of plasma
constituents that did not favour survival. Two (Nos 294 and 371) had high glucose
concentrations that were commonly associated with a poor prognosis for spontaneous
recovery. Ewe No. 294 and another ewe (No. 356) had high packed cell volumes,
indicative of severe dehydration. Three of the ewes (Nos 26, 38 and 178) had high
urea and creatinine concentrations indicative of severe renal failure. Several ewes
(Nos 363, 371, 26, 33 and 178) also had low serum CO 2 contents, indicating a severe
metabolic acidosis. The most important consideration of all, however, was that all
the sheep in this group were recumbent and/or comatose. There was thus virtually no
prospect of them making a spontaneous recovery if offered a palatable food of high
energy content.
Table 2.
Mean±s.d. ewe Iiveweights, foetal weights, liver weights and liver composition of ewes at
slaughter
Ewes were susceptible (S) or non-susceptible (NS) to pregnancy toxaemia with live (L) or dead (D)
lambs in utero at slaughter. Foetal weight, combined weights of foetuses; fat and glycogen are
expressed as gjl00 g offresh tissue. Glycogen values are means from 1, 4 and II animals in the NS-L,
S-L and NS-D and S-D groups respectively. Means with different superscripts are significantly
different ("h, P<0'05; AB, P<O·Ol). Data for individual animals are available as an Accessory
Publication and copies may be obtained on request from the Editor-in-Chief, Editorial and Publications Service, CSIRO, 314 Albert Street, East Melbourne, Vic. 3002
Group
n
Weight of
ewe
(kg)
Foetal
weight
(kg)
NS-L
SoL
NS-D and
SoD
7
II
46±2 a
45±7 a
5·1±0·7 a
5'1 ±0·9 a
736±75 a
775±162 a
41'2±3'3 A
33'2±5'4B
21'08±3'4I aA
13 '25±6'79 bAB
0·18
0'20±0'04 a
15
43±7 a
5'3±I'Oa
828±122 a
29'8±4'3 B
8'17±5'67 bB
I'OO± 1'05"
Liver
weight
(g)
Liver
dry matter
(g/IOO g)
Liver
fat
(g/IOO g)
Liver
glycogen
(g/IOO g)
The third group, designated S-D and NS-D, were characterized by the existence of
dead foetuses in utero when slaughtered. Those slaughtered before 10 days of starvation were designated susceptible by the same criteria used for the S-L group. Many of
the ewes, slaughtered after 10, 11 or 12 days of starvation, however, showed only
mild neurological signs of pregnancy toxaemia. On the basis of such clinical observations, one might predict that the ewes had a reasonable chance of survival once they
aborted their foetuses. Data from their blood chemistry, however, show that these
ewes were hyperglycaemic (Nos 84, 93, 132 and 172), dehydrated (Nos 300, 368, 93,
132 and 174), uraemic (Nos 300, 368, 6, 57,107,164,172 and 174), or acidotic (Nos
300, 6, 107, 164, 172 and 174). Their prospects of survival thus appeared low and it
was only by sUbjective judgment on the overall neurological condition that the so-called
Sand NS ewes could be distinguished. In view of the obvious heterogeneity in clinical
manifestation within both the S-D and NS-D classifications, the data have been
combined.
Data presented in Table 2 show that there were no significant differences between
the groups at slaughter for ewe liveweights or combined weight of the twin foetuses.
Liver Analyses
Also shown in Table 2 are the liver weights, the percentage of dry matter in the
liver and liver fat contents for the ewes at slaughter. Liver weights were comparable
277
Glucose Production in Starved Pregnant Sheep
for all the groups, but the NS-L ewes had significantly higher dry matter values than
the other two groups and higher fat contents than the combined NS-D and S-D
group. Nearly all the variation in liver dry matter (D, g/lOO g) between animals
could be accounted for by the increase in fat content (F, g/lOO g). The equation was
D
=
23 ·20(±0·58)+0·80(±0·04)F;
r
=
0·96 (P < 0·001).
Liver fat content was also significantly correlated with ketone concentrations
(K, mM), given in Table 1. The common regression equation was
K
=
1·21(± 1·02)+0·34(±0·07)F;
r
=
0·67 (P < 0'001),
with no apparent differences existing between the groups.
Liver glycogen analyses (see Table 2) showed that all four of the S-L ewes in the
second trial year had livers that were severely depleted of glycogen. Values for the
NS-D and S-D ewes, however, were extremely variable. Overall, there was no significant relationship between the glucose concentrations given in Table I and the
glycogen content of the livers.
Table 3. Mean ± s.d. glucose production rates (Ilmol!g dry weight per min) by hepatocytes from
starved pregnant sheep in the presence and absence of 10- 8 M glucagon
Ewes were susceptible (S) or non-susceptible (NS) to pregnancy toxaemia with live (L) or dead (D)
lambs in utero at slaughter. The substrates added, each at a concentration of 10 mM to the basal
medium, were: Prop, propionate; Lact, lactate; Ala, alanine; GIn, glutamine and Gly, glycerol.
Means based upon fewer animals are presented with n in parentheses. Data for individual animals
are available as an Accessory Publication and copies may be obtained on request from the Editorin-Chief, Editorial and Publications Service, CSIRO, 314 Albert Street, East Melbourne, Vic. 3002
Group
n
Basal
+ Prop
+ Lact
+Ala
+Gln
+Gly
Without glucagon
NS-L
SoL
NS-D and SoD
7
10
9
0-56±0-39
1-06±0-81
0-14±0-17(9) 0-31±0-55
1-47±0-03
3 -02±3 -21
NS-L
SoL
NS-D and SoD
7
10
9
1-21±1-00
1-36±1-09
0-15±0-19(9) 0-28±0-44
2-86±2-95
3-89±3-58
1-19±0-79
0-29±0-52
2-21±2-39
0-73±0-69(6) 0-77±0-56(3) 0-04(1)
0-12±0-31
0-15±0-33(5) 0-25±0-16(3)
5 -30±3 -12(3) 1-40±2-34(6)
1-94±2-21
With glucagon
1-34±0-96
0-98±0-91(4) 0-91±0-51 -0-02(1)
0-21 ±0-46
0-16±0-36
0-04±0-53(5) 0-16±0-10(3)
2 -89 ±2 -85(8) 3-85±3-37
4-85±3-88(4) 2-15±3-81(4)
Hepatocyte Glucose Production Rates
Table 3 presents mean data from all the incubations undertaken with viable
hepatocytes. On the basis of the results from the first year, which showed little
difference in the glucose production rates between propionate, lactate, alanine and
glutamine, glycerol was introduced in place of glutamine for the studies carried out
in the second year. There was a general pattern of low glucose production rates in
the SoL ewes, medium production rates in the NS-L ewes and high production rates
in the heterogeneous S-D and NS-D group. Five animals (Nos 193, 349, 38, 84 and
155) gave results which did not conform to this general pattern, and two of these
(Nos 84 and 155) have been excluded as outliers from the analysis of variance given
in Table 4. This analysis on the log transformed data from the basal, propionate,
lactate and alanine incubations shows that there was a highly significant classification x
glucagon interaction. Hepatocytes from the S-L ewes that produced little glucose
showed no response to glucagon, those from the NS-L ewes that produced slightly
278
M. E. Wastney et al.
Table 4. Analysis of variance on hepatocyte glucose production rates
Data from the basal, + propionate, + lactate and + alanine incubations
were analysed as In (rate + 1) to stabilize variances
Category
Between animals
Classification
Residual
Total
Within animals
Glucagon
Classification x glucagon
Medium
Residual
Total
Grand total
d.f.
S.S.
M.S.
P
2
23
25
29·16
50·19
79·35
14'58
2·18
<0·01
1
2
3
172
178
203
0'59
0·58
1·04
7·24
9·45
88·8
0'59
0·29
0·35
0·04
<0·001
<0·005
<0·005
Table 5. Glucose turnover rates on the day of slaughter for starved pregnant ewes
Ewes were susceptible (S) or non-susceptible (NS) to pregnancy toxaemia with live (L)
or dead (D) Iambs in utero at slaughter. Values for plasma glucose are means±s.d.
from all samples taken during the infusion. Turnover rates were calculated from plateau
specific activities. Means with different superscripts are significantly different by the
(-test cab, P < 0·05; AB, P < 0'01)
Group
and
animal No.
NS-L
193
S-L
26
33
38
178
Mean
s.d.
NS-D and S-D
6
57
61
84
93
107
132
155
164
174
182
Mean
s.d.
Glucose turnover
(mmol/min)
[U_ 14C]_
[6- 3 HJglucose
glucose
Live
weight
(kg)
Plasma
glucose
(mM)
Glucose
recycled
47
2'61±0'27
0·776
0·805
8·6
43
34
39
40
39
4
1·79±0·19
2·08±0·13
2·65±0·14
2'87±0'07
2·34"
0·50
0·536
0·338
0·298
0·443
0'404A
0·107
0·565
0·362
0·304
0·455
0'422A
0·114
5·3
6·5
2·0
2'7
4·1
2·1
36
35
36
40
45
35
38
43
52
51
51
42
7
3'51±0'46
5·44±0·80
2·87±0·31
12·1O±1·04
11·64±0·20
5·84±0·35
11'18±0'52
1·86±0·11
7·92±1·62
6·33±1·01
3·49±0·94
6'56 b
3·69
0·403
0·636
0·672
1·813
1·486
1·037
1·739
1·073
0·792
0·929
1·102
1.0628
0·454
0·411
0·658
0'732
1·802
1·688
1·094
1·859
1·299
0·792
0·893
1·230
1.133 8
0·490
2·0
3·3
8·2
-0,6
11·9
5·2
6·5
17·4
0·0
-4,0
10·4
5·5
6·2
(%)
279
Glucose Production in Starved Pregnant Sheep
more glucose showed a small but significant glucagon response, while those from the
ewes with dead foetuses produced most glucose with a large and highly significant
glucagon response. Of the three substrates added to the basal medium only propionate
and lactate produced significant increases in glucose production, with alanine being
intermediate. The use of glycerol as substrate failed to stimulate the glucose production rates over those measured for other substrates. Considered together, these
results demonstrate an impaired process of gluconeogenesis in hepatocytes obtained
from ewes moribund with pregnancy toxaemia. Upon death of the foetuses, however,
there was an increase in hepatocyte glucose production.
Glucose Turnover Rates
Measurement of glucose turnover rates were undertaken in the second-year studies
to obtain corroborating evidence of the defective hepatic gluconeogenesis seen in
ewes moribund with pregnancy toxaemia. The data shown in Table 5 confirm the
existence of the differences seen in hepatocyte glucose production rates. Unfortunately, only one NS-L ewe was studied. Ewes in the S-L category had significantly
lower glucose turnover rates than those in the mixed NS-D and S-D classification.
Data obtained using both tracers were in general agreement, and there was no evidence
of increased recycling of the 14C tracer in either group. The overall regression of
glucose turnover rate (Y, mmol/min, estimated with the 14C tracer) on glucose
concentration (X, mM) was
Y = 0·34(±0·12)+0·1O(±0·02)X;
r =
0·85 (P < 0·01).
Discussion
Experimental investigations into the pathogenesis of ovine pregnancy toxaemia
have been hampered by a number of problems which, to some extent, still existed in
the work reported here. The first problem is the variability between sheep in the
course of the disease and the nature of complications which can develop. Previous
experience (Wolff et al. 1974; O'Hara et al. 1975; J. E. Wolff, unpublished data)
showed that uraemia, acidaemia, dehydration, hyperglycaemia and foetal death were
the complications commonly associated with death during 10 days of starvation in
the last month of gestation. Table 1 shows that these complications were prevalent
in the present study, with much variability between animals. Seldom, however, did
all complications occur in anyone ewe.
There was also a wide variation in the neurological disturbances that developed
during starvation. Some sheep exhibited few neurological signs of the disease while
others exhibited many of the signs described by McClymont and Setchell (1955).
So far, we have not been able to find an association between the neurological signs
and the variations in blood chemistry.
A second problem, consequent upon the large between-animal variability in the
different attributes, is the uncertainty in classifying all individual sheep. The system
adopted for this study was an overall assessment of the ewe's chances of making a
spontaneous recovery. This permitted us to search for metabolic attributes associated
with survival. Ewes with dead foetuses were grouped separately because of the extra
variability associated with this factor and the uncertainty attached to their chances of
survival had they been able to abort their foetuses. This system of classification is
280
M. E. Wastney et
at.
different from the three syndromes proposed by Reid (1968) to accommodate his
observations on the varying aetiology of the disease. The conditions imposed upon
our ewes would correspond to Reid's syndromes 2 or 3, depending upon an assessment
of the stress factors. In the absence of data on the blood chemistry of ewes in Reid's
syndromes, we found it impossible to apply the subtle distinctions of his classification
criteria to the varying manifestations we observed.
A third problem faced in this type of investigation is the need to monitor additional
metabolic and physiological entities to provide better information on the ewe's clinical
condition. For example, arterial blood pressure could be monitored to diagnose the
possible existence of circulatory shock in the recumbent comatose ewe (Kronfeld
1972) and the levels of circulating hormones to ascertain the possible causes of the
hyperglycaemia. Simple techniques to assess foetal well-being are also needed to
ascertain the time and cause of the foetal death.
Despite these several limitations, the study has provided a number of new and
pertinent observations on the pathogenesis of ovine pregnancy toxaemia. Analysis
of liver samples showed that there was an increased dry matter (or a decreased water
content) of livers in the NS-L ewes, which can be accounted for by the increase in
fat content. A fatty liver has been a principal diagnostic criterion for field cases of
ovine pregnancy toxaemia (Davis 1974) but, in our study, livers from the NS-L ewes
contained a higher fat level than the other groups (Table 2). We also found a strong
correlation between the levels of liver fat and the concentration of plasma ketones,
suggesting that both changes are a consequence of fat mobilization caused by undernutrition. The high fat content of livers from the NS-L ewes suggests that a fatty
liver may not be of any pathological consequence. Similar considerations could apply
to the ketotic state, as we have not been able to find significant differences in ketone
concentrations between Sand NS ewes.
A major discovery of the present work has been the poor glucose production rates
by hepatocytes of the SoL ewes. These low rates occurred with all the substrates
tested, and no stimulation was apparent following addition of 10- 8 M glucagon.
For some ewes, it was possible to compare hepatocyte glucose production rates with
glucose turnover rates measured before slaughter. This was done by extrapolating
hepatocyte production rates to 85% of the total dry liver weight, this being the
fraction of the liver consisting of hepatocytes or parenchymal tissue (Krebs et al.
1974). For the SoL ewes slaughtered in a moribund state, the comparison suggests
either that these hepatocytes were not able to express the gluconeogenic capacity of
the liver in vivo or that there was some other source of glucose production. A large
glucose production by the kidneys appear& unlikely, as the renal production of glucose
has never been shown to be more than a small fraction of the glucose turnover rate
(Kaufman and Bergman 1974; McIntosh et al. 1973). Alternatively, the isolated cells
may have been unable to take up substrates or they may have lacked essential
cofactors and a suitable energy source. Evidence in support of the latter suggestion
is that Gallagher (1959) found liver tissue from sheep with pregnancy toxaemia was
less able to oxidize long chain fatty acids and Ferre et al. (1978) have shown that
neonatal hypo glycaemia in the rat was due to a lack of energy from the oxidation of
fat. Another possibility is that the cells may have undergone changes, in vitro, which
predisposed them to some form of damage not detected by either of the two viability
assessments. This possibility merits consideration as Kronfeld (1972) has argued
---,------------Glucose Production in Starved Pregnant Sheep
281
that circulatory shock from endotoxin may be a common terminal condition in the
moribund ewe and Hand et al. (1981) have reported a failure of hepatocytes to synthesize glucose when the cells were isolated from pigs in endotoxic shock.
The glucose turnover rates shown in Table 5 for the NS-L and S-L ewes are somewhat higher than those reported by others. For example, Kronfeld and Simeson (1961)
reported values of 0·3 and 0·2 mmol/min for fasted and toxaemic pregnant ewes
respectively that were about 1·5 times heavier than the ewes in our study, while Ford
(1965) obtained values of 0·4 and 0·7 mmol/min for spontaneous cases of pregnancy
toxaemia in ewes that were twice as heavy. From a regression equation relating
glucose turnover rates to plasma glucose concentrations, Bergman (1973) predicted
values of O' 3 mmol/min for ketotic pregnant ewes weighing 40 kg. The reasons for
these differences are not known, but the longer period of starvation in our study could
have been one major factor. Indeed, all but one of the ewes with live foetuses had
plasma glucose concentrations that were higher than those found during the peak of
ketosis, which frequently occurs about the fifth day of starvation. Another factor
could be the non-attainment of a true steady-state specific activity. In ewes with the
high glucose production rates, a plateau specific activity was rapidly attained, but, in
animals with slow rates, the ratio of primary dose to infusion rate (Kronfeld and
Ramberg 1981) was probably insufficient for a true plateau to be reached within the
experimental time and the production rates could thus have been overestimated.
With a six-fold range in turnover rates, an accurate prediction of the optimal ratio
for the primary dose to infusion rate was almost impossible. Thus, to reconcile the
differences between laboratories, glucose turnover rates need to be measured throughout the starvation period with specific activities measured after 300 min of infusion,
as this minimizes the effect of a poorly chosen priming dose (Kronfeld and Ramberg
1981).
Our regression equation relating glucose turnover rate to plasma concentration is
different from the regression equations published previously for pregnant sheep by
Bergman (1964, 1973) or Steel and Leng (1973). Their data included observations on
fed and starved pregnant ewes bearing twins, with none of the starved animals being
in a moribund or hyperglycaemic state. If we exclude data from the ewes with dead
foetuses, there was no significant linear regression. As we have already exposed
considerable variability between ewes bearing twins in their glucose-insulin relationships with the intravenous glucose tolerance test (Wastney et al. 1982), there appears
little reason to expect individual sheep to conform to a regression calculated from
observations made on different animals.
The other major discovery has been the relatively high glucose turnover rates and
hepatocyte glucose production rates seen in most of the ewes with dead foetuses in
utero at the time of slaughter. In some of these ewes, there was reasonable concordance between the two measurements when undertaken on the same ewe. The increase
in glucose production rate would thus contribute to the severe hyperglycaemia seen in
many of these ewes at the time of slaughter. The hyperglycaemia also indicated a
frank loss of glucose homeostasis. Other contributing factors could include an
inhibition of peripheral glucose utilization caused by high levels of circulating cortisol
(1. E. Wolff, unpublished data) and an excessive rate of glucose production caused
by the hyperglucagonaemia after renal failure (Bilbrey et al. 1974). A further common
282
M. E. Wastney et al.
factor is the reduced uptake of glucose by the pregnant uterus, but the deletion of a
site of utilization should not, by itself, cause both a loss of glucose homeostasis and
a higher glucose production rate by isolated hepatocytes.
A possible reason for the high hepatocyte production rates is that the gluconeogenic potential of these cells increased at the time of foetal death. This suggestion
leads to the hypothesis that the ultimate gluconeogenic potential of hepatocytes
increases during pregnancy but is constrained by the presence of live foetuses. The
constraint is lifted after parturition when much higher rates of glucose production are
needed to support milk production (Bergman 1973). Support for this hypothesis is
that the poor rates of milk production seen after the artificial induction of lactation
(Gow et al. 1981) can be increased by an intravenous glucose infusion of 0·22
mmoljmin (Leenanuruksa and McDowell 1982). We therefore suggest that, during
pregnancy, the liver is prepared for high rates of glucose production during lactation
but its manifestation is somehow constrained by the presence of a live foetus. A
placental hormone such as somatomammotrophin (placental lactogen) could be a
candidate for this inhibitory role and would be partly consistent with its diabetogenic
and lipolytic role as proposed by Grumbach et al. (1968). The elevated insulin and
decreased glucose concentrations observed by Handwerger et al. (1976) during infusion
of an ovine placental lactogen preparation into pregnant and non-pregnant sheep are
also consistent with somatomammotrophin restraining hepatic gluconeogenesis during pregnancy.
Acknowledgments
We are particularly grateful to W. M. Aitken, B. Crane, P. M. Dobbie, P. Edginton,
P. Gill, C. Harris, P. Harvey, D. Kerr, C. Larsen, R. R. Lasenby, V. McGuire, K.
Mitchell, A. L. Sim and S. P. Stuart for their skilled technical assistance. Biometric
assistance was rendered by H. V. Henderson and I. Gravett. M.E.W. was supported
by a New Zealand University Grants Committee Postgraduate Fellowship, a Shirtcliffe Fellowship and the Sir James Gunson Scholarship.
References
Ash, R., and Pogson, C. I. (1977). Preparation and biochemical characterization of isolated parenchymal cells from adult sheep liver. Biochim. Biophys. Acta 496, 475-83.
Baur, H., Kasperek, S., and Pfaff, E. (1975). Criteria of viability of isolated liver cells. HoppeSeyler's Z. Physiol. Chern. 356, 827-38.
Bergman, E. N. (1965). Glucose turnover rates in pregnant and non-pregnant sheep. Nature (London)
202,1333.
Bergman, E. N. (1973). Glucose metabolism in ruminants as related to hypoglycemia and ketosis.
Cornell Vet. 63, 341-82.
Bergman, E. N., Katz, M. L., and Kaufman, C. F. (1970). Quantitative aspects of hepatic and
portal glucose metabolism and turnover in sheep. Am. J. Physiol. 219, 785-93.
Berry, M. N. (1976). The development of techniques for the preparation of hepatic parenchymal
cell suspensions-a history and rationale. In 'Use of Isolated Liver Cells and Kidney Tubules
in Metabolic Studies'. (Eds J. M. Tager, H. D. Soeling and J.R. Williamson.) pp.131-8. (North
Holland: Amsterdam.)
Bilbrey, G. L., Faloona, G. R., White, M. G., and Knochel, J. P. (1974). Hyperglucagonemia ofrenal failure. J. Clin. Invest. 53, 841-7.
Blair, A., and Segal, S. (1960). The isolation of blood glucose as potassium gluconate. J. Lab. Clin.
Med. 55, 959-64.
Glucose Production in Starved Pregnant Sheep
283
Clark, M. G., Filsell, O. R., and Jarrett, I. G. (l976a). Gluconeogenesis in isolated lamb liver cells.
In 'Use of Isolated Liver Cells and Kidney Tubules in Metabolic Studies'. (Eds J. M. Tager,
H. D. Soeling and J. R. Williamson.) pp. 398-401. (North Holland: Amsterdam.)
Clark, M. G., Filsell, O. H., and Jarrett, I. G. (l976b). Gluconeogenesis in isolated intact lamb liver
cells. Effects of glucagon and butyrate. Biochem. J. 156, 671-80.
Davis, G. B. (1974). A sheep mortality survey in Hawke's Bay. N.Z. Vet. J. 22, 39-42.
Dawson, R. M. C., Elliott, D. C., Elliott, W. R., and Jones, K. M. (1969). 'Data for Biochemical
Research.' p.507. (Clarendon Press: Oxford.)
Faloona, G. R., and Unger, R. H. (1974). Glucagon. In 'Methods of Hormone Radioimmunoassay'.
(Eds B. M. Jaffe and H. R. Behrman.) pp.317-30. (Academic Press: New York.)
Ferre, P., Pegorier, J. P., Assan, R., Mariiss, E. B., and Girard, J. (1978). Influence of exogenous
cortisol and triglyceride feeding on glucose homeostasis in the fasted newborn rat. Pediat. Res.
12, 751-6.
Ford, E. J. H. (1965). Glucose utilization in spontaneous pregnancy toxaemia of sheep. Br. Vet. J
121, 139-44.
Gallagher, C. H. (1959). Biochemical studies on ovine pregnancy toxaemia. I. Enzymic activities
of liver and brain. Aust. J. Agric. Res. 10, 854-64.
Gow, C. B., McDowell, G. H., and Annison, E. F. (1981). Comparison of glucose biokinetics in
parturient ewes and ewes induced to lactate artificially. Aust. J. Bioi. Sci. 34, 463-8.
Grumbach, M. M., Kaplan, S. L., Sciarra, J. J., and Burr, I. M. (1968). Chorionic growth hormoneprolactin (CGP): secretion, disposition, biologic activity in man, and postulated function as the
"growth hormone" of the second half of pregnancy. Ann. N. Y. Acad. Sci. 148, 501-31.
Hand, M. S., Phillips, R. W., Chandrasena, L. G., and Cheek, J. L. (1981). Metabolic alterations
related to reverse portal vein flow in an endotoxic pig. Adv. Shock Res. 6, 131-40.
Handwerger, S., Fellows, R. E., Crenshaw, M. c., Huriey, T., Barrett, J., and Maurer, W. F. (1976).
Ovine placental lactogen : acute effects on intermediary metabolism in pregnant and non-pregnant
sheep. J. Endocrinol. 69, 133-7.
Howard, R. B., and Pesch, L. A. (1968). Respiratory activity of intact, isolated parenchymal cells
from rat liver. J. Bioi. Chem. 243, 3105-9.
Kaufman, C. F., and Bergman, E. N. (1974). Renal ketone body metabolism and gluconeogenesis
in normal and hypoglycemic sheep. Am. J. Physiol. 226, 827-32.
Krebs, H. A., Cornell, N. W., Lund, P., and Hems, R. (1974). Isolated liver cells as experimental
material. In 'Regulation of Hepatic Metabolism'. Proc. Alfred Benzon Symp. VI. (Eds F.
Lindquist and N. Tygstrup.) pp.726-50. (Academic Press: New York.)
Krisman, C. R. (1962). A method for the colorimetric estimation of glycogen with iodine. Anal.
Biochem. 4, 17-23.
Kronfeld, D. S. (1970). Ketone body metabolism, its control, and its implications in pregnancy
toxaemia, acetonaemia, and feeding standards. In 'Physiology of Digestion and Metabolism in
the Ruminant'. Third Int. Symp. Ruminant Physiology. (Ed. A. T. Phillipson.) pp. 566-83.
(Oriel Press: NewcastIe-Upon-Tyne, G.B.)
Kronfeld, D. S. (1972). Ketosis in pregnant sheep and lactating cows. A review. Aust. Vet. J. 48,
680--7.
Kronfeld, D. S., and Ramberg, C. F. (1981). Whole animal studies using tracer kinetics. Proc.
Nutr. Soc. 40, 129-38.
Kronfeld, D. S., and Simesen, M. G. (1961). Glucose biokinetics in ovine pregnancy toxemia.
Cornell Vet. 51, 478-88.
Laskov, R., and Margoliash, E. (1963). Properties of high molecular weight glycogen from rat liver.
Bull. Res. Coun. [sr. Sect. A 11, 351-62.
Leenanuruksa, D., and McDowell, G. H. (1982). The importance of glucose supply at the initiation
of artificial lactation in the ewe. Aust. J. Bioi. Sci. 35, 629-36.
McClymont, G. L., and Setchell, B. P. (1955). Ovine pregnancy toxaemia. 1. Tentative identification
as a hypoglycaemic encephalopathy. Aust. Vet. J. 31, 53-68.
McIntosh, G. H., Filsell, O. H., and Jarrett, I. G. (1973). Kidney function and net glucose production
in normal and acidotic sheep. Aust. J. Bioi. Sci. 26, 1389-94.
Morton, J. L., Buttery, P. J., and Lindsay, D. B. (1977). Gluconeogenesis from amino acids by
isolated sheep liver cells. Proc. Nutr. Soc. 36, 20A.
284
M. E. Wastney et al.
O'Hara, P. J., Wolff, J. E., and Oliver, R. E. (1975). The effect of starvation on the pregnant ewe.
Proc. N.Z. Vet. Assoc. Sheep Soc. 5th Seminar, pp. 22-31.
Reid, R. L. (1968). The physiopathology of undernourishment in pregnant sheep, with particular
reference to pregnancy toxaemia. Adv. Vet. Res. 12, 163-238.
Richardson, R. I., and Livesey, A. F. (1979). Gluconeogenesis in isolated sheep hepatocytes. Proc.
Nutr. Soc. 38, 2A.
Seglen, P. O. (1976). Preparation of isolated rat liver cells. In 'Methods in CelI Biology'. (Ed. D. M.
Prescott.) Vol. XIII. pp.29-83. (Academic Press: New York.)
Somogyi, M. (1945). Determination of blood sugar. J. BioI. Chern. 160, 69-73.
Steel, J. W., and Leng, R. A. (1973). Effects of plane of nutrition and pregnancy on gluconeogenesis
in sheep. 1. The kinetics of glucose metabolism. Br. J. Nutr. 30, 451'-73.
Wastney, M. E., Arcus, A. C., Bickerstaffe, R., and Wolff, J. E. (1982). Glucose tolerance in ewes
and susceptibility to pregnancy toxaemia. Aust. J. BioI. Sci. 35, 381-92.
Weekes, T. E. C., Richardson, R. I., and Geddes, N. (1979). The effect of ammonia on gluconeogenesis by isolated sheep liver cells. Proc. Nutr. Soc. 38, 3A.
Wolff, J. E., O'Hara, P. J., and Kronfeld, D. S. (1974). Effect of starvation on blood metabolites in
pregnant sheep. Proc. N.Z. Soc. Anim. Prod. 34, 66.
Manuscript received 19 October 1982, accepted 17 March 1983