- Email: [email protected]
Hepatic glucokinase activity in human diabetics and nondiabetics
Hepatic Glucokinase Activity in Human Diabetics and Nondiabetics By SAMUEL A. FERRIS Hepatic glucokinase activity was determined in surgical liver biopsies in human diabetics and nondiabetics. Our
data suggest this enzyme diabetes.
a relative exists in
insufficiency of maturity onset
T
HE BASIC UNDERLYING METABOLIC DEFECTS in diabetes are unknown, Chaikoffl demonstrated that alloxanized diabetic rats had deficiencies in hepatic glucokinase activity and an inability of the liver to synthesize encies in hepatic glucokinase activity and an inability of the liver to synthesize fatty acids from glucose. Renold2 showed that these deficiencies were reversed by the administration of insulin for 1 to 4 days prior to sacrifice. Similarly, if the diabetic syndrome in man were basically due to a relative or absolute insulin deficiency state, one might expect to find a normal hepatic glucokinase activity upon correction of the hyperglycemia with exogenous insulin or dietary restriction. However, if in contrast to the above-mentioned insulin deficiency states in animals, a genetically determined deficiency of hepatic glucokinase exists in certain types of human diabetes, then insulin or dietary therapy would not be expected to reverse this deficit. This study was designed to compare the hepatic glucokinase activity in human nondiabetics and in maturity-onset diabetics who were controlled by insulin therapy or dietary restriction. MATERIAL Five diabetic
and 6 nondiabetic
AND METHODS
patients subjected
to surgery
for various reasons served
as the source of the liver tissue analyzed in this experiment (see table 1). None of the patients selected had clinical or histologic evidence of liver disease. At the time of surgery a variety of anesthetic agents were used and between 10 and 15 Gm. of glucose were given intravenously prior to open liver biopsy. Approximately 250 mg. of liver was obtained and immediately placed in ice water after a small portion was saved for histologic study. Processing was begun within 15 to 20 minutes after biopsy. Glucokinase activity was determined by a modification of the method described by Long.3 The liver was blotted dry with filter paper, weighed and homogenized in an ice cold tissue grinder* with ice cold 0.12 M phosphate buffer, pH 7.8, containing 0.15 M KF (0.5 cc. buffer/100 mg. tissue); so that the homogenate contained 0.10 M phosphate and 0.125 M fluoride. A solution containing appropriate amounts of chromatographically pure uniformly la-
From the Division of En&mine and Cancer Research and the Departments of Medicine and of Physiology, J~erson Medical College, Tulsa, Oklahoma Th& work was done during a Fellowship in Endocrinology and Diabetes under the U.S.P.H.S. Training Grant No. 2A-5184, National Institute of Arthritis and Metabolic Diseases. Received for publication June 24, 1964. ‘A. H. Thomas Co., Philadelphia, Pa. 1478 METABOLISM, VOL. 13, No. 12 (DECEMBER), 1964
HEPATIC
1479
GLUCOICINASE ACTIVITY
belled 04 glucose+ and appropriate amounts of potassium pipetted into 10 ml. lusteroid centrifuge tubes’ and immersed
ATF’, Mg Cl,, KC 1 were in a water bath at 38 C. The
homogenate was prewarmed to 38 C. and 0.2 ml. (containing 33 mg. of wet liver) was pipetted into the centrifuge tube at zero time and the contents gently mixed. After incubation for 10 minutes, 0.5 ml. of 0.3 N Ba( OH)2 was added to arrest enzymatic activity. After removal from the bath 0.5 ml. of 5 per cent ZnSO, was added to precipitate proteins and absorb glucose-&phosphate and other phosphate esters. Exactly 3.5 cc. of distilled water was added to the tubes and the contents were thoroughly shaken and centrifuged for 5 minutes at 2000 r.p.m. An enzymatically inactivated
control was prepared by pipetting 0.3 ml. of the glucose into another centrifuge tube. After this, 0.2 ml. solution and 0.5 ml. of 0.3 N Ba(OH), of homogenate was added and thereby immediately inactivated; finally, 0.5 ml. of 5 per cent ZnSO, was added. This control tube was carried through the same procedure except that incubation was omitted. Duplicate samples and controls were run on each biopsy specimen. The standard test system had a total volume of 0.5 ml. and contained the following composition: Liver, 33 mg. (wet weight); uniformly labelled Cl4 glucose, 0.0022 M; potassium ATP, 0.005 M; KCL, 0.042 M. An aliquot and counted
M; mg. Cl,,
of the supematant on a RCL
water
mixture
M; KF, 0.05 M; phosphate
fluid was appropriately
windowless
Another aliquot of supernatant chromatography paper. A solvent and distilled
0.005
preflush
with a TGC-2
and 100 FL.
model
in a ratio
of 2:5:7
was used.
0.04
was plated
10200.
G-M tube)
to determine
The
chromatograms
weight) liver/hour. were determined by Technicon
were
de-
20 hours. They were removed, dried on a paper chromatography scanner
if any other significant
appeared in addition to glucose on the chromatogram strips. The glucose uptake was determined by comparing the difference trols and in the incubated samples and transposing this difference take/Gm. (wet Blood sugars per cent.
(pH 7.8),
was spotted on a 7 x 22 inch strip of Whatman No. I system using the upper phase of pyridine, ethyl acetate,
veloped in a descending direction for approximately in air, and cut into strips. The strips were scanned (equipped
diluted
flow detector,
buffer
Auto Analyzer;
radioactive
spots
in c.p.m. in the coninto mg. glucose up-
normal
range
80-100
mg.
RFSULTS The results indicate that there is a statistically significant difference in glucose uptake in diabetics as compared to nondiabetics (see table 2). The chromatograms of the supematant failed to reveal any consistent or significant radioactive spots other than glucose. The histologic examination was kindly carried out by Dr. J. Goddard, research pathologist in the Department of Endocrine and Cancer Research, and failed to reveal significant alteration. DISCUSSION
Our data suggest that there is a relative hepatic glucokinase deficiency in maturity-onset diabetes. It is interesting to speculate how such a deficit might result in inefficient utilization of glucose presented to the liver, thereby causing hyperglycemia which would result in increased insulin production. This sequence of events persisting over a number of years might result in -exhaustion” of the pancreatic islets and the appearanqe of the diabetic syndrome. fNuclear-Chicago
Corporation
S. A.
(1.55
,c./mg.).
F
F
Y
F
Y
Y
72
68
61
41
24
38
61
68
diabetes
A.
Y.
N. W.
D. 6.
L. c.
It. F.
A. R.
J. P.
F. B.j/
‘History
blood
to
=
nitrous
F
= sdmiasion;
Auothane; on
histologically.
fasting,
oxide;
Ca of colon
after
meal:
cyelopropane;
2 hr.
C =
A
oral
anectine.
standard
=
P.C.A P.Cb
resection
Colon resection
P.N.A
P.N.C,A
P.N,C.A
P.N.C.A
CLXttrols
P,N.A
P.NJ2.A
P3.A P3.A
P.N.F.A
test:
102
minutes
80
68
168
162
P.P.
2 hr.
preoperative.
Duration:
46
66
96
40 45
100
102
102
100
FBS
Adm.
--
45
60
120
30
120
30
60
80
Anestbesiat Duration Agents
Colon
Lymphadenectomy
Ca of colon Ca of colon
Cholecystectomy
Cholelitbiasis
pancreas
Reported:
normai
ml.
N
revealed
mg./lOO
admission.
Non-diabetic Cholecystectomy Cholecystectomy
Cholelithissis
NO NO NO NO NO NO
Colon re%ection
Cholelithiasis
cs of colon
Colon lwx!ction
Colon resection
Ca of c&n
of colon
Cholecystectomy
Cholecystectomy
Diabetic8
Operation
Data on Cases Studied
Cholelitbiash
Cholelithiasis
Diagnosis
NO
N-0 NOIll? NOIN? None NOtW NOM
Diet
Rxt
Ca
l
Y.5
No
Ya,
NO
IYrYbe
pentothal:
values
P =
examination
nuxar
/~Postmortem
PAlI
prior
M
preoperatively.
:Abbreviationa:
tTreatment
F
72
3. c.
F
F
II
40
66
L. w.
rd. s.
of
Sex
Ape
Pt.
Table I.-Clinical
prior
13
86
100
146
FBS
to liver
190
168
‘k
biopsy.
128
190
220
207
1 hr. after
Blood Supnri Glucose Tol. Test
104
134
222
220
=;“a?.
96
126
238
171
8
80
94
70
82
86
100
84
161
92
128
102
Pm.
HEPATIC
GLUCOKINASE
1481
ACTJMTY
Table 2.-Hepatic
Glucokinme Activity
Diabetic
---
NOUdiSb&iC
NUllIf
GluooeeUptake mo./Gm. (wet) Liver/h.
M. S. M. A. N. W.
+0.73 +0.73 +0.44 f0.44 f1.69
Mean SD.
Name
GlucceeUDtSkC mzJGm. (wet) Liver/‘hr.
D. S. L. M. C. R. F. ;*; .
. Mean SD.
0.81 +0.51 Difference
of means P(t)
<
+2.76 +1.06 f2.18 f1.80 f1.55 +2&I 2.0 +0.72
1.19
0.02
The role of pancreatic “exhaustion” in the production of permanent diabetes has been well documented in animals.*-’ The possibility exists that a genetically determined insufficiency of hepatic glucokinase is responsibr$ for some cases of maturity-onset diabetes. ACKNOWLEDGMENTS The author expresses his gratitude for generous advice and encouragement to the late Karl E. Paschkis, M.D., former Director of the Division of Endocrine and Cancer Research, and his deep appreciation to W. Bahinger, M.D., Associate in Surgery, Department of Surgery, Jefferson Medical College, who by supplying the biopsy specimens, has made this study possible. REFERENCES 1. Chaikoff, I. L.: Metabolic blocks in carbohydrate metabolism in diabetes. Harvey Lect. 4:99, 1951-1952. 2. Renold, A. E., Hastings, A. B., Nesbett, F. B., and Ashmore, J.: Studies on carbohydrate metabolism in rat liver slices. IV. Biochemical sequence of events after insulin administration. J. Biol. Chem. 213:135, 1955. 3. Long, C.: The hexokinase activity of rat tissue. Biochem. J. 50:407, 1952. 4. Young, G. F.: The diabetogenic action of crude anterior pituitary extracts. Bio-
them. J. 32:513, 1938. 5. Dohan, F. C., and Lukens, F. D. W.: Experimental diabetes produced by administration of glucose. Endocrinology 42244, 1948. 6. Campbell, J.: The Hypophyseal Growth Hormone, Nature and Actions. Edited by Smith, R. W., Gaebler, 0. H., and Long, C. N. H. New York, McGrawHill, 1955. 7. Houssay, B. A.: The thyroid and diabetes. Vit. & Hormones 4:187, 1946.
Samuel A. Ferris, M.D., Formerly Division of Endocrine and Cancer Research, Departments of Medicine and of Physiology, Jeferson Medical College, Tulsa, Okla. Present address: 2021 S. Lewis, Tulsa, Okla.
data suggest this enzyme diabetes.
a relative exists in
insufficiency of maturity onset
T
HE BASIC UNDERLYING METABOLIC DEFECTS in diabetes are unknown, Chaikoffl demonstrated that alloxanized diabetic rats had deficiencies in hepatic glucokinase activity and an inability of the liver to synthesize encies in hepatic glucokinase activity and an inability of the liver to synthesize fatty acids from glucose. Renold2 showed that these deficiencies were reversed by the administration of insulin for 1 to 4 days prior to sacrifice. Similarly, if the diabetic syndrome in man were basically due to a relative or absolute insulin deficiency state, one might expect to find a normal hepatic glucokinase activity upon correction of the hyperglycemia with exogenous insulin or dietary restriction. However, if in contrast to the above-mentioned insulin deficiency states in animals, a genetically determined deficiency of hepatic glucokinase exists in certain types of human diabetes, then insulin or dietary therapy would not be expected to reverse this deficit. This study was designed to compare the hepatic glucokinase activity in human nondiabetics and in maturity-onset diabetics who were controlled by insulin therapy or dietary restriction. MATERIAL Five diabetic
and 6 nondiabetic
AND METHODS
patients subjected
to surgery
for various reasons served
as the source of the liver tissue analyzed in this experiment (see table 1). None of the patients selected had clinical or histologic evidence of liver disease. At the time of surgery a variety of anesthetic agents were used and between 10 and 15 Gm. of glucose were given intravenously prior to open liver biopsy. Approximately 250 mg. of liver was obtained and immediately placed in ice water after a small portion was saved for histologic study. Processing was begun within 15 to 20 minutes after biopsy. Glucokinase activity was determined by a modification of the method described by Long.3 The liver was blotted dry with filter paper, weighed and homogenized in an ice cold tissue grinder* with ice cold 0.12 M phosphate buffer, pH 7.8, containing 0.15 M KF (0.5 cc. buffer/100 mg. tissue); so that the homogenate contained 0.10 M phosphate and 0.125 M fluoride. A solution containing appropriate amounts of chromatographically pure uniformly la-
From the Division of En&mine and Cancer Research and the Departments of Medicine and of Physiology, J~erson Medical College, Tulsa, Oklahoma Th& work was done during a Fellowship in Endocrinology and Diabetes under the U.S.P.H.S. Training Grant No. 2A-5184, National Institute of Arthritis and Metabolic Diseases. Received for publication June 24, 1964. ‘A. H. Thomas Co., Philadelphia, Pa. 1478 METABOLISM, VOL. 13, No. 12 (DECEMBER), 1964
HEPATIC
1479
GLUCOICINASE ACTIVITY
belled 04 glucose+ and appropriate amounts of potassium pipetted into 10 ml. lusteroid centrifuge tubes’ and immersed
ATF’, Mg Cl,, KC 1 were in a water bath at 38 C. The
homogenate was prewarmed to 38 C. and 0.2 ml. (containing 33 mg. of wet liver) was pipetted into the centrifuge tube at zero time and the contents gently mixed. After incubation for 10 minutes, 0.5 ml. of 0.3 N Ba( OH)2 was added to arrest enzymatic activity. After removal from the bath 0.5 ml. of 5 per cent ZnSO, was added to precipitate proteins and absorb glucose-&phosphate and other phosphate esters. Exactly 3.5 cc. of distilled water was added to the tubes and the contents were thoroughly shaken and centrifuged for 5 minutes at 2000 r.p.m. An enzymatically inactivated
control was prepared by pipetting 0.3 ml. of the glucose into another centrifuge tube. After this, 0.2 ml. solution and 0.5 ml. of 0.3 N Ba(OH), of homogenate was added and thereby immediately inactivated; finally, 0.5 ml. of 5 per cent ZnSO, was added. This control tube was carried through the same procedure except that incubation was omitted. Duplicate samples and controls were run on each biopsy specimen. The standard test system had a total volume of 0.5 ml. and contained the following composition: Liver, 33 mg. (wet weight); uniformly labelled Cl4 glucose, 0.0022 M; potassium ATP, 0.005 M; KCL, 0.042 M. An aliquot and counted
M; mg. Cl,,
of the supematant on a RCL
water
mixture
M; KF, 0.05 M; phosphate
fluid was appropriately
windowless
Another aliquot of supernatant chromatography paper. A solvent and distilled
0.005
preflush
with a TGC-2
and 100 FL.
model
in a ratio
of 2:5:7
was used.
0.04
was plated
10200.
G-M tube)
to determine
The
chromatograms
weight) liver/hour. were determined by Technicon
were
de-
20 hours. They were removed, dried on a paper chromatography scanner
if any other significant
appeared in addition to glucose on the chromatogram strips. The glucose uptake was determined by comparing the difference trols and in the incubated samples and transposing this difference take/Gm. (wet Blood sugars per cent.
(pH 7.8),
was spotted on a 7 x 22 inch strip of Whatman No. I system using the upper phase of pyridine, ethyl acetate,
veloped in a descending direction for approximately in air, and cut into strips. The strips were scanned (equipped
diluted
flow detector,
buffer
Auto Analyzer;
radioactive
spots
in c.p.m. in the coninto mg. glucose up-
normal
range
80-100
mg.
RFSULTS The results indicate that there is a statistically significant difference in glucose uptake in diabetics as compared to nondiabetics (see table 2). The chromatograms of the supematant failed to reveal any consistent or significant radioactive spots other than glucose. The histologic examination was kindly carried out by Dr. J. Goddard, research pathologist in the Department of Endocrine and Cancer Research, and failed to reveal significant alteration. DISCUSSION
Our data suggest that there is a relative hepatic glucokinase deficiency in maturity-onset diabetes. It is interesting to speculate how such a deficit might result in inefficient utilization of glucose presented to the liver, thereby causing hyperglycemia which would result in increased insulin production. This sequence of events persisting over a number of years might result in -exhaustion” of the pancreatic islets and the appearanqe of the diabetic syndrome. fNuclear-Chicago
Corporation
S. A.
(1.55
,c./mg.).
F
F
Y
F
Y
Y
72
68
61
41
24
38
61
68
diabetes
A.
Y.
N. W.
D. 6.
L. c.
It. F.
A. R.
J. P.
F. B.j/
‘History
blood
to
=
nitrous
F
= sdmiasion;
Auothane; on
histologically.
fasting,
oxide;
Ca of colon
after
meal:
cyelopropane;
2 hr.
C =
A
oral
anectine.
standard
=
P.C.A P.Cb
resection
Colon resection
P.N.A
P.N.C,A
P.N,C.A
P.N.C.A
CLXttrols
P,N.A
P.NJ2.A
P3.A P3.A
P.N.F.A
test:
102
minutes
80
68
168
162
P.P.
2 hr.
preoperative.
Duration:
46
66
96
40 45
100
102
102
100
FBS
Adm.
--
45
60
120
30
120
30
60
80
Anestbesiat Duration Agents
Colon
Lymphadenectomy
Ca of colon Ca of colon
Cholecystectomy
Cholelitbiasis
pancreas
Reported:
normai
ml.
N
revealed
mg./lOO
admission.
Non-diabetic Cholecystectomy Cholecystectomy
Cholelithissis
NO NO NO NO NO NO
Colon re%ection
Cholelithiasis
cs of colon
Colon lwx!ction
Colon resection
Ca of c&n
of colon
Cholecystectomy
Cholecystectomy
Diabetic8
Operation
Data on Cases Studied
Cholelitbiash
Cholelithiasis
Diagnosis
NO
N-0 NOIll? NOIN? None NOtW NOM
Diet
Rxt
Ca
l
Y.5
No
Ya,
NO
IYrYbe
pentothal:
values
P =
examination
nuxar
/~Postmortem
PAlI
prior
M
preoperatively.
:Abbreviationa:
tTreatment
F
72
3. c.
F
F
II
40
66
L. w.
rd. s.
of
Sex
Ape
Pt.
Table I.-Clinical
prior
13
86
100
146
FBS
to liver
190
168
‘k
biopsy.
128
190
220
207
1 hr. after
Blood Supnri Glucose Tol. Test
104
134
222
220
=;“a?.
96
126
238
171
8
80
94
70
82
86
100
84
161
92
128
102
Pm.
HEPATIC
GLUCOKINASE
1481
ACTJMTY
Table 2.-Hepatic
Glucokinme Activity
Diabetic
---
NOUdiSb&iC
NUllIf
GluooeeUptake mo./Gm. (wet) Liver/h.
M. S. M. A. N. W.
+0.73 +0.73 +0.44 f0.44 f1.69
Mean SD.
Name
GlucceeUDtSkC mzJGm. (wet) Liver/‘hr.
D. S. L. M. C. R. F. ;*; .
. Mean SD.
0.81 +0.51 Difference
of means P(t)
<
+2.76 +1.06 f2.18 f1.80 f1.55 +2&I 2.0 +0.72
1.19
0.02
The role of pancreatic “exhaustion” in the production of permanent diabetes has been well documented in animals.*-’ The possibility exists that a genetically determined insufficiency of hepatic glucokinase is responsibr$ for some cases of maturity-onset diabetes. ACKNOWLEDGMENTS The author expresses his gratitude for generous advice and encouragement to the late Karl E. Paschkis, M.D., former Director of the Division of Endocrine and Cancer Research, and his deep appreciation to W. Bahinger, M.D., Associate in Surgery, Department of Surgery, Jefferson Medical College, who by supplying the biopsy specimens, has made this study possible. REFERENCES 1. Chaikoff, I. L.: Metabolic blocks in carbohydrate metabolism in diabetes. Harvey Lect. 4:99, 1951-1952. 2. Renold, A. E., Hastings, A. B., Nesbett, F. B., and Ashmore, J.: Studies on carbohydrate metabolism in rat liver slices. IV. Biochemical sequence of events after insulin administration. J. Biol. Chem. 213:135, 1955. 3. Long, C.: The hexokinase activity of rat tissue. Biochem. J. 50:407, 1952. 4. Young, G. F.: The diabetogenic action of crude anterior pituitary extracts. Bio-
them. J. 32:513, 1938. 5. Dohan, F. C., and Lukens, F. D. W.: Experimental diabetes produced by administration of glucose. Endocrinology 42244, 1948. 6. Campbell, J.: The Hypophyseal Growth Hormone, Nature and Actions. Edited by Smith, R. W., Gaebler, 0. H., and Long, C. N. H. New York, McGrawHill, 1955. 7. Houssay, B. A.: The thyroid and diabetes. Vit. & Hormones 4:187, 1946.
Samuel A. Ferris, M.D., Formerly Division of Endocrine and Cancer Research, Departments of Medicine and of Physiology, Jeferson Medical College, Tulsa, Okla. Present address: 2021 S. Lewis, Tulsa, Okla.