MOLECULAR CLONING OF CHICKEN HEPATIC HISTIDASE AND PROTEIN

REGULATION OF THE mRNA EXPRESSION OF CHICKEN HEPATIC HISTIDASE

AND MALIC ENZYME

by

THIMMAIAH PONNAPPA CHENDRIMADA

(Under the Direction of Adam J. Davis)

ABSTRACT

After obtaining a cDNA clone for chicken hepatic histidase, experiments were conducted to study the regulation of histidase mRNA expression by dietary protein concentrations. Histidase mRNA expression was increased within 3 h when chicks consumed higher levels of dietary protein. Increasing the dietary concentration of histidine did not alter hepatic histidase mRNA expression. The rapid increase in histidase mRNA levels in response to dietary protein intake is similar to the rapid decrease seen in malic enzyme mRNA levels in chicks fed the high protein diets. Glucagon was shown to regulate the mRNA expression of both the enzymes, and could act as a mediator for the effect of dietary protein on histidase and malic enzyme mRNA expression since an increase in dietary protein intake elevated plasma glucagon concentration within 1 h.

While histidase mRNA expression seems to be regulated by concentrations of specific amino acids in the diet, malic enzyme mRNA expression seems to be regulated by the total protein or nitrogen level of the diet. Finally, addition of synthetic glutamic acid to a

practical corn-soy poultry diet reduced the amount of abdominal fat present in broiler chicks at slaughter.

INDEX WORDS: Chickens, Dietary Protein, mRNA Expression, Histidase, Malic Enzyme, Abdominal Fat

MOLECULAR CLONING OF CHICKEN HEPATIC HISTIDASE AND PROTEIN

REGULATION OF THE mRNA EXPRESSION OF CHICKEN HEPATIC HISTIDASE

AND MALIC ENZYME

by

THIMMAIAH PONNAPPA CHENDRIMADA

B.S., Arkansas State University, 1998

M.S., The University of Georgia, 2000

A Dissertation Submitted to the Graduate Faculty of The University of Georgia in Partial

Fulfillment of the Requirements for the Degree

DOCTOR OF PHILOSOPHY

ATHENS, GEORGIA

2003

© 2003

Thimmaiah Ponnappa Chendrimada

All Rights Reserved

MOLECULAR CLONING OF CHICKEN HEPATIC HISTIDASE AND PROTEIN

REGULATION OF THE mRNA EXPRESSION OF CHICKEN HEPATIC HISTIDASE

AND MALIC ENZYME

by

THIMMAIAH PONNAPPA CHENDRIMADA

Major Professor: Adam J. Davis

Committee: Nicholas Dale Mark Compton Brain D. Fairchild Michael J. Azain

Electronic Version Approved:

Maureen Grasso Dean of the Graduate School The University of Georgia August 2003

iv

DEDICATION

To my Amma and Dada v

ACKNOWLEDGEMENTS

I like to express my gratitude to my advisor Dr. Adam Davis for his support,

knowledge and most importantly for imparting on me the value of the power of

reasoning. His ability to listen and make sound judgments will be of great help to me as I

pursue a career in scientific research. I sincerely believe that his strong work ethic and

his outstanding dedication to the field of science will earn him great rewards in the future.

I am proud to have worked with him as I pursued my Master’s and Doctorate degrees

here in the Department of Poultry Science. My sincere thanks to Dr. Henry Marks for granting me an opportunity to work towards my Doctorate degree in this esteemed department

I am honored to have had a committee dedicated to my success. Dr. Nicholas

Dale, for making me think beyond the obvious; Dr. Mark Compton, for his enthusiasm in my research and his advice; Dr. Brain Fairchild and Dr. Michael Azain for their support and encouragement. I sincerely thank all of my committee members for their invaluable contributions towards my research. A very special thank you to Dr. Roger Wyatt for believing in me, and for his scholarly advice.

The Davis’s lab (The Fun Lab) second in command, Liz Freeman, deserves special mention. Without her help and exemplary planning, it would not have been possible to conduct and coordinate the experiments. Thanks a lot Liz. The farm crew lead by David Perry has been of great help to me in conducting the experiments. I thank my friends and fellow graduate students Madalena Lordelo, Marcelo Hidalgo and vi

Andrew Benson for helping me in my research. My special thanks to Madalena for being

the best office mate I ever had.

For making sure I have fulfilled everything that was required for completing the

program, I thank Kerry Banks. My sincere gratitude to the Department of Poultry

Science for all the encouragement that was bestowed upon me, and I hope it continues to

flourish as the most accomplished poultry science department in the world. The U.S.

Poultry and Egg association have been instrumental in funding this research program.

Finally, a well deserved thank you to my friends and roommates for making my stay in this university memorable.

vii

TABLE OF CONTENTS

Page

ACKNOWLEDGEMENTS...... v

LIST OF TABLES...... x

LIST OF FIGURES ...... xii

CHAPTER

1 INTRODUCTION ...... 1

1.1 REGULATION OF FOOD CONSUMPTION IN CHICKENS...... 1

1.2 RECOGNITION OF PROTEIN INTAKE ...... 2

1.3 CONCLUSION...... 4

2 MALIC ENZYME...... 5

2.1 DE NOVO FAT SYNTHESIS AND MALIC ENZYME ...... 5

2.2 FATTY ACID SYNTHESIS PATHWAY ...... 6

2.3 REGULATION OF MALIC ENZYME ...... 9

2.4 SUMMARY...... 15

3 HISTIDASE...... 16

3.1 L-HISTIDINE...... 16

3.2 HISTIDINE CATABOLISM...... 17

3.3 TISSUE DISTRIBUTION OF HISTIDASE AND HISTIDASE

ACTIVITY ...... 19

3.4 HISTIDASE ACTIVITY IN THE LIVER ...... 19 viii

3.5 HISTIDASE ACTIVITY IN THE SKIN...... 20

3.6 HISTIDINEMIA...... 21

3.7 MOLECULAR CHARACTERISTICS OF HEPATIC HISTIDASE...... 22

3.8 REGULATION OF HEPATIC HISTIDASE ...... 23

3.9 SUMMARY...... 27

4 STATEMENT OF PURPOSE ...... 28

5 MATERIALS AND METHODS...... 29

5.1 CLONING CHICKEN HEPATIC HISTIDASE ...... 29

5.2 DIETARY EXPERIMENTS ...... 35

5.3 TISSUE COLLECTION AND RNA EXTRACTION ...... 48

5.4 NORTHERN ANALYSIS...... 48

5.5 STATISTICS ...... 51

6 RESULTS ...... 52

6.1 CLONING CHICKEN HEPATIC HISTIDASE ...... 52

6.2 DIETARY EXPERIMENTS ...... 63

7 DISCUSSION ...... 90

7.1 CLONING CHICKEN HEPATIC HISTIDASE ...... 90

7.2 DIETARY EXPERIMENTS ...... 91

7.3 GLUCAGON ...... 93

7.4 SPECIFIC AMINO ACIDS...... 95

7.5 PRACTICAL IMPLICATIONS ...... 100

7.6 SUMMARY...... 102

8 REFERENCES ...... 103 ix

LIST OF TABLES

Page

Table 1: COMPOSITION OF THE UNIVERSITY OF GEORGIA STARTER DIET ....36

Table 2: COMPOSITION OF TWO EXPERIMENTAL DIETS DURING

EXPERIMENT 1 – 12 ...... 37

Table 3: COMPOSITION OF THE ESSENTIAL (EAA) AND THE NON-ESSENTIAL

(NEAA) AMINO ACID SUPPLEMENTED DIETS (EXPERIMENTS 9 AND

10)...... 41

Table 4: COMPOSITION OF THE BASAL SUPPLEMENTED DIETS FED IN

EXPERIMENT 11 ...... 43

Table 5: COMPOSITION OF THE BASAL SUPPLEMNTED DIETS FED DURING

EXPERIMENT 12 ...... 44

Table 6: COMPOSITION OF THE EXPERIMENTAL DIETS FED DURING THE

STARTER PHASE (0 TO 21 D) IN EXPERIMENT 13...... 46

Table 7: COMPOSITION OF THE EXPERIMENTAL DIETS FED DURING THE

GROWER/FINISHER PHASE (22 – 40 D) IN EXPERIMENT 13 ...... 47

Table 8: NUCLEOTIDE AND AMINO ACID SEQUENCES OF THE 442 BP RT- PCR

PRODUCT...... 54

Table 9: HOMOLOGY OF THE NUCLEOTIDE AND PROTEIN SEQUENCES OF

THE 442-BP RT-PCR PRODUCT...... 55

x

Table 10: NUCLEOTIDE SEQUENCE OF CHICKEN HEPATIC HISTIDASE...... 60

Table 11: HOMOLOGY OF THE DEDUCED AMINO ACID SEQUENCE OF

CHICKEN HEPATIC HISTIDASE WITH THE HISTIDASE SEQUENCES

OF MOUSE, RAT AND HUMAN...... 61

Table 12: PLASMA GLUCAGON CONCENTRATION AND FOOD CONSUMPTION

OF CHICKS FED THE BASAL OR HIGH PROTEIN DIET FOR EITHER 0,

1, 2, 3 OR 6 H ...... 73

Table 13: FOOD CONSUMPTION (G/CHICK) FOR 24 H IN CHICKS FED THE

BASAL, HIGH PROTEIN OR THE BASAL DIET SUPPLEMENTED WITH

EITHER L-GLUTAMIC ACID, GLYCINE, L-ALANINE OR

DIAMMONIUM CITRATE (DAC)...... 80

Table 14: BODY WEIGHT GAIN (BWG), FEED INTAKE AND FEED CONVERSION

RATIO (FCR), IN BROILERS FED THE BASAL, THE BASAL DIET

SUPPLEMENTED WITH EITHER 2.3, 4.7 AND 9.5% GLUTAMIC ACID

AND CONTROL1 DIET DURING THE GROWER (0 – 21D), FINISHER (21

– 40 D), AND THE ENTIRE GROW-OUT PERIOD (OVERALL, 0 – 40 D) 86

Table 15: THE CALCULATED AMOUNT OF TOTAL NITROGEN PRESENT IN

THE EXPERIMENTAL DIETS IN THE EAA AND THE NEAA

EXPERIMENTS...... 96

xi

LIST OF FIGURES

Page

Figure 1: TRANSLOCATION OF ACETYL CoA ACROSS THE MITOCHONDRIAL

MEMBRANE ...... 7

Figure 2: THE PATHWAYS OF HISTIDINE CATABOLISM ...... 18

Figure 3: ETHIDIUM BROMIDE STAINED AGAROSE GEL CONTAINING

CHICKEN HISTIDASE RT-PCR PRODUCTS ...... 53

Figure 4: ETHIDIUM BROMIDE STAINED AGAROSE GEL CONTAINING

CHICKEN HISTIDASE RT-PCR PRODUCTS ...... 57

Figure 5: SCHEMATIC RELATIONSHIP AMONG THE CHICKEN HEPATIC

HISTIDASE CLONES ...... 58

Figure 6: AUTORADIOGRAM FROM A NORTHERN ANALYSIS OF HISTIDASE

WITH TOTAL RNA FROM CHICKEN LIVER...... 62

Figure 7: THE RELATIVE DENSITY OF HEPATIC HISTIDASE mRNA OF CHICKS

FED DIFFERENT DIETARY PROTEIN CONCENTRATIONS

[EXPERIMENTS 1 (6 AND 24 H) AND 2 (1.5 AND 3 H)] ...... 64

Figure 8: THE RELATIVE DENSITY OF HEPATIC HISTIDASE mRNA OF CHICKS

FED DIFFERENT DIETARY PROTEIN CONCENTRATIONS

[EXPERIMENTS 3 (6 AND 24 H) AND 4 (1.5 AND 3 H)] ...... 65

xii

Figure 9: THE RELATIVE DENSITY OF HEPATIC HISTIDASE mRNA OF CHICKS

FED THE BASAL DIET, HIGH PROTEIN DIET OR THE BASAL DIET

SUPPLEMENTED WITH EITHER 0.22 G/100G DIET (H1), 0.43 G/100G

DIET (H2), OR 0.86 G/100G DIET (H3), L-HISTIDINE ...... 67

Figure 10: THE RELATIVE DENSITY OF HEPATIC MALIC ENZYME mRNA OF

CHICKS AT 1.5 OR 3 H AFTER BRACHIAL VEIN INJECTION OF

EITHER GLUCAGON OR SALINE ...... 68

Figure 11: THE RELATIVE DENSITY OF HEPATIC HISTIDASE mRNA OF

CHICKS AT 1.5 OR 3 H AFTER BRACHIAL VEIN INJECTION OF

EITHER GLUCAGON OR SALINE ...... 69

Figure 12: THE RELATIVE DENSITY OF HEPATIC MALIC ENZYME mRNA OF

CHICKS 3 H AFTER BRACHIAL VEIN INJECTION OF EITHER

GLUCAGON OR SALINE ...... 70

Figure 13: THE RELATIVE DENSITY OF HEPATIC HISTIDASE ENZYME mRNA

OF CHICKS 3 H AFTER BRACHIAL VEIN INJECTIONS OF

GLUCAGON OR SALINE ...... 71

Figure 14: THE RELATIVE DENSITY OF HEPATIC MALIC ENZYME (PANEL A.)

AND HEPATIC HISTIDASE (PANEL B.) mRNA OF CHICKS AT 3 H OR

6 H AFTER CONSUMPTION OF EITHER THE BASAL...... 74

Figure 15: THE RELATIVE DENSITY OF HEPATIC MALIC ENZYME mRNA OF

CHICKS FED FOR 24 H EITHER THE BASAL DIET, HIGH PROTEIN

DIET, OR THE BASAL DIET WITH NEAA OR EAA...... 75

xiii

Figure 16: THE RELATIVE DENSITY OF HEPATIC HISTIDASE mRNA OF

CHICKS FED FOR 24 H EITHER THE BASAL DIET, HIGH PROTEIN

DIET, OR THE BASAL DIET WITH NEAA OR EAA...... 77

Figure 17: THE RELATIVE DENSITY OF HEPATIC MALIC ENZYME mRNA OF

CHICKS FED FOR 6 H EITHER THE BASAL DIET, HIGH PROTEIN

DIET, OR THE BASAL DIET WITH NEAA OR EAA...... 78

Figure 18: THE RELATIVE DENSITY OF HEPATIC HISTIDASE mRNA OF

CHICKS FED FOR 6 H EITHER THE BASAL DIET, HIGH PROTEIN

DIET, OR THE BASAL DIET WITH NEAA OR EAA...... 79

Figure 19: THE RELATIVE DENSITY OF MALIC ENZYME mRNA OF CHICKS

FED FOR 24 H EITHER THE BASAL DIET, HIGH PROTEIN DIET OR

THE BASAL DIET SUPPLEMENTED WITH L- GLUTAMIC ACID,

GLYCINE, L-ALANINE OR DIAMMONIUM CITRATE ...... 82

Figure 20: THE RELATIVE DENSITY OF HISTIDASE mRNA OF CHICKS

FED FOR 24 H EITHER THE BASAL DIET, HIGH PROTEIN DIET OR

THE BASAL DIET SUPPLEMENTED WITH L- GLUTAMIC ACID,

GLYCINE, L-ALANINE OR DIAMMONIUM CITRATE ...... 83

Figure 21: THE RELATIVE DENSITY OF MALIC ENZYME mRNA OF CHICKS

FED FOR 24 H EITHER THE BASAL DIET, HIGH PROTEIN DIET OR

THE BASAL DIET SUPPLEMENTED WITH EITHER AMMONIUM

BICARBONATE, AMMONIUM PHOSPHATE OR BOTH...... 85

xiv

Figure 22: THE RELATIVE DENSITY OF HEPATIC MALIC ENZYME mRNA OF

CHICKS FED THE BASAL CONTROL, AND THE BASAL DIET

SUPPLEMENTED WITH EITHER 2.3, 4.7 OR 9.5% GLUTAMIC ACID

DIETS AT 18 D (PANEL A) AND 37 D (PANEL B.)...... 88

Figure 23: ABDOMINAL FAT PAD ( G/ 100 G LIVE BODY WEIGHT) IN CHICKS

FED THE BASAL, CONTROL, AND THE BASAL DIET

SUPPLEMENTED WITH EITHER 2.3, 4.7 OR 9.5% GLUTAMIC ACID

DIETS ...... 89

CHAPTER 1

INTRODUCTION

1.1 REGULATION OF FOOD CONSUMPTION IN CHICKENS

Regulation of physiological systems in complex living organisms involves

transmission of signals from an external source (ultimate signal) through various intermediates in the body, until a final response is obtained (Ferraris et al. 1988). Food is

one such ultimate signal, which triggers a complex set of signals in living systems which

in turn regulates its intake. Regulatory mechanisms to control food consumption in

chickens have been shown to exist. When birds which have been previously feed

restricted are provided feed ad libitum, they will consume more food than ad libitum fed

controls until they have obtained body weights similar to the ad libitum fed controls

(Plavnik and Hurwitz, 1990). In addition, chickens that have been force fed to gain

excessive body weight compared to ad libitum fed controls will when given free access to

food, consume less food than the control birds until they obtain a body weight similar to

the controls (Lepkovsky, 1973).

More specifically, the role of carbohydrates and lipids in controlling food

consumption is well established in chickens. Lacy et al. (1985) showed that infusion of

glucose into the hepatic circulation decreased food consumption in birds that were fed ad

libitum, but had no effect on fasted chicks. Additionally, Matei-Vladescu et al. (1977)

injected glucose into the cerebral lateral ventricle, and found that it decreased food

consumption. Increasing the energy of diets by increasing the amount of dietary fat also

1 2

reduces food consumption in chickens (Scott et al., 1982). In addition to these

highlighted general research reports, numerous other scientific papers have helped

establish how carbohydrates and lipids specifically regulate food consumption. Meier et

al. (2002) recently reviewed all of the known pathways and mechanisms by which the

body recognizes and regulates dietary carbohydrate and fat consumption.

1.2 RECOGNITION OF PROTEIN INTAKE

1.2.1 Amino Acids

Similar to the case with fat and carbohydrates, there is evidence to show that non-

ruminant animals are also able to recognize dietary protein intake. It is essential for birds

to have a constant and balanced supply of amino acids. Cellular proteins consist of

combinations of 20 common amino acids. About half of these amino acids are essential

dietary amino acids, because they cannot be synthesized by birds from other molecules or

synthesized at rates necessary to meet daily demands. Since all of the amino acids are

used in making proteins, a deficiency of just one of the essential amino acids will prevent

net accruement of protein. Additionally, there is no true storage reserve of amino acids to

utilize when there is an amino acid deficiency. Although protein, especially in the form

of muscle, is a major constituent of animal bodies, the amino acids from these proteins

can only be used for synthesis of new protein as old protein is degraded. Dietary supplementation of an amino acid above the bird’s requirement results in the elimination of the amino acid in the form of uric acid, with a loss of energy and water. On the other hand, a less than adequate supply of an amino acid, results in a negative nitrogen balance.

Therefore, from an evolutionary standpoint, it would be an adaptive advantage for a bird 3 to have the ability to distinguish and select a diet that provides it with a constant and balanced supply of amino acids (Gietzen, 1993).

1.2.2 Selection of Dietary Protein and Amino Acids

Given the importance of obtaining an adequate and balanced supply of all the essential amino acids for supporting growth and metabolic efficiency, it is not surprising that chickens show an incredible natural ability to select food based on amino acid needs.

Broiler chicks given a choice between a diet containing a lower concentration and one containing a higher concentration of protein than required will eat amounts of the two diets that is optimum for growth in terms of protein content (Shariatmadari and Forbes,

1993). Strains of chickens selected for high fat deposition, if given the choice will select a lower protein diet which metabolically supports fat deposition (Forbes and

Shariatmadari, 1994). Similarly, methionine deficient broilers will eat more of a methionine supplemented diet than control birds (Leclercq and Guy, 1991). If no other food choice is available, chickens will not eat or will drastically reduce their intake of diets, if the diet does not provide a proper balance of amino acids (Davis and Austic,

1982; Davis and Austic, 1994).

Further evidence that the dietary intake of amino acids can be sensed is provided by studies which report that ingestion of high protein diets increases the activity of many rate limiting enzymes of amino acid catabolism while low protein or protein free diets have the opposite effect in chickens and other animals (Ashida and Harper, 1961;

Schemke, 1962; Sanahuja and Harper, 1963; Featherston and Horn , 1973; Davis and

Austic, 1982; Dixon and Harper, 1984; Davis and Austic, 1997; Torres et al., 1998). 4

More recently some of these enzymes have even been shown to respond to the addition of

just a few specific dietary amino acids, and to respond within hours of feeding the

supplemented diets (Davis and Austic, 1994; Yuan et al., 2000). Finally, the

concentration of brush border amino acid transporters are quickly increased in animals

fed a high protein diet (Karasov et al., 1987). Interestingly, just a few specific amino

acids seem to up-regulate all of the amino acid transporters, including the transporters

which cannot even bind and translocate the very amino acid up-regulating their

expression (Stein et al., 1987).

1.3 CONCLUSION

Although, it is clear that mechanisms must exist for chickens to sense and regulate their amino acid intake, there is minimal information about how this occurs. The present research is aimed at developing a research model that begins to elucidate these mechanisms. To establish this research model, our goal was to identify one metabolic enzyme whose activity quickly increased, and one metabolic enzyme whose activity quickly decreased in response to changes in dietary protein intake. Determining when the activities of these enzymes initially changed in response to the alteration in dietary protein intake would establish a time-frame for when a chicken has sensed the change in amino acid intake. Perhaps more importantly, establishing the mechanisms by which the activity of these enzymes are changed could potentially provide information on what organ or tissue detects amino acid intake, and the nature of this biochemical response.

The enzyme systems identified for potential investigation in establishing this research model were malic enzyme and histidase. CHAPTER 2

MALIC ENZYME

2.1 DE NOVO FAT SYNTHESIS AND MALIC ENZYME

When excess amounts of calories are ingested, they can be converted and stored

as fat. This stored fat is broken down and utilized when insufficient calories are taken

into the body. The de novo synthesis of fat in animals occurs either in adipose or hepatic

tissue. In ruminants and rats, fatty acid synthesis occurs in the adipose tissue (Ballard et

al., 1969), whereas in the primate and avian species, the liver is the primary site for fatty

acid synthesis (Shargo et al., 1971). The overall reaction for fatty acid synthesis is as

follows:

8 Acetyl CoA + 14 NADPH + 14 H+ + 7 ATP →

Palmitate + 8 CoA + 14 NADP + 7 (ADP + Pi) + 6 H2O

Two essential enzymes for fatty acid synthesis are acetyl CoA carboxylase and

the multifunctional fatty acid synthase. Acetyl CoA carboxylase catalyzes the energy

dependent conversion of acetyl CoA to malonyl CoA. The multifunctional fatty acid

synthase catalyzes the conversion of malonyl CoA to palmitate. De novo fatty acid

synthesis takes place in the cytosol, and requires 14 reduced nicotinamide-adenine

dinucleotide phosphate, (NADPH) molecules. Malic enzyme [L-malate-NADP+ oxidoreductase (decarboxylating), EC 1.1.1.40] catalyzes the cytosolic conversion of malate to pyruvate while simultaneously generating one molecule of NADPH from

NADP+. The generated NADPH then can be used for fatty acid synthesis. Therefore, the

5 6

activity of hepatic malic enzyme is highly positively correlated with the rate of fatty acid

synthesis, percent body fat and percent abdominal fat in chicks (Yeh and Leveille, 1969;

Pfaff, 1977; Tanaka et al., 1983a; Grisoni et al., 1991;).

2.2 FATTY ACID SYNTHESIS PATHWAY

Pyruvate, the product of glycolysis, is converted to acetyl CoA in the

mitochondria by the enzyme pyruvate dehydrogenase. If an animal’s energy requirement

is satisfied, acetyl CoA does not enter the Kreb’s cycle, but is used for fatty acid

synthesis. However, fatty acid synthesis takes place in the cytosol, and acetyl CoA

cannot be translocated across the mitochondrial membrane. Therefore, acetyl CoA has to

be first converted to citrate, which is able to cross the membrane using a specific

exchange transporter. Once in the cytosol, citrate lyase cleaves citrate to acetyl CoA and

oxaloacetate (OAA). The carbons of OAA in the cytosol have to be returned to the

mitochondria. However, the mitochondrial membrane is also impermeable to OAA. The

OAA is therefore reduced to malate using cytoplasmic NAD malate dehydrogenase. This

reaction uses a NADH molecule, which is produced during glycolysis. Malate is then

oxidized to pyruvate, which is transported back into the mitochondria. The oxidation of

malate to pyruvate is catalyzed by malic enzyme. During this process, a NADPH

molecule is produced, which can then be used for fatty acid synthesis. Therefore, for

each acetyl CoA that is used for fatty acid synthesis, one NAPDH molecule is generated

by the activity of malic enzyme. The series of reactions for the translocation of acetyl

CoA from the mitochondria to the cytosol and the production of NADPH are summarized in Figure 1. 7

Pyruvate Palmitate NADP Malic Enzyme NADPH Malate Acetyl CoA

NAD

malate dehydrogenase Pyruvate OAA Citrate

Inner Mitochondrial Membrane

Pyruvate Acetyl CoA Citrate

Figure 1. Translocation of acetyl CoA across the mitochondrial membrane for participation in fatty acid synthesis and the generation of NADPH by malic enzyme. Abbreviations: OAA, oxyloacetate, NAD, nicotinamide adenine nucleotide, NADP, nicotinamide adenine nucleotide phosphate, NADPH, reduced nicotinamide

8

Overall, fatty acid synthesis uses 8 acetyl CoA molecules, resulting in the

production of 8 NAPDH molecules to be used for fatty acid synthesis. The 6 additional

NADPH molecules that are needed for the synthesis of 1 molecule of palmitate are

obtained either from the hexose monophosphate dehydrogenase shunt or from further actions of malic enzyme. In rats, the hexose monophosphate dehydrogenase shunt provides the 6 molecules of NADPH for fatty acid synthesis (Tepperman and Tepperman,

1964). In avian species, the activity of the hexose monophosphate dehydrogenase shunt is very low (Ball and Merrill, 1961). Furthermore, NADPH production capacity by malic enzyme in the avian liver is 20 times greater than in rats (Goodridge and Ball, 1966).

Therefore, it has been concluded that the activity of malic enzyme provides almost all the reducing equivalents necessary for fat synthesis in the bird (Goodridge and Ball 1967,

Goodridge 1968 and Tanaka et al. 1983a). However, if malic enzyme activity generates all 14 NADPH molecules, then there would be an excess of 6 acetyl CoA molecules in the cytosol for every molecule of palmitate formed. Since only 8 molecules of acetyl

CoA are used for fatty acid synthesis, the disposition of these extra acetyl CoA molecules is unclear and suggests further research is warranted on this topic.

The fatty acids synthesized in the liver, esterified to triglycerides, and secreted into circulation as very low density lipoproteins (VLDL). Lipoprotein lipase present at the terminal surface of capillary endothelial cells of various tissues including the liver, adipose tissue, muscle, heart, lungs, adrenal cortex, kidney and spleen catalyzes the hydrolytic release of nonesterified fatty acids (NEFA) from the lipoprotein triglycerides.

The NEFA then enter the adipocyte by passive transport. Once in the adipose tissue, the

NEFA are activated to CoA esters which are then esterified into stored triglycerides. 9

2.3 REGULATION OF MALIC ENZYME

2.3.1 Starvation and Refeeding

Studies performed in rats and birds have shown that starvation decreases malic enzyme activity, and that refeeding these animals increases malic enzyme activity. When rats were fasted for 48 h and then allowed to refeed for either 5, 12, 18 or 24 h,

Tepperman and Tepperman (1964) reported that malic enzyme activity was low during starvation, and was increased upon refeeding at each time point. In fact, a fast followed

by subsequent refeeding, can raise malic enzyme activity levels 300 percent above the

normal activity found in rats fed ad libitum (Young et al., 1964). Romsos and Leveille

(1974) confirmed that malic enzyme activity is depressed in rats by fasting. In chicks,

Goodridge (1968) reported that malic enzyme activity and fatty acid synthesis were depressed by fasting, and were increased by refeeding. However, unlike the situation in rats, malic enzyme activity did not increase to a level greater than that found in ad libitum fed birds. Findings similar to those found in the chick were also observed in starved and

refed pigeons (Goodridge and Ball, 1967).

In chicks, starvation also decreased the level of malic enzyme mRNA, and upon

refeeding the chicks, malic enzyme mRNA levels increased 40 to 50 fold (Winberry et

al., 1983). By measuring the steady state levels of nuclear precursors for hepatic malic

enzyme mRNA in starved and refed chicks, Ma et al.(1990), found that refeeding

regulated the transcription of the malic enzyme gene in the chick liver. Transcription rate

increased slightly at 1.5 h after refeeding, but was stimulated to the full extent after 3 h of

feeding (Ma et al., 1990). According to Ma et al.(1990), the magnitude and rapidity of

the refeeding-induced increase in transcription of the malic enzyme gene fully accounted 10

for the 40 to 50 fold increase in the steady-state level of malic enzyme mRNA detected

previously by Wineberry et al. (1983).

2.3.2 Dietary Carbohydrate and Fat

The quantity of carbohydrate consumed is an important factor in the regulation of fatty acid synthesis and of the enzymes involved in this biological process. Several reports have indicated that increasing the level of dietary carbohydrate increases fatty acid synthesis and the activities of the enzymes associated with fatty acid synthesis in the chick (Shargo et al. 1963, Clarke et al. 1979, Tanaka et al. 1983a). Tanaka et al. (1983a) reported a linear relationship between the level of carbohydrate consumed and hepatic fatty acid synthesis. Increased carbohydrate levels in the diet of the chick also increases the activities of glycolytic enzymes (Tanaka et al., 1983a). The increase in the glycolytic enzymes is due to the increased flux of glucose through glycolysis and this results in an increased level of acetyl CoA available for fatty acid synthesis. The increased availability of acetyl CoA may be responsible for the increased activities of citrate lyase and malic enzyme. Although dietary carbohydrates provide a substrate for fatty acid

synthesis, the true effects of carbohydrates on fatty acid synthesis are still uncertain to some extent, since alterations in the level of dietary carbohydrate concentration have been altered at the expense of dietary fat or protein in most of the research conducted.

Yeh and Leveille (1969) reported that hepatic fatty acid synthesis was significantly reduced when a high fat diet was fed to chicks. The high fat diet was created at the expense of dietary carbohydrate, but a constant calorie to protein ratio was maintained. The question that arises from this report is whether the alteration in fatty acid synthesis was due to changes in dietary fat or due to the changes in dietary 11

carbohydrate. The slopes of dietary fat’s and dietary carbohydrate’s effect on fatty acid

synthesis plotted by Yeh and Leveille (1969) were different, indicating that fat has a

specific affect on fatty acid synthesis. Yeh et al. (1970) force fed corn oil to chicks and

found that it reduced fatty acid synthesis in 1 h, however, there was no change in the

activity of malic enzyme. In subsequent experiments conducted by Tanaka et al. (1983b),

an increase in dietary fat also had no effect on the activity of hepatic malic enzyme even

though fatty acid synthesis was decreased in those chicks.

Fatty acids may, however, play a role in regulating malic enzyme activity and

mRNA levels depending on their chain length. Using hepatocyte cultures, Roncero and

Goodridge (1992) demonstrated that palmitate and sterate failed to inhibit thyroid

hormone (T3) induced malic enzyme activity and transcription of the malic enzyme gene.

On the other hand, hexanoate and octanoate, two fatty acids not typically present in

poultry diets, did inhibit T3 induced malic enzyme activity and inhibited the accumulation

of malic enzyme mRNA (Roncero and Goodridge, 1992). Although dietary fat appears

to have a limited effect on malic enzyme activity, it probably does influence fatty acid

synthesis. Yeh et al. (1970) proposed that an increase in circulating free fatty acids from

dietary sources would result in an increased conversion rate of fatty acids to fatty acid

CoA derivatives which would in turn reduce availability of free CoA necessary for de

novo fatty acid synthesis. In fact, chicks fed a high fat diet for 24 h had an increased level of hepatic long chain acyl-CoA and a decreased level of free CoA and fatty acid

synthesis (Yeh and Leveille 1971).

12

2.3.3 Dietary Protein

Dietary protein content also alters fatty acid synthesis and malic enzyme activity.

Increasing dietary protein was found to depress fatty acid synthesis (Yeh and Leveille,

1969), while feeding a protein free diet markedly stimulated fatty acid synthesis and

malic enzyme activity (Leveille and Yeh, 1972). To investigate the affect of dietary

protein on the rate of fatty acid synthesis, glucose-U-14C, pyruvate-2-14C, and acetate-1-

14C were injected into chicks fed different levels of protein. The rate of incorporation of

glucose-U-14C, pyruvate-2-14C, and acetate-1-14C into liver fatty acids was depressed by increasing the dietary protein level (Yeh and Leveille, 1969). The activity of malic enzyme was also found to be positively correlated with the rate of fatty acid synthesis, which led Yeh and Leveille (1969) to hypothesize that malic enzyme may be a major factor limiting fatty acid synthesis in chicks fed a high protein diet. Yeh and Leveille

(1971) subsequently reported that increasing the dietary protein concentration from 15 to

35% decreased hepatic malic enzyme activity by 90% and reduced fatty acid synthesis in chicks. Even when the level of dietary protein was increased while maintaining a constant level of dietary carbohydrate, the level of fatty acid synthesis and malic enzyme activity was decreased (Tanaka et al., 1983b).

Subsequent research by Rosebrough et al. (1985, 1986a, 1988, 1990, 1996 and

1999) confirmed the original finding that the activity of malic enzyme and the synthesis of liver fatty acids are decreased by increasing dietary levels of protein. Rosebrough and

Steele (1985) also reported an inverse relationship between dietary protein and body fat content.

13

The alteration in malic enzyme activity due to dietary protein may actually be due to the concentrations of specific amino acids in the diet rather than the whole protein content. Sulfur amino acids alter lipid metabolism (Rukaj and Serougne 1983, Yagasaki et al., 1986), and decrease the activity of malic enzyme in rats (Ayala et al. 1991 and Ide et al. 1992). In chickens, percentage of abdominal fat pad declined linearly as sulfur amino acids were added in increasing amounts to a low protein diet (Medonca and

Jensen, 1989). Similar results with sulfur amino acids and abdominal fat were obtained by Pesti et al. (1996) in experiments with normal and naked neck chickens. In addition to sulfur amino acids, Yeh and Leveille (1969) and Rosebrough et al. (1986b) both reported that the addition of lysine to a low protein diet depressed fatty acid synthesis in chicks.

Tanaka et.al (1992) reported lower malic enzyme activity levels in chicks fed either soybean or corn gluten protein than those fed diets based on casein or a fishmeal based protein diet.

The regulation of hepatic malic enzyme activity and fatty acid synthesis by dietary protein has been shown to occur at the pre-translational level in chicks. Adams and Davis (2001) reported that birds fed a high (40%) protein diet had significantly lower malic enzyme mRNA levels when compared to birds fed a 22% or 13% protein diet. The changes in malic enzyme mRNA expression in response to dietary protein occurred as early as 3 h after initial intake of the diets (Adams and Davis, 2001). The decrease in malic enzyme mRNA levels in chicks fed a high protein diet observed at 3 h subsequently lead to a decrease in hepatic malic enzyme activity at 6 h, and a decrease in total hepatic lipid concentrations at 24 h (Adams and Davis, 2001).

14

2.3.4 Hormonal Regulation

2.3.4.1 Thyroid Hormone

The effect T3 has on avian malic enzyme activity has been examined in vitro and

in vivo. Goodridge and Adelman (1976) reported that when liver cells were incubated in

the presence of T3, a 23 fold increase was seen in malic enzyme activity. Malic enzyme

activity returned to a baseline level when T3 was removed from the cell culture medium.

The effect of thyroid hormone on malic enzyme activity appears to be mediated through

changes in mRNA levels. Hepatocytes incubated for 48 h in the presence of exogenous

T3 had a mRNA level which was 21 times greater than the level found before adding the

T3 and 14 times the level found in cells incubated for 48 h without T3 (Back et al., 1986).

Subsequently, Salati et al. (1991) reported that malic enzyme mRNA was elevated within

1 h, and reached a maximal transcription rate at 6 h in hepatocytes cultured in the presence of T3 compared to those cultured in a media not containing T3.

2.3.4.2 Glucagon

Glucagon also has an effect on malic enzyme activity and fatty acid synthesis.

The typical stimulation of malic enzyme activity by incubating hepatocytes with T3, was inhibited by 97% when glucagon was added to the cell culture media along with T3

(Goodridge and Adelman, 1976). When hepatocytes were incubated with T3, the

subsequent addition of glucagon did not inhibit malic enzyme activity within the first 7 h

of incubation suggesting that the glucagon has to be added prior to the addition of T3 for the inhibition to occur (Goodridge and Adelman, 1976). Back et al. (1986) also reported that the typical increase of malic enzyme mRNA levels by the addition of T3 was

inhibited by 93% with the addition of glucagon. They also concluded that glucagon 15

regulated malic enzyme activity entirely by decreasing the cellular level of malic enzyme mRNA.

The effects of glucagon on malic enzyme activity and mRNA levels appear to be

mediated by cyclic AMP (cAMP). When cAMP is added to hepatic cell cultures, it

inhibits malic enzyme activity and decreases mRNA levels (Cramb et al., 1982). Cramb

et al. (1982) reported that glucagon increased cAMP levels within a few minutes after its

addition to cultured chicken hepatocytes. Mounier et al. (1997) demonstrated that

glucagon did increase cAMP levels which subsequently inhibited the transcription of the

malic enzyme mRNA in chick embryo hepatocytes. The 5’ flanking region of the

chicken malic enzyme gene contains at least four sequences involved in cAMP

responsiveness (Mounier et al., 1997). Goodridge and Adelman (1976) suggested that

plasma glucagon may be the hormone that communicates the nutritional state of the bird

to the liver, since an increase in plasma glucagon is associated with a decrease in malic

enzyme activity when an animal is starved.

2.4 SUMMARY

The activity of malic enzyme is essential for de novo fatty acid synthesis. In

chickens, activity of hepatic malic enzyme is positively correlated with the rate of fatty

acid synthesis, percent body fat, and percent abdominal fat. Dietary protein is a key

regulator of malic enzyme activity. The regulation of malic enzyme by dietary protein

occurs at the pretranslational level and alterations in protein intake change malic enzyme

mRNA expression within hours.

CHAPTER 3

HISTIDASE

3.1 L-HISTIDINE

L-histidine is an essential amino acid for rats, young chickens and human infants.

Adult humans and chickens can synthesize enough histidine to meet their requirements.

However, Kopple and Swendseid (1975) reported that even in adult humans the capacity to synthesize histidine is inadequate, if none is provided in the diet. According to the

National Research Council (NRC) (1994), the histidine requirement for broilers is 0.32%.

This value is based on the reports of Woodham and Deans (1975) and Han et al. (1991) which established the dietary histidine requirement at 0.34% (1.9% of dietary protein) and 0.32% of the diet respectively.

Histidine plays an important role in many proteins, since the imidizole group contributes to the buffering capacity of tissue and plasma proteins. Among the amino acids found in free or protein bound forms in organisms, only histidine has a high buffering capacity near the pH of intracellular fluids and blood. Hemoglobin, which has a high histidine content, has a high buffering capacity (Edsall and Wyman, 1958).

Histidine residues in hemoglobin also play a role in the release of oxygen to the tissues when the concentration of hydrogen ions becomes large (Busch and Ho, 1990). In addition to its buffering capability, histidine is also reactive at the catalytic sites of many enzymes, including ribonuclease A (Flogel and Biltonen,1975) and carbonic anhydrases

(Gupta and Pesando, 1975).

16 17

3.2 HISTIDINE CATABOLISM

Histamine is the primary amine formed by the decarboxylation of histidine. This

reaction is catalyzed by the enzyme histidine decarboxylase. Histamine is a

neurotransmitter both in the nervous system of insects and in the mammalian central

nervous system (Marshall, 1981). Apart from its neurotransmitter role, histamine is a

major causative factor of atopic diseases such as allergy, asthma, anaphylaxis, and atopic dermatitis (Kumar et al., 1968). In addition, histamine acts on the gastric mucosa to stimulate acid secretion, and plays an important role in regulating food consumption

(Kumar et al., 1968; Mercer et al., 1989; Mercer et al., 1994).

Histidase, the primary catabolic enzyme for histidine, catalyzes the non-oxidative deamination of L-histidine to urocanic acid (Mehler and Tabor, 1953). In the skin, as

reviewed by Taylor et al. (1991), the metabolic product of histidine, urocanic acid plays

an important role in ultra violet ray (u.v.) protection and immunosuppresion. Histidine

also undergoes transamination through the activity of histidine-pyruvate transaminase to

form imidazolepyruvate, which can then be broken down to imidazolelactic acid and

imidazoleacetic acid (Spolter and Baldridge, 1963). The different pathways of histidine

catabolism are shown in Figure 2. Histidine is also an important precursor for the

modified amino acids carnosine (β-adenyl-histidine), homocarnosine (γ-amino butyryl-

histidine) and methyl histidine which are found in high levels in the skeletal muscle

(Boldyrev et al., 1988). However, the exact functions of these molecules are unknown.

18

A. Histidine Decarboxylase L-Histidine Histamine +CO2

B1.(Liver) Histidase L-Histidine Trans-urocanic acid

Urocanase NH3

Glutamic acid Imidazolonepropionic acid

B2.(Skin) Histidase L-Histidine Trans-urocanic acid U.V. NH3 Cis-urocanic acid

C. histidine pyruvate transaminase L-Histidine Imidazolepyruvic acid

Figure 2. The Pathways of Histidine Catabolism. Panel A: Histidine decarboxylase pathway. Panel B: Histidase pathway; B1: Occurs in the liver, B2: Occurs in the skin. Since urocanase is not present in the skin, trans-urocanic acid accumulates, and is isomerized to cis-urocanic acid upon stimulation by u.v. light. Panel C: Histidine-pyruvate transaminase pathway.

19

3.3 TISSUE DISTRIBUTION OF HISTIDASE AND HISTIDASE ACTIVITY

Dhanam and Radhakrishnan (1976) compared histidase activity in two avian and five mammalian species. They found histidase activity in the liver and skin from chicken and pigeon. In rats, histidase activity was found in the liver, skin, as well as, in the kidney.

Guinea pig, rabbit, monkey and man exhibited histidase activity in the liver, skin, kidney, spleen and intestine. In all species examined, histidase activity could not be detected in the brain, heart or adrenals, and the highest activity was found in the liver followed by the skin.

Baden et al. (1969) conducted a comparative study of histidase activity in amphibian, reptilian, avian and mammalian epidermis. They found that histidase activity was present in the epidermis of most eutherian mammals including man, mouse, rat and goat. However, epidermal histidase activity was not found in samples from cow, crocodile, alligator or two species of frog (Baden et al., 1969). Contrary to the findings of Dhanam and Radhakrishnan (1976), histidase activity was not found in chicken epidermis (Baden et al., 1969). This difference may be attributed to the type of histidase assay employed by the investigators.

3.4 HISTIDASE ACTIVITY IN THE LIVER

The trans-urocanic acid formed from histidine by histidase in the liver is degraded by the action of urocanase to form 4-imidazolone-4-propionic acid. Imidazolone propionic acid is chemically unstable. Therefore, it immediately undergoes enzymic cleavage of the imidazolone ring to yield formimino glutamate (FIGLU). FIGLU is then converted to glutamate by transfer of the formimino group to tetrahydrofolate (Figure 2). 20

HISTIDASE ACTIVITY IN THE SKIN

Stratum corneum, the upper most layer of the skin, is the only layer of the

epidermis where histidase activity is detected (Scott, 1981). Since the cells of this layer

of the epidermis have no nuclei, histidase mRNA must be synthesized in one of the lower

cell layers of the epidermis such as the stratum granulosum or the stratum spinosum

(Taylor et al. 1991). Therefore, until the epidermis cells from the lower layers are pushed

up to form the stratum corneum, either histidase mRNA remains untranslated or the

histidase protein is inactive (Taylor et al., 1991). Similar to the liver, trans-urocanic acid

is formed in the skin from the deamination of L-histidine by histidase. However, the

enzyme urocanase that catalyzes the conversion of urocanic acid to imidazolone

propionic acid is absent in the skin. This leads to the accumulation of trans-urocanic acid

in the skin. Trans-urocanic acid undergoes isomerization to cis-urocanic acid upon

stimulation by u.v. rays (λmax = 275 nm). The proportion of cis and trans isomers in the

skin varies depending on the amount of exposure to u.v. (Taylor et al., 1991). Both forms

of urocanic acid have been suggested to be a natural “sun-screen” for the skin (Zenisek et

al., 1955) since they are the major absorbers of u.v. light in the skin. Urocanic acid’s

ability to protect skin from sunburn has been well documented (Everett et al., 1961;

Baden and Pathak, 1967; Zenisek et al., 1969; Wadia et al. 1975). Morrison et al. (1980) also reported that urocanic acid protects DNA from photomutagenesis, since the absorption spectra of both the cis and trans isomers overlap with the absorption spectrum of DNA.

There is also evidence to suggest that urocanic acid can act as an immunosuppressor. Rats treated with urocanic acid (0.8 mg/kg body weight) for seven 21

days following a heart allograft transplantation had 40% permanent graft acceptance

compared to a zero survival rate in untreated controls (Oesterwitz et al.,1990). In

addition, Reeve et al. (1989) reported that topical administration of 0.2% trans-urocanic

acid followed by u.v. irradiation of hairless mice resulted in a two to three fold increase

in the number of skin tumors compared to untreated controls, suggesting that while urocanic acid may protect the skin from u.v. damage, once damage has occurred, it may exacerbate problems. Finally, topical or intravenous administration of cis-urocanic acid from one to three day prior to the application of a contact sensitizer to the skin of rats suppresses delayed type hypersensitivity (Ross et al., 1986, 1988; Norval et al., 1989).

3.5 HISTIDINEMIA

Histidinemia is an inborn error of metabolism that has been detected in rats, mice and humans (Bulfield and Kacser, 1974). This disorder is caused by a deficiency of histidase activity in both the liver and skin which results in the accumulation of histidine in body fluids (Suchi et al., 1993). Clinically, patients with histidinemia have high blood and urine concentrations of histidine (La Du, 1978), imidazolepyruvate, imidazolelactic acid and imidazoleacetic acid (Auerbach et al., 1962; Ghadimi et al., 1962; La Du et al.,

1962; La Du, 1978), histamine (Tanabe and Sakura, 1989) and undetectable blood levels of urocanic acid (Wilcken and Brown, 1975). Histidinemia appears to be a generally benign disorder in humans (Levy, 1989). However, speech defects (Ghadimi et al., 1961) and mental retardation have been associated with this condition in some patients (Levy,

1989). Understanding why some patients with histidinemia have speech defects and mental retardation while others do not is an area of ongoing research. Finally, it has been 22 hypothesized by Taylor et al., (1991) that since histidinemic patients have reduced epidermal urocanic acid, it is possible that the incidence of cancer or skin disease could be altered in these patients.

3.6 MOLECULAR CHARECTERSITICS OF HEPATIC HISTIDASE

Rat histidase has a molecular weight of about 200 kDa, and appears to consist of three identical subunits of 75 kDa each (Okamura et al.,1974; Dhanam and

Radhakrishnan ,1974). The pH optimum for maximal activity is between 8.8 and 9.0

(Okamura et al., 1974). There is strong evidence for the presence of a modified amino acid, dehydroalanine, in the active site of histidase, and it is presumed that it plays a role in the activation of the amino group of histidine (Taylor et al., 1991). However, it is not known whether dehydroalanine formation occurs spontaneously or via a modifying enzyme (Taylor et al., 1990). In addition, little is known about the precursor of dehydroalanine. There has been a suggestion that the precursor could be the amino acid serine, but more recent research disputes this finding (Recsei et al., 1983). The only other enzyme known to have dehydroalanine at its active site is phenylalanine ammonia- lyase (Taylor et al., 1991).

Taylor et al. (1990) isolated the hepatic histidase gene from rat liver. The rat histidase cDNA contains a 1971 bp open reading frame that can be translated into a polypeptide of 657 amino acids. Using Northern analysis methodology, Taylor et al.

(1990), detected a 2.5 kb mRNA transcript in samples of total RNA from rat liver and skin. No histidase mRNA was detected by Northern analysis in the kidney, spleen, heart, 23

lung and brain. Taylor et al. (1990) found that histidase mRNA content of the liver was

ten times greater than that found in the skin.

Subsequently, cDNA clones for hepatic histidase from mouse (Taylor et al., 1993)

and human (Suchi et al., 1993) have been obtained. The nucleotide sequences of the

three histidase cDNA clones share greater than 87% identity. The predicted amino acid

sequences of histidase for all three species have greater than 93% homology. There are

four N-glycosylation sites (Asn-X-Ser/Thr) which are conserved across all species (Suchi

et al., 1993).

3.7 REGULATION OF HEPATIC HISTIDASE

3.7.1 Dietary Protein

Sahib and Murti (1969) fed rats 6, 18 and 40% casein diets, and found that the activity of hepatic histidase increased as dietary protein concentrations increased. Rats fed the 6% casein diet had only trace amounts of hepatic histidase activity while the rats fed the 18 and 40% casein diets had histidase activities which were 2 and 4 times greater, respectively, than the activity found in the rats fed the 6% casein diets. In addition, the activity of urocanase was also increased as dietary protein concentrations increased

(Sahib and Murti, 1969). Similarly, Schiremer and Harper (1970), reported a linear relationship between the activity of both histidase and urocanase of rat liver and the casein content of the diet. In addition, Kang-Lee and Harper (1979) demonstrated that hepatic histidase activity increased in the liver in response to an increase in protein intake, and declined in activity when protein intake was low. Interestingly, there was no increase in histidase activity detected in the epidermis in any of these reports (Sahib and 24

Murti, 1969; Schiremer and Harper, 1970; Kang-Lee and Harper, 1979). In fact, Sahib

and Murti (1969) reported that epidermal histidase activity decreased when rats were fed increasing levels of dietary protein.

The regulation of hepatic histidase by dietary protein in the rat has been shown to

occur at the pretranslational or messenger RNA level. Torres et al. (1998) reported that

hepatic histidase mRNA levels in rats increased as the dietary protein concentration

increased. Similar to the previously mentioned reports, Torres et al. (1998) did not see

any change in the activity of histidase in the skin.

3.7.2 Dietary Histidine

Unlike dietary protein, increasing the concentration of dietary histidine does not

increase hepatic histidase activity in the rat (Sahib and Murti, 1969). Schiremer and

Harper (1970), reported that the high hepatic histidase activity in rats caused by the

ingestion of a high protein diet could not be maintained once the rats were switched to a

diet containing high levels of histidine. Kang-Lee and Harper (1977) reported that

ingestion of a diet with a histidine level above its requirement increased the oxidation of

this amino acid without increasing histidase activity.

In contrast to rats, increasing dietary histidine can increase histidase activity in

chicks and fish. Addition of 6% histidine to a histidine adequate control diet nearly

doubled the activity of chick hepatic histidase when compared to the control diet (Scott

and Austic, 1982). Subsequently, Keene and Austic (2001) reported that increasing

dietary histidine levels elevated chick hepatic histidase activity, but that the addition of

dietary protein had a more pronounced effect on histidase activity than dietary histidine.

Histidase activity also increased significantly in rainbow trout when the dietary 25 concentration of histidine was doubled (Chiu et al., 1984). It should be noted that the levels of histidine supplemented to the diets in the reports by Scott and Austic (1982) and

Chiu et al. (1984) were extremely high and could be considered toxic. In fact, body weight gain was significantly reduced in the chicks and fish fed the diet supplemented with 6 and 2.6% histidine respectively. Interestingly, histidine is generally accepted as the most toxic of all amino acids when fed in excess (Muramatsu et al. 1971, Peng et al.

1973, Benevenga and Steele 1984).

3.7.3 Histidine Imbalance

Feeding an imbalanced amino acid diet will cause an increase in catabolism of the imbalanced amino acid (Davis and Austic 1994, Yuan et al. 2000). Rats (Torres et al.,

1999) and chicks (Keene and Austic, 2001) fed a histidine imbalanced diet have increased hepatic histidase activity. Research with rats indicates that the increase in histidase activity caused by feeding a histidine imbalanced diet is associated with an increase in the levels of hepatic histidase mRNA (Torres et al., 1999).

3.8.4 Hormones

3.8.4.1 Estrogen

In rats detectable hepatic histidase activity appears a few days after birth and continues to rise until it plateaus during the post weaning period (Lamartiniere, 1979). At puberty, histidase activity increases again, and the increase is greater in females than in males (Lamartiniere, 1979). Feigelson (1968) reported in rats that after 21 d of age, histidase activity rose 7 fold in females but only 3 fold in males. This dramatic increase in histidase activity in females is due to the hormone estrogen (Feigelson, 1968).

Ovariectomized female rats do not have a sharp increase in histidase activity at puberty, 26

but if they are given daily doses of 17 β-estradiol, a dramatic increase in the activity of

histidase is seen (Feigelson 1968, Lamartiniere and Feigelson 1977). Armstrong and

Feigelson (1968) suggested that hormones like growth and thyroid hormones may play a

role in the induction of hepatic histidase by estrogen, since estrogen is unable to enhance hepatic histidase activity in hypophysectomized animals. The physiological significance of estrogen regulation of histidase activity is not yet understood.

3.8.4.2 Glucocorticoids

Schiemer and Harper (1970) found that daily administration of hydrocortisone to adrenalectomized rats for 14 d increased histidase and urocanase activity by 3 and 2 fold respectively. Similar results were obtained by Alemán et al. (1998) when they injected rats with hydrocortisone or saline daily for 7 d and then measured histidase activity at 3,

6 and 12 h after the last injection. Hepatic histidase activity was increased at 3 h in the rats injected with hydrocortisone. Interestingly, injection of hydrocortisone did not increase histidase acitivity in rats fed a high carbohydrate-protein free diet (Alemán et al.,

1998).

3.8.4.3 Glucagon

The effect of glucagon on hepatic histidase and urocanase activity in rats was first reported by Kang-Lee and Harper (1971). Injection of rats with glucagon increased hepatic histidase activity 3 fold and urocanase activity 2 fold. The positive correlation between glucagon and hepatic histidase activity in rats was also confirmed by Fiegelson

(1973). Alemán et al. (1998), compared how glucagon or hydrocortisone effected hepatic histidase activity in rats and determined that glucagon had a more pronounced influence on histidase activity. 27

Lamartiniere and Fiegelson (1977) found that injection of dibutyryl cyclic AMP

(cAMP) increased hepatic histidase activity. The 5’ region of the human histidase gene

contains several cAMP responsive cis-acting elements including consensus sequences for

transcriptional factor AP-1 (activator protein-1) and ATF (activating transcription factor)

(Suchi et al., 1995). Thus, it is not surprising that Torres et al. (1998), reported that glucagon not only increased histidase activity in rats, but also increased histidase mRNA levels since the actions of glucagon in cells are mediated by cAMP.

3.9 SUMMARY

Although histidase is the primary catabolic enzyme of histidine, relatively little is known about it. The highest activity of this enzyme is found in the liver and skin of animals where it deaminates histidine to urocanic acid. Because of the absence of urocanase in the skin, urocanic acid continues to accumulate and acts as a u.v. protectant and an immunoregulator. The activity of hepatic histidase is positively correlated with the level of dietary protein intake in all species studied. The effect of dietary protein on histidase activity may be mediated by T3, glucocorticoids or glucagon since all of these

hormones have been reported to regulate histidase activity.

CHAPTER 4

STATEMENT OF PURPOSE

Chickens recognize and regulate dietary protein intake. However, the mechanisms by which chickens rapidly sense protein intake and respond metabolically are not well understood. To understand these mechanisms, our laboratory has been actively involved in establishing a research model which consists of two enzymes that respond differently to dietary protein intake. Determining when the activity of these two enzymes are first altered after feeding a high protein diet is essential, since it occurs after the chicken has sensed the higher amino acid intake and responded by triggering a biological response that includes altering the activity of these enzymes.

Previous work (Adams and Davis, 2001) in our laboratory helped establish part of this research model. In chickens, hepatic malic enzyme mRNA expression decreases when dietary protein concentrations increase. This change in mRNA expression takes place as early as 3 h after intake of a diet with an altered protein concentration.

The initial goal of the present research is to determine if histidase mRNA expression increases in response to an increase in dietary protein intake in chickens.

Another goal of the present research is to investigate whether glucagon or specific amino acids might be involved in mediating the response of malic enzyme and histidase mRNA expression to dietary protein intake. The final goal of this research is to determine if commercial broiler diets formulated based on the results obtained from the molecular research can reduce the abdominal fat pad weight of broilers.

28 CHAPTER 5

MATERIALS AND METHODS

5.1 CLONING CHICKEN HEPATIC HISTIDASE:

5.1.1 Primers for RT-PCR reactions

The nucleotide sequence of rat, mouse and human histidase mRNA were obtained

from GenBank™/ EMBL data bank and aligned. An upstream and a downstream primer

for RT-PCR were chosen from areas which had a high degree of homology between the

sequences of the three species. The RT-PCR primers predicted a product of 442 base

pairs. The forward primer sequence of 20 nucleotides was

5’AAGAGGGCCTGGCACTCATC 3’, and the reverse primer of 21 nucleotides was

5’CCTCCTTTGAAGGTACCACTT 3’. All primers were synthesized at the Molecular

Genetics Instrumentation Facility (MGIF) at the University of Georgia.

5.1.2 RT-PCR

The RT-PCR reaction was performed using total RNA obtained from the liver of

a ~3 week old broiler. Five micrograms of total RNA was added to 810 ng of random hexamers (Pharmacia Biotech, Piscataway, NJ), and sterile deionized water to make a final volume of 62 µl. The sample was heated at 94o C for 5 min and then immediately

frozen at -80o C for 20 min. After the sample was thawed on ice, RT-PCR buffer [final concentration 50 mM KCL, 20 mM Tris-HCl (pH : 8.3), and 2.5 mM MgCl2] dNTP’s

(final concentration of each 5 mM), 40 units of RNase inhibitor (Promega, Madison, WI),

and 70 units of avian myoblastosis virus reverse transcriptase (Promega, Madison, WI)

29 30

were added to the sample. The sample, with a final volume of 100 µl, was incubated at

42o C in a heating block for 2 h and then frozen at -80o C for future use.

For the PCR reaction, 20 ul of the RT reaction mix was added to 80 ul of a 50

mM KCl, 20 mM Tris-HCL (pH 8.3), and 2.5 mM MgCl2 solution containing 150 ng

each of the forward and reverse primer from Section 5.1.1. Control PCR reactions were

the same except that they contained no cDNA and had a 1 mM concentration of dNTP’s.

The reaction mix was heated to 95o C for 1 min prior to the addition of 2.5 units of Taq

Polymerase (Fisher Biotech, Pittsburgh, PA). Thirty three cycles of 94o C for 1 minute,

45o C for 1 minute and 72o C for 5 min was performed on a Amplitron® II Thermolyne

(Barnstead Thermolyne, Dubuque, IA) PCR-thermocycler. The annealing temperature

was lowered from 55oC to 50oC and finally to 45 oC for subsequent PCR reactions.

5.1.3 Cloning and Sequencing

Ten microliters of the PCR reaction volume was electrophoresed on a 1.5 % agarose gel to separate the PCR products. The gel was stained with ethidium bromide and visualized with ultraviolet light. For the RT-PCR reaction with an annealing temperature of 45oC, a band corresponding to the predicted 442 base pairs product was

excised from the gel, and the DNA was extracted using a Spinex column (Corning

Costar, Cambridge, MA) following the manufacturer’s recommendation.

To produce sufficient quantities of the PCR product for sequencing, library

screening and potential Northern analysis, the isolated PCR product was ligated into a

pCR II vector using the TA cloning kit (Invitrogen, San Diego, CA), following

manufacturer’s instructions. A portion of the ligation reaction was then used to transform

competent INVα E. Coli cells supplied by Invitrogen (San Diego, CA) following the 31

manufacturer’s protocol. Five colonies resulting from the transformation were then selected and grown in 5 ml of Luria-Bertani (LB) broth. The plasmid was isolated from

each of the broth preparations using the Wizard Plus Miniprep Plasmid Isolation Kit

(Promega, Madison, WI). The isolated plasmid was then digested with EcoRI (Gibco,

Life Technologies, Paisley, PA) to confirm that the plasmid contained the expected PCR

product. Sequence analysis of a positive plasmid preparation was performed using a T7

primer at the MGIF at the University of Georgia.

5.1.4 Rapid Amplification of cDNA Ends (RACE) PCR

In an effort to obtain the full length cDNA sequence of chicken histidase, RACE-

PCR was conducted. Total RNA extracted from chicken liver was used to make the

cDNA for the RACE-PCR reaction. Clontech’s (Palo Alto, CA) SMART® RACE cDNA

Amplification Kit was used for the RACE reactions. This kit provided two primers for

the PCR reactions, one for the 5’end amplification (Smart II™ oligonucleotide), and another for the 3’ end amplification (Oligo(dT) primer). Gene specific primers for the

RACE reactions were designed based upon the results of sequence analysis of the 442 bp

PCR product. The forward primer contained 26 nucleotides with the following sequence,

5’TGCGAAGCAGTTGAAAGAGCTAGCGC3’, and the reverse primer contained 26

nucleotides with the following sequence,

5’TGAGGGCAGCAGCGCATCGTATATGC3’. The primers were made at the MGIF at

the University of Georgia.

32

PCR reactions were carried out for the amplification of the 5’ and 3’ cDNA ends

of the histidase gene. For the 5’ end amplification, the Smart II™ oligonucleotide and the

newly constructed reverse primer were used, while the 3’ end amplification was

performed using the Oligo(dT) primer and the new forward primer. All procedures

followed the manufacturer’s instructions. The PCR reaction protocol consisted of 35

cycles of 94o C for 10 sec, 45o C for 20 sec and 72o C for 3 min.

5.1.5 RT-PCR using Nested Primers

Since no products were obtained from the original RACE PCR-reactions, another

3’ RACE reaction was performed using an alternative primer. In this RACE reaction, the

original forward primer (section 5.1.1) and the oligo(dT) primer provided by the kit were

used. In order to determine if any of the products from the 3’ RACE reaction were

actually chicken histidase, ‘nested’ PCR reactions were conducted. For this procedure, 1

and 5 µl aliquots from the RACE-PCR reaction were reamplified using a “nested”

primer, which was the forward primer of the original RACE reaction plus the oligo(dT)

primer. The conditions for the PCR reactions were the same as that was used in the

RACE protocol. Ten microliters of the reactions were then run on a 1.5% agarose gel,

and stained with ethidium bromide.

5.1.6 Additional Primers

In an additional effort to obtain more of the chicken histidase cDNA sequence,

two additional RT-PCR primers were made based upon two additional regions of the

nucleotide sequences of rat, mouse and human histidase that showed a high degree of

homology. The new forward primer consisted of 20 nucleotides with the following

sequence 5’AGGTGTCGGATGCTCTTGGC3’, and the new reverse primer contained 22 33 nucleotides with a sequence of 5’ATCCTCCCATGGAGACGTGGTC3’. The primers were made at the MGIF at the University of Georgia.

Three RT-PCR reactions were set up, with one reaction having the gene specific forward and reverse primer from Section 5.1.4. The second reaction had the gene specific forward primer (Section 5.1.4) and the newly constructed reverse primer. The third reaction had the newly constructed forward primer and the gene specific reverse primer (Section 5.1.4). The first PCR reaction was used as a control, and the primers predicted a 254 bp product. The primers for the second PCR reaction predicted a 661 bp product, whereas the primers for the third reaction predicted a 726 bp product.

Conditions for the PCR reactions were the same as those presented in section 5.1.2, except that the annealing temperature was 52o C.

A 10 µl aliquot of each PCR reaction was then electrophoresed on a 1.5% agarose gel and stained with ethidium bromide. A band obtained from the second reaction was excised from the gel and the DNA was extracted from the gel slice using Spinex columns

(Corning Costar, Cambridge, MA) following manufacturer’s recommendation. The cloning, transformation, plasmid purification, and sequence analysis of this RT-PCR product was as previously described (Section 5.1.3).

5.1.7 Chicken Liver cDNA Library Screening

A chicken liver Lamda Zap® cDNA library was purchased from Stratagene (La

Jolla, CA). The library contained approximately 2.0 x 106 independent clones. The cDNA library was constructed from RNA obtained from the liver of a 7 week old broiler breeder. For the primary screening, 450,000 plaque forming units (pfu) were screened from a total of ten 150 mm agar plates. Duplicate membrane lifts (Plaque Screen 34

Hybridization Transfer Membranes, NEN, Boston, MA) were made from each of the plates following the manufacturer’s instructions. The membranes were then hybridized with a 32P preparation of the 442 bp PCR product. The hybridization and membrane

washing protocol were the same as those previously described by Chen and Johnson

(1996). Putative positive colonies were cored from the agar plates for subsequent

screening and isolation. For each positive core isolated, the pBluescript® SK(+/-)

phagemid containing the insert was excised from the Lamda ZAP® vector using the single

clone excision method as recommended by Stratagene (LaJolla, CA). The excised phagemid was inserted into competent cells (XL1-Blue-MRF’ strain) using helper phage

(R408 interference-resistant), and then grown on LB-ampicillin agar plates. Colonies

appearing on the plate contained the pBluescript® double stranded phagemid with the

cloned DNA insert. Colonies were picked from each plate and grown in LB-ampicillin

broth. Plasmid from each preparation was then isolated using the Wizard Plus Miniprep

Plasmid Isolation Kit (Promega, Madison, WI).

To determine if the plasmid isolated had the cDNA insert of interest, it was

digested with EcoRI and then run on a 1.5% agarose gel. The contents of the gel were

then transferred to a nylon membrane for Southern blot analysis. The membranes were

hybridized with a 32P preparation of the 442 bp PCR product, for the detection of positive plasmids. Sequence analysis of two positive plasmids containing the largest cDNA inserts was conducted using T7, T3, M13 primers, and one internal primer

(5’TCTCTTTAGATGGCAACAGC3’) at the MGIF at the University of Georgia.

35

5.2 DIETARY EXPERIMENTS

5.2.1 Battery Experiments

5.2.1.1 General Bird Care, Experiments (1 – 12)

Day old broiler chicks (Ross X Ross) obtained from ConAgra (Athens, GA) were

housed in thermostatically controlled, electrically heated battery brooder cages with wire

floors. The cages were lighted for 24 h, and the birds had free access to feed and water.

For the first seven days, the birds were fed a practical UGA starter diet (Table 1). After

this period, the birds were sorted by weight, and those with extreme weights discarded.

The remaining chicks were randomly assigned to experimental pens with each pen containing two birds. The chicks were then fed a semi-purified 22% (basal) protein diet

(Table 2) for an acclimation period of 4 or 5 days, after which they were given access to

the experimental diets. The semi-purified basal diet used in the experiments was the

“adjusted basal diet” established previously by Adams and Davis (2001). For the sake of convenience, in this dissertation, the adjusted basal diet will be designated as the basal diet. At the end of the experiments, feed intake and the body weight of the birds were determined. The chicks were killed by cervical dislocation, and liver samples were

immediately taken for RNA extraction. The Instructional Animal Care and Use

Committee of The University of Georgia approved all animal procedures.

5.2.1.2 Experiment 1-4

Total RNA isolated from four previous experiments (Adams and Davis, 2001)

was used to determine the effect of dietary protein on chicken hepatic histidase mRNA expression. In these previous experiments, chicks were fed either a low (13 g/100g diet), basal (22 g/ 100g diet) or a high (40 g/100 g diet) protein diet. In Experiment 1, these 36

Table 1. Composition of the University of Georgia unmedicated chick starter diet

INGREDIENT g/100 g diet

Corn 57.3

Soybean Meal 33.5

Poultry Fat 3.15

Poultry by Product 3.00

Defluronated Phosphate 1.54

Limestone 0.79

Vitamin Mix1 0.25

Salt 0.22

DL-Methionine 0.19

Mineral Mix2 0.08

1 Vitamin Mix provided the following per 100 g of diet: Choline 19.13 mg, Thiamine HCl 2.2 mg, Riboflavin 0.44 mg, Nicotine acid amide 4.41 mg, Folic acid 0.55 mg, Pyridoxine HCl 0.47 mg, D-Biotin 0.011 mg, Vitamin B12 0.001 mg, d-Calcium pantothenate 1.12 mg, Menadione sodium bisufite 0.33 mg, Vitamin E 1.1 IU, Vitamin D3 110 IU, Vitamin A 551 IU, Ethoxyquin 12.5 mg. 2 Mineral Mix provided the following in mg/100g diet: Mn 6.0, Zn 5.0, Fe 3.0, I 1.5, and Se 0.5

37

Table 2. Composition of the two experimental diets fed during Experiments 5-12.

INGREDIENT DIETARY TREATMENTS

Basal diet High protein diet 22 % protein 40% protein g/100g diet

Isolated soybean protein 22.950 41.730

Glucose Monohydrate 40.958 40.958

Cellulose 2.1000 1.8000

Corn Oil 4.1200 4.200

Sand (Filler) 11.951 0.000

Glycine 0.4000 0.7300

L-Methionine 0.6000 1.0900

Choline Chloride 0.1540 0.1540

Vitamin Mix1 0.8326 0.8326

Mineral Mix2 7.9300 7.9300

Potassium Bicarbonate 0.6680 0.6680

1Vitamin Mix provided the following in mg/kg diet: Thiamine HCl 7.5, Riboflavin 7.5, Nicotine acid amide 75, Folic acid 3, Pyridoxine

HCl 6, D-Biotin 0.3, Vitamin B12 10, d-Calcium pantothenate 20, Menadione sodium bisufite 4.5, Vitamin E 114, Vitamin D3 750, Vitamin A 14, Ethoxyquin 50, I-Inositol 125, Dextrose 3811 2 Mineral Mix provided the following in g/100g diet: CaHPO42.H20

2.07, CaCO3 1.48, KH2PO4 1.00, NaSeO4 0.0002, MnSO4H2O 0.035, FeSO47H20 0.05, MgSO4(anhydrous) 0.30, KIO3 0.001, NaCl 0.60,

CuSO4.5H20 0.003, ZnCO3 0.015, CoCl2 0.00017, NaMoO4 0.00083, KCl 0.10

38

diets were fed for 6 or 24 h, while in Experiment 2, the diets were fed for 1.5 and 3 h.

These two initial experiments were then duplicated in Experiments 3 and 4. For each experimental time period, feed intake and body weight were determined, and liver samples were taken for subsequent RNA extraction and Northern blot analysis.

5.2.1.3 Experiment 5

This experiment was conducted to determine if the observed effects of the high protein diet on the expression of histidase mRNA were due to the increased histidine content of this diet, to the increased protein level of this diet or to a combination of both of these factors. Sixty birds were randomly distributed to 30 pens of two birds each.

After the adjustment period, the 30 pens were split into five groups, and the chicks were then fed either the basal diet, a high protein diet (40 g/100 g diet) (Table 2) or the basal diet supplemented with 0.22 g/100g diet (H1), 0.43 g/100g diet (H2), or 0.86 g/100g diet

(H3) of L-histidine (Dyets, Inc. Bethlehem, PA). The final calculated total histidine content of each diet was 0.53 g/100g diet, 0.75 g/100g diet, 0.96 g/100g diet, 1.39 g/100g diet and 0.96 g/100g diet for the basal, H1, H2, H3 and high protein diets respectively.

The composition of the basal and high protein diets was the same as those used in

Experiments 1 – 4. Additions of histidine to the basal diet were at the expense of cellulose. The chicks had access to these diets for 24 h, after which feed consumption and body weight were determined for each pen. The chicks were killed by cervical

dislocation at the end of the experiment to obtain liver samples for RNA extraction.

39

5.2.1.4 Experiment 6-7

The goal of these experiments was to determine if glucagon altered hepatic malic enzyme and histidase mRNA expression. In Experiment 6, 48 birds were sorted into 24 experimental pens with 2 birds each. Day-old chicks were fed a regular starter diet for one week, after which they were fed the basal diet for an acclimation period of 4 days.

The birds were then divided into 2 groups of 12 pens. The birds in half of the pens were injected (brachial vein) with 240 µg glucagon/kg body weight, while the birds in other pens were injected with an equivalent volume of an avian saline solution. The glucagon used in these experiments was obtained from porcine pancreas cells (Sigma, St. Louis,

MO). After the injection, the birds continued to have free access to the basal diet and water for either 1.5 h or 3 h. At the end of each time period, liver samples were collected and pooled by pen for RNA extraction for subsequent Northern analysis of malic enzyme and histidase.

Experiment 7 was similar in protocol to that of Experiment 6, except that the effect of glucagon on the expression of malic enzyme and histidase was determined only for the 3 h time period and there were only 4 replicate pens per treatment.

5.2.1.5. Experiment 8

This experiment was conducted to determine if changes in dietary protein intake alters the plasma glucagon concentration in chicks. For this experiment, at the end of the acclimation period for the basal diet the birds were fasted for 2 h, after which they were fed either a high protein diet or the basal protein diet for 0, 1, 2, 3 or 6 h. For both treatments, there were a total of 6 replicate pens of two birds each, for time 0, 3 and 6 h, and 9 replicate pens for time 1 and 2 h. 40

At the end of each time period, food intake was recorded. Blood was collected

from the brachial vein and immediately placed into glass test tubes containing EDTA as

an anti-coagulant. Blood samples were placed on ice until they were centrifuged at 1000

x g for 10 minutes at 4oC. Plasma samples were collected and then frozen at -80oC. To

measure plasma glucagon concentration, 100 µl of plasma from each bird of each pen

was pooled for radio-immuno assay (RIA) using the DA Glucagon Kit (ICN

Biochemicals, Irvine, CA). The RIA was performed following manufacturer’s

instructions on duplicate samples from each pen.

Liver samples (six replicate pens per treatment) were collected for subsequent

RNA extraction from both birds of each pen at 0, 3 and 6 h after access was given to the

diets.

5.2.1.6 Experiment 9-10

These experiments were conducted to investigate the role of specific groups of amino acids in the regulation of malic enzyme and histidase mRNA expression. In these experiments, chicks were fed one of four different diets. The four diets were the basal diet, the high protein diet or the basal diet supplemented with either essential (EAA) or non-essential amino acids (NEAA) to equal the concentrations found in the high protein diet. The composition of the EAA and the NEAA diets is shown in Table 3. There were a total of six replicate pens of two birds each for each treatment. The birds were given access to these diets for either 24 h (Experiment 9) or 6 h (Experiment 10). At the end of each experiment, feed intake and body weight were measured and liver samples were collected for RNA extraction.

41

Table 3. Composition of the essential (EAA) and the non-essential (NEAA) amino acid supplemented diets (Experiments 9 and 10).

INGREDIENT DIETARY TREATMENTS

EAA NEAA Supplemented Supplemented diet diet g/100g diet

Isolated soybean protein 22.950 22.950 Glucose Monohydrate 40.985 40.985 Cellulose 2.1000 2.100 Corn Oil 8.100 8.100

Sand (Filler) 6.200 7.031

Glycine 0.730 0.400 L-Methionine 1.090 0.600

Choline Chloride 0.154 0.154 Vitamin Mix 0.833 0.833

Mineral Mix 7.930 7.930 Potassium Bicarbonate 0.668 0.668 Essential Amino Acid Mix1 8.338 2 Non-Essential Amino Acid Mix 8.320 Total Nitrogen (From AA supplements) 1.377 0.903

1 EAA Mix provided the following in g/100g diet: Arginine 1.258, Glycine 0.695, Histidine 0.432, Isoleucine 0.808, Leucine 1.352, Lysine 1.033, Methionine 0.225, Phenylalanine 0.864, Threonine 0.620, Tryptophan 0.225, and Valine 0.826. 2 NEAA Mix provided the following in g/100g diet: Alanine 0.714, Aspartic acid 1.916, Cysteine 0.207, Glutamic acid 3.155, Proline 0.845, Serine 0.864 and Tyrosine 0.620

42

5.2.1.7 Experiment 11

The goal of this experiment was to determine if the total nitrogen concentration in

the diet regulated malic enzyme and histidase mRNA expression. After the acclimation

period, the chicks were divided into 5 groups of six replicate pens of 2 birds each. The five groups of birds were fed either the basal diet, high protein diet or the basal diet

supplemented with either 9.5% glutamic acid, 6% alanine, 5% glycine or 7.5%

diammonium citrate (DAC). The composition of these experimental diets is shown in

Table 4. The amount of total nitrogen present in the basal supplemented diets was

equivalent to the nitrogen present in the NEAA supplemented diet used in Experiment 9.

The birds had access to these diets for 24 h after which feed intake and body weight were

collected. Liver samples from the birds of each pen were pooled for total RNA

extraction.

5.2.1.8 Experiment 12

The goal of this experiment was to determine if dietary additions of ammonium

phosphate and ammonium bicarbonate would alter malic enzyme mRNA expression in a

manner similar to DAC. There were 5 dietary treatments consisting of 6 replicate pens of

2 birds each. One group of birds was maintained on the basal protein diet, while the

other groups were fed either the high protein diet, or the basal diet supplemented with

either ammonium phosphate (4.25%), ammonium bicarbonate (5.08%) or a combination

of both ammonium phosphate (2.13%) and ammonium bicarbonate (2.54%). The

composition of the diets is shown in Table 5. The birds were given access to these diets

for 24 h after which feed intake and body weight gain were determined. Liver samples

were collected and pooled by pen for RNA extraction. 43

Table 4. Composition of the basal supplemented diets fed in Experiment 11.

INGREDIENT DIETARY TREATMENTS

Glutamic Glycine Alanine DAC Acid

g/100g diet Isolated soybean protein 22.950 22.950 22.950 22.950

Glucose Monohydrate 40.985 40.985 40.985 40.985

Cellulose 2.100 2.100 2.100 2.100

Corn Oil 8.100 10.600 9.400 8.100

Sand 5.826 7.800 8.000 7.031

Glycine 0.400 0.400 0.400 0.400

L-Methionine 0.600 0.600 0.600 0.600

Choline Chloride 0.154 0.154 0.154 0.154

Vitamin Mix 0.833 0.833 0.833 0.833

Mineral Mix 7.930 7.930 7.930 7.930

Potassium Bicarbonate 0.668 0.668 0.668 0.668

L-Glutamic acid 9.496

Glycine 5.000

L-Alanine 6.000

Diammonium citrate 7.500

Total N (From AA 0.903 0.903 0.903 0.903 supplements)

44

Table 5. Composition of the basal supplemnted diets fed during Experiment 12.

INGREDIENT DIETARY TREATMENTS Ammonium Ammonium Both Phosphate Bicarbonate g/100g diet

Isolated soybean protein 22.950 22.950 22.950

Glucose Monohydrate 40.985 40.985 40.985

Cellulose 2.100 2.100 2.100

Corn Oil 11.500 11.500 11.500

Sand 7.690 6.860 7.270

Glycine 0.400 0.400 0.400

L-Methionine 0.600 0.600 0.600

Choline Chloride 0.154 0.154 0.154

Vitamin Mix 0.833 0.833 0.833

Mineral Mix 7.930 7.930 7.930

Potassium Bicarbonate 0.668 0.668 0.668

Ammonium Phosphate 4.250 2.130

Ammonium Bicarbonate 5.080 2.540

Total N (From supplements) 0.903 0.903 0.903

45

5.2.2 Floor Pen Experiment (Dietary Experiment 13)

A grow-out experiment was conducted to determine if supplementing a standard corn soy diet with glutamic acid would reduce the amount of abdominal fat in broilers. A total of 500 (Ross X Ross) straight run broiler chicks were obtained from ConAgra,

Athens, GA and distributed to 24 floor pens containing pine shaving litter. The pens

were maintained in an environmentally controlled room under a 24 h light cycle. Twenty

pens with 20 chicks each were randomly assigned to 4 diets (5 replicates per treatment).

From 0 to 21 d, the chicks in these pens were fed either a basal 22% protein practical

corn-soybean diet or this diet supplemented with 2.3%, 4.7% or 9.5% L-glutamic acid

(Table 6). The remaining 4 pens had 25 chicks each, and were fed a diet, which was

similar to the 9.5% glutamic supplemented diet, except that the glutamic acid was

replaced by glucose monohydrate and sand. This final diet was included as a control diet

to ensure that any differences between the practical corn-soybean diet and the diet

supplemented with 9.5% glutamic acid were related to the glutamic acid supplementation

and not due to the increase in the fat content of this diet to keep it isocaloric to the basal

diet. All diets were isocaloric (Table 6). After 21 d, the chicks were fed grower/finisher

diets (Table 7).

Feed intake and body weight were measured on 21 and 40 d. Liver samples were

taken on day 18 and 37 from 12 randomly selected birds per treatment. The 12 liver

samples per treatment were pooled in groups of two birds (n = 6 samples per treatment)

for total RNA extraction. 46

Table 6. Composition of the experimental diets fed during the starter phase (0 to 21 d) in Experiment 13

INGREDIENT DIETARY TREATMENTS

Basal 2.3% 4.7% 9.5% Control L-Glutamic L-Glutamic L-Glutamic acid acid acid g/100g diet

Corn 58.522 55.226 51.930 45.454 45.454

Soybean Meal 35.683 36.273 36.864 38.000 38.000

Poultry Fat 2.253 2.595 2.936 3.585 3.585

Glucose Monohydrate 0.000 0.000 0.000 0.000 8.290

Sand 0.000 0.000 0.000 0.000 1.210

Dicalcium Phosphate 2.400 2.500 2.500 2.650 2.650

Limestone 0.914 0.902 0.890 0.827 0.827

Salt 0.294 0.294 0.293 0.291 0.291

Vitamins 0.250 0.250 0.250 0.250 0.250

DL-Methionine 0.202 0.206 0.211 0.220 0.220

Trace Minerals 0.070 0.070 0.070 0.070 0.070

L-Lysine 0.052 0.039 0.025 0.000 0.000

L-Glutamic acid 0.000 2.375 4.750 9.500 0.000

CALCULATED ANALYSIS

Metabolizable Energy, kcal/kg 3050 3050 3050 3050 3050

Crude Protein (%) 21.99 21.99 22.01 22.00 22.00

Calcium (%) 1.00 1.00 1.00 0.98 0.98

Available Phosphorus (%) 0.45 0.45 0.45 0.45 0.45

47

Table 7. Composition of the experimental diets fed during the grower/finisher phase (22 – 40 d) in Experiment 13.

INGREDIENT DIETARY TREATMENTS

Basal 2.3% 4.7% 9.5% Control L-Glutamic L-Glutamic L-Glutamic acid acid acid g/100g diet

Corn 63.586 60.281 56.976 50.366 50.366

Soybean Meal 29.544 30.136 30.728 31.911 31.911

Poultry Fat 3.161 3.505 3.849 4.538 4.538

Glucose Monohydrate 0.000 0.000 0.000 0.000 8.290

Sand 0.000 0.000 0.000 0.000 1.210

Dicalcium Phosphate 1.483 1.494 1.505 1.528 1.528

Limestone 1.167 1.158 1.150 1.132 1.132

Salt 0.503 0.504 0.504 0.505 0.505

Vitamins 0.250 0.250 0.250 0.250 0.250

DL-Methionine 0.168 0.172 0.177 0.186 0.186

Trace Minerals 0.070 0.070 0.070 0.070 0.070

L-Lysine 0.069 0.055 0.042 0.015 0.015

L-Glutamic acid 0.000 2.375 4.750 9.500 0.000

CALCULATED ANALYSIS

Metabolizable Energy, kcal/kg 3150 3150 3150 3150 3150

Crude Protein (%) 19.50 19.50 19.50 19.50 19.50

Calcium (%) 0.85 0.85 0.85 0.85 0.85

Available Phosphorus (%) 0.40 0.40 0.40 0.40 0.40

48

On 40 d, 10 birds (5 males and 5 females) were randomly selected from each pen for abdominal fat pad measurements. In addition to this, 5 more birds were randomly selected from each pen to obtain fresh prechilled carcass weights. All selected birds were

fasted overnight, and processed the next day. To measure abdominal fat pad, New York

dressed broilers were chilled overnight at 4oC. The following day, fat pads were removed

from each bird and weighed.

5.3 TISSUE COLLECTION AND RNA EXTRACTION:

The surgical instruments used to collect the liver samples were soaked in a solution containing 2% Absolve (NEN Inc., Boston, MA) to eliminate RNase activity.

Approximately 150 mg of liver tissue from the left lobe from each bird was collected and pooled per pen in a 15 ml siliconized corex tube containing 3 ml of ice cold working solution D (Chomczynski and Sacchi, 1987). The samples were kept on ice until all the samples were collected. Samples were homogenized using a PowerGen 700 (Fisher

Scientific, Pittsburg, PA) tissue disrupter. Total RNA was extracted using the guanidine isothiocynate/phenol-chloroform method as described by Chomczynski and Sacchi

(1987).

5.4 NORTHERN ANALYSIS

All electrophoresis equipment (Hoefer Biotech Inc. San Francisco, CA) including the combs and gel tray were soaked with a solution containing 2% Absolve for 45 minutes to eliminate RNase activity. Forty micrograms of total RNA from each sample was electrophoresed on a 1.5% agarose/formaldehyde gel. Ethidium bromide (6 µg/ 100 49

ml of running buffer) was added to the running buffer (1 X MOPS) to stain the ribosomal RNA of each sample. The gel was electrophoresed either overnight at 29 V or at 75 V for 8 h. The integrity of the RNA samples in the gel was assessed by viewing the

ethidium bromide stained ribosomal bands under UV light. The RNA from the gel was transferred to GeneScreen Plus nylon membrane (NEN, Boston, MA) using a Stratagene

(LaJolla, CA) PosiBlot pressure blotter. After transfer, the RNA was cross-linked to the membrane using a FB-UVXL-1000 Cross Linker (Fisher-Biotech, Pittsburgh, PA).

Northern analysis was performed using the following radiolabeled probes: 442 bp

chicken histidase RT-PCR clone (Section 5.1.2), a duck malic enzyme cDNA clone

(graciously provided by Dr. Susan Stapleton, Western Michigan University), and a

chicken GAPDH RT-PCR clone (Davis and Johnson, 1998). The procedure for making the probes was as described previously (Chen and Johnson, 1996). In brief, a total of 100 ng of cDNA was labeled with 5 µCi 32P-dCTP (ICN Biochemicals, Irvine, CA) using a

random primer-kit (Primer-It, Stratagene, LaJolla, CA) to a specific activity of 1.0-5.0 x

108 cpm/µg. Membranes were pre-hybridized for at least an hour at 42oC in a

hybridization bottle (Fischer Scientific, Forbes, PA) containing 20 ml of formamide, 10

ml of 20X SSPE, 4 ml of 5X Denharts solution and 4 ml of 10% SDS and 0.4 ml of 10

mg/ml herring sperm DNA. The herring sperm DNA was denatured at 95oC for 5 min

prior to its addition to the pre-hybridization solution. After the prehybridization period,

the radioactive labeled probe was denatured for 5 minutes at 95oC and added to the

prehybridization solution. The membranes were then incubated for at least 16 h at 42oC.

The Northern blots from each experiment were hybridized with separate 32P labeled preparations of the malic enzyme and histidase probes. The blots were stripped of the 50

previously hybridized probe before being hybridized with the subsequent probe. To

verify and correct for equality of RNA loading and transfer, the final hybridization of the

blots was done with the GAPDH radiolabelled probe.

After each hybridization, the membranes were washed with a solution containing

1% SDS and 2X SSC. The washing protocol included two five-minute washes at room

temperature, followed by two thirty minute washes at 65oC. The washed membranes

were then exposed to X-ray film (Classic CX single emulsion blue base film, Laboratory

Product Sales, Rochester, NY and Kodak BioMax MS film, Eastman, Kodak, Rochester,

NY) at -80oC in cassettes containing intensifying screens. The films for the histidase

blots were exposed for 6-7 d, malic enzyme blots were exposed for 4-5 d and GAPDH

blots were exposed for 45 – 60 min. The films were tray developed using GBX

developer and fixer (Eastman Kodak, Rochester, NY).

Autoradiographs were then scanned using a HP ScanJet 5100 scanner (Hewlett

Packard, Corvallis, OR). The intensity of the autoradiograph signals was quantified

using Image Quant (Version 3.3, Molecular Dynamics, Sunnyvale, CA) densitometry

software. Relative mRNA expression of malic enzyme and histidase was determined for

the samples of each blot by calculating the signal intensity for each sample relative to the

strongest malic enzyme or histidase signal, which was assigned a value of 1. Before

calculation of relative malic enzyme or hisitdase mRNA levels, GAPDH mRNA was

used to correct the malic enzyme and histidase values for equality of RNA loading and

transfer.

51

5.5 STATISTICS

ANOVA was performed using the General Linear Model Procedures of SAS

(SAS Institute, Cary, NC) with replicate and dietary treatment as factors. Duncan’s multiple range test was used to determine significant differences between the treatments.

Differences were considered significant when P values were <0.05.

CHAPTER 6

RESULTS

6.1 CLONING CHICKEN HEPATIC HISTIDASE:

6.1.1 RT-PCR Product

The RT-PCR reaction with primers selected based upon the conserved nucleotide

sequences of rat, mouse and human histidase yielded a 442 bp product when a low PCR

annealing temperature of 45oC was used (Figure 3). The nucleotide sequence and the

derived amino acid sequence are shown in Table 8. The homology of the nucleotide and

protein sequences was greater than 80% and 95%, respectively, with the corresponding

cDNA sequences of rat, human and mouse histidase (Table 9).

6.1.2 RACE PCR

No RACE-PCR products were obtained for the original 3’ and 5’ reactions.

However, products were obtained for the 3’ RACE-PCR when the original forward

primer was used with the poly T primer.

6.1.3 Nested – PCR

Further investigation of the 3’ RACE products using a nested primer revealed that

the products were not portions of the cDNA for chicken histidase since none of the

products were amplified using the nested primer with the poly T primer.

52 53

Lane

1 2 3

872 bp →

603 bp →

310 bp →

Figure 3. Ethidium bromide stained agarose gel containing chicken histidase RT-PCR products. Lane 1: φX174 – DNA marker (Promega, Madison WI). Lane 2: control PCR reaction containing glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and histidase RT-PCR product. In this PCR reaction, there were 4 primers in the tube (GAPDH forward and reverse, original histidase forward and reverse). Lane 3: RT-PCR product (442 bp) obtained when using the original forward and reverse primer with a 45oC PCR annealing temperature.

54

Table 8. Nucleotide and amino acid sequences of the 442 bp RT- PCR product.

Nucleotide Sequence 5’aagagggcctggcactcatcaatgggacacaaatgatcacctcgctgggatgcgaagcagttga aagagctagcgccattgctaggcaagcagacatagtcgctgcccttacacttgaagtcctgaagggt acaactaaggcctttgatactgatatccatgctgtacgtccacatcgagggcaggctgaagtggcattt cgattcangtcccttctggattctgaccatcacccatcagaaatagcagagagccaccgattttgtgac cgagttcaggacgcatatacgatgcgctgctgccctcaggtccacggagtagtaaatgatacaatcg cttttgtgaaggacatcatgacaactgaaatcaacagcgccacagacaaccctatggtgtttgctgaa agagcagagaccatttctggaggaaattttcacggtga3’

Protein Sequence EGLALINGTQMITSLGCEAVERASAIARQADIVAALTLEVLKGT TKAFDTDIHAVRPHRGQAEVAFRFXSLLDSDHHPSEIAESHRFC DRVQDAYTMRCCPQVHGVVNDTIAFVKDIMTTEINSATDNPM VFAERAETISGGNFHVE

55

Table 9: Homology of the nucleotide and protein sequences of the 442-bp RT-PCR product with the corresponding sequences of rat, mouse and human histidase.

Rat MouseHuman

Nucleotide Identity (%) 80 81 81

Protein Identity (%) 96 95 95

56

6.1.4 Use of Additional Primers

A 661 bp RT-PCR product was obtained using the gene specific forward primer

(Section 5.1.4) and the new reverse primer (Section 5.1.6) (Figure 4). Sequence analysis of this new product resulted in extending the original 442 bp RT-PCR product sequence an additional 296 bp towards the 3’ end. The combined nucleotide and derived amino acid sequences obtained from the two RT-PCR products had about 83% and 93% sequence homology with the corresponding nucleotide and amino acid sequences of rat, mouse and human histidase.

6.1.5 Chicken Liver cDNA Library Screening

Twenty positive histidase cDNA clones were identified and isolated after screening 3 x 105 pfu. Two of the clones (cHAL-8 and cHAL-10) with the largest cDNA inserts, were selected for sequence analysis. The reading frame for the cHAL-8 and cHAL-10 sequences was established by comparison to the published nucleotide sequences of rat (Taylor et al., 1990), mouse (Taylor et al., 1993) and human (Suchi et al., 1993) histidase. The schematic relationship among our sequenced histidase cDNA clones and the original RT-PCR product is depicted in Figure 5. Sequence analysis revealed that the cHAL-8 clone contained the start codon, but lacked 541 bp of the terminal 3’ coding region, while the cHAL-10 clone contained the entire 3’ terminal end including the poly A tail, but lacked 196 bp of the coding sequence from the 5’ end. The two clones had an overlapping region of 1497 bp, and together contain the complete coding sequence for chicken hepatic histidase.

57

Lane

1 2 3 4

872 bp →

603 bp →

310 bp →

Figure 4. Ethidium bromide stained agarose gel containing chicken histidase RT-PCR products. Lane 1: φX174 – DNA marker (Promega, Madison WI). Lane 2: RT-PCR product obtained from using the forward and reverse gene specific RACE primers. It was used as a positive control reaction. Lane 3: RT-PCR product obtained from gene specific RACE forward primer and a new reverse primer. Lane 4: RT-PCR reaction mix of the new forward primer and the gene specific RACE reverse primer.

58

cHAL-8

RT-PCR Product

cHAL-10

ATG TAA

-28 1 205907 1348 1701 1983 2234

Figure 5: Schematic relationship among the chicken hepatic histidase clones (cHAL-8 and cHAL-10) and the RT-PCR product used to screen the chicken hepatic cDNA library. Numbers correspond to nucleotide bases. The start of the protein coding sequence is indicated by the nucleotide 1 (ATG, start codon), and the end of the protein coding sequence is indicated by nucletide 1983 (TAA, stop codon).

59

The full length coding sequence of chicken hepatic histidase (Table 10) has 83

percent nucleotide identity with the full length coding sequence of rat, mouse and human histidase. The 3’- untranslated region of chicken histidase cDNA contains a variant of the polyadenylation hexamer (AATAAA), which starts 18 bp before the beginning of the poly A tail. The complete cDNA sequence and the derived amino acid sequence are part of the GenBank database (Accession number: AY227348).

The 1983 bp coding sequence of chicken histidase predicts a protein of 660 amino

acids. The deduced amino acid sequence of chicken histidase has 88, 88 and 87 percent

identity with the amino acid sequences of rat, mouse and human histidase, respectively.

The chicken histidase sequence has three additional amino acids at the C-terminus end,

compared to the histidase sequences of rat, mouse and human (Table 11). Chicken

histidase has four N-glycosylation consensus sequences (Asn-X-Ser/Thr) beginning at amino acids 238, 307, 408 and 476. The position of these four N-glycosylation sites in histidase are conserved across all species studied thus far.

In the Northern analysis of total RNA derived from a liver sample of a 2 week broiler chick, the chicken histidase cDNA probe hybridized to a major band of histidase mRNA at approximately 2.9 kb and to a minor band slightly below it (Figure 6).

60

Table 10. Nucleotide sequence of chicken hepatic histidase from 5’- 3 ’. Nucletoides are numbered on the left. The start (ATG) and stop (TAA) codons for the protein coding sequence are underlined. The GenBank® accession number is AY227348.

1 caggagagga acagtctggg gaggaaacAT Gccaagatac acagtgcacg tccgaggaga 61 atggctggca gtgccgtgcc cacacggcac aaacacggtc ggatggctgg gaaaggaggc 121 tgtgaggcgg tacatgaaga acaaacctga taacggcgga tttacctcag tggaagaagt 181 aaagtttttt gttcggaggt gcaagggtct cggcttgctg gatcttgatg atacagtgga 241 ggatgccctg gaggacaatg agtttgttga agttgttata gagggagata taatgtctcc 301 agacttcata ccatctcagc cagaaggagt tcatttatat agcaagtatc gagaaccaga 361 acagtatatc tctttagatg gcaacagctt aacaacgcag gacttggtca acttaggaaa 421 agggctctac aagataaagc tcacccctga agctgaagct aaagtcaagc aatcacgaga 481 agtgattgaa aggattgtaa aggaacagac agttgtttat ggaatcacca cggggtttgg 541 gaagtttgcc agaactgtca ttccaaacag caaactgatg gagcttcaaa tgaacttggt 601 tcgttcacat tctgcaggtg tggggaaacc tttaacccca gagaggtccc gcatgctgct 661 ggcactgagg atcaatgtcc tagcaaaggg ctacagtgga atatccctag aaaccctcca 721 gcaagttatt gaagcattta atgcttcctg cctgccttat atccctgaga agggaacggt 781 tggagccagc ggagacttgg cccccctctc tcatcttgcg ttgggattaa caggagaggg 841 aaagatgtgg tccccaaaga gtggctgggc tgacgctaaa tatgtcctcg aagcccatgg 901 tctgaaacca attaccttga aaccaaaaga gggtctggct ctcatcaatg ggacacaaat 961 gatcacctcg ctgggatgcg aagcagttga aagagctagc gccattgcta ggcaagcaga 1021 catagtcgct gcccttacac ttgaagtcct gaagggtaca actaaggcct ttgatactga 1081 tatccatgct gtacgtccac atcgagggca ggctgaagtg gcatttcgat tcaggtccct 1141 tctggattct gaccatcacc catcagaaat agcagagagc caccgatttt gtgaccgagt 1201 tcaggacgca tatacgatgc gctgctgccc tcaggtccac ggagtagtaa atgatacaat 1261 cgcttttgtg aaggacatca tgacaactga aatcaacagc gccacagaca acccaatggt 1321 gtttgctgaa agagcagaga ccatttctgg aggaaatttt cacggtgaat accctgcaaa 1381 ggctttggac tacttggcaa tcggtgtgca cgaactcgct gcaattagtg aaagaagaat 1441 tgagaggctc tgcaaccctt ccctcagcga actgccagca tttttagtca ccgaaggagg 1501 tctgaactct ggcttcatga tagcacactg cacagcagct gccctggttt cggagaacaa 1561 agccttgtgc cacccctcct ctgtggattc tctgtccacc agcgctgcta cggaggacca 1621 cgtgtccatg ggaggatggt ctgcaagaaa agcactgaga gtcattgagc atgtggaaca 1681 agttctggcc atagagctgc tcgctgcctg ccagggcatt gaattcctac gccccctgag 1741 gacgaccacc ccattggaga aggtctacga cctcgtgcgc tcagtggtga ggccttggat 1801 gaaggaccgc ttcatggccc cggatattga agctgctcac aggttgctgg tggagcagaa 1861 ggtgtgggaa gtagccgaac cttacattga aaaatacagg agagaacaca tccctgaatc 1921 cagacctggt tcaccaacag ccttctccct gggatcgctg gaaaggaaaa cacacgatgg 1981 ccacaaccac aggcatcaca atgaactgta acaattgctc tcctcagcac caggcacatc 2041 ttatgtgaag catacagatt cttgtataac aaaagcgttt tggagacagg tgcttcataa 2101 atcaaaagag ctctgtgcgT AAgaacacgc tgtggtgatt actcctccag aacaaaaaca 2161 ctgcattctg atgatggact acatttaatg aagatttcca agacctatgg gctcacacct 2221 gcaataaatg cagaggctcc aaaaaaaaaa aaaaaaaaaa aa

61

Table 11. Homology of the deduced amino acid sequence of chicken hepatic histidase with the histidase sequences of mouse, rat and human. For the mouse, rat and human sequences, only those amino acids which are different from the chicken sequence are shown.

Chicken 1 MPRYTVHVRGEWLAVPCPHGTNTVGWLGKEAVRRYMKNKPDNGGFTSVEEVKFFVRRCKG Mouse 1 ______QDGKLT_____R______M______DEVQ_L_H____ Rat 1 ______QDGKLS_____R______M______DEVR_L_R____ Human 1 ______QDAQLT_____R______I______DDAH_L_R____

Chicken 61 LGLLDLDDTVEDALEDNEFVEVVIEGDIMSPDFIPSQPEGVHLYSKYREPEQYISLDGNS Mouse 61 _____NE_EL_V______V______FL______K__A___DS Rat 61 _____NE_LL_V______V______FL______K__A___DS Human 61 _____NE_RL_V______A______YL______K__E___DR

Chicken 121 LTTQDLVNLGKGLYKIKLTPEAEAKVKQSREVIERIVKEQTVVYGITTGFGKFARTVIPN Mouse 121 _S_E______R______SI__KK_QQ_____DS_I__R______A Rat 121 _S_E______H______SI__KK_QQ_____DS_I__R______A Human 121 _T_E______R______PT__KR_QK_____DS_I__K______I

Chicken 181 SKLMELQMNLVRSHSAGVGKPLTPERSRMLLALRINVLAKGYSGISLETLQQVIEAFNAS Mouse 181 N__Q______S______S______K____A____ Rat 181 N__Q______S______S______K____V____ Human 181 N__Q______S______S______K____M____

Chicken 241 CLPYIPEKGTVGASGDLAPLSHLALGLTGEGKMWSPKSGWADAKYVLEAHGLKPITLKPK Mouse 241 __S_V______I______IV____ Rat 241 __S_V______I______IV____ Human 241 __P_V______V______VI____

Chicken 301 EGLALINGTQMITSLGCEAVERASAIARQADIVAALTLEVLKGTTKAFDTDIHAVRPHRG Mouse 301 ______L______V_____ Rat 301 ______V______V_____ Human 301 ______V______L_____

Chicken 361 QAEVAFRFRSLLDSDHHPSEIAESHRFCDRVQDAYTMRCCPQVHGVVNDTIAFVKDIMTT Mouse 361 _I______L______D_I__ Rat 361 _I______L______D_I__ Human 361 _I______L______N_I__

Chicken 421 EINSATDNPMVFAERAETISGGNFHGEYPAKALDYLAIGVHELAAISERRIERLCNPSLS Mouse 421 _L______S_G__I______V______Rat 421 _L______S_G__I______V______Human 421 _L______N_G__V______I______

Chicken 481 ELPAFLVTEGGLNSGFMIAHCTAAALVSENKALCHPSSVDSLSTSAATEDHVSMGGWSAR Mouse 481 ______A______S______A__ Rat 481 ______A______S______A__ Human 481 ______A______N______A__

Chicken 541 KALRVIEHVEQVLAIELLAACQGIEFLRPLRTTTPLEKVYDLVRSVVRPWMKDRFMAPDI Mouse 541 _____V______I______Rat 541 _____I______I______Human 541 _____I______I______

Chicken 601 EAAHRLLVEQKVWEVAEPYIEKYRREHIPESRPGSPTAFSLGSLERKTHDGHNHRHHNEL Mouse 601 ______LD______A______M______L______ES_RKNSATIPESDDL Rat 601 ______LD______A______M______L______ES_RKNSATIPESDDL Human 601 ______LE______A______M______L______QF_HKKSTKIPESEDL 62

9.49

7.46

4.40

2.37

1.35

Figure 6. Autoradiogram from a Northern Analysis of histidase with total RNA from chicken liver. A 0.24- to 9.5-kb RNA ladder was run in the line adjacent to the liver sample, and the kilobase sizes of the RNA bands are shown with arrows.

63

6.2 DIETARY EXPERIMENTS

6.2.1 EXPERIMENTS 1-4

Food consumption data for these four experiments have been published previously (Adams and Davis, 2001). In brief, food intake in chicks fed either the low, basal or the high protein diet did not differ from each other at 1.5, 3 and 24 h. The only significant difference in food consumption was between chicks fed the high and the low protein diet at the 6 h experimental time period in Experiment 1 (Adams and Davis,

2001).

In Experiment 1, hepatic histidase mRNA expression was significantly higher in chicks fed the high protein diet, when compared to chicks fed the basal or low protein diet at both the 24 and 6 h experimental time periods (Figure 7). Chicks fed the low protein diet had significantly lower histidase mRNA expression when compared to those fed the basal protein diet at both the 24 and 6 h experimental time periods (Figure 7).

In Experiment 2, histidase mRNA expression was significantly higher in chicks fed the basal and the high protein diet when compared to chicks fed the low protein diet at 3 h (Figure 7). At 1.5 h there were no significant differences in histidase mRNA expression between the three dietary treatments.

The Northern analysis results in Experiment 3 and 4 were the same as in

Experiment 1 and 2 respectively, with the expression of histidase mRNA increasing as dietary protein concentrations increased (Figure 8).

64

Figure 7. The relative density of hepatic histidase mRNA of chicks fed different dietary protein concentrations [Experiments 1 (6 and 24 h) and 2 (1.5 and 3 h)]. Values are means ± SEM, n = 6 replicate pens. Means at a time with different letters differ, P < 0.05. Note that the relative densities of histidase mRNA to one another are specific for each time point and that all statistical comparisons are within a given time period.

65

Figure 8. The relative density of hepatic histidase mRNA of chicks fed different dietary protein concentrations [Experiments 3 (6 and 24 h) and 4 (1.5 and 3 h)]. Values are means ± SEM, n = 6 replicate pens. Means at a time with different letters differ, P < 0.05. Note that the relative densities of histidase mRNA to one another are specific for each time point and that all statistical comparisons are within a given time period. 66

6.2.2 EXPERIMENT 5

Food consumption (mean ± SEM, grams of feed consumed per chick) for 24 h for

the basal, high and the histidine supplemented diets was 38 ± 2, 35 ± 1, 37 ± 2 (H1), 37 ±

4 (H2), and 39 ± 2 (H3), respectively. There were no significant differences in food intake among the birds fed the five different diets.

Histidase mRNA expression was greater in chicks fed the high protein diet than in chicks fed the basal diet or the basal diet supplemented with histidine (Figure 9).

Addition of L-histidine to the basal diet did not increase hepatic histidase mRNA

expression (Figure 9).

6.2.3 EXPERIMENTS 6-7

The relative expression of hepatic malic enzyme and histidase mRNA at 1.5 and 3 h after chicks were injected with either glucagon or saline are shown in Figure 10 and 11, respectively. Malic enzyme and histidase mRNA expression were not significantly

different between the birds injected with either glucagon or saline at 1.5 h. At 3 h post

injection, malic enzyme mRNA expression in the glucagon injected birds tended (p value

= 0.077) to be lower than the saline injected birds (Figure 10). Hepatic histidase mRNA expression was significantly higher in the glucagon injected birds than in the saline injected controls at 3 h (Figure 11).

In Experiment 7, glucagon injected birds had significantly lower expression of malic enzyme mRNA and significantly higher expression of histidase mRNA than saline injected birds (Figures 12 – 13).

67

b

a

a a

a

Figure 9. The relative density of hepatic histidase mRNA of chicks fed the basal diet, high protein diet or the basal diet supplemented with either 0.22 g/100g diet (H1), 0.43 g/100g diet (H2), or 0.86 g/100g diet (H3), L-histidine. Values are means ± SEM, n = 6 replicate pens. Means with different letters differ, P < 0.05.

68

Figure 10. The relative density of hepatic malic enzyme mRNA of chicks at 1.5 or 3 h after brachial vein injection of either glucagon or saline. Values are means ± SEM, n = 6 replicate pens for glucagon or saline injected birds.

69

Figure 11. The relative density of hepatic histidase mRNA of chicks at 1.5 or 3 h after

brachial vein injection of either glucagon or saline. Values are means ± SEM, n = 6 replicate pens. Means at a time with different letters differ, P < 0.05. Note that the relative densities of histidase mRNA to one another are specific for each time point and that all statistical comparisons are within a given time period.

70

c

Figure 12. The relative density of hepatic malic enzyme mRNA of chicks 3 h after brachial vein injection of either glucagon or saline. Values are means ± SEM, n = 4 replicate pens. Means with different letters differ, P < 0.05.

71

c

Figure 13. The relative density of hepatic histidase enzyme mRNA of chicks 3 h after brachial vein injections of glucagon or saline. Values are means ± SEM, n = 4 replicate pens. Means with different letters differ, P < 0.05.

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6.2.4 EXPERIMENT 8

Food consumption (mean ± SEM, grams of feed consumed per chick) for chicks

fed the basal or high protein diets for either 1, 2, 3 or 6 h is shown in Table 12. The birds

fed the basal diet had significantly higher food consumption than the birds fed the high

protein diet at all the time periods.

The concentration of plasma glucagon was significantly higher at 1 and 3 h in

birds fed the high protein diet when compared to birds fed the basal protein diet (Table

12). Expression of malic enzyme mRNA was lower and expression of histidase mRNA

was higher in chicks fed the high protein diet when compared to chicks fed the basal diet after 3 and 6 h of feeding (Figure 14).

6.2.5 EXPERIMENT 9 (NEAA and EAA supplements, 24 h feeding)

Food consumption (mean ± SEM, grams of feed consumed per chick) at 24 h in chicks fed the basal diet, high protein diet or the basal diet supplemented with EAA or

NEAA was 49.5 ± 3.5, 40.3 ± 3.9, 43.7 ± 2.2 and 53.0 ± 1.2 respectively. The only

significant difference in food consumption was for the birds fed the NEAA supplemented

diet which consumed more food than the birds fed the high protein diet.

Due to limitations in the number of RNA samples that could be electrophoresed in one gel, the EAA and the NEAA RNA samples were each electrophoresed with the basal and high protein RNA samples on separate gels. Malic enzyme mRNA expression was significantly lower in birds fed the EAA supplemented diet, when compared to chicks fed the basal diet (Figure 15). Supplementing the basal diet with NEAA did not alter malic enzyme mRNA expression (Figure 15).

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Table 12. Plasma glucagon concentration and food consumption of chicks fed the basal or high protein diet for either 0, 1, 2, 3 or 6 h. Values are mean ± SEM, n = 9 replicate pens for each treatment for times 1 and 2 h, and n = 6 replicate pens for each treatment for times 0, 3 and 6 h. Values with different letters differ, P < 0.05. Note that the plasma glucagon and food consumption values are specific for each time point and that all statistical comparisons are within a given time period

Time Treatment Plasma Glucagon Intake (h) (pg/ml) (g/chick) 0 1599.3 ± 241.8

1 Basal 784.9 ± 97.9a 3.33 ± 0.19a High 1288.5 ± 197.1b 2.10 ± 0.22b

2 Basal 1503.2 ± 82.8a 5.00 ± 0.33a High 1481.7 ± 129.9a 3.22 ± 0.22b

3 Basal 1385.5 ± 153.2a 6.00 ± 0.66a High 2060.9 ± 187.9b 4.00 ± 0.50b

6 Basal 1992.3 ± 186.5a 11.17 ± 0.50a a b High 1846.9 ± 264.2 9.17 ± 0.17

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Figure 14. The relative density of hepatic malic enzyme (panel A.) and hepatic histidase (panel B.) mRNA of chicks at 3 h or 6 h after consumption of either the basal or high protein diet. Values are means ± SEM, n = 6 replicate pens. Means at a time with different letters differ, P < 0.05.

75

Figure 15. The relative density of hepatic malic enzyme mRNA of chicks fed for 24 h either the basal diet, high protein diet, or the basal diet supplemented with NEAA or EAA. Values are means ± SEM, n = 6 replicate pens. Means with different letters differ, P < 0.05. Note that the relative densities of malic enzyme mRNA to one another are specific for each dietary block and that all statistical comparisons are within a given dietary block.

76

In chicks fed the basal diet supplemented with EAA or NEAA, expression of

histidase mRNA was not significantly different from the expression found in chicks fed

the basal or high protein diets (Figure 16).

6.2.6 EXPERIMENT 10 (NEAA and EAA supplements, 6 h feeding)

Food consumption (mean ± SEM, grams of feed consumed per chick) for chicks fed the basal, high, EAA supplemented and the NEAA supplemented diets was 14.0 ±

1.5, 10.7 ± 0.3, 11.5 ± 0.7 and 10.4 ± 0.4, respectively. Chicks fed the basal diet had significantly higher food intake than the birds fed the high and NEAA supplemented diets.

At 6 h, malic enzyme mRNA expression in chicks fed the EAA and the NEAA supplemented diets was significantly lower than in chicks fed the basal diet, but not as low as in birds fed the high protein diet (Figure 17). Feeding the high protein diet, the

EAA supplemented diet, or the NEAA supplemented diet significantly increased hepatic histidase mRNA expression compared to feeding the basal protein diet (Figure 18).

6.2.7 EXPERIMENT 11 (Nitrogen supplements)

Food consumption (mean ± SEM, g/chick) for chicks fed for 24 h the basal protein diet, high protein diet or the basal diet supplemented with either glutamic acid, glycine, alanine, glutamine or DAC is shown in Table 13. Chicks fed the glycine and the

DAC supplemented diets had significantly lower food consumption than birds fed the other treatment diets (Table 13).

As previously mentioned, all of the samples could not be electrophoresed together on one gel. Thus, RNA samples from birds fed the basal supplemented diets were each

electrophoresed in separate gels along with the RNA samples obtained from birds fed the 77

Figure 16. The relative density of hepatic histidase mRNA of chicks fed for 24 h either the basal diet, high protein diet, or the basal diet supplemented with NEAA or EAA. Values are means ± SEM, n = 6 replicate pens. Means with different letters differ, P < 0.05. Note that the relative densities of histidase mRNA to one another are specific for each dietary block and that all statistical comparisons are within a given dietary block.

78

Figure 17. The relative density of hepatic malic enzyme mRNA of chicks fed for 6 h either the basal diet, high protein diet, or the basal diet supplemented with NEAA or EAA. Values are means ± SEM, n = 6 replicate pens. Means with different letters differ, P < 0.05. Note that the relative densities of malic enzyme mRNA to one another are specific for each dietary block and that all statistical comparisons are within a given dietary block.

79

Figure 18. The relative density of hepatic histidase mRNA of chicks fed for 6 h either the basal diet, high protein diet, or the basal diet supplemented with NEAA or EAA. Values are means ± SEM, n = 6 replicate pens. Means with different letters differ, P < 0.05. Note that the relative densities of histidase mRNA to one another are specific for each dietary block and that all statistical comparisons are within a given dietary block.

80

Table 13. Food consumption (g/chick) for 24 h in chicks fed the basal, high protein or the basal diet supplemented with either L-glutamic acid, glycine, L-alanine or diammonium citrate (DAC). Values are means ± SEM, n = 6 replicate pens for each treatment. Values with different letters differ, P < 0.05

Treatments Food Consumption (g / chick)

Basal 52.00 ± 1.80 a

High 49.17 ± 2.46 a

Basal + L-glutamic Acid 50.33 ± 3.56 a

Basal + glycine 32.33 ± 2.13 c

Basal + L-alanine 48.50 ± 3.21 a

b Basal + DAC 41.83 ± 1.01

81

basal and the high protein diets. Feeding any of the basal supplemented diets significantly lowered malic enzyme mRNA expression when compared to feeding the basal protein diet (Figure 19). Birds fed the high protein diets had significantly lower malic enzyme mRNA expression then birds fed the basal or the basal diet supplemented with glutamic acid, alanine, glutamine or DAC (Figure 19).

Feeding the basal diet supplemented with either glutamic acid, alanine, glutamine or DAC did not alter histidase mRNA expression (Figure 20). Feeding a glycine supplemented basal diet, however did increase histidase mRNA expression (Figure 20).

6.2.8 EXPERIMENT 12 (Non Protein Nitrogen supplements)

After 24 h of feeding, food consumption (mean ± SEM, g/chick) in birds fed the basal diet, high protein diet, or the basal diet supplemented with either ammonium phosphate, ammonium bicarbonate or a mixture of both ammonium phosphate and ammonium bicarbonate was 58.5 ± 3.61, 38.5 ± 2.89, 23.0 ± 3.05, 50.1 ± 3.31, 38.7 ±

1.59, respectively. Birds fed ammonium phosphate supplemented diets had significantly lower food consumption than birds fed the other diets. In addition to this, birds fed the

basal and ammonium bicarbonate supplemented diets had significantly higher food intake

than the birds fed the high protein diet and the mixture (combination of both ammonium

phosphate and ammonium bicarbonate) diet. There was no significant difference in food consumption in birds fed the basal and the ammonium bicarbonate supplemented basal

diet.

82

Figure 19. The relative density of malic enzyme mRNA of chicks fed for 24 h either

the basal diet, high protein diet or the basal diet supplemented with L- glutamic acid, glycine, L-alanine or diammonium citrate (DAC). Values are means ± SEM, n = 6 replicate pens. Means with different letters differ, P < 0.05. Note that the relative densities of malic enzyme mRNA to one another are specific for each dietary block and that all statistical comparisons are within a given dietary block

83

Figure 20. The relative density of histidase mRNA of chicks fed for 24 h either the basal diet, high protein diet or the basal diet supplemented with L- glutamic acid, glycine, L-alanine or diammonium citrate (DAC). Values are means ± SEM, n = 6 replicate pens. Means with different letters differ, P < 0.05. Note that the relative densities of histidase mRNA to one another are specific for each dietary block and that all statistical comparisons are within a given dietary block.

84

Malic enzyme mRNA expression was significantly lower in birds fed the non protein nitrogen supplemented diets and high protein diets when compared to birds fed the basal protein diet (Figure 21).

6.2.9 EXPERIMENT 13 (Grow-out experiment)

6.2.9.1 Body weight gain (BWG), Food consumption, Feed Conversion Ratio (FCR)

and carcass yield

During the starter period (0 – 21d), birds fed the 4.7 and 9.5% glutamic acid supplemented diets had significantly lower BWG and food consumption than the birds fed the basal, control or the 2.3% glutamic acid supplemented diets (Table 14).

Compared to all the other dietary treatments, food consumption was significantly lower in the birds fed the diet containing the highest level of glutamic acid supplement (Table

14). However, there were no significant differences in the FCR among the treatment diets (Table 14).

In the finisher period (21 – 40 d), BWG and food consumption in birds fed the diet supplemented with 2.3% glutamic acid was lower than in the birds fed the basal corn/soy diet (Table 14). The FCR value in birds fed the control diet was significantly lower than any of the values obtained for birds fed any of the other diets (Table 14).

For the entire experimental period (0 – 40 d), birds fed the basal corn/soy diet gained more weight than the birds fed the diet supplemented with any level of glutamic acid (Table 14). Birds fed the control and the 9.5% glutamic acid supplemented diets had the lowest FCR (Table 14). The percent carcass yield (mean ± SEM) for the basal, basal diet supplemented with either 2.3, 4.7 or 9.5% glutamic acid and the control diets was

85

Figure 21. The relative density of malic enzyme mRNA of chicks fed for 24 h either the basal diet, high protein diet or the basal diet supplemented with either ammonium bicarbonate, ammonium phosphate or both. Values are means ± SEM, n = 6 replicate pens. Means with different letters differ, P < 0.05. Note that the relative densities of malic enzyme mRNA to one another are specific or each dietary block and that all statistical comparisons are within a given dietary block. 86

Table 14. Body weight gain (BWG), feed intake and feed conversion ratio (FCR), in broilers fed the basal, the basal diet supplemented with either 2.3, 4.7 and 9.5% glutamic acid and control1 diet during the grower (0 – 21d), finisher (21 – 40 d), and the entire grow-out period (overall, 0 – 40 d). Values are means ± SEM, n = 5 replicate pens for the basal, 2.3, 4.7 and 9.5 % glutamic acid supplemented diet, and n = 4 replicate pens for the control diet. Means within a column with different letters differ, P < 0.05.

Starter (0 – 21 d) Finisher (21 – 40 d) Overall (0 – 40 d)

Glutamic BWG Intake FCR BWG Intake FCR BWG Intake FCR acid (%) (g) (g/bd) (feed : gain) (g) (g/bd) (feed : gain) (g) (g/bd) (feed : gain)

Basal 639 ± 07 a 957 ± 18 a 1.50 ± 0.04 a 1657 ± 38 a 2563 ± 46 a 1.55 ± 0.01 a 2296 ± 36 a 3674 ± 54 a 1.60 ± 0.03 a

2.3 615 ± 19 a 927 ± 31 a 1.50 ± 0.01 a 1540 ± 30 b 2382 ± 07 b 1.55 ± 0.03 a 2157 ± 13 c 3464 ± 95 a 1.61 ± 0.05 a

1.56 ± 0.03 4.7 566 ± 08 b 861 ± 12 b 1.52 ± 0.03 a 1633 ± 34 ab 2457 ± 35 ab 1.51 ± 0.02 ab 2200 ± 29 bc 3430 ± 63 a ab 1.48 ± 0.04 9.5 531 ± 11 b 801 ± 17 c 1.51 ± 0.02 a 1614 ± 19 ab 2328 ± 60 bc 1.44 ± 0.03 b 2145 ± 28 c 3167 ± 110 b bc

Control 630 ± 10 a 923 ± 14 a 1.47 ± 0.02 a 1648 ± 39 a 2220 ± 56 c 1.35 ± 0.01 c 2278 ± 37 ab 3159 ± 67 b 1.39 ± 0.01 c

1 The control diet was similar to the 9.5% glutamic supplemented diet, except that the glutamic acid was replaced by glucose monohydrate and sand 87

70.5 ± 0.4, 70.9 ± 0.5, 70.9 ± 0.2, 69.8 ± 0.3 and 71.1 ± 0.3, respectively. The only

significant difference in carcass yield was seen between birds fed the control versus that

of the 9.5% glutamic acid supplemented diet.

6.2.9.2 Malic Enzyme mRNA expression and Abdominal Fat Pad

Malic enzyme mRNA expression was not significantly different among the treatment groups at both 18 and 37 d (Figure 22). Birds fed the 2.3, 4.7 and 9.5 % glutamic acid supplemented diets had significantly lower abdominal fat pad weights than birds fed the corn/soy basal diet or the control diet (Figure 23).

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Figure 22. The relative density of hepatic malic enzyme mRNA of chicks fed the basal, control, and the basal diet supplemented with either 2.3, 4.7 or 9.5% glutamic acid diets at 18 d (panel A) and 37 d (panel B.). Values are means ± SEM, n = 6 replicates per treatment.

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Figure 23. Abdominal fat pad ( g/ 100 g live body weight) in chicks fed the basal, control, and the basal diet supplemented with either 2.3, 4.7 or 9.5% glutamic acid diets. Values are means ± SEM, n = 5 replicate pens. Means with different letters differ, P < 0.05. CHAPTER 7

DISCUSSION

7.1 CLONING CHICKEN HEPATIC HISTIDASE

A 442-bp RT-PCR product was obtained when the annealing temperature of the

PCR reaction was reduced from 55 oC to 45oC, which indicated that the primers did not

have a 100% homology with the cDNA sequence of chicken histidase. Sequence analysis

of the 442 bp product, revealed a high degree of nucleotide (80%) and protein (95%)

homology with the histidase sequences of rat, mouse and human. This high degree of

homology with the histidase sequences of other species was a clear indication that the obtained PCR product was part of chicken hepatic histidase.

Using the determined nucleotide sequence of the RT-PCR product, we were able to construct gene specific primers for the RACE-PCR technique which was utilized in an attempt to obtain the full length cDNA sequence of chicken hepatic histidase. The RACE technique has been used previously to clone cDNAs obtained from various tissues in chickens (Kohchi and Tsutsui, 2000). In addition, the RACE technique is a relatively quick and inexpensive method for cloning genes of interest. In the present research, however, utilization of the RACE-PCR technique was not successful. Therefore, a premade cDNA library of chicken liver was purchased and screened with radio-labelled preparations of the 442 bp RT-PCR product.

90 91

Screening of the chicken liver cDNA library resulted in obtaining the full length cDNA sequence of chicken hepatic histidase. The nucleotide and predicted amino acid sequences of chicken hepatic histidase were 80 and 89 percent identical with the respective nucleotide and amino acid sequences of rat, mouse and human histidase.

Interestingly, the nucleotide sequence of chicken histidase has nine additional nucleotides at the 3’ end compared to the nucleotide sequences of rat, mouse and human histidase.

The reason for the additional nucleotides which code for 3 extra amino acids is unclear, however, even the last 16 amino acids of the C-terminal end of the human histidase protein have only about 55% identity with the corresponding rat and mouse sequences.

This percent identity is less than the >90% identity seen for the remainder of the human amino acid sequence with the sequences from rat and mouse.

By Northern analysis, a major histidase mRNA transcript of approximately 2.9 kb was detected. A minor transcript was detected just below the major histidase transcript.

Minor transcripts have also been detected in the rat. A major transcript of 2.5 kb and minor transcripts of 4.4 and 7.3 kb have been reported from Northern analysis of rat liver

(Taylor et al., 1990).

7.2 DIETARY EXPERIMENTS

Keene and Austic (2001) reported that when chickens are fed increasing levels of dietary protein, hepatic histidase activity also increases. The present research indicates that the effect of dietary protein on hepatic histidase is mediated by changes in histidase mRNA levels. This finding agrees with the report from Torres et al. (1998) that ingestion of a high protein diet by rats increased hepatic histidase activity, and was associated with 92

an increase in the mRNA levels of histidase. This alteration in the mRNA concentration

takes place as early as 3 h after a change is made in the dietary protein intake of chicks.

Such a rapid change in the mRNA levels of a metabolic enzyme due to changes in dietary

protein concentrations has been reported previously. The mRNA expression of malic

enzyme decreases within 3 h of feeding chicks a high protein diet (Adams and Davis,

2001).

Increasing dietary histidine levels in a histidine adequate chick diet has been reported to increase hepatic histidase activity (Keene and Austic, 2001). The present

research indicates that supplementing a histidine adequate diet with more histidine does

not increase histidase mRNA expression. Even when the basal diet was supplemented

with 1.5 times more histidine than was found in the high protein diet, histidase mRNA

expression was not altered. This suggests that high levels of dietary histidine do not regulate histidase activity at the pretranslational level. It could mean that the dietary

histidine regulation of histidase activity in the liver noted by Keene and Austic (2001)

was due to an allosteric mechanism or a decrease in the rate of histidase protein

degradation.

The mechanism by which dietary protein intake rapidly modifies the mRNA levels of malic enzyme and histidase are not understood. Clearly, in the case of histidase,

dietary concentrations of its substrate do not seem to play a role. One metabolic mediator

that may participate in the effect of dietary protein intake on the activity of these two enzymes is glucagon.

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7.3 GLUCAGON

An increase in the concentration of dietary protein or amino acids in mammalian

species stimulates the secretion of glucagon by the pancreas (Ohneda et.al, 1968; Munro,

1970; Hoffer, 1994 and Charlton et al., 1996). In red tailed hawks, an oral infusion of amino acids caused a significant increase in the plasma glucagon concentration ten minutes after the infusion (Minick et al., 1996). In vivo studies by Goodridge and

Adelman (1976) using chick liver cells indicate that glucagon inhibits malic enzyme activity. Regulation of malic enzyme activity by glucagon takes place at the pretranslational level (Lefevre, et al., 1999). The 5’ region of the chicken malic enzyme gene contains response elements for cAMP which mediate the effect of glucagon on malic enzyme mRNA synthesis (Mounier et al., 1997). Torres et.al (1998) reported that glucagon regulates histidase activity in rat liver, and that this regulation takes place at the mRNA level. A report on the human hepatic histidase gene also indicates that the promoter sequence of the 5’ region has several cAMP responsive elements (Suchi et al.

1995), which would be necessary for the cAMP mediated action of glucagon. The results of the present research indicate that intravenous injection of glucagon alters the mRNA expression of hepatic malic enzyme and histidase within 3 h in broiler chicks. This alteration in the mRNA expression of both enzymes by glucagon was similar to what had been observed previously when chicks were fed increasing levels of dietary protein.

Comparing the plasma glucagon concentrations of chicks fed the basal protein

diet or the high protein diet failed to definitively indicate that glucagon is responsible for

altering the activities of malic enzyme and histidase when chicks consume increasing

levels of dietary protein. After 1 h of feeding the two diets, birds fed the high protein diet 94

had a significantly higher plasma glucagon level than those fed the basal diet. A similar

difference was observed at 3 h, but not at 2 and 6 h. The lack of consistency in the results

makes it difficult to assume a definite relationship between protein intake and plasma

glucagon levels.

The birds were fasted 2 h before access was given to the basal and high protein

diets to ensure immediate consumption of the diets. This fast would elevate plasma glucagon levels, and thus explain why plasma glucagon levels were higher at the

initiation of the experiment than after feeding for 1 h. The fast followed by the

immediate consumption of food also may make the 1 h plasma glucagon values more

reflective of the potential affect of dietary protein.

Glucagon secretion from the pancreas has been shown to be affected by numerous

factors, including stress (Freeman and Manning, 1976), circadian rhythm (Apfelbaum et

al., 1972), and food consumption (Sonoda, 1983). Since food consumption decreases

plasma glucagon levels, it would have been ideal if the birds fed the basal and the high

protein diet had consumed the same amount of food. But, birds fed the high protein diet

consumed less food than birds fed the basal diet at all time periods. Hence, it is likely

that this difference in food consumption played a significant role in the plasma glucagon

concentrations obtained between the two diets.

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7.4 SPECIFIC AMINO ACIDS

Specific amino acids affect the transcription of specific genes. It has been shown that cells recognize alterations in amino acid pools and respond by either upregulating or

downregulating the initiation of protein translation (Bastians and Ponstingl, 1996).

Straus and Takemoto (1988) reported that the limitation of the essential amino acids;

leucine, methionine, phenylalanine or tryptophan caused a decrease in the abundance of

albumin mRNA in rat heptoma cells. Several other reports have implicated specific

amino acids in regulating the transcription of genes (Varga et al., 1994; Quillard et al.,

1996; Kimball et.al, 1999). There exist a number of mechanisms by which the amino

acids are able to play a role in transcriptional regulation, including regulating the

eukaryotic initiation factors (eIF) essential for transcription (Vary et al. 1999).

The results obtained from supplementing the basal diet with mixtures of NEAA or

EAA indicate that if malic enzyme and histidase mRNA expression are regulated by

specific amino acids, then it would have to be a mixture of specific essential and

nonessential amino acids since the NEAA and the EAA mixtures failed to elicit a

response equivalent to feeding the high protein diet. Of course, another interpretation of

these results could be that the regulation of the mRNA expression of these two enzymes

may be due to the total nitrogen content of the diet. The total nitrogen content of the

NEAA and the EAA diets was intermediate to the levels found in the basal and high

protein diets (Table 15). Therefore, it may not be surprising that the level of malic

enzyme and histidase mRNA expression was also intermediate, for chicks fed the basal

96

Table 15. The calculated amount of total nitrogen present in the experimental diets in the EAA and the NEAA experiments.

Type of Diet Nitrogen (g/100g)

Basal (22% Protein) 2.88

EAA (22% + EAA equal to high) 4.45

NEAA(22% + NEAA equal to high) 3.91

High (40% Protein) 5.23

97

diet supplemented with either NEAA or EAA compared to those fed the basal or high

protein diets.

Experiment 11 was unique in that it was designed to determine if individual amino acids as well as dietary nitrogen from a NPN source had an effect on the levels of mRNA for malic enzyme and histidase. The decision to use individual NEAA was made because dietary excesses of NEAA tend to have reduced effect on food intake when compared to feeding a diet containing high levels of an individual EAA. L-glutamic acid,

L-alanine and glycine were the amino acid supplements chosen, and DAC was used as the NPN supplement. Maruyama et al. (1975) supplemented chick diets with up to 10% of L-glutamic acid, and did not observe any decline in food consumption. Addition of

3% L-alanine (Maruyama et al., 1975) or 1.6% glycine (Lee et al., 1972) to a semi- purified basal diet also did not depress food consumption by chicks. Davis and Austic

(1997) did report a decrease in food consumption in chicks over a 9 d period when 4% glycine was added to the diet. However, since the current experiment was to be conducted for only 24 h period, no difference in food consumption was expected.

DAC was used as the NPN source, as previous research by Allen and Baker

(1974) had shown that chicks utilize DAC much better than urea or diammonium

phosphate as nitrogen sources. In addition, DAC levels as high as 11% had been used in

chick diets without any detrimental effect on food consumption (Lee et al., 1972).

98

Interestingly, the results from Experiment 11 indicate that both dietary nitrogen and specific amino acids are involved in the regulation of malic enzyme and histidase mRNA expression. Malic enzyme mRNA expression was reduced by each one of the nitrogen sources suggesting that dietary nitrogen content rather than specific dietary amino acids is an important regulator of malic enzyme mRNA expression. It must be noted that food consumption was lower in chicks fed the basal diet supplemented with

either glycine, DAC and in a subsequent experiment, ammonium phosphate. Goldman et

al. (1985) found that when well fed ducklings were starved, the level of malic enzyme

mRNA rapidly decreased. The maximum inhibition of the transcription of malic enzyme

mRNA by starvation was estimated to be 3 h (Goldman et al., 1985). Even though the

situation for the birds fed the basal diet supplemented with glycine, DAC or ammonium

phosphate is not equivalent to fasting, the reduced food consumption could have

contributed to the reduced levels of malic enzyme mRNA expression in chicks fed these

diets. But more importantly, chicks fed the basal diet supplemented with glutamic acid,

alanine or ammonium bicarbonate did not have reduced food intake and still had

significantly lower malic enzyme mRNA expression.

In contrast to the results for malic enzyme, not all of the supplements to the basal

diet altered histidase mRNA expression. Only the glycine supplement increased histidase

mRNA expression which indicates that histidase mRNA expression is probably regulated

by concentrations of specific amino acids in the diet. The regulation of histidase and

malic enzyme by two different mechanisms potentially negates the role of glucagon as

the sole mediator for the effect of dietary protein on malic enzyme and histidase mRNA

expression. 99

Although it appears that malic enzyme mRNA levels are regulated by dietary

nitrogen, it should be noted that the nitrogen from the NPN sources eventually are transferred to other amino acids. Studies in neonatal pigs have shown that 15N from

dietary [15N] DAC was eventually found in each of the essential and non-essential amino

acid body pools of both germ-free and specific pathogen-free pigs (Deguchi et al., 1980).

In addition, the highest concentration of 15N concentrations was found in glutamic acid

(Deguchi et al., 1980). Similarly, Lee et al. (1972) reported that a substantial amount of

the nitrogen from DAC was also incorporated into glutamic acid in germ-free chicks.

Therefore, the effect of DAC on the mRNA expression of the malic enzyme may have

been due to its preferential conversion to glutamic acid or another amino acid and not a

direct effect of the nitrogen from DAC.

Considering the potential importance of using NPN sources in commercial poultry

diets to reduce fat synthesis, further studies were conducted with other NPN sources.

When added to the basal diet, both ammonium phosphate and ammonium bicarbonate

decreased malic enzyme mRNA expression. The severe reduction in food consumption

in birds fed the ammonium phosphate supplemented diet may have been due to a

calcium/phosphorus imbalance. When ammonium phosphate was added to the basal diet,

the calcium content of the diet was not adjusted. Holcombe et al. (1976) reported that

laying hens exhibit an aversion to a 2.43% P diet when compared to a 1.00% P diet, where both diets contained 3% Ca. However, when the calcium level of the 2.43% P diet was elevated to 6% percent, the hens increased the consumption of this diet (Holcombe et al., 1976). Since our experiment was to last for only 24 h, we assumed incorrectly that 100

the poor Ca : P ratio would not affect food consumption, however, based on our results the birds sensed the imbalance in the Ca : P ratio within 6 h (data not shown).

Although the ammonium bicarbonate supplement did not decrease food

consumption, it still will not be a practical dietary ingredient. After it is added to the diet,

it becomes unstable, and this results in the release of ammonia from the diet over time.

Furthur research will be needed to find a NPN source that can be incorporated into the

diet that does not have any negative aspects.

7.5 PRACTICAL IMPLICATIONS

The problem of excess body fat continues to be a financial liability for the poultry

industry. Continuous genetic selection for improved performance characteristics has

inadvertently lead to strains of broilers which tend to accumulate excessive body fat, especially in the abdominal area. As an ever increasing proportion of poultry products

are sold as “cut up” and deboned products, it is no longer possible to include these fat

pads with the whole carcass. Thus they may enter the rendering chain at a drastic

decrease in value.

The current molecular nutrition research may help in formulating diets that reduce

abdominal fat pads, and hence be of enormous financial benefit to the broiler sector. It is

well known that feeding a high protein diet to a commercial broiler reduces abdominal fat

pad weight (Cahaner et al., 1995; Deschepper and De Groote, 1995). However, the

current research indicates that there may be no need to increase dietary protein intake per

se, but just increase the nitrogen content in the diet. An increase in the nitrogen content

in the diet will reduce malic enzyme mRNA expression, which will subsequently reduce 101

malic enzyme activity and the amount of fat synthesized by the liver to be transported to

other parts of the body.

L-glutamic acid supplementation of a practical corn/soy broiler diet significantly

reduced abdominal fat pad weights at slaughter. The difference in abdominal fat pad

weight between a bird fed the basal diet supplemented with 2.3% glutamic acid and one

fed the basal diet was about 7 g. When calculated for a typical large scale poultry

processing facility processing over 1 million birds per week, the reduction in the amount of fat entering the rendering chain would be enormous.

Future research will be focused on finding a relatively inexpensive dietary NPN source

that can reduce malic enzyme activity as glutamic acid did in the present research. The

NPN sources studied in the current research have practical problems, from reduced food

consumption to instability in poultry diets. Future research can also focus on the feeding

duration of the supplements, as it might not be necessary to feed these diets for the entire

broiler production cycle. Cabel et.al (1988) supplemented a practical corn-soy diet with

either poultry feather meal as a NPN source or the amino acid glycine during the

finishing stages (35 – 49 d or 42 – 49 d), and obtained a decrease in abdominal fat pads.

The use of an extra nitrogen source in the finisher or grower diets only, might also help

alleviate the problems of reduced body weight gain observed in birds fed high levels of

glutamic acid during the starter diets.

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7.6 SUMMARY

By obtaining a cDNA clone for chicken hepatic histidase, we were able to

determine that feeding broilers increased levels of protein rapidly increased the mRNA expression of hepatic histidase. This finding allowed for the completion of our research model. This model will facilitate our ability to study how protein and amino acid intake is detected by the bird. Interestingly, our initial findings with the model already indicate that the mechanisms involved in the dietary protein regulation of malic enzyme and histidase mRNA expression differ. It appears that specific dietary amino acids other than histidine regulate the mRNA expression of histidase, while the mRNA expression of malic enzyme is regulated by dietary nitrogen content. Finally, it appears that our increased understanding of how malic enzyme activity is regulated by dietary protein intake could have significant practical implications in producing a leaner broiler for market.

CHAPTER 8

REFERENCES

Adams K. A. and Davis A. J. (2001) Dietary protein concentration regulates the mRNA expression of chicken hepatic malic enzyme. J. Nutr. Sep; 131(9): 2269 – 2274.

Aleman G., Torres N., Bourges H., Tovar A. R. (1998) Regulation of histidase gene expression by glucagon, hydrocortisone and protein-free/high carbohydrate diet in the rat. Life Sci. 63(18): 1663 – 1672.

Allen N. K., Baker D. H. (1974) Quantitative evaluation of nonspecific nitrogen sources for the growing chick. Poult Sci. Jan; 53(1): 258 – 264.

Apfelbaum M., Reinberg A., Assan R., Lacatis D. (1972) Hormonal and metabolic circadian rhythms before and during a low protein diet. Isr. J. Med. Sci. Jun; 8(6): 867 – 873.

Ashida, K., and Harper, A. E. (1961) Metabolic adaptations in higher animals. IV. Liver arginase activity during adaptation to high protein Proc. Soc. Exp. Med. 107: 151 – 156.

Auerbach V. H., DiGeorge, A. M., Baldridge, R. C., Tourtellotte, C. D., and Brigham, M. P. (1962) Histidinemia. A deficiency in histidase resulting in the excretion of histidine and imidazolepyruvic acid. J. Pedia. 60: 487 – 497.

Ayala A, Gordillo E, Castano A, Lobato MF, Machado A. (1991) Involvement of diminution of glutathione, produced by deficiency of methionine in the diet, in the elevation of malic enzyme level in rat liver. Biochim Biophys Acta. Jun 19; 1084(1): 48 – 52.

Back, D. W., Goldman, M. J., Fisch, J. E., Ochs, R. S., Goodridge, A. G. (1986). The fatty acid synthase gene in avian liver two mRNA are expressed and regulated in parallel by feeding, primarily at the level of transcription. J. Biol. Chem. 261: 4190 – 4197.

Baden H. P., Sviokla S., Maderson P. F. (1969) A comparative study of histidase activity in amphibian, avian, reptilian and mammalian epidermis. Comp Biochem Physiol. Sep 1; 30(5): 889 – 894.

103 104

Baden H. P., and Pathak M. A. (1967) The metabolism and function of urocanic acid in skin. J. Invest Dermatol. Jan; 48(1): 11 –17.

Ball, E. G. and Merrill, M. A. (1961) A monometric assay of insulin and some results of the application of the method to sera and islet containing tissues. Endocrinol. 69: 596 – 607.

Ballard F. J., Hanson R. W., Kronfeld D. S. (1969) Gluconeogenesis and lipogenesis in tissue from ruminant and nonruminant animals. Fed. Proc. Jan-Feb; 28(1): 218 – 231.

Bastians H., Ponstingl H. (1996) The novel human protein serine/threonine phosphatase 6 is a functional homologue of budding yeast Sit4p and fission yeast ppe1, which are involved in cell cycle regulation. J. Cell. Sci. Dec;109 ( Pt 12): 2865 – 2874.

Benevenga N. J., Steele R. D. (1984) Adverse effects of excessive consumption of amino acids. Annu Rev Nutr.4: 157 – 181.

Boldyrev A. A., Dupin A. M., Pindel E. V., Severin S. E. (1988) Antioxidative properties of histidine-containing dipeptides from skeletal muscles of vertebrates. Comp Biochem Physiol B. 89(2): 245 – 250.

Bulfield G., Kacser, H. (1974) Histidinemia in mouse and man. Arch. Dis. Child. 49. 545 – 552.

Busch M. R., Ho C. E. (1990) Effects of anions on the molecular basis of the Bohr effect of hemoglobin. Biophys Chem. Aug 31; 37(1-3): 313 – 322.

Cabel M. C., Goodwin T. L., Waldroup P. W. (1988) Feather meal as a nonspecific nitrogen source for abdominal fat reduction in broilers during the finishing period. Poult Sci. Feb; 67(2): 300 – 306.

Cahaner A., Pinchasov Y., Nir I., Nitsan Z. (1995) Effects of dietary protein under high ambient temperature on body weight, breast meat yield, and abdominal fat deposition of broiler stocks differing in growth rate and fatness. Poult. Sci. Jun; 74(6): 968 – 975.

Charlton M. R, Adey D. B, Nair K. S. (1996) Evidence for a catabolic role of glucagon during an amino acid load. J. Clin. Invest. Jul 1; 98(1): 90 – 99.

Chen, C.C., Johnson, P. (1996) Expression of inhibin α and inhibin/activin βA subunits in the granulose layer of the large preovulatory follicles of the hen. Biol. Reprod. 55: 450 – 454.

105

Chiu Y. U., Austic R. E., Rumsey G. L. (1984) Effect of dietary electrolytes and histidine on histidine metabolism and acid-base balance in rainbow trout (Salmo gairdneri). Comp. Biochem. Physiol. 78(b), no.4, pp. 777 – 784.

Chomczynski P., Sacchi N. (1987) Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem. Apr; 162(1): 156 – 159.

Clarke S. D., Watkins P. A., Lane M. D. (1979) Acute control of fatty acid synthesis by cyclic AMP in the chick liver cell: possible site of inhibition of citrate formation. J. Lipid Res. Nov; 20(8): 974 – 85.

Cramb G., Langslow D. R., Phillips J. H.(1982) Hormonal effects on cyclic nucleotides and carbohydrate and lipid metabolism in isolated chicken hepatocytes. Gen. Comp. Endocrinol. Mar; 46(3): 310 – 321.

Davis A. T., Austic R.E. (1982) Threonine metabolism of chicks fed threonine- imbalanced diets. J. Nutr. Nov; 112(11): 2177 – 2186.

Davis A. J., Johnson P. A. (1998) Expression pattern of messenger ribonucleic acid for follistatin and the inhibin/activin subunits during follicular and testicular development in gallus domesticus. Biol. Reprod. Aug; 59(2): 271 – 277.

Davis A. J., Austic R. E. (1997) Dietary protein and amino acid levels alter threonine dehydrogenase activity in hepatic mitochondria of Gallus domesticus. J. Nutr. May; 127(5): 738 – 44.

Davis A. J., Austic R. E. (1994) Dietary threonine imbalance alters threonine dehydrogenase activity in isolated hepatic mitochondria of chicks and rats. J. Nutr. Sep; 124(9): 1667 – 77.

Deguchi E., Niiyama M., Kagota K., Namioka S. (1980) Incorporation of nitrogen-15 from dietary [15N] diammonium citrate into amino acids of liver and muscle proteins in germfree and specific-pathogen-free neonatal pigs. Am. J. Vet. Res. Feb; 41(2): 212 – 214.

Deschepper K., De Groote G. (1995) Effect of dietary protein, essential and non-essential amino acids on the performance and carcase composition of male broiler chickens. Br. Poult. Sci. May; 36(2): 229 – 245.

Dhanam M., and Radhakrishnan A. N. (1976) Comparative studies on hstidase: distribution in tissues, properties of liver histidase and its development in rat. Indian J. Exp. Biol. Mar; 14(2): 103 – 106.

106

Dhanam M., and Radhakrishnan A. N. (1974) Purification and properties of histidine ammonia-lyase from monkey liver. Indian J. Biochem. Biophys. Mar; 11(1): 1 – 6.

Dixon J. L and Harper A. E. (1984) Effects on plasma amino acid concentrations and hepatic branched-chain alpha-keto acid dehydrogenase activity of feeding rats diets containing 9 or 50% casein. J. Nutr. Jun; 114(6): 1025 – 1034.

Edsall, J. T. and Wyman, J. (1958) Biophysical chemistry, Vol. 1, pp. 550 – 590, Academic Press, New York.

Everett, M. A., Anglin, J. H., Jr and Bever, A. T. (1961) Ultraviolet induced biochemical alterations in skin. I. Urocanic acid. Arch. Dermatol. 84: 717 – 719.

Featherston W. R., and Horn G. W. (1973) Dietary influences on the activities of enzymes involved in branched-chain amino acid catabolism in the chick. J. Nutr. May; 103(5): 757 – 765.

Feigelson M. (1973) Studies on the postnatal development of responses of hepatic histidase to endocrine control. Biochim. Biophys. Acta. May 28; 304(3): 669 – 685.

Feigelson M. (1968) Estrogenic regulation of hepatic histidase during postnatal development and adulthood. J Biol Chem. Oct 10; 243(19): 5088 – 5093.

Ferraris, R. P., Kwan, W. W., Diamond, J. (1988) Regulatory signals for intestinal amino acid transporters and peptidases. Am. J. Physiol. 255 (Gastrointest. Liver Physiol. 18): G151 – G157.

Flogel M., Biltonen R. L. (1975) The pH dependence of the thermodynamics of the interaction of 3'-cytidine monophosphate with ribonuclease A. Biochemistry. Jun 17; 14(12): 2610 – 2615.

Forbes, J. M., and Shariatmadari, F. (1994) Diet selection for protein by poultry. World's Poultry Science Journal 50: 7-24.

Freeman B. M., Manning A. C.(1976) Mediation of glucagon in the response of the domestic fowl to stress. Comp Biochem Physiol A. 53(2): 169 – 171.

Ghadimi H., Partington, M. W., Hunter, A. (1962) Inborn error of histidine metabolism. Pediatrics. 29: 714 – 728.

Ghadimi H., Partington, M. W., Hunter, A. (1961) A familial disturbance of histidine metabolism. N. Eng. J. Med. 265: 221 – 224.

107

Gietzen, D. W. (1993) Neural mechanisms in the responses to amino acid deficiency. J. Nutr. Apr; 123(4): 610 – 625.

Goldman M. J., Back D. W., Goodridge A. G. (1985) Nutritional regulation of the synthesis and degradation of malic enzyme messenger RNA in duck liver. J. Biol. Chem. Apr 10; 260(7): 4404 – 4408.

Goodridge A. G. (1968) Citrate-cleavage enzyme, 'malic' enzyme and certain dehydrogenases in embryonic and growing chicks. Biochem. J. Jul; 108(4): 663 – 666.

Goodridge A. G., and Adelman T. G.(1976) Regulation of malic enzyme synthesis by insulin triiodothyronine, and glucagon in liver cells in culture. J Biol Chem. May 25; 251(10): 3027 – 3032.

Goodridge A. G., and Ball E. G. (1966) Lipogenesis in the pigeon: in vitro studies. Am. J. Physiol. Sep; 211(3): 803 – 808.

Goodridge A. G., and Ball E. G. (1967) Lipogenesis in the pigeon: in vivo studies. Am. J. Physiol. Jul; 213(1): 245 – 249.

Grisoni M. L., Uzu G., Larbier M., Geraert P. A. (1991) Effect of dietary lysine level on lipogenesis in broilers. Reprod. Nutr. Dev. 31(6): 683 – 90.

Gupta R. K, and Pesando J. M. (1975) Magnetic resonance study of exchangeable protons in human carbonic anhydrases. J. Biol. Chem. Apr 10; 250(7): 2630 – 2634.

Han Y. M., Suzuki H., Baker D. H. (1991) Histidine and tryptophan requirement of growing chicks. Poult Sci. Oct;70(10): 2148 – 53.

Hoffer, L. J. (1994) Starvation. In “Modern nutrition in health and disease” (Shils M. E., Olson J. A., and Shike, M. eds.) vol 2, pp. 927 – 949. Lea and Febiger, Philadelphia, PA.

Holcombe D. J., Roland D. A. Sr, Harms R. H. (1976) The ability of hens to regulate phosphorus intake when offered diets containing different levels of phosphorus. Poult. Sci. Jan; 55(1): 308 – 317.

Ide T, Murata M, Sunada Y.(1992) Soybean protein-dependent changes in triacylglycerol synthesis and concentration of diacylglycerol in the liver microsomes of fasted- refed rats. Ann Nutr Metab. 36(2): 87 – 96.

Kang-Lee Y. A., Harper A. E. (1979) Effect of induction of histidase on histidine metabolism in vivo. J Nutr. Feb; 109(2): 291 – 299.

108

Karasov W. H., Solberg D. H., Diamond J. M. (1987) Dependence of intestinal amino acid uptake on dietary protein or amino acid levels. Am. J. Physiol. May;252 (5 Pt 1):G614-25.

Keene J. C, Austic R. E. (2001) Dietary supplements of mixtures of indispensable amino acids lacking threonine, phenylalanine or histidine increase the activity of hepatic threonine dehydrogenase, phenylalanine hydroxylase or histidase, respectively, and prevent growth depressions in chicks caused by dietary excesses of threonine, phenylalanine, or histidine. J. Nutr. Biochem. May; 12(5): 274 – 284.

Kimball S. R., Shantz L. M., Horetsky R. L., Jefferson L. S. (1999) Leucine regulates translation of specific mRNAs in L6 myoblasts through mTOR-mediated changes in availability of eIF4E and phosphorylation of ribosomal protein S6. J. Biol. Chem. Apr 23; 274(17):11647 – 11652.

Kohchi C., Tsutsui K. (2000) Avian galanin: cloning of complementary DNAs and characterization of transcripts in different tissues. J. Exp. Zool. Jul 1; 287(2): 183 – 190.

Kopple JD, Swendseid ME.(1975) Evidence that histidine is an essential amino acid in normal and chronically uremic man. J. Clin. Invest. May; 55(5): 881 – 891.

Kumar V, Laddu A. R., Lahiri P. K., Sanyal R. K. (1968) The physiological levels of histamine in human tissues. Int. Arch. Allergy. Appl. Immunol. 34(3): 233 – 236.

La Du B. N. (1978) Histidinemia. In “The metabolic basis of inherited disease”. (Stanbury, J. B., Wyngaarden, J. B., and Fredrickson, D. S., eds), McGraw-Hill, New York. pp. 317 – 327.

La Du B. N., Howell R. R., Jacoby G. A., Seegmiller J. E., Zannoni V. G., Canby J. P., Ziegler, L. K. (1963) Clinical and biochemical studies on two cases of histidinemia. Pediatrics. 32: 216 – 217.

Lacy, M. P., Van Krey, H. P., Skewes, P. A., Denbow, D. M. (1985) Effect of intrahepatic glucose infusions on feeding in heavy and light breed chicks. Poult. Sci. Apr; 64(4): 751 – 756.

Lamartiniere C. A. (1979) Neonatal estrogen treatment alters sexual differentiation of hepatic histidase. Endocrinology. Oct; 105(4): 1031 – 1035.

Lamartiniere C. A., Feigelson M. (1977) Effects of estrogen, glucocorticoid, glucagon, and adenosine 3':5'-monophosphate on catalytic activity, amount, and rate of de novo synthesis of hepatic histidase. J. Biol. Chem. May 25; 252(10): 3234 – 3239.

109

Leclercq B., Guy G. (1991) Further investigations on protein requirement of genetically lean and fat chickens. Br. Poult. Sci. Sep; 32(4): 789 – 798.

Lee D. J., McNab J. M., Shannon D. W., Blair R. (1972) Enzyme studies with the livers of chicks fed semi-synthetic diets containing crystalline amino acids and diammonium citrate. Br Poult Sci. 1972 May;13(3):229-35

Lefevre P., Diot C., Legrand P., Douaire M. (1999) Hormonal regulation of stearoyl coenzyme-A desaturase 1 activity and gene expression in primary cultures of chicken hepatocytes. Arch. Biochem. Biophys. Aug 15; 368(2): 329 – 337.

Lepkovsky, S. (1973) Hypothalamic-adipose tissue interrelationships. Fed. Proc. Jun; 32(6): 1705 – 1708.

Leveille G. A., and Yeh Y. Y. (1972) Influence of intermittent fasting or protein-free feeding on lipid metabolism in young cockerels. J Nutr. Jun; 102(6): 733 – 740.

Levy, H. L. (1989) Disorders of histidine metabolism. In “The metabolic basis of inherited disease” (Scriver C. R., Beaudet A. L., Sly W. S., Valle, D., eds) McGraw-Hill, New York, pp. 563 – 576.

Ma, X. –J., Salati, L. M., Ash, S. E., Mitchell, D. A., Klautky, S. A., Fantozzi, D. A., Goodridge, A. G. (1990) Nutritional regulation and tissue-specific expression of the malic enzyme gene in the chicken transcriptional control and chromatin structure. J. Biol. Chem. 265: 18435 – 18441.

Marshall I. (1981) Histamine modulation of neurotransmission in the sympathetic nervous system. J. Auton. Pharmacol. Jun;1(3): 235 – 250.

Maruyama K., Harper A. E., Sunde M. L. (1975) Effects of D-, DL-and L-glutamic acid on chicks. J. Nutr. Aug; 105(8): 1012 – 1019.

Matei-Vladescu, C., Apostol, G., Popescu, V. (1977) Reduced food intake following cerebral intraventricular infusion of glucose in Gallus domesticus. Physiol. Behav. Jul; 19(1): 7 – 10.

Mehler A. H., and Tabor H., (1953) Deamination of histidine to form urocanic acid in liver. J. Biol. Chem. 201: 775

Meier, J. J., Gallwitz, B., Schmidt, W. E., Nauck, M. A. (2002) Glucagon-like peptide 1 as a regulator of food intake and body weight: therapeutic perspectives. Eur. J. Pharmacol. Apr 12; 440(2-3) : 269 – 279.

Mendonca, C. X., and Jensen, L. S. (1989) Influence of protein concentration on the sulfur containing amino acid requirement of broiler chickens. Brit. Poul. Sci. 30: 889 – 898.

110

Mercer, L. P., Kelley, D. S., Humphries, L. L., Dunn, J. D. (1994) Manipulation of central nervous center system histamine or histaminergic receptors affect food intake in rats. J. Nutr. 124: 1029 – 1036. Mercer, L. P., Dodds, S. J., Schweisthal, M. R.., Dunn, J. D. (1989) Brain histidine and food intake in rats fed diets deficient in single amino acids. J. Nutr. 119: 66 – 74.

Minick M. C., Duke G. E., Fishman S. W., Harvey, R. B. (1996) Effects of intragastric nutrients upon simultaneously determined plasma insulin, glucagon, insulin/glucagon ratios and glucose in red-tailed hawks, Buteo jamaicensis gmelin. Comp. Biochem. Physiol. 113: 173 – 179.

Mounier C., Chen W., Klautky S. A., Goodridge A. G. (1997) Cyclic AMP-mediated inhibition of transcription of the malic enzyme gene in chick embryo hepatocytes in culture. Characterization of a cis-acting element far upstream of the promoter. J. Biol. Chem. Sep 19; 272(38): 23606 – 23615.

Morrison H., Avnir D., Zarrella T. (1980) Analysis of Z and E isomers of urocanic acid by high-performance liquid chromatography. J Chromatogr. Jul 11; 183(1): 83 – 86.

Munroe, H. (1970). Free amino acid pools and their role in regulation. In: “Mammalian Protein Metabolism”, pp: 229 – 386. Academic Press, New York, NY

Muramatsu K., Odagiri H., Morishita S., Takeuchi H. (1971) Effect of excess levels of individual amino acids on growth of rats fed casein diets. J Nutr. Sep; 101(9): 1117 – 1125.

National Research Council (1994). The nutrient requirements of poultry, 9th rev. ed. National Academy of Science, Washington, D.C.

Norval M., Simpson T. J., Bardshiri E., Howie S. E. (1989) Urocanic acid analogues and the suppression of the delayed type hypersensitivity response to Herpes simplex virus. Photochem Photobiol. May; 49(5): 633 – 639.

Oesterwitz H., Gruner S., Diezel W., Schneider W. (1990) Inhibition of rat heart allograft rejection by a PUVA treatment of the graft recipient. Role of cisurocanic acid. Transpl Int. May; 3(1): 8 – 11.

Ohneda A., Parada E., Eisentraut A. M., Unger R. H. (1968) Characterization of response of circulating glucagon to intraduodenal and intravenous administration of amino acids. J. Clin. Invest. Oct; 47(10): 2305 – 2022.

Okamura H, Nishida T, Nakagawa H. (1974) L-histidine ammonia-lyase in rat liver. I. Purification and general characteristics. J Biochem (Tokyo). Jan;75(1):139-52.

111

Peng Y., Gubin J., Harper A. E., Vavich M. G., Kemmerer A. R. (1973) Food intake regulation: amino acid toxicity and changes in rat brain and plasma amino acids. J. Nutr. Apr; 103(4): 608 – 617.

Pesti G. M., Leclercq B, Chagneau A. M., Cochard T.(1996) Effects of the naked neck (Na) gene on the sulfur-containing amino acid requirements of broilers. Poult Sci. Mar; 75(3): 375 – 380.

Pfaff F.E., Jr. (1977) Effect of dietary protein and amino acids on carcass composition and lipogensis in the growing chick. Ph.D. Thesis, Cornell University, Ithaca, NY. 1 – 159.

Plavnik I. and Hurwitz, S. (1990) Performance of broiler chickens and turkey poults subjected to feed restriction or to feeding of low-protein or low-sodium diets at an early age. Poult. Sci. 69: 945 – 952.

Quillard M., Husson A., Lavoinne A. (1996) Glutamine increases argininosuccinate synthetase mRNA levels in rat hepatocytes. The involvement of cell swelling. Eur. J. Biochem. Feb 15; 236(1): 56 – 59.

Recsei P. A., Huynh Q. K., Snell E. E. (1983) Conversion of prohistidine decarboxylase to histidine decarboxylase: peptide chain cleavage by nonhydrolytic serinolysis. Proc Natl Acad Sci U S A. Feb; 80(4): 973 – 977.

Reeve V. E., Greenoak G. E., Canfield P. J., Boehm-Wilcox C., Gallagher C. H. (1989) Topical urocanic acid enhances UV-induced tumour yield and malignancy in the hairless mouse. Photochem Photobiol. Apr; 49(4): 459 – 64.

Romsos D. R., and Leveille G. A. (1974) Effect of dietary fructose on in vitro and in vivo fatty acid synthesis in the rat. Biochim Biophys Acta. Jul 26; 360 (1): 1 – 11.

Roncero C., and Goodridge A. G. (1992) Hexanoate and octanoate inhibit transcription of the malic enzyme and fatty acid synthase genes in chick embryo hepatocytes in culture. J Biol Chem. Jul 25; 267(21): 14918 – 14927.

Rosebrough R. W., and Steele N. C. (1990) Dietary crude protein levels and the effect of isoproterenol on in vitro lipogenesis in the chicken. J Nutr. Dec; 120(12): 1684 – 1691.

Rosebrough R. W., and Steele N. C. (1985) Energy and protein relations in the broiler chicken. 2. Effect of varied protein and constant carbohydrate levels on body composition and lipid metabolism. Growth. Winter; 49(4): 479-489.

Rosebrough R. W., McMurtry J. P., Vasilatos-Younken R.(1999) Dietary fat and protein interactions in the broiler. Poult Sci. Jul;78(7):992 – 998.

112

Rosebrough R. W., Mitchell A. D., McMurtry J. P. (1996) Dietary crude protein changes rapidly alter metabolism and plasma insulin-like growth factor I concentrations in broiler chickens. J Nutr. Nov;126(11):2888 – 2898.

Rosebrough R.W., Mitchell A.D., Von Vleck M.F., Steele N.C. (1990) Protein and energy relations in the broiler chicken. 8. Comparison involving protein- and lysine-adequate and inadequate diets on lipid metabolism. Br. J. Nutr. Sep; 64(2): 515 – 23.

Rosebrough R. W., McMurtry J. P., Mitchell A. D., Steele N. C. (1988) Chicken hepatic metabolism in vitro. Protein and energy relations in the broiler chicken--VI. Effect of dietary protein and energy restrictions on in vitro carbohydrate and lipid metabolism and metabolic hormone profiles. Comp Biochem Physiol B.; 90(2): 311 – 316.

Rosebrough R. W, Steele N. C., McMurtry J. P., Richards M. P., Mitchell A. D., Calvert C. C. (1986a) Energy and protein relations in the broiler chicken. 4. Role of sex, line and substrate on in vitro lipogenesis. Growth. Winter; 50(4): 461 – 471.

Rosebrough RW, Steele NC, McMurtry JP, Plavnik I. (1986b) Effect of early feed restriction in broilers. II. Lipid metabolism. Growth. Summer; 50(2): 217 – 227.

Ross J. A., Howie S. E., Norval M., Maingay J. (1988) Systemic administration of urocanic acid generates suppression of the delayed type hypersensitivity response to herpes simplex virus in a murine model of infection. Photodermatol. Feb; 5(1): 9 – 14.

Ross J. A, Howie S. E., Norval M., Maingay J., Simpson T. J. (1986) Ultraviolet- irradiated urocanic acid suppresses delayed-type hypersensitivity to herpes simplex virus in mice. J Invest Dermatol. Nov; 87(5): 630 – 633.

Rukaj A and Serougne C. (1983) Effect of excess dietary cystine on the biodynamics of cholesterol in the rat. Biochim Biophys Acta. Aug 29; 753(1): 1 – 5.

Sahib M. K., Murti C. R. (1969) Induction of histidine-degrading enzymes in protein- starved rats and regulation of histidine metabolism. J. Biol. Chem. Sep 10; 244(17): 4730 – 4734.

Salati LM, Ma XJ, McCormick CC, Stapleton SR, Goodridge AG.(1991) Triiodothyronine stimulates and cyclic AMP inhibits transcription of the gene for malic enzyme in chick embryo hepatocytes in culture. J Biol Chem. 1991 Feb 25; 266(6): 4010 – 4016.

Sanahuja, J. C., and Harper, A. E. (1963) Effect of dietary amino acid pattern on plasma amino acid pattern and food intake. Amer. J. Physiol. 204: 686 – 690.

113

SAS Institute. 1999. SAS User’s Guide: Statistics. Version 8 Ed. SAS Institute Inc., Cary, NC.

Schemke, R. T. (1962) Adaptive characteristics of urea cycle enzymes in the rat. J. Biol. Chem. 237: 459 – 468.

Schirmer, M. D., Harper, A. E. (1970) Adaptive responses of mammalian histidine- degrading enzymes. J. Biol. Chem. 245: 1201 – 1211.

Scott I. R. (1981) Factors controlling the expressed activity of histidine ammonia-lyase in the epidermis and the resulting accumulation of urocanic acid. Biochem J. Mar 15; 194(3): 829 – 838. Scott R. L., Austic, R. E. (1982) Effect of dietary histidine on histidine-degrading enzymes in the chicken (Gallus domesticus). Nutr. Res. 2, 185 – 192.

Scott, M. L., Nesheim, M. C. & Young, R. J. (1982) Nutrition of the chicken. 3rd ed. M. L. Scott and Associates, Ithaca, New York.

Shargo, E., Glennon, J. A. Gordon, E. S. (1971) Comparative aspects of lipogenesis in mammalian tissues. Metabolism 20, 54-62.

Shargo, R., Lardy, H. A., Nordlie, R. C. (1963) Hormonal and metabolic control of hepatic phosphoenolpyruvate carboxykinase and the malic enzyme. Fed. Proc. 22: 410 – 415.

Shariatmadari F., Forbes J.M. (1993) Growth and food intake responses to diets of different protein contents and a choice between diets containing two concentrations of protein in broiler and layer strains of chicken. Br. Poult. Sci. Dec; 34(5): 959 – 70.

Sonoda T. (1983) Hyperinsulinemia and its role in maintaining the hypothalamic hyperphagia in chickens. Physiol. Behav. Mar;30(3):325-9.

Spolter, P. D., and Baldridge, R. C. (1963) The metabolism of histidine. V. On the assay of enzymes in rat liver. J. Biol. Chem. 238(6): 2071 – 2074.

Stein E. D., Chang S. D., Diamond J. M. (1987) Comparison of different dietary amino acids as inducers of intestinal amino acid transport. Am. J. Physiol. May; 252(5 Pt 1): G626 – G635.

Straus D. S., Takemoto C. D. (1988) Regulation of albumin mRNA in H4 rat hepatoma cells by the availability of essential amino acids. Biochim. Biophys. Acta. Oct 28; 972(1): 33 – 36.

114

Suchi M, Sano H, Mizuno H, Wada Y. (1995) Molecular cloning and structural characterization of the human histidase gene (HAL). Genomics. Sep 1;29(1):98- 104.

Suchi M., Harada N., Wada Y., Takagi Y. (1993) Molecular cloning of a cDNA encoding human histidase. Biochim Biophys Acta. Nov 16; 1216(2): 293 – 295.

Tanabe M., Sakura N. (1989) Hyperhistaminemia in patients with histidinemia due to increased decarboxylation of histidine. Clin. Chim. Acta. Dec 29; 186(1): 11 – 7.

Tanaka, K., Okamoto, T. Ohtani, S. (1992) Effects of dietary protein on the activities of lipogenic enzymes in the liver of growing chicks. Asian-Australas. J. Anim. Sci. 5: 123 – 128.

Tanaka K, Ohtani S, Shigeno K. (1983a) Effect of increasing dietary energy on hepatic lipogenesis in growing chicks. I. Increasing energy by carbohydrate supplementation. Poult Sci. Mar; 62(3): 445 – 451.

Tanaka K, Ohtani S, Shigeno K. (1983b) Effect of increasing dietary energy on hepatic lipogenesis in growing chicks. II. Increasing energy by fat or protein supplementation. Poult Sci. Mar; 62(3): 452 – 458.

Taylor R. G., Grieco D., Clarke G. A., McInnes R. R., Taylor B. A.(1993) Identification of the mutation in murine histidinemia (his) and genetic mapping of the murine histidase locus (Hal) on chromosome 10. Genomics. Apr;16(1):231-40.

Taylor R. G., Lambert M. A., Sexsmith E., Sadler S. J., Ray P. N., Mahuran D. J., McInnes R. R. (1990) Cloning and expression of rat histidase. Homology to two bacterial histidases and four phenylalanine ammonia-lyases. J. Biol. Chem. Oct 25; 265(30): 18192 – 18199.

Taylor R. G., Levy H. L., McInnes R. R. (1991) Histidase and histidinemia. Clinical and molecular considerations. Mol Biol Med. Feb; 8(1): 101 – 116.

Tepperman, H. M. and Tepperman, J. (1964) Patterns of dietary and hormonal induction of certain NADP-linked liver enzymes. Am. J. Physiol. 206: 357 – 361.

Torres N., Beristain L., Bourges H., Tovar A. R. (1999) Histidine-imbalanced diets stimulate hepatic histidase gene expression in rats. J Nutr. Nov; 129(11): 1979 – 1783.

Torres N, Martinez L, Aleman G, Bourges H, Tovar A. R. (1998) Histidase expression is regulated by dietary protein at the pretranslational level in rat liver. J. Nutr. May; 128(5): 818 – 824.

115

Varga J, Li L, Mauviel A, Jeffrey J, Jimenez SA. (1994) L-Tryptophan in supraphysiologic concentrations stimulates collagenase gene expression in human skin fibroblasts. Lab Invest. Feb;70(2):183-91.

Vary T. C., Jefferson L. S., Kimball S. R. (1999) Amino acid-induced stimulation of translation initiation in rat skeletal muscle. Am J Physiol. Dec; 277(6 Pt 1): E1077 – E1086.

Wadia A. S., Sule S. M., Mathur G. P.(1975) Epidermal urocanic acid & histidase of albino guineapig following total body UV-irradiation. Indian J Exp Biol. May; 13(3): 234 – 237.

Wilcken B., Brown D. A. (1975) Histidinemia – evaluation of an improved method for confirmation, and the implications of the diagnosis. Aus. Pediat. J. 11: 126 – 127.

Winberry L. K., Morris S. M. Jr, Fisch J. E., Glynias M. J., Jenik R. A., Goodridge A. G. (1983) Molecular cloning of cDNA sequences for avian malic enzyme. Nutritional and hormonal regulation of malic enzyme mRNA levels in avian liver cells in vivo and in culture. J Biol Chem. Jan 25; 258(2): 1337 – 1342.

Woodham A. A., Deans P. S. (1975) Amino acid requirements of growing chickens. Br. Poult. Sci. May; 16(3): 269 – 287.

Yagasaki K, Machida M, Funabiki R. (1986) Effects of dietary methionine, cystine, and glycine on endogenous hypercholesterolemia in hepatoma-bearing rats. J. Nutr. Sci. Vitaminol. (Tokyo). 1986 Dec;32 (6): 643 – 651.

Yeh Y. Y., and Leveille G. A. (1971) Studies on the relationship between lipogenesis and the level of coenzyme A derivatives, lactate and pyruvate in chick liver. J Nutr. Jul;101(7):911 – 918.

Yeh Y. Y., and Leveille G. A., Wiley J. H. (1970) Influence of dietary lipid on lipogenesis and on the activity of malic enzyme and citrate cleavage enzyme in liver of the growing chick. J. Nutr. Aug;100(8): 917 – 924.

Yeh Y. Y., and Leveille G. A. (1969) Effect of dietary protein on hepatic lipogenesis in the growing chick. J. Nutr. Jul; 98(3): 356 – 366.

Young, J. W., Shargo E., Lardy, H. A. (1964) Metabolic control of enzymes involved in lipogensis and gluconeogenesis. Biochemisty 11: 1687 – 1692.

Yuan J. H, Davis A. J., Austic R. E. (2000) Temporal response of hepatic threonine dehydrogenase in chickens to the initial consumption of a threonine-imbalanced diet. J. Nutr. Nov; 130(11): 2746-52.

116

Zenisek, A., Kral, J. A. and Hais. I. M. (1955) “Sun-screening” effect of urocanic acid. Biochim. Biophys. Acta. 18: 589 – 591.

Zenisek, A., Hais. I. M., Strych, A., and Kral, J. A. (1969) Does solar irradiation affect the natural antisunburn properties of the skin? Fr. Ses. Pasfums. 12: 131 – 133.