EFFECT OF REMEP} EEVELGFMEHT 0N
KEY GLUCOREBGEREC ENZYME
ACT’WF'HES {N CALVES

Thesis E09 i120 Degree of M. S.
MICHIGAN STEVE “WERE-HY
Rodney" Kenneth McGuffey
{9‘74

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ABSTRACT

EFFECT OF RUMEN DEVELOPMENT ON KEY
GLUCONEOGENIC ENZYME ACTIVITIES
IN CALVES

BY

Rodney Kenneth McGuffey

Two experiments were conducted to determine the
effects of rumen development on gluconeogenic enzyme activity
in young caIves.

. In the first experiment, three diets designed to
stimulate rumen development at different rates were fed to
12 calves from birth to 12 weeks of age. Diet I consisted
of milk for 12 weeks; Diet II consisted of milk for 8 weeks
and hay and grain for 4 weeks; and Diet III consisted of
milk for 3 weeks and hay and grain for 9 weeks.

Enzyme activities were measured from liver obtained
by biopsy at 10, 30, 56, and 84 days of age. Pyruvate
carboxylase (PC), phosphoenolpyruvate carboxykinase (PEPCK),
and glucose-G-phosphatase (G6Pase) were determined on each
calf at each age.

Calves receiving Diet I showed little change in
enzyme activity for PC, PEPCK, and GGPase during the

experiment. Diet II calves showed little change for PC and

G6Pase but PEPCK was elevated at Day
showed no change for G6Pase over the
was increased by more than 100% over
ing period (Day 30) but decreased to
Day 56 and again increased by 40% at
ity increased steadily after weaning

activity at Day 84 compared to 0.975

Rodney Kenneth McGuffey

84. Diet III calves

84 day period. PC

Diet I during the wean-
Diet I values at

Day 84. PEPCK activ-
to 2.134 units of“

units for Diet I.

In the second experiment, 3 calves were fed milk

for 42 days, weaned within 7 days, and fed hay and grain

for 14 days. BiOpsies were at 42, 56, and 63 days of age.

PC and PEPCK increased steadily from 0.287 and 0.646 units,

respectively, at Day 42 to 0.400 and 1.227 at Day 63 when

activity was expressed on a per gram wet tissue basis.

G6Pase decreased from 34.6 units at Day 42 to 22.4 units

at Day 63 when activity was expressed on a per gram wet

tissue basis.

For both experiments, the pattern of enzyme activity

was the same regardless of whether activity was expressed on

a protein or wet tissue basis.

EFFECT OF RUMEN DEVELOPMENT ON KEY
GLUCONEOGENIC ENZYME ACTIVITIES

IN CALVES

Rodney Kenneth McGuffey

A THESIS

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

MASTER OF SCIENCE
Department of Dairy

1974

(O )6,»

6‘90

ACKNOWLEDGMENTS

The writer extends his sincere appreciation to
Dr. J. W. Thomas for his encouragement, advice, and friendly
nature throughout his graduate program.

The writer would like to thank Dr. L. L. Bieber and
Dr. J. H. Britt as members of his graduate committee for
their advice and participation.

The writer would like to thank Dr. G. A. Leveille

.for advice in develOping the assay for pyruvate carboxylase.

The writer wishes to thank Dr. C. A. Lassiter and
Dr. J. A. Hoefer for making the facilities of Michigan State
University and the Michigan Agricultural Experiment Station
available for this research.
Special thanks go to Drs. R. E. Lichtenwalner,
J. E. Shirley, and D. G. Britt, to Mr. J. T. Johns, and to
Mrs. Sue Heisner for special assistance during this research.
Finally, to his wife, Patty, a very special thank-you

for her never-ending smile of encouragement.

ii

TABLE

LIST OF TABLES . . . . . .

LIST OF FIGURES . . . . .

INTRODUCTION . . . . . . .

LITERATURE REVIEW . . . .

OF CONTENTS

Anatomical Deve10pment of the Rumen . .
Physiological and Metabolic DeveloPment of

the Rumen . . . . . .

Pathway of Glucose Synthesis . . . . . . .

Pyruvate Carboxylase .

Phosphoenolpyruvate Carboxykinase . . .
Fructose-1,6 Diphosphatase . . . . . . .

Control of PEP Formation

Citrate Cleavage Pathway . . . . . . . .

Pyruvate Carboxylase .
Malate Dehydrogenase .

Initiation of Gluconeogenesis . . . . . .
Glucose Kineticijlthe Ruminant . . . . .
Gluconeogenesis in the Ruminant . . . . .
Substrate Availability and Gluconeogenesis

Propionate . . . . . .
Amino Acids . . . . .
Glycerol . . . . . . .
Lactate . . . . . . .

MATERIALS AND METHODS

Experiment I . . . . . .
Biopsy Technique . . .
Enzyme Assays . . . .

Experiment II . . . . .

RESULTS AND DISCUSSION . .

Results of Experiment I

Pattern of Pyruvate Carboxylase . . . .

Pattern of PEPCK . . .

iii

Page

vii

12
14
18'
19
20
21
22
23
24
25
27
28
28
3O
32
33

36
37
39
41

43
43

47
51

Page

Pattern of Glucose-6-Phosphatase . . . . . . . . 53
Overall Considerations in Experiment I . . . . . 54
Results of Experiment II . . . . . . . . . . . . . 57
SUMMARY 0 0 O O O O O O O O O O O O O O I O O O O O 6 0
APPENDIX . . . . . . . . . . . . . . . . . . . . . 62
LITERATURE CITED . . . . . . . . . . . . . . . . . . 68

iv

LIST OF TABLES

Table Page
1. Properties of Three Key Gluconeogenic

Enzymes . . . . . . . . . . . . . . . . . . 15

2. Glycogenic Amino Acids . . . . . . . . . . . . 30

3. Glucose Precursors and Their Contribution to
Glucose Synthesis in Ruminants During
Different Physiological States . . . . . . . 35

4. Feeding and BiOpsy Schedule for Calves in
Experiment I . . . . . . . . . . . . . . . . 38

5. Serum Glucose at Day 84 and Average Daily
Gain of Calves in Experiment I . . . . . . . 43

6. Average Daily Dry Feed Consumption for Calves
in Experiments I and II . . . . . . . . . . 45

7. Activity of Pyruvate Carboxylase in
Experiment I . . . . . . . . . . . . . . . . 48

8. Individual Feed Intakes and Pyruvate

Carboxylase Activity for Calves 30 Days

of Age in Diet III . . . . . . . . . . . . . 48
9. Activity of PEPCK in Experiment I . . . . . . 51

10. Activity of G1ucose-6-Phosphatase in
Experiment I . . . . . . . . . . . . . . . . 54

11. Correlation Coefficients of Several Variables
in Experiments I and II . . . . . . . . . . 56

12. Activity of Pyruvate Carboxylase, PEPCK and
Glucose-6-Phosphatase in Experiment II . . . 58

13. Individual Calf Enzyme Activities and Liver
Protein at Day 10 . . . . . . . . . . . . . 63

Table Page

14. Individual Calf Enzyme Activities and Liver
Protein at Day 30 . . . . . . . . . . . . . 64

15. Individual Calf Enzyme Activities and Liver
Protein at Day 56 . . . . . . . . . . . . . 65

16. Individual Calf Enzyme Activities and Liver
Protein at Day 84 . . . . . . . . . . . . . 66

17. Individual Calf Enzyme Activities and Liver
Protein in Experiment II . . . . . . . . . . 67

vi

LIST OF FIGURES

Figure Page

1. Pathway of Gluconeogenesis . . . . . . . . . 13

vii

INTRODUCTION

Energy metabolism in the ruminant animal differs
markedly from that of the monogastric animal. Monogastric
animals derive the majority of their energy from glucose
and long-chain fatty acids. In contrast, the ruminant
animal derives from 50 to 80 percent of its energy from
volatile fatty acids resulting from the fermentation of
dietary carbohydrates within the rumen by anaerobic micro-
.organisms unique to the rumen environment.

At birth the young ruminant animal is essentially
a monogastric animal in terms of digestive function. Like
monogastric animals the pre-ruminant animal depends entirely
upon simple sugars, especially glucose, and long-chain fatty
acids as a source of energy. During the weaning period,
the pre-ruminant animal begins the transition to a func-
tional ruminant. Rumen development brought about by the
consumption of dry feed begins to supply the animal with a
new source of energy.

With the establishment of an active rumen fermen-
tation little glucose becomes available to the lower portion
of the digestive tract for absorption. The animal requires
more glucose than is available from absorption; thus the
animal is dependent on gluconeogenesis to meet these needs.

1

The purpose of this study was to determine changes
of key gluconeogenic enzyme activities in the liver of
animals fed diets designed to stimulate rumen development

at different rates during the first three months of life.

LITERATURE REVIEW

Anatomical Development of the Rumen

 

The gut of the bovine embryo begins to differentiate
at about 36 days after fertilization (93). By 50 days, all
pouches are visible. Becker §E_§l. (14) reported the rumen
to account for most of the total stomach weight at 120
days, but by term the abomasum accounted for 50 percent of
the total stomach weight.

At birth, rumen papillae are present and may be
'2.6 mm in length, probably owing to the influence of vola-
tile fatty acids from the maternal blood supply (83).

With the consumption of milk, these papillae regress until
the proper stimulus is supplied.

Several workers, in an attempt to evaluate rumen
anatomical development, have used physiological and morpho-
logical criteria as a basis for description. Lengemann and
Allen (59) measured rumen function by:

1. an ability of the rumen liquor to digest cel-
lulose

2. a description and estimate of the more char-
acteristic types of bacteria

3. number of protozoa, and
4. measurement of indigenous rumen acids.

Other criteria used by workers are:

1. tissue weights

2. position and capacity of the various compart-
ments

3. relative shape
4. gross and histological structure
5. weight of contents, and
6. blood glucose levels (95).
Leat (57) classified rumen development into three stages:

1. birth to three weeks, when the rumen is non-
functional

2. three to eight weeks, when the rumen is begin-
ning to develop, and

3. eight weeks of age onward when the rumen is
‘fully developed.

Wardrop (92) reported lower weights of the rumen,
reticulum, and small intestines and heavier abomasal weight
in nine-week old lambs fed only milk as compared to lambs
of similar age fed hay and milk. Introduction of hay into
the diets of these milk-fed lambs caused rapid structural
change of muscle and mucosa of the rumen and reticulum.

Brownlee (20) and Harrison g£_al. (44) have shown
rumen muscle development is dependent on ingestion of solid
feed. The latter group showed the growth of rumen muscle
to be independent of rumen papillae growth.

The reticulo-rumen of the young ruminant has irregu-
lar contractions atypical of the mature stomach (15). Hay-
fed calves showed much earlier mature-type movements than

their milk-fed counterparts (31). Vagal stimulation of

calves fed either milk or hay resulted in similar type
movements of the reticulo-rumen.

The approximate volume of the rumino-reticulum of
newborn calves is between 0.5 and 1.6 liters (94). Calves
fed hay, milk, and grain approach a rumino-reticulum volume
to body weight ratio similar to that of mature ruminants
by 12 to 16 weeks (83, 95). The rumino-reticulum as a per-
centage of total stomach tissue reaches a maximum at 12
weeks (94). Calves receiving milk only had less capacity
in the reticulo-rumen at 12 weeks of age than similarly
aged animals receiving hay and grain (83). Warner et_§l.
_(95) reported 13-week old calves fed either milk, grain,
hay, or hay and grain to have reticulo-rumen volumes of
3.2, 13.3, 31.4, and 13.6 liters per 45.4 kg body weight.

In young ruminants raised on an all-milk diet,
milk passes directly to the omasum by way of the eSOphageal
groove with very little appearing in the rumen. Solid feed
reaches the rumen sac causing initiation of structural
development.

.Development of rumen papillae is stimulated by end-
products of fermentation (20,75,76,83). Administration of
salts of acetate, propionate and butyrate into the rumen
of young, milk-fed calves resulted in stimulation of papil-
lary growth (75,76,83). Butyrate was most effective, while
pr0pionate was more effective than acetate. The rumen

epithelium of these animals did not exhibit the degree of

cornification as did that of animals receiving hay, milk
and grain. The color of the rumen wall was a much lighter
brown in the animals receiving acids than those fed hay,
grain and milk.

Early workers noted a gradual decline in blood
reducing-sugar concentration in young calves reaching adult
levels 6 to 9 weeks after birth (45,50,62,63).' McCandless
and Dye (67) suggested a reciprocal relationship between
blood glucose and rumen activity. Some workers have
reported an all-milk diet maintained blood glucose level in
young calves (5,45,46,63). Inclusion of hay and/or grain
.into the diet caused a rapid decline in blood glucose.
Other workers have shown blood glucose declined regardless
of the type of diet fed, though the age when adult levels
were attained may be related to diet (49,69,99).

Physiological and Metabolic
DevelOpment of the Rumen

 

 

Absorption of end—products of fermentation occurs
readily through adult rumen mucosa (2,47,80,9l,96,97).
Sutton gt_al. (80) showed no significant change in the
rate of absorption of acetate in calves at l and 13 weeks
of age when fed milk-only diets. Calves receiving hay,
milk and grain showed progressive increases in rates of
acetate absorption to 13 weeks. At one week of age, 25 mg
acetate was absorbed from a test solution (pH6.6) admin-

istered into the rumen at the rate of 10 ml per hour.

Acetate absorption rates for calves fed a ration of hay,
milk and grain were 162, 179, and 401 mg acetate per 100 ml
per hour at 4, 8, and 13 weeks. Milk-fed calves averaged
26, 21, and 22 mg acetate per 100 ml of administered solu-
tion per hour at 4, 8, and 13 weeks.

Several intraruminal factors affect the absorption
of fermentation end-products. The single most important
factor is ruminal pH. Gray (41) showed absorption to be
inhibited when rumen pH was near 7.0. Pfander and Phillip-
son (73) and Sutton et_al. (80) found absorption to be
greater with acid as compared to alkaline condition.

Lowered rumen pH increases absorption by rumen
epithelium. Stevens and Stettler (79) postulated the
increased absorption resulting from a lower pH was due to:

l. a greater transepithelial concentration

gradient, and

2. an increased lipid soluble form of the VFAs.

Danielli et_al. (28) demonstrated an increased
absorption rate of VFA as rumen pH was lowered, indicating
a greater permeability of the rumen epithelium to the
undisociated ion. Acetic, propionic and butyric acids have
disociation constants of 4.75, 4.87, and 4.81, respectively.
Since rumen pH rarely falls below 6.0, most absorption of the
VFAs occurs in the ionized specie.

Concentration and interactions among VFAs affect

absorption rates. With absolute absorption rate of acetate

as 1.0 for a 60:25:15 molar percent test solution of ace-
tate, propionate, and butyrate, and a pH of 4.8, butyrate
absorption was 1.84 times greater and prOpionate 1.45 times
greater than acetate. At pH 7.2, absorption of the volatile
fatty acids occurred at equal rates. Increased concentra-
tions of ruminal VFAs led to increased rates of absorption.
Doubling the concentration of a test solution from 75 to
150 mmol/liter doubled the rate of absorption. Increasing
the test solution concentration to 225 mmol/liter further
increased the rate of absorption (96,97). This probably
results from increased blood flow to the rumen epithelium
.produced by~a local anoxia from tissue metabolism (30).

During absorption and translocation of the VFAs to
the liver, the rumen epithelium actively metabolizes to
ketone bodies about 75 percent of the butyrate and 30 per-
cent of the acetate. Little or no propionate produced in
the rumen is metabolized to detones by rumen epithelium
(96,97).

At one week of age, the rumen wall of lambs con-
verts small quantities of butyrate to ketone bodies (91).
At 11 weeks of age animals reared on pasture converted
70 percent of the butyric acid to ketones, while adult sheep
converted 60 percent. Little change in utilization of
acetate and propionate for ketone production occurred with
age. The rumen wall of milk—fed lambs, 7 to 9 weeks of age,
converted less than 1 percent of butyric acid metabolized

to ketones (91).

Sutton §t_al, (81) showed the rumen mucosa of milk-
fed calves produced less acetone, acetoacetate and B-
hydroxybutyrate than the mucosa from calves fed hay, milk
and grain from an equimolar mixture of acetic, propionic
and butyric acids. Mucosa from milk-fed calves 16 weeks
old produced 0.1 u moles each of acetone, acetoacetate and
B-OH-butyrate per 100 mg dry tissue when acetate was the
substrate metabolized in yitrg. Mucosa from calves the same
age but fed hay, milk and grain produced 1.7 p moles acetone
and acetoacetate per 100 mg dry tissue and 0.2 u moles B-OH
butyrate per 100 mg dry tissue using acetate as substrate.
_Pr0pionate and glucose did not result in the formation of
ketones by the ruminal mucosa from either group of calves.
When butyrate was used as substrate, mucosa from animals
fed a ration of hay, milk and grain produced 29.7 n moles
of acetone and acetoacetate and 8.6 u moles B-OH-butyrate
per 100 mg dry tissue. Their milk-fed counterparts produced
1.2 u moles acetone and acetoacetate and 0.3 u moles B-OH-
butyrate per 100 mg dry tissue (81). These restuls point to
the extent and importance of metabolic changes in energy
producing metabolites that occur in yiyg during rumen
develOpment.

Cook §E_gl, (26) showed the activity of butyrl
co-enzymelksynthetase to be high in rumen epithelium, liver
and kidney. PrOpionyl co-enzyme A synthetase activity was

high in all tissues studied. The activity of acetyl co-enzyme

10

A synthetase was highest in extrahepatic tissues that are
highly aerobic (i.e. muscle and heart).

Stevens and Stettler (79) proposed the enzyme sys-
tem for butyrate metabolism in rumen mucosa to be easily
saturated. Annison et_al. (2) found little or no butyrate
but a high level of ketones in portal blood when rumen
butyrate was 40 mM. Even if butyrate enters portal circu-
lation, the liver rapidly converts this to B-OH-butyrate(24).

The propionate absorbed by the rumen is converted
to glucose by the liver. Pennington and Sutherland (72)
showed lactate to be the major end product of propionate
,metabolism by rumen epithelium in yitrg. Whe l-C14 pro-
pionate was incubated with rumen epithelium, the greatest

portion of the isot0pe appeared as CO Leng et a1. (58)

2.
concluded the incorporation of 14C from propionate provides
quantitative information concerning Synthesized glucose and
lactate only if propionate was labeled in positions 2 and 3.
They concluded 70 percent of the propionate of rumen origin
was converted to lactate by rumen epithelium before conver-
sion to glucose by the liver. Cook and Miller (25) concluded
that propionate is absorbed from the rumen and appears in
portal blood as such.

Weigand g£_al.(7) in a more recent study presented
conclusive evidence that 85 to 90 percent of isotope appear-

ing in lactate from 2-14C prepionate first must be synthe-

sized to glucose. This synthesis of glucose must occur in

11

the liver and kidney. Propionate conversion into lactate
by rumen epithelium ranged from 2.5 to 9.1 percent, depend-
ing on concentration and pH of the test solution admin-
istered.

'The transition of the young ruminant is an important
event for the animal and occurs early in life with the
proper stimulus. This transition involves changing from
essentially a monogastric animal dependent on simple sugars
and long-chain fatty acids as the major sources of energy
to a functional ruminant whose primary source of energy
becomes short-chain fatty acids arising as degradation pro-
_ducts of complex carbohydrates from the rumen fermentation.
This change results in a lowered amount of glucose that is
directly supplied by the diet as such. Consequently, the
animal must utilize the pathways of gluconeogenesis more
than before and this places a premium upon the endogenous
glucose produced. The following section covers pathways
and control mechanisms involved in the maintenance of a
functional glucose producing pathway. Work from both mono-
gastrics and ruminants will be included and comparisons
made. Many points included will be from monograstric spe-
cies due to the lack of such work in ruminants.

The simple-stomached animals depend to a large
extent on dietary sugars as an energy source. These
sugars, especially glucose, are important in maintenance

of neural. tissues, the formation of complex macromolecules

12

such as glycoproteins, and lactose synthesis in the lactat-
ing animal.

Mature ruminants fed maintenance type diets derive
50 to 80 percent of their energy from volatile fatty acids
produced as a result of the fermentation in the rumen of
dietary carbohydrates (10). On high roughage diets, the
amount of carbohydrate escaping rumen fermentation is low.
This leaves little or no glucose to be absorbed in the lower
tract. Consequently, the ruminant on a high roughage diet
must synthesize most all of its glucose from glucogenic
precursors.

The low blood glucose found in adult ruminants gives
a partial indication to the high premium for its use. Gly-'
colytic enzyme activities decrease as the ruminant matures
(40). Like the monogastric, ruminants need glucose for
maintenance of neural tissue, formation of complex glyco-
molecules, lactose and glycerol synthesis in the lactating
animal. The volatile fatty acids are used in place of glu-

cose as a major source of energy for the ruminant (10).

Pathway of Glucose Synthesis
Figure 1 illustrates the pathway for synthesis of
glucose from glucogenic precursors. The synthesis of
glucose is the direct reversal of the glycolytic pathway
with the exception of four enzymes. These enzymes--
pyruvate carboxylase (E.C. 6.4.1.1.), phosphoenolpyruvate

carboxykinase (E.C. 4.1.1.32), fructose 1,6 diphosphatase

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(E.C. 3.1.3.11) and glucose-G-phosphatase (E.C. 3.1.3.9)—-
provide for alternative reactions to the thermodynamically
unfavorable reaction for reverse glycolysis (54).

A thorough review of the properties controlling the
activity of key gluconeogenic enzymes is beyond the sc0pe
of this thesis. Table 1 lists enzyme properties for pyruvate
carboxylase, phosphoenolpyruvate carboxykinase, and fructose
diphosphatase with selected references for the reader.

The following part of this thesis will discuss con-
trol mechanisms involved in the regulation of gluconeo-
genesis. Areas of control to be discussed include factors
,at the enzyme level, subcellular level, cellular level,

and dietary level.

Pyruvate Carboxylase

 

Pyruvate carboxylase catalyzes the reaction:

Pyruvate + C0 + ATP Acetyl COA\

2 Mg++ 447 Oxaloacetate + ADP + Pi

 

Taylor gt_§l. (84) found pyruvate carboxylase to
be completely localized in mitochondria of well-fed mature
sheep. In fasted and diabetic states, up to 40 percent of
the activity of pyruvate carboxylase was found in the
cytosol, possibly indicating membrane fragility.

Pyruvate carboxylase was first isolated from avian
liver by Utter and Keech (87). It is now well known that

the enzyme is activated by acetyl-CoA. Recent work by

15

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17

McClure §t_al. (65) has shown the enzyme to have a molecular
weight of about 1.3 x 105. The protomer contained biotin and
manganese and dissociated into three to four polypeptide
chains. In addition to acetyl-CoA and pyruvate, enzyme activ-
ity required magnesium (Mg) in excess of Mg=ATP complex and
a monovalent cation (usually K+). Maximal reaction velocity
was between pH 8.0 and 8.5 (65). The enzyme is inhibited by
a number of products, including magnesium chelates of nucleo-
side triphosphates, MgADP, orthOphosphate, and adenosine;
the latter being most potent (66).

McClure §t_§l, (66) have prOposed a two-site ping
_pong mechanism for the carboxylation of pyruvate. The first

site (Site 1) involves binding of MgATP and HCO3 to the
enzyme in a random nature. The second site (Site 2) binds
pyruvate at a separate site possibly near the Mn of the
enzyme and carboxylation occurs.

From their exhaustive studies into the properties of
pyruvate carboxylase, McClure and Lardy (64) presented a
relatively simple explanation to in 3139 regulation. First,
the amount of active enzyme present in mitochondria is con-
trolled by the level of acetyl-CoA and second by the amount
of substrate available,ix1this case pyruvate. At high
acetyl-CoA concentrations with constant concentrations of
MgATP, HCO3-, and pyruvate sufficient active enzyme is pres-

ent to readily bind these reactants to the enzyme. In this

case, the controlling factor would be the amount of pyruvate

18

available for oxaloacetate formation. At rate-limiting
acetyl-CoA concentrations, the same enzyme saturation curve
of pyruvate would result, but the maximal reaction rate
would be lower due to a greater proportion of inactive

enzyme (64).

Phosphoenolpyruvate Carboxykinasel

PEPCK catalyzes the_freely reversible reaction:

PEPCK~ PEP2 + co + IDP

+
Oxaloacetate ITP W 2

The enzyme was first isolated from chicken liver by Utter
.and Kurahashi (88).

PEPCK is found in liver mitochondria of pigeon (38)'
and chicken (86), in the cytosol of rat and mouse (70), and
near evenly divided between mitochondria and cytosol in
guinea pig (70), rabbit (70), pig (82), cow (8,9), and
sheep (84). PEPCK is ITP or GTP dependent, requires the
divalent cation manganese, and is apparently coupled with
succinic thiokinase for use of generated GTP (or ITP). The
molecular weight of PEPCK is approximately 73,000 and
requires free sulfhydryl groups for activity. The decar-

boxylating reaction (0AA to PEP) is competitively inhibited

3 , GMP, and GDP (or IMP and IDP) (22).

by HCO

 

lPhosphoenolpyruvate Carboxykinase abbreviated PEPCK.

2Phosphoenolpyruvate abbreviated PEP.

19

The enzyme appears to act in a two-step sequence with
the mechanism to be explained for the carboxylation reaction
(PEP to 0AA). In the first step, PEP and IDP are bound to
the enzyme, resulting in an enzyme-—IDP--enolpyruvate com-
plex. In the second step, oxygen from bicarbonate makes a
nucleophilic attack on the enolphosphorylphosphorus atoms.
Subsequent exchange at the bicarbonate carbon and phosphoryl-
phosphorus leads to formation of oxaloacetate and ITP (23).
Since this reaction is freely reversible, the formation of
PEP from oxaloacetate would proceed by the reverse mechanism.

Once PEP is formed, gluconeogenesis is the reversal

.of glycolysis until fructose diphosphate (FDP) is reached.

Fructose-1,6 Diphosphatase

Fructose diphosphatase catalyzes the unidirectional

reaction:
FDP + H20 ——> F-6-P + Pi

At this level, the interactions of FDPase and phos-
phofructokinase (PFK) become very important in regulating
the flow of carbon to either glucose or pyruvate. PFK is
inhibited by citrate and ATP, and stimulated by AMP, ADP,
and FDP (53). FDPase is stimulated by low concentrations of
FDP with a Km for the purified form of 2.6 x 10-6 M at
pH 7.5. At 0.4 mM FDP, the enzyme activity is reduced by
50 percent of maximum. FDPase shows a complete inhibition

4

by AMP at a concentration of 10’ M (as). This inhibition

20

can be lowered by increasing the concentration of Mg which
apparently chelates AMP (85).

Geller gt_al. (37) showed highly purified bovine
FDPase activity to have a pH Optimum of about 9.0. Crude
extracts showed a pH optima at 6.0-6.5 and one at 8.0-9.0.
The acid pH activity could be destroyed with proteolytic
enzymes which resulted in an increase in alkaline activity.
Similar results were obtained using sulfhydryl reagents such
as P-mercuribenzoate and N-ethylmaleimide. This implies
small changes in conformation may have occurred.

Byrne §£;§l, (21), using a bovine preparation of
,Geller §t_al. (37), estimated the molecular weight of the
enzyme to be about 130,000. This molecular weight was sim-'
ilar to reported values for the rabbit enzyme. Traniello
gt_§l. (85), using the rabbit enzyme, were able to isolate
the enzyme with Optimal activity at neutral pH. Its molecu-
lar weight was about 143,000, indicating some modification
of the enzyme from previous isolation procedures. This
neutral form is more sensitive to AMP inhibition and acti-

vated by sulfhydryl compounds such as CoA and homocysteine.

Control of PEP Formation

 

With the exception of glycerol, all glucogenic pre-
cursors to be discussed converge through various pathways
to form oxaloacetate. The conversion of oxaloacetate to PEP

is generally accepted to be the fine control point in

21

gluconeogenesis. Each pathway leading to oxaloacetate has
some secondary control.

Figure l is a detailed description of the pathways
leading to oxaloacetate formation. The mitochondria is
included to emphasize the importance of this cell organelle
in regulation of substrate flux. Control points leading to
oxaloacetate formation are identified with capital letters
with the following discussion to center on these control

points.

Citrate Cleavage Pathway
The enzyme ATP-citrate lyase (E.C. 4.1.3.8) catalyzes

.the reaction:
Citrate + CoA -———e> Acetyl CoA + oxaloacetate

The enzyme is located in the cytosol of non-ruminant liver
and absent in ruminant liver (105).

Under conditions where an excess of acetyl CoA is
formed in the mitochondria, citrate readily diffuses out of
the mitochondria to the cytosol. Here ATP-citrate lyase
cleaves citrate to form oxaloacetate and acetyl CoA. The
acetyl CoA is readily used in fatty acid synthesis. The
oxaloacetate could be used for PEP formation since PEPCK is
also located in the cytosol. More likely, the oxaloacetate
is converted into pyruvate sequentially by NAD-malate
dehydrogenase and NADP-malate dehydrogenase (also absent

in the ruminant liver) (48). This mechanism allows for

22

resynthesis of oxaloacetate within the mitochondria by pyru-
vate carboxylase and a net loss of oxaloacetate from the
mitochondria is prevented. The probability that this is the
preferred mechanism of oxaloacetate is the fact that

under conditions of lipogenesis the activity of NADP-malate
dehydrogenase increases but does not in gluconeogenesis.
Therefore, this reaction does not appear to be important

in gluconeogenesis (105).

Pyruvate Carboxylase

 

The second reaction leading to the formation of
oxaloacetate is catalyzed by pyruvate carboxylase. Alanine,
.serine and lactate enter the gluconeogenic pathway through
pyruvate. In the ruminant liver, this enzyme is activated
by butyryl CoA and propionyl CoA in addition to acetyl CoA
(10). Since ruminant liver concentration of these CoA
derivatives is high, this could explain the glucose sparing
effect some pe0ple have reported.

The metabolism of pyruvate between ruminant and
non-ruminant species is preferentially different with
respect to the pathway which pyruvate follows. In non-
ruminant species, such as the rat, pyruvate is preferen-
tially decarboxylated to acetyl CoA. The acetyl CoA enters
the TCA cycle by combining with oxaloacetate to form citrate.
Citrate can also diffuse to the cytOplasm and undergo the

reactions described in the previous section.

23

In ruminant liver pryuvate carboxylase and pyruvate
dehydrogenase are sufficiently active to allow for flow of
pyruvate carbon through either carboxylation to oxaloacetate
or decarboxylation to acetyl CoA. In ketosis, the carboxy-
lation pathway is the preferred pathway evidenced by an
increase in the activity of this enzyme during this metabolic
aberration (ll). Lactation causes an increase in pryuvate
carboxylase activity over the non-lactating state (11). The
increased demand for glucose by lactation is, therefore,
partially met by an increase in pyruvate carboxylase activ-
ity. Fasting has been shown to increase the activity of

,pyruvate carboxylase (9,11,35).

Malate Dehydrogenase

 

The third reaction leading to the formation of oxalo-
acetate is catalyzed by NAD-malate dehydrogenase (E.C. 1.1.1.
37). The enzyme is located in both the mitochondria and the
cytoplasm. The equilibrium for the reaction is toward mal-
ate with the malate to oxaloacetate ratio approximately 95:5.
NAD-malate dehydrogenase is more active in ruminant liver
than non-ruminant liver. Since many glucose precursors in
ruminants pass through malate, this may account for the
increased activity of NAD-malate dehydrogenase found in rum-
inant animals (42).

Ballard gt_al. (11) have postulated two pathways of
gluconeogenesis in the ruminant. Each revolves around the

metabolism of malate in the mitochondria. First, malate

24

formed from propionate or amino acids can diffuse out of the
cytoplasm and there be converted into oxaloacetate and PEP

by NAD-malate dehydrogenase and PEPCK (Figure l). The sec-
ond pathway involves conversion of mitochondrial malate
sequentially into oxaloacetate and PEP. The PEP thus formed
diffuses across the mitochondrial membrane and becomes avail-
able to the remainder of the gluconeogenic pathway.

Control of gluconeogenesis thus occurs at several
levels. At the enzyme level many aforementioned factors
exert influences to regulate enzyme activity. Whether these
factors stimulate or inhibit an enzyme, thus a pathway, is

,dependent on the phySiological state of the animal.

Initiation of Gluconeogenesis

Before birth, fetal glycogen levels are high, reach-
ing levels up to 10 mgs glycogen per gram of liver in many
species. After birth, glycogen stores are depleted rapidly
and the neonate must rely on synthesis as the major source
of glucose (77).

In fetal rats, all enzymes for gluconeogenesis
exhibit activity by Day 20 of gestation with the exception
of PEPCK (7,89,104). Immediately after birth, the activity
of PEPCK increases, reaching a maximum about two days after
birth. Other key gluconeogenic enzymes increase in activity,
but the increases in their activities are less dramatic

(7,68,89,104).

25

During the suckling period, the activities of all
gluconeogenic enzymes remain high (90). During this period
of life, the animal is consuming a milk diet high in fat and
protein. Amino acid utilization for gluconeogenesis reaches
a maximum at about the fifth day of postnatal life. Aspar-
tate amino-tranSferase shows a similar postnatal development
for amino acid incorporation into glucose (103).

During the weaning period, the diet begins to provide
the animal with a source of glucose (90). Enzymes involved
in gluconeogenesis decrease in activity, reaching adult lev-
els at about 20 days of age. If the animal is weaned to a
,high fat and/or a high protein diet, a delayed decrease in
gluconeogenic enzyme activity is seen. Glycolytic enzymes,’
especially those involved in regulation of the pathway, are
regulated in the reverse manner; i.e., they increase in

activity when gluconeogenic enzymes are decreasing (68,89,90).

Glucose Kinetics in the Ruminant

In discussing kinetic data for a substrate, terms
must be used to define properties of that substrate within
the animal. Glucose pool can be defined as the amount of
exchangeable glucose available to the animal per unit body
weight. Glucose space is defined as the amount of fluid
necessary to dissolve the glucose pool giving the same con-
centration as that found in plasma. Glucose entry rate is

defined as the rate of entry of glucose into the body pool.

26

Glucose turnover is defined as the rate of removal of glu-
cose from the body pool (29).

Davis and Brown (29) found the glucose pool size in
two-week old calves to be 1.4 times that of the glucose pool
of six-month old steers. Jarrett gt_al. (50), using young
lambs and adult sheep, found the glucose pool of young lambs
to be 2.5 times greater than that of adult animals.

The glucose space in young animals is larger than
that of adult animals. The glucose space in two-week old
lambs is about 70 percent of body weight, while adult values
range from 24 to 28 percent (50). Calves two weeks of age
.had a larger glucose space than six—month old steers (29).

6 The concentration of a metabolite in the blood is
not indicative of its importance in the overall metabolism
of the animal. If a substance has a high plasma concentra-
tion but a low turnover rate, the substance may not be as
important as one found with a low plasma concentration but
having a high turnover rate (29). Davis and Brown found
glucose turnover in two—week old calves to be 8.30 g/hr/

100 pounds body weight and that in six-month old steers to

be 4.71 g/hr/100 pounds body weight, indicating glucose
utilization to be dependent on the amount available.

Jarrett gt_al. (50) found the same trend in mature sheep, but
the decrease in glucose utilization was 75 percent of that

of young lambs. Bergman and Hogue (16) reported the glucose
turnover rates in lactating sheep to be three times that of

non-lactating sheep.

27

Fasting decreases the glucose entry rate in rumi-
nants (55,56). Kronfeld and Raggi (55) found the entry rate
to be reduced 50 percent in fasting cows and increased 35
percent in insulin-treated cows. Spontaneous ketotic cows
had an entry rate equal to normal cows. Wiltrout and Satter
(98) reported the entry rate for dry and lactating cows to
be 7.6 and 14.0 moles/day (0.44 and 0.81 g/hr/Kg B.w.°°75),

respectively.

Gluconeogenesis in the Ruminant

 

Practically all the dietary carbohydrates fed to
ruminants are fermented in the rumen to volatile fatty acids.
.On a high roughage diet, the amount of digestible carbohy-
drate escaping the rumen is low, and these acids provide
as much as 80 percent of the animal's maintenance require-
ment for energy (10). Karr §£_gl. (52) and McRae and
Armstrong (67) have found high concentrations of monosac-
charides in the abomasum of ruminants receiving high starch
diets.

Of the volatile fatty acids produced in the rumen,
only propionate provides a net synthesis of glucose. Pro-
pionate is rapidly cleared from portal circulation by the
liver of ruminants where it is converted into glucose
(17,51,58). Acetate and butyrate, though not providing a net
glucose synthesis, are thought to spare glucose by acting as

an extrahepatic energy source. Virtually no acetate is

28

cleared by the liver (3,25) and butyrate appears in periph-
eral circulation as B—hydroxybutyrate (24).

Other important precursors of glucose include amino
acids, glycerol and lactate. Many amino acids are gluco-
genic as illustrated in Table l, but the relative importance
of the alanine cycle (34) remains to be proven. Glycerol
released during lipolysis is a precursor of glucose. About
5 percent of propionate produced in the rumen is converted
to lactate by the rumen epithelium during absorption (97).
Lactate produced as a result of tissue glycolysis is another
carbon source of glucose, but a net synthesis of glucose

,from lactate is not possible (4,27).

Substrate Availability and Gluconeogenesis

Propionate

 

Propionate produced in the rumen has been considered
by many workers (3,17,51,58,98) to be the major precursor of
glucose in the ruminant animal. These workers estimate
25 to 60 percent of the synthesized glucose is derived from
prOpionate, depending on the diet and metabolic state of the
animal.

When animals consume high concentrate diets, about
27 percent of the glucose synthesized originates from
propionate (51). On high roughage diets, prOpionate may
contribute up to 54 percent of the synthesized glucose (58).

Pr0pionate production in the lactating cow is about

one Kg per day when fed a ration to meet NRC requirements

29

for TDN with a 60:40 concentrate to hay ratio (98). Wiltrout
and Satter estimated that one- to two-thirds of the pro-
pionate produced is converted to glucose with a mean of

45 percent for the lactating cow and 32 percent in the dry
cow. However, glucose entry rates for these animals averaged
14.0 and 7.4 moles/day for lactating and dry cows, respec-
tively. This amounts to 6.3 and 2.4 moles of glucose pro-
duced per day from propionate. Thus, glucose derived from
propionate can give rise to 4,536 Kcal per day for a lactat-
ing cow and 1,728 Kcal per day for a dry cow. For a mature
500 Kg cow this accounts for 12.9% of metabolizable energy
,during the dry period and 13.7% metabolizable energy with

a production of 20 Kg of milk per day at 3 percent butter-
fat (71).

Smith and Osborne-White (78) have shown tflua major
product of propionate metabolism by sheep liver mitochondria
to be malate with less than 20 percent of the pr0pionate
converted to PEP by intramitochondrial PEPCK. Presumably,
the malate formed readily diffuses into the cytosol where
cytosolic NAB-malate dehydrogenase and PEPCK convert malate
to oxaloacetate and PEP, respectively..-

When 2414C pr0pionate and pyruvate were incubated
with mitochondria, the major product of propionate metabolism
was citrate. This indicates the oxaloacetate formed in the
mitochondria from pr0pionate may be used for replenishing

TCA cycle intermediates in the ruminant (78). These findings

30

would support the former pathway of gluconeogenesis proposed

by Ballard et a1. (11) alluded to earlier.

Amino Acids

The other major precursors for gluconeogenesis are
amino acids. This source has received little attention
until recently. Of the amino acids, about 18 are gluco-
genic (Table 2), of which only about five are considered to
be of any major importance. These amino acids enter the
gluconeogenic pathway at various points with a common inter-
mediate of oxaloacetate and/or malate. The transaminases
involved in supplying intermediates for gluconeogenesis

.occur in cytoplasm and mitochondria (103).

Table 2.--G1ycogenic amino acids.

 

Alanine Glutamine Proline
Arginine Glycine Serine
Aspartate Isoleucine Threonine
Asparagine Lysine TryptOphan
Cysteine Methionine Tyrosine
Glutamate Phenylalanine Valine

 

Egan and Black (32) have shown glutamic acid to
account for over 50 percent of the lactose carbon appearing
in milk after the I.V. injection of uniformly labeled 14C
glutamic acid. These workers found more carbon from gluta-

mate appeared in lactose than in milk protein even when

propionate was simultaneously infused intraveneously.

31

Aikawa §E_al. (1), using rats, have shown alanine
aspartate and glutamate to be incorporated into blood glue
cose at a more rapid rate than any other amino acids. Quan-
titatively, asparate and alanine were most important, fol-
lowed by glutamate and serine under physiological plasma
concentrations. When plasma concentration reached 3mM for
alanine and serine, the incorporation ratio (ratio of pro-
duct radioactivity to glucose after two injections of
radioactive substrate) did not change, indicating a linear
increase in production of glucose. Raising the plasma con-
centrations of aspartate and glutamate decreased the incor-
_poration ratio, indicating a smaller fraction of these sub-
strates were incorporated into glucose.

In sheep, about one-half of the alpha-amino nitrogen
removed by the liver can be accounted for by glycine, ala-
nine, and glutamine (100). Wolff and Bergman (102) have
estimated the mean turnover rate (u moles/hr) of alanine,
glutamate, glycine, and serine to be 11.5, 8.5, 13.5 and 6.7,
respectively. About one-third of alanine, glycine and ser-
ine was utilized by the liver with the remainder utilized by
extrahepatic tissues (102).

The relative importance of five individual amino
acids as glucose precursors in sheep receiving a near main-
tenance ration have been determined (101). Alanine and
glutamate contributed the largest amount of carbon to glucose

synthesis with serine and aspartate the least. Glycine

32

contribution was intermediate. These amino acids contributed
between 11 and 29 percent of the glucose synthesized. This
estimate of amino acid contribution to glucose synthesis 7
agrees with that of Reilly and Ford of 28.2:t5.l percent

(74).

Glycerol

Glycerol is released from adipose tissue during fast-
ing, ketosis, or other periods of fat mobilization, and its
glucogenicity is well recognized. Most of the glycerol
(80-90 percent) is removed from circulation by the liver.
This, plus the high activity of liver glycerokinase, suggest
.the importance of glycerol in gluconeogenesis (18).

Aikawa gt_al. (l) have shown glycerol to be more
rapidly incorporated into glucose than lactate or alanine in
the rat. Since glycerol is not regulated at the level of
PEPCK, less control on glycerol conversion to glucose has
been proposed by these workers.

Bergman gt_§l. (18) have estimated the conversion of
glycerol to glucose in sheep under various physiological
conditions. Nonpregnant ewes receiving 800 g of alfalfa
per day derived about 5 percent of their glucose from
glycerol. Nonpregnant, fasted animals (fasted for 3-5 days)
have elevated plasma glycerol levels with 23 percent of
their glucose originating from glycerol. Pregnant fasted
animals derived as much as 40 percent (mean 28 percent) of

their glucose from glycerol. In the absence of feed, no

33

propionate was produced, and the amount of glycerol con-
verted to glucose could not account for the discrepancy

between glucose metabolized and glucose synthesized.

Lactate
In 1928, Cori and Cori reported on the importance
of lactate in replenishing muscle glycogen (27). The Cori

cycle, as it came to be known, went as follows:

Muscle glycogen ——5 lactate ——>'1iver glycogen ——>»glucoes
-—<> 'muscle glycogen. Muscle glycogen could be used as a
source of energy and converted to lactate which was released
.into the bloodstream and traveled to the liver. At the
liver, the lactate was converted back into liver glycogen
which upon demand releases glucose into the circulation.

The released glucose would be removed by muscle and con-
verted into muscle glycogen completing the cycle. Although I
no net synthesis of glucose occurred, the Cori cycle would
provide the body a mechanism to reutilized carbon atoms

for glucose synthesis.

The extent of the Cori cycle in ruminants in unknown
although it is probably not a major supplier of glucose due
to the relatively low activity of glycolytic enzymes in
mature ruminants. This is not to say it is not important,
however, since it provides a glucose sparing mechanism.

Annison et_gl. (4), using mature wethers on a daily

ration of 500 g lucerne and 100 g corn, had a lactate entry

34

rate of 1.7 mg/min/Kg. Animals fasted for 24 hours had a
decreased entry rate of 1.2 mg/min/Kg. The percentage
glucose derived from lactate was 16 percent for fed animals
and 13 percent for fasted animals. These values were for
resting animals. Since glucose is probably used to some
extent in lactating animals as an energy source, the 16 per-
cent value could underestimate the contribution of lactate
to glucose synthesis. If glucose was completely oxidized to

CO the 16 percent value would overestimate the contribu-

2,
tion of lactate (16).

Summation of results of the research discussed in
_this section-gives the results shown in Table 3. Listed
are the glucose precursors, contribution to total synthe-
sized glucose, and reference. Much of the work reported in
Table 3 was done under different dietary conditions. Some
animals were offered feed twice daily, while others were
given feed at hourly intervals. This, along with some car—
bohydrate escaping rumen fermentation, may help in explain-

ing a value of less than 100 percent when the percent contri-

bution of each glucose precursor is added.

35

Table 3.--G1ucose precursors and their contribution to
glucose synthesis in ruminants during different
physiological states.

 

 

 

Precursor Specie Statea %b References

PrOpionate Bovine NL 32 98
L 45 98
Ovine Fed 27-40 17
Fasted 0 17

Amino Acids Ovine Fed 11-30 74,101
Glycerol Ovine Fed 5-10 18
Fasted 23-34 18
Lactate Ovine Fed 15 4

a

° NL'= non-lactating; L = lactating; animals fasted
3 days.

bFirst number in % column represents the conversion
of 14C precursor to glucose. Latter number in % column
represents net hepatic uptake of precursor. Rows with one
number are based of 14C data.

MATERIALS AND METHODS

Experiment I

 

Twelve male Holstein calves were separated from
their dams at three days of age and placed in individual
pens using wood shavings as bedding. Calves were randomly
assigned to one of three diets designed to stimulate rumen
develOpment at different rates during an 84-day feeding
period.

Dieth: Diet I consisted of whole milk fed at the
rate of 12 percent body weight, twice daily. At four weeks.
of age intake was reduced to 8 percent body weight for the
remainder of the experimental period.

Diet II: Diet II consisted of milk fed at 12 per-
cent body weight for 49 days at which time milk intake was
reduced by 50 percent and grain and alfalfa hay offered at
1.35 Kg each per day. Milk feeding ceased at Day 56 with

grain and hay offered ad libitum for the remainder of the

 

experimental period.

Diet III: Diet III consisted of milk fed similar
to Diets I and II. Grain and alfalfa hay were fed initially
at 1.0 Kg per day, beginning on Day 15. On Day 21, milk
intake was reduced to one-half and discontinued on Day 30.

Calves received grain and hay ad libitum for the remainder

 

of the experimental period.

36

37

Calves were weighed periodically for intake adjust-
ments and at the end of the experiment to determine growth
rate. The experimental design is shown in graphic form in

Table 4.

BiopsyfiTechnique

 

Liver biopsies were taken at 11:00 a.m. on Days 10,
30, 56, and 84 according to the technique described by
Erwin gt_al. (33). The right posterior rib area was shaved
and scrubbed twice with an iodine solution and blotted dry
with cotton. A scapel with a 20 gauge blade was used to
make a dorsoventral incision at either the tenth or eleventh
'intercostal space approximately 15 centimeters from the
dorsal midline.

The trocar and cannula were placed through the inci-
sion and pointed anteriorly toward the junction of the left
front leg and body cavity. After entering the body cavity
the trocar was removed and a 50 m1 syringe attached to the
cannula. The liver was located by touch and the cannula
bored into the liver to a depth of approximately 10 centi-
meters. Vacuum from the syringe was developed simultaneously
with the removal of the cannula from the animal.

The liver sample was forced onto a piece of cheese-
cloth, blotted gently to remove any blOod, placed in a 6
whirlpak and dipped into a styrofoam contained containing

liquid nitrogen.

38

Table 4.--Feeding and biopsya schedule for calves in
Experiment I. ‘

 

Age (days)
3

10

21

30

49

56

84

Diet I

T

Milk 2x/day
12% BW

 

Milk 2x/day
8% BW

 

Diet II

T

Milk 2x/day
12% BW

 

._L__

Milk 2x/day
8% BW

 

Milk lx/day
4% BW, Alf,
Hay, Grain

 

 

Alf, Hay
Grain-
Ad Lib

_L

Diet III

Milk 2x/day
12% BW

Milk lx/day
6% BW, Alf,
Hay, Grain

 

Alf, Hay, Grain
Ad Lib

 

 

aCalves were biopsied on Days 10, 30, 56, and 84.

39

The incision was packed with "Ureka" Sulmidel powder
and closed using a wound clip. All instruments were'kept.
sterile by placing in 80 percent ethyl alcohol until used.
Calves received Sec of Procaine Penicillin i.m.

The sample was stored in the laboratory freezer at

-60° C for future analysis.

Enzyme Assays

 

Phosphoenolpyruvate carboxykinase (PEPCK) was
assayed by the method of Utter and Kurahashi (88) as modified
by Barnes and Keech (13). Frozen tissue was homogenized in
six volumes of 40 mM Tris-citrate pH 6.5 and 0.6 volumes of
'10 mM dithiothreitol with a teflon homogenizer. Each tissue
sample was homogenized and then assayed in duplicate. Tubes
for each assay contained in p moles: Tris-citrate (pH 6.5),

50; (PEP), 2.4; (IDP), 1.0; MnCl 1.5; glutathione, 0.8;

NaH14CO

2.
3, 5.0 (cpm/umole-4.oX105) and 0.02 ml of tissue
homogenate (total volume 0.6 ml). The reaction mixture was
incubated for 10 minutes at 30° C and terminated by addition
of 0.1 ml of 20 percent trichloroacetic acid. Assay tubes
were placed on ice and allowed to stand 30 minutes. Solid
CO2 was added to each tube 5 times to dilute out and release
14

any unreacted CO Control tubes with no PEP were used

2.
for all tissue homogenates. The precipitated protein was

removed by centrifugation and 0.1 ml aliquot of the

 

1Jensen-Salsbery Laboratories, Kansas City, Mo.

40

supernatant used to determine radioactivity in a Nuclear-
Chicago Model 6770 liquid scintilation spectrOphotometer.
The efficiency of counting was 75 percent to 80 percent
throughout the experiment. The scintilation fluid consisted
of: p-dioxane, 770 ml; xylene, 770 ml; absolute ethanol,
460 ml; napthalene, 160 g; PPO (2,5-diphenyloxazole), 10g;
and dimethyl POPOP 1,4 -bis (2-4-methyl-g-phenyloxazolyl)
-benzene, 0.lg.

Pyruvate carboxylase was assayed by the procedure
of Yeung gt;al. (103). Frozen tissue was homogenized in
6 volumes of 40mM Tris-acetate pH 7.4 and 0.6 volues of lOmM
,dithiothreitol, with a teflon homogenizer. Homogenized tis-
sue was assayed in duplicate. Tubes for each assay contained
in u moles: Tris-HCl (pH 7.4), 40; pyruvate, 5.0; ATP, 5.0;

14

acetyl CoA, 0.67; MgCl 5.0; NaH CO 5.0 (cpm/pmole 4.0 X

2' 3'
105) and 0.02 ml of tissue homogenate (total volume 0.6 ml).
The reaction mixture was incubated for 10 minutes at 30° C,
terminated by addition of 0.1 ml of 20 percent TCA, and
handled in the same manner as PEPCK. Each assay was cor-
rected for blank activity by omitting acetyl CoA.
G1ucose-6-phosphatase was determined by the method
of Harper (43). Frozen tissue was homogenized in 20 volumes
of 10 mM Citric acid adjusted to pH 6.5 with 30 percent NaOH.
The homogenate was prepared with a Polytron homogenizer at

a rheostat setting of 4. 780.1 m1 aliquot of the homogenate

was preincubated at 37° C for 5 minutes after which 0.1 ml

41

of 80 mM glucose-6-phosphate was added. Enzyme activity was
measured initially and after 15 minute incubations at 37.0 C.
The reaction was st0pped by addition of 2.0rmlof 5 percent
TCA, the precipitate was removed by centrifugation and a
1.0 ml aliquot of the supernatant used for Pi determination
(36).

Protein in all supernatant fractions was determined
by the method of Lowry (60).

ATP was obtained from Nutritional Biochemical,
Cleveland, Ohio. All other special chemicals were obtained
from Sigma Chemical Co., St. Louis, Mo. Radioactive

14

.NaH CO was obtained from Amersham-Searle Corporation,

3
Arlington Heights, Illinois. The remaining chemicals used

were of the highest obtainable purity.

Experiment II

 

To study the effects of dietary transition on key
gluconeogenic enzyme activity, three male Holstein calves
received milk in the same manner as those in Experiment I
until 42 days of age, when milk intake was reduced by 50 per-
cent and hay and grain offered at the rate of 1.35 kg each
per day. On Day 49 milk was removed from the daily feeding
regime, with calves receiving hay and grain for the remain-
ing experimental period. Biopsies were obtained at 42, 56,
and 63 days of age.

Frozen liver samples were subjected to the same

procedures as those in Experiment I.

42

Analysis of variance as described by Gill (39) was
used to test significance among days, diets, and day X time
interaction for Experiment I. Similarly, analysis of vari-

ance was used to test days and animals for Experiment II.

RESULTS AND DISCUSSION

 

Results of Experiment I

The three diets were fed to allow for rumen develop-
ment at different rates. In this study, calves fed milk for
84 days had a significantly higher serum glucose level than
did calves raised on diets designed to stimulate rumen
develOpment (Table 5). This would seem to indicate animals
in Diet I remained non-functional ruminants during the
experimental period while animals in Diet II and Diet III

apparently became functional ruminants.

Table 5.--Serum glucose at Day 84 and average daily gain of
calves in Experiment I.

 

 

Diet I Diet II Diet III
Serum Glucosea 139.54b 84.94c 89.79c
SEMd 22.27 5.80 4.22
ADGe 454 _ 513 503

 

aMilligrams glucose per 100 milliliters serum.

b’cValues with different superscripts are signifi-
cantly different (p < .05) as determined by Tukey's "HSD"
test.

dStandard error of the mean.

eAverage Daily Gain in grams per day.

43

44

The extent to which these animals approached the
mature ruminant state though not determined is a very impor-
tant factor for consideration. If the animals were partially
functioning as ruminants, carbohydrate bypassing rumen fer-
mentation would decrease the need for gluconeogenesis by the
liver, resulting in a decreased enzyme activity.

Regardless of rumen develOpment, animals consuming
large amounts of concentrates may have as much as 40 percent
of dietary carbohydrates appearing in the small intestines
(52). In the present study, animals initially received a
pelleted concentrate and alfalfa hay each at the rate of
_0.5 Kg per day. Feed consumption by calves receiving diets
designed to stimulate rumen development is shown in Table 6.
Animals receiving Diet III, the diet for early rumen develop-
ment, consumed 60 percent of the daily feed as concentrate
initially. After the second week, concentrate averaged
80 percent of total dry matter intake.

Animals in Diet II, the diet for the intermediate
rate of rumen development, began to eat dry feed readily
consuming about one Kg of feed per day by the second week,
of which 66 percent was concentrate. The percentage of
concentrate consumed increased to 76 percent during the
fourth week of hay and grain feeding. Between 9 and 12
weeks of age, total dry matter consumption between groups
did not differ. However, concentrate made a greater con-

tribution to the total ration for calves in Diet III.

45

Table 6.--Average daily dry feed consumption for calves in
Experiments I and II.

 

Experiment I Experiment II

 

 

 

Dieta
II III
Week Conc.b Hayb Conc. Hay Conc. Hay
4 -- -- 0.18 0.10
5 -- -- 0.25 0.15
6 -- -- 0.51 0.34
7c -- -- 0.64 0.15 0.70 0.18
8 0.35 0.27 0.64 0.22 0.83 0.19
9 0.64 0.33 0.86 0.18 1.20 0.19
- 10 0.88 0.47 0.96 0.26
11 1.49 0.46 1.02 0.20
12 1.74 0.37 1.69 0.40

 

aDiet I consisted of milk for a 12-week period.
bConcentrate and hay in Kg. of feed per day.

CIncludes Days 42 through 49 for Experiment II.

Animals consuming a ration with a greater proportion
consisting of concentrates would probably have a greater
amount of carbohydrate escaping rumen fermentation, espec-
ially young ruminants. This bypass of rumen fermentation
by carbohydrates would allow hexoses to be absorbed in the

small intestine, thereby decreasing the need for gluconeo-

geneSis.

46

The concentrate that did not escape rumen fermen-
tation would give rise to volatile fatty acids. Pr0pionate
production in the rumen increases as the percentage of con-
centrate in the ration increases. Since propionate is the
only VFA to give rise to a net synthesis of glucose, it may
be the major glucose precursor in young animals since amino
acids should be spared for growth.

All animals received milk prior to the first biOpsy
at 10 days of age. No difference in enzyme activities was
expected between the three groups. Day 10 values for pyru-
vate carboxylase (Table 7) were similar for all groups when
.activity was expressed on a per gram tissue or per mg pro-
tein basis. Day 10 PEPCK activity (Table 9) ranged from
0.53 to 1.19 units and 3.90 to 9.12 units when expressed
on a per gram or per mg protein basis, respectively. This
difference was not significant. Glucose-6-phosphatase
activity (Table 10) at Day 10 was similar for all groups.

The enzyme activities of the animals receiving Diet I
remained at a fairly constant level of activity throughout
the experimental period. The range of activities for pyru-
vate carboxylase, PEPCK, and glucose-6-phosphatase varied
less than 35 percent between periods in animals receiving
Diet I. Thus, Diet I was a suitable control diet to compare
changes in enzyme activity related to dietary change.

No previous reports have been found to indicate

changes in PEPCK activity in ruminant animals with change in

47

diet, lactation, and/or spontaneous ketosis. Pyruvate
carboxylase has been reported to rise during fasting,
ketosis, and lactation (9,35). In the rat, the situation of
PEPCK and pyruvate carboxylase is opposite to that found in
the bovine during fasting (63,90).

Analysis of variance indicated a significant increase
in liver protein per gram of wet tissue from Day 10 to Day
30. Day 10 and Day 30 values averaged 135 and 172 mgs pro-
tein per gram of tissue. Thereafter, protein content did
not differ among periods. Dietary effects and diet X time
interactions were not significant.

.Pattern of Pyruvate
Carboxylase

 

 

Many precursors may give rise to glucose synthesis.
Almost all of these precursors must pass through PEP while
proceeding to glucose. The reversibility of pyruvate kin—
ase is thermodynamically unfavorable. Pyruvate carboxylase
in conjunction with PEPCK supplants pyruvate kinase.

The activity of pyruvate carboxylase is shownin
Table 7. Overall analysis of variance gave a highly sig-
nificant (p < 0.01) diet X time interaction for pyruvate
carboxylase when activity was expressed on a per gram tissue
basis. When expressed on a specific activity basis, all
parameters were non-significant.

No appreciable differences were found between the

three diets on Day 10 as expected. At 20 days of age,

48

Table 7.--Activity of pyruvate carboxylase in Experiment I.

 

 

 

 

Day
10 30 ‘56 84
Diet a b a b a b a b
I 0.223 1.57. 0.262 1.52 0.246 1.29 0.245 1.39
SEMC 0.04 0.34 0.06 0.34 0.03 0.17 0.08 0.36
II 0.210 1.62 0.128 0.77 0.296 1.73 0.214 1.26
SEM 0.02 0.18 0.02 0.13 0.02 0.09 0.03 0.18
III 0.221 1.62 0.518 3.04 0.214 1.16 0.332 1.76
SEM 0.02 0.18 0.20 1.17 0.02 0.09 10.06 0.33
aUnits of activity: u moles CO2 fixed per minute

per gram wet tissue.

bUnits of activity: milli u moles CO
minute per milligram hepatic protein.

2 fixed per

cStandard error of the mean.

Table 8.--Individual feed intakes and pyruvate carboxylase
activity for calves 30 days of age in Diet III.

 

 

Calf Feed Intakea Enzyme Activity
(Kg)
1 0.57 .064
2 0.43 .308
3 0.14 .846
4 0.21 .854

 

. aFeed intake is the average daily intake of hay and
grain for the 7-day period preceding the bi0psy date (Day 30);
correlation coefficient = -0.99; p < .01.

49

animals on Diet III began their change in diet. Animals
on Diet II began the change at 49 days of age. In each
case, the transition was completed (i.e. no further milk)
by the next biopsy date.

Feed intake levels for calves at Day 30 receiving
Diet III, shown in Table 6, indicate the animals to be par-
tially fasting. After one week on the diet the animals were
eating an adequate amount of feed.

The activity of pyruvate carboxylase at Day 30 for
animals receiving Diet I was not different from the previous.
sample. The activity for animals in Diet II decreased by
-39 percent and 52 percent when activity was expressed on a
per gram tissue and per milligram protein basis, respec-
tively, from the previous sample (Day 10).

Animals receiving Diet III had a highly variable
response which could be related to feed intake (Table 7).
Pyruvate carboxylase activities for individual calves were
0.064, 0.308, 0.846, and 0.854 units and feed intake was
0.57, 0.43, 0.14, and 0.21, respectively. These values
resulted in a correlation coefficient of r = 0.99, which was
significant (p < .01) (Table 8). These data are comparable
to earlier reports showing pyruvate carboxylase to increase
in activity when ruminants were fasted (9,11,35).

The activity of pyruvate carboxylase at Day 56 for
animals receiving Diet I was similar to each of the previous

biopsy samples. Animals receiving Diet III had completed

50

their change in diet and were now eating about one Kg
of feed per day. Pyruvate carboxylase had returned to an
activity comparable to Diet I at Day 56.

Animals receiving Diet II at Day 56 were just com-
pleting their change in diet similar to the change that
occurred between Day 10 and Day 30 for Diet III. With this
change came a peak in enzyme activity which was 2.31 and 2.25
times greater than the previous sample date (Day 30), when
activity was expressed on a per gram of tissue and per mg
protein basis. This magnitude of change was similar in
Diet III, when the change occurred between the first and
.second biopsy date (Table 7).

At Day 84, pyruvate carboxylase activity for animals
receiving Diet I was similar to all previOus values for
that group. Activity of animals receiving Diet II had
decreased to a level equal to the activity found for the
Day 56 value of Diet III. :This would seem to indicate that
a change in diet causes an initial increase in pyruvate
carboxylase activity (probably a result of decreased feed
intake). After adjustment to the new diet, the enzyme
activity would decrease back to a level similar to the
activity before the change.

Animals receiving Diet III had an increase in pyru-
vate carboxylase activity on Day 84. If this was an actual
representative increase, this could represent the animal
actually approaching a mature ruminant. This value compares

with adult ruminants.

51

 

Pattern of PEPCK

As alluded to in an earlier part of the discussion,
the activity of PEPCK on Day 10 (Table 9) was unique to each
experimental diet. Ballard has noted differences in PEPCK
activity in newborn rats due to weight and litter size (6).
Animals receiving Diet II weighed 4.5 Kg more at birth than
did those in the other groups and had PEPCK values 1.6 to
2.2 times those of Diets I and II. The effect weight could

have on PEPCK 10 days after birth is unknown.

Table 9.--Activity of PEPCK in Experiment I.

 

Day
10 30 56 84
Diet a b a b a b a b

 

 

I 0.767 5.53 0.794 4.61 0.945 4.00 0.975 5.60
SEM 0.16 1.15 0.15 0.76 0.18 1.00 0.27 1.07

II 1.186 9.12 0.959 5.73 1.298 7.62 1.244 7.45
SEM 0.05 0.37 0.09 1.60 0.51 2.99 .22 1.40

III 0.530 3.90 0.754 4.24 1.494 9.29 2.134 11.21
SEM 0.06 0.40 0.14 0.58 0.32 2.21 0.34 1.66

 

aUnits of activity: u moles CO fixed per minute per

gram wet tissue.

bUnits of activity: milli u moles CO
minute per milligram hepatic protein.

2

2 fixed per

cStandard error of the mean.

52

By Day 30, all groups approached an equal activity
for PEPCK. Diet II retained the highest activity of the
groups at 30 days of age with Diets I and III nearly equal
in activity.

As with pyruvate carboxylase, PEPCK activity from
animals receiving Diet I was similar at all ages. When PEPCK
activity was expressed on a per gram tissue basis (Diet I),
Days 10 and 30 were nearly equal as were Days 56 and 84.

The specific activity of PEPCK remained nearly equal for each
day in Diet I.

Unlike pyruvate carboxylase, PEPCK is not affected
,by fasting (1,35). As noted in the section on pyruvate carboxy-
lase, the change in diet at Day 21 was followed by an increase
in pyruvate carboxylase activity attributable to reduced food
intake for animals receiving Diet III. While PEPCK activity
increased between Day 10 and Day 30 for Diet III, this
increase was less than the increase in pyruvate carboxylase.

On Day 56, PEPCK activity had increased for all
diets. This represented a 19 percent, 26 percent, and 98
percent increase (on a per gram tissue basis) for Diets I,
II, and III, respectively, from the previous sampling period.

Day 84 values for Diets I and II remained at approxi-
mately the same level as Day 56 but Diet III animals had a
great increase in PEPCK activity. The activity noted here
approaches that of mature ruminants and like pyruvate car-

boxylase for this group at Day 84 may represent an actual

53

mature (as far as rumen function is concerned) animal.
Activity in Diet III at 84 days was significantly higher
(p < .05) than Diet I.

Pattern of Glucose-6-
Phosphatase

 

 

The activity of glucose-6-phosphatase (Table 10)
when expressed on a per gram tissue basis was similar for
diets and days. There was no dramatic change in activity
when dietary changes were imposed. All diets had an increase
in activity from Day 10 to Day 30, a decrease from Day 30
to Day 56, and an increase from Day 56 to Day 84. Day 30
,and Day 84 values were similar, as were Day 10 and Day 56.
Glucose-6-phosphate is a metabolite with many competing path-
wahs, and significant amounts of glucose-6-phosphatase are
found only in liver and kidney. The high activity of glucose-
6-phosphatase found in the liver, regardless of diet, indi-
cates the importance of the liver in regulating blood
glucose.

The specific activity of glucose-G-phosphatase had
a highly significant diet X time interaction (p < .001).
The activity for animals receiving Diet I was lower initially
than the other two diets, and the activity declined during
the middle of the experiment but at the end of the experi-
ment glucose-6-phosphatase activity had actually increased.
Diets II and III had a steady decline in specific activity
from the initial sample. The rate of decrease was faster,

however, in Diet III. In both groups, the rate of decline

54

Table 10.—-Activity of g1ucose-6-phosphatase in Experiment I.

 

Day ‘
10 30 56 84
Diet a b a b a b a b

 

 

I 18.6 0.134 21.3 0.125 17.8 0.095 23.7 0.141
SEM 0.5 0.009 2.0 0.008 5.2 0.029 4.8 0.021

II 19.2 0.148 24.4 0.144 21.2 0.126 21.8 0.131
SEM 0.4 0.008 1.4 0.009 1.53 0.012 1.1 0.012

III 19.5 0.144 22.4 0.128 19.5 0.121 22.6 0.119
SEM 1.1 0.009 2.4 0.011 2.0 0.012 10.8 .004

 

a . . . . . .
Units of act1V1ty: u moles P1 liberated per minute
per gram wet tissue.

bUnits of activity: p moles Pi liberated per minute
per milligram hepatic protein.

CStandard error of the mean.

was greatest on the biopsy date immediately after the

change in diet.

Overall Considerations
in Experiment I

 

 

With the knowledge that ingestion of solid feed
stimulates rumen development and subsequent fermentation of
available carbohydrates, the rates of gluconeogenesis would
be expected to increase in animals consuming solid feed.
Measurement of key enzymes involved in the control of a par-
ticular pathway is a first approximation in determining the

importance of a pathway, and may give insight for future

55

investigations. In the present study, the amount of tissue
obtained by the method used did not allow for more detailed
study of gluconeogenesis such as substrate flux or hepatic
concentrations of pathway metabolites.

Correlation coefficients for parameters measured are
in Table 11. Coefficients between serum glucose and the
activity of pyruvate carboxylase and PEPCK at Day 84 were
negative and approached significance (p < .05). The corre-
lation between serum glucose and glucose-6-phosphatase was
negative also, but was not significant. These correlations
indicate an increased gluconeogenesis with a decreased serum)
.glucose (increased rumen function).

Correlation coefficients between the enzymes assayed
were calculated for each diet to prevent dietary effects in
one diet affecting other diets. Several coefficients were
significant but the important point to note is the trend
established for PEPCK and pyruvate carboxylase for the three
diets. The correlation between PEPCK and pyruvate carboxy-
lase was positive and significant for Diet I, the all milk
diet. Since little rumen fermentation is present for pro-
pionate production, most glucose precursors would arise from
amino acids and lactate. Consequently, pyruvate carboxylase
and PEPCK would be required for the formation of PEP.

The correlation between PEPCK and pyruvate carboxy-
lase for Diet II was less than the correlation for Diet I

yet still positive. This diet was designed to stimulate

56

Table 11.--Correlation coefficientsa of several variables in

Experiments I and II.

 

 

PEPCKf PCf G-6-Pasef
b ' c b c b 0
Serum Glucose -0.52 --- -0.49 --- -0.35 --—
Diet PEPCK --- --- 0.66e 0.55d 0.59 0.42
I PCBL --- --- --- --- 0.22 -0.01
Diet PEPCK --- --- 0.29 0.37 -0.09 0.41
11 PCBL --- --— --- --- -0.51d -0.15
Diet PEPCK --- --- -0.24 -0.39 0.23 0.29
III PCBL --- —-- --- --- 0.50d 0.31
Exp PEPCK --- —-- 0.61 0.47 -0.65 -0.30
II PCBL --- --- --- —-- -0.82e

-0.33

 

aCorrelation coefficients between enzyme activity
and serum glucose based on Day 84 values of each parameter.
Correlations between enzyme activities are within diets over
the entire experimental period.

bCorrelation coefficients based on enzyme
per gram of tissue.

activity

cCorrelation coefficients based on enzyme
per milligram hepatic protein.

activity

dValues with superscript significant (p < .05).

eValues with superscript significant (p < .01).

fPEPCK: Phosphoenolpyruvate carboxykinase
PC : Pyruvate carboxylase
G-6-Pase: Glucose-G-phosphatase

57

rumen development at a slow rate. In these animals some
propionate would be produced by rumen fermentation. The
carbon skeleton of prOpionate would have to go through PEPCK
only for PEP formation (Figure 1), thereby decreasing the
correlation as shown.

The correlation between PEPCK and pyruvate carboxy-
lase for Diet III was negative but not significant. Animals
receiving this diet were weaned early for early rumen devel-
Opment. These animals then utilized prOpionate earlier than
animals receiving either Diets I or II, resulting in an
increased PEPCK activity for Diet III without a necessary
.increase in‘pyruvate carboxylase.

The magnitude of the difference of the correlation
coefficient for Diet I and Diet III was similar when expressed

either on a per gram tissue or per milligram protein basis.

Results of Experiment II

 

Experiment II was designed to study the change in
enzyme activity during the period immediately before, during,
and after the change in diet. A mature-type rumen fermenta-
tion would not be expected to occur during the 21-day period.
However, some fermentation should begin to occur, especially
near the end of the experimental period.

In the previous experiment, a change in diet resulted
in an increase in pyruvate carboxylase activity. This
increase was attributed to the fasting caused by the change

in diet. In the present experiment, pyruvate carboxylase

58

activity increased from 0.29 at Day 42 to 0.40 at Day 56
and Day 63 (Table 12). The activity at Day 42 was for
animals that had been consuming milk since birth and was

comparable to Day 30 values for Diet I in Experiment I.

Table 12.--Activity of pyruvate carboxylase, PEPCK and
glucose-6-phosphatase in Experiment II.

 

 

 

Day
42 56 63
a b a b a b
Pyruvate c _
Carboxylase 0.287 2.06 0.403 2.34 0.400 2.25
1"“
_PEPCKd - 0.646 4.71 1.421 8.64 1.227 6.99
1,:& LI?

G1ucose-6- e '
Phosphatase 34.6 2.46 26.8 1.60 22.4 1.25
.7? ,6»:

 

aUnits of activity per gram wet tissue.

bUnits of activity per milligram hepatic protein.
cFor Units of Enzyme Activity, see Table 7.

dFor Units of Enzyme Activity, see Table 9.

eFor Units of Enzyme Activity, see Table 10.

After this biOpsy, the animals began receiving a
diet of hay and grain plus milk. Milk was withdrawn at
Day 49. Weekly feed consumption is shown in Table 6. There
was no difference by week in feed consumption after the

dietary change.

59

The activity of PEPCK increased 120 percent from
Day 42 to Day 56 but then decreased 14 percent at Day 63.
The correlation coefficient between PEPCK and pyruvate car—
boxylase for the experiment was 0.61 (Table 11). This posi-
tive correlation was similar to that for Diet I in Experi-
ment 1. Correlations for Diet II and Diet III in Experiment
I between PEPCK and pyruvate carboxylase were positive and
negative, respectively. Since Experiment II was over a
shorter period (21 days), a rumen fermentation producing
propionate is questionable. Too, of the large amount of
concentrate eaten, much carbohydrate would bypass rumen
.fermentation, resulting in the absorption of glucose in the
lower tract.

The activity of glucose-G-phosphatase steadily
decreased during Experiment II. Howarth et_al. (48) found
glucose-6-phosphatase activity to be higher in steers than
calves fed whole milk, indicating absorption of glucose from
the intestines influences glucose-6-phosphatase activity in
a negative manner. No such interpretation of the present
data can be made. However, the activity of glucose-6-
phosphatase was approximately 15 times greater than PEPCK
activity, which would be sufficient to convert PEP carbon

to glucose carbon.

SUMMARY

1. An all-milk diet fed to calves for 84 days
resulted in similar activities of the liver enzymes phos-
phoenolpyruvate carboxykinase (PEPCK), pyruvate carboxylase
(PC), and glucose-6-phosphatase (G-6-Pase) on Days 10, 30,
56, and 84.

2. Liver protein concentration increased from Day 10
to Day 30 in all diets. No changes in liver protein con- 6
centration were observed between diets on Days 30, 56, and 84.

3. Liver PEPCK from calves fed to stimulate early
rumen function had increasing activity with each sample date,
and approached mature values at 84 days of age.

4. Changes in PC activity were associated with
weaning. PC activity increased when calVes were changed
from milk to hay and grain feeding, and this change in
activity was associated with a decrease in feed intake.

When feed intake returned to normal, enzyme activity returned
to control levels. I

5. The activity of G-6-Pase did not differ between
diets at any age.

6. Correlation coefficients between PEPCK and PC
changed from positive (r2 = .66) for milk-fed calves to

negative (r2 2-424) for calves fed to stimulate early rumen

function.
60

61

7. The increase in PEPCK activity, the small
increase in PC, and the signs of correlation coefficients
indicate pr0pionate to be the major glucose precursor in

the calf.

APPENDIX

RAW DATA

62

63

Table 13.--Individual calf enzyme activities and liver

protein at Day 10.

 

 

Diet PCL PEPCK G-6-P Protein
I 0.189 0.665 20.15 126.67
0.212 0.402 18.26 155.72
0.117 0.801 17.87 130.23
0.372 1.194 18.11 146.82
II 0.221 1.240 19.16 135.54
0.223 1.266 18.76 142.16
0.152 1.053 18.54 126.75
0.242 1.186 20.14 117.49
III 0.146 0.543 21.26 126.67
0.258 0.543 16.42 128.50
0.226 0.370 20.45 136.74
0.253 0.670 19.77 153.18

 

64

Table l4.--Individua1 calf enzyme activities and liver

protein at Day 30.

 

 

Diet PCL PEPCK G-6-P Protein
I 0.340 1.099 27.26 185.30
0.120 0.551 19.78 153.24
0.392 1.012 19.22 171.40
0.197 0.513 19.08 169.95
II 0.138 1.676 25.56 160.17
0.075 0.878 23.72 193.87
0.142 0.710 20.78 155.72
0.157 0.572 27.60 169.95
III 0.064 1.058 22.41 211.65
0.308 0.588 16.14 141.95
0.854 0.466 28.01 176.10
0.846 0.904 22.96 174.71

 

65

Table 15.--Individual calf enzyme activities and liver

protein at Day 56.

 

 

Diet PCL PEPCK G-6—P Protein
I 0.246 0.596 9.54 190.01
0.306 0.665 10.29 193.01
0.282 1.277 19.58 196.17
0.149 1.253 31.96 180.86
II 0.296 0.777 23.51 174.40
0.364 1.000 16.70 182.65
0.238 0.609 22.30 152.17
0.285 2.806 22.35 169.74
III 0.205 1.494 23.24 172.06
0.231 1.452 18.50 185.81
0.130 2.288 21.80 147.46
0.189 0.742 14.45 143.78

 

66

Table 16.--Individual calf enzyme activities and liver

protein at Day 84.

 

 

Diet PCL PEPCK G-6-P Protein
I 0.476 1.705 34.82 201.90
0.082 0.497 18.53 137.39
0.212 1.008 13.61 166.56
0.210 0.688 28.11 160.38
II 0.194 0.873 22.18 198.45
0.314 1.691 18.67 174.16
0.162 0.859 22.55 150.00
0.186 1.548 23.85 155.81
III 0.356 2.043 24.12 187.79
0.173 3.003 20.51 194.62
0.332 2.134 23.78 189.90
0.466 1.357 22.08 186.44

 

67

Table 17.--Individua1 calf enzyme activities and liver
protein in Experiment II.

 

 

Day PCL PEPCK G-6—P Protein
42 0.314 0.479 34.07 130.17
0.242 0.583 35.43 156.99
0.306 0.928 34.34 137.39
56 0.473 1.530 23.30 172.72
0.333 1.918 26.67 153.81
0.372 0.814 30.28 177.35
63 0.460 1.519 17.20 174.71
0.399 1.354 26.95 181.32
0.340 0.809 23.00 178.30

 

LITERATURE CITED

68

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Annison, E. F., R. A. Leng, D. B. Lindsay, and R. R.
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