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A comparison of glucose tolerance in normal rats and those with fatty livers

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A comparison of glucose tolerance in normal rats and those with fatty livers

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Content A COMPARISON OF GLUCOSE TOLERANCE IN NORMAL MTS AND THOSE WITH FATTY LIVERS A Thesis Presented to the Faculty of the Department of Biochemistry University of Southern California School of Medicine In Partial Fulfillment of the Requirements for the Degree Master of Science hy Daisie Adelle Davis May 1959 O' This thesis, written by Daisie Adelle Davis under the direction of h..fF Faculty Committee, and app ro ved by all its members, has been presented to and accepted by the Council on Graduate Study and Research in partial fulfill ment of the requirements for the degree of c \ Master of Science D ean Secretary 1939 D ate. F acu lty C om m ittee ..... 1 " C hairm an r e ....... fbco ^ 9 TABLE OF CONTENTS CHAPTER PAGE I. THE PROBLEM . . . . ................. 1 II, REVIEW OF THE LITERATURE....................... 5 III, GENERAL PROCEDURE.......................... 8 IV. RESULTS....................................... 12 V. DISCUSSION.................................... 22 VI. SUMMARY....................................... 28 BIBLIOGRAPHY................................... 29 LIST OF TABLES TABLE PAGE I. THE GLUCOSE TOLERANCE CURVES AND LIVER ANALYSES OF MALE RATS PREVIOUSLY FED THE STOCK DIET..... 13 II. THE GLUCOSE TOLERANCE CURVES AND LIVER ANALYSES OF FEMALE RATS PREVIOUSLY FED THE STOCK DIET .... 14 III. THE GLUCOSE TOLERANCE CURVES AND LIVER ANALYSES OF MALE RATS PREVIOUSLY FED A HIGH-FAT DIET .... 15 IV. THE GLUG08E TOLERANCE CURVES AND LIVER ANALYSES OF FEMALE RATS PREVIOUSLY FED A HIGH-FAT DIET ... 16 V. ANALYSES OF LIVERS OF RATS KILLED AFTER 1 DAY FAST - STOCK DIET......................... 17 VI. ANALYSES OF LIVERS OF RATS KILLED AFTER 1 DAY FAST - HIGH-FAT DIET................... 18 VII. SUMMARY OF GLUCOSE TOLERANCE CURVES OF FOUR GROUPS OF RATS.......................... 19 VIII. SUMMARY TABLE SHOWING THE COMPARATIVE ANALYSES OF LIVER................................ 20 CHAPTER I THE PROBLEM There are a variety of pathological conditions in which a fatty infiltration of the liver occurs. Such phenomena have long been observed after chloroform or phosphorus poisoning, alcoholism, and experimental and clinical diabetes, where the fat infiltration is not prevented by the use of insulin. Pernicious anemia, yellow fever, acute yellow atrophy, eclampsia, and poisoning from such chemi cals as carbon tetrachloride, phenylhydrazine, and phloridzin, are among the conditions in which fatty livers are encountered. Probably the most severe condition can be produced experimentally by dietary means. In the latter instance, fatty infiltration takes place to such an extent that the livers are almost white in color, extremely friable, and show such little color from blood that one is led to sus pect an interference with circulation in the abnormal organ. Although the liver possesses such varied functions as de amination, urea synthesis, and the production of thrombin, serum proteins, and bile, probably one of the most important functions is the regulation of blood sugar. Not only are the processes of glyco- genesis and of glycogenolysis concerned with the control of the blood sugar level, but also of gluconeogenesis, which takes place largely in this organ. That such alteration of liver composition may be re lated to an abnormality in carbohydrate metabolism seems to be evi dent from the work of Best and his associates (3,4). If the liver function is severely damaged, it would seem highly probable that its function in storing glycogen might be one of the abnormalities first 2 observed. In order to test the liver function of these fatty livers and to determine their ability and the speed with which they can re move glucose from the blood, we have followed the glucose tolerance curves, comparing these to the curves of normal animals. In view of the fact that the deposition of fat takes place more rapidly in the female than in the male (14), it seems probably- that greater abnormalities might be found in the females; therefore we have compared the glucose tolerance both of males and females hav ing fatty livers with that of normal animals. CHAPTER II REVIEW OF THE LITERATURE Early in the work on experimental diabetes, depancreatized dogs, receiving insulin, were found not to survive longer than one to eight months on a meat and sugar diet (l)• Post-mortem examina tion of these dogs revealed that the liver was the chief and in most cases the only organ obviously affected, marked fatty infiltration up to 35.5 per cent being observed. The animals kept on this diet were characterized by an extreme sensitiveness to insulin. The dose of this hormone had to be reduced in order to prevent hypoglycemia, and in many cases insulin was discontinued without glycosuria. The experimental production of fatty livers in normal rats was first produced by Best, Hershey, and Huntsman (4), by the use of a daily ration containing 2.5 grams of beef drippings for a three weeks' period. Channon, Jenkins, and Smith (10) investigated the degree to which beef drippings, palm oil, olive oil, cod liver oil, butter, and coconut oil caused the accumulation of fat in the livers of rats when fed as 40 per cent of the diet. Intense fatty livers resulted, varying from 30.7 per cent of liver weight in the case of butter fat and ranging in descending order through beef drippings, palm oil, coconut oil, and olive oil to 7.2 per cent for the cod liver oil. Deuel, Hallman, and Murray (16) likewise investigated the degree of fatty infiltration of rat livers when 40 per cent of the diet was made up of butter fat, cod liver oil, coconut oil, and 40 per cent lard together with 2 per cent cholesterol. The greatest infiltration resulted from the lard-cholesterol diet; in other res- 4 pects their findings were those of Channon in that butter fat gave the highest and cod liver oil the lowest fatty deposits. That cholesterol feeding, together with a diet otherwise low in fat, can produce fatty livers has been shown by the work of Blatherwick (8), Channon and Wilkinson (12), and Best and Ridout (7). The fatty livers caused by cholesterol feeding are character ized by the appearance of excessive amounts of cholesterol esters together with a large increase of neutral fat. Not only can fatty livers be produced experimentally by a high-fat diet, but forced feeding of carbohydrate results in a similar fatty infiltration. In the goose (18) such forced feeding causes the deposition of a liver fat characterized by a larger proportion of sat urated fatty acids, even more so than depot fat. It would be interest ing to determine whether such liver fat behaved metabolically similar to that deposited after a high-fat diet. Best and co-workers (5) found that after fatty livers had developed in dogs receiving a high- fat diet, the fat infiltration increased still further when a diet of sucrose alone was given. The influence of the endocrines on the production of fatty livers has been emphasized by a number of workers. Verzar and Laszt (22) have shown that adrenalectomy abolishes the fatty livers produced by phosphorus poisoning and by fasting in the rat. These workers also showed that when the adrenal cortical hormone was injected into adrenal- ectomized animals, fat again accumulated in the livers. Long and Lukens (21) found that adrenalectomy also abolishes the fatty liver re sulting from pancreatectomy in cats. 5 Shortly after the discovery of insulin, Allen, Bowie, Mac Leod, and Robinson (l) found that depancreatized dogs adequately treated with insulin could be kept alive and the development of liver changes could be prevented by the addition of raw pancreas to the diet. Although this effect was first thought to be due to the pancreatic juice in the pancreas fed and later to the increased in take of lecithin and choline supplied by the pancreas, Dragstedt (17) has shown that neither of these conceptions are likely. He has isolated from beef pancreas a hormone called "lipocaic" which, when fed to depancreatized dogs treated with insulin, permits sur vival and prevents and relieves the fatty degeneration and infiltra tion of the livers. The work of Dragstedt was confirmed by MacKay (26) who used rats as his experimental animals. MacKay and Barnes (25) have shown that fatty livers can also be produced by injections of ketogenic anterior pituitary ex tracts, and that these can be abolished by adrenalectomy. They con clude that the fat-liver-producing extracts of the anterior pituitary act through a stimulation of the adrenal cortex. However, Chaikoff and co-workers (9) have found that if hypophysectomy in dogs is follow ed by pancreatomy, fatty livers occur. The sex glands likewise effect the infiltration of fat into the liver. MacKay (26) found that more fat was deposited in the livers of female than male rats when kept on a low-protein, high-fat diet. Deuel, Hallman, and Murray (14) analyzed the livers of male and female rats fed a high-fat diet varying lengths of time ranging from one to sixteen days; they found that the females deposited not 6 only more fat in their livers, but that the fat was deposited in the livers of the females more rapidly than the males. There are a number of other factors which alter the depos its of fat in the liver. Hershey and Soskin (20) showed that lecithin when fed to depancreatized dogs treated with insulin, prevented the in filtration of fat in the liver. Best and his co-workers (4) showed that lecithin likewise prevented the increase in liver fat of rats fed high-fat diets. Later these workers, feeding the various components of lecithin to rats kept on high-fat diets, found that choline inhibit ed the deposition of fat in the livers. Betaine, chemically related to choline, likewise prevented the formation of fatty livers. Best and Ridout (7) further showed that either choline or betaine could prevent the fatty livers in rats resulting from cholesterol feeding. These same workers (6) found that choline increased the rate of disap pearance of fat from the liver during the recovery phase of phosphorus poisoning in rats, but did not inhibit the deposition which took place after the injection of large amounts of phosphorus. More recently, substances chemically related to choline have been investigated. Channon, Platt, and Smith (11) found that synthetic homocholine (trimethyl--hydroxypropyl ammonium hydroxide) is even more effective than choline in the prevention of fatty livers. The triethyl-y9 -hydroxyethyl ammonium hydroxide was much less active than choline, while tripropyl-/3 -hydroxyethyl ammonium hydroxide was without action. ?/hen rats are fed on a high-fat, low-choline diet, vitamin Bj, according to McHenry (24), markedly increases the amount of fat 7 in the liver, sometimes to values three to four times higher than in the livers of rats without B^. However, his animals given vitamin ate more of the high-fat diet than those whose diets were deficient. Best, Huntsman, and Ridout (5) found that the addition of protein to the diet of rats decreased the amount of fat deposited in the liver. Beeston and Channon (2) found that in rats on a diet containing 5 per cent casein and 40 per cent lard, supplemented with 0.5 per cent cystine, the liver fat was increased by 57 per cent. Conversely, Tucker and Eckstein (27), using the same diet but by re placing the cystine with 0.5 per cent methionine, found that the fat content of the liver decreased by 41 per cent. No explanation can be given for the action of these two amino acids which have been con sidered so similar in their function in metabolism. CHAPTER III GENERAL PROCEDURE Male and female rats, four to five months old, of known heredity, were divided into two groups, each consisting of ten males and ten females. The first group was fed the stock diet, which is as follows: Per cent Whole yellow corn-meal 43.0 Whole wheat flour 28.0 Dried skim milk powder 16.0 Desiccated liver 5.0 Ground alfalfa leaves 4.0 God liver oil 2.0 Irradiated yeast 1.0 CaC0_ o 0.5 NaCl 0.5 The second group was placed in separate cages and fed a high-fat diet, similar to that used by Channon and co-workers (12) for twelve days. This diet is as follows: Per cent Butter fat 40.0 Glucose (cerelose) 43.0 Cellu-flour 5.0 Salt mixture 5.0 Casein 5.0 Yeast 2.0 8 All animals were allowed free access to water* Before the glucose tolerance tests were made the animals were fasted for twenty-four hours to deplete the liver glycogen* Samples of blood to determine the fasting glucose level were taken. Immediately afterward, 200 milligrams of glucose per 100 grams of body weight of rat were given by stomach tube. Other samples of blood were taken, 30, 60, 90 and 120 minutes after the glucose solu tion was given. The blood was drawn from the caudal vein. The very tip of the tail was cut with a sharp knife, quickly forced down with a hammer, making a clean cut. A drop of blood was allowed to drain onto a clean paraffin block, and measured by 0.025 cc. micro-pipettes. Preliminary studies showed that samples of blood taken when the rat was moving or struggling gave glucose values as much as 25 milligrams higher than samples taken three to four minutes later when the animal was quiet. It was necessary that some technique should be employed whereby the animals movements would be restricted. Thick glass tubing, large enough to permit comfortable entrance of an animal but no turning, was cut the length of a rat's body; heavy wire netting was clamped to one end of the tube, and the opposite end was closed with a large cork through which holes were cut for the ani mal's tail. The animals were put into these tubes fifteen minutes before the first blood sample was taken. By this time they had be come accustomed to their surroundings and normal blood sugars were ob tained. They were kept in the tubes during the determination of the 9 glucose tolerance. In order to increase circulation, the tubes were wanned moderately for two or three minutes before the tail was cut. No more than one-sixteenth of the tail was cut at one time. Since the animal is likely to struggle each time the tail is cut, the cut ting was done a few minutes before the sample of blood was to be taken and the animal allowed to become quiet again. If only a small amount of the tail is cut, and the animal is-kept away from the heat after the blood sample is taken, no bleeding follows. Duplicate samples of blood for glucose determinations were taken throughout the study. Blood glucose determinations were made by a modification of the Folin micro blood-sugar method (25) and readings taken on a Klett-Summerson photoelectric colorimeter. The colorimeter was cali brated with solutions of 50, 75, 100, 125, 175, and 200 milligrams per cent of U.S. Bureau of Standards glucose. The chart showing the calibration is given on page 10. The color given by the modifica tion of the Folin micro blood-sugar method was found to be too intense to allow the maximum efficiency of the colorimeter, and the method was still further modified by dilution of the total volume from 12.5 cc. to 25 cc. Immediately after the termination of the glucose tolerance tests, sodium amytal was given to the animals. The liver was removed, and one-half was used for the determination of glycogen by the method of Good, Kramer, and Somogyi (19). After hydrolysis of the glycogen, the glucose was determined by the Shaffer-Hartman method. The other half of the liver was used to determine both water and fat. Liver -JS-ODn-fQ JO &WVZ/9/T7/IA/ O pt 0 3 / 0 0 / 0 9 ■ a a i s M M E M ■ r + 1 + • f + - r hnul ■ SSI — .603 h s 11 water was ascertained by drying the liver to a constant weight in a vacuum oven heated to 50°. The determination of liver lipid was made on the dried sample by gross extraction with ether on the Bailey- Walker apparatus. In order to determine normal liver glycogen, water and lipid, two groups of control animals, both male and female, were kept on the stock and high-fat diets and sacrificed without glucose. Glycogen, water and lipid determinations were made by the methods listed above. CHAPTER IV RESULTS The glucose tolerance curves and the liver analyses show ing water, glycogen, and lipid content of male rats previously fed the stock diet is given on Table I. Table II gives the glucose tolerance eurves and liver analyses of female rats previously fed the stock diet. Tables III and IV give the body weights and the amount of food eaten, as well as the glucose tolerance curves and liver analyses of male and female rats previously fed a high-fat diet. Table V shows the age, body weight, and the liver analyses of male and female rats killed after one day fast, having previously been fed the stock diet. The age, differences in body weight, the amount of food eaten, and the liver analyses of male and female rats previously fed a high-fat diet are given on Table VI. A summary of the glucose tolerance curves of four groups of rats is given in Table VII, together with the probable error of the mean which is calculated from the standard deviation. Table VIII is also a summary chart, showing the analyses of the livers of eighty-two animals used in the experiments. A summary of the glucose tolerance curves of four groups of rats is plotted on Chart I. TABLE I THE GLUCOSE TOLERANCE CURVES AND LIVER ANALYSES OF MALE MTS PREVIOUSLY FED THE STOCK DIET Expt. No. Rat No. Age Body wt» Blood sugar in mgs. per cent after glucose Liver analyses days gms. Control 30 min. 60 min. 90 min. 120 min. Water Glyco Lipid gen 1 4578 130 210 97 124 132 106 % % % 2 4579 136 210 61 97 77 76 71 5 4580 135 250 60 88 100 89 81 4 4586 129 180 99 124 116 125 90 5 5008 94 215 125 156 140 135 134 69.4 0.63 3.56 6 5000 95 225 113 134 138 122 116 69.0 0.75 3.48 7 4888 115 220 88 131 138 129 122 68.1 0.71 4.39 8 4789 116 230 92 143 138 129 110 68.9 0.70 4.91 9 5187 88 210 91 143 144 127 109 66.6 0.84 5.17 10 5189 88 240 100 139 138 124 93 66.9 0.81 4.31 Average 113 219 92.6 127.9 126.1 116.6 103.0 68.1 0.74 4.30 TABLE II THE GLUCOSE TOLERANCE CURVES AND LIVER ANALYSES OF FEMALE RATS PREVIOUSLY FED THE STOCK DIET Expt. No. nat No. Age Body wt. .'moo'd' sugar in nigs, per cent after glucose Liver analyses days gms. Control 30 min. 60 min. 90 min. 120 min. Water Glyco Lipid gen 21 5872 105 130 117 174 172 137 123 % 65.4 % 1.28 % 4.13 22 5881 99 130 104 171 139 127 113 69.1 1.76 5.32 25 5885 99 155 86 134 154 137 108 70.3 0.66 3.86 24 5954 103 140 88 118 110 106 111 71.8 0.36 5.33 25 5991 93 152 no 143 131 118 110 69.2 0.31 5.80 26 5952 92 135 98 143 150 96 102 69.0 1.52 4.34 27 5961 86 145 103 155 131 130 118 68.6 1.41 4/40 28 5965 92 170 93 125 121 116 111 68.6 0.96 4.86 29 5974 86 130 100 150 131 122 108 70.1 1.16 2.97 30 5985 86 105 109 167 151 132 116 70.4 1.08 3.08 Average 94 137,.. 100.8 148.0 139.0 122.1 112.0 69.2 1.05 4.21 TABLE III THE GLUCOSE TOLERANCE CURVES AND LIVER ANALYSES OF MALE RATS PREVIOUSLY FED A HEGH-FAT DIET Expt. Rat Age Body Weight Food Eaten Blood sugar in mgs. per cent after glucose Liver analyses No. No. Initial Final Total per 100 days gm. gm. gm. Control 30 min. 60 min. . 90 min. 120 min. Water Glyco- Lipid gen 41 4576 123 194 160 gm. 138 gm. 72.6 100 178 140 158 170 % ' i % 42 4577 123 225 196 113 51.4 79 132 123 127 167 43 4996 96 170 150 83 55.3 124 159 165 154 103 63.5 0.92 14.2 44 4997 96 225 205 125 56.8 121 180 154 140 123 63.8 0.58 14.0 45 8 96 272 260 170 62.9 118 156 147 130 130 55.8 0.84 26.4 46 9 96 195 190 105 55.3 120 154 150 115 92 55.3 1.22 21.6 47 4899 113 207 200 105 52.4 125 150 160 156 157 54.8 0.78 25.9 48 4900 113 185 185 98 53.0 130 156 161 140 116 59.7 1.42 20.4 49 4889 116 225 210 90 40.8 124 148 145 140 134 52.8 0.62 29.9 50 4880 116 175 150 75 44.1 129 154 149 125 113 59.7 1.72 13.9 51 4709 135 180 155 82 45.5 131 150 145 159 136 58.1 1.19 17.2 52 4746 134 190 160 103 54.3 129 152 161 161 151 50.0 0.43 33.4 Average 116 204 185 107 53.6 119 156.1 150 140.5 132.7 57.4 0.97 21.7 TABLE IV THE GLUCOSE TOLERANCE CURVES AND LIVER ANALYSES OF FEMALE RATS PREVIOUSLY FED A HIGH-FAT DIET Expt. No. Rat No. Age Body Weight Initial Final Food Eaten Total per 100 Blood sugar in mgs. per cent after glucose Liver analyses Gays gnu gm. gm. Control 05 o P 60 min. 90 min. 120 min. Water Glycogen Lipid 63 5874 112 140 130 gm. 78 gm. 55.7 116 156 140 184 128 % 55.8 % 0.37 % 18.5 64 5883 108 140 130 77 55.0 97 142 160 138 116 56.0 0.37 27.3 65 5932 119 160 150 101 65.2 109 155 137 142 140 53.5 0.37 33.8 66 5941 105 150 140 110 73.4 94 150 164 210 155 47.3 0.62 29.2 67 5945 104 142 130 110 78.7 104 174 162 172 143 42.4 0.62 35.9 68 5954 102 125 150 105 84.1 113 169 198 198 158 40.8 0.60 38.2 69 5963 99 165 140 107 64.8 101 177 162 181 143 39.5 0.36 43.2 70 5972 99 140 135 93 66.4 97 175 156 154 130 40.8 0.64 35.2 71 5981 100 150 140 95 65.3 94 162 146 142 130 51.1 0.74 31.4 72 5985 100 130 125 95 73.1 122 169 155 145 156 49.8 0.47 34.5 Average 104 144 137 97 67.7 104.7 162.9 158.0 166.6 139.9 51.0 0.52 32.7 TABLE V ANALYSES OF LIVERS OF RATS KILLED AFTER 1 DAY FAST STOCK DIET Male Hats Expt. Rat Age Body Liver Analyses No. No. Wt. Water Glycoeen Lipid days gms. % % % 11 11 121 220 68.6 0.41 4.42 -12 77 93 215 68.9 0.22 4.06 13 46 108 240 70.7 0.38 4.35 14 41 108 250 68.7 0.26 4.37 IS 54 111 255 68.2 0.20 5.00 16 84 91 165 69.8 0.32 4.10 17 90 91 210 71.3 0.31 3.74 18 102 91 140 70.4 0.37 2.59 19 124 88 210 70.0 0.33 3.93 20 120 88 180 69.7 0.49 3.82 Average 99 207 69.6 0.35 4.04 Female Hats 31 5871 103 135 70.5 0.63 4.39 32 5875 103 130 71.8 0.82 4.32 33 5884 99 155 70.0 0.34 4.05 34 5933 97 150 71.3 0.29 3.44 35 5942 95 135 72.9 0.33 2.53 36 5955 88 155 70.2 0.43 4.13 37 5964 85 135 69.9 0.28 3.51 38 5992 85 150 70.1 0.37 4.55 39 5993 85 155 71.5 0.35 3.52 40 5994 85 155 70.2 0.30 3.82 Average 96 143 70.8 0.41 3.83 TABLE VI ANALYSES OF LIVERS OF RATS KILLED AFTER 1 DAI FAST HIGH FAT DIET Male Rats Expt., Rat Age Body Weight Food Eaten Liver Analyses No. No. Initial Final Total per 100 sms. Water Glycogen Lipid days gms. gms. gms. gms. % % % 53 5086 92 150 135 70 46.5 50.7 0.30 32.9 54 5087 91 190 185 107 56.3 58.3 0.51 22.9 55 5088 91 201 185 86 43.0 54.7 0.34 28.1 56 5089 91 210 180 96 45.7 48.0 0.25 36.1 57 5090 91 190 175 91 47.7 42.1 0.41 31.3 58 5096 90 205 175 90 43.7 60.3 0.42 14.8 59 5097 90 145 135 97 67.0 55.0 0.26 29.4 60 5098 90 145 120 78 53.7 55.8 0.17 27.1 61 5099 90 170 160 104 61.2 60.5 0.28 20.2 62 5100 90 265 250 121 45.7 56.0 0.22 25,5 Average 90.0 187 170 94 51.0 54.1 0.32 27.5 Female Rats 73 5873 110 150 130 67 44.7 55.2 0.28 28.5 74 5882 106 140 112 70 50.0 67.3 0.96 7.7 75 5931 116 160 145 108 67.7 53.9 0.18 28.0 76 5935 102 150 132 105 70.0 57.8 0.34 23.7 77 5944 99 170 155 125 73.5 47.5 0.31 34.2 78 5953 97 165 140 96 58.0 54.3 0.33 29.1 79 5962 94 150 138 115 77.0 62.8 0.69 11.5 80 5971 94 155 130 95 61.3 52.3 0.18 28.3 81 5975 94 145 130 103 71.2 58.0 0.21 26.6 82 5984 94 155 140 100 64.6 60.8 0.27 18.9 Average 100.6 154 134 98 63.8 56.4 0.37 23.6 TABLE VII SUMMARY OF GLUCOSE TOLERANCE CURVES OF FOUR GROUPS OF RATS Stock diet Male No. of AverageFood EatenBlood glucose in mg. per cent1 Tests Weight per 100 gms_____________________________ 10 gms. gms. Control SO min 60 min 90 ^nin 120 min 219 92.6 127*9- 126.1 116.1 103.0 +5.8 +3.6 +3.2 +3.3 +4.3 Female 10 137 100.8 +2.0 148.0 +3.8 139.0 +3.6 122.1 +2.7 112.0 +1*3 High-fat diet Male 12 185 53.6 119.0 +2.8 156.1 +2.3 150.0 +2.1 140.5 +2.5 132.7 +4.7 Female 10 137 76.7 104.7 +2.0 162.9 +2.4 158.0 +2.3 166.6 +4.9 139.9 +2.8 "^Including the probable error of the mean. This is calculated as follows: (f (standard deviation)= \id^/n. The probable error (individual determina tion) = <f x 0.6745; the probable error (mean) -probable error (individual) VF. TABLE VIII SUMMARY TABLE SHOWING THE COMPARATIVE ANALYSIS OF LIVER Fasted rats receiving no glucose - No. of Tests Body wt. Water Liver Analyses Glycogen Lipid gms. % % % Stock diet Males 10 207 69.6 0.33 4.04 Females 10 143 70.8 0.41 3.83 High-fat diet Males 10 170 54.1 0.32 27.5 Females 10 134 56.4 0.37 23.6 Fasted rats 2 hours after glucose Stock diet Males 10 219 68.1 0.74 4.30 Females 10 137 69.2 1.05 4.21 High-fat diet Males 12 185 57.4 0.97 21.7 Females 10 137 51.0 0.52 32.7 160 140 /20 too 8o 60 I2Z 212 40 eo _ 60 90 T/ME /// M/ n u t e s CHAPTER V DISCUSSION In the post-absorptive state, the blood glucose normally lies between 70 to 90 milligrams per cent. If glucose is then given to a healthy animal or person, the blood sugar rises to a maxi mum during the first half-hour or hour and returns to the normal level by the end of the second hour. The maximum, rise in blood glucose may fall between 140 and 160 milligrams per cent, but does not exceed 180 milligrams per cent. It is well known that a number of factors can alter the tolerance for glucose. Perhaps the most outstanding of these is found in clinical or experimental diabetes. Here the fasting level of blood sugar is higher than normal; following glucose feeding, the blood sugar rises slowly, reaching a peak of 200 to 400 milligrams per cent, perhaps higher, at the end of two or more hours; the blood glucose then drops slowly and may not reach the fasting level for sev eral hours. After injections of phlorhizin, we again find an abnormal glucose tolerance curve (IS). Despite the fact that the blood glu cose may be as low as 50 or even 40 milligrams per cent before the glucose feeding is given, the glucose tolerance curve reaches an ab normally high maximum in about two hours, and returns to normal very slowly, approximating the curve of the diabetic. If carbohydrate is given a normal individual who has fast ed for some time, the glucose tolerance curve again approaches that of the diabetic; the maximum value is not reached as quickly as nor- 23 many, and the blood sugar remains higher for a longer time. This reaction depends, of course, upon the length of the fast; the long er the fast, 1^e more nearly the curve resembles that of the diabetic. During a fast, the insulin mechanism is not stimulated and it becomes less efficient in changing glucose into glycogen. The reverse is seen when the insulin secretion is repeatedly stimulated by a carbohydrate meal followed by other carbohydrate meals. Here the subsequent feedings cause scarcely any rise in blood glucose. A similar "leveling" of the blood sugar curve after glucose is given is found in hypopituitarism. In this case, glucose is chang ed into glycogen more rapidly than normally, and the level of glucose in the blood is low in fasting and after feeding alike. It is known that glucose tolerance is reduced in phosphorus and chloroform poisoning, and in other conditions where the liver is injured. The greater the liver damage, the more difficult it is for glucose to be changed into glycogen. This condition, we believe, applies to our present studies. In our experiments, the glucose tolerance curves of the males and females on the stock diet fall well within the normal range. The males have a fasting blood glucose of 92.6 milligrams per cent, compar ed to 100.8 for the females. In each case the peak of the curve is reached one-half hour after the glucose is given, being 127.9 milli grams per cent for the males and 148.0 milligrams per cent for the fe males. Although the high point in the curve is greater for the females than for the males, the glucose disappears from the blood somewhat more 24 rapidly in the females. One hour after the glucose was given, the females show a drop of 9 milligrams per cent below the maximum value, whereas the males have dropped only 1.8 milligrams. Both males and females show the blood glucose dropping rapidly toward normal values 90 minutes after the glucose was given. The values at this period were 116 milligrams per cent for the males and 122 for the females. The curve of each group continues to drop, and at 120 minutes after the glucose feeding, both groups are only about 11 milligrams above the fasting level. Two male rats, numbers 4586 and 5189, show blood glucose values at the end of the two hour period below the fasting level, whereas the blood glucose of only one female, number 5991, has returned to the fasting level. Deuel, Hallman, Murray, and Samuels (15) found that glyco gen was formed somewhat more rapidly in normal males than in normal female rats. This may be the explanation for the higher peak reach ed by the females and for the fact that fewer females showed blood glucose values approximating that of the fasting level at the end of two hours. The male rats previously fed the high-fat diet showed aver age blood glucose values of 119.0, 156.1, 150.0, 140.5, and 132.7 milligrams per cent for the fasting level and 30, 60, 90, and 120 minutes respectively after the feeding of glucose. For the fasting level and the same time intervals, the females gave glucose values of 104.7, 162.9, 158.0, 166.6, and 139.9 milligrams per cent. The fast ing glucose level was lower for the females than the males, due, we believe, to the fact that males*store larger amounts of glycogen than 25 females. The males on the high-fat diet reached a peak in one- half hour following the glucose feeding, after which the blood glu cose declines, thereby resembling that of the normal curve. On the other hand, the females show a rapid rise in blood sugar at the end of one-half hour, a slight fall at 60 minutes, and a maximum second ary peak at the end of 90 minutes. Comparing the curve of the ani mals previously fed the high-fat diet, we find that the females show increases of 58.2, 55.5, 61.9, and 55.2 milligrams per cent above the fasting level for the intervals of 50, 60, 90, and 120 minutes after the glucose meal, whereas the males show increases of only 57.1, 50.0, 21.5, and 15.7 milligrams per cent at these same periods. The glucose tolerance curve of the females is much more abnormal than that of the males on the same diet, and it more nearly resembles that of the diabetic curve. The abnormality of the curve becomes even more obvious when the females previously fed a high-fat diet are compared with the females previously fed the stock diet. The normal females show increases in blood sugar of 47.2, 58.2, 21.5, and 11.2 milligrams per cent above the fasting level, whereas the females fed the high- fat diet show much greater increases of 58.2, 55.5, 61.9, and 55.2. The latter will be seen to be 11.0, 15.1, 40.6, and 24.0 milligrams higher than the former, the increase becoming greater near the end of the test period. A similar comparison between the females previously fed the high-fat diet and the males fed the stock diet show increases of 22.9, 19.8, 58.4, and 24.8 milligrams per cent in the rise of the female curve over the fasting level above the same rise in the male 26 curve. Of the animals given glucose previously fed the high-fat diet, the females had an average liver-fat of 52.7 per cent, compared to 21.7 per cent fat for the males. Since the females showed a low er glucose tolerance than the males, this indicates that the higher the liver-fat, the less ability the liver has of changing glucose efficiently into glycogen. This conclusion is also indicated by several individual experiments. Of the males, rats number 4746, 4889, and 4899 had livers containing 35.4, 29.9, and 25.9 per cent fat, and these animals show the lowest tolerance for glucose. Simi larly female rats number 5965, 5954, and 5941 had liver fat of 45.2, 38.2, and 29.2 per cent respectively, and again these animals showed the highest glucose tolerance curves. It is of interest to note that the normal females showed a liver glycogen of 1.05 per cent after glucose had been given. The females previously fed the high-fat diet had a liver glycogen of only 0.52 per cent after the glucose meal. This indicates a greater ability of the normal females to form glycogen from the blood glucose, and probably accounts for the more rapid fall of blood sugar found in the normal females. The fasting level of liver glycogen of the rats in both the normal and fat-fed groups were approximately the same in all cases. It seems probable that the difference in glucose tolerance curves is not to be traced to a variation in the insulin stimulating mechanism at the start of the test, for both groups had fasted the same 27 length of time. We believe that the explanation of these results is that the high-fat feeding, by causing the infiltration of such large amounts of fat into the liver, injures the liver cells to such an extent that they cannot perform their function of glycogenesis rapidly and efficiently. CHAPTER VI SUMMARY A technic has been worked out whereby glucose tolerance tests may be made satisfactorily on rats. Male and female rats, previously fed a stock diet, show ed normal glucose tolerance curves, the blood sugar reaching a peak in one-half hour after the glucose meal and falling to nearly fast ing level at the end of two hours. Male and female rats, previously fed a high-fat diet, showed glucose tolerance curves approaching that of a diabetic curve. The glucose tolerance curve of the female fed a high-fat diet was more abnormal than that of the male on the same diet. The high point of the curve was not reached until 90 minutes after glu cose was given, and in 120 minutes the blood sugar showed slight return to the normal level. BIBLIOGRAPHY 1. Allan, F.H., D.J. Bowie, J.J.R. MacLeod, and W.L. Robinson, Brit. J. Exper. Path.. 5: 75, 1924. 2. Beeston, A.W., and H.J. Ghannon, Biochem. J., 30: 280, 1936. 3. Best, G.H., G.C. Ferguson, and J.M. Hershey, J. Physiol.. 79: 94, 1933. 4. Best, C.H., J.M. Hershey, and M.E. Huntsman, J. Physiol.. 75: 59, 1932. 5. Best, C.H., M.E. Huntsman, and J.H. Ridout, Mature. 135: 821, 1935. 6. Best, C.H., D.L. Maclean, and J.H. Ridout, J. Physiol.. 83: 275, 1934-35. 7. Best, C.H., and J.H. Ridout, J. Physiol.. 78: 415, 1933. 8. Blatherwick, N.R., E.M. Medlar, P.J. Bradshaw, A.L. Post, and S.D. Sawyer, J. Biol. Chem.. 103: 93, 1933. 9. Chaikoff, I.L., G.E. Gibbs, G.F. Hoitorn, and F.L. Reichert, Am. J. Physiol.. 116: 543, 1936. 10. Channon, H.J., G.N. Jenkins, and J.A.B. Smith, Biochem. J., 31: 1736, 1937. 11. Channon, H.J., A.P. Platt, and J.A.B. Smith, Biochem. J., 31: 1736, 1937. 12. Channon, H.J., and H. Wilkinson, Biochem. J., 29: 2659, 1935. 13. Deuel, H.J., Jr. J. Biol. Chem.. 89: 77, 1930. 14. Deuel, H.J., Jr., L.F. Hallman, and S. Murray, In Print. 15. Deuel, H.J., Jr., L.F. Hallman, S. Murray, and L.T. Samuels, J. Biol. Chem.. 119: 607, 1937. 16. Deuel, H.J., Jr., S. Murray, L.F. Hallman, and D.B. Tyler, J. Biol. Chem.. 120: 277, 1937. 17. Dragstedt, L.R., J. Van Prohaska, and H.F. Harms, Am. J. Physiol.. 117: 175, 1936. 18. Flock, E.V., and H.R. Hester, Proc. Staff Meetings Mayo Clinic. 12: 676, 1937. 19. Good, C.A., H. Kramer, and M. Somogyi, J. Biol. Chem.. 100: 485, 1933. 30 20. Hershey, J.M., and S. Soskin, Am. J. Physiol.. 98: 75, 19S1. 21. Long, G.N.H., and F.D.W. Lukens, Am. J. Physiol.. 116: 96, 1936. 22. Luck, J.M., and C.R. Holler, ed., Annual Review of Biochemistry. Palo Alto: Stanford University Press, 1938. 23. Jegher, H.J., and V.C. Myers, J. Labor, and Clin. Med.. 15: 982, 1929-30. 24. McHenry, E.W., J. Physiol.. 89: 287, 1937; Biochem. J., 31: 1621, 1937. 25. MacKay, E.M., and R.H. Barnes, Am. J. Physiol.. 118: 184, 525, 1937; 120: 362, 1937. 26. MacKay, E.M., and R.H. Barnes, Am. J. Physiol.. 119: 783, 1937. 27. Tucker, H.F., and H.C. Eckstein, J. Biol. Chem.. 121: 479, 1937. UMI Number: E P41274 All rights reserved INFORMATION TO ALL U SER S The quality of this reproduction' is d e p en d en t upon the quality of the copy subm itted. In the unlikely event that the author did not sen d a com plete m anuscript and there a re m issing p ag es, th e se will be noted. Also, if m aterial had to be rem oved, a note will indicate the deletion. UMI Oiwartffiftw PiiisIiiMng UMI E P41274 Published by P roQ uest LLC (2014). Copyright in th e D issertation held by the Author. Microform Edition © P roQ uest LLC. All rights reserved. This work is protected against unauthorized copying under Title 17, United S ta te s C ode P roQ uest LLC. 789 E ast E isenhow er Parkw ay P.O. Box 1346 Ann Arbor, Ml 4 8 1 0 6 -1 3 4 6

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Asset Metadata

Creator Davis, Daisie Adelle (author)

Core Title A comparison of glucose tolerance in normal rats and those with fatty livers

School Department of Biochemistry

Degree Master of Science

Degree Program Biochemistry

Degree Conferral Date 1939-05

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag chemistry, biochemistry,OAI-PMH Harvest

Language English

Contributor Digitized by ProQuest (provenance)

Advisor Deuel, H.J. (committee chair), [illegible] (committee member)

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c17-775157

Unique identifier UC11345577

Identifier EP41274.pdf (filename),usctheses-c17-775157 (legacy record id)

Legacy Identifier EP41274.pdf

Dmrecord 775157

Document Type Thesis

Rights Davis, D. A.

Type texts

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Access Conditions The author retains rights to his/her dissertation, thesis or other graduate work according to U.S. copyright law. Electronic access is being provided by the USC Libraries in agreement with the au...

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