THE EFFECT OF CALOREE SOURCE ON THE FATTY LIVER WNDROME AND SELECTED ENZYME SYSTEMS OF MALE ALBiNO RATS

Transcription

1 THE EFFECT OF CALOREE SOURCE ON THE FATTY LIVER WNDROME AND SELECTED ENZYME SYSTEMS OF MALE ALBiNO RATS Thesis for 19 Dogma a; M. S. MlCHEGfiN STATE UNIVERSKTY June E. DeHate 1962

2 it.. M" 1.. L 113 RA R Y Michigan State Univcrsity I - -

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4 ABSTRACT Several groups of workers have observed that the consumption of a 9% casein diet supplemented with methionine and tryptophan but not with threonine caused fatty livers in weanling rats. In almost every experiment, sucrose was the carbohydrate source in these low protein, threonine deficient diets. The studies presented here were undertaken in an effort to determine the effect of altering certain dietary components, particularly carbohydrate, on the fatty liver syndrome associated with a deficiency of threonine. Male weanling rats of the Sprague-Dawley strain were fed various experimental diets deficient in threonine. All diets were isocaloric. Nine percent casein diets were used with one of the following modifications: 1) different carbohydrates were used in the diets; 2) the source of dietary fat was changes; or 3) the ratio of carbohydrate to fat calories was adjusted to a given percent of the total carbohydrate-fat calories. Food intake, weight gain, fresh liver weight, liver moisture, liver nitrogen and liver fat were determined in all animals. In addition, liver pyridine nucleotides, activity of the fatty acid oxidase system and endogenous respiration in liver tissues were measured in cm experiment. Fatty livers did not develop in rats fed a threonine

5 June DeHate deficient basal diet (81.2% carbohydrate) if the carbohydrate was glucose, fructose or maltose; fatty livers did occur when the carbohydrate source was sucrose or a combination of glucose and fructose simulating sucrose. There were no differences between glucose-fed rats and sucrose-fed rats with respect to : 1) activity of the fatty acid oxidase system; 2) concentration of liver pyridine nucleotides; or 3) rate of endogenous oxidation by liver tissues. The chemical nature of dietary fat (saturated versus unsaturated) when present as 5% of the diet had no influence on the deposition of fat in the livers of rats fed threonine deficient diets containing glucose or sucrose. If 90, 75 or 50% of the carbohydrate-fat calories of a threonine deficient diet were present as glucose, liver fat levels of rats were near normal. If 25 or 10% of these calories were present as glucose, liver fat concentrations increased proportionately with the decrease in dietary glucose. When 90, 75, 50 or 25% of the carbohydrate-fat calories of a threonine deficient diet came from sucrose, fatty livers occurred in all rats.

6 THE EFFECT OF CALORIE SOURCE ON THE FATTY LIVER SYNDROME AND SELECTED ENZYME SYSTEMS OF MALE ALBINO RATS BY June E. DeHate A THESIS Submitted to Michigan State University - in partial fulfillment of the requirements ' for the degree of MASTER OF SCIENCE Department of Foods and Nutrition 1962

7 ACKNOWLEDGMENTS Grateful appreciation is extended to Dr. Dorothy Arata of the Department of Foods and Nutrition for her guidance and counsel on matters both personal and scientific, which led to the conclusion of these studies presented here. For her technical assistance and ready good will, the author wishes to thank Bette Smith. To the author's parents go her deepest feelings of gratitude for their understanding and patience.

8 TABLE or CONTENTS. PAGE RWIEW OF LITERATUBEOOO l GWEML MATERIAE AND METHODSCOOOOOOOO OOOOOOOOOOOQOOO 14 PART I. THE EFFECT OF VARYING THE CARBOHYDRATE SOURCE ON THE SEVERITY OF FATTY LIVERS IN RATS FED THREONINE DEFICIENT DIETS INTRODUCTIONOOOOOOOIOOOOOOOOOOOOOOOOCOOO MATERIALS AND MTHODSOOOOOOOCOOO-O...00" RESULTSOOOOO DISCUSSIONOOOOOOOO~OOOOOODOOCOOOOOOOOOOOOOOOCCOO 24 PART II. THE EFFECT OF TYPE OF DIETARY FAT ON THE APPEARANCE OF FATTY LIVERS IN THREONINE DEFICIENT RATS FED DIETS CONTAINING GLUCOSE OR SUCROSE INTRODUCTIONOOOOOO OOOOOOOOOOOOOO 30 MATERIALS AND MTHODSOOOOOOOOOOOOOOOOOOOOOOOOOO. 31 RESULTSOOOOOOOOOOOOOOO OOOOOOOOOOOOOOOOOOO 32 DISCUSSION...COO...O00...OOOOOOOOOOOOOOOOOOOOOO 34 PART III. THE EFFECT OF A DIFFERENTIAL APPORTIONMENT OF CALORIES FROM GLUCOSE AND FAT IN ISOCALORIC DIETS ON THE APPEARANCE OF FATTY LIVERS IN THREONINE DEFICIENT RATS INTRODUCTIONOOOO OOOOOOOOOOOOIOOOOOOOO 37 MATEIALS AND METHODS... OOOOOOOOCOOOOOOOOOOOCOO 38 RESULTS DISCUSSION GENERAL SUMMARY AND CONCLUSION BIBLIOGRAPHYOOOOOOOOOOOOOOOOOOO-OOOOOOOOOOOOOOOOOOOOOO 50

9 Table 1. Table 2. Table 3. Table 4. Table 5. Tflfle6. Table 7. Figure 1. LIST OF TABLE AND FIGURES Food intake and weight gain of rats fed threonine deficient diets in which the source of the carbohydrate varied... Liver composition of rats fed threonine deficient diets in which the source of the carbohydrate varied... Liver pyridine nucleotides, fatty acid oxidase activity and endogenous oxidation of rats fed threonine deficient diets in which the source of carbohydrate varied oooooooooooooooooooo00... Food intake and weight gain of rats fed threonine deficient diets containing glucose or sucrose and saturated or unsaturated fat... Liver composition of rats fed threonine deficient diets containing glucose or sucrose and saturated or unsaturated fat... Food intake and weight gain of rats fed threonine deficient diets in which the calories from carbohydrate and fat variedoo coooo Liver composition of rats fed threonine deficient diets in which the calories from carbohydrate and fat varied... The effect of various levels of glucose and sucrose in isocaloric diets on the percent liver fat (dry weight basis) in threonine def1c1ent rats PAGE

10 REVIEW OF LITERATURE When rats were fed a diet of mixed grains and 40% saturated fat, approximately three times as much fat accumulated in their livers as compared to the livers of rats fed a diet of mixed grains and bone meal (Best and Huntsman, 1932). This infiltration of fat was prevented by the addition of choline to the diet. In a more extensive study, Best and Huntsman (1935) Proved that choline was lipotropic under a variety of dietary conditions. Specifically, fatty livers were induced in rats by feeding a diet of mixed grains and 40% fat. Under these conditions, liver fat accumulated to the extent of 14% of the fresh weight of tissue. The rats were then divided into 4 groups and each group was fed one of the following diets: 1) 100% sucrose; 2) 100% sucrose plus 75 mg/day choline; 3) 80% sucrose and 20% casein; and 4) 80% sucrose and 20% casein plus 75 mg/day choline. The fat content of livers from rats fed 100% sucrose increased from 14% to 23% in 13 days; when choline was included in the 100% sucrose diet, liver fat levels decreased from 14% to 5% in the same time period. When 20% of the sucrose was replaced by casein, liver fat levels remained constant over the 13 day study; inclusion of choline in the 80% sucrose- 20% casein diet caused a reduction of liver fat to 3%, a level lower than that established by choline on the diet 1

11 free of protein. Since casein prevented the increase in liver fat which occurred when sucrose alone was fed, a lipotropic role was postulated for casein. However, the lipotropic action of casein was thought to be due to impurities such as betaines or other established lipotropic factors. These observations stimulated a concerted effort by many workers to determine whether or not casein pgg'gg was lipotropic. In 1935, Channon and Wilkinson fed rats a 40% fat diet with increasing levels of protein (at the expense of glucose) and observed as the level of protein increased, the percent fat in the liver decreased. In this study rats fed a 5% casein diet developed fatty livers to the extent of 13% on a fresh weight basis. When the casein was increased to 20%, liver fat was reduced to 7% and further reduced to 6% when the casein was increased to 50% of the diet. From these results, the authors proposed a lipotropic action for casein on the basis of its amino acid content rather than any impurities it contained. The observations made by these two groups led to the following distinct but related fields of investigation: 1) the study of the metabolism of methyl groups and the possible relationship between methyl metabolism and lipotropic activity; and 2) the study of the lipotropdc effect of protein. It is the latter aspect with which this present review is primarily concerned. Beeston et a1., (1936) compared the liver fat concentratlion of rats fed a 5% casein-40% fat diet containing

12 various levels of choline, to the concentration of liver fat in rats fed a 30% casein 40% fat diet. Livers of rats fed the low protein diet to which no choline was added contained 20% fat (on a fresh weight basis). Inclusion of 0.1% or 0.2% choline in the low protein diet caused liver fat levels to fall to 9% and 6% respectively. However, the concentration of fat in the livers of rats fed the 30% casein diet was 6%. From these data, the authors proposed a lipotropic activity for casein over and above that of choline and began a methodical study of the effects of individual amino acids. In 1936, Beeston and Channon reported the livers of rats fed a 5% casein-40% fat diet deficient in choline contained fat to the extent of 26% of the dry weight of the tissue. When this diet was supplemented with 0.2% or 0.6% cystine, the fat content of the livers was increased to 36 or 37%. When the level of casein was increased to 30%, liver fat accumulation due to choline deficiency was prevented and the addition of cystine did not increase the level of fat in the liver. without effect on liver fat. Other amino acids1 tested were Thus it was found that cystine exerted an antilipotropic effect which could be reversed by feeding additional casein. The antilipotropic activity of cystine stimulated interest in the sulfur-containing amino acids and in 1937, # ' 1 lysine, glutamic acid, aspartic acid, serine, glycine and phenylalanine.

13 Tucker and Eckstein found methionine effective in preventing fatty livers in rats consuming choline deficient diets. The lipotropic effect of methionine was attributed to its choline-sparing action. That is, with an excess of methionine in the diet (0.5% in this case), less exogenous choline was needed because of its synthesis from the excess methionine by way of transmethylation. In 1948, Singal et al., noted supplementing a 9% casein diet with histidine, valine, threonine and lysine produced a marked growth retardation in rats. or tryptophan reversed this effect. The addition of niacin The addition of niacin or tryptophan to an unsupplemented 9% casein diet did not stimulate growth. Therefore, the authors concluded one or more of the above amino acids was limiting. By a process of elimination, they found threonine was the limiting factor for growth of rats fed a 9% casein ration supplemented with niacin or tryptophan. Simultaneously, they observed the livers of rats fed the latter diet appeared yellow in color which led the authors to believe these livers contained more fat than normal. Subsequently, Singal et al., (1949) fed rats a 9% casein-5% fat ration supplemented with niacin or tryptophan and choline. The average concentration of fat in livers from these animals reached 14% of the dry weight of the liver. The inclusion of threonine in the diet reduced liver fat to 6%. Similar results were observed when an amino acid

14 mixture simulating 9% casein, supplemented with tryptophan, was fed to rats. Without additional threonine, liver fat was 17%; with additional threonine, liver fat was 5%. That threonine is lipotropic and its lipotropic activity is dependent upon the quantity of other amino acids and the presence of choline in the diet has been substantiated by many other workers (Dick et al., 1952; Litwack et al., 1952; Harper et al., 1953a, b; Singal et al., 1953a, b; Harper, Benton, Winje and Elvehjem, 1954; Harper, Benton, Winje, Monson and Elvehjem, 1954; and Harper, Monson, Benton, Winje and Elvehjem, 1954). Singal et al., (1953a) observed a relationship between the rate of growth and liver fat deposition in threonine deficient rats. Various amounts of threonine were added to an amino acid diet simulating 9% casein supplemented with tryptophan. The rate of growth and level of liver fat increased as the level of threonine increased until the animals were no longer deficient in threonine at which time liver fat levels were restored to normal although the growth rate was still considerably lower than that supported by 25% casein. Thus, fatty livers can be produced in rats fed a threonine deficient diet virtually irrespective of the growth rate (Singal et al., 1953a and Harper et al., 1953a). However, in order to produce the maximum deposition of fat in livers of threonine deficient animals, a sufficient quantity of threonine must be included in the ration to support

15 6 growth at a level 80% of that supported by the 9% casein control diet (Singal et al., 1953a). Harper, Monson, Benton, Winje and Elvehjem (1954) investigated the possible influence of type and amount of dietary fat on liver fat content of threonine deficient rats. Butter fat or corn oil at the 5 or 20% level of the diet did not affect the lipotropic activity of threonine. Time, however, was found to be a factor. Harper, Benton, Winje, Monson and Elvehjem (1954) found liver fat deposition in weanling rats reached a peak during the first two weeks on a threonine deficient diet and then began to taper off. They reported 30 to 40% fat at the end of two weeks and about 15% fat at the end of 10 weeks. The effect of a deficiency of threonine on the activities of certain liver enzymes was studied by Harper, Monson, Litwack, Benton, Williams and Elvehjem (1953). The activi? ties of the mitochondrial enzymes, succinic oxidase and choline oxidase, were higher in liver homogenates from threonine deficient animals as compared with threonine supplemented animals. In contrast, the activities of the cytoplasmic enzymes xanthine oxidase and tyrosine oxidase were lower in the livers of threonine deficient animals as compared with control animals. Reduced endogenous respiration observed in liver homogenates from animals receiving the 9% casein (threonine deficient) diet, as compared to controls was not linked directly with changes in liver fat deposition

16 since factors such as supply of respiratory substrates and activities of oxidative enzymes could affect the rate of endogenous respiration. Arata et al., (1954) also found endogenous oxidation reduced in the livers of threonine deficient rats. This observation led them to a study of pyridine nucleotides on the basis that a large percentage of endogenous respiration probably occurred by way of the pyridine nucleotide-linked systems (Arata et al., 1956). In addition, diphosphopyridine nucleotide (DPN) was known to be intimately connected with fatty acid oxidation. The results of their study showed both a defect in DPN synthesis and improper metabolism of endogenous DPN in the liver. The authors suggested this disruption in the metabolism of pyridine nucleotides could represent at least one major factor in causing accumulation of fat in the livers of threonine deficient animals. In 1960, Carroll et al., reported enzyme activity and fat deposition in the livers of rats fed a 9% casein diet was a function of time (in agreement with Harper, Benton, Winje, Monson and Elvehjem, 1954). Fat deposition reached a maximum at 24 days and decreased thereafter while the amount of liver fat in the 9% casein, threonine supplemented controls remained fairly constant. Correspondingly, the activities of xanthine oxidase and malic dehydrogenase decreased, as compared with the controls, reaching minimum activity on the 19th day of the experiment. This decrease

17 in activity was followed by a period of recovery. A second experiment of the same design verified the above results and led the authors to the observation that fat was not mobilized out of the livers of threonine deficient rats until after the enzymes had begun to recover. Yoshida and Harper (1960) studied the metabolism of 014-labeled acetate and palmitate in threonine deficient versus threonine supplemented rats. Labeled acetate was injected intraperitoneally into both groups of rats and expired 002 was collected for three hours, after which the~ animals were killed. Labeled palmitate was also injected into threonine deficient and threonine supplemented rats and these rats were killed 5 hours after the injection. Radioactivity of liver and carcass lipids was counted directly after a series of extractions. The amounts of Cl4 from labeled acetate incorporated into body fat and liver neutral fat were significantly greater in threonine deficient rats than in the controls, suggesting an increased fat synthesis. While the accumulation of liver fat in threonine deficient rats could have resulted from migration of body fat to the liver, this possibility was discounted by the authors since the specific activity of liver neutral fat greatly exceeded that of carcass fat after injection of both labeled acetate and palmitate. It also seemed unlikely to the authors that transport of fat from the liver to the body was impaired because after administration of either labeled compound, the

18 amount of radioactivity in the carcass fat of rats fed the threonine deficient diet was as great or greater than that in the controls. No evidence was obtained that threonine deficiency impaired the ability of the rat to oxidize either acetate or palmitate, but the possibility that fatty acid oxidation in the liver alone was impaired could not be eliminated since this might not be detected in intact animals. Hence, the authors concluded that fat accumulation in the livers and carcasses of rats fed a low protein,threonineékficient diet was caused primarily by a stimulation of fat synthesis. Using manometric techniques to measure the activity of the fatty acid oxidase system in liver homogenates from threonine deficient and threonine supplemented rats, Arata (1960) found fatty acid oxidase activity was significantly lower in the threonine deficient group than in the control group after two weeks. The activity continued to decline throughout the experimental period of 4 weeks and this decline was associated with profound disturbances in the metabolism of adenosine triphosphate and pyridine nucleotides which are necessary cofactors for the fatty acid oxidases. 0n the basis of these data, the author concluded that one reason for the abnormally high quantities of fat accumulating in the livers of rats fed a diet deficient in threonine could be the liver's loss of ability to oxidize fatty acids. Litwack et al., (1952) compared the effect on liver fat

19 10 and growth of feeding rats a 9% casein~sucrose diet to feeding rats a 9% casein-dextrin diet. Rats fed the latter diet grew at a better rate and had less severe fatty livers than rats fed the former diet. The authors suggested carbohydrate may influence liver fat and growth of rats on a low protein diet by controlling the rate of absorption of the necessary amino acids from the gut. Harper and Katayma (1953) studied the utilization of a 9% casein diet in the presence of sucrose or cornstarch. Better growth was supported by cornstarch than by sucrose but any growth differences could be eliminated by increasing either the level of casein or the levels of 5 essential amino acids (lysine, valine, threonine, tryptophan and histidine). They suggested the growth differences observed were due to the availability of greater quantities of amino acids when sucrose was replaced by cornstarch. Unfortunately, there were no liver fat data reported in this study. Harper, Monson, Arata, Benton and Elvehjem (1953) confirmed the observation by Litwack et al., (1952) that less fat was deposited in the livers of rats fed a 9% casein diet when sucrose was replaced by dextrin. In addition, a reduction in liver fat was observed when cerelose replaced sucrose whereas if fructose replaauisucrose, an increase in liver fat occurred. The decrease in liver fat resulting from the replacement of sucrose with dextrin or glucose in a 9% casein diet was attributed to improved utilization of dietary

20 11 protein. Sidransky and Clark (1961) forcewfed rats a high carbohydrate (28-40 cal/day) or low carbohydrate (l6 cal/day) synthetic diet deficient in threonine. Higher levels of fat were deposited in the periportal area of the livers of rats fed the high carbohydrate diet as compared with the low carbohydrate diet. This was true whether the source of carbohydrate was sucrose or dextrin. In a similar study, Yoshida et al., (1961) used a 9% casein-sucrose diet. The caloric value of the diet was lowered at the expense of sucrose with the protein intake held constant in all groups of rats. Liver fat decreased as the number of calories sup plied by sucrose in the diet decreased. This observation suggested the reduction in diet calories from carbohydrate decreased the quantity of precursors for fat synthesis. If fatty livers induced by a threonine deficiency were caused by an increase in fat synthesis as suggested by Yoshida and Harper (1960), then a reduction in liver fat with a corresponding decrease in carbohydrate calories would be expected. A later study by Yoshida and Ashida (1962) confirmed the above observation. One group of rats was fed a threonine deficient basal diet ad libitum. The other groups of rats were fed the same amount of casein and methionine as the basal group received but caloric intake was restricted to 90, 80 and 70% of the basal. A control group was fed the basal diet supplemented with threonine and allowed to eat

21 12 ad libitum. The level of liver fat of rats fed the threonine deficient diet decreased with each decrease in caloric intake. Moreover, the amount of-nitrogen retained by calorie restricted animals deficient in threonine decreased but the ratio of calories to retained nitrogen increased. That is, as the level of calories from carbohydrate in the diet decreased, the amount of nitrogen retained by the animals increased suggesting the lower the level of carbohydrate, the more efficient the utilization of dietary protein. Since this inverse relationship was not observed in the threonine supplemented animals, the authors suggested fatty livers caused by feeding rats an imbalanced amino acid diet wwsdue to a disproportionately high intake of calories in relation to the intake of a balanced protein. Litwack et al., (1952) demonstrated the balance of amino acids required for good growth of rats was not the same as that required for preventing fatty livers. Supplementing a 9% casein ration with tryptophan or niacin promoted good growth of the animals but fatty livers developed. The addition of 0.3% threonine to the above diet promoted growth and prevented fat from accumulating in the liver; addition of 0.15% threonine also promoted growth but did not depress fat content of the liver. Supplementing a 9% casein diet (without additional niacin or tryptophan) with 0.15% threonine reduced the liver fat level but also lowered the growth rate. Similar results were reported by Harper,

22 13 Monson, Benton and Elvehjem (1953) and Elvehjem and Krehl (1955). From the above observations it is evident that feeding rats a 9% casein diet results in a classical" example of amino acid imbalancel. In order to ensure good growth of the animals being fed a 9% casein diet, the diet must be supplemented with tryptophan; if it is desirable to prevent fatty livers from occurring in rats fed a 9% casein diet, the diet must be supplemented with threonine; if both good growth and normal liver fat are to be achieved, the 9% casein diet must be supplemented with both threonine and tryptophan. In the studies reported here, 9% casein diets supplemented with tryptophan and niacin were used to measure the effects of various dietary modifications on the fatty liver syndrome associated with threonine deficiency. 1 An amino acid imbalance has been defined as: "... the addition of a relatively small amount of an indispensable amino acid or a mixture of such amino acids, or of an unbalanced protein, [pausing] a retardation of growth or some other adverse effect that can be prevented by the addition of small quantities of the most limiting amino acid to the diet. (Harper, 1958)

23 GENERAL MATERIALS AND METHODS Male weanling rats of the Sprague-Dawley strain were used in all experiments. The animals were divided into groups on the basis of weight; the mean weight of all groups in the same experiment did not vary by more than one gram. No fewer than 10 animals were used in any one group. The rats were housed in individual cages with raised screen bottoms and allowed food and water ad libitum. All animals were weighed weekly during the experimental period of two weeks. Food consumption was recorded for each animal and any food spilled by the animal was recovered and weighed to ensure more accuracy of food intake measurements. The percent composition of the basal diet (A) was as follows: Casein 9.00 Salt Mixture w Hydrogenated Fat Glucose Choline 0.15 Methionine 0.30 Tryptophan 0.10 Vitamin Mix Obtained from Nutritional Biochemicals Corp. 2 Containing 7.5 mg of artocopheral acetate. The terms hydrogenated or unhydrogenated and saturated or unsaturated fat refer to fats with iodine numbers of 46 and 85, respectivegi.

24 15 The composition of the vitamin mix in mg/kg of diet was: ' Thiamine HCl 5.0 Riboflavin 5.0 Niacin 10.0 Pyridoxine HCl 2.5 Ca pantothenate 20.0 Inositol Para-aminobenzoic acid 10.0 Folic acid 0.2" B12 (0.1% trituration with mannitol) 20.0 Biotin 0.1 Vitamin A powder (20,000 IU/g) Calciferol 1.8 Menadione 3.8 Sucrose All other diets were identical to the basal with one of the following modifications: 1) a different sugar was used in place of glucose; 2) the type of fat was changed from saturated to unsaturated; or 3) the number of calories provided by dietary carbohydrate versus dietary fat were adjusted to be a given percent of the total calories from fat and carbohydrate combined (calories provided by dietary protein were not calculated since the protein content did not change in any of the diets). All diets used in these studies were isocaloric and deficient in threonine. Alpha 061, a non-nutritive filler, was used when necessary to adjust weight differences in the diets. For ease of presentation and discussion, this paper has been divided into three parts. Each part is devoted to a study of one of the above dietary modifications. The

25 16 description of the diets used in each study is presented in the appropriate section. At the end of the two week experimental period, the animals were stunned by a sharp blow on the head and decapitated. The livers were excised, weighed and homogenized with water in a Potter Elvehjem homogenizer. The homogenates were quantitatively transferred to tared evaporating dishes and dried at loo-110 C for 12 hours. The dried livers were weighed and ground to a fine powder in a small Wiley mill. One to two grams of the dried ground liver were subjected to continuous ether extraction for three hours in a Goldfisch fat extractor. The ether soluble lipoidal material was weighed and calculated as percent dry weight of tissue. Nitrogen analyses were done in duplicate on 0.3 g of dry, fat-extracted tissue using the macro Kjeldahl procedure. Results were expressed as percent nitrogen based on the fresh weight of liver. Standard errors of the means were calculated for all data and Student s "t test was used as a measure of significance. Differences_between groups were considered significant if the probamlflw'wxe less than 0.01 unless otherwise indicated.

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27 PART I THE EFFECT OF VARYING THE DIETARY CARBOHYDRATE SOURCE ON THE SEVERITY OF FATTY LIVERS IN RATS FED THREONINE DEFICIENT DIETS

28 INTRODUCTION The consumption of a 9% casein diet supplemented with methionine and tryptophan, but not with threonine, causes the appearance of fatty livers in weanling albino rats (Dick et al., 1952; Singal et al., 1949, 19533, b; Litwack et al., 1952; Harper, Monson, Benton and Elvehjem, 1953a; Harper, Benton, Winje, Monson and Elvehjem, 1954; Arata et al., 1954). This type of fatty liver has been intensively investigated by many workers. In virtually every instance, sucrose has supplied the carbohydrate in these low protein, threonine deficient rations. Little attention has been devoted to studies designed to measure the effect of the type of carbohydrate in the diet on fatty livers associated with a threonine deficiency. A pilot study was conducted in this laboratory wherein glucose replaced sucrose in a 9% casein diet deficient in threonine. The expected fatty livers did not appear in glucose-fed, threonine deficient rate. This preliminary observation led to a more specific study of the effect of the dietary source of carbohydrate. 1?

29 MATERIALS AND METHODS The composition of the diets used in this study was identical to the basal diet as described on page 14 except the carbohydrate source was varied as follows: Diet A % glucose (basal) Diet B % sucrose Diet % glucose and 40.6% fructose Diet D - Diet E - Diet F 81.2% fructose 81.2% maltose 81.2% lactose A level of 81.2% lactose (Diet F) proved to be highly toxic to the weanling rat. Therefore, no data from group F are included in this report. A concentration in the diet of about 50% lactose approaches the upper limit of this sugar which can be metabolized by the rat (Tomarelli et al., 1960). At the close of the two week experimental period, the animals were sacrificed as previously described (p. 16) except when fatty acid oxidase activity, endogenous oxidation and liver pyridine nucleotides were determined. The procedure was then modified as follows: Beginning on the 14th day of the experimental period, two animals from each group were sacrificed daily by decapitation; the livers were removed as quickly as possible and immediately chilled in ice. A sample ( mg) was placed into a tared weighing 18

30 bottle containing 20 mg Ce(504)2 in 10 ml of 2% nicotinamide 19 solution. The exact weight of the sample was obtained by difference and liver pyridine nucleotides determined according to the method of Robinson et al., (1947). The activity of the fatty acid oxidase system in liver homogenates was measured manometrically using the Warburg apparatus. The method used was essentially that of Lehninger (1955) except that one ml of whole homogenate (33.3%) was used in place of 0.5 m1 of mitochondrial suspension. The remaining liver was weighed and saved for fat, nitrogen and moisture analyses as described on page 16. When the residual liver from a given rat was inadequate in size for these analyses, it was combined with the residual liver from one or more rats in the same group until approximately 2.5 g of liver were collected. This entire procedure was repeated every day until the 18th day when all of the animals had been sacrificed.

31 RESULTS A. Food Intake and Weight Gain. These data are summarized in table 1. The correlation between weight gain and food intake is obvious; an increaafl.food intake resulted in an increase in weight of the animals whereas a lower food intake resulted in a lesser weight gain. The fact that animals consuming the sucrose diet (B) or the fructose diet (D) ate significantly less than the other groups could possibly be explained on the basis of palatability since fructose and sucrose are the sweetest of the sugars used here. However, the observation that animals receiving the diet of half glucose and half fructose (C) consumed a quantity comparable with the glucosegyxup (A) or the maltose group (E) tends to nullify this explanation. B. Fresh Liver Weight. A pronounced increase in the fresh weight of liver tissues was observed in the groups fed fructose-containing diets as compared with the glucose controls (table 2). Livers from rats fed equal parts of glucose and fructose (group C) as the carbohydrate source were significantly heavier in weight than those from rats fed either glucose (group A) or sucrose (group B). The liver weights of the maltose-fed rats (group E) and of the sucrosefed rats (group B) were roughly comparable to the control group A (glucose). 20

32 21 The observation that the livers of rats fed a diet containing fructose were significantly greater in weight could be explained on the basis of the work reported by Cori (1926) and Reinhold and Karr (1927). These workers demonstrated that fructose was a more rapid and effective glycogen former than was glucose. Although glycogen analyses were not done in the experiments reported here, glycogen is the only other major component of the liver not measured. Since the increased liver weights of groups C and D could not be correlated with increased concentrations of moisture, protein or fat (table 2) the increased weight could be reflective of an increased glycogen storage in these tissues. C. Moisture Content. Significant variation in the moisture content of the livers of rats fed diets containing different sources of carbohydrate was observed (table 2). These variations were closely correlated with the amount of fat present in the livers. As the amount of fat deposited in the liver increased, the percent moisture decreased. Therefore, fat deposition occurred to some extent, at the expense of moisture. D. Nitrogen Content. These data are summarized in table 2. An interesting trend in liver nitrogen content was observed with a change in dietary carbohydrate. No significant difference was noted between groups A and E (glucose and maltose). Likewise, no significant differences were observed among the fructose-containing groups B, C and

33 22 D (sucrose, glucose and fructose, and fructose). The nitrogen content of livers from rats fed the fructose diets (either as free fructose or combined in the sucrose molecule) was lower than that of livers from rats fed the glucose diets (either as free glucose or combined in the maltose molecule). The meaning of these data is not clear. E. Fat Content. Significant differences were observed in the liver fat content of rats fed the various carbohydrate diets (table 2). The sucrose diet caused a significantly greater deposition of liver fat than did any other diet. When the dietary carbohydrate was provided by fructose (group D) or maltose (group E) the concentration of liver fat in animals consuming these diets was comparable to that in the glucose control group (A). Liver fat levels in these three groups were approximately one-half those in the sucrose-fed group (B). When the sucrose in the diet was replaced with equal portions of the constituent monosaccharides, liver fat levels were not similar (groups B and 0). Animals fed equal parts of glucose and fructose (group C) deposited significantly less fat in the livers than did animals fed diet B (sucrose). This experiment was conducted twice and similar results were obtained each time. On the other hand, feeding a diet composed of equal parts of glucose and fructose (0) caused fatty livers to develop which contained significantly more fat than the glucose (A), fructose(d) or maltose (E)

34 23 groups. F. Liver Pyridine Nucleotides, Fatty Acid Oxidase Activity and Endogenous Oxidation. These data are summarized in table 3. There were no significant differences among any of the groups in concentration of liver pyridine nucleotides. Replacement of the carbohydrate in the control group A (glucose) with sucrose (group B) did not affect the activity of the fatty acid oxidase system or endogenous oxidation. However, when the carbohydrate in the diet contained free fructose (groups C and D) the oxidation of octanoate was increased and endogenous oxidation was decreased when compared to the glucose controls. Comparison of the data from the group of rats fed the _ diet simulating sucrose (C) with that from the sucrose-fed group (B) groups. revealed metabolic differences between these two Although both dieuainduced fatty livers in the rats consuming them, the fatty acid oxidase activity of group C was significantly greater than the activity of group B. There was no significant difference in endogenous oxidation between these two groups.

35 DISCUSSION The results of this study indicate carbohydrate has a very definite effect on liver fat deposition in threonine deficient animals. Glucose, fructose or maltose, if the sole carbohydrate source in the diet, exerts a "protective" action on threonine deficient animals since liver fat levels in these animals are only slightly above the normal level of 8 to 10% (dry weight basis). When threonine deficient diets containing sucrose as the carbohydrate source are fed to weanling rats, roughly twice as much fat is deposited in the livers as compared to control animals. Neither the fructose nor the glucose part of the sucrose molecule appears to be responsible for this effect since neither of these monosaccharides caused the appearance of fatty livers. These data are in conflict with an observation made by Harper, Monson, Arata, Benton and Elvehjem (1953). They noted the appearance of fatty livers in rats fed a 9% casein diet deficient in threonine when fructose provided the carbohydrate source. In the study reported here, animals fed a 9% casein diet deficient in threonine and containing fructose as the carbohydrate source did ggtdevelop fatty livers. The experiment was conducted on three separate occasions and involved different lot numbers of fructose. On each occasion, our results were 24

36 25 identical. The source of the discrepancy between these data and those collected by Harper and his coworkers is unknown. Another perplexing aspect of this study is the effect produced by the diet containing equal parts of glucose and fructose (C) as compared with that produced by the sucrose diet (B). In combination, these two monosaccharides cause fat accumulation in the livers of threonine deficient rats whereas separately they do not, under the conditions previously described. The fact that not as much fat is deposited in the livers when rats consume a diet containing equal parts of glucose and fructose (C) as compared with sucrose (group B) is not a function of growth, since the sucrose-fed rats grow more slowly. Further, in group C there is an increased activity of the fatty acid oxidase system as compared with group B. Clearly, the metabolism of a equal parts of glucose and fructose is not comparable with the metabolism of sucrose (Reinhold and Karr, 1927; Rabinowitch, 1947). It has previously been reported that the occurrence of fatty livers in rats fed a threonine deficient diet containing sucrose as the carbohydrate source cmfld.be attributed to: 1) reduced activity of the fatty acid oxidase system (Arata, 1960); 2) a defect in diphosphopyridine nucleotide (DPN) production; and/or 3) the impr0per metabolism of endogenous DPN in the liver (Arata et al., 1956). These aberrations could be corrected by the addition of threonine to the diet.

37 26 In the present study, fatty livers were observed as expected in rats fed a threonine deficient diet with sucrose as the carbohydrate source. However, when glucose was substituted for sucrose in the threonine deficient diet, fatty livers did ggt develop. Furthermore, there was no difference between the glucose and sucrose groups with respect to: l) activity of the fatty acid oxidase system; 2) concentration of liver pyridine nucleotides; or 3) rate of endogenous oxidation. Therefore, the mechanism whereby glucose prevents accumulation of fat in the livers of rats fed a 9% casein diet supplemented with methionine and tryptophan is not the same as the mechanism involved when threonine is added to such a diet. The above observations necessarily lead to the conclusion that fatty livers in rats fed a 9% casein diet are not the result of a threonine deficiency Instead, these fatty livers must be reflective of some intricate balance or interaction between threonine and the kind of dietary carbohydrate.

38 27 Table 1. Food intake and weight gain of rats fed threonine deficient diets1 in which the source of the carbohydrate2 varied. Group2 N Food Intake Weight Gain grams/week A B :2.1 l9.2il : D il E tl.2 1 All diets were isocaloric. 2 A - glucose (basal diet) B - sucrose 0 - half glucose and half fructose D - fructose E - maltose 3 Standard error of the mean.

39 28 Table 2. Liver composition of rats fed threonine deficient diets1 in which the source of the carbohydrate2 varied. Group? N 322$? WW 5 F F 3 A : : s : tl.l c : D : : E :0.22 lo IO.4 1 All diets were isocaloric 2 A - glucose (basal diet) B sucrose C - half glucose and half fructose D - fructose E - maltose 3 Expressed as % fresh weight of tissue Expressed as % dry weight of tissue 5 Standard error of the mean

40 29 Table 3. Liver pyridine nucleotides, fatty acid oxidase activity and endogenous oxidation of rats fed threonine deficient diets1 in which the source of the carbohydrate2 varied. Fatty Acid Endogenousfi Group2 Liver PN's Oxidase Oxidation mcg_ N/g tissue ul 02(hrggm tissue A (10) (9) (9)4 B (10) (8) (8) C (10) (6) (6) D (10) (7) 1234:82 (9) 1 All diets were isocaloric 2 A - glucose B - sucrose C - half glucose and half fructose D - fructose 3 Standard error of the mean 4 Number of observations Table 3a. Significance of differences between comparable groups. (Percent indicates level of significance.) -_ Groups Liver Fatty Acid Endogenous Compared ggy's Oxidase Oxidatign A-B none none none A-C none 2% none A-D none 1% 2% B-C none 2% none B-Q none 1% 1%

41 PART II THE EFFECT OF TYPE OF DIETARY FAT ON THE APPEARANCE OF FATTY LIVERS IN THREONINE DEFICIENT RATS FED DIETS CONTAINING GLUCOSE OR SUCROSE.

42 INTRODUCTION In the preceding section it was established that dietary carbohydrate markedly influenced the extent of liver fat deposition in threonine deficient animals. Before further investigation of this phenomenon was undertaken, of other dietary components had to be measured. the influence Two studies were designed to explore this subject. One was directed toward an evaluation of the effect of increasing the vitamin concentration of the basal diet, and the other was aimed toward determining the effect of the fat source in the basal diet. No effect on liver fat deposition in threonine deficient animals as compared with controls was observed as a result of increasing the B vitamin content of the diet or increasing the concentration of a single B vitamin, niacinl. In the experiment reported here, animals were fed 9% casein diets deficient in threonine, containing either glucose or sucrose and either saturated or unsaturated fat. The extent to which the two different fats altered the se verity of fatty livers under the conditions stated was measured. 1 Unpublished data. 30

43 MATERIALS AND METHODS The composition of the diets used in this study was identical to the basal diet as described on page 14 with the following modifications: Diet A % glucose and 5% saturated fat (basal) Diet B - Diet G - Diet H % sucrose 81.2% glucose 81.2% sucrose and5% saturated fat and 5% unsaturated fat and 5% unsaturated fat. 31

44 RESULTS A. Food Intake and Weight Gain. The animals fed the sucrose diets (B and H), regardless of the fat source, consumed significantly less diet and grew more slowly (table 4) than the animals fed the glucose diets (A and G). Significantly less food was consumed by the animals fed the glucosehydrogenated fat diet (A) as compared with animals fed the glucose-unhydrogenated fat diet (G) but this difference in food consumption was not reflected in the growth rates of the animals. B. Fresh Liver Weight. There was no significant difference in fresh weight of the liver between any group and its control (table 5). The substitution of sucrose for glucose in a diet containing saturated fat (groups A'vs B) or unsaturated fat (groups G vs H) did not alter the liver weights. Similarly, when the fat source was changed from saturated to unsaturated in the glucose diet (groups A vs G) or the sucrose diet (groups B vs H), no difference in liver weights was observed. 0.. Moisture and Nitrogen Content. These data are summarized in table 5. The livers of rats fed the sucrosehydrogenated fat diet (group B) contained less moisture and nitrogen than did those of rats fed the glucose-hydrogenated fat diet (group A). However, when the saturated fat in the 32

45 33 sucrose-containing diet was replaced with saturated fat (groups_b and H), the nitrogen and moieture content of the livers was significantly increased. As a result, when the fat source was unsaturated, no'differences were observed in nitrogen or moisture concentrations between livers from sucrose- and glucose-fed animals (groups C and H). There were no differences in moisture or nitrogen content of livers from rats fed glucose diets, regardless of the nature of the fat source. D. Fat Content. These data are presented in table 5. Percent liver fat of animals fed the sucrose diets (B and H) was significantly higher than that of the glucose-fed animals (groups A and G). Whether the dietary fat was hydrogenated or unhydrogenated did not significantly affect liver fat deposition in either the sucrose groups or the glucose groups.

46 DISCUSSION From the results presented here, it is evident that the kind of fat, at the level of 5% of the diet, has no influence on the amount of fat deposited in the livers of threonine deficient rats. Harper, Monson, Benton, Winje and Elvehjem (1954) reported similar results. Some preliminary data from this laboratory have been collected which suggested a marked difference in ability of threonine deficient animals to metabolize saturated versus unsaturated fat when the fat content of the diet was elevated to 40%. However, this difference was not evident when the fat level of the diet was 5%. Since data in all portions of the study reported here were collected using diets containing 5% fat, one must conclude that the alleviation of fatty livers by replacing sucrose with glucose, fructose, or maltose (Part I) is not altered by the nature of the fat source in the diet. Thus, the fatty livers observed in threonine deficient animals fed diets containing sucrose and 5% fat reflect an apparent inability of those animals to metabolize sucrose. 34

47 PART III THE EFFECT OF A DIFFERENTIAL APPORTIONMENT OF CALORIES FROM GLUCOSE AND FAT IN ISOCALORIC DIETS ON THE APPEAR- ANCE OF FATTY LIVERS IN THREONINE DEFICIENT RATS.

48 35 Table 4. Food intake and weight gain of rats fed threonine deficient diets1 containing glucose or sucrose and saturated or unsaturated fat. *1 < 1* Group? N. Fgod Intake Weight Gain ' grams7week ' A s : ti.2 H : to.9 J :1.7 l7.6tl.0 1 All diets were isocaloric cameo» llll glucose and saturated fat sucrose and saturated fat glucose and unsaturated fat sucrose and unsaturated fat 3 Standard error of the mean

49 36 Table 5. Liver composition of rats fed threonine deficient diet containing glucose or sucrose and saturated or unsaturated fat. Fresh Liver r *7 Liver Com osition Group2 N Weight N (Mgisture Nitro en3 Fatz l s % "' FL""""75""" A : : B tO.2l liO O.O l H oio io to.6 J :0.19 lo t tl.l 1 All diets were isocaloric 2 A - glucose and saturated fat B - H - J - sucrose and saturated fat glucose and unsaturated fat sucrose and unsaturated fat 3 Expressed as % fresh weight of tissue 4 Expressed as % dry weight of tissue 5 Standard error of the mean

50 INTRODUCTION Rats fed a 9% casein diet supplemented with methionine and tryptophan but not with threonine develop fatty livers when the carbohydrate source of the diet is sucrose and the fat level of the diet is 5%. The data collected in Part I of this study established the importance of the carbohydrate source in determining whether or not a deficiency of threonine resulted in the appearance of fatty livers. When sucrose was replaced with glucose in threonine deficient diets, the deficiency of this essential amino acid was no longer manifested by the appearance of fatty livers. The chemical nature of the dietary fat, when provided as 5% of the diet did not represent a major factor in the appearance of fatty livers (Part II). An experiment was designed to study the effect of different levels of glucose in the diet on the severity of the fatty liver syndrome. All diets used in this study were isocaloric to avoid introducing another variable. 37

51 MATERIALS AND METHODS The composition of the diets used in this study was identical to the basal diet as described on page 14 except the percent glucose and fat varied as follows: Diet % Glucose % Fat % Alpha Cel Caloric Ratiol J :1O K :25 L :50 M :75 N :90 1 Caloric ratio : percent of calories from glucose compared to the percent of calories from saturated fat. Since the protein content of these diets did not vary, calories from this source were not calculated in the total. 38

52 RESULTS A. Food Intake and Weight Gain. The average food intake from group to group covered a wide range (table 6). The differences in food intake did not appear to be directly correlated with the percent carbohydrate in the diet. However, as noted in Part I, a correlation exists between food consumption and weight gain. The differences in growth were relatively small, but a tentative trend mightbe noted. Animals grew best when the diet contained a 25:75 ratio of either fat:g1ucose or glucose:fat calories (groups K and M). The poorest growth and lowest food consumption was observed in group J (90:10 glucose:fat caloric ratio). No reason for this trend is apparent. These differences, though statistically significant, may be an artifact. A larger number of animals must be studied before this suggestion can be verified. B. Fresh Liver Weight. These data are summarized in table 7. Although there were some differences in fresh liver weights between groups, these differences were relatively small and no correlation between liver weight and dietary modification was evident. C. Moisture and Nitrogen Content. Some variation was observed in moisture and nitrogen content from group to group (table 7) but, again, these differences were small and 39

53 40 did not describe any trend with respect to modifications in the diet. D. Fat Content. These data are presented in table 7. There were no differences in liver fat levels of rats fed diets containing 90, 75 or 50% of the glucose-fat calories from glucose (groups J, K and L). However, a reduction of glucose calories to 25% resulted in a significantly greater level of liver fat (group M); a further reduction to 10% increased liver fat levels still more (group N).

54 DISCUSSION The relationship between the caloric level of carbohydrate in the diet and the amount of fat deposited in the livers of rats consuming the various diets can be seen in figure 1. Two carbohydrate sources, glucose and sucrose, were compared. There apparently was an Optimum range of glucose concentration in the diet which prevented the occurrence of fatty livers in threonine deficient animals. This range was 50 to 90% of the calories from glucose. When 50% or more of the fatzcarbohydrate calories were provided by glucose fatty livers did not appear; when more than 50% of the calories were provided by glucose liver fat levels increased concomitantly. However, as the level of glucose decreased in these isocaloric diets, the level of dietary fat necessarily increased. Thus, when 10% of the carbohydrate-fat calories were present as glucose and 90% as fat, the fat content of the diet approached 40%. Since this constituted a high fat diet, the suggestion arose that the fatty livers observed in rats fed diets J or K resulted from an induced choline deficiency. Griffith (1948) observed the choline requirement increased as the level of dietary fat increased. The sucrose curve in figure 1 was constructed from unpublished data collected in this laboratory, of which this 41

55 42 study is an extension. None of the liver fat values represented in the sucrose curvevnnssignificantly different from any other9 thus the values for liver fat observed when the level of sucrose was varied in a threonine deficient diet lay essentially on a straight line. If the increased liver fats observed in glucose groups M and N were reflections of a choline deficiency induced by a high fat diet, then a similar result would be expected in the sucrose-fed animals. This was not the case. There is no adequate explanation at present for this paradox. The effect of dietary calories on liver fat accumulation was also studied by Yoshida et al., (1961) in rats fed a 9% caseinmsucrose diet supplemented with methionine and choline. The protein and fat intake of the experimental groups was kept constant but the total caloric intake was restricted by limiting the intake of sucrose. They observed when the caloric intake was 70% of the controls, liver fat was decreased from 20% in the controls to 13% in the restricted animals. Weight gain of the two groups was comparable. The authors suggested the restriction in total caloric intake decreased the quantity of precursors for fat synthesis, thus accounting for the reduction in liver fat in calorie restricted rats. A subsequent study by Yoshida and Ashida (1962) similarly constructed and results were comparable to was those from the above study. Since the fat and protein intake of

56 43 the animals was held constant, and the caloric restrictions were accomplished by adjusting the sucrose intake, the authors Concluded the restriction of calories from carbohydrate was responsible for preventing fatty livers in threonine deficient animals. In order to establish the fact that a reduction in liver fat was caused by a decreased intake of carbohydrate calories as opposed to a decreased intake of total calories, an experiment would have to be done in which the carbohydrate calories remained constant and the fat calories restricted. If fatty livers were not observed, then it could readily be said that excess carbohydrate calories were responsible for fatty livers. However, if fatty livers did occur, the concept that carbohydrate calories were responsible for fatty livers would be negated, thus indicating the total caloric intake, regardless of source, was responsible for fat deposition in the livers of threonine deficient rats. The results of the study reported here do not support the hypothesis of Yoshida and Ashida (1962) that the restriction of calories from carbohydrate is responsible for fatty livers in threonine deficient animals. In this study, when animals were fed sucrose-containing diets which were isocaloric, no reduction in liver fat levels was observed as the sucrose content decreased. In addition, the data presented here support the suggestion that the effect of glucose is markedly different from the effect of sucrose

57 44 on fatty livers associated with a threonine deficiency. Therefore, the use of the term "carbohydrate is contraindicated in discussing the fatty liver syndrome in connection with a deficiency of threonine.

58 45 Table 6. Food intake and weight gain of rats fed threonine deficient diets1 in which the calories from glucose and fat varied. Caloric Group N Ratio2 Food Intake Weight Gain grams/week ' J 10 90: K 20 75: il L 20 50: l 24.7il.2 M 19 25: i :o.9 N 19 10: i All diets were isocaloric 2 Caloric ratio 2 percent of calories from glucose compared to the percent of calories from saturated fat. Since the protein content of these diets did not vary, calories from this source were not calculated in the total. 3 Standard error of the mean

59 46 Table 7. Liver composition of rats fed threonine deficient diets1 in which the calories from glucose and fat varied. Caloric Fresh Liver Liver Com osition Group N Ratio2 Weight Moisture Nitro en3 Fat, 5 % 3 % J 10 90: : * o K 20 75: l.ltO ll.3to.5 L 20 50: :0.10 7o.oio.4 2.6iio.2o l3.2io.2 M 19 25: iO N 19 10: : :o : :o.2 All diets were isocaloric 2 Caloric ratio : percent of calories from glucose compared to the percent of calories from saturated fat. Since the protein content of these diets did not vary, calories from this source were not calculated in the total. 3 Expressed as % fresh weight of tissue 4 Expressed as % dry weight of tissue 5 Standard error of the mean

60 47 Figure 1. The effect of various levels of glucose and sucrose in isocaloric diets on the percent liver fat (dry weight basis) in threonine deficient rats SUCROSE 20 liver fat GLUCOSE d 75 9b Percent of fat-carbohydrate calories from carbohydrate

61 GENERAL SUMMARY AND CONCLUSIONS Male weanling rats of the Sprague-Dawley strain were fed various experimental diets deficient in threonine. All diets were isocaloric. Nine percent casein diets were used with one of the following modifications: 1) different carbohydrates were used in the diets; 2) the source of dietary fat was changed; or 3) the ratio of carbohydrate to fat calories was adjusted to a given percent of the total carbohydrate-fat calories. Food intake, weight gain, fresh liver weight, liver moisture, liver nitrogen and liver fat were determined in all animals. In addition, liver pyridine nucleotides, activity of the fatty acid oxidase system and endogenous reapiration in liver tissues were measured in one experiment. From these studies, the following observations were made: 1. Fatty livers do not develop in rats fed a threonine deficient diet if the sole source of carbohydrate is glucose, fructose or maltose. 2. Fatty livers are observed in rats fed a threonine deficient diet when the carbohydrate of the diet is sucrose or a combination of equal parts of glucose and fructose, the former causing more sever fatty livers than the latter. 3. There are no differences between glucose-fed rats 48

62 49 and sucrose-fed rats with respect to: a) activity of the fatty acid oxidase system; b) Concentration of liver pyridine nucleotides; or 0) rate of endogenous oxidation by liver tissues. 4. The chemical nature of dietary fat (saturated versus unsaturated) at the 5% level of the diet has no influence on the deposition of fat in the livers of rats fed threonine deficient diets. This is true whether the dietary carbohydrate is glucose or sucrose. 5. When 90, 75 or 50% of the carbohydrate-fat calories of a threonine deficient diet are present as glucose, liver fat levels of rats are near normal. When 25 or 10% of the carbohydrate-fat calories of a threonine deficient diet are present as glucose, fatty livers in rats are observed which could be due to an induced choline deficiency. 6. If 90, or 25% of the carbohydrate-fat ca1ories of a threonine deficient diet are present as sucrose, fatty livers are observed in rats. 7. Fatty livers in rats fed a 9% casein diet, supplemented with methionine and tryptophan, are not the result of a threonine deficiency peg. g, Instead, these fatty livers are closely associated with the metabolism of sucrose, which cannot be accounted for by the metabolism of either glucose or fructose.

63 BIBLIOGRAPHY Arata, D Mechanism of adaptation to a threonine deficient diet. I. Biochemical aspects of a threonine deficiency. U.S. Dept. Comm., Office Tech. Serv., PB Rept. 153: pp. Arata, D., A.E. Harper, G. Svenneby, J.N. Williams, Jr. and C.A. Elvehjem 1954 Some effects of dietary threonine, tryptophan, and choline on liver enzymes and fat. Proc. Soc. Exp. Biol. Med., 91:544. Arata, D., G. Svenneby, J.N. Williams, Jr. and C.A. Elvehjem 1956 Metabolic factors and the development of fatty livers in partial threonine deficiency. J. Biol. Chem., 219,327. Beeston, A.W. and H.J. Channon 1936 Cystine and the dietary production of fatty livers. Biochem. J., fi9:280. Beeston, A.W., H.J. Channon, J.V. Loach and H. Wilkinson 1936 Further observations on the effect of dietary caseinogen in the prevention of fatty livers. Ibid., 39:1040. Best, C.H. and M.E. Huntsman 1932 The effects of the components of lecithin upon deposition of fat in the liver. Jo Phy8101e 15:405e Best, C.H. and M.E. Huntsman 1935 Choline and fat metabolism. Ibid., 91:255. Carroll, 0., D. Arata and D.C. Cederquist 1960 Effect of threonine deficiency on changes in enzyme activity and liver fat deposition with time. J. Nutrition, 19:502. Channon, H.J. and H. Wilkinson 1935 Protein and dietary production of fatty livers. Biochem. J., 99:350. Cori, C.F The fate of sugar in the animal body. III. The rate of glycogen formation in the liver of normal and insulinized rats during the absorption of glucose, fructose and galactose. J. Biol. Chem., 19:577. Dick, F. Jr., W.K. Hall, V.P. Sydenstricker, W. McCollum and L.L. Bowles 1952 Accumulation of fat in the liver with deficiencies of threonine and of lysine. A-M.A. Arch. Path., 51:

64

65 51 Elvehjem, C.A. and W.A. Krehl 1955 Dietary interrelationships and imbalance in nutrition. Borden's Rev. Nutr. Res., 19:69. ' ' Griffith, W.H Choline metabolism. IV. The relation of age, weight and sex of young rats to the occurrence of hemorrhagic degeneration on a low choline diet. J. Nutrition, 19:437. Harper, A.E Balance and imbalance of amino acids. Ann. N. Y. Acad. Sci., 99:1025. Harper, A.E Amino acid balance and imbalance. 1. Dietary level of protein and amino acid imbalance. J. Nutrition, 99:405. Harper, A.E., D.A. Benton, M.E. WinJe and C.A. Elvehjem 1954 On the lipotropic action of protein. J. Biol. Chem., 209:171. Harper, A.E., D.A. Benton. M.E. Winje, W.J. Monson and C.A. Elvehjem 1954 Effect of threonine on fat deposition in the liver of mature rats. Ibid., 209:165. Harper, A.E. and M.C. Katayma 1953 The influence of various carbohydrates on the utilization of low protein rations by the white rat. I. Comparison of sucrose and cornstarch in 9% casein rations. J. Nutrition, 9:26l. Harper5 A.E., W.J. Monson, D.A. Arata, D.A. Benton and C.A. Elvehjem 1953 Influence of various carbohydrates on the utilization of low protein rations by the white rat. II. Comparison of several proteins and carbohydrates, growth and liver fat. Ibid., 51:523. Harper, A.E., W.J. Monson, D.A. Benton and C.A. Elvehjem 1953a The influence of protein and certain amino acids, particularly threonine, on the deposition of fat in the liver of the rat. Ibid., 59: b Influence of carbohydrates and pro teins on liver fat deposition in rats. Fed. Proc., 12:416. Harper, A.E., W.J. Monson, D.A. Benton, M.E. Winje and C.A. Elvehjem Factors other than choline which affect the deposition of liver fat. J. Biol. Chem., 206:151.

66 52 Harper, A.E., W.J. Monson, G. Litwack, D.A. Benton, J.N. Williams, Jr. and C.A. Elvehjem 1953 Effect of partial deficiency of threonine on enzyme activity and fat deposition in the liver. Proc. Soc. Exp. Biol. Med., 93:414. Lehninger, A.L Methods 12 Enzymology. Edited by S.P. Colowick and N.0. Kaplan. :5 5. Litwack, G., L.V. Hankes and C.A. Elvehjem 1952 Effect of factors other than choline on liver fat deposition. Proc. Soc. Exp. Biol. Med., 91:441. Rabinowitch, I.M Metabolism of sucrose. II. Am. J. Digest. Dis., 15:315. Reinhold, J.G. and W.G. Karr 1927 Carbohydrate utilization. II. Rate of disappearance of various carbohydrates from the blood. J. Biol. Chem., 19:345. Robinson, J., N. Levitas, F. Rosen and W.A. Perlzweig 1947 The fluoreqcent condensation product of N1-methylnicotinamide and acetone. IV. A rapid method for the determination of the pyridine nucleotides in animal tissues. The coenzyme content of rat tissues. Ibid., 119:653. Sidransky, H. and S. Clark 1961 Chemical pathology of acute amino acid deficiencies. IV. Influence of carbohydrate intake on the morphologic and biochemical changes in young rats fed threonine- or valine-devoid diets. Arch. Path., 19: Singal, S.A., V.P. Sydenstricker and J.M. Littlejohn 1948 Further studies on the effect of some amino acids on the growth and nicotinic acid storage of rats on low casein diets. J. Biol. Chem., 119: The lipotropic action of threonine. Fed. Proc., 9:251. Singal, S.A., J.J. Hazan, V.P. Sydenstricker and J.M. Littlejohn 1953a The production of fatty livers in rats on threonine and lysine deficient diets. J. Biol. Chem., 999: b The lipotropic action of threonine and related substances in the rat. Ibid., 200:883.

67 ' 53 Tomarelli, R.M., R. Hartz and F.W. Bernhart 1960 The effect of lactose feeding on the body fat of the rat. J. Nutrition,,11:22l. Tucker, H.F. and H.C. Eckstein 1937 The effect of supplementary methionine and cystine on the production of fatty livers by diet. J. Biol. Chem., 121:479. Yoshida, A. and K. Ashida 1962 Protein utilization and caloric intake of rats with fatty liver due to an amino acid imbalance. Agr. Biol. Chem. (Tokyo),'99:56. Yoshida, 1., K. Ashida and A.E. Harper 1961' Prevention of fatty liver due to threonine deficiency by moderate caloric restriction. Nature, 189:917. Yoshida, A. and A.E. Harper 1960 Effect of threonine and choline deficiencies on the metabolism of 614-1abeled acetate and palmitate in the intact rat. J. Biol. Chem., 925:2586.

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