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THE EFFECT OF INSULIN AND OTHER FACTORS ON CARBOHYDRATE METABOLISM
A Dissertation Presented to the Faculty of the Graduate School University of Southern California
In Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy
by Richard Wolcott Bancroft December 1950
UMI Number: DP30162
All rights reserved INFORMATION TO ALL USERS The quality of this reproduction is dependent upon the quality of the copy submitted. In the unlikely event that the author did not send a complete manuscript and there are missing pages, these will be noted. Also, if material had to be removed, a note will indicate the deletion.
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T h is d is s e rta tio n , w r i t t e n by
BI.CilABD...WOLCO.TT..MJSQPLQF.T............ u n d e r the g u id a n c e o f /t.i.S..F a c u l t y C o m m itte e on S tudies, a n d a p p r o v e d by a l l its m e m b e rs, has been p resen ted to a n d accep ted by the C o u n c i l on G ra d u a te S tu d y a n d R e search, in p a r t i a l f u l f i l l m e n t o f re q u ire m e n ts f o r the degree o f DOCTOR
P H IL O S O P H Y
........... / /
Committee on Studies
Grateful acknowledgment Is made to Professor Douglas R. Drury for his helpful direction and criticism during this study.
Gratitude is also
expressed to Doctor Arne N. Wick and the staff of the Research Laboratories of the Scripps Metabolic Clinic for the radioactive analyses used in this work.
TABLE OF CONTENTS CHAPTER
I. THE PROBLEM................................
Statement of the p r o b l e m ................
The significance of investigating the glu cose utilization and the action of insulin on extra-hepatic tissues
, i .. . .
The significance of further investigating the glucose equivalent of protein. . . .
The significance of investigating the basal Insulin requirements of depancreatized dogs under anoxic conditions II. HISTORICAL DISCUSSION
Literature regarding the utilization of glucose and the action of hormones on carbohydrate metabolism ................
Review of the literature regarding anoxia and carbohydrate metabolism ............
Review of the literature on the D:N ratio and phlorizin diabetes III.
THE UTILIZATION OF GLUCOSE BY EVISCERATED R A B B I T S ................................. The general plan of the experiments on glu cose utilization by the extra-hepatic
Methods used for the tissue analysis \U The measurement of C radioactivity Plasma glucose determination
Plasma glucose specific activity
Collection of the expired carbon dioxide
The preparation of the carcass for analyses
Total crude fat and fatty a c i d s ..........
G l y c o g e n ............................. . .
Body carbon diox i d e ......................
The results of the experiments on glucose utilization by eviscerated rabbits
Discussion of the results on glucose utili zation by the eviscerated rabbit IV.
THE ACTION OP INSULIN IN EVISCERATED RABBITS
The general plan and methods of the
Results of the studies on the action of
insulin on the extra-hepatic tissues
Discussion of the results on the action of
PAGE Insulin inthe eviscerated rabbit
. *. .
THE CARE AND MAINTENANCE OF DEPANCREATIZED D O G S .................................. The postoperative care ofdiabetic dogs
VI . THE EFFECT OF MILD ANOXIA ON THE BASAL INSULIN REQUIREMENTS OF DEPANCREATIZED D O G S .............
The experimental methods used for deter mining the basal insulin requirements of depancreatized dogs under anoxic c o n d i t i o n s ............ ♦ .............
The results of the basal insulin deter minations on depancreatized dogs at an altitude of 15*000 f e e t ........
Discussion of the results that were ob tained by exposing depancreatized dogs to an altitude of 15*000 f e e t .. VII.
THE GLUCOSE EQUIVALENT OF PROTEIN . . . . . . The experimental procedure
The results of feeding a protein diet to phlorlzlnlzed-depancreatlzed dogs
•• • •
The results of feeding only sugar to phlor izin! zed-depancreatized dogs ...........
PAGE The glucose equivalent of fed protein
Discussion of the r e s u l t s ................
SUMMARY AND C O N C L U S I O N S ....................
S u m m a r y ..................................
C o n c l u s i o n s ..............................
B I B L I O G R A P H Y ......................................
LIST OF TABLES TABLE I*
PAGE 1It The Distribution of Cx^ in Eviscerated Rabbits after an 8-hour Period of lii Constantly Injected C -labeled Glucose
The Hourly Specific Activities of the Expired COg from Eviscerated Rabbits
during an 8-hour Period of Constantly 14 Injected C -labeled Glucose ............ ik The Hourly C Content of the Expired COg
from Eviscerated Rabbits during an 8-hour 14 Period of Constantly Injected C -labeled G l u c o s e ............................... IV.
The Comparison of Glucose Disappearance and Oxidation Rates in Eviscerated Rabbits . .
Comparison of Total Plasma Radioactivity by Fermentation and Combustion (Expressed as Count a/M.1. /fain.) ........................
The Blood Sugar Level, Rate of Glucose Injec tion, and the Specific Activity of the Expired COg from an Eviscerated, Insulinized Rabbit during 18 Hours of Constantly 14 Injected C -labeled Glucose ...........
VII. The Hourly Specific Activities of the i
Expired COg from Eviscerated Rabbits during an 8-hour Period of Constantly Injected C
-labeled Glucose with and
without I n s u l i n ........................ VIII.
Content of the Expired COg from
Eviscerated Rabbits during an 8-hour 14 Period of Constantly Injected C -labeled Glucose with and without Insulin Adminis tration IX.
........................ . . . .
Comparison of Glucose Disappearance and Oxidation Rates in Eviscerated Rabbits during an 8-hour Period of Constantly 14 Injected C -labeled Glucose with and without I n s u l i n .........
The Effect of Insulin on the Distribution of 14 C in Eviscerated Rabbits after a Period of Constantly Injected C^-labeled Glucose
A Comparison of the Amount of Glucose that 14 Is Equivalent to the C Distribution in Eviscerated Rabbits after a Period of 14 Constantly Injected C -labeled Glucose with and without Insulin................
ix TABLE XII.
PAGE The Insulin Requirements of Two Depancrea tized Dogs following the Ingestion of 50 Gm. Sucrose and 225 Gm. of Meat . . . .
The Daily Amount of Sugar and Nitrogen Excreted by a Phlorizinized-Depancreatized Dog (#1) while on a Tuna Pish Protein Diet for Six D a y s ............................
The Daily Amount of Sugar and Nitrogen Excreted by a Phlorizinized-Depancreatized Dog (#2) while on a Tuna Fish Protein Diet for Six D a y s ............. ..............
The Daily Amount of Sugar and Nitrogen Excreted by Two Phlorizinized-Depancrea tized Dogs while on a Tuna Protein and Casein Diet for Five Days
. . . . . . . .
The Glucose and Nitrogen Excretion during 24-hour Periods by a PhlorizinizedDepancreatized Dog (#1) after the Ingestion of 100 and 200 Grams of Sucrose and G l u c o s e ....................
The Glucose and Nitrogen Excretion during 24-hour Periods by a PhlorizinizedDepancreatized Dog (#2) after the
PAGE Ingestion of 100 and 200 Grams of Sucrose and G l u c o s e ....................
LIST OF FIGURES FIGURE 1*
Blood Sugar Levels of a Diabetic Dog Resulting from Insulin Injections at Three Different R a t e s ....................
The Blood Sugar Level of a Diabetic Dog with a Basal Insulin Injection Rate of 0.15 Unit per Hour
The Blood Sugar Levels of Two Diabetic Dogs during a Basal Insulin Test at 15,000 Feet with Insulin Injections of 0.10 Unit per Hour .
The Comparison of the Basal Insulin Require ments of a Diabetic Dog (Dog 1) at Ground Level and at 15,000 F e e t ..................
The Comparison of the Basal Insulin Require ments of a Diabetic Dog (Dog 2) at Ground Level and at 15,000 F e e t ..................
6 . The Comparison of the Basal Insulin Require ments Repeated with Dog 1 at Ground Level and at 15,000 Feet 7.
The Comparison of the Basal Insulin Require ments Repeated with Dog 2 at Ground Level and at 15,000 Feet
8 . The Sugar and Nitrogen Excreted In 24 Hours by Two Phlorizinized-Depancreatized Dogs Following the Ingestion of 100 Grams of Sucrose or G l u co s e ..........................
CHAPTER I THE PROBLEM Statement of the problem. The original purpose of this dissertation was to investigate the insulin require ments of the diabetic animal under anoxic conditions and to j evaluate some of the effects of anoxia on carbohydrate metabolism.
However, it became apparent early in the study r
that before this problem could be adequately approached and studied, several debatable points regarding insulin and glucose utilization per se should first be clarified.
utilization of glucose by diabetic tissues and the influence of insulin on glucose oxidation under normal conditions are still open to discussion.
A further consideration of car
bohydrate utilization, especially by the diabetic animal, requires a re-examination of the glucose equivalent of body protein as a source of carbohydrate and an analysis of the significance of the urinary D:N ratio as a tool for measuring this phase of carbohydrate metabolism. Consequently, the main portion of the dissertation is primarily concerned with the results of several investi gations which are a necessary basis for future work on the effects of an abnormal environment on the metabolism of carbohydrates.
The utilization of glucose and the action
of insulin on the extra-hepatic tissues-have been studied
2 by means of radioactive glucose administered to the evis cerated rabbit.
The question of the urinary D:N ratio as
an indication of the amount of glucose that is available
from protein sources has been investigated with the phlorizin!zed-depancreatized dog, with particular consider-; atlon being given to the glucose that is utilized by this i
type of animal preparation.
Finally, the effects of mild
anoxia on the basal insulin requirements of the fasting depancreatized dog were investigated. It was necessary to use a different experimental approach to each phase of this series of investigations, and several types of animal preparations have been used in order to limit the variables and to control the experimen tal conditions.
Also, techniques and measurements that were
best suited for each series of experiments were employed. The significance of investigating the glucose utili zation and the action of insulin on extra-hepatic tissues. The utilization of glucose by the tissues has been an important measurement in the study of carbohydrate metabo lism, and it is a basis for establishing important metabolic concepts.
These measurements have usually been made by
determining the rate at which glucose must be injected into an eviscerated animal in order to maintain a normal blood sugar level.
Although there is evidence that some glucose
may be formed by the kidney under certain conditions, it has been generally assumed in the past that the liver and gastro-intestinal tract are the sole sources of glucose in !the body.
The eviscerated preparation eliminates these two
variables, as well as the action of the pancreas, all of
which would be difficult to control with the intact animal. However, this method only measures the rate at which the
injected glucose disappears from the circulation, and it affords no indication as to how this disappearing glucose is being utilized by the tissues— at what rate it is being oxidized to carbon dioxide and water, stored as glycogen and fat, or converted to other body constituents and inter- ; mediary products.
With the absence of the liver, it is
possible that many of the partial breakdown products, which would normally be reconverted to glucose by this organ, accumulate in the eviscerated animal as Bdead end” deriva tives or are excreted by the kidneys. With the availability Ik of labeled C glucose, a method is possible by which injected radioactive
glucosein the eviscerated animal can
be traced through its metabolic course, and direct experi mental data can be obtained concerning the intermediary and final utilization of
glucoseby the extra-hepatic tissues.
The action of
insulinon the utilization of glucose
by animal tissue is in many respects not completely agreed
upon by authorities in the field.
The fact that insulin
lowers the blood sugar and prevents it from rising to abnormal levels is of course unquestioned, but whether, in the Intact animal, this is accomplished predominantly by an ;increased oxidation of glucose by the tissues, or by the storage of exeess blood sugar as glycogen and fat, or by
the inhibition of gluconeogenesis from non-carbohydrate sources is still open to discussion.
There are experimental
data to support all of these possibilities, although the interpretation of much of the evidence on which the glucose oxidation theory is based is open to criticism (Soskln, 19^1).
It can be argued that metabolic R.Q.*s, which have
been used to show that the oxidation of carbohydrate Increases with insulin, can be interpreted quite different ly.
As yet there is no direct evidence which indicates
that Insulin can increase the oxidation of glucose in the liver-less animal.
Here again, the eviscerated animal, 14 maintained with labeled C glucose, offers a method for directly investigating the way in which the extra-hepatic tissues utilize glucose when under the influence of maximum doses of insulin. The significance of further investigating the glucose equivalent of protein.
The amount of glucose that is
available from catabolized protein has been calculated on
5 the basis of urinary D:N ratios of fasting phlorizinized dogs*
The classical D:N ratio of 3.65 is most generally
used to show that 100 grams of protein can yield 58 grams of glucose*
Other estimations maintain that a more correct
urinary D:N ratio cannot be much higher than about 3*1 if the possible conversion of glycerol to glucose is taken into account*
t Still other workers have reported D:N ratios,1
with apparently well phlorizinized dogs, that have been well, below 3*63*
Revised calculations from all these data are 1
now used to show that only about 48 grams of glueose can be derived from 100 grams of animal protein.
However, in order;
to reach these conclusions it is necessary to assume that all of the glucose formed by the phlorizinized animal is excreted through the kidneys and that none is utilized by the tissues*
But the action of phlorizin appears mainly to
prevent the reabsorption of glucose by the renal tubules, 1
and there are no indications for assuming that it also inhibits the utilization of glucose.
Only a few workers on J
this problem have considered the possibility that a certain • ’amount of the glucose that is formed from protein sources In the phlorizinized animal may be burned during the period of the experiment and that this utilized sugar can never appear in the urine for direct measurement. Although the D:N ratio of the depancreatized dog has >
6 never been found to be as high as 3 *65* nevertheless the assumption has also been made that the completely diabetic animal cannot utilize glucose, despite the fact that there is much evidence to show that the tissues of both the phlorizinized and depancreatized animals can and do burn glucose.
In fact, certain tissues of the body must be
supplied with glucose under any conditions for their sur vival.
Since important concepts of carbohydrate metabolism
have been based on the observed D:N ratios of the phlori zinized and the depancreatized dog, particularly where it is concerned with the etiology and control of diabetes, the true DsN ratio should be established and its real signifi cance interpreted correctly. Most of the previous work on this problem has been done on either the phlorizinized or the depancreatized animal.
Because of this, additional data have been obtained
,by studying depancreatized dogs that were also phlorizin ized.
The dogs were fed large quantities of protein under
these conditions in order to insure a significant increase in both the nitrogen and glucose excretion above the fasting amounts.
Also by feeding the dogs known quantities
,of either sucrose or glucose, an attempt was made to determine the amount of sugar utilized by the animals.
The significance of Investigating the basal insulin requirements of depancreatized dogs under anoxic conditions. Concerning the effect of anoxia on carbohydrate metabolism, : there are very little data available at the present time as to how the diabetic animal may be affected.
conducted on normal humans and animals indicate that anoxic conditions may produce complex physiological effects, many of which are reflected in the blood glucose levels.
Interest have been the possible chemical changes caused by oxygen insufficiency itself and the compensatory reactions that are initiated to meet the stress of the abnormal environment.
The adrenal and pituitary glands particularly
have been shown to play important roles in these physio logical readjustments.
An investigation of the effects of
anoxia on the depancreatized animal and an evaluation of its insulin needs would also be of value, both in estimating the effects that might be expected in a diabetic Individual when exposed to anoxia, and as a means of further studying some of the mechanisms that are involved in carbohydrate metabolism.
CHAPTER II I
Literature regarding the utilization of glucose and the action of hormones on carbohydrate metabolism. The utilization rate of glucose by the body tissues has been found to be dependent on several factors.
The nutritional i;
condition of the animal and the prevailing blood sugar
'level both exert important effects on the rate at which the itissue cells take up glucose.
The liver itself, in the
intact animal, is the prime regulating organ of the body for I
the storage, supply and formation of glucose.
The liver is
the important center for maintaining the dynamic homeo stasis that normally exists in the process of metabolism. Above all, the endocrine system has a direct control over the utilization of carbohydrate and the functions of the liver, particularly in maintaining the blood sugar level and in regulating the requirements of the tissues and the supply of available glucose.
Tissue cells by themselves
apparently have an uninhibited glucose utilization rate that is far greater than the physiological rates of the body, which are normally under hormonal control. The possibility that the utilization rate of glucose decreases with starvation was reported by Hofmeister (1889)
9 when he found that animals that had been fasted were more susceptible to alimentary glycosuria than when they were well fed.
Since then, this "hunger diabetes" has been
demonstrated by many others, and it has been shown by Sweeney (1927) that it is the carbohydrate starvation that causes the decreased utilization rate of glucose by the tissues, rather than the lack of protein and fat in the diet.
Vigneaud and Karr (1925) found that the ability to
utilize carbohydrate diminishes progressively throughout the fasting period when they administered glucose to a series of rabbits that were previously fasted for as long as 20 days. However, in order to prove that it is actually the tissues themselves that have a decreased rate of glucose utilization, Drury (1942) has pointed out that it is necessary to demonstrate this effect when the blood sugar level is noimal and to eliminate the action of the liver from storing and forming sugar.
By using the eviscerated
rabbit, Bergman and Drury (1937) found that a pre-operative fast of four days decreased the glucose utilization rate from 206 mg. per kg. per hour to 110 mg. per kg. per hour. Also Mirsky (1938), while working with eviscerated dogs that had previously been fasted 96 hours, maintained them with 140 mg. of glucose per kg. per hour, while the non-
lo: -fasted hepatectomized dog, as shown by Yater, et al. (1933)y
i requires 250 mg. per kg. per hour.
Soskin and Levine
I(1937) also obtained similar rates for the well fed evis-
! cerated dog.
The effect of fasting on the oxidative action of insulin has been recently demonstrated by Drury, Wick and
They have shown that the oxidation of glu-
cose to carbon dioxide and water under the influence of 'insulin by the extra-hepatic tissues of the eviscerated rabbit is most marked in the non-fasted, fed animal.
oxidation rate of glucose decreases from about 300 mg. per kg. per hour in the fed, insulin-treated rabbit to about 50 mg. per k g . per hour in the four-day fasted insulin14 treated rabbit. Radioactive C glucose was used as a tracer in these experiments. In this connection, the observation of Himsworth (1949) that one action of the anterior pituitary is to adapt the animal to withstand starvation conditions and to prevent the accelerating effect of insulin is suggestive of a possible mechanism for the decreased utilization rate of glucose caused by fasting. The importance of the blood sugar level when consid ering glucose utilization rates is stressed by Bouckaert and de Duve (1947), regardless of the conditions under
which the rates are being measured.
They state that “the
rate of glucose consumption is directly related to its concentration in the blood."
After careful consideration
of previous work, they are of the opinion that much of the ;conflicting data that have been accumulated in the past regarding glucose utilization, both with and without the action of insulin, can be reconciled by the proper inter pretation of the blood sugar levels.
There is experimental
evidence that shows that both the storage and oxidation of carbohydrate vary directly with the blood sugar level with in the limits of the animal to utilize sugar.
showed that when glucose was slowly injected intravenously, the blood sugar at first rose rapidly and then decreased while the injection continued.
The body was able to handle
the glucose faster than it was injected.
With much larger
injections of glucose in the dog, Butsch (193*0 also obtained the initial rise in the blood sugar, which remained at a constant concentration for a period of time, and then suddenly began to increase still further.
increase was interpreted to indicate the point at which the body had stored the maximum amount of glycogen, after which the glucose then disappeared from the blood only as fast as it could be oxidized.
Wierzuchowski (1936), with large
constant injections of glucose in the resting dog, obtained
.results similar to those of Bang and Butsch, and he was 1able to show that the maximum rate of glucose utilization was 6 gm. per kg. per hour*
Soskin and Levine (1937)*
while studying the utilization of carbohydrate in totally eviscerated dogs, found that between certain limits, the ;rate of sugar utilization varied directly with the height of the blood sugar level.
When the blood sugar was main
tained at 60-80 mg. per cent, the utilization rate was about 24 mg./kg./hr.
When the blood sugar level was raised
as high as 450 mg. per cent, the utilization rate increased progressively to a maximum of about 500 mg. per kg. per hour.
Higher blood sugar levels did not increase the
utilization rate further.
They also showed that the
diabetic (depancreatized) dog could utilize carbohydrates as well as the normal dog providing the blood sugar was maintained at a high level. The investigations of Mann and his collaborators on the physiology of the liver have firmly established the prime importance of this organ in the storage and formation of glucose and in maintaining a normal blood sugar level under changing conditions in the body.
first demonstrated the liberation of sugar into the blood from the liver, but it was not until Mann (1921) developed a method for the complete removal of the liver in animals
1 that the way was made clear for evaluating the true role of this organ in metabolism and for studying the extra-hepatic phases of carbohydrate utilization.
Mann and Magath (1922)
demonstrated that a steady and profound fall in blood sugar occurred when the liver was removed and also that the administration of sugar would alleviate the symptoms of 'hypoglycemia. These investigators further demonstrated the vital functions of the liver in maintaining the blood sugar iby crossed circulation experiments with normal and hepatectomized dogs (1922).
Evidence that the liver is the
most important source of blood sugar in the fasted animal was obtained by Boliman, Mann and Magath (1925) with studies on muscle glycogen following removal of the liver. They found that with dehepatized dogs, as the blood sugar fell, the glycogen in the muscles was not greatly reduced, and there were no indications that muscle glycogen was capable of being converted to glucose to maintain the blood sugar levels in the absence of the liver.
further showed that epinephrine and other hyperglycemic agents failed to affect the falling blood sugar level of hepatectomized dogs, while Houssay and Poglia (1936) found that in liverless dogs, anterior pituitary extracts were ;also without effect in preventing the steady fall in blood sugar.
All of this evidence, and much more by other
:workers, has demonstrated that without the liver the main tenance of the blood sugar is impossible in the fasting or eviscerated organism unless glucose is administered from external sources* More recently, evidence from several sources has shown that the kidneys are able to form and to contribute i some glucose to the circulation under certain conditions. Himsworth and Scott (1938), with their studies on the action of adrenalin and the utilization of glucose by the peripheral tissues, suggest that there is an extra-hepatic source of carbohydrate that can supply glucose to the blood.
Bergman and Drury (1938), working with eviscerated
rabbits, showed that when the rabbits were also nephrectomized, more glucose was required to maintain the blood sugar at a normal level than when the kidneys were left intact.
Russell and Wilhelmj (1941), with in vitro studies,
found that kidney slices could form carbohydrate from succinate, pyruvate, a-ketoglutarate, alanine and glutamic acid.
This evidence suggests that the kidney is capable of
reforming sugar from glucose fragments and also from protein sources*
Shipley (1944), working with kidney slices
that were incubated in serum, found that glucose was formed more rapidly by kidney than by liver slices.
and Relnecke (1943) also showed that the blood sugar falls
*5 ; more rapidly in rats that are nephreetomized and eviscer ated than in rats that are just eviscerated.
Samuels (1944) made analyses of the arterial and venous renal blood of fasted eviscerated rats and found that the !blood sugar was higher in the renal vein*
which averaged 18 mg. per cent, was not found in the intact 'rat nor in fed eviscerated rats.
Also the blood amino
acids were not changed, so that the authors concluded that the glucose formed by the kidney must have originated from some other source.
Reinecke and Hauser (1948) made simul
taneous analyses of arterial and venous renal blood in the eviscerated dog, and they too found that the sugar concen tration in the renal vein was higher than the arterial blood sugar.
By determinations made on hepatectomized dogs, Cohn
and Kolinsky (1949) also found that the decline in blood sugar was more rapid if the dogs were nephreetomized than if the kidneys were intact.
This observation was true for
both normal and diabetic dogs.
Moreover, the formation of
sugar occurred irrespective of the blood sugar levels.
rate of fall in the blood sugar was estimated to be about twice as rapid in the nephreetomized animals.
and MacKay (1950), using radioactive glucose as a tracer, studied the production of glucose by the kidney in the eviscerated rabbit.
Determinations on the specific activity
of the circulating glucose, after one injection of the tagged glucose, showed that the specific activity remained unchanged if the kidneys had been removed, but that the activity decreased with time if the kidneys were left intact. On the other hand, when the blood sugar was main tained at a normal level for eight hours with radioactive glucose injections, the specific activity of the terminal blood glucose was about the same as that of the injected glucose, indicating that the kidneys had formed no new sugar.
However, evidence suggests that the kidney, when
under the conditions of a normal blood sugar level, may reform glucose from fragments of the circulating glucose, and if this is true, no change would be expected in the plasma specific activity. The influence of the endocrine system on carbohydrate metabolism is now well established.
The state of metabolism
in the body at any given instant has been shown to depend a great deal on the resultant effect of at least three ductless glands and the delicate interplay between their hormonal secretions.
These endocrine glands, especially
the adrenal cortex, the anterior pituitary, the beta cells of the pancreas, and possibly the thyroid, not only exert a controlling force on carbohydrate metabolism, but they have a pronounced effect on protein and fat metabolism as well.
A shift in the balance of the internal secretions of these ' glands will not only alter the state of carbohydrate metabolism, but in so doing, the metabolism of both protein and fat will consequently change. Although the administration of epinephrine will
i cause a rapid rise in the blood sugar level, by converting hepatic glycogen to glucose, this hormone does not appear to play as important a role in carbohydrate metabolism as was believed at one time.
Collens, et al. (1926) showed
that epinephrine did not cause a hyperglycemic response when the liver and its glycogen supply was tied off from the circulation, while Soskin (1927) found that epinephrine, when administered to hepatectomized dogs, did not prevent the steady fall in the blood sugar.
Geiger and Schmidt
(1928, 1929) have shown that adrenalin will cause the break down of muscle glycogen to lactic acid, which in turn is reconverted to glycogen in the liver.
Cori and Cori (1928)
found that adrenalin administered to rats at first had a glycogenolytic effect, but that eventually the lactic acid from the muscle glycogen actually increased the glycogen in the liver.
Bollman, Mann and Wilhelmj (1931) found that
in hepatectomized animals the administration of epinephrine caused neither the breakdown of muscle glycogen nor the production of lactic acid.
Thus the chief action of
18 ,epinephrine appears to be the rapid mobilization of carbo hydrate to meet emergency conditions, and it requires the presence of the liver for this action to be effective* Himsworth and Scott (1938) found evidence that the adminis tration of adrenalin to rabbits which had their livers cut ioff from the circulation caused the peripheral, extrahepatic tissues to utilize carbohydrate at an increased rate.
The blood sugar decreased faster in adrenalin-
treated "dehepatized" rabbits than in rabbits under the same condition that received no adrenalin* Likewise, the thyroid hormone, aside from generally increasing the metabolic rate of the tissues, appears to have no specific effect on carbohydrate metabolism.
shall and Greene (1933) observed that fasted hyperthyroid rats, when administered glucose, stored less glycogen than normal rats, indicating that they oxidized the sugar at a faster rate.
Also, Mirsky and Broh-Kahn (1936) found that
the blood sugar of eviscerated hyperthyroid rats decreased more rapidly than in normal eviscerated rats.
tion of sugars through the intestinal wall was found by Althausen (1937) to be faster in hyperthyroid rats than in normal ones.
Thus the effect of the thyroid secretion is
|to accelerate the uptake of carbohydrate by the tissues and to deplete carbohydrate stores.
In many respects the
Iaction of the thyroid gland is similar to the increased utilization of sugar caused by exercise (Peters and Van Slyke, 1946). Under normal physiological conditions, the main con trol of carbohydrate utilization in the body is the result ant effect of the adrenal cortical and anterior pituitary hormones balanced against the secretion of insulin from the pancreas.
In separately evaluating the actions of the
hormones, it must be realized that many of the effects that are observed when the source of any one of these hormones is removed from the body may actually be an accentuation of the remaining secretions. The influence of the adrenal cortex on carbohydrate metabolism is particularly well demonstrated by the work of Long, Katzin and Fry (1940), as well as the earlier work by other Investigators.
When adrenalectomized rats and mice
were maintained with an adequate supply of sodium salts and carbohydrates, their blood sugar and glycogen levels were practically normal, but starvation rapidly led to hypo glycemia and depletion of liver and muscle glycogen. Conversely, the administration of adrenal cortical extracts *
,to normal or adrenalectomized rats caused an increase in liver glycogen and a rise in blood sugar.
The excretion of
nitrogen also increased, and the newly formed carbohydrates
20 in the blood and liver could be accounted for by this accelerated protein catabolism*
Long* Katzin and Fry also
obtained evidence that demonstrates the synergistic rela1tlonshlp between the adrenal cortex and the anterior pituitary.
They found that injections of adrenal cortical 1
extract in fasted hypophysectomlzed rats not only greatly improved their general condition, but that also their
carbohydrate levels and nitrogen excretion increased i
Long and Lukens (1936) have shown that
adrenalectomy in depancreatlzed cats will greatly ameliorate the severity of the diabetes, and Long, Lukens and Dohans (1937) obtained the same results with dogs.
This effect on
diabetes is similar to that produced by hypophysectomy in the depancreatlzed animal as demonstrated by Houssay and Biasotti (1931)*
Russell (19*10) showed that the adminis
tration of adrenal cortical extract and glucose to normal animals resulted in a retardation of the oxidation of carbohydrate and an Increased amount of liver glycogen. Corey and Britton (1939) also found that the injection of adrenal cortical extracts into hypophysectomized rats would build up the glycogen stores in the liver.
On the other
hand, Russell (1940) administered anterior pituitary extracts to adrenalectomized animals and found that the muscle glycogen was replenished without much effect on the
These experiments Indicate that an impor
tant effect of the adrenal cortex is the formation of liver glycogen, in contrast to the anterior pituitary which tends to maintain the supply of muscle glycogen.
This action of
the adrenal cortical secretions on carbohydrate metabolism appears to be brought about by an inhibition of glucose utilization by the tissues and an increased conversion of 'protein to glycogen in the liver. The anterior pituitary gland, in many respects, acts synergistleally with the adrenal cortex, and there is evi dence that part of its action is mediated through the adrenal cortex by its adrenocorticotrophic principle.
herblvora the utilization of glucose by the tissues appears to be held in check by the action of the anterior pituitary. Greeley (1940) showed that in the hypophysectorn!zed rabbit the glucose utilization rate averaged 700 rag. per kg. per hour and that this rapid disappearance of glucose could not be accounted for by Increased glycogen or fat in the liver. Russell (1938) showed this same rapid utilization in hypophysectomized rats.
Peters and Van Slyke (1946) suggest
that part of this effect can result from the release of the pancreas from the inhibitory effect of the anterior pituitary and may be regarded as the uncontrolled action of insulin.
However, Greeley (1940) showed that the high
glucose requirement of the hypophysectomized rabbit remained unchanged after evisceration when the source of insulin was also removed*
On the other hand, Soskin,
Levine and Heller (1933) have shown that the eviscerated hypophysectomized dog has a decreased glucose utilization rate*
Also, Crandall and Cherry (1939)# using the London
Cannula technique, have estimated that the sugar output from the liver of the hypophysectomized dog is about 50 per cent less than the output from livers of normal dogs* Soskln (1941) has reported that the meat-fed hypophysectomlzed dog is able to maintain an adequate blood sugar level from the ingested protein, but that when the dog is fasted, gluconeogenesis from protein sources is diminished to such an extent that the blood sugar falls to hypoglycemic levels*
Soskin concludes that this fasting
hypoglycemia in the hypophysectomized dog is evidence for retarded gluconeogenesis in the absence of the pituitary hormones* The evidence obtained by Price, Cori and Colowick (1945) that extracts of the anterior pituitary inhibit the action of the enzyme hexokinase in the reaction of glucose to glucose-6-phosphate affords a basis for the opinion that the oxidation of glucose is retarded by the anterior pitui tary gland*
Reiss, Badrick and Halkerston (1949) have
23 32 found that the uptake by rat brain slices of PJ was increased 50 to 400 per cent after hypophysectomy.
workers attribute this to an increased rate of phosphoryla;tion and an increased rate of carbohydrate combustion after hypophysectomy.
This is further evidence in favor of a
pituitary principle that inhibits the oxidation of glucose.
P ric e , O ori and Golowick, s fu r th e r o b se rva tio n (1945) th a t in s u lin w i l l a b o lis h t h is in h ib it o r y e ffe c t o f the a n te rio r p it u it a r y also o ffe r s a p o ssib le e xp la n a tio n f o r a t le a s t one o f the a c tio n s o f in s u lin . In evaluating the effect of the anterior pituitary secretions on the metabolism of carbohydrate and of the administration of anterior pituitary extracts, the chief actions appear to be a diminution in the glucose needs of the tissues, a conservation of muscle glycogen and the formation of glycogen by the liver from protein sources. Hlmsworth (1949) is of the opinion that the anterior pituitary gland is primarily concerned with adaptation of the body to starvation conditions by retarding the utili zation of sugar by the tissues and in preventing the accelerating effect of insulin. According to Soskin (1941), the two main factors in the endocrine balance affecting the blood sugar level and carbohydrate metabolism are the secretions from the
24 anterior hypophysis and the pancreas* Since diabetes has been the most commonly recognized endocrine disorder that primarily affects carbohydrate metabolism, the study of the pancreas and the actions of insulin have received a great deal of consideration in the past*
The possibility that the pancreas secretes a speci
fic hormone that facilitates the utilization of carbohydrate by the tissues was first suggested by the classical experi ments of von Mering and Minkowski (1890), in which they showed that the removal of the pancreas in the dog caused a typical diabetic condition*
However, it was not until the
discovery of insulin by Banting and Best (1922) that the actual existence of the hormone was established*
then, experimental attempts to demonstrate the main action of insulin in the body have resulted in at least two schools of thought by authorities in the field* Until more recent times, it was generally concluded by workers in the field that a diabetic condition was exemplified by the inability of the organism to utilize carbohydrate and that insulin was necessary for the utili zation of glucose, particularly for the oxidation of glucose by the tissues. With the early investigations on the depancreatized dog and the observation that sugar was excreted more or
less quantitatively, as first shown by Minkowski (1893)» the conclusion was drawn that diabetics could not utilize ;carbohydrate#
Measurements of the oxygen consumption and
respiratory quotients of experimental animals have also ;been used to show that a lack of insulin decreases the oxidation of carbohydrate, and that the completely depan creatlzed animal is utilizing only fat.
.critically discusses the postulates on which R.Q.*s of the Intact animal are based and challenges the validity of this method of measurement.
He points out that the R.Q., as
measured, is actually a composite of many different R.Q.fs in the body, and that the oxidation of energy materials is a complicated process into which the respiratory quotient can give little insight.
Soskin1s recalculation of the
data that were obtained by Best, et al. (1926), which was purported to show that insulin increases the oxidation of carbohydrate in the tissues, actually demonstrates that sugar was oxidized more rapidly before the administration of insulin.
Prom such considerations as these and from his
own experimental observations, Soskin attributes the main action of insulin as being primarily the prevention of an overproduction of sugar by the liver and the facilitation of the tissues to utilize carbohydrate when the blood sugar is at a relatively low normal level.
The work of Soskin
26 ;and Levine (1937) has shown that the depancreatlzed dog with a high blood sugar level can utilize as much or more sugar than a normal dog.
Bouekaert and de Duve (1947)* in ,
discussing the action of insulin, also attack the evidence that has been used to support the “oxidation theory” in peripheral tissues.
Bouekaert is of the opinion that the
true action of insulin is often masked by the abnormal blood sugar levels that have been maintained during many of the insulin experiments.
Blood sugar levels that are above
normal may in themselves cause an increased utilization of glucose, while blood sugars that are too low may induce secondary reactions that antagonize the action of insulin. In this regard, the “glycogenolytic” action of insulin can often be accounted for by the release of epinephrine to mobilize the liver glycogen.
Evidence is also accumulating
that many Insulin preparations contain a hyperglycemic factor (Glukagon), which is possibly formed by the alpha cells of the pancreatic islets.
This substance may explain
some of the conflicting results that have been obtained in insulin and diabetic studies, and may be the cause of the hyperglycemic reaction that is seen in alloxan diabetes, but which is absent in the depancreatlzed animal, as well as i
account for the difference between the insulin requirements of the human diabetic and that of the totally depancreatlzed
Bouekaert and de Duve conclude that uncontaminated
insulin Increases hepatic glycogen, providing the blood sugar is maintained at a normal level.
Soskin (1941) and
Bouekaert and de Duve (1947) are unable to reconcile all the experimental evidence on insulin and diabetes with the ‘’utilization” or “oxidation” theory for the action of insulin.
Careful consideration and interpretation of the
experimental data, in their opinion, can point only to an overproduction of carbohydrates as the main effect of diabetes, and that insulin is necessary to inhibit this overproduction. In this regard, Drury (1940) has shown by feeding experiments with both depancreatlzed dogs and rats that one of the primary actions of insulin is to promote the storage of carbohydrates as glycogen and fat, particularly at the time it is ingested and absorbed.
Depancreatlzed dogs that
were fed and fasted on alternate days required additional insulin above their basal needs only during the four or five hours after they were fed, while the food was being absorbed.
The source of their energy needs during the
fasting days was the food that was stored under the influ ence of the extra insulin during the feeding periods. There were no indications that the basal insulin was neces sary for the oxidation of the carbohydrate, but rather that
it prevented the excessive breakdown of body protein. I With studies on the partially depancreatlzed rat, ,which requires no basal insulin and which can be maintained on a high carbohydrate diet, it was shown that Insulin is necessary for the deposition of fat and for the animal to 'gain weight.
During alternate feeding and fasting days,
1 the depancreatlzed rat will lose weight and the lost weight j cannot be regained unless insulin is administered with the ;food. Further studies on the depancreatlzed rat by Pauls and Drury (1942) also showed that insulin caused the diabet ic rat to store large quantities of sugar.
Only 24 per
cent of the stored carbohydrate could be accounted for by extra glycogen in the liver and muscles, and it was con cluded that most of the sugar that is stored under the influence of Insulin is in the form of fat. Stetten and Klein (1946) demonstrated the effect of insulin in rabbits by tracing the fate of deuterium that had first been concentrated in the body fluids in the form of
With alloxan diabetic rabbits on a high carbohy
drate, low fat diet, the rate of lipogenesis was found to be well below normal.
This was interpreted to indicate a
general impairment of glucose utilization under this dia betic condition.
In response to insulin the muscle and
liver glycogen appeared to arise mainly by a direct process; from dietary glucose.
When insulin was given to a normal
rabbit, a large increase in the rate of hepatic lipogenesis: was observed. Zilversmit, et al. (1948) investigated the rate that 14 administered C glucose was oxidized by normal and by alloxan diabetic rats.
They found that the Cl402 produced ;
in six hours accounted for the oxidation of about 50 P©1* cent of the injected glucose in normal rats, while only about 20 per cent was oxidized by the diabetic rats and 40 14 per cent was excreted in the same time. However, the C 0^ appeared in the expired air of both the normal and diabetic 14 rats during the first 30 minutes, and studies on the C 0g production by the rats after nephrectomy indicated that the over-all rate of oxidation by the diabetic rats did not differ significantly from that of the normal rats. The main site for the action of insulin has generally been considered by many workers to be in the muscles and peripheral tissues.
Mann and Magath (1923) and Mann and
Boliman (1933) found that insulin produced as rapid a decrease in the blood sugar of the hepatectomized dog as in the intact animal and concluded that the liver could not possibly be essential for the hypoglycemic action of insulin.
Contrary to the conclusions of Mann and Magath
30 (1923) and Mann and Bollraan (1933)» Bouekaert maintains that the liver is the main site of insulin action and that insulin is necessary for the inhibition of the overproduc tion of glucose from the liver* Prom the foregoing experimental studies regarding the influence of insulin on carbohydrate metabolism, it appears that insulin exerts several actions, of which the inhibition of gluconeogenesls from protein and the increased rate in the storage of carbohydrate as glycogen and fat are by no means the least important.
in the oxidation of glucose by the tissues has generally been accepted and taught as the main action of insulin, but as yet no direct experimental proof has been obtained to support this assumption.
The antagonistic relationship
between insulin and the secretions of the anterior pitui tary and the adrenal cortex must be considered when evaluating the effect of insulin. Review of the literature regarding anoxia and carbo hydrate metabolism. Experimental evidence that anoxia affects the carbohydrate metabolism of animals and influ ences the hormonal balance of the body has been obtained by several investigators. Evans (1934) found that rats, which were kept in an environment .of.1/2 an atmosphere (380 m m • Hg) showed an
Increase in their carbohydrate stores.
This was most
striking at the end of a 24-hour exposure.
glycogen was found to have increased as much as 34 per cent, and the other carbohydrate sources in the body had ,either increased or were undiminished. Similar experiments i with adrenalectomized rats showed that the ability to increase or to maintain the liver glycogen was entirely lacking.
Further work by Evans (1936) showed that no
intact rat failed to increase the liver glycogen when exposed to 1/2 atmospheric pressure, either with high, nor mal, or low temperature, or even when sick or prostrated. The intact rats were found to excrete 30 per cent more nitrogen during a 24-hour exposure than the ground level controls.
The adrenalectomized rats at normal atmospheric
pressures excreted 21 per cent less nitrogen than the intact control rats and they showed no Increase in nitrogen excretion when exposed to 1/2 an atmosphere.
cluded from these data that with the intact anoxic rat, the newly formed liver glycogen probably originates from pro tein and that the adrenal gland is concerned with this conversion of protein to carbohydrate.
suggests that when rats are exposed to anoxic conditions, they can be considered in the same category as the depancreatized or phlorizinized animalj that is, the increased
32 ;carbohydrate originates largely from protein, except that the sugar is not excreted, but is stored as glycogen, Langley (19^2) confirmed these experiments by Evans, except that he failed to find a rise in blood sugar when the animals had been fasted for 2k hours before the expo sure to low atmospheric pressure.
However, even in these i
cases the liver glycogen was markedly augmented.
other hand, if the animals were exposed to the anoxia for several days, to become acclimatized before fasting, the ’liver glycogen decreased.
This indicated that possibly the
'adrenal gland is activated only during the period of accli matization, Lewis, et al. (19^2), using rats, rabbits, dogs, monkeys, and human subjects, further investigated the role of the adrenal cortex in acute anoxia.
animals and subjects were exposed to similated altitudes that ranged between the equivalent of 11,000 to 3^*000 feet by breathing appropriate gas mixtures.
With rats, rabbits
and monkeys, the results were similar to those found by Evans (1936).
The dog, on the other hand, did not show the
increase in liver glycogen nor the rise in blood sugar with 2^-hour exposure.
However, there was a definite increase
in the excretion of nitrogen and phosphorus.
sugar levels of human subjects were not particularly
33 altered with short exposures to anoxia.
An over-all anal
ysis of the results of the experiment indicated that during the initial phase of the anoxic periods, there appeared to be an increased utilization of carbohydrates, with a normal blood sugar maintained at the expense of the liver glycogen. Continued exposure resulted in an increase in protein catabolism with an increase in carbohydrate stores, and an increased nitrogen excretion.
In the absence of the adrenal
cortex, these changes did not occur. Van Middlesworth, Kline and Britton (19W) investi gated the blood sugar regulation under severe anoxic condi tions in rats by exposing them to simulated altitudes of 27*000 to 37*000 feet (260-160 mm. Hg).
The results, in
general, showed that during the first 60 to 90 minutes at 260 mm. Hg pressure (27*000 feet), all the normal rats developed hyperglycemia, followed by a moderate hypoglycemia during the next two to four hours.
However, if the rats
were well fed or acclimatized, the later hypoglycemic phase did not occur.
Adrenalectomized rats, under the same condi
tions, developed a progressive hypoglycemia.
fasted and well fed rats were subject to convulsions which seemed to occur at any blood sugar level.
Anoxic and hypo
glycemic convulsions were considered to be fundamentally similar, both possibly depending on the reduced ability of
34 the tissue to utilize carbohydrate material. The effect of moderately low altitudes on the blood sugar levels of human subjects was shown by D*Angelo (1945) to produce no significant changes.
Exposures to altitudes
of 8,000 to 10,500 feet in a decompression chamber caused the blood sugar levels to remain essentially the same as at ground level.
Glucose toleranee curves after the ingestion
of glucose were also essentially the same as the control curves.
However, D*Angelo suggests that even at these low
altitudes, homeostatic mechanisms for controlling blood sugar levels are brought into play. Smith and Oster (1946) have shown that cats, after exposures to low oxygen tensions, have marked increases in the blood sugar levels.
Fasted cats were able to remain
conscious longer than well fed cats.
Van Liere (1948)
showed that when dogs were exposed to a simulated altitude of 28,000 feet, the blood sugar level increased, but that after a period of daily four-hour exposures at 24,000 feet, a second exposure to 28,000 feet resulted in no increase in the blood sugar level.
Increasing the altitude to 32,000
feet, however, caused the blood sugar to rise.
Northup and Van Liere (1948) have subsequently shown that the blood sugar levels in the dog progressively increase with the degree of anoxia, but that the change is not
35 significant below an altitude of 24,000 feet.
tolerance tests indicated that exposures to anoxia resulted in a decreased tolerance to glucose*
The glucose tolerance
curves that were determined at altitude stayed at a higher level longer than control curves at ground level.
minutes at altitude, the blood sugar curves were still 50 per cent above normal, while at ground level conditions the blood sugars had returned to normal within 90 minutes. These workers believe that both the adrenal medulla and the pancreatic island cells may be involved.
Dogs exposed to
28,000 feet for 30 minutes normally show a decline in their blood sugar levels, suggesting that the secretion of epi nephrine may be exhausted.
On the other hand, Gellhorn and
Packer (1940) found that, although adrenalin caused a greater increase in the blood sugar of rabbits during the first hour at altitude than it did at ground level, the adrenalin almost completely lost its efficiency during the second hour of anoxia.
In this case, the liver was not
depleted of its glycogen and the adrenals still contained adrenalin.
The conclusions were drawn that adrenalin fails
to act on the liver under prolonged periods of anoxia. The possibility that anoxia may affect the pancreas and the secretion of insulin was demonstrated by McQuarrie, Ziegler and Hay (1942).
They not only showed that anoxia
36 caused a rise in the blood sugar of the normal dog and a fall In the blood sugar of the adrenalectomized dog, but they also found that with either condition there were decreases in the plasma potassium and inorganic phosphate which were similar to the decreases resulting from the administration of insulin in small doses.
when the adrenalectomized-depancreatized dog was exposed to anoxia, the blood sugar level remained essentially un changed and there was no significant decrease in the potassium and phosphate levels.
From this evidence the
authors concluded that anoxia excites the vago-insulin system as well as the sympathico-adrenal system, although in the intact animal the effect of the adrenals overshadows the insulin effect. Earlier work by McQuarrie and Ziegler (1938) showed that dogs that were subjected to anoxia and insulin exhi bited a greater fall in the blood sugar than when the same amount of insulin was administered at ground level.
the dogs did not show the convulsive symptoms that ordinar ily resulted from the anoxia.
Gellhorn and Packer (1940)
found that anoxia tended to antagonize the effect of insulin administered to rabbits and that the blood sugar returned to its normal value faster than at ground level. Sundstroem and Michaels (1942) have shown that the
37 sugar tolerance curves of rats are high during the first week of exposure to 300 mm. Hg, after which time the toler ance curves return to approximately normal.
along this line by Van Middlesworth (1946) shows that rats which have been exposed to severe anoxia (258 mm. Hg for four hours), and which have been fed glucose, develop a prolonged hyperglycemia and a pronounced glycosuria during the period of anoxia.
The amount of heart and liver glyco
gen was found to be less than in glucose-fed controls at ground level.
This evidence suggests that even under severe
anoxia the rate of glucose absorption through the intestine was sufficient to produce the hyperglycemia while the rate of glycogenesis was reduced.
The result was an eventual
glycosuria which Van Middlesworth terms an "anoxic dia betes."
Keyes and Kelly (1949) have studied the effect of
anoxia on the glucose tolerance of dogs as well as further studies on the role of the adrenals under anoxic conditions. They conclude that the sympathico-adrenal system exerts only a minor effect on the glucose tolerance of dogs at 24,000 feet, providing they are decompressed gradually, while the predominant factor is an increased secretion from the adrenal cortex.
Careful analysis of the glucose toler
ance curves indicates that there is a more rapid formation of glycogen than at ground level, and that there is
actually an Increase In the glucose tolerance of dogs at 24,000 feet.
Although the tolerance curves at altitude
persisted at a high level for a longer period of time than the control curves, the increased blood sugar is the result of gluconeogenesis from fat and protein brought about by the increased production of the adrenal cortical hormones. Langley (1950) has recently reported that when the adrenal gland is transplanted in the rat, an accumulation of liver glycogen does not occur after a 24-hour exposure at 20,000 feet, in contrast to the increased liver glycogen found in intact rats under the same conditions.
the transplant rats showed a lower blood urea concentration than the intact animals.
These findings have been used to
support the theory that the adrenal cortical reaction to stress is dependent upon the activation of the adrenal medulla through its sympathetic innervation.
The theory is
that the stress causes a release of adrenalin which in turn causes the anterior pituitary to release ACTH. Review of the literature on the D:N ratio and phlor izin diabetes.
The work of Long, Katzin and Fry (1940) on
the significance of the adrenal cortex in carbohydrate metabolism has shown that injections of the adrenal cortical extract into fasted normal and adrenalectomized rats caused
39 an Increase in the liver glycogen that is from ten to forty times that of the untreated animals*
The blood sugar
levels were elevated, and a marked increase in the urinary nitrogen excretion also followed the administration of the cortical extract*
Long has estimated that the urinary
nitrogen from the accelerated protein catabolism was suffi cient to account for the extra carbohydrate.
results were obtained with fasted hypophysectomized and depancreatized rats following cortical extract injections. It was concluded that an adrenal cortical principle causes the maintenance of the blood glucose and liver glycogen by gluconeogenesis at the expense of the body protein. The earlier studies by Evans (1936) have shown that fasted phlorizinized-adrenalectomized rats excrete less sugar and nitrogen than do normal phlorizinized rats.
urinary D:N ratio decreases from 3.18 for the intact phlor izinized rat to 2.65 for the adrenalectomized animal. Also, when normal rats were exposed to 1/2 an atmosphere for 24 hours, both the liver glycogen and the urinary nitrogen increased, but this failed to occur when adrenal ectomized rats were similarly treated.
that the increased nitrogen excretion that resulted from the anoxia was more than sufficient to account for the newly formed glycogen in the intact animal.
ko These results suggest a possible relationship in the diabetic animal between the relative excess of adrenal cortical secretions and the lack of insulin*
hibited cortical hormones not only depress the utilization of glucose by the tissues but also promote gluconeogenesis from protein sources, causing the increase in urinary nitrogen and glycosuria.
The added stress of anoxia on an
animal in the diabetic state could conceivably cause a change in its insulin requirements. A measure of the degree1 of glycosuria and the excretion of nitrogen that occurs in a diabetic animal when exposed to anoxic conditions could be useful for evaluating the changes in carbohydrate metabolism that is caused by the abnormal environment*
this respect, the D:N ratio under these conditions might serve as an index of the physiological changes in the body. Although the results of Evans (1936), as well as the work of Keyes and Kelley (19*19)» are indicative of an increased gluconeogenesis from body protein under anoxic conditions, a true estimate of the conversion of protein to carbohydrate must also take into consideration the total amount of sugar that is utilized by the animals, rather than just the amount of stored glycogen and the increase in the blood sugar levels.
The work of Lewis, et al. (19*12)
suggests that at least during the initial phase of an
41 anoxic exposure, there is an increased utilization of car*
If it is accepted that the excreted nitrogen can
account for the Increased amount of stored glycogen, then it follows that at least part of the glucose that is utilIized by the tissues during the anoxic period must also be derived from protein.
With a diabetic animal, the observed :
urinary D:N ratio alone could not indicate the true proteinto-carbohydrate conversion. A re-evaluation of the D:N ratio as a measure of the carbohydrate equivalent of nitrogen or protein is necessary before the urinary nitrogen excretion can be most effective ly used as an indication of changes in carbohydrate metabo lism under both normal and abnormal environments. Studies on the depancreatized or phlorizinized animal have been one of the chief methods for investigating the possible precursors of sugar in the body.
work of von Mering and Minkowski (1890) on depancreatized and on phlorizinized dogs led to the conclusion that the diabetic animal was unable to utilize carbohydrate and that ingested sugar was quantitatively excreted in the urine. Further studies in Lusk*s laboratory supported this conclu sion, particularly when Lusk (1915) reported that the respiratory quotients of the phlorizinized dog remained essentially unchanged after the Ingestion of glucose and
The work of Ringer (1912) also indicated that
glucose, when administered to the phlorizinized dog, was completely excreted as extra sugar, although the ingested carbohydrate did cause a protein-sparing effect.
work by Ringer and Frankel (1914), which was concerned with , the rate that ingested glucose was excreted by phlorizinized animals, led them to conclude that the kidneys of diabetic animals excrete glucose as fast as it enters the blood stream. The possibility that blood sugar could be formed from non-carbohydrate sources in the body also was first suggested by Minkowski’s work (1893) when he found that the amount of glucose and nitrogen excreted by the depanereatized dog seemed to show a constant relationship, even when various quantities of protein were ingested.
ratio was extensively investigated by Reilly, Nolan and Lusk (1898) and Janney and Csonka (1913) with fasting phlorizinized dogs, and the glucose to nitrogen ratio of 3 .65:1 has generally been used for estimating the amount of
glucose that can be derived from protein.
is reason to doubt the validity of the calculations that are based on Lusk’s ratio of 3.65:1.
dogs that were studied by Minkowski had on the average a D:N ratio of 2.8:1.
Soskin (1941) points out that either
the depancreatized animals were utilizing some of the sugar that was formed from protein, or the phlorizinized animals were excreting glucose that was derived from other sources as well as from protein. Janney and Csonka (1915) investigated the formation of glucose from body protein and estimated that most animal proteins are capable of yielding between 58-60 per cent glucose, or a theoretical D:N ratio of 3.6-3.8:l.
When the ,
observed D:N ratios of several other investigators were averaged together, a D:N ratio of 3.43:1 was obtained. Janney points out that the total nitrogen of the urine can only contain about 95 per cent of the nitrogen from glucogenetic proteins, because about 5 per cent of the nitrogen is derived from creatine, creatinine and purines.
average D:N ratio of 3*43:1 Is corrected for this nitrogen, the D:N ratio then becomes 3.6:1. Although Janney and Csonka do not take into account the utilization of newly formed glucose by the animal tissues, they do, however, suggest that this might be possible in some cases.
They note that rabbit body pro
teins can theoretically yield 60 per cent glucose or create a D:N ratio of 3.8:1.
Actually, the phlorizinized rabbit
has been observed to have a urinary D:N ratio of 2.8:1, which is much lower than the ratio of the phlorizinized
Because of this, they conclude that the rabbit must
utilize some of the glucose. The fact that both the depancreatized and the phlor izinized animal can utilize carbohydrate has been estab lished by several investigators.
Wierzuchowski (1926) and
others, contrary to the earlier work, were only able to recover between 50-80 per cent of administered glucose in the urine of the phlorizinized dog.
These results indi
cated that part of the ingested glucose must have been utilized by the tissues.
demonstrated that after the administration of glucose to the phlorizinized dog, the respiratory quotient increased from the diabetic level of 0.70 to 0.80, and he calculated that the animal must have oxidized between 14-20 per cent of the ingested carbohydrate.
Deuel, Wilson and Milhorat
(1927)> investigating the mechanism of phlorizin diabetes, could find no impairment in the ability of phlorizinized animals to oxidize normal quantities of glucose.
ticular interest were the findings of these workers that the phlorizinized-nephrectomized dogs were not diabetic. The blood sugar levels remained constant, and the non protein nitrogen of the blood changed in the same manner as in the normal nephrectomized dog.
Drury, Bergman and
Greeley (1936) have shown that the tissues of the
phlorizinized dog are able to utilize the blood sugar that is derived only from body sources as well as from exogenous foodstuffs.
The glucose utilization rate of the fasted
phlorizinized dog after hepatectomy was found to be 75 per kg. per hour.
When this amount of glucose is added to
the excreted glucose, the D:N ratio becomes about 6:1, an almost complete conversion of protein to glucose, if it be assumed that there is no conversion of fatty acids to glucose. Soskin, Levine and Lehman (1938) similarly found that the eviscerated phlorizinized dog was able to utilize sugar at the same rate as the tissues of normal animals at comparable blood sugar levels. Greeley and Drury (1940) investigated the glucose utilization rate of the eviscerated diabetic rabbit and found that under basal conditions the diabetic extrahepatic tissue utilized glucose at the same rate as that of the normal. Soskin (1941) is not only of the opinion that there is no justification for assuming that the phlorizin-treated animal cannot utilize glucose, but that also the sugar that is excreted is not necessarily formed entirely from protein alone.
Soskin feels that the possible conversion of fatty
acids to glucose must be considered.
However, until there is clear-cut proof that a fatty acid-to-glucose transformation actually occurs in the animal body under diabetic conditions, both the glucose that is excreted and that which is used by the tissues of the fasting diabetic animal can be accounted for by accept ing a D:N ratio that is higher than 3*65*
Drury (1942) has
critically considered the possibility that body protein is capable of furnishing larger quantities of glucose than has generally been assumed.
He has calculated from the results
of protein and sugar feeding experiments with the depan creatized dog that, when the sugar that is utilized by the tissues during the experimental period is added to the excreted glucose, a D?N ratio of between 5 and 6 is obtained, or a conversion of protein to glucose that approaches 100 per cent.
That such a complete conversion
is possible rests, first of all, on the possibility that protein is at least theoretically capable of being trans formed to such an extent in the body.
From among the amino
acids, lysine appears to be the only one that has defi nitely been shown to be incapable of forming glycogen.
has been shown that it is possible for such supposedly doubtful glycogen-formers as histidine and tyrosine to be converted to carbohydrate by the body (Peters and Van Slyke, 1946).
Todd and Talman (1949) have estimated that
47 glycine, when fed to rats, will cause an increase in gluco neogenesis that results in about six times as much addi tional carbohydrate as can be accounted for by a complete conversion of the fed glycine. Recently, Sanadi and Greenberg (1950) have reported 14 that following the administration of C -labeled tyrosine and phenylalanine to phlorizinized rats, radioactive glucose and ketone bodies were excreted in the urine. Although a small part of the glucose that is formed in the body is derived at least from the glycerol portion of fat, when the glucose utilization rate is considered in the D:N calculations, a much larger quantity of the glucose can be accounted for as derived from protein than has generally been assumed in the past.
In this connection the
D:N ratio can be a useful tool for studying and evaluating the effects of various factors on carbohydrate metabolism.
CHAPTER III THE UTILIZATION OF GLUCOSE BY EVISCERATED RABBITS The general plan of the experiments on glucose uti lization by the extra-hepatic tissues.
rabbit and three rabbits that were fasted three to four days were eviscerated and, after recovery from the ether anesthesia, were maintained by a constant infusion of radio active glucose through a jugular vein cannula. were left intact.
The method for the evisceration and the
maintenance of the rabbits were performed according to the technique described by Drury (1935).
A preliminary partial
ligation of the Inferior vena cava was performed some time prior to the evisceration for the establishment of a col lateral system sufficient to take care of the circulation when the vena cava was tied off and the liver, together with the intestines, spleen and pancreas, was completely removed.
A plastic cannula was tied securely in the jugu
lar vein for the administration of the radioactive glucose solution and a tracheal cannula was secured in place for the collection of the expired carbon dioxide.
recovered rapidly from the operation and were usually sitting up in a normal manner within an hour after the ether administration had been stopped. Blood sugars were determined frequently during the
course of the experiments so that the infusion rates of the radioactive glucose eould be adjusted and the blood sugar levels could be maintained within a normal range.
to establish quickly a concentration of radioactive glucose in the rabbits that was comparable to the specific activity of the glucose that was to be injected continuously, a small priming dose of potent radioactive glucose was first admin istered to the animals at the beginning of the experiment. 14 Within 30 minutes this C glucose was thoroughly mixed throughout the body fluids and was diluted with the pre vailing glucose content of the animal.
Thus, when the less
potent infusion glucose was injected into the circulation, its specific activity remained essentially unchanged.
specific activity of the body glucose that resulted from the single injection of the priming dose was calculated by first determining the blood glucose level of the animal and then multiplying this glucose concentration by the volume of body fluid that contains glucose.
This "glucose space”
has previously been found by Wick, Drury and MacKay (1950) to be about 25 per cent of the body weight of the eviscer ated rabbit.
The specific activity of the body fluids is
equal to the specific activity of the radioactive glucose multiplied by the number of milligrams in the priming dose, and then divided by the milligrams of glucose in the
A radioactive glucose solution with the same
specific activity as in the glucose space was then prepared iand infused continuously at a rate designed to maintain the blood sugar within a normal range.
Urine was collected
throughout the experiments, and the collection of the expired carbon dioxide was started as soon as the radio active glucose was injected.
The carbon dioxide was
absorbed by 2 normal sodium hydroxide in collection vessels which were changed every hour during the course of the experiments.
In this way the carbon dioxide production
could be calculated on an hourly basis.
At the termination
of each experiment, which lasted at least eight hours, the animal was heavily anesthetized with nembutal and the abdomen was quickly opened.
After the collection of a
large blood sample the organs and carcass were immediately frozen in either liquid nitrogen or carbon dioxide snow. The plasma was collected from the heparinized blood sample after centrifugation and then frozen. Methods used for the tissue analysis. All of the analyses that were made on the tissues and organs of the experimental animals were carried out by the personnel in the biochemistry laboratories of the Scripps Metabolic Clinic, under the direction of Dr. Arne Wick. T he .-labeled glucose was also.prepared at the
51 Scripps Metabolic Clinic by a method similar to that of Putman, et al. (1948).
was uniformly incorpor
ated into the leaf starch of plants by photosynthesis.
glucose was obtained by acid hydrolysis of the starch which had been extracted from the leaf tissue with perchloric acid and isolated as the starch-iodine complex.
The speci14 fic activity of the carbon dioxide obtained from the C labeled glucose by baker*s yeast fermentation and by total combustion was the same• The measurement of C
radioactivity. All the
radioactive determinations, except on the fat fractions, were performed on BaCO^ as described by Wick, Barnet and Ackerman (1949).
The BaCO^ was obtained by either fermen
tation or by combustion of the sample material.
activity of the fat fractions was counted directly.
cases the samples were of infinite thickness. Plasma glucose determination.
The determination of
plasma glucose was carried out by a combination of micro fermentation with Fleischmann*s baker*s yeast as described by Reinecke (1943) and the ferricyanide-ceric-sulfate macro procedure of Miller and Van Slyke (1936).
between the glucose equivalent of the non-fermentable reducing substance and the conventional plasma sugar value
52 was taken as the glucose equivalent of the fermentable i
reducing substance in the blood. i
The determination of the total
radioactivity (other than carbon dioxide) per unit volume of plasma was performed on 1 ml. of plasma that was mixed with 1 ml. of 0.5 M phosphate buffer.
This mixture was ! 14 ' frozen, lyophilized and weighed. The BaC 0o that was o obtained from dry combustion was measured for radioactivity. Plasma glucose specific activity.
activity of the plasma glucose was determined from the carbon dioxide produced by the fermentation with Fleischmannfs bakerfs yeast of 1 ml. of plasma and 60 mg. of glucose added as a carrier.
The carbon dioxide was pre
cipitated as BaCO^. Collection of the expired carbon dioxide.
expired carbon dioxide was collected from the animal through a tracheal cannula.
The exhalation side of the
cannula was attached by rubber tubing to the collection flasks that contained 250 ml. of 2 N NaOH.
The flow of the
expired carbon dioxide from the animal to the collection flasks was facilitated by the aid of suction on the exhala tion side of the cannula and by pure oxygen pressure on the inhalation side.
The collected carbon dioxide was
was precipitated as BaCO^. The preparation of the carcass for analyses.
frozen carcass, including the bones and skin (heart, lung and kidneys were kept separate), were broken into several f
large pieces and then disintegrated by passing the pieces through a hand-operated ice crusher.
This finely chopped
material was kept frozen at all times during the crushing i operation, and it was dehydrated in the frozen state in a large lyophillzation apparatus.
The dehydrated tissue was
subjected to the following treatment and determinations. Total crude fat and fatty acids. The dehydrated tissue was extracted in a Soxhlet-type extractor for 24 hours with an ethyl ether-petroleum ether (1:1) mixture. The partially defatted residue was removed from the extractor, and after removal of the solvent the residue was finely ground in a Raymond pulverizer (hammer mill).
material was replaced in the Soxhlet extractor and reextracted for another 24 hours.
An aliquot of the extract
was used for determination of total solids and radioactive measurements.
For the fatty acid determination another
aliquot was saponified by refluxing for six hours with 30 per cent potassium hydroxide in 50 per cent alcohol.
alcohol was removed and the alkaline mixture was extracted
54 five times with an equal volume of petroleum ether.
fatty acid solution was acidified and re-extracted with petroleum ether.
The petroleum ether solution was thor
oughly washed with water, dried with magnesium sulfate, and the solvent removed for fatty acid determinations. Water-soluble fraction. An aliquot (25 per cent) of the defatted carcass was extracted three times with gener ous quantities of boiling water.
In this procedure the
residue was added to the boiling water and stirred before filtering through a large Buchner funnel.
extracts were dried by lophilization. Protein fraction.
The residue from the water ex
traction was dried and weighed for radioactivity determina tions.
In order to remove glycogen and other poly
saccharides, an aliquot was hydrolyzed at 100°C. for three hours with 2.2 per cent hydrochloric acid (2.2 ml. of con centrated hydrochloric acid diluted to 37 ml.).
Insoluble residue, called the "true protein" fraction, was washed in order to remove sugar fragments. Glycogen.
The glycogen of the defatted carcass was
determined by the method of Good, Kramer and Somogyi (1933)• For Isolation, an aliquot of the defatted carcass was refluxed with 30 per cent potassium hydroxide for three
55 hours and the glycogen precipitated with alcohol.
obtain pure glycogen for counting purposes, it was neces sary to redissolve the glycogen in trichloracetic acid solution, filter, and reprecipitate the glycogen with alcohol. Body carbon dioxide♦ The body carbon dioxide was determined by acidifying an alkaline digest of the carcass and collecting the carbon dioxide as BaCO^. The results of the experiments on glucose utiliza tion by eviscerated rabbits. The routes of disposal for the infused glucose in the body of the eviscerated rabbit and the relative magnitude of each, as indicated by the tissue analysis, are shown in Table I.
Rabbit #1 was non
fasted before evisceration, rabbit #2 was fasted three days, and rabbits #3 and #4 were previously fasted four days.
The expired carbon dioxide for all of the rabbits,
at the most, did not contain much more than 20 per cent of the injected counts.
However, it is significant to note
that the expired carbon dioxide from the four-day fasted rabbits (#3 and #4) contained the least amount of radio active carbon, while the non-fasted rabbit, #1, produced 14 the greatest amount of C Og. The water-soluble fraction of the carcass accounted for nearly 50 per cent of the
TABLE I THE DISTRIBUTION OP C1* IN EVISCERATED RABBITS AFTER AN 8-HOUR PERIOD OF CONSTANTLY INJECTED C -LABELED OLUCOSE
Fraction Expired COg Body CO2 Water soluble As glucose Glycogen Crude protein True protein Crude fat Fatty acids Urine Kidneys-lungs-heart Total recovery
Rabbit #1 Non-fasted
Per cent of injected counts Rabbit #2 Rabbit #3 4-day fast 3-day fast
Rabbit #4 4-day fast
24.0 5.3 4.7 6.1 5-1 1.7 0.8 4.3 5-0
12.7 3.* 32.-7 10. Q 3.2 6.1 5.6 0.9 0.2 4.2 5.0
17.7 4.4 50.3 9.0 2.6 7.5 1.9 2.0 0.4 None 5.0
19.0 3.8 46.0 6.4 2.5 4.8 3.6 1.0 0.1 4.0 5.0
22.5 • •
57 counts in the four-day fasted animals, but in the nonfasted rabbit only about one half as much activity was found in the water-soluble material. rabbit, #2, fell intermediately.
Again, the three-day fasted However, in any event,
the water-soluble fraction in all the rabbits contained the greatest percentage of the injected counts.
Also of inter-
est is the small amount of radioactivity that was found in .the glycogen and fat, with a relatively larger percentage appearing in the body protein.
The fatty acids contained
a particularly small amount of the radioactive C
An appreciable amount of activity appeared in the urine, but further analysis showed that less than 10 per cent of these counts could be accounted for as carbonate, urea and fermentable substances.
Also, it is significant
that in the water-soluble fraction most of the radioactive carbon was in material other than glucose. The hourly changes in the specific activity of the expired carbon dioxide during the eight-hour experimental periods are shown in Table II, as well as the specific activities of the plasma glucose at the beginning and end of each experiment and the specific activity of the Infused glucose In each case.
It can be seen that the specific
activity of the expired carbon dioxide increased rather sharply during the first three hours of the experiments and
TABLE II THE HOURLY SPECIFIC ACTIVITIES* OF THE EXPIRED C02 FROM EVISCERATED RABBITS DURING AN 8-HOUR PERIOD OF CONSTANTLY INJECTED C^-LABELED GLUCOSE
Hours 1 2 3 k 5 6 7 8 S. A. of plasma glucose 30 min. after priming dose S. A. of terminal plasma glucose S. A. of glucose soIn. Injected during run
Expired COg specific activity* Rabbit #1 Rabbit #2 Rabbit #3 Rabbit #4 Not fasted 3-day fast 4-day fast 4-day fast 150 243 323 344 353 411 436 425
148 197 266 290 314 346 375 380
89 137 172 206 239 275 321 390
40 123 154 174 182 209 224 237 3330
♦Specific activity is cts./rain./mg. carbon.
59 then tended to level off during the next five hours, although It was still rising slowly at the end of this time. It is also interesting to note in Table II that with the fasted rabbits the maximum specific activity of the expired * carbon dioxide during the last hour of the experiments was only about one tenth of the specific activity of the infused glucose or of the circulating plasma. Table III shows the per cent of the injected counts that were contained in each hourfs collection of the expired carbon dioxide during the eight-hour experimental periods.
Here again, the hourly increase in the counts was
similar to the increase in the specific activity of the expired carbon dioxide (Table II), The data concerning the rates of glucose oxidation and of the glucose disappearance are shown in Table IV, order to take into account the slight difference between the specific activities of the circulating and injected glucose, the glucose oxidation rates per hour were esti mated by two methods, the results of which are both given in Table IV.
In the first method (#1) the average number
of counts of carbon dioxide that was expired per hour was divided by the counts per milligram of plasma glucose as determined by the fermentation procedure.
In the other
method (#2) the average counts in the expired carbon
TABLE III THE HOURLY C1^ CONTENT OP THE EXPIRED C02 FROM EVISCERATED RABBITS DURING AN 8-HOUR PERIOD OP CONSTANTLY INJECTED C^-LABELED GLUCOSE
Hours 1 2 3 4 5 6 7 8 Total percentage
Per cent of injected counts In expired COo Rabbit #1 Rabbit #2 Rabbit #4 Rabbit #3 Not fasted 4-day fast 4-day fast 3-day fast 1.0 2.1 3-4 3.1 2.6 3.5 3.5 3.4
1.0 1.0 1.1 1.3 1.5 1.8 2.1 2.9
1.2 1.3 1.7 1.8 2.1 2.0 2.3 5.3
1.0 2.1 2.0 2.6 2.6 2.7 3.0 3.0
TABLE IV THE COMPARISON OF GLUCOSE DISAPPEARANCE AND OXIDATION RATES IN EVISCERATED RABBITS
Glucose dlsappearance m g ./kg./hr.
1 2 3 4
128 84 124 113
Glucose oxidation rate rag./kg./hr. #1 #2
18 32 26
34 17 28 28
fdioxide were divided by the counts per milligram of the injected glucose.
In both cases, the results are expressed
in milligrams per kilogram per hour.
The apparent glucose
disappearance rate is the average rate at which the glucose had to be injected in order to maintain the blood sugar at a constant level.
Since the specific activities of the
plasma glucose and the injected glucose were similar, the
oxidation rates given by the two methods agree fairly closely.
It can be seen that only 20-30 per cent of the
iglucose that disappeared in the animals was undergoing rapid oxidation to carbon dioxide.
Most of the glucose
that was utilized went into other disposal routes such as i
glycogen, fat and protein, and particularly into the watersoluble fraction. A comparison of the plasma radioactivity when it is measured by the fermentation procedure and by the combus tion method is shown in Table V.
These data afford an
indication of the degree of radioactivity in the fluid space of the animal.
It can be seen that the total activ
ity of the plasma during the first hour of the experiment is essentially the same with the two methods.
when the two methods are used on terminal plasma samples, a comparison of the results shows that the fermentation method gives a radioactivity that is appreciably lower than that
TABLE V COMPARISON OP TOTAL PLASMA RADIOACTIVITY BY FERMENTATION AND COMBUSTION (EXPRESSED AS COUNTS/ML./folN.)
Rabbit #2 #3 #4
Time of sampling during constant infusion of glucose During 1st hour After 8 hours Ferment, Combust, Combust, Ferment, 1470 1610 1270
1450 1450 1480
1560 1140 920
1930 1930 1500
obtained with the combustion method♦ Discussion of the results on glucose utilization by the eviscerated rabbit. The tracer analysis of the evis;cerated rabbit following constant infusion of radioactive glucose indicated that only a small proportion of the circulating glucose underwent rapid oxidation to carbon dioxide during the course of the experiment.
still smaller amount of the disappearing glucose could be accounted for in the form of glycogen and fat.
component of the utilized glucose was found in the watersoluble fraction of the carcass and, of this£ only about 20-30 per cent was in the form of glucose, which was the circulating glucose in the body fluids.
Thus, the main
bulk of the injected glucose was found in the non-glucose water-soluble fraction in the eviscerated rabbit.
non-glucose portion, a certain amount of it probably represented glucose derivatives that were in the process of metabolism and which would continue on to completion with time.
Also, it is possible that part of the non-glucose
material consisted of glucose derivatives and breakdown products that were unable to proceed in their normal chain of reactions in the absence of the liver.
Because of this,
these ‘’dead end1* fragments accumulated in the body during the course of the experiment.
Evidence that suggests this
65 possibility can be found in the comparison of the plasma radioactivity as determined by combustion and fermentation (Table V)*
At the end of eight hours, the counts by com-
:bustion became considerably greater than the counts by fermentation, indicating an accumulation of glucose deriva tives in the circulating fluids.
Preliminary work on the
fractionation of the non-glucose water-soluble material indicates that less than 10 per cent of the C*1* of this fraction can be accounted for by pyruvic and lactic acids and the phosphate esters of fructose, glucose, pyruvic, dihydroxyacetone, and 3-glyceraldehyde. A further suggestion that glucose fragments may tend to accumulate in the body of the eviscerated animal is found by a consideration of the relatively large excretion in the urine of radioactive material that is not glucose carbonate or urea.
In the absence of the liver, some of
these products may pile up to abnormal proportions in the circulation and be excreted by the kidneys. Although only a comparatively small amount of labeled glucose was rapidly oxidized, very little of the sugar was changed to glycogen and fat and deposited in the conven tional storage depots in the body.
This in part may be due
to the absence of both the liver and a source of insulin. Also, the short duration of eight hours may not have been
sufficient time for the conversion of glucose to glycogen and fat in an animal the size of the rabbit. From the evidence, the largest proportion of the glucose that was utilized by the eviscerated rabbit appears jto have been deposited in a water-soluble metabolic pool. !This may represent a type of storage which contains glucose fragments that are in a slow process of metabolism. Further evidence that indicates that the rate of glucose utilization is decreased by starvation can be found by comparing the radioactivity of both the expired carbon dioxide and the water-soluble fractions of the fasted and non-fasted rabbits.
The expired carbon dioxide of the non
fasted rabbit showed the highest percentage of injected counts and the greatest specific activity throughout the course of the experiment, indicating that its ability to oxidize glucose rapidly was greater than that of the fasted animals.
Also, the ability of the fed rabbit to mobilize
the injected glucose can be seen by the relatively small amount of radioactivity in the water-soluble fraction.
CHAPTER IV THE ACTION OP INSULIN IN EVISCERATED RABBITS The general plan and methods of the experiments. These experiments were carried out in the same manner that has already been described in the foregoing chapter, except that ten units of regular insulin were given intravenously each hour.
Four rabbits were used for the eight-hour
experiments and one rabbit was maintained for 24 hours.
the group of four rabbits, rabbits #5 and #6 were nonfasted, rabbit #7 was fasted for one day and rabbit #8 was fasted four days.
For comparison purposes, the rabbits
that were used for the previously described glucose utili zation studies served as the control group for the insulinized rabbits. The preparation of the labeled glucose and the methods for the tissue analyses have also been described. Results of the studies on the action of insulin on the extra-hepatic tissues*
Data from the insulinized,
eviscerated rabbit that was maintained for 24 hours with 14 radioactive C glucose are shown in Table VI. Just the results for the first 18 hours are given since there were no further changes during the rest of the 24-hour period. During this time the blood sugar was kept within a normal
TABLE VI THE BLOOD SUGAR LEVEL, RATE OF GLUCOSE INJECTION, AND THE SPECIFIC ACTIVITY OF THE EXPIRED C02 FROM AN EVISCERATED, INSULINIZED RABBIT DURING 18 HOURS OF CONSTANTLY INJECTED C -LABELED GLUCOSE Hours
Blood sugar level at start of period
Glucose inj. rate mg./kg./hr.
Specific activity of C02
S. A. inj. glucose* S . A * COg
*S. A . of injected glucose * 330 counts/min./mg. carbon.
range by the constant glucose Infusion, which had to be increased somewhat as the experiment progressed.
It can be
seen that, unlike the rabbits that received no insulin, the specific activity of the expired carbon dioxide was more than 50 per cent of the specific activity of the injected 1glucose by the end of the first eight hours, and during the last half of the experiment the expired carbon dioxide showed a specific activity that was almost two thirds of .that of the injected glucose. In Table VII, the specific activity of the expired carbon dioxide from the insulinized rabbits is compared to the specific activity of the carbon dioxide from the con trol group.
Again, it can be seen that by the end of the
eighth hour of the experiments, the specific activity of the expired carbon dioxide from
the insulinized rabbits was
:between 33 and 88 per cent of the specific activity of the injected glucose, while the control rabbits showed carbon dioxide specific activities that were only 10-15 per cent. Most of the carbon dioxide from
the control rabbits must
have come from non-glucose sources.
On the other hand, the
administration of insulin Increased the oxidation of the injected glucose from two- to sixfold above the controls. The nutritional condition of the animals appears to affect the glucose oxidation rate.
This can be seen
TABLE VII THE HOURLY SPECIFIC ACTIVITIES OF THE EXPIRED C02 FROM EVISCERATED RABBITS DURING AN 8-HOUR PERIOD OF CONSTANTLY INJECTED C■'■^-LABELED GLUCOSE WITH AND WITHOUT INSULIN
Hours 1 2 3 4 5 6 7 8 S. A. of inj. glucose
Specific activity of expired COg Control rabbits Insulinized rabbits Not fasted Pasted 3-^ days Not fasted Fasted #4 #2 #6 #8 #1 #3 #5 #7 150 243 323 344 353 411 436 425
148 197 266 290 314 346 375 380
89 137 172 206 239 275 321 390
40 123 154 174 182 209 224 237
377 680 960 1080 1290 1330 1370
282 654 1100 1500 1760 1920 2090 2970
88 221 378 500 640 780 920 1020
122 213 348 440 607 773 840 983
particularly in the data for the non-fasted rabbit #6 and the four-day fasted rabbit #8.
The well fed rabbit through
out the experiment oxidized almost three times as much of the infused glucose as the starved animal as judged by the radioactivity of the expired carbon dioxide. However, even I the fasted insulinized rabbits oxidized the injected glu cose at more than twice the rate of the well fed control rabbit. Table VIII shows a comparison of the C ^
the carbon dioxide that was expired by the insulinized and non-insulinized rabbits.
Of particular interest is the
comparatively low count in the carbon dioxide of the insu linized rabbits during the first hour of the experiments and the steady increase during the following hours.
carbon dioxide from the insulin-free rabbits appears to 14 approach its maximum hourly C content by the third hour with a slow increase thereafter.
Again, the difference
between the fasted and non-fasted animals is apparent. Greeley (1940) has shown that the effect of a single dose of insulin on sugar disappearance is maximal immediately after the injection.
However, the disappearing glucose
does not go immediately to carbon dioxide, but instead it apparently undergoes a preliminary transfer to a metabolic pool from where the complete metabolism of the sugar then
TABLE VIII THE C1^ CONTENT OP THE EXPIRED C02 FROM EVISCERATED RABBITS DURING AN 8-HOUR PERIOD OP CONSTANTLY INJECTED C^-LABELED GLUCOSE WITH AND WITHOUT INSULIN ADMINISTRATION
Per cent of total Injected C Control rabbits Not fasted Pasted 3-4 days #1 #2 #3 #4
1 2 3 H 5 6 7 8 Total %, C ^ O 2
in expired C02 Insulinized rabbits Not fasted Fasted #5 #6 #7 #8
0.7 1.7 3.2 4.0 4.6 5.0 6.0 9.9
0.3 0.9 1.6 1.7 2.7 3.3 4.0 6.6
0.1 0.6 1.6 1.9 1.8 2.7 4.4 3.6
1.0 2.1 3.4 3.1 2.6 3.5 3.5 3.4
1.0 1.0 1.1 1.3 1.5 1.8 2.1 2.9
1.2 1.3 1.7 1.8 2.1 2.0 2.3 5-3
1.0 2.1 2.0 2.6 2.6 2.7 3.0 3.0
1.6 2.7 4.5 4.9 5-4 5.8 6.4
73 ) proceeds to carbon dioxide.
Since the insulinized rabbits
were given hourly doses of insulin, the maximum effect was maintained throughout the experiments, so that after the initial lag during the first hour, the
began to appear
;in the expired carbon dioxide in increasing amounts as more and more of the injected glucose went into oxidation routes through the metabolic pool. The hourly rates at which the injected glucose was t
oxidized under the influence of insulin and the average
1rates at which the injected glucose disappeared are shown in Table IX.
The hourly oxidation rates were determined
from the specific activity of the injected glucose and the total carbon dioxide expired.
The marked and steady
increase in the amount of glucose that was oxidized by the ■insulinized animals can be readily seen when compared with the animals that did not receive insulin.
average oxidation rate with insulin was 4.5 times as great as without insulin.
However, if the average rates at the
end of the seventh hour are compared, the insulinized group oxidized six times as much glucose as the control rabbits. The average rate at which the glucose had to be injected in order to maintain the blood sugar within a normal range, i.e., the "utilization" rate, when compared with the amount of glucose that underwent complete
TABLE IX COMPARISON OF GLUCOSE DISAPPEARANCE AND OXIDATION RATES IN EVISCERATED RABBITS DURING AN 8-HOUR PERIOD OF CONSTANTLY INJECTED Cl4-LABELED GLUCOSE WITH AND WITHOUT INSULIN
Hours 1 2 3 4 5 6 7 8 Average/hr. Glucose inj. mg./kg/hr.
Mg. glucose oxidized to CC^Ag-Aour Control rabbits Insulinized rabbits Not fasted Pasted 3-^ days Not fasted Pasted #1 #2 #3 #4 #5 #6 #7 #8 13 29 *7 43 37 48 49 47 34
10 10 11 13 15 17 20 29 19
15 17 21 22 27 25 29 68 28
22 29 29 31 33 33 36 27
38 95 177 220 255 275 330 546 242
15 38 67 71 113 138 167 276 112
3 15 38 45 42 64 102 85 49
53 92 153 166 184 198 217 • ♦
oxidation, indicates that in both the insulinized and noninsulinized animals a large part of the injected glucose disappeared into disposal routes that did not lead to oxidation during the eight-hour period. The effect of insulin on the distribution of the in the body of the eviscerated rabbit is shown in Table X. The water-soluble fraction of the insulinized animals accounts for a lower percentage of radioactivity than in the control group, but it still represents a significant part of the injected counts.
Insulin appears to have
greatly increased the mobilization of glucose through this water-soluble pool into both oxidative and storage routes. The average glucose equivalent of the
tion in both the insulinized and control groups is shown in Table XI.
The influence of insulin on the utilization of
glucose is apparent in every fraction of the analyses.
glucose in the water-soluble fraction in both groups repre sents the glucose in the body fluids.
Not only did the
oxidation of glucose increase fourfold with insulin, but the conversion of glucose to glycogen and protein increased nearly six times, and the conversion to fat ten times above the non-insulin level. Discussion of the results on the action of insulin in the eviscerated rabbit.
By tracing the. injected
TABLE X THE EFFECT OF INSULIN ON THE DISTRIBUTION OF C1* IN EVISCERATED RABBITS AFTER A PERIOD OF CONSTANTLY INJECTED C1^-LABELED GLUCOSE
Fraction Expired COg Body COg Water soluble As glucose Glycogen Crude protein True protein Crude fat Fatty acids Urine Kidneys-heartlungs
Per cent of injected counts Control rabbits Insulinized rabbits Not fasted Pasted 3-4 days Not fasted Pasted #1 #2 #3 #4 #5* #6 #7 #8
24.0 5.3 *.7 6.1 5.1 1.7 0.8 4.3
12.8 3-4 32.7 10.0 3.2 6.1 5.6 0.9 0.2 4.2
17.7 4.4 50.3 9.0 2.6 7.5 1.9 2.0 0.4 None
19.0 3.8 46.0 6.4 2.5 4.8 3.6 1.0 0.1 4.0
22.5 • •
21.8 1.6 10.0 5.8 1.4 7.7 2.5 4.0
21.1 4.5 22.4 5-0 10.6 16.7 4.5 4.2 0.3 2.1
21.7 2.8 11.5 2.5 2.5 2.8 0.2 0.4
31.3 *.3 • • ♦ .♦ • • • • ♦ • • • • •
35.2 • •
16.8 • •
*The method used for the carcass analysis of Rabbit #5 is not comparable with the method used for the other rabbits.
TABLE XI A COMPARISON OF THE AMOUNT OF GLUCOSE THAT IS EQUIVALENT TO THE C14 DISTRIBUTION IN EVISCERATED RABBITS AFTER A PERIOD OF CONSTANTLY INJECTED Cl4-LABELED GLUCOSE WITH AND WITHOUT INSULIN
Fraction Expired COg Body COg Water soluble As glucose Glycogen Crude protein True protein Crude fat Fatty acids Urine Kidneys-heartlungs
Average glucose equivalent of the C (mg.) Control rabbits Insulinized rabbits 5*3 93 980 205 127 172 112 41 13 us
2144 360 1857 265 704 980 690 446 127 209
-labeled glucose through its metabolic course in the
eviscerated rabbit, the direct experimental evidence shows that insulin increases the oxidation of glucose by the extra-hepatic tissues as well as promotes glucose utiliza tion along storage routes.
The previous indirect evidence
which suggested that insulin accelerates the oxidation of glucose has been generally accepted, but some authorities in the field have maintained that it was unsatisfactory for positive proof.
In this regard, Soskin (1941) has dis
cussed the inadequacy of the earlier work. The results show that under the influence of insulin the blood glucose immediately begins to disappear at an accelerated rate, but there is a lag during the first hour of Insulin administration before evidence for the oxidation of the glucose appears as expired carbon dioxide in signi ficant quantities.
Apparently, one of the main actions of
insulin on the extra-hepatic tissues is to facilitate the transfer of glucose into routes that lead to oxidation and carbon dioxide.
One of the first steps is possibly an
almost immediate breakdown of the glucose into watersoluble derivatives that soon accumulate into a sizeable metabolic pool.
In the absence of the liver, a part of
these glucose fragments may remain in the water-soluble fraction as “dead end" products, which in the intact animal
would normally be reconverted to glucose or other body constituents.
The action of the insulin then continues to
direct the partially metabolized glucose into different I ichains of reactions that result in an increased oxidation ,rate and also an increased conversion to glycogen and protein.
A slower process may be involved in the conver\
sion of glucose to fat, which would account for the rela-
tively small amount of radioactivity in the fat fractions.
Drury (1940) studied the effect of insulin on the storage of carbohydrate in rats with feeding and fasting periods that lasted for several days.
It was found that large
quantities of carbohydrate were stored with the aid of insulin, but that only a small part could be accounted for as glycogen.
It was concluded that most of the stored
carbohydrate must have been in the form of fat.
study by Pauls and Drury (1942) again showed that only about 25 per cent of the stored glucose was in the form of glyco gen.
It was felt that the main bulk of the sugar was
stored as fat, but that the transient storage as carbohy drate metabolites should also be considered.
Klein (1946) have shown by the use of deuterium that insulin will greatly increase the rate of hepatic lipogenesis in the normal rabbit. after a 48-hour feeding period.
This effect was observed In the present study, the
large amount of water-soluble material that is derived from the injected glucose indicates that this pool of interme diate breakdown products may serve as a temporary storage ;depot for carbohydrate, a part of which is continually in process toward further metabolism. Further work by Wick, Drury and MacKay (1950) on the water-soluble fraction shows that under the influence of i
insulin a large part of the material is fermentable, but is non-reducible.
This is in contrast to the water-soluble
substances that accumulate in the non-insulinized eviscer ated rabbit.
CHAPTER V THE CARE AND MAINTENANCE OF DEPANCREATIZED DOGS Before describing the experimental work concerning the insulin requirements of the diabetic dog under anoxic conditions and the study of the glucose equivalent of fed protein, a brief discussion of the postoperative care of the two depancreatized dogs that were used in these inves tigations is first presented.
The eare and maintenance of
this type of animal preparation, both during the experi mental work itself and for the day-to-day survival of the animal, requires an understanding of their diabetic condi tion and the procedures that are necessary for keeping them in good health and for evaluating the degree of diabetes. The postoperative care of diabetic dogs.
dogs, weight approximately 9 kg« and 7 kg, respectively, were depancreatized according to the method described by Markowitz (19^9).
The dogs were maintained In good condi
tion for many months after the operation on a measured diet that consisted of rabbit meat, sucrose and raw beef pancreas with daily insulin injections. Without the addition of raw pancreas to the diet, the depancreatized dog gradually becomes jaundiced, refuses
:to eat, and eventually dies In a coma due to hepatic fail ure, The same beneficial effect of the raw pancreas, as i ishown by Hershey and Soskin (1931), can be obtained by the addition of lecithin in the diet.
Best, et al, (193*0
investigated the dietary properties of lecithin further and established the fact that choline is the responsible prin ciple in both the lecithin and raw pancreas.
acid, methionine, is also effective in preventing fatty livers in rats.
The pancreatic juices in the raw pancreas
are necessary for the release of choline during digestion ;which insures the proper functioning of the liver. The addition of carbohydrate In the diet, in the form of sucrose, is a good dietary constituent for main taining the body weight of the depancreatized animal, providing an adequate amount of Insulin Is administered to facilitate its storage as glycogen and particularly as fat. Just enough regular Insulin to prevent glycosuria was injected subcutaneously at the time of each feeding.
#2, before pancreatectomy, had a body weight of 15 pounds. During the month following the operation the dog*s weight increased to 20 pounds, a weight gain of 25 per cent, despite the fact that the animal was totally depancreatized. The amount of insulin that was necessary to keep the urine relatively free of sugar was determined by studies
that were based on the methods described by Drury (19^0) i 1 \ ‘ and Greeley, Tyler and Drury (1939)• This method consisted! '
!essentially of feeding the dogs the regular diet of rabbit meat, 200 gm.; sucrose, 50 gm.; and raw pancreas, 25 gm« ■About three hours before the feeding, one unit of regular insulin was injected intravenously in order to bring the blood sugar levels within a normal range.
At the time of
feeding, 2 units of regular insulin were injected intra venously and the blood sugar levels were determined at hourly intervals thereafter, at which times doses of insu lin were injected that were calculated to keep the blood sugar at a normal level.
The course of the blood sugar
levels of the two dogs following the feeding and subsequent injections of insulin is shown by the data in Table XII. It can be seen that during a five-hour period following the ingestion of the food, the dogs required 5 and 5*5 units of Insulin to keep the blood sugar from rising too high.
largest doses of insulin were needed immediately after eating.
Within three or four hours following the meal, the
insulin requirements had decreased to 1/2 unit per hour. The total quantity of insulin administered in this manner at hourly intervals amounted to 5 and 5.5 units, following the ingestion of 50 gm. of sucrose in the diet, or one unit of Insulin per 10 gm. of sucrose.
Greeley, Tyler and Drury
TABLE XII THE INSULIN REQUIREMENTS OP TWO DEPANCREATIZED DOGS FOLLOWING THE INGESTION OF 50 GM. SUCROSE AND 225 GM. OF MEAT
Time 8:30 10:00 11:00 12:15* 1:15 2:00 3:00 4:20 5:15
DOG #1 Blood sugar Insulin mg, units 200 122 115 153 160 107 120 120 128
♦Food fed at 12:15.
1.0 • * • •
2.0 1.0 1.0 0.5 0.5 • *
DOG #2 Blood sugar Insulin mg. % units ♦ •
122 112 128 220 150 125 100 128
1.0 • • ♦ •
2.0 1.0 1.0 1.0 0.5 • +
(1939) have shown that about two times more sucrose than :glucose can be tolerated by the depancreatized dog with the same amount of insulin. During the routine care of diabetic dogs, however, it is not necessary to administer hourly doses of insulin, but rather to inject an adequate amount of regular insulin at the time of feeding.
This amount is somewhat larger
than the sum of the hourly Injections since a single dose of insulin, while it is acting on the ingested carbohydrate, is continually being destroyed by the body.
has investigated the duration of insulin action in the depancreatized dog and has found that the time of activity of an intravenous dose of insulin depends on the size of the dose.
However, the duration of action is not directly
proportional to the size of the dose, but rather it is directly proportional to the amount of insulin in the body at any given instant, or it is proportional to the loga rithm of the original dose.
Greeley found experimentally
that the hourly rate of insulin decay is about 0.4 of the amount of insulin present in the body at any given instant. When insulin has decreased below a basal minimum concentration in the body, the apparent action of the insu lin dose ends and the blood sugar begins to rise.
fasting and postabsorptive periods, a basal amount of
86 Insulin Is required to prevent the excessive formation of * ,new glucose, particularly from body protein. This basal
,insulin requirement has been studied by Greeley (1937) the depancreatized dog, and a method for determining this requirement Is described by him.
The basal insulin need of
one dog is not necessarily the same for all depancreatized dogs but varies with the individual differences of each animal• Consequently, in addition to the regular Insulin that was given to the depancreatized dogs at the time of feeding, a certain amount of protamine zinc insulin was also administered to meet the basal needs during the postabsorptive periods. The basal insulin requirements of the two dogs were determined according to the method described by Greeley (1937)•
The test consists essentially of determining the
amount of insulin that Is needed each hour to keep the blood sugar level within a normal range when the depancrea tized dogs are In a fasting condition.
The dogs received
their last food, with regular Insulin, 24 hours before the start of the test.
A small amount of regular insulin
(about 2 units) was administered subcutaneously the evening before the test and 1-2 units were injected intravenously on the morning of the test in order to bring the blood
I I I
sugar to a normal level.
Blood sugar determinations were
made every 30 minutes by the Folin-Malmros micro method following the morning injection of insulin*
When the blood
1sugar levels were in a normal range and were beginning to 1increase, a dilute solution of insulin in distilled water
(1 unit per ml.) was injected intravenously each hour
during the tests in amounts that were estimated to maintain ia constant blood sugar level*
An hourly insulin rate that
is too high will cause the blood sugar level to decrease, 1while an insufficient rate will cause a rise in the blood sugar level*
When just enough insulin is injected to meet
the needs of the fasting animal, the blood sugar will stay at a steady level*
Figure 1 illustrates the course of the
blood sugar during three basal insulin tests on the same dog.
Curve A shows the blood sugar trend when insulin was
injected at more than an adequate rate.
at an hourly rate of 0.15 unit and 0.12 unit caused a decline in the blood sugar.
Curve B indicates that 0.08
unit per hour is not quite enough to maintain the blood sugar at a constant level.
A more optimum rate is shown by
Curve C when insulin was injected at an hourly rate of 0.10 unit.
The basal insulin requirement of this dog (#1)
appears to be between 0.1 unit per hour and 0.12 unit per hour.
0,15 UNIT 0.1.2 UNIT
/ 0 . 0 8 UNIT INSULIN
100 75 0.10 UNIT INSULIN PER
FIGURE 1 BLOOD SUGAR LEVELS OF A DIABETIC DOG RESULTING FROM INSULIN INJECTIONS AT THREE DIFFERENT RATES
89 The basal insulin need for dog #2 is about 0.15 unit per hour, as illustrated in Figure 2.
175 150 0.15
100 75 50 25 “
FIGURE 2 THE BLOOD SUGAR LEVEL OF A DIABETIC DOG WITH A BASAL INSULIN INJECTION RATE OF 0.15 UNIT PER HOUR
CHAPTER VI THE EFFECT OF MILD ANOXIA ON THE BASAL INSULIN i
REQUIREMENTS OF DEPANCREATIZED DOGS
At the present time there are very little experimen tal data concerning the effect of anoxia on the diabetic organism*
The work of Evans (1936) and McQuarrie, et al.
(1942), as well as other investigations, on normal and :adrenalectomized animals suggest that the stress of anoxia may influence the secretion of insulin by the pancreas. In order to determine whether or not exposures to mild anoxic conditions, as might be encountered in presentday aircraft, might alter the basal insulin requirements of diabetic animals, two depancreatized dogs were subjected to a simulated altitude of 15,000 feet in a decompression chamber for periods of at least four hours.
insulin needs of the dogs during this time were compared with their ground level requirements. The experimental methods used for determining the basal insulin requirements of depancreatized dogs under anoxic conditions.
The procedure that was followed for
measuring the basal insulin needs of the diabetic dogs when exposed to a simulated altitude was essentially the same as has already been described for the basal insulin
92 determinations under normal environmental conditions, which 1 is based on the method of Greeley (1937).
The dogs re
ceived their last food with regular insulin 24 hours before the start of the test and were given 2 units of regular insulin the evening before the test*
On the morning of the
test, 1 to 2 units of regular insulin were injected intra venously in order to bring the blood sugar levels within a normal range*
Blood sugar determinations in this series of
experiments were determined every hour by the Folin-Wu micro method as described by Bray (1944).
When the blood
sugars were at a normal level and were beginning to increase, the previously determined basal dose of diluted insulin was injected intravenously into each dog and the animals were placed in a decompression chamber*
sure was reduced and the animals remained at a simulated altitude of 13*000 feet for four hours*
At hourly inter
vals during this period, the dogs were given their basal dose of insulin Intravenously and blood samples were collected for sugar determinations.
One half ml. of blood
was collected in 2 ml. syringes by venepuncture with sodium oxalate as an anticoagulant.
The blood was then imme
diately lysed and the protein precipitated according to the Folin-Wu method.
The actual sugar determinations were
performed on the filtrate following the collecting period.
The decompression chamber was arranged in such a
manner that the basal insulin injections could be adminis1tered and the blood samples obtained while the dogs remained at the simulated altitude.
This was done by
placing an animal decompression chamber, which was large enough for two dogs, inside a large human decompression chamber.
Suitable hose connections from the small chamber
led through the large chamber to a separate pump and the manometers on the outside.
In this way, the large chamber
was used as a lock system for the smaller chamber.
was necessary to administer the insulin and collect blood samples, the large chamber, with the experimenter, was decompressed to the same altitude as the animal chamber. The dogs could then be removed from their chamber and the necessary work performed on them.
They were then replaced
in the small chamber and the large chamber recompressed to ground level* A sufficient amount of the diluted insulin was pre pared so that the basal insulin test could also be per formed at ground level with the same insulin solution that was used for the test at altitude.
These ground level
tests were performed two days either before or after the altitude tests so that the dogs could be fed and could recuperate for a day between the tests.
In order to
94 prevent the dogs from becoming acclimatized, they were exposed to altitude not more than once each week. The results of the basal insulin determinations on depancreatized dogs at an altitude of 15>000 feet.
parison of the basal insulin needs of the two dogs (#1 and #2) while they were both at a simulated altitude of 15,000 !
feet for four hours is shown in Figure 3*
experiment the dogs were both given 0.1 unit of insulin per hour.
It can be seen that the blood sugar level for dog #1
remained at a constant level with this amount of insulin, while with the same insulin dosage, the blood sugar level of dog #2 increased above 200 mg. per cent.
insulin requirement for dog #1 under ground level condi tions had previously been determined to be about 0.1 unit per hour (Figure 1), and the exposure to an atmosphere of 425 mm. Hg pressure did not alter this requirement.
ground level insulin needs of dog #2 had been found to be about 0.15 unit per hour.
With an insufficient insulin
supply of 0.1 unit per hour under anoxic conditions, the blood sugar level increased steadily to hyperglycemic levels as it would have under normal conditions. Figure 4 graphically illustrates the hourly blood sugar levels of dog #1 during a four-hour exposure to 15>000 feet, with a comparison o f .the_blood sugar levels
AT 1 5 ,0 0 0
75 50 25
FIGURE 3 THE BLOOD SUGAR LEVELS OF TWO DIABETIC DOGS DURING A BASAL INSULIN TEST AT 15,000 FEET WITH INSULIN INJECTIONS OF 0.10 UNIT PER HOUR
AT GROUND 0.10 U. 0.10 U.
LEVEL 0.10 U.
175 150 125
0.12 U. AT
1 5 ,0 0 0
FIGURE 4 THE COMPARISON OF THE BASAL INSULIN REQUIREMENTS OF A DIABETIC DOG (DOG 1) AT GROUND LEVEL AND AT 15,000 FEET
for a similar period of time at ground level.
dilute insulin solution was used for both tests.
It can be
noted that, during the ground level test, 0.10 unit of insulin per hour was not quite sufficient to prevent the blood sugar from increasing and that in two instances 0.12 unit was administered in an attempt to bring the blood sugar back to normal.
This suggests that in this ease an
hourly rate of 0.12 unit would have maintained the blood sugar at a constant level.
When the dog was at 15*000
feet, a steady hourly rate of 0.12 unit did maintain the blood sugar at a constant level.
It appears that during
these two tests the basal insulin needs were the same both at ground level and at an altitude of 15*000 feet. On the other hand, during the same altitude and control experiments and using the same insulin solution, different results were obtained for dog #2. shown in Figure 5,
This dog was given insulin at an hourly
rate of 0.15 unit during both the ground level and altitude tests.
In contrast to dog #1 (Figure 4), the blood sugar
tended to rise to an abnormal level with the exposure to 15,000 feet, but was maintained at a constant level during the control period.
The insulin requirement of dog #2
appears to have been slightly increased by the anoxic exposure.
ALTITUDE 0.15 U.
200 175 150 125
AT 1 5 ,0 0 0
0.15 U. AT
75 50 25
FIGURE 5 THE COMPARISON OF THE BASAL INSULIN REQUIREMENTS OF A DIABETIC DOG (DOG 2) AT GROUND LEVEL AND AT 15,000 FEET
These experiments were repeated Tor the two dogs,
the results of which are shown in Figure 6 for dog #1 and ;in Figure 7 for dog #2.
During these tests, insulin was
'administered to dog #1 at a rate of 0.12 unit per hour and i to dog #2 at an hourly rate of 0.15 unit, both at altitude
*and at ground level.
The results indicate that, for both
dogs, the basal insulin needs were increased during the period at 15,000 feet. The blood sugar level for dog #1 i (Figure 6) was maintained fairly constant with 0.12 unit of ;insulin per hour at ground level, but the blood sugar ,tended to increase slightly as soon as the dog was decom pressed.
Dog #2 (Figure 7) showed a definite and steady
increase in the blood sugar level with exposure to alti tude.
Again, 0.15 unit of insulin per hour was an adequate
rate during the control period at ground level. Discussion of the results that were obtained by exposing depancreatized dogs to an altitude of 15*000 feet. The results of these experiments tend to indicate that the insulin requirements of the diabetic dog may be increased during exposures to moderately low altitudes.
before definite conclusions can be drawn, several other factors should first be considered. Dog #1 showed only a slight tendency toward an .increased insulin need at altitude, and_during^two
250 3 .0
1 5 ,0 0 0
200 0.12 U.
A L TIT U D E 0.12 U.
175 150 UNITS
FIGURE 6 THE COMPARISON OF THE BASAL INSULIN REQUIREMENTS REPEATED WITH DOG 1 AT GROUND LEVEL AND AT 15,000 FEET
2 2 5 - 3 .0 UNITS INSULIN
200 AT 1 5 ,0 0 0
A L T I T U D E --------
125 0 .1 5 U.
G RO UND
0 .1 5 U.
0 .1 5 U.
FIGURE 7 THE COMPARISON OF THE BASAL INSULIN REQUIREMENTS REPEATED WITH DOG 2 AT GROUND LEVEL AND AT 15,000 FEET
102 experiments (Figures 3 and 4) the insulin requirements appeared to be the same both at ground level and at 15,000 feet.
Dog #2 was more consistent in showing an increased
need for insulin under anoxic conditions (Figures 5 and 7). This dog*s basal insulin requirement in a normal environ-
ment was considerably higher than the needs of dog #1, and it may be that dog #2 showed a greater inability to adapt
quickly to the stress of the reduced atmospheric pressure. The dogs were well trained and usually behaved quietly when they were handled during the basal insulin tests at ground level.
However, during the tests at alti
tude^ they appeared to be in a continual state of excite ment.
The strangeness and confinement of the small
altitude chamber and the constant noise of the decompression may have been a contributing factor in the increased blood sugars, as well as the anoxia.
When the dogs were released
into the large chamber for their hourly insulin injections and blood samples, they immediately calmed down and could be handled as easily as at ground level. Stickney, Northup and Van Liere (1948) have shown that the normal intact dog does not show a significant blood sugar response below 24,000 feet.
However, they also
indicate that the pancreatic islet cells are involved in the animalfs readjustment to the abnormal environment as
103 well as the adrenals.
The depancreatized dog could be
expected to be more sensitive to environmental changes than i
,the normal animal.
The work of McQuarrie, Ziegler and Hay
(1942) on adrenalectomized-depancreatized dogs also indi cates that the pancreas is activated under the stress of anoxia, but in the normal dog the sympathico-adrenal system tends to overshadow the insulin effect.
This also suggests
that the diabetic dog without an insulin reserve would tend :to be affected by a less severe stress than the intact animal• The experiments of Evans (1936) with normal and adrenalectomized rats that were decompressed to 18,000 feet for 24 hours have shown that the nitrogen excretion and liver glycogen of the normal animals were greatly increased by the anoxic environment.
Evans considered the normal rat
under low oxygen tensions in the same category as the depancreatized or phlorizinized rat.
The increased carbo
hydrate originated chiefly from the body protein, except that it was not excreted but was stored as glycogen.
adrenalectomized or iiypophysectomized rat failed to show this effect.
This work suggests that an already existing
diabetic condition might be intensified by anoxia. In view of these previous experiments by other investigators, the results of the present work were not as
104 striking as might have been expected.
However, the tend
ency for an increased insulin need even at a comparatively low altitude is evident.
Lewis, et al. (1942), have
pointed out that, during the first phase of anoxic expo sures, the glucose utilization rate appears to increase and the blood sugar is maintained at a normal level by the •glycogen stores and gluconeogenesis.
This may well be a
factor that should be considered in analyzing the results of the present experiments. Future experiments along this line are suggested by the work of Evans (1936) on the nitrogen excretion and glycogen formation from endogenous protein.
indicate that the D:N ratio of the anoxic diabetic animal !might be a useful tool for determining the effects of the abnormal environment.
However, the estimations by Evans of
the amount of glycogen that was derived from protein did not include all of the carbohydrate that was utilized by the animals during the anoxic exposures.
excreted nitrogen or the urinary D:N ratio can be a signi ficant indication of the amount of carbohydrate that is iderived from protein, the glucose utilization rate of the animal must also be considered. In view of these possibilities, the amount of carbo- ; hydrate that can be formed from protein was re-estimated
105 by using the phlorizinized-depancreatized dog under normal >
CHAPTER VII THE GLUCOSE EQUIVALENT OF PROTEIN In order to study further the conversion of protein to glucose in the diabetic animal, corrected D:N ratios were calculated for two phlorizinized-depancreatized dogs by taking into consideration their glucose utilization rates as well as their exeretion of glucose and nitrogen. The experimental procedure.
The method for this
study was not only to measure the amount of glucose and nitrogen that was excreted when the phlorizinized-depan creatized dogs were fed large quantities of protein, but also to obtain an indication of the amount of glucose that was utilized during the protein feeding periods.
done with control periods when the dogs were fed only sucrose or glucose.
The difference between the quantity of
fed sugar and the excreted sugar indicated the amount of sugar that was utilized by the tissues.
The amount of sugar
that was utilized when the glycosuria was of the same degree as during the protein feedings was used for cor recting the observed urinary D:N ratios. Drury (1941) has shown that for studies of this type It Is necessary to use depancreatized animals.
utilization rate of the intact phlorizinized dog definitely
107 increases with protein feeding so that the urinary D:N ratio is lower than during fasting conditions.
creatized dog does not show this marked increased utiliza tion rate with feeding and it is possible to estimate the sugar that is actually derived from fed protein by cor recting for the small amount that is used by the tissues. In order to prevent a severe ketosis in the dogs, with vomiting and anorexia, a small dose of protamine zinc insulin was administered daily.
The urine sugar was
determined by the Shaffer-Hartman method as modified by Shaffer and Somogyi (1933)*
The macro-Kjeldahl method was
used for the urine nitrogen measurements, as described by Hawk, et al. (1947). The dogs were kept in individual metabolism cages and were fed the protein meal or the sugar solution each morning during the course of the experiments.
At the time
of feeding the dogs were also given one gram of phlorizin, suspended in olive oil, subcutaneously.
Also at this time
the protamine zinc insulin was administered subcutaneously. During the first protein feeding period, 2 units of insulin were injected daily, but this amount was not sufficient to i
prevent a progressive ketonuria.
Consequently, 4 units
were injected daily during all of the other periods. The urine was collected every 24 hours and a few ml. ,
108 of 50 pe**
cent sulfuric acid were used as
The high protein diet that sisted of essentially pure protein.
a preservative. to the dogscon
that contained at least 28 per cent protein and
1 per cent carbohydrate and fat
made up thediet
during the first experimental period of six days.
■were then allowed to recuperate from the phlorizin injec-
tions for two weeks because the injection sites began to break open and the health of the dogs appeared to be failing.
The amount of protein was increased during the
second protein feeding period by supplementing 50 to 100 grams of casein to the dietary tuna fish.
Increased the excretion of both sugar and nitrogen. During the control periods the dogs were given only a sugar solution for their daily diet, which consisted of 100 grams and 200 grams of either sucrose or glucose.
,weighed amounts of sugar were dissolved in water and diluted, to 400 ml.
If the sugar solutions were diluted to larger
volumes the dogs failed to drink the entire amount. Usually during these sugar feeding periods the dogs con-
sumed the sugar solutions fairly eagerly, especially for •the first few days. The results of feeding a protein diet to phlorlzin'lzed-depancreatized dogs.
The amounts of sugar a n d ____
109 nitrogen that were excreted by the dogs during the periods when they were fed a diet that consisted essentially of ,pure protein are shown in Tables XIII, XIV and XV.
determinations for the first day of each period have been omitted from all the tables because of the well known readjustments to the new diet that take place at this time. j t It can be seen in Tables XIII and XIV that, when the ! !two dogs were fed about 110 grams of protein each day, they ;excreted an average of 31*3 and 37*5 grams of sugar per day, and the urinary D:N ratios averaged 3*74:1 and 3.95:1 respectively.
When the amount of fed protein was increased,
as shown in Table XV, sugar and nitrogen were excreted in considerably greater quantities.
Dog #1 excreted an aver
age of 70 grams of sugar per day and dog #2 showed a con sistent daily excretion of 63 grams.
During this period
;the urinary D:N ratios decreased somewhat, as compared with the previous period, to an average of 3 *68:1 and 3 *52:1 respectively for the two dogs.
However, it can be noted
that all of these average D:N ratios are within the same range that has been reported for the fasting phlorizinized dog (Lusk, 1928), and they are well above the D:N ratios ■that have been observed in the fed phlorizinized dog (Drury, 1941).
TABLE XIII THE DAILY AMOUNT OP SUGAR AND NITROGEN EXCRETED BY A PHLORIZINIZED-DEPANCREATIZED DOG (#1) WHILE ON A TUNA PISH PROTEIN DIET FOR SIX DAYS Daily food intake gm. protein
Insulin units per day
Ketones excreted gm.
Sugar excreted gm.
65 115 110 110 110
2 2 2 2 2
0.12 0.15 0.87 1.66 1.12
22.1 40.5 28.9 31.4 36.1
6.8 9.7 8.5 8.6 9.0
3.25 4.18 3.41 3.65 4.02
Average (last 5 days)
Nitrogen excreted gm.
THE DAILY AMOUNT OF SUGAR AND NITROGEN EXCRETED BY A PHLQRIZINIZED-DEPANCREATIZED DOG (#2) WHILE ON A TUNA FISH PROTEIN DIET FOR SIX DAYS Daily food intake gnu protein
Insulin units per day
Ketones excreted gm.
Sugar excreted gm.
65 115 110 110 110
2 2 2 2 2
0.13 0.50 0.86 0.91 0.83
20.5 47.3 38.4 36.6 44.6
5.1 9.8 10.3 10.3 12.0
4.03 4.83 3.74 3.55 3.72
Average (last 5 days)
Nitrogen excreted gm.
THE DAILY AMOUNT OF SUGAR AND NITROGEN EXCRETED BY TWO PHLORIZINIZEDDEPANCREATIZED DOGS WHILE ON A TUNA PROTEIN AND CASEIN DIET FOR FIVE DAYS DOG #2
DOG #1 Daily food intake gm. protein
Insulin units per day
Sugar excreted gm*
Nitrogen excreted gm.
210 210 190 165
4 4 4 4
71.2 83.8 76.2 50.8
15-5 25.1 21*3 14.2
Average (last 4 days)
Sugar excreted gm.
Nitrogen excreted gm.
4.62 3.35 3.57 3.58
63.1 63.6 63.1 62.3
16.9 19.6 18.7 16.4
3-74 3.25 3.38 3.80
113 The results of feeding only sugar to phlorlzlnlzeddepancreatlzed dogs. In order to determine the amount of *sugar that the dogs were utilizing during the protein feeding periods, the dogs were then given 100 and 200 grams of sucrose or glucose for their daily feedings.
of sugar that was excreted by dog #1 under these condi tions, as well as the difference between the fed and ;recovered sugar, and the nitrogen excretion are shown in Table XVI.
The results for dog #2 are given In Table XVII.
The glycosuria that resulted during the periods when 100 grams of either sucrose or glucose were fed to the dogs appears to approximate most closely the glycosuria that occurred when large quantities of protein were fed.
100-gram sucrose feedings caused an average daily sugar ;excretion of 76.9 grams and ?6.1 grams for dogs #1 and #2 respectively.
The 100-gram glucose feedings resulted in an
average excretion of 81.1 grams for dog #1 and 68.6 grams for dog #2.
With the high protein feedings, the two dogs
excreted daily an average of 70.0 grams and 63*0 grams of .sugar respectively (see Table XV). !
The 200-gram sugar feedings resulted in a glycosuria i
that was greatly in excess of the amount of sugar excreted during the protein feedings. The amount of sugar that was not recovered in the
THE GLUCOSE AND NITROGEN EXCRETION DURING 24-HOUR PERIODS BY A PHLORIZINIZED-DEPANCREATIZED DOG (#1) AFTER THE INGESTION OF 100 AND 200 GRAMS OF SUCROSE AND GLUCOSE Sugar ingested gm./24 hr. Sucrose 1G0 gm. Average Glucose 100 gm. Average Sucrose 200 gm.
Average Glucose 200 gm.
Sugar excreted gm./24 hr.
Nitrogen excreted gm./24 hr.
Sugar utilized gm./24 hr.
51.9 89.7 89.0
1.74 3.00 3.28
48.1 10.3 11.0
29.8 29.9 27.1
121.9 137.1 180.7 115.6
3.23 2.50 2.34 1.53
78.1 62.9 19.3 84.4
37.7 54.8 77.4 75.6
TABLE XVII THE GLUCOSE AND NITROGEN EXCRETION DURING 24-HOUR PERIODS BY A PHLORIZINIZED-DEPANCREATIZED DOG (#2) AFTER THE INGESTION OF 100 AND 200 GRAMS OF SUCROSE AND GLUCOSE Sugar ingested gm./24 hr. Sucrose 100 gm.
Average Glucose 100 gm. Average Sucrose 200 gm. Average Glucose 200 gm. Average
Sugar excreted gm./24 hr.
Nitrogen excreted gm./24 hr.
Sugar utilized gm./24 hr.
88.9 92.2 73.4 60.1
3.30 2.82 2.79
11.1 7.8 26.6 39-9
119.1 120.0 128.9
2.56 2.20 2.34
80.3 80.0 71.1
46.5 54.6 55.1
D/H 27.0 32.7 • •
116 urine, or which was utilized by the dogs, during the peri;ods when they were fed 100 grams of sugar per day, on an over-all average was 23«7 grams per day. Regarding the comparatively wide fluctuations in the daily amounts of excreted sugar, there appears to be a direct relationship between the amount of sugar excreted and the amount of nitrogen excreted.
This relationship is
graphically shown in Figure 8 for the sugar and nitrogen excretions during the periods when both the dogs were fed 100 grams of sucrose or glucose.
It can be seen that the
amount of sugar excreted each day varies directly with the amount of excreted nitrogen.
As a result, the daily D:N
ratios are comparatively constant within a narrow range between 20:1 and 355 1 (Tables XVI and XVII).
'relationship exists between the daily sugar and nitrogen excretions with the 200-gram sugar feedings, with the possible exception of the 200-gram sucrose feedings, for dog #1.
However, the D:N ratios with these larger sugar
feedings again tend to show a definite grouping, although not quite as marked as with the 100-gram feedings. i
This apparent relationship between the excreted sugar and nitrogen suggests a possible explanation for the daily fluctuations in the amounts of excreted glucose following the ingestion of constant quantities of sugar.
(0 2 <
With this in view, the significance of the D:N ratio
of the phlorizlnized-depancreatized dog under normal envi-
ronmental conditions was investigated as an indication of the amount of glucose that is available from protein
Particular consideration was given to the amount
of glucose that was utilized by the animals under these
conditions as well as the amounts of sugar and nitrogen .that were excreted in the urine*
When the urinary D:N
ratios were corrected for this utilized glucose and for the ; i
nitrogen that originated from non-glucose forming compounds,
icalculated D:N ratios of 5*65:1
5*93:1 were obtained,
Instead of the classical ratio of 3 *65*1 *
a 90-95 per cent conversion of protein to glucose instead 1of the 58 per cent conversion that has generally been ;assumed. I
From the results of this series of
studies on carbohydrate metabolism, the following conclu
sions can be drawn: 1. i
Only a small proportion of the glucose that is
utilized by the insulin-free eviscerated rabbit is oxidized within eight hours to carbon dioxide.
Most of the radio
activity of the glucose that is utilized by this prepar ation is found in the water-soluble fraction of the carcass 1 in the form of non-glucose material, with lesser amounts in the glycogen and protein fractions. 2.
The administration of insulin to the eviscerated
:rabbit greatly increases the oxidation of glucose to carbon j dioxide.
Again, a large proportion of the disappearing
glucose is found in the water-soluble fraction in the form of glucose derivatives which appear to be en route to com plete oxidation or to the formation of glycogen and protein. 3*
The basal insulin requirements of fasting
depancreatized dogs tended to increase during exposures at La simulated_altitude„of_15,000 feet_as_ ^compared with their
! j i ■
129 basal insulin needs at ground level. 4.
When the urinary D:N ratios of the phlorizinized-
depancreatized dogs are corrected for the amount of glucose that is utilized by the tissues of this type of animal preparation, the estimated conversion of protein to glucose approaches 100 per cent.
B I B L I O G R A P H Y
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