- Email: [email protected]
Studies on carbohydrate metabolism in rat kidney slices
Studies on Carbohydrate Metabolism in Rat Kidney Slices.1 II. Effect of Alloxan Diabetes and Insulin Administration on Glucose Uptake and Glucose Formation Ching Tseng Teng
Received
July
20, 1953
INTI~oDUCTIO~
In the preceding paper (1) the formation of glucose by kidney slices from normal rats has been confirmed. It was felt that the kidney slice might be a useful tissue for the study of the effect of diabetes and insulin administration on the formation and utilization of glucose in vitro. Preliminary experiments showed, however, that in comparison with kidney slices from normal rats those from unrorttroHed diabetic. rats rontained six fold more glycogen and put out five fold more glucose during blank incubation. Consequently, with diabetic kidney slices measurements of glucose uptake and glucose formabion from an added substrate could not be made with any accuracy. To overrome these difficulties, a method of preliminary incubation was devised, whic*h effectively removed all of the glucose output before the slices were used in t)hr metabolic experiment during a subsequent incubation. By applying t.his procedure t,o all groups of rats, comparable results OJI glu(%e uptake and glucose formation from pyruvate have been obtained. ‘l’hese results indicate that glutaose uptake is decreased and glurose formation from pyruvate is increased in kidney slices from uncontrolled diabetic rats, and that insulin treatment restores glucose formation to, and raises glucose uptake above, normal levels. 1 Aided [Inivcrsit,v
by a Research Grant (bllege of Medicine.
from the M. I). .4nclerson 4 I .-I
Foundation
to Baylor
416
CHING
TSENG
TABLE Insulin
Group
Treatment
Rats
TENG
I
and Average Blood Glucose Concentration
of Rats Used in Study
Insulin treatment ~__.-~. - -
Blood glucose
Dosage and duration --~~
t : e
Normal Normal Diabetic Diabetic Diabetic
None 24 units 3@40 min. before sacrifice. None None 3 units once daily or 2 units twice daily for 4-7 days.
Before insulin
At time of sacrifice
w. %
w. %
I--
90 ~ 530
/
90 56 144 480 69
EXPERIMENTAL Five groups of male Sprague-Dawley rats were employed: (a) normal rats, (b) normal rats receiving 2-4 units of crystalline insulin2 30-40 min. before sacrifice, (c) mildly diabetic rats, (d) severely diabetic rats, and (e) severely diabetic rats treated with protamine-zinc insulin, 2 units twice daily or 3 units once daily for 4-7 days prior to sacrifice. Diabetes was induced by the intravenous injection of alloxan monohydrate, 50 mg./kg. body weight,. These rats were used from 1 to 5 weeks after the development of diabetes. Blood glucose concentrations were determined before the institution of insulin therapy in the last group and at the time of sacrifice in all groups of animals. These data are shown in Table I. The media and the preparation of kidney slices have been described in the preceding paper (1). The kidney glycogen content was determined (2) on sample slices at zero time and on slices after 1 hr. of incubation in a medium without added substrate. The difference in glycogen values represented net glycogenolysis. Aliquots of the substrate-free media used for slicing and incubation in these and other experiments were analyzed for glucose. The appearance of glucose under these circumstances (i.e., slicing and blank incubation) was designated as gZu.cose output, while the appearance of glucose during incubation in the presence of glycogenic substrate had been designated its glucose jormation. In metabolic experiments in which the uptake and formation of glucose were measured, a preliminary incubat.ion was carried out. The procedure was as follows: Kidney slices were first incubated in the absence of added substrate for 15 min. This time was experimentally determined aa sufficient for the kidney slices to release all of their preformed and potential glucose (Fig. 1). Then the slices were thoroughly rinsed in fresh, chilled slicing medium, then blotted, weighed, and transferred to experimental vessels, and then further incubated in the presence similar of the desired substrate for 1 hr. With glucose as substrate (20 mmoles/l.), aliquots of the medium taken at the beginning and at the end of incubation were 2 The insulin preparations used in this work were gift.s from the Lilly Laboratory, Indianapolis, Ind.
Rreertrph
CARBOHYDRATE METABOLISM. II
417
TIME OF INCUBATION - MINUTES E’rc;. 1, Glucose CJUtlJUt l,y kidney slices from four untreated diabetic rats plotted nK;tinst time of inwkttion in :I medium containing no added SUbstrate. The :rver;~gr I)lootf glucose concentration was 595 mg. %.
:malyzed for glucosr, ant1 ylucose uptake M:+Scsl~lsi~ed from the difference bet ween these values. With pyruvate as substrate (10 mmoles/l.), the amount of ylurose farmed was determined at the end of the incubation. All experiments were carried out under aerobic conditions. RESULTS
Glwose Output (Table 1I) The glucaose output from normal kidney slices was small, the amount a,ppearing during blank incubation being negligible. It was greatly illcareased in slices from untreated, diabetic rats (group d), which had a pronounced hyperglycemia averaging 480 mg. yO (see Table I). This incareased glucose output was reduced to norma. levels in a similar group of severely diabetic rats which had received insulin treatment and had a mean blood glucose concentration of 69 mg. yO at the time of death (group e). With the mildly diabetic rats (group c), whose hyperglycemia was not, marked, the glucose output was only slightly above normal. h’id~rw.y Glycoyen. and Glycogenolysis (Table III) ysis
III the rlormal rats (group a) kidney glyvogen was low and glycogenolwas negligible. In the untreated, severely diabetic rats (group 13)
418
CHING
TSENG
TABLE
TENG
II
Glucose 0utpu.t by Kidney Slices Result,8 expressed in micromoles of glucose/g. of wet kidney. Number of observa.tions given in parentheses. For details of insulin treatment and blood glucose response see Table I. Glucose
output
____
-__--
Insulin
Rats
Group
Slicing Meall
Blank incubation -____ -~~-...~
S.E.
MeaIl ____
.~___
Normal 0 3.5 f 0.34 (7) 1.6 b” Normal + Diabetic 0 3.4 i 0.15 (5) 2.9 15.7 i 1.40 (12) Diabetic 0 Fl 10.5 1.1 f 0.25 (4) e Diabetic 1.8 I + I -___ _______~_~ 0 Probability that the difference from group a is less TABLE Kidney
a
b i e
I
Normal Normal Diabetic Diabetic Diabetic
a Probability * Probability
0 +
0 0 +
f
1 _Mean
0.19 (8)
f i f
0.25 (8)” 0.86 (22) 0.49 (4) -I___ than 0.01.
5.1 6.3 26.2 2.9
III
=
Insulin
Rats
Group
SE. -_--
Glycogen and Glycogenolysis
Same as in Table II
--
Total
- .__Mean 1.7 2.0 3.7 10.3 2.3
Glycogen
Glycogenolysis
S.E. -~__--
f f f f f
0.18 0.10 0.78” 2.25 0.15
= Meall
(6) (6) (7) (5) (4)
SE. ____--
-0.2
f
0.14
-1.3 -4.6
f f
0.59* (7) 0.79 (5)
that the difference from group a is less than 0.05. that the difference from group a is greater than 0.05.
the kidney glycogen concentration was sixfold higher, and glycogenolysis was considerably greater than normal. It was assumed that this would yield glucose to the medium and thus account for a part of the large glucose output shown in Table II. In the mild diabetics (group c) the results were intermediate. In the insulin-treated diabetics (group e) and normals (group b), kidney glycogen was close to the normal value; glycogenolysis was not measured.
CARBOIIYDRITl2
MET~~BOLIS~l.
Preliminary
11
41!1
Incubation
The effective duration of preliminary incubation was determined in four experiments with kidney slices from untreated, severely diabetic rats. The glucose output from these slices reached a maximum within the first lo-15 min. of incubation, and thereafter no more glucose appeared (Fig. 1). Tn other experiments, the preincubated slices were thoroughly rinsed and subsequently incubated in the presence of glucose substrate for 1 hr. The uptake of glucose of these slices was compared ivith that of slices from the same rats which had not been preincubated. With normal kidney slices no difference in results was observed. This indicates that the small glucose output (1.C pmoles/g.) from normal kidney slices did not materially mask the disappearance of glucose from the medium, and that the brief period of the preliminary incubation did not significantly alter the rate of glucose uptake of the slices during the subsequent incubation. With diabetic kidney slices the results were different. Without the preliminary incubation the disappearance of glucose from the medium was apparently largely masked by the output of glucose into the medium from the slices. Obviously the glucose uptake could not be measured accurately and the measured figure was much smaller than the actual uptake. This source of error uxs eliminated h\the preliminary incubation whic*h rffectively removed all of the glucose output from the slices. This was indicated by the substantially greater glucose uptake during the subsequent incubation. These data are shown in Table TV. Tn all subsequent experiments a 15-min. preliminary incuTABLE E.flwt
of Prcliminnry
Zncubaflott
IV
011 Glucose
lJpta.ke hi/ k’rdnc.~/ Slices
I_)ur:tt,ion of prelimirmry incubation (P.1.) : 15 min. Gl~~~osc wncentration in incubation medium: 20 mmoIes/l. 1Cwh experimental vrwvl contained O.&O.6 g. of kidney alices in 3-1 ml. of medium. Results expwswd irk mivromnles of glucase/g. wet kidne.y/hr. Glucose
Group
i I
Rats
Without
Insulin
uptake
P.I. -~~~-
,I
tl
1 Normal Diabetic
I-.-
~-
0 0
With P.I. ______
420
CHING
TSENG
TABLE Glucose Uptake
TENG
V
by Kidney
Slices
Incubation in all experiments preceded by a ELmin. preliminary Other details same as in Tahle IV. --
incubation.
Glucose uptake Rats
Insulin’ --
__-
pb No. of observ. __-
Meall __
PC
S.E. _
.-.--
-11.9
f
-15.4 -10.7 -7.3 -17.1
f 1.62 -f 0.62 f 0.76 f 1.25
___-~
..
0.34 <0.05
0 For details of insulin dosage and blood glucose response, see Table I. b Probability that the difference from group a is due to chance. c Probability t,hat the difference from group d is due t,o chance. bation was carried out before metabolic measurements were made after another hour of incubation in a fresh medium containing the desired
substrate. Glucose Uptake (Table V) Glucose uptake was reduced in kidney slices from diabetic rats and increased in those from insulin-treated animals. The reduction of glucose uptake varied with the severity of the diabetes. While it was almost 40 % below normal in one group of diabetic rats with an average blood glucose concentration of 480 mg. 7’ (group d), it was not significantly reduced in another group of diabetic rats with a mean blood glucose concentration of 144 mg. % (group c). Insulin administration, on the other hand, caused kidney slices to use more glucose than normal. This was seen in the diabetic (group e) as well as in normal rats (group b). In both groups the blood glucose concentration was lowered t,o hypoglycemic levels following insulin treatment (see Table I).
Glucose Formation from Pyruvate (Table VI) Glucose formation was increased in kidney slices from diabetic rats. Again, the severity of the diabetes was an important factor. While an increase of 68 % above normal was seen in severely diabetic rats (group d), no change was observed in the mildly diabetic group (group c). Insulin treatment restored glucose formation to normal levels in diabetic
Pb
so. of
PC
observ. __.~ I,
b c d I
..~.
..~
___~-
Normal Sormal J)ia.lwtic Diabetic TXahetic
‘1 For details o t’rol~:rl~ilit)r I’rc,l~bility
0 5.3 * 0.57 (3) 3.3 zt 0.39 <0.05 -t (1) 0 5.1 f 0.53 > (J (J;i 0.05
24
100
200
300
BLOOD GLUCOSE
400 MG%
500
FIG. 2. The mean values of total glucose output 1)~ kidney slices during slicing and during blank incubation (see Table II) and 1he mean values of kitlney gl~.wgen at zero time (see Table III) are plotted against the mean vnlucs of I~looci glucose concentration at the time of death (see Tahlc I). I’oinis from left to right repro?sent: insulin-treated diabetic rats (group e), normal rats (group (I), mildly diatwt ic rats (group c), and severely diabetic rats (group d).
422
CHING TSENG TENG
kidney slices (group e) and caused a 38% decrease in normal kidney slices (group b). DISCUSSION
The data presented in Tables I, II, and III are compared in Fig. 2. The effects of diabetes, of the severity of diabetes, and of insulin administration upon the glucose output from kidney slices and upon kidney glycogen are clearly shown. It appears that kidney glycogen and glucose output from kidney slices are directly related to the blood glucose concentration of the rat. It is assumed that blood glucose in the glucose space (3) of the kidney and kidney glycogen are two of the sources of glucose output from the kidney slices. The question remains, however, as to whether these are sufficient to account for all of the glucose appearing in the media, particularly in the case of the untreated, severely diabetic rats. The following calculation provides a negative answer: Observed Data
1. 2. 3. 4. 5. 6.
Glucose output, during slicing Glucose output, during blank incubation Glucose output, total Glycogenolysis Blood glucose concentration Glucose space Derived
15.7 10.5 26.2 4.6 480 mg. y0 = 26.6 amoles/ml. blood 31 % = 0.3 ml./g. kidney Data
7. Preformed glucose in kidney = 26.6 X 0.3 = 8.0 rmoles/g. kidney 8. Glucose output unaccounted for = (3) - (4) - (7) = 13.6 pmoles/g. kidney
These results indicate that the conversion of other glucose precursors to glucose may be increased in the diabetic kidney. The observation of increased kidney glycogen in untreated diabetic rats is consistent with the finding of increased liver glycogen in fasting alloxan diabetic rats.3 (4-6). Both findings are interpreted as manifestations of increased gluconeogenesis. From the results recorded in Tables V and VI it is evident that the metabolic defects of alloxan diabetes in the rat are decreased utilization as well as increased production of glucose. Lending support to this conclusion are the observations that the extent of these defects varied directly with the intensity of the diabetes, and that prior administration of large doses of insulin led to opposite results. The present observations 2 An increase of liver glyeogen has also been found in fasting pancreatectomized rats having blood glucose levels around 300 mg. 70 (Renold, A. E., Hastings, A. B., and Nesbett, F. B., personal communication).
with those of other investigat.ors working with intact &abetic animals (7-9) or with liver slices from alloxan diabetic rats (10). are consistent
.iCKNOWLEDGMESTS
I am indebted to Prof. A. Baird Hastings .4. Happoport for their interest and advice
and Drs. P. MarotA in t,his work.
Sinex and Donald
SSUMRIAKY
The kidney glycogen concentrat,ion and the glucose output by kidney slices from normal and diabetic rats wit,h or without prior insulin treatment have been studied. It was found that both were increased in diabetes and that insulin administration reduced bot,h to normal or subnormal levels. -1 close direct corrrelation between these values and the blood glucose levels at the time of death wa,s demonstrated The large output of glucose from diahet,ic kidney slices interfered with accurate measurements of the uptake of added glucose and the formation of new glucose from added pyruvete. This difficulty was obviated by carrying out a preliminary incbubation in the absence of added substrate, which effectively removed all of t,he glucose output from t’he slices. Then the slices were used for metabolic2 measuremer& in a subs+ quent incubation in the presence of the desired substrate. From the data obtained it. was concluded that the metabolic defec%s in diabetes lvere both decreased utilization atld inc&reased production of glucose. The extent of these defects varied direr’tly with the int,ensity of t,he diabetes. Insulin administration to t,hrl rats before the removal of the kidneys led to opposite results. REFERENCES
1. TENC, C. T., Arch. Biochem. a& Biophys. 48. 409 (1954). 2. GOOD, C. A., KRAMER, H., AND SOMOOYI, Lt., J. Biol. phem. 100, 485 (1933). 3. WICK, .4. S., DRIJRY, 11. R., AND MACKAY, FZ. M., :Inc J. Ph~ysiol. 163, 224 (19.50). 1. ‘h?i(;, t:. T.,
&ZEX,
F. xl.,
I)EANE,
H. F-5’., .+%I) hSTI%S,
z\. II.,
J. fdhhzr
Camp. Physiol. 39, 73 (1952). 5. MOHITA, T., AND ORTEN, J. M., Am. J. I’kysiol. 161, 545 (1950). 6. WEBER, H., fiature 168, 627 (1946). i. FELLER, D. D., STRISOWER, l?. H., AND CHAIKOFF, I. I,., J. Biol. Chem. 187, 571 (1950). 8. WELT, 1. I)., STETTEN, D., JR., INCLE, D. J., AND MORLEY, E. H., J. Biol. Chcm. 197, 57 (1952). $4. ~TIWI’~;..:.*, D., JR., WEI;I’, I. I)., Ir;om, 1). J., .4~‘1) MORLEY, lt. H., J. Hid. (‘hrvn.
192,
s17
(39.51).
IO. Fl \~UYC:S, :2. I(., TENT:, (‘. T., .ANI) SINEX, I*‘. ,\I., 18111 Ittl~~rtt. .\lMl rwts, 1). 2cl!4 (19.5t)~.
(‘ougt-.
I’Il>~siol.
Received
July
20, 1953
INTI~oDUCTIO~
In the preceding paper (1) the formation of glucose by kidney slices from normal rats has been confirmed. It was felt that the kidney slice might be a useful tissue for the study of the effect of diabetes and insulin administration on the formation and utilization of glucose in vitro. Preliminary experiments showed, however, that in comparison with kidney slices from normal rats those from unrorttroHed diabetic. rats rontained six fold more glycogen and put out five fold more glucose during blank incubation. Consequently, with diabetic kidney slices measurements of glucose uptake and glucose formabion from an added substrate could not be made with any accuracy. To overrome these difficulties, a method of preliminary incubation was devised, whic*h effectively removed all of the glucose output before the slices were used in t)hr metabolic experiment during a subsequent incubation. By applying t.his procedure t,o all groups of rats, comparable results OJI glu(%e uptake and glucose formation from pyruvate have been obtained. ‘l’hese results indicate that glutaose uptake is decreased and glurose formation from pyruvate is increased in kidney slices from uncontrolled diabetic rats, and that insulin treatment restores glucose formation to, and raises glucose uptake above, normal levels. 1 Aided [Inivcrsit,v
by a Research Grant (bllege of Medicine.
from the M. I). .4nclerson 4 I .-I
Foundation
to Baylor
416
CHING
TSENG
TABLE Insulin
Group
Treatment
Rats
TENG
I
and Average Blood Glucose Concentration
of Rats Used in Study
Insulin treatment ~__.-~. - -
Blood glucose
Dosage and duration --~~
t : e
Normal Normal Diabetic Diabetic Diabetic
None 24 units 3@40 min. before sacrifice. None None 3 units once daily or 2 units twice daily for 4-7 days.
Before insulin
At time of sacrifice
w. %
w. %
I--
90 ~ 530
/
90 56 144 480 69
EXPERIMENTAL Five groups of male Sprague-Dawley rats were employed: (a) normal rats, (b) normal rats receiving 2-4 units of crystalline insulin2 30-40 min. before sacrifice, (c) mildly diabetic rats, (d) severely diabetic rats, and (e) severely diabetic rats treated with protamine-zinc insulin, 2 units twice daily or 3 units once daily for 4-7 days prior to sacrifice. Diabetes was induced by the intravenous injection of alloxan monohydrate, 50 mg./kg. body weight,. These rats were used from 1 to 5 weeks after the development of diabetes. Blood glucose concentrations were determined before the institution of insulin therapy in the last group and at the time of sacrifice in all groups of animals. These data are shown in Table I. The media and the preparation of kidney slices have been described in the preceding paper (1). The kidney glycogen content was determined (2) on sample slices at zero time and on slices after 1 hr. of incubation in a medium without added substrate. The difference in glycogen values represented net glycogenolysis. Aliquots of the substrate-free media used for slicing and incubation in these and other experiments were analyzed for glucose. The appearance of glucose under these circumstances (i.e., slicing and blank incubation) was designated as gZu.cose output, while the appearance of glucose during incubation in the presence of glycogenic substrate had been designated its glucose jormation. In metabolic experiments in which the uptake and formation of glucose were measured, a preliminary incubat.ion was carried out. The procedure was as follows: Kidney slices were first incubated in the absence of added substrate for 15 min. This time was experimentally determined aa sufficient for the kidney slices to release all of their preformed and potential glucose (Fig. 1). Then the slices were thoroughly rinsed in fresh, chilled slicing medium, then blotted, weighed, and transferred to experimental vessels, and then further incubated in the presence similar of the desired substrate for 1 hr. With glucose as substrate (20 mmoles/l.), aliquots of the medium taken at the beginning and at the end of incubation were 2 The insulin preparations used in this work were gift.s from the Lilly Laboratory, Indianapolis, Ind.
Rreertrph
CARBOHYDRATE METABOLISM. II
417
TIME OF INCUBATION - MINUTES E’rc;. 1, Glucose CJUtlJUt l,y kidney slices from four untreated diabetic rats plotted nK;tinst time of inwkttion in :I medium containing no added SUbstrate. The :rver;~gr I)lootf glucose concentration was 595 mg. %.
:malyzed for glucosr, ant1 ylucose uptake M:+Scsl~lsi~ed from the difference bet ween these values. With pyruvate as substrate (10 mmoles/l.), the amount of ylurose farmed was determined at the end of the incubation. All experiments were carried out under aerobic conditions. RESULTS
Glwose Output (Table 1I) The glucaose output from normal kidney slices was small, the amount a,ppearing during blank incubation being negligible. It was greatly illcareased in slices from untreated, diabetic rats (group d), which had a pronounced hyperglycemia averaging 480 mg. yO (see Table I). This incareased glucose output was reduced to norma. levels in a similar group of severely diabetic rats which had received insulin treatment and had a mean blood glucose concentration of 69 mg. yO at the time of death (group e). With the mildly diabetic rats (group c), whose hyperglycemia was not, marked, the glucose output was only slightly above normal. h’id~rw.y Glycoyen. and Glycogenolysis (Table III) ysis
III the rlormal rats (group a) kidney glyvogen was low and glycogenolwas negligible. In the untreated, severely diabetic rats (group 13)
418
CHING
TSENG
TABLE
TENG
II
Glucose 0utpu.t by Kidney Slices Result,8 expressed in micromoles of glucose/g. of wet kidney. Number of observa.tions given in parentheses. For details of insulin treatment and blood glucose response see Table I. Glucose
output
____
-__--
Insulin
Rats
Group
Slicing Meall
Blank incubation -____ -~~-...~
S.E.
MeaIl ____
.~___
Normal 0 3.5 f 0.34 (7) 1.6 b” Normal + Diabetic 0 3.4 i 0.15 (5) 2.9 15.7 i 1.40 (12) Diabetic 0 Fl 10.5 1.1 f 0.25 (4) e Diabetic 1.8 I + I -___ _______~_~ 0 Probability that the difference from group a is less TABLE Kidney
a
b i e
I
Normal Normal Diabetic Diabetic Diabetic
a Probability * Probability
0 +
0 0 +
f
1 _Mean
0.19 (8)
f i f
0.25 (8)” 0.86 (22) 0.49 (4) -I___ than 0.01.
5.1 6.3 26.2 2.9
III
=
Insulin
Rats
Group
SE. -_--
Glycogen and Glycogenolysis
Same as in Table II
--
Total
- .__Mean 1.7 2.0 3.7 10.3 2.3
Glycogen
Glycogenolysis
S.E. -~__--
f f f f f
0.18 0.10 0.78” 2.25 0.15
= Meall
(6) (6) (7) (5) (4)
SE. ____--
-0.2
f
0.14
-1.3 -4.6
f f
0.59* (7) 0.79 (5)
that the difference from group a is less than 0.05. that the difference from group a is greater than 0.05.
the kidney glycogen concentration was sixfold higher, and glycogenolysis was considerably greater than normal. It was assumed that this would yield glucose to the medium and thus account for a part of the large glucose output shown in Table II. In the mild diabetics (group c) the results were intermediate. In the insulin-treated diabetics (group e) and normals (group b), kidney glycogen was close to the normal value; glycogenolysis was not measured.
CARBOIIYDRITl2
MET~~BOLIS~l.
Preliminary
11
41!1
Incubation
The effective duration of preliminary incubation was determined in four experiments with kidney slices from untreated, severely diabetic rats. The glucose output from these slices reached a maximum within the first lo-15 min. of incubation, and thereafter no more glucose appeared (Fig. 1). Tn other experiments, the preincubated slices were thoroughly rinsed and subsequently incubated in the presence of glucose substrate for 1 hr. The uptake of glucose of these slices was compared ivith that of slices from the same rats which had not been preincubated. With normal kidney slices no difference in results was observed. This indicates that the small glucose output (1.C pmoles/g.) from normal kidney slices did not materially mask the disappearance of glucose from the medium, and that the brief period of the preliminary incubation did not significantly alter the rate of glucose uptake of the slices during the subsequent incubation. With diabetic kidney slices the results were different. Without the preliminary incubation the disappearance of glucose from the medium was apparently largely masked by the output of glucose into the medium from the slices. Obviously the glucose uptake could not be measured accurately and the measured figure was much smaller than the actual uptake. This source of error uxs eliminated h\the preliminary incubation whic*h rffectively removed all of the glucose output from the slices. This was indicated by the substantially greater glucose uptake during the subsequent incubation. These data are shown in Table TV. Tn all subsequent experiments a 15-min. preliminary incuTABLE E.flwt
of Prcliminnry
Zncubaflott
IV
011 Glucose
lJpta.ke hi/ k’rdnc.~/ Slices
I_)ur:tt,ion of prelimirmry incubation (P.1.) : 15 min. Gl~~~osc wncentration in incubation medium: 20 mmoIes/l. 1Cwh experimental vrwvl contained O.&O.6 g. of kidney alices in 3-1 ml. of medium. Results expwswd irk mivromnles of glucase/g. wet kidne.y/hr. Glucose
Group
i I
Rats
Without
Insulin
uptake
P.I. -~~~-
,I
tl
1 Normal Diabetic
I-.-
~-
0 0
With P.I. ______
420
CHING
TSENG
TABLE Glucose Uptake
TENG
V
by Kidney
Slices
Incubation in all experiments preceded by a ELmin. preliminary Other details same as in Tahle IV. --
incubation.
Glucose uptake Rats
Insulin’ --
__-
pb No. of observ. __-
Meall __
PC
S.E. _
.-.--
-11.9
f
-15.4 -10.7 -7.3 -17.1
f 1.62 -f 0.62 f 0.76 f 1.25
___-~
..
0.34 <0.05
0 For details of insulin dosage and blood glucose response, see Table I. b Probability that the difference from group a is due to chance. c Probability t,hat the difference from group d is due t,o chance. bation was carried out before metabolic measurements were made after another hour of incubation in a fresh medium containing the desired
substrate. Glucose Uptake (Table V) Glucose uptake was reduced in kidney slices from diabetic rats and increased in those from insulin-treated animals. The reduction of glucose uptake varied with the severity of the diabetes. While it was almost 40 % below normal in one group of diabetic rats with an average blood glucose concentration of 480 mg. 7’ (group d), it was not significantly reduced in another group of diabetic rats with a mean blood glucose concentration of 144 mg. % (group c). Insulin administration, on the other hand, caused kidney slices to use more glucose than normal. This was seen in the diabetic (group e) as well as in normal rats (group b). In both groups the blood glucose concentration was lowered t,o hypoglycemic levels following insulin treatment (see Table I).
Glucose Formation from Pyruvate (Table VI) Glucose formation was increased in kidney slices from diabetic rats. Again, the severity of the diabetes was an important factor. While an increase of 68 % above normal was seen in severely diabetic rats (group d), no change was observed in the mildly diabetic group (group c). Insulin treatment restored glucose formation to normal levels in diabetic
Pb
so. of
PC
observ. __.~ I,
b c d I
..~.
..~
___~-
Normal Sormal J)ia.lwtic Diabetic TXahetic
‘1 For details o t’rol~:rl~ilit)r I’rc,l~bility
0 5.3 * 0.57 (3) 3.3 zt 0.39 <0.05 -t (1) 0 5.1 f 0.53 > (J (J;i
24
100
200
300
BLOOD GLUCOSE
400 MG%
500
FIG. 2. The mean values of total glucose output 1)~ kidney slices during slicing and during blank incubation (see Table II) and 1he mean values of kitlney gl~.wgen at zero time (see Table III) are plotted against the mean vnlucs of I~looci glucose concentration at the time of death (see Tahlc I). I’oinis from left to right repro?sent: insulin-treated diabetic rats (group e), normal rats (group (I), mildly diatwt ic rats (group c), and severely diabetic rats (group d).
422
CHING TSENG TENG
kidney slices (group e) and caused a 38% decrease in normal kidney slices (group b). DISCUSSION
The data presented in Tables I, II, and III are compared in Fig. 2. The effects of diabetes, of the severity of diabetes, and of insulin administration upon the glucose output from kidney slices and upon kidney glycogen are clearly shown. It appears that kidney glycogen and glucose output from kidney slices are directly related to the blood glucose concentration of the rat. It is assumed that blood glucose in the glucose space (3) of the kidney and kidney glycogen are two of the sources of glucose output from the kidney slices. The question remains, however, as to whether these are sufficient to account for all of the glucose appearing in the media, particularly in the case of the untreated, severely diabetic rats. The following calculation provides a negative answer: Observed Data
1. 2. 3. 4. 5. 6.
Glucose output, during slicing Glucose output, during blank incubation Glucose output, total Glycogenolysis Blood glucose concentration Glucose space Derived
15.7 10.5 26.2 4.6 480 mg. y0 = 26.6 amoles/ml. blood 31 % = 0.3 ml./g. kidney Data
7. Preformed glucose in kidney = 26.6 X 0.3 = 8.0 rmoles/g. kidney 8. Glucose output unaccounted for = (3) - (4) - (7) = 13.6 pmoles/g. kidney
These results indicate that the conversion of other glucose precursors to glucose may be increased in the diabetic kidney. The observation of increased kidney glycogen in untreated diabetic rats is consistent with the finding of increased liver glycogen in fasting alloxan diabetic rats.3 (4-6). Both findings are interpreted as manifestations of increased gluconeogenesis. From the results recorded in Tables V and VI it is evident that the metabolic defects of alloxan diabetes in the rat are decreased utilization as well as increased production of glucose. Lending support to this conclusion are the observations that the extent of these defects varied directly with the intensity of the diabetes, and that prior administration of large doses of insulin led to opposite results. The present observations 2 An increase of liver glyeogen has also been found in fasting pancreatectomized rats having blood glucose levels around 300 mg. 70 (Renold, A. E., Hastings, A. B., and Nesbett, F. B., personal communication).
with those of other investigat.ors working with intact &abetic animals (7-9) or with liver slices from alloxan diabetic rats (10). are consistent
.iCKNOWLEDGMESTS
I am indebted to Prof. A. Baird Hastings .4. Happoport for their interest and advice
and Drs. P. MarotA in t,his work.
Sinex and Donald
SSUMRIAKY
The kidney glycogen concentrat,ion and the glucose output by kidney slices from normal and diabetic rats wit,h or without prior insulin treatment have been studied. It was found that both were increased in diabetes and that insulin administration reduced bot,h to normal or subnormal levels. -1 close direct corrrelation between these values and the blood glucose levels at the time of death wa,s demonstrated The large output of glucose from diahet,ic kidney slices interfered with accurate measurements of the uptake of added glucose and the formation of new glucose from added pyruvete. This difficulty was obviated by carrying out a preliminary incbubation in the absence of added substrate, which effectively removed all of t,he glucose output from t’he slices. Then the slices were used for metabolic2 measuremer& in a subs+ quent incubation in the presence of the desired substrate. From the data obtained it. was concluded that the metabolic defec%s in diabetes lvere both decreased utilization atld inc&reased production of glucose. The extent of these defects varied direr’tly with the int,ensity of t,he diabetes. Insulin administration to t,hrl rats before the removal of the kidneys led to opposite results. REFERENCES
1. TENC, C. T., Arch. Biochem. a& Biophys. 48. 409 (1954). 2. GOOD, C. A., KRAMER, H., AND SOMOOYI, Lt., J. Biol. phem. 100, 485 (1933). 3. WICK, .4. S., DRIJRY, 11. R., AND MACKAY, FZ. M., :Inc J. Ph~ysiol. 163, 224 (19.50). 1. ‘h?i(;, t:. T.,
&ZEX,
F. xl.,
I)EANE,
H. F-5’., .+%I) hSTI%S,
z\. II.,
J. fdhhzr
Camp. Physiol. 39, 73 (1952). 5. MOHITA, T., AND ORTEN, J. M., Am. J. I’kysiol. 161, 545 (1950). 6. WEBER, H., fiature 168, 627 (1946). i. FELLER, D. D., STRISOWER, l?. H., AND CHAIKOFF, I. I,., J. Biol. Chem. 187, 571 (1950). 8. WELT, 1. I)., STETTEN, D., JR., INCLE, D. J., AND MORLEY, E. H., J. Biol. Chcm. 197, 57 (1952). $4. ~TIWI’~;..:.*, D., JR., WEI;I’, I. I)., Ir;om, 1). J., .4~‘1) MORLEY, lt. H., J. Hid. (‘hrvn.
192,
s17
(39.51).
IO. Fl \~UYC:S, :2. I(., TENT:, (‘. T., .ANI) SINEX, I*‘. ,\I., 18111 Ittl~~rtt. .\lMl rwts, 1). 2cl!4 (19.5t)~.
(‘ougt-.
I’Il>~siol.