...... ..... - . . V o . . - ......... .......... ..... SOHE DEVELGPMENTHL .ESHEcEs :0?! Moms. ' 31'." AHD Hommz FATTY A023 museum-1H EHE ' a f 357 :1r35‘3if‘fi55-i CON‘ENTEGML MD ufl‘fiTREi HUMENHNT ThS‘SIS f8? zhe DE fig? €23 0T, “#1. S ; PM HM STWT A... C'TEVTPEITY ' ' KIM 3” “NT“ ' LI B R/ R V MichxsE State ‘ University ABSTRACT SOME DEVELOPMENTAL ASPECTS OF GLUCOSE AND VOLATILE FATTY ACID METABOLISM IN THE CONVENTIONAL AND GERMFREE RUMINANT By Kim H. Ponto Plasma glucose levels and glucose and acetate half- lives, hepatic glutamic-pyruvate transaminase, glucose-6- phosPhatase, pyruvate kinase and glucose-6-phosphate and 6-phosphogluconate dehydrogenase activities plus hepatic concentrations of glucose-6-phosphate and phosphoenol- pyruvate were investigated in conventional and germfree pygmy goats fed either milk, milk plus glucose or milk plus volatile fatty acids. A number of conventional animals fed a pelleted grain ration served as controls. Plasma glucose concentrations decreased from 98.4 to 71.6 mg/100 ml, glucose half-lives increased from 16.8 to 30.3 minutes and acetate half-lives increased from 2.58 to 4.50 minutes between the ages of 60 and 180 days. In all cases, differ- ences due to age were highly significant. In general, the activities of glutamic-pyruvic transaminase and glucose-6- phosphatase increased with age while pyruvate kinase activity was quite variable. A correlation coefficient of -O.7O (P<:0.05) was obtained for the relationship between pyruvate kinase activity and glucose half-life. Relation- ships between hepatic concentrations of glucose-6-phosphate and phosphoenolpyruvate and overall patterns of metabolic Kim H. Ponto activity were inconclusive. Effects due to dietary treat- ment and germfree versus conventional status of the animal were not observed. SOME DEVELOPMENTAL ASPECTS OF GLUCOSE AND VOLATILE FATTY ACID METABOLISM IN THE CONVENTIONAL AND GERMFREE RUMINANT by Kim H. Ponto A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Animal Husbandry 1973 (:57 '21? “l E. This manuscript is dedicated to the memory of my father Hilton A. Ponto ii ACKNOWLEDGEMENTS The author would like to extend his great appreciation to Dr. Werner G. Bergen for his personal interest and guidance so freely given him while preparing for this degree. Appreciation is also due Dr. Elwyn R. Miller and Dr. Roy S. Emery for their critical reading and constructive criticism during the preparation of this manuscript. The Animal Husbandry Department and the National Institute of Health are acknowledged for their financial support during the author's graduate program. Technical assistance given by Paul Hassinger and laboratory analyses performed by Elizabeth Rimpau and Colleen Patterson are acknowledged and appreciated. A Finally the author is indebted to his parents, Mr. and Mrs. Hilton Ponto for their continual encouragement through- out his graduate career. iii TABLE OF CONTENTS .EAEE List of Tables ...... ................. v List of Figures ..... ..... ............ Vi Introduction ... ......... .... ........ . 1 Review of Literature ................. 3 Experimental Procedure ....... ... ..... 27 Results and Discussion ............... 42 General Discussion ............. . ..... 91 Conclusions ....... ........ ........... 93 Bibliography ....... ........... ..... .. 95 iv LIST OF TABLES TABLE PAGE 1 Composition of Basal Diet ..................... 29 Composition of Experimental Diets ............. 29 Composition of Pelleted Germfree Animal Diet .. 30 -F‘\NN 1969 T Glucose and Glucose and Acetate Half- Lgves ................... ..... ............ 1+8 5 1970 T Glucose and Glucose and Acetate Half- LTves at Approximately 60 Days of Age .... 49 6 1970 TO Glucose and Glucose and Acetate Half- Lives at Approximately 180 Days of Age ... 50 7 1969 Glutamic-Pyruvic Transaminase Activity ... 61 1969 G1ucose-6-Phosphatase Activity ........... 6A 9 1970 Glucose-6-Phosphatase Activity ........... 65 10 1969 Pyruvate Kinase Activity ................ . 68 11 1970 Pyruvate Kinase Activity ................. 69 12 1970 G1ucose-6-Phosphate Dehydrogenase Activity ...... .......................... . 74 13 1970 6-Phosphogluconate Dehydrogenase Activity ................... ............. . 75 14 1970 Hepatic Metabolic Substrates ....... ..... . 78 15 Plasma Essential Amino Acids ........... ....... 80 16 Plasma Non-Essential Amino Acids ...... ...... .. 81 1? Liver Free Essential Amino Acids .............. 82 18 Liver Free Non-Essential Amino Acids .......... 83 19 Plasma Non-Essential to Essential Amino Acid Ratio .....OOOOOOOOOOOO 00000000000 00...... 00000 87 LIST OF FIGURES FIGURE PAGE 1 Average fasting plasma glucose ............... 52 2 Average glucose half-lives ................... 56 vi I INTRODUCTION At different stages from fetal to adult life, the rumi- nant utilizes various substrates for energy metabolism. Car- bohydrate for the develoPing fetus is provided by placental transfer of glucose. At birth, this energy SQLK2 is re- placed by a milk diet which supplies both carbohydrate and lipid. In the ruminant, rumen fermentation begins at weanhag and provides volatile fatty acids (VFA) as the major energy source. As the ruminant adapts first from high carbohydrate utilization prenatally to a high carbohydrate and lipid diet postnatally and finally to high VFA utilization at the adult stage, there are many metabolic changes which must take place. Adult patterns of VFA metabolism in the ruminant have been investigated to a much greater extent than the metabo- lism of carbohydrate by the prenatal and postnatal animal. Because the carbohydrate metabolism of the young ruminant resembles that of the non-ruminant animal (Ballardlgg‘a;., 1969) and then adapts to the metabolism typical of the adult ruminant, enzyme activities and metabolite concentrations in the tissues of the ruminant muSt be altered with successive changes of the substrates for energy metabolism. Metabolic activity may be altered by substrate composition and avail- ability or age dependent, constitutive changes in tissue enzyme levels. 2 Most research published to date would support the view, that changes in enzyme activities are substrate dependent whereas this conclusion has been questioned by Purser and Bergen (1969). This study was initiated to determine whether changes in ruminant metabolism are age or substrate dependent. Germfree goats, which should retain a carbohydrate (glucose) dependent energy metabolism because of the absence of ruminal microbiota, were used to test the above hypotheses. To this end, metabolic changes, as monitored by glucose and acetate tolerance tests, metabolic intermediates, and key enzyme activities were studied employing germfree and conventional animals. II REVIEW OF LITERATURE A. Glycolytic and Gluconeogenic Enzyme Alterations During DevelOpment 1. Patterns of glycolysis Hepatic glucose phosphorylating activity has been found to be high in adult guinea pigs, rats and rabbits but mar- ginal in fetal and newborn livers of these same animals (Walker, 1962). Walker (1965) showed that glucokinase [E.C.2.7.1.2] activity was absent in the fetal liver of the guinea pig but activity develoPed immediately after birth. Hepatic glucokinase activity in the rat however, appeared only after 16 days of life and reached adult levels 10-12 days later (Ballard and Oliver, 1964a). This increase in rat glucokinase activity was accounted for by.gg.pgzg synthesis of the enzyme (Walker, 1965). Ballard and Oliver (1964b) showed that the activity of the high Km’ insulin dependent hexokinase (glucokinase) was absent in fetal and adult sheep liver. It has also been shown that the activity of the low Km’ insulin independent hexokinase [E.C.2.7.l.1] was much lower in the liver of post- natal sheep than in rat liver (Ballard and Oliver, 1965). These authors concluded that negligible glucokinase activity was a metabolic consequence of maternal and placental regu- lation of fetal glucose concentrations and that lower 3 n hexokinase activity in the adult ruminant was due to the animal's own gluconeogenic regulatory systems (Ballard‘gp.§l, 1969)- In the liver of the deve10ping rat and sheep, total nicotinamide nucleotide coenzymes increased steadily and reached a maximum when growth ceased (Craiger.ggial., 1962). The ratio of oxidized and reduced forms of nicotinamide adenine dinucleotide (NAD) and nicotinamide adenine dinucleo- tide phosphate (NADP), ( [NAD] + [NADH] ) : ([NADP] + [NADPHZ] ) in the adult liver of the rat and sheep has been determined and a Species difference existed. In the young rat and sheep the ratio was 2.4 and 1.6 respectively. At maturity this ratio was 1.3 for the rat and 2.5 for the sheep, demonstrathng a relative increase in NADP during the develOpment of the rat and NAD during the development of the sheep. Increased NADP levels may reflect the importance of the oxidative pentose phosphate pathway for reductive synthesis in rat liver, while increased NAD levels in the liver of sheep may reflect the level of acetate oxidation via the tricarboxylic acid cycle. That is, greater emphasis is placed on the oxidative metabo- lism of acetate by the sheep in comparison to the rat (Craiger.gtial., 1962). Filsell 2£.§l- (1963) found slowing rates of reduction of NADP in the presence of glucose-6- phosphate, 6-phosphogluconate and glucose plus AIT’(adenosine triphosphate) in lamb liver homogenates and concluded that the lower coenzyme ratio observed in the adult sheep was 5 consistant with changing patterns of carbohydrate metabolism with age, i.e., less glycolytic activity in the maturing ruminant. . Other investigators have discussed similar decreases in the glycolytic metabolism of other species. Nemeth and Dickerman (1960) showed decreased g1ucose-6-phosphate dehydrogenase [E.C.1.l.l.49] levels in the liver of the developing guinea pig and Howarth'gpial. (1968) found a de- creased activity in this same enzyme plus fructose-l, 6- diphosphate aldolase [E.C.4.l.2.6] and glyceraldehyde-3- phosphate dehydrogenase [E.C.l.2.l.12] when hepatic activity of finished steers was compared to the activity exhibited by calves. Goetsch (1966) found a general decrease in the glycolytic enzymes of the adult ruminant and implied that these changes were due to deve10ping rumen function with less carbohydrate (substrate) available to the glycolytic pathway. 2. Patterns of gluconeogenesis and glycogen release from the liver While develOpmental changes are occurring in the glycolytic pathway, concurrent changes are also taking place in hepatic enzymes concerned with gluconeogenesis and glycogen release. Shelley (1960 and 1961) determined that in the liver of the fetal sheep, approximately 2 mg of glycogen per gram wet liver weight were laid down daily over the last half of gestation, which resulted in a total store 6 of approximately 90 mg glycogen per gram wet liver weight at birth. This onset of glycogen accumulation late in gestatkn1 was also observed in the guinea pig by Nemeth (1954). Imme- diately after birth, about 90 percent of the liver glycogen stores were mobilized. This rapid decline takes from two to three hours and even occurs in animals which suckled one-half hour after birth (Shelley, 1960). Adult glycogen levels of approximately 40 mg/gm wet liver weight are reached by two to three weeks post partum. Kornfeld and Brown (1963) examined the enzymes related to glycogen synthesis in the fetal and neonatal guinea pig. The activity of UDP glucose-glycogen-glucosyltransferase [E.C.2.4.1.ll] and UDP-glucose perphosphorylase [E.c.2.7.7.9] increased from approximately 50 percent of the adult level in the 45 to 60 day old fetus to the full adult level at term. CX-glucan phosphorylase [E.C.2.4.l.l] activity in- creased from 75 Percent of the adult level in the 45 to 60 day fetus to twice the adult level at term. Normal activity was reached on the sixth day of life. Glycogen however was only found to increase after the 67th day of the 68 day gestation period. The characteristic decrease in liver glycogen was also noted at birth in the guinea pig (Kornfeld and Brown, 1963). Enzymes involved in the degradation of glycogen appeared late in gestation only after glycogen synthesizing enzymes had been established in the rat liver (Jacquot, 1964). Ballard and Oliver (1965) found that during the develOpment of the sheep, UDP glucose-glycogen- 7 glucosyltransferase, UDP-glucose-perphosphorylase and gly- cogen content were highest in two week old lambs, while con- siderably lower levels were found in the adult sheep liver. When compared to the adult rat, the activity of hexo- kinase and the rate of incorporation of 140] -glucose into glycogen were much lower in liver of postnatal sheep (Ballard and Oliver, 1965). These workers found that the activity of hexosediphosphatase [E.C.3.l.3.1l , glucose-6-phosphatase [E.C.3.l.3.9] and the incorporation rates of [140] -pyruvate and [lTC] -pr0pionate into glycogen increased from low levels in the liver of fetal sheep and were at the highest levels a few weeks after birth. The corresponding rates of these enzymes were slightly lower in adult sheep. Krebs (1954) first recognized that gluconeogenesis was not a simple reversal of glycolysis but that certain disthnt enzymes existed to accomplish the synthesis of glucose from non-hexose precursors. Enzymes which are considered rate- limiting in gluconeogenesis are pyruvate carboxylase [E.C.6.4.l.l], phosphoenolpyruvate carboxykinase [E.C.4.l.l. 32], fructose diphosphatase [E.C.3.l.3.11] and glucose-6- phosphatase. In addition for the ruminant, prOpionyl carboxylase [E.C.6.4.l.3] should also be included as a gluconeogenic control enzyme (Baird.g§.a;., 1968). During conditions which stimulate gluconeogenesis, such as fasting and diabetes, these enzymes increased in activity and when glucose was available their activities declined (Shargo‘gt ‘al., 1963; Weber.g§'§1., 1964). These key enzymes of 8 gluconeogenesis metabolically Oppose the glycolytic enzymes pyruvate kinase [E.C.2.7.l.40] phosphofructokinase [E.C.2.7. 1.11] and glucokinase (Krebs, 1963; Nordlie and Lardy, 1963; Seubert and Huth, 1965; and Utter and Keech, 1963). Ballard and Oliver (1963) showed that in the fetal rat liver, synthesis of phosphoenolpyruvate from malate could not be detected and that [140] -glucose and[14C] -pyruvate could be gluconeogenic only after the birth of the animal. It was also shown that gluconeogenesis from pyruvate was absent from the liver of fetal rats and appeared first in neonatal rats (Ballard and Oliver, 1965). Vernon'gt.al. (1968) using L-lactate, casein hydrolysate, L-serine, L-threonine, L-alanine and glycerol as substrates showed that gluconeogenesis occurred only after birth in rats. Other work with neonatal rat liver (hang and Oliver, 1967) has shown that utilization of aspartate, glutamate and alanine for gluconeogenesis was initiated only during post natal life, reaching a maximum at the fifth day. It was further shown that overall gluconeogenic activity followed the activity pattern of aspartate transaminase. When comparing sheep with rats a different gluconeo- geneic pattern is found. Ballard and Oliver (1965) showed that liver of fetal sheep in the fourth month of gestation, converted pyruvate to glucose at 20 times the rate of the liver of a fetal rat in its 19th day of gestation. The rat showed rapid increases in this pathway at birth, while con- comitant gluconeogenic increases in the sneep were not as 9 dramatic. Enzymatic work (Ballard and Hanson, 1967) has shown that the activity of pyruvate carboxylase and phos- phoenolpyruvate carboxykinase have reached adult levels by the time of birth in the rat. Wagle (1966) compared gluco- neogenic activities of the adult rat and sheep by deter- mining the activity of key gluconeogenic enzymes. He showed that the enzymes pyruvate carboxylase, phosphoenolpyruvate carboxykinase, glucose-6-phosphatase and fructose-l, 6- diphosphatase had twice the activity of the same enzyme in rat liver. It was concluded that this greater gluconeogenic activity exhibited by the ruminant was reasonable since the ruminant must synthesize the major portion of its own glucose. The gluconeogenic enzyme glucose-6-phosphatase has been shown to develop before birth in the guinea pig (Lea and Walker, 1964; Nemeth, 1954) and Dawkins (1961) noted in- creases in the activity of this enzyme in the liver and kidney of the lamb, rat and guinea pig only 24 hours after birth. Hepatic glucose-6-phosphatase activity was found to be greater in steers than in calves. Calves reached adult enzymatic activity 8-12 weeks post partum (Bartley gt al., 1966). These metabolic changes in gluconeogenesis coincided with the develOpment of a functional rumen (Bartley 2£.§l-9 1966). Intracellular distribution of the gluconeogenic enzymes pyruvate carboxylase and phosphoenolpyruvate carboxykinase differs between species. In most animals, pyruvate 10 carboxylase was found to be located within liver mito- chondria, while phosphoenolpyruvate carboxykinase occurs in the cytosol of liver cells (Ballard and Hanson, 1967; Freedman and Kohn, 1964; Keech and Utter, 1963). Oxalacetate formed by carboxylation of pyruvate by the mitochondrial pyruvate carboxylase must migrate into the cellular cytosol to be converted to phosphoenolpyruvate and finally glucose. Studies on the rates of gluconeogenesis have shown that oxal- acetate cannot pass out of the mitochondria and into the cytosol fast enough to be a main precursor of glucose (Lardy .22.2i-2 1965). Thus it has been suggested that oxalacetate must be converted to aspartate and/or malate in the mito- chondrion. These metabolites can then pass into the cytosol and be reconverted to oxalacetate, which is then transformed to phosphoenolpyruvate (Haynes, 1965; Shargo and Lardy,l966). Ballardlgt'al. (1969) however cautioned that the dis- tinction between intra- and extramitochondrial enzymes are not as distinct in the bovine as in the rat. They found pyruvate carboxylase activity in the cytosol and this activity increased in response to physiological stress i.e., lactation, ketosis, and starvation. Phosphoenolpyruvate carboxykinase activity appeared in significant amounts in the mitochrondrion of bovine liver but did not exhibit the same adaptive prOperties as pyruvate carboxylase (Ballard _e_1:, 91-. 1969). Extramitochondrial pyruvate carboxylase enzyme found in the ruminant may participate in the control of gluconeogenesis 11 from pyruvate. Pyruvate carboxylase has been found to be activated by acetyl CoA and propionyl CoA, respectively. It has more recently been found that purified calf liver pyru- vate carboxylase is activated (cooperativity) by butyryl CoA (Ballard'g§'§1., 1969), the first product of butyrate metab- olism. These volatile fatty acids arising from rumen fer- mentation can be converted to the corresponding coenzyme A derivatives in the cytosol (Hanson and Ballard, 1967) and activate the extramitochondrial pyruvate carboxylase. Thus at a time when volatile fatty acids are plentiful and can be used fbrenergy purposes, pyruvate and gluconeogenic pre- cursors arising from the rumen are used for gluconeogenesis. As further evidence for this control, Black (1963) has shown .22.!112 that during spontaneous ketosis, when acyl CoA con- centrations may be elevated, the pathway for the carboxy- lation of pyruvate to oxalacetate was favored over decarboxv- lation of pyruvate to acetyl CoA. Enzymes associated with glycolysis may also be inhibited by increased concentrations of acetyl CoA (Randle §£,§l., 1966). B. Dietary Glucose and Gluconeogenic Substrates For The Ruminant Absorption of glucose from the gastrointestinal tract of the ruminant fed high-roughage diets is minimal (Heald, 1951; Annisonigplal., 1957; Bensadoun.g§,§1., 1962; and Roe .25.§l°2 1966). Fries and Conner (1960) estimated from portib- carotid blood glucose differences that glucose could provide 12 roughly 10 percent of the available energy for a young bull calf when fed a diet of alfalfa hay and corn.' For diets high in concentrates, investigations have shown that appreciable amounts of starch escape rumen fermen- tation, reach the small intestine and may be absorbed as glucose. Approximately 331 g of starch disappeared from the small intestine of steers consuming 1002 g of starch from a 20 percent ground corn ration and 624 g of starch disappeared from the small intestine of these same steers consuming 2684 g of starch from an 80 percent corn ration (Karr,g§.§l., 1966). Experiments with sheep have provided similar results. About 16 g of starch disappeared posterior to the rumen of sheep fed 172 g of starch from 20 percent ground corn ration while 101 g of starch disappeared in the small intestine of these sheep consuming 576 g of starch from an 80 percent ground corn ration (TuckerIggial., 1968). MacRae and Armstrong (1966 and 1969) have reported intermediate values (to those of Tucker.gp.§l.) for amounts of CX-linked glucose polymer presented to the duodenum when high barley diets were fed to sheep. Thus, when quantatively assessing the requirement of gluconeogenic precursors for the ruminant, account must be taken of diets which are high in grain, since an appreciable amount of glucose is passed to the duo- denum for absorption. Since the extent to which dietary glucose escapes rumen fermentation depends on the diet itself, the diet influences the importance of gluconeogenic precursors required by the 13 ruminant. Under dietary conditions which increase the im- portance of the gluconeogenic capacity of the ruminant, pr0pionic acid and gluconeogenic amino acids become the important precursors for this process. Acetic, pr0pionic and butyric acids which are the major and products of rumen fermentation account for 70-80 percent of the ruminant's energy requirements (Bergman.g£flgl., 1965 and Warner, 1964). Of these three acids, only propionic acid can be gluconeogenic (Annison.§t,al., 1963 and Leng and Annison, 1963). The first direct quantitative measurements of the role of pr0pionate in gluconeogenesis was made by Bergman‘gplal. (1966). In sheep fed a maintenance ration of 800 g of alfalfa per day, it was determined by infusion of pr0pionate- 2-17C into the rumen vein, that pr0pionate accounted for about 27 percent (range for four animals 24-33) of the glucose turnover and this contribution could be raised to 50 percent by carrier pr0pionate infusion. Metabolized protein accounted for 63 percent of the glucose turnover and the remaining 10 percent probably came from dietary glucose and other carbohydrate precursors i.e., lactate. The contri- bution of protein for gluconeogenesis was calculated from urinary excretion of nitrogen by methods which strictly apply to the non-ruminant (West and Todd, 1961). Bergman .g§.§1.(1966) overlooked the fact that urea produced by the liver from rumen ammonia can pass into the urine dfthe ruminant. Such a large contribution from protein for 14 gluconeogenesis was obviously an overestimate. Recently Wolff and Bergman (1972) reevaluated the amonn; of blood glucose derived from plasma amino acids using U -th - labeled alanine, aspartate, glutamate, glycine and serine. In sheep fed a near-maintenance ration, at least 11 percent of the blood glucose was derived from amino acids. .A maximum of 30 percent of the blood glucose could be derived from amino acids based on net hepatic uptake of these amino acids but part of this uptake was undoubtedly diverted into synthesis of plasma proteins and other amino acid metabolism pathways. It was concluded that only between 11 and 30 percent of the blood glucose in the fed sheep was derived from plasma amino acids. Leng 2£.él- (1967) found by the intraruminal infusion of [ITGI-prOpionate into sheep on a diet of 800 g lucerne per day, that 54 percent of both glucose and lactate arose from pr0pionate carbon. The intraruminal infusion of labelled[lz+C] -propionateprovided evidence that there was appreciable conversion of pr0pionate to lactate at the rumen wall. This phenomenon had not been allowed for by Bergman .££.§l- (1966) because they had measured only the gluconeo- genic contribution of absorbed pr0pionate. Judson gg‘gl. (1968) have examined the effect of in- creased dietary crushed maize on the contribution of pr0pi- onate for gluconeogenesis. In sheep fed 400 g of crushed maize per day, only 27 percent of the animals' glucose was derived from pr0pionate. They explained their unaltered 15 levels of pr0pionate production from such a ration (compared to all roughage diets) by increased amounts of starch es- caping rumen fermentation. They suggested that glucose ab- sorption from the gastrointestinal tract may have reduced gluconeogenesis from pr0pionate. Other evidence has shown that the gluconeogenic needs of sheep may be entirely met by pr0pionate alone. Armstrong (1965) has calculated that the requirements for energy absorbed as pr0pionate to meet glucose needs for the non- pregnant sheep and pregnant sheep to be 18 and 28 kcal/ kg0’75/day. Annison and Lindsay (1962) found that sheep fed a diet of 400 g lucerne and 500 g maize absorbed 64 kcal/ kgo'75/day as pr0pionate. If 50 percent of this pr0pionate is converted to glucose, the glucose requirements of these animals would be adequately met. The once great emphasis placed on the role of amino acids as precursors of the process of gluconeogenesis, a process of more importance to the ruminant than the non- ruminant, has lessened. Purser (1970) pointed out the paradox that the ruminant which apparently is in great need of amino acids for protein synthesis would have at the same time a great requirement for these amino acids as precursors of’gluconeogenesis. In the non-pregnant ruminant with only maintenance requirements, gluconeogenesis from amino acids may be minimal but may become increasingly more important to the animal under the stress of growth, pregnancy or lactathnu V 16 C. Glucose Tolerance, Blood Glucose Levels And Dietary Effects On These Parameters Previous work involving the intravenous glucose toler- ance test in man showed that blood glucose disappearence followed an exponential decrease with time and this decrease has been mathematically defined. The relationship between log blood glucose increment and time after injection is linear (Greville, 1943; Amatuzio 23.31., 1953; Duncan, 1956; and Hlad.g§'al., 1956). The constant rate of removal of injected glucose indicated that glucose utilization follows first order kinetics and is a linear function of the log arterial blood glucose concentration. This relationship was found to be constant over a wide range of blood glucose con- centrations in the dog (Lang's; al., 1954; Soskin and Levine, 1937). The glucose tolerance test, applied to the adult rumi- nant, has shown that the rate of removal of injected glucose by ruminant tissues is much slower than that observed in the non-ruminant (McCandless g§,al., 1948; McCandless and Dye, 1950; Reid, 1952; Jarrett and Potter, 1957; and Reid, 1958). Fasted calves and calves made diabetic either by pancre- atectomy or alloxan treatment had lowered glucose tolerance (Cook'gt‘gl., 1949; and McCandless and Dye, 1949). Six week old calves were found to clear blood glucose as rapidly as the normal dog or human since blood glucose levels of 200 to 300 mg% returned to normal values in one to two hours. This same glucose load required four to six hours to reach normal ”slut-‘1 .4 17 levels in the adult sheep (McCandless and Dye, 1949; McCandless and Dye, 1950; Jarrett and Potter, 1957). Equine glucose tolerance rates fell between those of the ruminant and non-ruminant animal (Mehring and Tyznik, 1970). Fasting blood glucose level of the young calf was found to be about 97 mg% or about twice the level of the adult animal (McCandless and Dye, 1950). Young lambs had about the same fasting blood glucose levels as the calf and these levels declined steadily until at five to six weeks of age, when rumination was observed, they fell to 50-65 mg% (McCandless and Dye, 1950). This general trend in decreasing blood glucose has been observed in other ruminants by many workers (Lambert gl'al., 1955; McCarthy and Kesler, 1956; Wing ggflgl., 1955; Attebery and Colvin, 1963; Hodgson 23,§1., 1932; and Murley g; 21., 1952). Factors causing the decreased glucose tolerance and plasma glucose levels have been investigated with conflicting results. McCarthy and Kesler (1956) fed calves either whole milk or a milk replacer during which time hay was fed ad libitum. By eight weeks, blood glucose levels fell from 90 mg% to 50 mg% and a concomitant increase in blood VFA due to increased rumen cellulose fermentation was observed. They concluded that the diet caused the decreasing blood glucose levels. Attebery and Colvin (1963) using calves over a 13 week feeding trial period were able to maintain a constant fasting blood glucose level by feeding a diet of milk solids, but the inclusion of grain and hay resulted in 18 gradual decline in fasting blood glucose. The effect of diet on glucose tolerance was determined by Harry 3; a1. (1967) by feeding calves essentially an all milk and milk solid diet or a diet of milk and milk solids plus grain and/ or hay. They found that the glucose tolerance, performed after a 12-14 hour fast, was constant for the calves fed the milk diet while decreased glucose tolerance was observed in the treatments which included hay and/or grain. They stated, "extrinsic stimuli many be more important than intrinsic stimuli in the transition of the ruminant energy metabolism from one involving glucose primarily to one which uses pre- dominantly volatile fatty acids." Others (McCandless and Dye, 1950; Hale and King, 1958; and Omari and Nakajima, 1961) have concluded that glucose tolerance is dependent on diet and nature of rumen fermentation. Reid (1958) also showed that the diet of the mature ruminant had a pronounced effect on glucose tolerance. Adult sheep, fed a diet containing 50 percent cracked maize lag libitum, showed the shortest glucose half-life; those on a restricted maize diet, an intermediate half-life and the longest glucose half-life was recorded for sheep on a re- stricted roughage diet. Thus he showed that the shortest .half-life or fastest rate of glucose utilization occurred on diets which supplied the greatest amount of pr0pionate for gluconeogenesis. This contradicts McCandless and Dye (1950) who postulated that decreased glucose tolerance reflected quantitative differences in metabolism coincident to the U 19 fermentation of carbohydrate in the rumen. Low glucose tolerance characteristic of the adult ruminant was believed to be due to increased rates of gluconeogenesis. Just the Opposite dietary effects have also been ob- served. Murley 33 al. (1952) and Wing 2£.§l° (1955) em- ploying calves and feeding reconstituted skimmed milk diets found that at four days of age fasting blood glucose levels were approximately 85 mg% and fell to approximately 55 mg% by the fifth week. The fasting blood glucose decreased even though composition of the diet was unchanged, i.e. only skimmed milk. Jacobson it; _a_1_. (1951) compared calves fed milk diets with calves on varying roughage diets and found similar trends in blood glucose levels regardless of treat- ment. Other workers (Hodgson.g£,al., 1932 and Lambert, 1955) have found that diet has no effect on fasting blood glucose in maturing dairy cattle. In an interesting study by Reid (1953), the major portion of the decrease in blood glucose during the first four to five weeks of life of the lamb was due to a decrease in corpuscle glucose. Post-absorptive blood glucose levels fell steadily with advancing age from levels of 85 - 90 mg% in the first week of life and stable adult levels were .reached at the age of six to nine weeks. Since 50 percent of the total decrease in blood glucose occurred before rumi- nation had started to any appreciable amount, diet was not the factor influencing blood glucose levels. Few intravenous acetate tolerance tests have been 20 reported in the literature. Ciaranfi and Fonnesu (1945) found that a load of 0.25 g acetic acid per kg body weight was rapidly removed from the blood of the dog and disa- pearance occurred at a constant rate depending on the initial blood concentration. Disappearance was markedly reduced in the diabetic dog (Ciaranfi and Fonnesu, 1945). Acetate tolerance in the cat was found to be similar to that of the dog (Smyth, 1947) whereas the rate of disappearance in the adult sheep was considerably slower (Jarrett'g§.a1., 1952; Reid, 1958). Jarrett and Filsell (1960) showed that after the injection of acetate at the rate of 5 m moles per kg body weight, blood acetate levels returned to pre-injection levels within 30 minutes in lambs under four months of age. Lambs over four months of age required 60 minutes, the same time required by adult sheep. This observation supported the hypothesis that the young lamb metabolically behaves much like the non-ruminant and gradually develops the metabolic characteristics of the adult ruminant. Reid (1958) showed that the diet of the adult sheep had a marked effect on acetate tolerance. An acetate half-life of approximately 10 minutes was observed for sheep consuming a diet containing 50 percent cracked maize and increased to approximately 25 minutes for those animals consuming a re- stricted high-roughage diet. Reid suggested that the faster rate of acetate utilization in those sheep fed the 50 percent maize ration was related to an increased rate of entry of acetate into the pathways of oxidation and fat synthesis. 21 Support for this suggestion stemmed from blood acetate levels which were considerably lower in relation to rumen levels on diets containing increased amounts of wheat or maize when compared to relative blood acetate levels of sheep fed a roughage ration. D. Gross Dietary Effects on the Gluconeogenic and Glycolytic Pathways as Assessed by Activities of Key Enzymes Alterations of major metabolic pathways (gluconeogenesis and glycolysis) in response to dietary and hormonal manipu- lations have been extensively investigated. These investi- gations, however, have been made employing the rat and, to a lesser extent, other nonruminants, while the ruminant has been largely neglected. Because of this lack of knowledge concerning changes in the carbohydrate metabolism of the ruminant due to the effects of dietary regime, most current information as to the assessment of major metabolic pathways by key enzyme activities proceeds from the nonruminant. Changes in the activity of the key enzymes associated with gluconeogenesis have been observed in response nadistary and hormonal treatments. Starvation, diabetes, high protein- 1ow carbohydrate diets, and adrenocortical hormones have all been shown to increase the activity of hepatic glucose-6- phosphatase (Langdon and Weakley, 1955; Freedland and Harper, 1957; Freedland and Harper, 1958a and 'b; Kvam and Parks,l960; Weber 2; al., 1961; Weber 2; al.,1963; Weber g£,al., 1965a,b and c; Freedland 31; _a_1_., 1966; Freedland and Harper, 1966; Szepesi and Freedland, 1967; and Szepesi and Freedland,]£MBcL 22 fructose-1, 6-diphosphatase (Mokrasch 23‘a1., 1956; Kvam and Parks, 1960; Weber g3 al., 1961; Weberugp'a1., l965a,b and c; Szepesi and Freedland, 1967; Szepesi and Freedland, 1968c), pyruvate carboxylase and phosphoenolpyruvate carboxykinase (Henning 3;,al., 1963; Shrago g; a;., 1963; Weber 3; al., 1965a; and Tepperman g£_al., 1970). Decreased activity due to starvation, diabetes and low carbohydrate diets have been shown for the glycolytic enzymes glucokinase (Salas'gpla1., 1963; Vinuela.g£[al., 1963; Blumenthal‘§£.al., 1964; Oliver and Cooke, 1964; Sharma.g£lal., 1964; and Sillero gp‘al., 1969) and pyruvate kinase (Krebs and Eggleston, 1965; Weber .2£.§l°9 l965d; Yudkin and Krauss, 1967; Sillero g§.al., 1969; Szepesi and Freedland, 1968a; and Szepesi and Freedland, 1969a). Activity of glutamic-pyruvic transaminase [E.C.2.6. 1.2], an enzyme closely related to gluconeogenesis, has been shown to increase in response to the same factors which increase the gluconeogenic capacity of the rat. The factors have been found to be starvation, diabetes, high protein-low carbohydrate diets and glucocorticoids (Rosen.gtla1., 1958; Rosen.2£‘al., 1959; Segal g$_a1., 1962; Szepesi and Freedland, 1968b and c; Szepesi and Freedland, 1969b). Glucose-6- phOSphate dehydrogenase [E.C.l.1.l.49], the initial enzyme of the pentose phosphate cycle, also has been shown to be influenced by the dietary regime of the rat and to be an in- dicator of capacity of the pathway as starvation, diabetes, low carbohydrate and high fat diets all have been shown to depress the activity of this enzyme (Niemeyer 33 a1., 1962; 25 Ono 23‘a1., 1963; Vaughn and Winders, 1964; Benevenga g; a;., 1964; McDonald and Johnson, 1965; Peraino, 1967; and Szepesi and Freedland, 1968c). Fitch and Chaikoff (1960) showed a three-to-ten fold increase in glucose-6-phosphate dehydro- genase activity in rats fed a 60 percent hexose diet and Peraino (1967) showed that protein was required in the diet for the induction of the enzyme by carbohydrate to be expressed. The effects of diet on overall metabolic pathways of the ruminant have been reported. Alterations in gluconeo- genic or glycolytic metabolism have been assessed to a limited extent in the ruminant by enzymatic studies but whether the changes have been adaptions to diets or were constitutive effects has not been clearly demonstrated. To study the effect of diet on the rate of glucose utilization, Ford (1965) fed rations with increasing amounts of crude protein to adult sheep and observed increasing rates of glucose utilization and percentages of exhaled carbon dioxide derived from glucose. Also (Ford and Reilly, 1969), increased glucose entry rates of 2.25 mg/min/kg were observed for sheep fed a pelleted grass diet as compared to 1.2 mg/min/ kg observed for animals consuming a hay diet. Concurrent increased rates of free amino acids utilization were also reported. Ulyatt g3 a1. (1970) presented work concerning the effect of diet on glucose entry rates in sheep which were largely at variance with Ford (1965) and Ford and Reilly (1970). Feeding an 85 percent barley, dried grass or hay 24 diet which supplied greatly differing amounts of CX-linked glucose polymer and crude protein, no differences in glucose entry rate, plasma glucose concentration or size of the total body glucose pool was observed. It would be eXpected that the diets high in starch may produce a higher pr0portion of pro- pionate during rumen fermentation and considerable amounts of dietary starch may escape fermentation and become avail- able for absorption in the lower gastrointestinal tract. Both factors would then be expected to alter the rate of glucose utilization. The effect of diet on glucose entry remains unsolved. Work has shown that the factors which increased the gluconeogenic capacity of the adult sheep, i.e., fasting and diabetes, have been reflected by increased activities of key gluconeogenic enzymes (Filsell et a1., 1969). It was shown that after a six to seven day fast, hepatic activities of glucose-6-phosphatase, fructose-1, 6-diposphatase and pyruvate carboxylase increased with pyruvate carboxylase exhibiting a four-fold increase. Phosphoenolpyruvate car- boxykinase showed about a two-fold increase only in sheep which were made diabetic by pancreatectomy or alloxan treatment. Wagle and Nelson (1966) compared the activities of gluconeogenic enzymes of adult sheep to those of the rat. The activity of glucose-6-phosphatase, fructose-1, 6-diphosphatase, phosphoenolpyruvate carboxykinase and pyruvate carboxylase in sheep was twice that observed in rats and they attributed the high enzyme activity to the greater 25 gluconeogenic capacity of the adult ruminant. Unfortunately, neither the diet of the sheep nor the rat were stated in the report. Goetsch (1966) observed a gradual decrease in activity enzymes associated with glycolysis as calves matured over an eight week period. The decrease was stated to be due to decreased rates of glucose absorption from the lower tract as a consequence of rumen develOpment. Bartley 33 al. (1966) found that the activities of phosphorylase and glucose-6-phosphatase increased in calves between eight to twelve weeks of age to levels found in the adult cow. The increased activities were cited as evidence that metabolic adaptations involving carbohydrate metabolism coincide with develOpment of rumen function (a response to a decrease in availability of glucose from the intestinal tract). Also, the intraduodenal infusion of massive doses of glucose into fifteen week old calves or mature cows de- creased the activity of glucose-6-phosphatase and phosphory- lase to the level of that observed in calves with non- functional rumens. Therefore, the enzymatic responses observed with maturity were related to the availability of glucose. Bartley 23 a1. (1966) thus provided evidence for the adaptability of metabolic processes of ruminants to substrate availability. Purser and Bergen (1969) compared the metabolic proc- esses of gnotobiotic goats, and conventional goats and rats, fed a diet containing high levels of easily digestible 26 carbohydrates. It was assumed that gluconeogenic activity was assessed by glucose-6-phosphatase activity and glycolytic activity by pyruvate kinase. Glucose-6-phosphatase activity was found to be greater in the gnotobiotic and conventional goat than the rat while pyruvate kinase activity was com- parable in all three. The glucose-6-phosphatase activity of the gnotobiotic and conventional goat was not different. This was contrary to the eXpectation that the gnotobiotic goat, with the absence of normal rumen development, would have exhibited lower glucose-6-phosphatase activity. No differences in pyruvate kinase activity were noted for the gnotobiotic or conventional goat, indicating no greater glycolytic activity in the gnotobiotic goat. This was also contraty to expectation. Purser and Bergen (1969) were able to show that a gnotobiotic animal fed the same diet only lower in protein, had an apparent reduction in the rate of glycolysis as measured by a substantial decrease in pyruvate kinase activity. Decreased plasma glucose concentration and increased glucose half-life with increasing age were repommm. for both the gnotobiotic and conventional animal. The evidence indicated that in contrast to Bartley‘g£,a1. (1966) and Goetsch (1966) changes in metabolic processes of the ruminant may not be limited to the inductive role of various nutrients. III EXPERIMENTAL PROCEDURE A. Introduction Experimentation consisted of two trials which were con- ducted over a period of two years, 1969 and 1970. Pygmy goats were obtained by hysterotomy and maintained as germ- free or conventional animals. Dietary treatments were imposed, consisting of a "glucose", "volatile fatty acid" or a commercial pelleted germfree animal diet. Changes in patterns of carbohydrate metabolism as a function of matu- rity of the animal were assessed by glucose and acetate tolerance tests, liver metabolite levels and activities of key gluconeogenic or glycolytic enzymes. B. General Conduct of Experiment 1. Procurement and maintenance of the pygmy goat. Because of their size and temperment, pygmy goats were the animal of choice for these germfree experiments. Male pygmy goats were obtained from the Primate Research Center, Oregon State University and bred to Toggenburg does. Off- spring of this cross were only part pygmy although they will be referred to as pygmy goats throughout this manuscript. The preparative, surgical, and pggg partum technique of the hysterotomy have been fully described (Oxenderig£.a;., 1971). Germfree preparative procedures, sterility control ’ 27 28 and isolator equipment also have been described (Meyer,gp a1., 1964). Kids taken by hysterotomy into germfree iso- lators will be referred to as germfree and those taken by this procedure but kept in nonsterile isolators will be called conventional goats. 2. Experimental diets and feeding regimes Composition of the basal diet is shown in Table 1. This diet was the major constituent of the glucose and volatile fatty acid diets which were made by adding iso- caloric amounts of glucose or volatile fatty acid salts in the amounts shown in Table 2 to this basal diet. These diam; were sterilized and fed on a dry matter basis at the rate of 3 percent on the animal's body weight. Feeding of this liquid diet was accomplished by using a regular human infant's baby bottle and nipple. The pelleted germfree diet was a commercial preparation and was fed.ad libitum. The composition of this diet appears in Table 3. 3. Experimental Protocol - 1969 Six germfree and three conventional animals were all maintained from birth on the basal diet with two of the conventional goat's diets supplemented (ad libitum) with the pelleted germfree animal diet. Glucose and acetate tolerance tests were performed in.§;§g twenty-four hours before the sacrifice of the animal. At the time of sacri- fice, the kid was quickly removed from the isolator, electrocuted and its liver was excised and chilled for enzyme analysis. Activities of the hepatic enzymes pyruvate 29 TABLE 1. Composition of Basal Diet Constituents Amount Cow's milk 19.0 L Glucose 227.0 g Solka Floca 213.0 9 Magnesium sulfate (M9804 7H20) 11.4 9 Vitamin mixb 22.7 g a Solka.Floc, Brown Company, Chicago, 111. b General Biochemicals, Chagrin.Falls, Ohio. Composition of the vitamin mixture in grams per 45.5 kg: Thiamine 4.36, ribo- flavin 10.90, niacin 72.64, Ca panto- thenate 43.58, choline chloride 3,632.00, 0.1% B12 in manitol 72.64, folic acid 0.91, PABA 181.60, biotin 0.36, pyri- doxine 3.63, and dextrose filler. TABLE 2. Composition of Experimental Diets Constituents Glucose Diet VFA Diet Basal diet 1.5 L 1.5 L Glucose 32.2 g Sodium acetate 30.0 g Sodium pr0pionate 11.1 g Sodium butyrate 2.1 g 3O TABLE 3. Composition of Pelleted Germfree Animal Diet1 Amount Constituents (g/kg) L-lysine HCl 5.000 DL-methionine 5.000 Soybean meal (50% 300.000 Alfalfa meal (17%) 35.000 Corn, whole yellow ground 589.625 Oil, corn 30.000 Salt mix (see below) 25.250 BHT (butylated hydroxytoluene) 0.125 Vitamin mix (see below) 10.000 Salt Mix g/kg Calcium carbonate (CaC03) 5.00000 Cobalt chloride (C0C12°6H20) 0.00266 Cupric sulfate (Cuso4-5H2o) 0.00553 Calcium phosphate dibasic (Ca2HP04) 10.00000 Ferric citrate (FeC6H705' 5H20) 0.13213 Manganese sulfate (MnSO4- 4H20) 0.20329 Potassium iodate (K103) 0.00222 Sodium chloride (NaCl) 10.00000 Zind sulfate (ZnSO4- 7H20) 0.06587 Vitamin Mix g/kg PABA 0.0506 B12 Vitamin 0.0044 Calcium pantothenate 0.2860 Choline chloride 1.9800 Folic acid 0.0110 Menadione 0.8800 Niacin 0.0660 Pyridoxine HCl 0.0220 Riboflavin 0.0308 Thiamine HCl 0.0660 IU/kg Vitamin A 26,400.0 Vitamin D3 1,200.0 Vitamin E acetate 100.0 1 General Biochemicals, Chagrin Falls, Ohio. 31 kinase, glutamic-pyruvic transaminase and g1ucose-6-phospha- tase were determined. Age of the animal at sacrifice varied. Two germfree animals were sacrificed at 49 days of age, four germfree animals at approximately 124 days and the conventional animals at approximately 130 days. 4. Experimental Protocal - 1970 Experimental protocol used in 1970 was slightly more detailed. Animals were procured and maintained by exactly the same techniques as those employed in 1969, however the number of animals used, the diets imposed, the number of tolerance tests given and the liver parameters investigated were altered. Six germfree goats were used three of which were fed the glucose diet (Table 2) and the rest were fed the VFA diet (Table 2). There were five conventional animals, two con- sumed the glucose diet, two the VFA diet and one, the pel- leted germfree animal diet. Two tolerance tests per animal were performed ip.§i£g; the first at two months and the second at six months of age. Twenty-four hours after the last tolerance test, each animal was removed from its germfree or conventional isolator and immobilized on its left side upon a surgical table. A 10 ml blood sample from the jugular vein was taken for plasma amino acids. Hair was carefully clipped from the rib cage area directly posterior to the axilla. After removal of the hair, the area between the third and fourth rib from the 32 the caudal section of the rib cage was anesthetized to permit a three centimeter incision, running dorsal to ven- tral between the two ribs and extending into the abdominal cavity. After this incision was made, the lobe of the liver closest to this Opening was exteriorized. Approximately three grams of liver were excised with surgical scissors and quickly plunged into liquid nitrogen. This area of liver was sampled each time and the time required for removing the hepatic tissue until plunging it into liquid nitrogen took consistently under 10 seconds. These samples were stored at -70°C until the determination of the concentration of liver metabolites i.e., glucose-6-phosphate and phosphoenolpyruwna, was conducted. After the excision of the liver tissue, the animal was immediately electrocuted and the remaining liver tissue was removed and quickly chilled. One 5-10 gram sample of the liver portion was frozen for liver free amino acid determination and the rest was used immediately for enzyme analysis. Activity of the hepatic enzymes, pyruvate kinase, glucose-6-phosphatase, glucose-6-phosphate and 6-phospho- gluconate dehydrogenase was determined. C. Procedures and Analytical Determinations 1. Performance of the tolerance tests All glucose and acetate tolerance tests were performed at eight o'clock in the morning on twenty-four fasted animals. Tolerance tests on germfree animals were performed under sterile conditions. The goat was physically restrained and the hair was clipped from the area over the external 33 jugular veins. An 18 gauge bleeding needle was inserted into the jugular vein and an initial 4 ml (To) blood sample was withdrawn. Teflon tubing of a diameter small enough to pass through the needle was then inserted into the vein and the needle was subsequently removed. Simultaneous infusion of glucose and acetate was ac- complished via this catheter by mixing these solutions in the syringe and then injecting the mixture over the period of one minute. A glucose solution (30% w/v) and acetate solution (25% w/v) were injected at the rate of 0.30 g/kg and 0.25 g/kg respectively. Both solutions were sterile. These methods and dosages are those of Reid (1958). At 5, 10, 15 and 20 minutes after infusion of the glucose-acetate solution, blood samples were taken. Four ml aliquots were withdrawn using a 10 m1 syringe and immediaely transferred the heparinized test tubes. Frequent flushing of the catheter with heparinized saline was required to prevent clots in the teflon tube. 2. Determination of blood glucose and acetate con- centrations and calculation of half-life The 4 ml blood samples were centrifuged at 10,000 x g for 10 minutes and 2 ml of the plasma was pipetted into another centrifuge tube. Then 0.4 m1 of 25% 5-sulf0- salicylic acid was added and the mixture was allowed to re- main on ice for 30 minutes while precipitation of plasma proteins ensued. After complete precipitation of proteins had been effected, the protein-free supernatant was stored frozen until analyzed for glucose and acetate. 3L. Glucose analysis was achieved by a modification of the glucose oxidase method of Hugget and Nixon (1957). A 0.2 ml aliquot of the protein-free supernatant was neutralized with 0.02 N sodium hydroxide solution which gives a final volume of 3.3 ml. One ml of sample, standard glucose solutions or blank was used in duplicate in the assay applying the prepared Glucostatl reagents and procedure. Blood acetate was determined by gas-liquid chroma- tography using the protein free supernatant just described. Approximately 5,» l aliquots were injected onto a 6.0 foot by 0.25 inch glass column packed with Chromosorb 1012 in a Packard Gas Chromatograph. Inlet, column and detector tem- peratures were 188, 195 and 215°C, respectively. Carrier gas was nitrogen, flowing at the rate of 40 ml per minute and detection was accomplished using a hydrogen flame ioni- zation detector. The concentration of acetate in these samples was determined by employing appropriate acetate standard solutions and comparing peak heights. Calculation of the half-life of glucose and acetate was by means of the method of Reid, 1958. If the relationship between log blood glucose or acetate concentration and time is linear, then the half-life of these metatolites can be represented by: T = 1. log 2 i k e 1 Worthington Biochemical Corporation, Free hold, New Jersey, 07728. 2 Varian Aerograph, Walnut Creek, California, 94598. 35 where T% is the half-life (min.) and k is the specific rate constant (minTl), equal to the fraction of the excess glucose or acetate removed per minute. The constant k is more simply the lepe of the line which delineates the relationship between log blood glucose or acetate concen- tration (pre minus post injection conc.) and the sampling time. 3. Liver free amino acids Thawed samples of liver (taken for liver free amino acid analysis) were prepared essentially according to Clark .g£'§;., 1966, using norleucine as an internal standard. One ml Of lmM norleucine was added to l g of tissue in 4 ml of deionized distilled water and homogenized in a Virtis homo- genizer at tOp speed for 3 minutes. The homogenates were mixed with 5 ml of 10% (w/v) 5-sulfosa1icylic acid and centrifuged at 32,000 x g for 10 minutes after precipitation of protein was complete. Five m1 of the protein-free supernatant were pipetted into a round bottom flask and concentrated in a rotary flash evaporator to near dryness. The residue was dissolved in 3 ml of 0.3N lithium-0.05M citrate buffers (pH 1.9). A dual column Technicon TSM amino acid analyzer performed the amino acid analyses on 0.4 ml aliquot of this solution (Bergen and Potter, 1971; Makdani 33 a1., 1971). 4. Plasma amino acid determination The 10 ml blood samples, taken at the time Of sacrifice, 36 were collected into heparinized syringes and the plasma separated by centrifugation as described above. To each ml of plasma thus obtained, 0.1 p.mole of norleucine was added as the internal standard. A 0.1 m1 aliquot of 50% (w/v) 5-su1fosalicylic acid then was added per m1 of plasma and the proteins were allowed to precipitate for 30 minutes on ice. The protein-free supernatant was decanted after centri- fugation of the mixture at 20,000 x g. The supernatant was stored frozen until plasma amino acid concentrations were determined with the Technicon Auto Analyzer. 5. Liver metabolite extraction and determination a. Tissue powdering procedure The tissue powdering procedure was performed in a cold-room at 4°C to reduce water uptake by the tissue from the air. Three gram samples of tissue were placed in a mortar and pestle, precooled by liquid nitrogen and ground. The samples were not allowed to thaw during this process. After the powdering was complete the ground tissue was added to 6% H010,+ (w/v) to effect precipitation of proteins and extraction of metabolic intermediates. The ratio of the final total liquid volume of the sample to its original weight was 1:6. For example, if 2 g of tissue were used, then 10.5 ml of HC10# were needed (based on 25% dry matter of liver tissue). A 15 ml centrifuge tube containing a glass rod and 5 ml of 6%IHClO4 was weighed. The powdered tissue was then added and the centrifuge tube reweighed. From the increase in h 37 weight the corresponding amount of 6% H0104 was calculated from the above ratio and added allowing for the original 5 ml in the tube. The glass rod was used to stir and grind tissue. After an extraction time of 10 minutes, the prepara- tion was centrifuged at 3000 Xig and the supernatant was de- canted into 50 ml beakers cooled in ice. Titration of this acidic supernatant to pH 6.0 was accomplished using 5 M K200} and a Corning pH meter. This pH provides maximum stability of the metabolites determined particularly phos- phoenolpyruvate. The amount of K2003 added was recorded and the dilution due to its addition was calculated into the final dilution factor. After the evolution of C02 stOpped the perchlorate salts settled, the supernatant was decanted and used immediately for the determination of liver meta- bolites. b. Determination of D-glucose-6-phosphate The determination of D-glucose-6-phosphate was performed by the method of Hohorst in Bergmeyer (1963). The method is based on the following reaction catalyzed by the enzyme, g1ucose-6-phosphate dehydrogenase: Glucose-6-phosphate+NADP2::6-phosphogluconolactone+NADPH2 Extent of the reaction is measured by the increase in optical density at 340 mll when NADP is reduced. The blank solution was 0.4 m triethanolamine buffer (pH 7.6) and the final volume of the assay was 1.025 ml. All spectrOphotometric measurements were made in duplicate on a Gilford Recording SpectrOphotometer, Model 2000. 38 0. Determination of phosphoenolpyruvate This determination was performed by the method of Czok and Eckert in Bergmeyer (1963). The concentration of phosphoenolpyruvate is measured by the following coupled reactions: Pyruvate Kinase PEP + ADP -—-—-—3 pyruvate + ATP F +2 + Mg,K LDH Pyruvate + NADI«12-.‘-?—-A lactate + NAD The decrease in optical density due to the oxidation of NADPH2 is pr0portiona1 to the amount of phosphoenolpyruvate present. The assay was carried out at 340 m}1 to increase the accuracy of analysis and the maximum amount of de- proteinized sample used in the assay was 1.50 ml. The assay was blanked against air and measurements were made in duplicate using the Gilford Recording Spectrophotometer, Model 2000. 6. Enzyme preparation and assay a. Liver preparation The whole liver was weighed and minced with surgical scissors on ice. This mincing procedure was done to obtain random samples of liver for assay, since enzyme activity for particular enzymes may be localized in hepatic tissue. ‘kua 39 One portion Of this liver was homogenized with 19 volumes of cold, 0.1 M sodium citrate buffer (pH 6.5) in a Potter-Elvehjem homogenizer, strained through two layers of cheesecloth and used the same day of preparation for the assay of glucose-6-phosphatase activity. Another sample of tissue was homogenized with 4 volumes of cold 0.14 M KCl in a Potter-Elvehjem homogenizer and was centrifuged for 20 minutes at 10,000 x g for the assay of glutamic-pyruvic transaminase, 30 minutes at 20,000 x g for the assay of glucose-6-phosphate and 6-phosphogluconate dehydrogenase, and 30 minutes at 30,000 x g for the assay of pyruvate kinase. All supernatants were filtered through cheesecloth and used the same day for assay. b. Glucose-6-phosphatase assay The method of assay Of glucose-6-phosphatase activity was that described by Harper in Bergmeyer (1963). The rate of the enzymatic hydrolysis of glucose-6-phosphate by glucose-6-phosphatase was measured by the increase in inorganic phosphate in a 15 min assay. Absorbance due to the liver homogenate was corrected for and citrate buffer (pH 6.5) was used as the blank. All SpectrOphotometric measurements were made using a Coleman Junior II. This method has been used in this laboratory in other studies (Purser and Bergen, 1968). c. Glutamic-pyruvic transaminase assay The assay of this enzyme activity was performed by the method of Bergmeyer and Bernt in Bergmeyer (1963). 40 The glutamic-pyruvic transaminase reaction was coupled with the lactic acid dehydrogenase reaction and glutamic-pyruvic transaminase activity was then measured by the decrease in Optical density at 340 m}l due to the oxidation of NADHZ. The blank used was 0.5 m phosphate buffer (pH 7.6) and SpectrOphotometric measurements were made using a Gilford Recording SpectrOphotometer, Model 2000. d. G1ucose-6-phosphate and 6-phosphogluconate dehydrogenase assay The assay of these enzyme activities was made following the method of Freedland (1967). The rate of the enzymatic reactions was followed by the increase in optical density at 340 m}1 due to the reduction of NADP. The blank solution was 0.017 m glycylglycine (pH 7.6) and all spectro- photometric measurements were made in duplicate using a Gilford Recording SpectrOphotometer. e. Pyruvate kinase assay This assay for enzymatic activity was performed by the method of Bficher and Pfleiderer in Colowick and Kaplan (1955). The pyruvate kinase reaction was coupled to the lactic acid dehydrogenase reaction and the decrease in Optical density at 340 mlldue to the oxidation Of NADH2 was used to measure enzymatic rate. The blank solution was lemn triethanolamine buffer (pH 7.5) and spectrOphotometric measurements were made in duplicate using a Gilford Recording SpectrOphotometer. This assay has been used in this laboratory for other metabolic studies (Purser and Bergen, 41 1968). f. Protein determination on liver enzyme preparations Protein determinations on liver homogenates were performed by the Folin-phenol method, modified by Miller, 1959. Bovine serum albumin was the standard refer- ence protein and spectrOphotometric measurements were per- formed on a Coleman Junior 11 SpectrOphotometer. D. Statistical Analysis The data from the 1969 trial were analyzed by a One-way analysis of variance. Least squares analysis of variance was employed to define the significant relationship in the 1970 trial. The inverse relationship between pyruvate kinase and glucose half-life was analyzed for statistical difference by the student's t-test. IV RESULTS AND DISCUSSION The experimental design differed between the 1969 and 1970 trials. The 1969 trial consisted of six germfree and three conventional pygmy goats. All six of the germfree goats and one of the conventional animals were fed the basal diet (Table l) and the two other conventional animals re- ceived the germfree pelleted diet (Table 3). These animals were sacrificed at various ages and all experimental parameters were determined at this time. The germfree animals, 20 and 21, were sacrificed at 49 days of age while animals 11, l7, l8 and 19 were sacrificed at 146, 118, 115 and 117 days respectively. Animal 11 was approximately 30 days older than the others, but since it was germfree it was included in this group. Animal 16, a conventional animal, fed the basal diet was sacrificed at 123 days while 14 and 15, conventional animals fed the pelleted diet, were sacrificed at 151 and 121 days. Statistical analysis of all the data of the 1969 trial was computed by a one-way analysis of variance. The effect of age on the metabolic parameters investigated was analyzed. The three groups by animal number were 20 and 21; ll, l7, l8 and 19; and l4, l5 and 16, representing the ages of 49, approximately 124 and 130 days. The reason for such an arrangement is that there was a lack of control animals if 1.2 1+3 one were to analyze on the basis of dietary regime or germ- free status. The experimental design of the 1970 trial consisted of six germfree pygmy goats, three of which received the gluana diet (Table 2) and the other three the VFA diet (Table 2). There were four conventional animals, two of which received the glucose diet and two the VFA regime. Finally there was one conventional goat which received the pelleted germfree diet (Table 3). Fasting plasma glucose levels and glucose and acetate half-lives were determined on all animals at approximately 60 days of age. At 180 days fasting plasma glucose levels, glucose and acetate half-lives were re- peated, the animals sacrificed and enzyme activities and metabolite determinations were made. These data were analyzed by the method of least squares. The effect of diet, germfree status and diet-germfree status interactions on the metabolic parameters investigated were determined. The one conventional animal receiving the pelleted diet was excluded from the statistical analysis. As the ruminant matures fasting blood glucose levels have been observed to decrease from approximately 100 mg% to 50-60 mg% (McCandless and Dye, 1950 and Reid, 1955) and glucose and acetate half-lives increase (Jarrett and Filsell, 1960 and Purser and Bergen, 1969). Experimental work with young ruminants has shown that fasting blood glucose level and glucose tolerance could be held constant over extended periods if a diet of milk and milk solids was fed to calves 44 (Attebery and Colvin, 1965 and Harry 2;.al., 1967). The inclusion of hay and/or grain in the diet (Harry.g§.al., 1967) and subsequently the initiation of rumen fermentation (McCarthy and Kesler, 1956; McCandless and Dye, 1950 and Hale and King, 1958) were postulated to be the cause of decreased blood glucose concentration and increased glucose tolerance. In the adult sheep, Reid (1958) showed that glucose and acetate tolerance could be greatly influenced by diet. Shortest half-lives (fastest rate of utilization) for both glucose and acetate were observed when a 50 percent maize diet was fed. Longest half-lives (slowest rate of utilization) were observed in those sheep consuming a re- stricted roughage diet. These studies indicate dietary control over blood glucose levels and glucose and acetate tolerance in both the young and adult ruminant. Others (Murley.g§‘§l., 1952 and Wing.gt'al., 1955) found that blood glucose levels decreased to adult levels by the fifth week of feeding a skimmed milk diet to calves. Hodgson.gt‘§1. (1952), Jacobson gt‘al. (1951) and Lambert (1955) also showed that various diets had no effect on fasting blood glucose levels in maturing cattle and Reid (1953) found blood glucose levels to fall in lambs before rumination could be detected. These results advocate some factor other than diet in control of fasting blood glucose. One of the objectives of the 1969 and 1970 trials was to determine the control of these processes. The 1969 trial employed germfree and conventional pygmy goats, fed a basal 45 diet which contained essentially cows milk with an extra 12:; of glucose per liter or a germfree pelleted diet which wasjfl) percent ground corn (Tables 1 and 5). In 1970 the dietary regime was changed from the basal diet of 1969 to the "glucose" and "VFA" diet. The glucose diet consisted of the basal diet to which an extra 21.4 g of glucose per liter had been added. The VFA diet consisted of 20 g, 7.4 g and 1.4 g of sodium acetate, propionate and butyrate per liter of basal diet. The glucose and VFA diets of 1970 were used with the expec- tation of exaggerating differences in certain metabolic patterns. The high carbohydrate diets of both 1969 and 1970 were eXpected to maintain the rate of glycolysis (especially in the germfree animal) at the rate observed in the very young ruminant, while the animals consuming the VFA diet of 1970 were expected to develop patterns of metabolism character- istic of the adult ruminant, i.e., decreased rates of glycolysis and increased rates of gluconeogenesis. Control animals were conventional pygmy goats fed either the basal, glucose or VFA diet. Also included in these experiments were conventional goats fed a high grain diet (germfree pelleted diet) in order to have a normal reference ruminant. The effect of the basal diet in 1969 on fasting plasma glucose concentration and glucose and acetate tolerance, if these parameters are under dietary control in the germfree goat, would be the maintenance of plasma glucose and glucose and acetate half-lives at those levels characteristic of the 46 very young ruminant. Since the absence of rumen fermen- tation would allow the direct absorption from the lower gut and hence, utilization of the glucose and other carbo- hydrates of the basal diet, plasma glucose concentration would be expected to be high and the rate of glucose and acetate utilization fast in comparison to those conventkmal goats fed the pelleted diet. Because rumen fermentation was allowed to develop in these conventional animals, de- creased plasma glucose concentration and a decreased rate of glucose and acetate utilization, all characteristic of the normal ruminant, was expected. The conventional animal fed the basal diet was a control to the germfree animals and was used to assess the effect of germfree status on the animal. Dietary changes of 1970 were made in order to increase differences in the parameters measured, however the same general trends were exPected. The glucose diet fed to both germfree and conventional (control) animals was expected to maintain plasma glucose levels and glucose and acetate utilization rates above those levels and rates seen in germ- free and conventional animals fed the VFA diet and the animal fed the germfree pelleted diet. The germfree animals fed the VFA diet, a diet which supplied acetate, propionate and butyrate, were expected to exhibit plasma glucose concentrations and glucose and acetate utilization rates typical of the animal having normal rumen function. Plasma glucose levels were expected to be lowered 47 and glucose and acetate tolerance decreased in comparison to the germfree goats fed the glucose diet and similar to the conventional animals fed the germfree pelleted diet. Tables 4, 5 and 6 present fasting plasma glucose con- centrations (To glucose) and half-lives for plasma glucose and acetate from the 1969 and 1970 trials. In 1969 (Table 4) the two germfree animals at 49 days of age had an average plasma glucose concentration of 132.5 mg/100 ml and the four germfree animals aged 124 days had an average To glucose concentration of 115.6 mg/100 ml. In comparison, the conventional animals at 150 days averaged 94.7 mg/100 ml. Since these differences approached statistical signif- icance at the P<:0.05 level, these data suggest decreased To glucose concentrations with advanced age. Any con- clusions as to the effect of age on fasting plasma glucose concentrations should be made by comparing the two groups of germfree animals. If this parameter (To glucose) is under dietary control, one would not expect a decrease. The decrease in plasma glucose levels seems to be due to advancing age and independent of diet, rumen development and volatile fatty acid production. Animal 16, a conventional animal, had a fasting plasma glucose concentration of 110.4 mg/100 ml which is comparable to those levels observed in germfree animals at 124 days of age. This animal may have been behaving more like a germ- free animal since the basal diet fed via a nipple-bottle passed directly to the abomasum since’drksov gtflgl. (1970) TABLE 4. 1969 To Glucose and Glucose and Acetate Half-lives Age of Glucose Acetate Animal Weight Animal To Glucose Half-life Half-life Number (kg) Status (days) (mg/lOOnfl) (min) (min) 20 2.27 GFa 49 127.3 32.4 47.5 21 2.77 GF 49 137.6 29.4 23.3 Mean :l: S.E.M. 132.51: 30.93: 35.44b 5.2 1.5 12.1 17 7.26 GF 118 133.9 18.0 6.8 18 6.80 GF 115 106.3 52.0 7.4 19 7.71 GP 117 127.5 35.8 9.7 11 7.71 GF 146 94.9 19.8 12.8 Mean i S.E.M. 115.6:I: 31.43: 9.21: 9.1 7.9 1.4 14 8.16 CONC’d 151 83.9 86.0 4.3 15 6.80 CONd 121 89.8 18.2 15.3 16 6.35 CON 123 110.4 16.6 16.8 Mean i S.E.M. 94.71 40.31: 12.14 8.0 22.8 3.9 a Animals designated GP were germfree. b Significantly different (P<:0.025) from means of other age groups in the acetate half-life trial. C Animals designated CON were conventional. d Fed the pelleted germfree animal diet gg libitum in addition to basal diet. 49 TABLE 5. 1970 To Glucose and Glucose and Acetate Half-lives at Approximately 60 Days of Age Age of Glucose Acetate Animal Weight Animal To Glucose Half-life Half-life Number (kg) (days) Status Diet (mg/ 100 ml) (min) (min) 22 10.9 111 GP Glucose 94.6 11.5 3.09 24 6.8 61 GP Glucose 144.0 6.9 1.20 27 6.8 55 GP Glucose 45.1 50.2 - Mean:i S.E.M. 94.&£ 22.8i 2.141 28.5 13.7 0.94 23 6.8 69 GP VPAb 153.4 - - 25 6.8 64 GP VFA. 99.0 9.7 - 26 6.8 63 GP VFA 99.2 18.5 2.67 Mean :1: S.E.M. 117.2}: 14.13: 18.1 4.4 30 9.1 61 CON Glucose 84.4 15.4 2.73 31 7.7 59 CON Glucose 85.0 17.3 3.54 Mean:i S.E.M. 84.7i 16.4i 3.131 0.3 0.9 0.39 28 9.1 67 CON VPA 105.6 13.4 1.69 32 7.7 59 CON VPA 89.0 14.7 3.04 fleanzf S.E.M. 97.3i 14.1i 2.36f 8.3 0.7 0.67 36 9.1 100 CON pelletsC 86.2 110.3 4.83 a Table No. 2. b Table No. 2. c Table No. 3. TABLE 6. 50 1970 To Glucose and Glucose and Acetate Half-lives at Approximately 180 Days of Age Age of Glucose Acetate Animal Weight Animal To Gluc°se Half-life Half-life Number (kg) (days) Status Diet (mg/IOOHU) (min) (min) 22 10.9 180 GP Glucosea 99.9 14.0 4.99 24 6.8 181 GP Glucose 78.5 14.7 7.11 27 6.8 183 GP Glucose 44.6 74.1 6.56 Mean i S.E.M. 74. 34.21 6.221 16.1 19.9 0.64 23 6.8 180 GP MFAb 81.9 17.1 4.73 25 6.8 180 GP VFA 79.4 37.2 4.86 26 6.8 183 GP VFA 91.5 11.9 3.74 Mean.i S.E.M. 84.3t 22.11 4.441 3.7 7.7 0.35 30 9.1 188 CON Glucose 62.2 - .47 31 7.7 178 CON Glucose 67.6 18.2 3.91 hfiean i S.E.M. 6409—4:- - 306% 207 "' 0022 28 9.1 183 CON VFA 61.3 40.6 3.84 32 7.7 183 CON VFA 64.8 52.8 3.42 Mean i:S.E.M. 63.1i 46.7i 3.631 1.8 6.1 0.21 36 9.1 182 CON pelletsC 75.7 25.4 5.32 a Table No. 2 b Table No. 2 C Table No. 3 51 have shown that complete closure of the esophageal groove continued to occur in lambs weaned onto a teat-bottle. The conventional animals 14 and 15 had To glucose levels of 85.9 and 89.8 mg/100 ml respectively. These animals were fed the pelleted germfree animal diet‘gg libitum in addition to the basal diet. This lowered TO glucose concentration may be a consequence of rumen fermen- tation and the production of volatile fatty acids thereby hastening the decrease in To glucose. Within the limits of the experimental design and large animal variability, it is difficult to draw definite conclusions as to the effect of age or diet on To glucose. The 1970 trial was designed to differentiate between age and dietary effects on To glucose. The T0 glucose results for the 1970 trial at 60 and 180 days are given in Tables 5 and 6 respectively. Analyzed individually at 60 and 180 days, there was no statistical difference demon- strated between treatment means of the four groups of germ- free and conventional animals fed the glucose or VFA diet. Animals fed the VFA diet did not exhibit lower To glucose concentrations in comparison to the animals fed the glucose diet. As illustrated by Figure 1, there was a decrease in average To glucose for each treatment group between 60 and 180 days and these decreases approached statistical signifi- cance. Average fasting plasma glucose concentrations for the germfree animals fed the glucose diet fell from 94.6 to Fig. l 1970 Trial 52 Average fasting plasma glucose concentrations at 60 and 180 days. Abbreviations: GF = germfree COM = conventional Composition of glucose and VFA diets found in Table 2. 120 Glucose D GF' CON Glucose I a 3‘ HO ; VFA F G \\ CON VFA .1: . . k. .. . .. L. . _ l- , - . . . . .. - v 1 2. v; s, a! i. .i. a- .-- 71L . _ . v... .. -7 .. - . . . . . e. . . s . . . .. 7. . . . o . . y _. .l A 14., D" 7" I I! IIIIDI All: {0 If O . O .0 9 oo 7 anu EEOC—\mEV mm002_0 oEnoE 180 days - ' 60 days AGE 55 74.5 mg/100 ml and this difference was significant at the P< 0.25 level. The decrease observed in the germfree animals fed the VFA diet was from 117.2 to 84.5 mg/100 ml (P<:0.10). For the conventional animals fed the glucose diet, To glucose levels fell from 84.7 to 64.9 mg/100 ml (P<:0.05) and for those animals fed the VFA diet, the decrease was frmn 97.5 to 65.1 mg/100 ml (P<:O.10). Although the levels of statistical significance for these mean differences in To glucose between 60 and 180 days were low, the trend was apparent and had more animals been included in the treatment groups, the higher level of significance might have been achieved. When To glucose values for all animals at 60 days were compared to the values at 180 days regardless of treatment, the decrease in average To glucose between the two times was 98.4 to 71.6 mg/100 ml and this decrease was highly signifi- cant at the P<:0.005 level. The decrease in To glucose with increasing age is in accordance with other workers (Hodgson _e_t _a_1_., 1952; Jacobson gt _a_1_., 1951; Murley 2.13. a_]_._., 1952; Reid, 1955 and Wing'g£.al., 1955) who have observed de- creased fasting glucose levels in ruminants with advanced age, in spite of widely varying dietary treatments. Also Purser and Bergen (1969) using germfree and conventional goats fed a diet similar to the 1969 basal diet, observed decreased fasting plasma glucose concentrations with in- creased age. The results of the 1969 and 1970 trials indicate that 54 high carbohydrate diets fed to germfree or conventional ruminants do not maintain fasting plasma glucose concen- trations typical of the young animal. Decreased To glucose with advanced age occurred regardless of diet. Glucose half-lives for the animals in the 1969 trial appear in Table 4. There was no statistical difference between the means of the treatment groups. The ability of the animals to remove an intravenous glucose load did not differ with age between the two groups of germfree animals. Furthermore, changes in glucose half-life due to diet and germfree or conventional status were obscured by animal variability in the conventional animals. Likewise, there were no statistical differences attributable to type of animal (germfree versus conventional) or diet (glucose versus VFA) at either 60 or 180 days in the 1970 trial (Tables 5 and 6). This is in contrast to the results of Reid (1958) who showed that the glucose half-life of adult sheep was affected by diet. Shortest glucose half- 1ives occurred in animals fed 1800 g of a 50 percent cracked maize diet while longest half-lives were seen in animals fed 700 g of wheaten chaff. Harry.gt.a1. (1967) found that glucose half-lives were constant in calves fed a diet of milk and milk solids while decreased glucose tolerance was observed when this diet included hay and/or grain. Other reports (McCandless and Dye, 1950; Hale and King, 1958 and Omari and Nakajima, 1961) also showed that the diet of the ruminant affects glucose tolerance. 55 Changes in average glucose half-lives for the four treatment groups between 60 and 180 days are illustrated in Figure 2. In general there was an increase with age in glucose half-life for each treatment group. However, only the increase in this parameter for the conventional animals fed the VFA diet was significant (P<:0.05). When the glucose half-lives for all animals at 60 days were compared to the glucose half-lives at 180 days regardless of treat- ment, the increase in average glucose half-life was from 16.8 to 50.5 minutes and this increase was significant at the P< 0.01 level. Webb gt,al. (1969) found that there was no difference in glucose tolerance among three groups of dairy calves fed either milk plus milk solids, milk plus high-starch concen- trate or milk plus high-fiber concentrate over 100 days. Glucose tolerance decreased markedly with advancing age in all treatment groups. Purser and Bergen (1969) found similar trends as glucose half-lives increased with in- creasing age and differences between germfree and conven- tional goats were not apparent. The results of this study indicate that glucose half- 1ife increases with advancing age and may require as long as 180 days for this increase to become apparent. Furthermore, this decreased tolerance to glucose does not depend upon animal type (germfree versus conventional) or diet (glucose versus‘VFA). Acetate half-lives for animals in the 1969 trial appear Fig. 2 1970 56 Average glucose half-lives at 60 and 180 days. Abbreviations: GF = germfree CON = conventional Composition of glucose and VFA diets found in Table 2. Glucose Half-Life (min) GF Glucose D GF VFA CON Glucose a CON VFA 50 40 7 3O 20 10 AGE 57 in Table 4. The two youngest (49 days) germfree goats had a mean acetate half-life of 55.4 minutes, which was signifi- cantly longer (P<:0.025) than the other half-lives. The fbur germfree animals at 124 days of age had an average acetate half-life of 9.2 minutes while the average half-life for the 150 day old conventional animals was 12.1 minutes. These means were not significantly different. Between the two groups of germfree animals, there was a large decrease in acetate half-life with advancing age. This decrease in acetate half-life is contrary to the obser- vation of Jarrett and Filsell (1960) who found that lambs under four months of age removed an intravenous acetate load considerably faster than adult sheep. Also, acetate half- lives of this length were not observed in 60 day old goats in the 1970 trial (Table 5). This decrease in acetate half- life with advancing age is difficult to explain and is most likely due to animal variation. Reid (1958) showed that the diet would affect acetate half-life. When sheep were fed a restricted high-roughage diet, the acetate half-life was approximately 25 minutes, whereas a diet of 50% maize fed.ad libitum resulted in a half-life of 10 minutes. The 124 day old germfree goats and the group of conventional animals both had acetate half- lives in the range of 10 minutes. The similarity of acetate half-life values of germfree and conventional animals may be due to diets which were quite similar in their effect on acetate half-life. The basal diet would not be fermented in 58 either the conventional animals, due to rumen bypass, or the germfree animals. Also, the germfree pelleted diet was a high grain diet. These factors decreased the half-life of acetate to those levels observed in adult sheep fed a high grain diet. Acetate half-lives for the animals in the 1970 trial appear in Tables 5 and 6. At 60 days of age there were no statistically significant differences among treatment means of the four groups of germfree and conventional animals fed the glucose or'VFA diets. However, at 180 days of age, the mean acetate half-life for both germfree and conventional animals fed the glucose diet was 4.9 minutes, while the mean for germfree and conventional animals fed the VFA diet was 4.0 minutes and this difference approached statistical significance at the P<:0.10 level. On the basis of the work by Reid (1958) the diet which supplied the VFA should have produced longer acetate half- lives while the glucose diet should have maintained shorter acetate half-lives. In either case, whether fed the glucose or VFA diet, the acetate half-lives observed in these animals were much shorter than those half lives reported by Reid (1958), indicating that the dietary differences in the 1970 trial did not exaggerate differences in acetate half- life. Both diets maintained low acetate half-life values, characteristic of a young ruminant or an adult ruminant fed a high concentrate diet. Interestingly, there was a statistically significant 59 difference in acetate half-life between germfree and con- ventional animals at 180 days. The mean acetate half-life for germfree animals was 5.5 minutes while the mean acetate half-life for conventional animals was 5.7 minutes and the difference was significant at the P<:0.025 level. Since the germfree and conventional groups of animals contained animals from the two dietary treatments, the effect on acetate half- life appeared to depend upon conventional versus germfree status. However, the reason that the absence of a normal complement of bacteria produced a significantly longer acetate half-life is difficult to explain and may warrant further research. There was an increase in average acetate half-life for each treatment group between 60 and 180 days. Average acetate half-life for the germfree animals fed the glucose diet increased from 2.14 to 6.22 minutes and this increase was significant at the P<:0.05 level. The increase observed in the germfree animals fed the VFA diet was from 2.67 to 4.44 minutes (P<:0.10). For the conventional animals fed the glucose diet, acetate half-life increased from 5.15 to 5.69 minutes (P<:0.10) and for those animals fed the VFA diet, the increase was from 2.56 to 5.65 minutes but this difference was not statistically significant. The increase in acetate half-life with advancing age became more apparent when the acetate half-lives of all animals at 60 days were statistically compared to the acetate half-lives of all animals at 180 days regardless of treatment. The increase 60 was from 2.58 to 4.50 minutes and this difference was signifi- cant at the P< 0.005 level. This increased acetate half-life or decreased ability to metabolize an intravenous acetate load with increased age was similar to the observations of Jarrett and Filsell (1960). These workers observed a sudden decrease in the rate of metabolism of an injected dose of acetate at four months of age in sheep and this decrease was independent of diet and the onset of rumination. Here, acetate half-lives increased with age and acetate half-lives were in general short, comparable to those half-lives observed in sheep fed a high grain diet (Reid, 1958). Hepatic glutamic-pyruvic transminase activity for the 1969 trial is presented in Table 7. The mean enzyme activity for the two groups of germfree pygmy goats imreased approximately two-fold between the ages of 49 and 124 days when enzyme activity was expressed on either a per milligram hepatic protein or a per gram liver basis. Likewise, there was an increase in mean transminase activity with advancing age, although not as pronounced, when activity was expressed on a body weight basis. The conventional animals had a mean glutamic-pyruvic transaminase activity comparable to the mean activity of the older group of germfree animals. There were no statistical differences demonstrated between treatment means for the various methods of calculation of enzyme activity. In the nonruminant animal, enhanced gluconeogenesis 61 TABLE 7. 1969 Glutamate-Pyruvate Transaminase Activity A98 of Glutamic-pyruvate transaminase Animal Animal NUmber (days) Status 1a 2b 3c 20 49 GP 7.48 0.568 199.2 21 49 GP 4.31 0.314 86.9 Mean:i S.E.M. 5.891 0.44ht 143.0i 1.58 0.127 56.2 17 118 GP 7.36 0.699 129.9 18 115 GP 13.27 0.995 203.1 19 117 GP 16.68 1.351 220.5 11 146 GP 3.81 0.305 80.9 Mean2i S.E.M. 10.281 0.83%t l58.é£ 2.89 0.222 32.5 14 151 CONd 6.34 0.634 162.9 15 121 cowd 6.25 0.663 143.1 16 123 CON 9.47 0.881 201.1 Ideal] i Sothlo 703% 00726:h 1690021: 1.06 0.080 17.0 Glutamate pyruvate transaminase activity: m u moles NADH oxidized per minute per milligram hepatic protein. Glutamate pyruvate transaminase activity: p moles NADH oxidized per minute per gram liver. Glutamate pyruvate transaminase activity: u moles NADH oxidized per minute per 10 kilograms body weight. d Fed pelleted germfree animal diet ag libitum in addition to sterile glucose diet. 62 from amino acids and parallel increases in glutamic-pyruvic transaminase activity have been found to occur when the utilization of glucose was limited by hormonal and dietary conditions such as diabetes, starvation, or administration of a high protein - low carbohydrate diet (Rosen.gp.§1., 1958; Rosen.g§.§1., 1959; Segal gt,gl., 1962; Szepesi and Freedland, 1958b and c; Szepesi and Freedland, 1969b). By extension to the ruminant animal, glutamic-pyruvic transaminase activity may indicate the initiation of increased rates of gluconeo- genesis in the maturing animal. This enzyme activity would increase at the onset of rumen function when lesser amounts of glucose become available for intestinal absorption and greater rates of gluconeogenesis ensue or the activity of this enzyme may be controlled by constitutive processes independent of substrate alterations. It would appear that since the germfree animals were fed the same diet, the in- creased glutamic-pyruvic activity exhibited by the older group of animals was due to processes other than the initiation of gluconeogenesis because of substrate alter- ations. Also, the conventional animals in which ruminal fermentation and consequently substrate alterations were allowed to occur, no greater glutamic-pyruvic transaminase activity was detected. From a statistical standpoint, definite conclusions as to the constitutive prOperties of this enzyme in the ruminant animal are indeed difficult to make. Further investigation into the factors controlling glutamic-pyruvic 63 transaminase activity may reveal the processes controlling the maturation of the ruminant animal. Glucose-6-phosphatase activity for the animals in the 1969 and 1970 trials appear in Tables 8 and 9 respectively. In 1969, there was a slight increase in mean enzyme activity in the germfree animals between the ages of 49 and 124 days of age when enzyme activity was calculated on a per gram hepatic protein or a per gram liver basis. On a body weight basis, however, there was a decrease in glucose-6-phosphatase activity in these animals. Conventional animals on the average showed higher enzyme activity but variability was great within this group. In 1970, these germfree pygmy goats fed the glucose diet had in general greater glucose-6- phosphatase activity than those germfree animals fed the VFA diet regardless of the method of enzyme activity calculation. The single conventional animal had the lowest activity of all the animals in the trial. In general, animals at 180 days of age from the 1970 trial had a higher g1ucose-6-phos- phatase activity than the younger animals in the 1969 trial. There were no statistically significant differences demon- strated between treatment means for either year, 1969 or 1970. The activity of g1ucose-6-phosphatase, a key enzyme in the gluconeogenic pathway, has been found to increase in response to starvation, diabetes and high protein-low carbohydrate diets (Langdon and Weakley, 1955; Freedland and Harper, 1957; Freedland and Harper, 1958a and b; Weber'gglal., l965a,b and c; Freedland and Harper, 1966 and Szepesi and 64 TABLE 8. 1969 G1ucose-6-Ph05phatase Activity Age of _ _ h Animal Animal Glucose 6 Phosp atase Number (days) Status 1a 2b 3C 20 49 GP 0.071 14.16 4967.3 21 49 GP 0.123 24.63 6820.0 Mean.i S.E.M. 0.097i 19.39i 5893.01 0.026 5.24 926.3 17 118 GP 0.102 20.53 3816.5 18 115 GP 0.093 18.65 3806.4 19 117 GP 0.110 22.05 3598.6 11 146 GP 0.133 26.55 7049.0 Mean i S.E.M. 0.110i 21.95i 4567.5: 0.008 1.68 828.6 14 151 CONd 0.434 86.79 22,305.0 15 121 CONd 0.054 10.88 2348.9 16 123 CON 0.079 15.81 3609.4 Mean i S.E.M. 0.189i 37.82i 9421.Lt 0.123 24.52 6452.2 a G-6-Pase activity: milligram hepatic protein. liver. kilogram body weight. n moles Pi cleaved per minute per G-6—Pase activity: p moles Pi cleaved per minute per gram G—6-Pase activity: p.moles Pi cleaved per minute per 10 Fed the pelleted germfree diet 2g libitum in addition to sterile glucose diet. TABLE 9. at Approximately 180 Days of Age 1970 G1ucose-6-Ph05phatase Activity G1ucose-6-Phosphatase Animal b Number Status Diet la 2 3C 22 GP Glucose 0.294 12.94 1953.9 24 GP Glucose 0.357 16.29 3616.4 27 GP Glucose 0.502 26.49 4662.2 Mean 1 S.E.M. 0.3841 18.571 3410.81 0.062 4.10 788.5 23 GP VFA 0.348 13.92 3354.7 25 GP VFA 0.305 14.14 2417.9 26 GP VFA 0.284 17.03 3255.7 Mean 1 S.E.M. 0.3121 15.031 3002.71 0.018 1.00 294.4 30 CON Glucose 0.352 19.14 3598.3 31 CON Glucose 0.334 18.14 3101.9 Mean 1 S.E.M. 0.3431 18.641 3350.11 0.009 0.50 248.2 28 CON VFA 0.356 22.19 3040.0 32 CON VFA 0.285 17.81 3223.6 Mean 1 S.E.M. 0.3211 20.001 3131.81 0.035 1.19 91.8 36 CON Pellets 0.093 13.12 1902.4 G-6-Pase activity: p moles P. cleaved per minute per milligram hepatic protein. G-6-Pase activity: per gram liver. per 10 kilogram body weight. n moles Pi cleaved per minute G-6-Pase activity: 1p moles Pi cleaved per minute 66 Freedland, 1968c). Fisell.g£.a1. (1969) showed using adult sheep that fasting and diabetes increased the gluconeogenic capacity of the ruminant animal and the increased rate of gluconeogenesis was reflected by increased activity of the key gluconeogenic enzymes, g1ucose-6-phosphatase, fructose-l, 6-diphosphatase and pyruvate carboxylase. Bartley‘gt'al. (1966) showed that glucose-6-phosphatase increased in calves between the ages of 8 and 12 weeks to levels typical of the adult cow. Intraduodenal infusion of glucose into 15 week old calves decreased the activity of glucose-6-phosphatase and provided evidence for the adaptability of metabolic processes in the ruminant by substrate availability (Bartley 21; a1_., 1966). In this study, low statistical significance of treat- ment means due to low animal numbers and large variability, make the interpretation of the results difficult. However, the slight increase in glucose-6-phosphatase activity with age in germfree animals fed the basal diet and the increased enzyme activity seen in the older animals in the 1970 trial compared to the animals in the 1969 trial suggest that' constitutive processes may have operated to control this enzyme activity. Also, the time at which these processes exerted their effect was between 124 and 180 days of age in the pygmy goat. This perhaps explains the reason for the oldest animal in 1969 to also have the greatest glucose-6- phosphatase activity. Animals, whether germfree or conventional, fed the 67 glucose diet had greater g1ucose-6-phosphatase activity than those raised on the VFA diet and this was opposite to the effect of intraduodenally infused glucose in 15 week-old calves, Bartley gtflal. (1966). On the basis of this work, the availability of dietary glucose was expected to decrease gluconeogenesis and consequently the activity of glucose-6- phosphatase, while the VFA diet was expected to increase gluconeogenesis if metabolic pathways are influenced by substrate availability. Explanations for the greater activity in those animals fed the glucose diet are difficult to make particularly since Purser and Bergen (1969) found that the g1ucose-6-phosphatase activity of their germfree and conventional pygmy goats was comparable. As in the case of glutamic-pyruvic transaminase activity, further work with glucose-6-phosphatase may reveal the controlling processes of maturation in the ruminant. Pyruvate kinase enzyme activity is presented in Tables 10 and 11 for the 1969 and 1970 trials respectively. In 1969, the group of germfree animals at 124 days of age had a greater mean pyruvate kinase enzyme activity compared to those germfree animals at 49 days, when enzyme activity was calculated on a per milligram hepatic protein or a per gram liver basis. The converse was true when activity was calculated on a body weight basis. Mean pyruvate kinase activity in the conventional animals was less than that observed in the older groups of germfree animals when activity was calculated on a per milligram hepatic protein TABLE 10. 1969 Pyruvate Kinase Activity Age Of Pyruvate Kinase Animal Animal a b c Number (days) Status 1 2 3 20 49 GP 0.104 8.00 2806.4 21 49 GP 0.099 6.45 1786.0 Mean 1 S.E.M. 0.1021 7.231 2296.21 0.003 0.78 510.2 17 118 GP 0.119 10.19 1894.3 18 115 GP 0.124 8.70 1775.7 19 117 GP 0.099 7.18 1171.7 11 146 GP 0.124 9.33 2477.1 Mean 1 S.E.M. 0.1161 8.851 1829.71 0.006 0.63 267.6 14 151 CONd 0.116 10.82 2780.7 15 121 CONd 0.109 10.46 2258.3 16 123 CON 0.119 9.52 2173.4 Mean.1 S.E.M. 0.1121 10.261 2404.11 0.002 0.38 189.8 a Pyruvate kinase activity: p moles NADH oxidized per minute per milligram hepatic protein. b Pyruvate kinase activity: p moles NADH oxidized per minute per gram liver. C Pyruvate kinase activity: pp moles NADH oxidized per minute per 10 kilogram body weight. d Ped the pelleted germfree animal diet 3g libitum in addition to sterile glucose diet. 69 TABLE 11. 1970 Pyruvate Kinase Activity at Approximately 180 days of age Pyruvate Kinase Animal b c Number Status Diet la 2 3 22 GP Glucose 0.072 14.4 2174.4 24 GP Glucose 0.123 24.6 5461.2 27 GP Glucose 0.143 28.6 5033.6 Mean 1 S.E.M. 0.1121 22.51 4223.11 0.021 4.2 1031.8 23 GP VPA 0.149 29.8 7181.8 25 GP VFA 0.145 29.0 4959.0 26 GP VPA 0.137 27.4 5206.0 Nlean It SoEoNlo 0.14% 28.73: 5782og: 0.004 0.7 703.4 30 CON Glucose 0.179 35.8 6730.4 31 CON Glucose 0.136 27.2 4651.2 Mean 1 S.E.M. 0.1581 31.51 5690.81 0.022 4.3 1039.6 28 CON VFA 0.097 19.4 2657.8 32 CON VPA 0.171 34. 6190.2 Mean 1 S.E.M. 0.1341 26.81 4424.01 0.037 7.4 1766.2 36 CON Pellets 0.099 19.96 2894.2 a Pyruvate kinase activity: p moles NADH oxidized per minute per milligram hepatic protein. b Pyruvate kinase activity: p moles NADH oxidized per minute per gram liver. c Pyruvate kinase activity: In moles NADH oxidized per minute per 10 kilogram body weight. H 70 basis but greater when the other two methods for the calcu- 1ation of enzyme activity were used. In 1970, there were no recognizable trends in the data in relation to the treat- ments imposed, i.e. germfree versus conventional status or dietary regime. There were no statistically significant differences demonstrated between treatment means in either the 1969 or 1970 trials. Pyruvate kinase has been shown to be a control enzyme in the glycolytic pathway as the activity of this enzyme is influenced by certain dietary and hormonal conditions. In the nonruminant animal, decreased enzymatic activity due to starvation, diabetes and low carbohydrate diets has been shown to occur (Krebs and Eggleston, 1965; Weber.gt,§l., l965d; Yudkin and Krauss, 1967; Szepesi and Freedland, 1968a; Sillero.gt‘al., 1969 and Szepesi and Freedland, 1969a). In the ruminant animal, Goetsch (1966) found a gradual decrease in glycolytic activity as calves matured over an eight week period. This decrease was thought to be a consequence of decreased availability of glucose for absorption as a consequence of rumen develOpment. 0n the basis of the preceding, it was thought that glycolytic activity in the pygmy goat would be controlled by the availability of dietary glucose. In the 1969 trial, germfree animals at any age would have similar pyruvate kinase enzyme activities and their activity would be greater than the activity determined in conventional animals in which rumen function was allowed to deve10p. Compared to L4 71 the younger group of germfree animals, average pyruvateldste activity was greater in the older group of germfree animals when activity was calculated on a per milligram hepatic pro- tein and per gram liver basis but lesser on a body weight basis. The method of calculation more indicative of true metabolic processes is questionable but more importantly, mean emzyme activity did not appear similar between these two groups fed the same diet. Pyruvate kinase activity in the conventional animals was slightly lesser or even greater than the activity in the older germfree animals, depending on the method of activity calculation. Again, the behavior of this enzyme activity was different than that which was expected. In the 1970 trial, the glucose diet was eXpected to maintain pyruvate kinase activity above the activity determined in animals fed the VFA diet. Also, the conven- tional animal was expected to have an activity comparable to the animals fed the VFA diet. The mean pyruvate activity between treatment groups varied but any relationships between the glucose and VFA diet or germfree and conventional status of the animal and the activity of this enzyme were not discernible. The lack of response of this enzyme to imposed treatments is reflected in low statistically significant differences between treatment means for both years and is similar to the findings of Purser and Bergen (1969). In these animals, pyruvate kinase activity may not be the appropriate measure of glycolytic activity as its 72 response seemed not to be influenced by the availability of dietary glucose. Also, within the limits of this experiment, other processes which act to control pyruvate kinase activity were not identified. Perhaps, a better means of assessing glycolytic activity in these animals must first be found before controlling factors may be determined. Since pyruvate kinase activity was assayed one day after glucose tolerance tests were given, correlation co- efficients between pyruvate kinase activity and glucose half-life were determined individually for the two years. The correlation coefficient between mean pyruvate kinase activity and mean glucose half-life for the three treatment groups in 1969 was -0.690 (P<:0.05) and in 1970 the correla- tion coefficient was -0.680 (P<:0.01). The animals with the greatest pyruvate kinase activity had the shortest glucose half-life or fastest rate of glucose utilization. This relationship between pyruvate kinase and glucose utilization in germfree and conventional goats was also reported by Purser and Bergen (1969), however they obtained a higher correlation coefficient of -0.949 (P<:0.01). Interestingly, there seems to be a relationship between this enzyme activity and the rate of glucose utilization in these animals, but this enzyme seems to be unaffected by the availability of dietary carbohydrate. It would seem that in the pygmy goat at least, differences in pyruvate kinase activity and therefore, the rate of glucose utilization occur between animals but the factors affecting individual 73 enzyme activity are not clear. Further elucidation of the processes controlling these changes in the ruminant may reveal more major metabolic differences between the ruminant and nonruminant and contribute to the better understanding of ruminant metabolism. Hepatic g1ucose-6-phosphate and 6-phosphog1uconate dehydrogenase activities for the 1970 trial are presented in Tables 12 and 15. In the case of glucose-6-phosphate dehy- drogenase, the mean activity for the animals fed the VFA diet was greater and significantly different from the mean activity of the animals fed the glucose diet. Mean enzyme activity (p moles/min) for the animals consuming the VFA diet was 2.41, 0.482 and 85.2 (per milligram hepatic protein, per gram liver and per 10 kilograms body weight) as compared to 1.65, 0.529 and 59.2 for the animals fed the glucose diet and these means were statistically different at the P<:0.025 level. There were no statistically significant differences between treatment means for 6-phosphogluconate dehydrogenase activity. Starvation, diabetes, low carbohydrate and high fat diets have been shown to decrease with activity of glucose- 6-phosphate dehydrogenase (Niemeyer'g£.§1., 1962; Ono 21.21-. 1965; Vaughn and Winders, 1964; Benevenga.g£'a1., 1964; McDonald and Johnson, 1965; Peraino, 1967 and Szepesi and Freedland, 1968c). Howarfll.g§.al. (1968) showed that the activities of glucose-6-phosphate and 6-phosphog1uconate dehydrogenase in adipose tissue were higher in steers than .F.‘ TABLE 12. 1970 Glucose-6-Phosphate Dehydrogenase Activity to Approximately 180 Days of Age Glucose-6—Phosphate Dehydrogenase Animal a b c Number Status Diet 1 2 3 22 GP Glucose 1.83 0.366 55.3 24 GP Glucose 1.53 0.306 67.9 27 GP Glucose 2.57 0.514 90.5 Mean 1 S.E.M. 1.981 0.3951 71.21 0.31 0.062 10.3 23 GP VFA 1.84 0.368 88.7 25 GP VPA 2.49 0.498 85.2 26 GP VPA 2.53 0.506 96.1 Mean 1 S.E.M. 2.281 0.4571 90.01 0.22 0.045 3.2 30 CON Glucose 1.19 0.238 44.7 31 CON Glucose 1.44 0.288 49.7 Mean.1 S.E.M. 1.321 0.2631 46.91 0.12 0.025 2.2 28 CON VFA 2.58 0.516 70.7 32 CON VPA 2.49 0.498 90.1 Mean.1-S.E.M. 2.541 0.50%1 80.41 .04 .009 9.7 36 CON Pellets 3.21 0.642 93.1 a Glucose-6-phosphate dehydrogenase activity: p moles NADP reduced per minute per milligram hepatic protein. b Glucose-6-phosphate dehydrogenase activity: ‘p moles NADP reduced per minute per gram liver. c Glucose—6-phosphate dehydrogenase activity: p moles NADP reduced per minute per 10 kilogram body weight. TABLE 13. '75 1970 6-Ph08phogluconate Dehydrogenase Activity at Approximately 180 Days of Age 6-Phosphogluconate Dehydrogenase Animal b c Number Status Diet la 2 3 22 GP Glucose 5.29 1.06 159.8 24 GP Glucose 5.90 1.18 261.9 27 GP Glucose 5.35 1.07 188.3 Mean 1 S.E.M. 5.511 1.101 203.31 0.19 0.04 30.4 23 GP VFA 6.84 1.36 329.7 25 GP VFA 4.96 0.99 169.6 26 GP VPA 6.77 1.35 257.3 Mean 1-S.E.M. 6.191 1.231 252.21 0.61 0.12 46.3 30 CON Glucose 5.91 1.18 222.2 31 CON Glucose 6.53 1.31 223.3 Mean 1 S.E.M. 6.221 1.241 222.81 0.31 .06 0.5 28 CON VFA 5.11 1.02 140.0 32 CON VFA 7.69 1.54 278.3 Mean 1 S.E.M. 6.401 1.281 209.21 1.29 0.26 69.2 36 CON Pellets 6.38 1.28 185.0 6-Ph05phogluconate dehydrogenase activity: m p moles NADP reduced per minute per milligram hepatic protein. NADP reduced per minute per gram liver. 6-Phosphog1uconate dehydrogenase activity: p moles 6-Ph05phogluconate dehydrogenase activity: p moles NADP reduced per minute per 10 kilogram body weight. 76 calves and that there were greater changes in these enzymes in adipose tissue than in liver and muscle tissues. They suggested that this was a reflection of a greater capacity for fat synthesis in the mature ruminant. The activities of these enzymes however were found to be lower in steers than in calves fed a whole milk diet twice daily (Howarth gt‘§1., 1968). In this experiment, animals fed the glucose diet had lower glucose-6-phosphate dehydrogenase activity than the animals fed the VFA diet which is difficult to reconcile as this is contrary to the observations of others. It is attractive to hypothesize that in these goats fed the VFA diet, enzyme changes have taken place which severely limit the amount of glucose that can be used for lipogenesis. This change is brought about by decreased activity of the citrate cleavage pathway (Ballard.g§'§l., 1969). Animals which were fed the VFA diet then had a source of acetate and butyrate which are efficient precursors for lipid synthesis and had a correspondingly greater need for NADPHZ, greater than the animals fed the glucose diet. Greater glucose-6- phosphate dehydrogenase activity then in the VFA diet-fed animals may reflect a greater need for NADPH2 due to greater rates of lipogenesis. If this is the case, volatile fatty acids may be acting to control which precursor for lipo- genesis is used predominantly. This is pure conjecture but may explain at least in part the observed relationship between dietary VFA and increased glucose-6-phosphate dehydrogenase. ta 77 Hepatic metabolic substrates for the 1970 trial are presented in Table 1h. Liver tissue concentrations of glucose-6-phosphate and phosphoenolpyruvate were determined, since changes in tissue levels of metabolic intermediates may occur which could produce metabolic changes in the absence of enzyme adaptions. Animals fed the glucose diet had a mean tissue phospho- enolpyruvate concentration of 0.0330 n mole/g liver which was significantly different at the P<:0.25 level from the mean concentration of 0.0229 u mole/g liver for those animals fed the VFA diet. This possibly indicates a limitation in pyruvate kinase activity of these pygmy goats fed the glucose diet compared to animals consuming the VFA diet and may be due to the greater amount of Carbohydrate in the glucose diet. It would appear then at least that greater amounts of dietary glucose may not increase glycolytic activity in these animals. This along with the fact that pyruvate kinase activity did not respond to the dietary treatments suggest that glycolytic activity in pygmy goats at this age (180 days) may become refractory to increased dietary carbohydrate. The concentrations of glucose-6-phosphate were variable and no consistent pattern was discernible. Increased con- centrations of glucose-6-phosphate in those animals with increased amounts of phosphoenolpyruvate would indicate a decreased rate of glycolytic activity and be consistent with the work of Bartley gt_gl. (1966). However, in these TABLE 14. 1970 Hepatic Metabolic Substrates Glucose-6 PhOSpho- Animal Phosphate enolpyruvate Number Status Diet (u moles/g liver) (p moles/g liver) 22 GN Glucosea - - 24 GN Glucose 0.250 0.0373 27 GN Glucose 0.035 0.0257 Mean 1 S.E.M. 0.1431 0.03151 0.108 0.0058 23 GN VPAb 0.792 0.0167 25 GN VFA 0.284 0.0244 26 GN VFA 0.485 0.0271 Mean 1 S.E.M. 0.5201 0.02211 0.148 .0031 30 CON Glucose 0.284 0.0258 31 CON Glucose 0.0690 0.0434 Mean:1 S.E.M. 0.1711 0.03461 0.108 0.0088 28 CON VFA 0.021 0.0200 32 CON VFA 0.064 0.0263 Mean 1 S.E.M. 0.0421 0.02311 0.022 0.0032 36 CON pelletsC 0.023 0.0312 a Table No. 2 b Table No. 2 Table No. 3 79 animals such a relationship did not appear.Conventional ani- mals had the lowest level of this metabolite and this cwld in- dicate a greater rate of gluconeogenesis. Animals with greater glucose-6-phosphatase activity did not consistently have less- er amounts of glucose-6—phosphate.A.better understanding of the overall metabolic processes in these animals would come from knowing the levels of more hepatic intermediates. Tables l5, l6, l7 and 18 present the essential and non- essential plasma and liver free amino acid patterns of the animals in the 1970 trial. Similar analyses were not per- formed in 1969. Plasma and liver samples were taken after a 16 hour fast, when the animal was approximately l80 days of age. The data were not analyzed statistically because of their consistent patterns. The major reason for these analyses stems from the work of Felig.gt'a;. (1970) which showed that in the post-absorp- tive human, plasma alanine concentration was 344 p moles/ liter and that this amino acid accounted for approximately 50 percent of the total net amino acid consumption of liver with glycine, serine and threonine supplying most of the remainder. This amino acid uptake by the liver was deter- mined by arteriohepatic venous (A-HV) difference. After a five to six week fast, the absolute amounts of the A-HV difference for the individual amino acids were greatly re- duced but alanine still accounted for approximately 50 percent of the total new amino acid consumption. 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0vm.a 0m0.0 550.0 H5H.0 500.0 05v.0 mmooSHo 00 mm H55 m>o ma< 5H0 can 5H0 cm< Hmm [mww 422 CH0 pmwo msvmpm amnesz Hmefic< A0\wwHoE 1V mvfio< ocfie< Hmflpcmmmmucoz mmnm nm>flq .ma mgm<5 84 were variable in their contribution to net amino acid con- sumption. The source of increased plasma alanine levels in the post-absorptive human was determined to be increased mobili- zation of peripheral muscle amino acids. This was shown by arterial-venous (AAV) difference across the forearm. Over a four to six week fast the amounts of all plasma acids tested by AAV difference were greatly reduced, indicating decreased mobilization of amino acids from muscle with duration of the stress. The results from the 1970 trial show that there are no large, apparent increases in plasma or liver free alanine, glycine, serine or threonine concentration nor are there increases for any other amino acid in the various treatment groups (Tables 15, 16, 17 and 18). It was expected that if the ruminant behaved like the human under conditions which increase the rate of gluconeogenesis, noticeable increases in certain plasma amino acids would have appeared partic- ularly in the germfree goats fed the "volatile fatty acid" diet and to a lesser extent in the conventional animals fed the "glucose" or "volatile fatty acid" diet and the con- ventional goat fed the pelleted diet.' Since Felig gt filo (1970) showed that, with time in the severely fasted human, the plasma concentrations of gluco- neogenic amino acids decreased until almost normal patterns were reattained, differences in amino acid patterns of these goats due to treatments which increase the rate of 85 gluconeogenesis would be greatly lessened after 180 days duration. Plasma amino acid concentrations would certainly be given enough time to readjust under the prolonged stress. If the ruminant reacts like the human to gluconeogenic stress, the increases in alanine or other gluconeogenic amino acids due to their mobilization from muscle would have been most apparent just after the initiation of the treat- ments expected to increase gluconeogenesis. Because of the type of liquid diet that was fed to the animals in this experiment and the fact that they were fed by nipple-bottle, differences between the post absorptive state of the germ- free or conventional goat and the human due to previous diet may be minimal. An explanation of the differences in plasma and liver amino acid patterns could be different rates of amino acid mobilization and utilization between the two species. Apparently in the post absorptive state, amino acid mobili- zation from muscle tissue in the human is faster than the rate of their utilization as gluconeogenic precursors and this accounts for the transitory elevation of plasma amino acids. In the post-absorptive ruminant, if the rate of mobilization of muscle tissue amino acids was faster or slower than the rate of their utilization for gluconeogemxfis, then increased or decreased levels of plasma amino acids would have been observed, respectively. Also, if the rate of utilization of amino acids was in equilibrium with the rate of amino acid mobilization no change would be observed. 86 Unfortunately, to make this kind of comparison of plasma amino acid levels the amino acid pattern of fed animals should have been determined but this was not done in this experiment. Comparisons can be made between groups of animals in which the rate of gluconeogenesis was expected to be greater than those groups where more normal metabolic patterns were expected. Since these comparisons show no difference in plasma amino acid levels, the lack of sufficient numbers of animals per group or diets which do not exaggerate differ- ences in overall patterns of metabolism are at fault. Another reason for the lack of apparent differences among the groups of animals may come from the supposed constitutive control of gluconeogenesis in the ruminant (Purser and Bergen, 1969). If there is an inherent rate of gluconeogenesis in the ruminant and if the choice of diets or germfree status in this experiment were not able to exaggerate the rate of utilization of amino acids as gluco- neogenic precursors, differences in the amino acid levels of the animals in the post-absorptive state between groups will not be apparent. These circumstances best explain the observed results. The plasma non-essential to essential amino acid ratios (NEAA/EAA) for each treatment group of the 1970 trial are shown in Table 19. These ratios were calculated from the mean amino acid concentrations of each treatment group. The samples upon which the plasma amino acid determinations were 87’ TABLE 19. Plasma Non-essential to Essential Amino Acid Ratioa Status Diet NEAAb/EAAC GP Glucose 1.93 GE VFA 2.05 CON Glucose 1.89 CON VFA 1.95 CON Pellets 1.09 a Calculated from the mean amino acid concentrations of each treatment group. b Non-essential amino acid. C Essential amino acid. Ly 88 made, were taken from the animals after a 16 hour fast at approximately 180 days of age. The NEAA/EAA ratio has been used clinically as an indicator of kwashiorkor or protein-calorie malnutrition in humans (Waterlow, 1969). These ratios were examined in this set of experiments to generally assess the effect of the various diets on the protein-calorie nutrition of the animal and to determine whether the overall results of the experi- ment must be discussed in terms of an additional dietary effect. The discussion of the results proceeds from the work of Oltjen 33 al. (1969). In their experiments, heifer and bull calves were maintained before and after weaning on three experimental diets fed.ad libitum. These diets were a natural diet, a purified diet containing urea and a purified diet containing protein. The natural diet con- sisted essentially of cracked corn, alfalfa and timothy hay and soybean meal. The purified diets contained in common wood pulp, corn starch and cerelose in comparable amounts but differed in that one purified diet contained 4.7 percent urea and the other 14.9 percent isolated soy protein. Besides studying blood protein patterns and blood glucose levels at 42 and 84 days of age and hair amino acid content at 210 days of age, blood plasma free amino acid patterns were determined at 42, 8A and 210 days. Although Oltjen.gt'al. (1969) did not calculate the NEAA /EAA ratios from their plasma amino acid data for the treatment 89 groups, this calculation yields interesting results. The NEAA/EAA ratios for the heifers consuming the natural diet were very consistent with age. These data were 1.68, 1.88 and 1.65 at 42 , 84 and 210 days of age (all subsequent ratios were calculated at these ages). NEAA/EAA ratios for those heifers consuming the soy diet (2.18, 2.36 and 1.88) were more variable but comparable to the above ratios. The ratios were consistently higher and increased steadily for those heifers consuming the urea diet (2.12, 2.61 and 3.29). Similar results were obtained from the data on bull calves, however, no bull calves consumed the natural diet. The NEAA/EAA ratios for bull calves consuming the soy diet were fairly consistent and comparable to those ratios for heifers on the same diet. They were 2.21, 2.34 and 1.24, while for those bull calves on the urea diet they were 2.42, 3.50 and 4.59. Again, as for the heifers on the urea diet, ratios were consistently higher and steadily increased with age. Since the natural diet contained only 10% more gross energy than the urea diet and only 5% more gross energy than the soy diet, these ratios were affected by differences in the amount of protein presented to the animal for utilizatiai The NEAA/EAA ratios which were consistently higher and steadily increased with age for both those heifers and bull calves consuming the urea diet reflected decreased amounts of microbial protein for utilization by the animal. These results showed that for the ruminant the NEAA/EAA ratio is an indicator of the state or protein-calorie nutrition. ring *1. 90 Normal ratios were approximately 2.0 as it is in the human (Waterlow, 1969) and as protein-calorie malnutrition ensued, these values increased. The NEAA/EAA ratios in Table 19 range from 1.09 to 2.05 over the treatment groups of the 1970 trial. Interpretation of these ratios from the work of Oltjen.g§.gl. would indicate that the animals in this eXperiment were normal with respect to protein-calorie nutrition. The importance of this con- clusion is that results of this experiment were not con- founded by diets which did not meet the protein-calorie requirements of the animal and complicate the interpretation of the results by an additional dietary stress. V GENERAL DISCUSSION The cause of the decrease in blood glucose and increase in glucose half-life in the ruminant animal has been investi- gated many times in the past. In general, these phenomenon have been attributed to either changes in ration and there- fore substrates at the onset of rumen develOpment or processes which proceed independent of ration and rumen function. The specific cause or causes for these changes are yet unknown. It was realized that as the ruminant adapted first from carbohydrate utilization prenatally to a high carbohydrate and lipid diet postnatally and finally to the utilization of VFA at maturity, many metabolic changes must take place. The increased dependence on gluconeogenesis in the mature animal compared to the immature ruminant must be made mani- fest in alterations in the activities of key gluconeogenic and glycolytic enzymes. Enzyme studies with the ruminant animal have in general shown that gluconeogenesis increased and glycolytic activity decreased as the ruminant matured and the changes were related to decreased availability of glucose for intestinal absorption. Most research published would support this view that alterations in enzyme activity were substrate dependent. Preliminary work with germfree goats questioned this conclusion (Purser and Bergen, 1969) 91 92 and this present work was initiated to further define the factors which effect the observed changes in ruminant metabolism. In this study with germfree and conventional pygmy goats, plasma glucose decreased and glucose and acetate half- lives increased significantly with advancing age of the animal and these changes were unrelated to dietary glucose or VFA. Glutamic-pyruvic transaminase and glucose-6- phosphatase activities increased with age while pyruvate kinase activity was variable. The activities of these enzymes were also unaffected by the imposed dietary treat- ments. In addition, an inverse relationship between pyruvate kinase activity and glucose half-life was found. In con- clusion, the metabolic values which were studied in this experiment underwent changes characteristic of the maturing ruminant, but these changes were constitutive and, unrelated to direct dietary effects. VI CONCLUSIONS In germfree and conventional pygmy goats, fasting plasma glucose concentrations decreased from 98.4 to 71.6 mg% (P<:0.005) between the ages of 60 and 180 days. Dietary glucose did not maintain fasting plasma glucose levels in germfree or conventional animals with time. Glucose half-life increased from 16.8 to 30.3 minutes in germfree and conventional animals between the ages of 60 and 180 days and the increase was significant at the P< 0.01 level. Decreased tolerance to glucose did not depend on animal type (germfree versus conventional) or diet (glucose versus VFA). Acetate half-life increased from 2.58 to 4.50 minutes between the ages of 60 and 180 days in germfree and conventional animals and this increase was significant at the P< 0.005 level. Decreased tolerance to acetate did not depend on animal type or diet. Hepatic glutamic-pyruvic transaminase activity increased 93 10. ll. 12. 13. 94 with age in germfree animals fed identical diets. 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