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A simple method for determining total glucose utilisation by isolated adipocytes using [5-3H]-glucose
ANALYTICAL
BIOCIlEMISTRY
A Simple
Method
by Isolated
61, 492-499 (1974)
for Determining Adipocytes D. BROWN
Department Heslington,
Using
Glucose
[ 5”H]
Utilisation
-Glucose
C. J. GARRATT
AND
of Chemistry, York YOI
Received February
Total
University 600,
Great
of York, Britain
25, 1974; accepted April 26, 1974
Glucose utilisation by adipocytes incubated with and without insulin and at two concentrations of extracellular glucose has been estimated by three different procedures. Glucose disappearance from the medium was calculated by using glucose oxidase to determine the glucose concentration remaining after incubation and comparing this with the glucose concentration in standard solutions made up by appropriate dilution of the original medium. [U-“Cl-glucose utilisation was calculated by summing the 14C found in CO,, triglycerides, and anions. [“HI-Ha0 formation from [5-‘HI-glucose was the third measure of glucose utilisation. All three methods gave similar answers, but the L5-3H1-glucose is simpler to use than NJ-“Cl-glucose and gives substantially more reproducible results than glucose oxidase.
In many studies of metabolism in isolated tissues it is necessary or useful to estimate exogenous glucose utilisation by the tissue. The classical method of doing this is to measure the difference between the glucose present at the beginning and at the end of an experiment. However, in order to measure glucose utilisation with precision by this technique it is necessary for a significant, fraction of the glucose to be used and this is not always desirable. An alternative approach is to measure the incorporation of radioactivity from glucose labeled either with 14C or 3H into all the metabolic products. With most tissues the number of products into which [I%]-glucose can be incorporated is so large that this label is too cumbersome for routine use. However, as mentioned by Ashcroft et al. (1) the use of [PHI -glucose avoids this problem because water is almost the only radioactive metabolic product of [5-3H]-glucose. The label from this position exchanges with water during triose phosphate isomerisation (1). If this reaction is not in complete equilibrium then further exchange can take place at the enolase reaction. Only glucose which is converted directly to glycogen does not pass through these steps. In adipose tissue where synthesis of glycogen is normally negligible, it follows that all the metabolised glucose should 492
Copyright @ 1974 by Academic Press, Inc. All rights of reproduction in any form reserved.
MEASURING
TOTAL
GLUCOSE
METABOLISM
493
lose its “H to water. Thus the measurement of “H in water should be sufficient to obtain a good estimate of total glucose utilisation. The experimental results reported in this paper show that the theoretical conclusion is valid at least with respect to the metabolism of adipocytes. Measurement of 3H in water therefore provides a simple technique for routine analysis of glucose utilisation by adipocytes. The measurement can be used in conjunction with studies of [l*C]-glucose metabolism. In these experiments incorporation of 3H int.o triglyceride and anions has been shown to be negligible. Furthermore the estimate of glucose utilisation obtained from the [3H] -water studies agrees well with the estimates made from glucose disappearance, and from [U-l&C] -glucose incorporation into metaholites. MATERIALS
AND
METHODS
[S3H]-glucose and [C-l”C]-glucose were obtained from the Radiochemical Centre, Amersham, and were rigorously purified before each experiment by ascending paper chromatography. The solvent used was t-butanol, methyl ethyl ketone, water, and formic acid (44:44:11:0.26 by vol) (21, the solvent front was allowed to run 15 cm and the glucose. which runs with an Rf of 0.10, was eluted with Krebs-Bicarbonate buffer. Purification of [3H]-glucose by repeated solution and lyophilisation and of [‘4C]-glucose by passage through an anion exchange resin is possible but in our view less effective. Adipocytes were prepared from rat epididymal fat pads and incubated essentially as described previously (31. Approximately 4 X lo5 cells were incubated in 0.5 ml of Krebs-Bicarbonate buffer containing glucose and 2% albumin and in addition either 75 nCi of ]5-3H]-glucose or 125 nCi of [‘%I-glucose, or both. The 6 X 1.5 cm plast.ic tubes in which incubation took place were fitted with suba seals from each of which was suspended a plastic well into which base could be injected for the trapping of co,. Incubation was terminated and protein precipitated by injection through t’he suba seal of 0.5 ml of 20% trichloracetic acid and this was followed by the injection of 0.1 ml of 1.0 M hyamine hydroxide into the hanging well. Control samples (known as cell blank samples’) were routinely set, up in which cells were added directly to acidified medium. When the radioactive glucose was properly purified the radioact,ivit! identified as metabolised glucose in these samples was very small. Tritiated water was separated from the incubation mixture by lyophilisation. A simple manifold was constructed which allowed a series of samples to be attached simultaneously to the vacuum pump and Iyophilised independently.
494
BROWN
AND
GARRATT
The lyophilised medium was adjusted to its original volume by the addition of water and, after centrifugation, the supernatant from replicate samples could be analysed for radioactive anions and residual glucose. Glucose was determined in 500 ~1 aliquots of the medium by the glucose oxidase technique. In the experiments reported here radioactive anions were measured in aliquots from samples obtained by pooling the acidified medium from cells incubated under identical conditions. The anions were separated from unreacted glucose and uncharged metabolites by applying 500 ~1 aliquots of the medium to 0.8 X 35 cm glass columns containing 0.6 g of DEAE-Sephadex A-25-120, washing the resin with 50 ml water to remove unbound molecules and eluting the anions with 15 ml of 0.5 M HCl. 1 ml Aliquots of this eluate were counted. Under these conditions mixtures of [ 14C] -glucose and [“Cl -lactate made up in acidified incubation medium were completely separated and the two components quantitatively recovered. Triglycerides and nonesterified fatty acids were extracted from the precipitate in the incubation vial using the method of Dole and Meinertz (4). No heptane-soluble radoactivity could be found in the supernat.ant. Radioactivity was counted in a Nuclear Chicago liquid scintillation counter. [“Cl -0, in hyamine and anions were counted in Bray’s scintillation fluid (5) triglycerides in toluene containing 4.0 g PPO and 0.2 g POPOP per litre, and [ 3H] -HZ0 in toluene-Triton X-100 scintillator (6). Internal standardisation was used to estimate quenching and all counts were corrected. Corrected counts were related to a standard obtained by counting a sample of the original radioactive glucose in the appropriate scintillation fluid. RESULTS
AND
DISCUSSION
An exploratory experiment showed that the specific activity of the lyophilised water was always the same as the specific activity of the solution from which it was derived. 1 ml Samples of tritiated water were taken and different volumes were removed by lyophilisation. Aliquots of the lyophilised water and of the residue were counted. Any difference in the rate of lyophilisation of the heavier tritiated water should result in a higher count in the residue. Table 1 shows that this was not found. The possibility that tritium might be incorporated into metabolites other than water was examined by incubating cells with [5-3H]-glucose and determining the amount of label trapped in hyamine, extracted as triglyceride and retained by an anion exchange resin. The radioactivity detected in the hyamine or in the anion fraction could not be distinguished from the cell blank samples. Table 2 shows that less than 5% of the total metabolised [5-SH]-glucose is found in the triglyceride fraction.
MEASURING
Comparison
TOTAL
GLUCOSE
METABOLISM
TABLE 1 of the Specific Activity of Lyophiliied Tritiated Water Specific Activitv of the Residual Tritiated Watera
with
the
Specific Wt of wat’er collected k)
Residue (cpm/d
0.17 0.20 0.26 0.27 0.30 0.84 0.35 0.35 0.40 0.45 0.46 0.50
4946 4888 5288 5171 4916 4833 4788 4865 4943 4724 4763 4827
Lyophilisate (wdg)
a 1 ml Samples of tritiated water were lyophiiised collected was determined by weighing. The specific and residual water was measured.
5275 5250 5106 5097 5051 4987 4807 4858 4922 4872 4817 4847
activity rat.io (residue/ lyophilisate) 0.94 0.93 1.04 1.01 0.97 0.97 1.00 1 00 1.00 0.98 0.99 1.00
for different times and the amount activity of aliquots of lyophilised
In a parallel experiment cells were incubated with [U-‘%I-glucose and incorporation into the various fractions was determined. When these samples were lyophilised no radioactivity could be detected in the water. The incorporations of label into other metabolites are shown in Table 2. The glucose utilisation determined from [5-3H]-glucose is rather higher than that determined from [U-W] -glucose. The observation that none of the [14C]-metabolites were detected in the lyophilised water means that cells can be exposed to [‘“Cl -glucose and r3H] -glucose simultaneously. The small incorporation of [“HI glucose int.o triglyceride does not affect the determination of [‘“Cl -glucose incorporation into triglyceride since it is possible to count 14C independently of 3H. Table 3 shows the results of an experiment in which glucose utilisation was estimated using [ 5-3H] -glucose and [ U-14C] -glucose in the same incubation vial. Glucose utilisation measured from [ 3H] -glucose will be slightly underestimated because of the incorporation of tritium into triglyceride. In spite of this the [‘*Cl-glucose incorporation into metabolites is consistently less than the incorporation of 3H from [5-3H]glucose into water. Since it is difficult to see how the [5-3H]-glucose can give an overestimate for glucose utilisation these results suggest that there is a systematic underestimation in glucose utilisation determined from 1‘“Cl-glucose metabolism. The overall variation in glucose uptake
+ +
lin
10-S Insu-
M
f + + f
Hz0
1.26 1.02 1.22 1.11
0.29 1.32 0.71 1.53
+ IL + t
0.04 0.20 0.34 0.62
Triglyceride f + + k
Total 1.33 1.77 2.14 1.43
Jncubat,ed
9.27 48.79 16.68 85.02
into
by Adipocytes
3H incorporat,ion
TJ tilisat,ion
2.37 14.83 3.28 21.35
+ f + z!z
CO8
TABLE 2 under Various
0.34 0.68 0.21 0.91
W
3.75 25.83 5.04 39.35
Eit,her
+ f * +
0.46 0.18 0.15 0.78
0.05 1.51 0.55 2.07
8.29 48.75 11.77 80.84
0.79 0.52 0.19 0.89
0.89 1.00 0.71 0.95
Incorporation ratio W/aH
No incorporation under identical
f +_ i f
Total
or v-I%]-Glucosea
products shown. fat cells incubated
+ f k +
Anions
into
[5-3HJ-Glucose
2.77 11.17 4.21 21.33
incorporation
wit,h
Triglyceride
Conditions
utilisation is taken to be the sum of the radioactivity incorporated into the metabolic in metabolites not shown. The figures are the means of three estimates (with SD) from are expressed as nmole glucose/hr/pmole of triglyceride.
8.88 47.47 15.96 83.49
of Glucose
a Total glucose could be detected conditions. Data
100 100 250 250
Glucose (PPl ml)
Estimation
5 $ g $ ~3
9
g
MEASURING
TOTAL
GLUCOSE
METABOLISM
497
M
are expressed
+
200 200
data
-+
100
n The
loInsulin
4 4
2.
192.6 150.4
71.3 96.9 f 5.1 + 9.5
2.0 if 2.7
Residual glucose concentration Wml)
No. of replicate incubations
34
TABLE Utihsation Obtained from Residual Glucose Determined
of Glucose
&J in Table
of Estimates
Glucose (m/ml)
Comparison
14.56 91.78
53.15 5.80
of the Metabolism OxidaseD
f f
10.27 17.68
3.71 f.f 5.03
Glucose disappearance
4 Measurement by Glucose
10.35 89.15
52.21 5.63
+ 1.04 f 7.09
+ 0.70 5.76 zk
aH-glucose metabolism
of [5-*HI-Glucose
1.40 1.02
1.02 1.03
Utilisation ratio diappearance/aH
and of
5
i
3
s b
E
MEASURING
TOTAL
GLUCOSE
METABOLISM
499
obtained by either method does not appear to differ much. However it should be remembered that 14C incorporation into anions was measured in a sample of pooled medium so that an average value for anion production was used for each of the different conditions of incubation. This could give rise to an artificially low estimate of variation for the [‘4C]-glucose measurements. The apparent variation may also be kept artificially low by the fortuitous combination of values obtained for CO2 and triglyceride production each of which is subject to its own error. In a final experiment glucose utilisation was measured wit,h [5-3H]glucose and by using glucose oxidase to determine the glucose concentration in the medium before and after the incubation. The results are shown in Table 4. In this experiment glucose utilisation was so low in t,he absence of insulin that it was not possible to get a reliable estimate of the utilisation by the glucose oxidase method. This is apparent from the reproducible values obtained for residual glucose which nevertheless give huge variations in values obtained for glucose utilisation in these samples. However in the presence of insulin there was close agreement between the values obtained for glucose utilisation by the two methods. We conclude that the incorporation of 3H from [5-3H]-glucose provides a simple and satisfactory method for determining glucose utilisation by isolated adipocytes. REFERENCES J. H., WEERASINGHE, L. C. C.. BASSETT, J. M., AND RANDLE, P. J. (1972) Biochem. J. 126, 525-532. FINK, Ii., CLINE, R. E., AND FINK, R. M. (1963) Anal. Chem. 35, 389. GARRATT. C. J., HARRISON, D. M., AND WICKS, M. P. (1972) Biochem. J. 126, 123. DOLE, V. P., AND MEINERTZ, H. (1960) J. Biol. Chem. 235, 2575. BRAY, G. A. (1960) Biochemidry 1, 279. PATTERSON, M. S., AND GREEN, R. C. (1965) And. Chem. 37, 854.
1. ASHCROFT,
2. 3. 4.
5. 6.
S.
BIOCIlEMISTRY
A Simple
Method
by Isolated
61, 492-499 (1974)
for Determining Adipocytes D. BROWN
Department Heslington,
Using
Glucose
[ 5”H]
Utilisation
-Glucose
C. J. GARRATT
AND
of Chemistry, York YOI
Received February
Total
University 600,
Great
of York, Britain
25, 1974; accepted April 26, 1974
Glucose utilisation by adipocytes incubated with and without insulin and at two concentrations of extracellular glucose has been estimated by three different procedures. Glucose disappearance from the medium was calculated by using glucose oxidase to determine the glucose concentration remaining after incubation and comparing this with the glucose concentration in standard solutions made up by appropriate dilution of the original medium. [U-“Cl-glucose utilisation was calculated by summing the 14C found in CO,, triglycerides, and anions. [“HI-Ha0 formation from [5-‘HI-glucose was the third measure of glucose utilisation. All three methods gave similar answers, but the L5-3H1-glucose is simpler to use than NJ-“Cl-glucose and gives substantially more reproducible results than glucose oxidase.
In many studies of metabolism in isolated tissues it is necessary or useful to estimate exogenous glucose utilisation by the tissue. The classical method of doing this is to measure the difference between the glucose present at the beginning and at the end of an experiment. However, in order to measure glucose utilisation with precision by this technique it is necessary for a significant, fraction of the glucose to be used and this is not always desirable. An alternative approach is to measure the incorporation of radioactivity from glucose labeled either with 14C or 3H into all the metabolic products. With most tissues the number of products into which [I%]-glucose can be incorporated is so large that this label is too cumbersome for routine use. However, as mentioned by Ashcroft et al. (1) the use of [PHI -glucose avoids this problem because water is almost the only radioactive metabolic product of [5-3H]-glucose. The label from this position exchanges with water during triose phosphate isomerisation (1). If this reaction is not in complete equilibrium then further exchange can take place at the enolase reaction. Only glucose which is converted directly to glycogen does not pass through these steps. In adipose tissue where synthesis of glycogen is normally negligible, it follows that all the metabolised glucose should 492
Copyright @ 1974 by Academic Press, Inc. All rights of reproduction in any form reserved.
MEASURING
TOTAL
GLUCOSE
METABOLISM
493
lose its “H to water. Thus the measurement of “H in water should be sufficient to obtain a good estimate of total glucose utilisation. The experimental results reported in this paper show that the theoretical conclusion is valid at least with respect to the metabolism of adipocytes. Measurement of 3H in water therefore provides a simple technique for routine analysis of glucose utilisation by adipocytes. The measurement can be used in conjunction with studies of [l*C]-glucose metabolism. In these experiments incorporation of 3H int.o triglyceride and anions has been shown to be negligible. Furthermore the estimate of glucose utilisation obtained from the [3H] -water studies agrees well with the estimates made from glucose disappearance, and from [U-l&C] -glucose incorporation into metaholites. MATERIALS
AND
METHODS
[S3H]-glucose and [C-l”C]-glucose were obtained from the Radiochemical Centre, Amersham, and were rigorously purified before each experiment by ascending paper chromatography. The solvent used was t-butanol, methyl ethyl ketone, water, and formic acid (44:44:11:0.26 by vol) (21, the solvent front was allowed to run 15 cm and the glucose. which runs with an Rf of 0.10, was eluted with Krebs-Bicarbonate buffer. Purification of [3H]-glucose by repeated solution and lyophilisation and of [‘4C]-glucose by passage through an anion exchange resin is possible but in our view less effective. Adipocytes were prepared from rat epididymal fat pads and incubated essentially as described previously (31. Approximately 4 X lo5 cells were incubated in 0.5 ml of Krebs-Bicarbonate buffer containing glucose and 2% albumin and in addition either 75 nCi of ]5-3H]-glucose or 125 nCi of [‘%I-glucose, or both. The 6 X 1.5 cm plast.ic tubes in which incubation took place were fitted with suba seals from each of which was suspended a plastic well into which base could be injected for the trapping of co,. Incubation was terminated and protein precipitated by injection through t’he suba seal of 0.5 ml of 20% trichloracetic acid and this was followed by the injection of 0.1 ml of 1.0 M hyamine hydroxide into the hanging well. Control samples (known as cell blank samples’) were routinely set, up in which cells were added directly to acidified medium. When the radioactive glucose was properly purified the radioact,ivit! identified as metabolised glucose in these samples was very small. Tritiated water was separated from the incubation mixture by lyophilisation. A simple manifold was constructed which allowed a series of samples to be attached simultaneously to the vacuum pump and Iyophilised independently.
494
BROWN
AND
GARRATT
The lyophilised medium was adjusted to its original volume by the addition of water and, after centrifugation, the supernatant from replicate samples could be analysed for radioactive anions and residual glucose. Glucose was determined in 500 ~1 aliquots of the medium by the glucose oxidase technique. In the experiments reported here radioactive anions were measured in aliquots from samples obtained by pooling the acidified medium from cells incubated under identical conditions. The anions were separated from unreacted glucose and uncharged metabolites by applying 500 ~1 aliquots of the medium to 0.8 X 35 cm glass columns containing 0.6 g of DEAE-Sephadex A-25-120, washing the resin with 50 ml water to remove unbound molecules and eluting the anions with 15 ml of 0.5 M HCl. 1 ml Aliquots of this eluate were counted. Under these conditions mixtures of [ 14C] -glucose and [“Cl -lactate made up in acidified incubation medium were completely separated and the two components quantitatively recovered. Triglycerides and nonesterified fatty acids were extracted from the precipitate in the incubation vial using the method of Dole and Meinertz (4). No heptane-soluble radoactivity could be found in the supernat.ant. Radioactivity was counted in a Nuclear Chicago liquid scintillation counter. [“Cl -0, in hyamine and anions were counted in Bray’s scintillation fluid (5) triglycerides in toluene containing 4.0 g PPO and 0.2 g POPOP per litre, and [ 3H] -HZ0 in toluene-Triton X-100 scintillator (6). Internal standardisation was used to estimate quenching and all counts were corrected. Corrected counts were related to a standard obtained by counting a sample of the original radioactive glucose in the appropriate scintillation fluid. RESULTS
AND
DISCUSSION
An exploratory experiment showed that the specific activity of the lyophilised water was always the same as the specific activity of the solution from which it was derived. 1 ml Samples of tritiated water were taken and different volumes were removed by lyophilisation. Aliquots of the lyophilised water and of the residue were counted. Any difference in the rate of lyophilisation of the heavier tritiated water should result in a higher count in the residue. Table 1 shows that this was not found. The possibility that tritium might be incorporated into metabolites other than water was examined by incubating cells with [5-3H]-glucose and determining the amount of label trapped in hyamine, extracted as triglyceride and retained by an anion exchange resin. The radioactivity detected in the hyamine or in the anion fraction could not be distinguished from the cell blank samples. Table 2 shows that less than 5% of the total metabolised [5-SH]-glucose is found in the triglyceride fraction.
MEASURING
Comparison
TOTAL
GLUCOSE
METABOLISM
TABLE 1 of the Specific Activity of Lyophiliied Tritiated Water Specific Activitv of the Residual Tritiated Watera
with
the
Specific Wt of wat’er collected k)
Residue (cpm/d
0.17 0.20 0.26 0.27 0.30 0.84 0.35 0.35 0.40 0.45 0.46 0.50
4946 4888 5288 5171 4916 4833 4788 4865 4943 4724 4763 4827
Lyophilisate (wdg)
a 1 ml Samples of tritiated water were lyophiiised collected was determined by weighing. The specific and residual water was measured.
5275 5250 5106 5097 5051 4987 4807 4858 4922 4872 4817 4847
activity rat.io (residue/ lyophilisate) 0.94 0.93 1.04 1.01 0.97 0.97 1.00 1 00 1.00 0.98 0.99 1.00
for different times and the amount activity of aliquots of lyophilised
In a parallel experiment cells were incubated with [U-‘%I-glucose and incorporation into the various fractions was determined. When these samples were lyophilised no radioactivity could be detected in the water. The incorporations of label into other metabolites are shown in Table 2. The glucose utilisation determined from [5-3H]-glucose is rather higher than that determined from [U-W] -glucose. The observation that none of the [14C]-metabolites were detected in the lyophilised water means that cells can be exposed to [‘“Cl -glucose and r3H] -glucose simultaneously. The small incorporation of [“HI glucose int.o triglyceride does not affect the determination of [‘“Cl -glucose incorporation into triglyceride since it is possible to count 14C independently of 3H. Table 3 shows the results of an experiment in which glucose utilisation was estimated using [ 5-3H] -glucose and [ U-14C] -glucose in the same incubation vial. Glucose utilisation measured from [ 3H] -glucose will be slightly underestimated because of the incorporation of tritium into triglyceride. In spite of this the [‘*Cl-glucose incorporation into metabolites is consistently less than the incorporation of 3H from [5-3H]glucose into water. Since it is difficult to see how the [5-3H]-glucose can give an overestimate for glucose utilisation these results suggest that there is a systematic underestimation in glucose utilisation determined from 1‘“Cl-glucose metabolism. The overall variation in glucose uptake
+ +
lin
10-S Insu-
M
f + + f
Hz0
1.26 1.02 1.22 1.11
0.29 1.32 0.71 1.53
+ IL + t
0.04 0.20 0.34 0.62
Triglyceride f + + k
Total 1.33 1.77 2.14 1.43
Jncubat,ed
9.27 48.79 16.68 85.02
into
by Adipocytes
3H incorporat,ion
TJ tilisat,ion
2.37 14.83 3.28 21.35
+ f + z!z
CO8
TABLE 2 under Various
0.34 0.68 0.21 0.91
W
3.75 25.83 5.04 39.35
Eit,her
+ f * +
0.46 0.18 0.15 0.78
0.05 1.51 0.55 2.07
8.29 48.75 11.77 80.84
0.79 0.52 0.19 0.89
0.89 1.00 0.71 0.95
Incorporation ratio W/aH
No incorporation under identical
f +_ i f
Total
or v-I%]-Glucosea
products shown. fat cells incubated
+ f k +
Anions
into
[5-3HJ-Glucose
2.77 11.17 4.21 21.33
incorporation
wit,h
Triglyceride
Conditions
utilisation is taken to be the sum of the radioactivity incorporated into the metabolic in metabolites not shown. The figures are the means of three estimates (with SD) from are expressed as nmole glucose/hr/pmole of triglyceride.
8.88 47.47 15.96 83.49
of Glucose
a Total glucose could be detected conditions. Data
100 100 250 250
Glucose (PPl ml)
Estimation
5 $ g $ ~3
9
g
MEASURING
TOTAL
GLUCOSE
METABOLISM
497
M
are expressed
+
200 200
data
-+
100
n The
loInsulin
4 4
2.
192.6 150.4
71.3 96.9 f 5.1 + 9.5
2.0 if 2.7
Residual glucose concentration Wml)
No. of replicate incubations
34
TABLE Utihsation Obtained from Residual Glucose Determined
of Glucose
&J in Table
of Estimates
Glucose (m/ml)
Comparison
14.56 91.78
53.15 5.80
of the Metabolism OxidaseD
f f
10.27 17.68
3.71 f.f 5.03
Glucose disappearance
4 Measurement by Glucose
10.35 89.15
52.21 5.63
+ 1.04 f 7.09
+ 0.70 5.76 zk
aH-glucose metabolism
of [5-*HI-Glucose
1.40 1.02
1.02 1.03
Utilisation ratio diappearance/aH
and of
5
i
3
s b
E
MEASURING
TOTAL
GLUCOSE
METABOLISM
499
obtained by either method does not appear to differ much. However it should be remembered that 14C incorporation into anions was measured in a sample of pooled medium so that an average value for anion production was used for each of the different conditions of incubation. This could give rise to an artificially low estimate of variation for the [‘4C]-glucose measurements. The apparent variation may also be kept artificially low by the fortuitous combination of values obtained for CO2 and triglyceride production each of which is subject to its own error. In a final experiment glucose utilisation was measured wit,h [5-3H]glucose and by using glucose oxidase to determine the glucose concentration in the medium before and after the incubation. The results are shown in Table 4. In this experiment glucose utilisation was so low in t,he absence of insulin that it was not possible to get a reliable estimate of the utilisation by the glucose oxidase method. This is apparent from the reproducible values obtained for residual glucose which nevertheless give huge variations in values obtained for glucose utilisation in these samples. However in the presence of insulin there was close agreement between the values obtained for glucose utilisation by the two methods. We conclude that the incorporation of 3H from [5-3H]-glucose provides a simple and satisfactory method for determining glucose utilisation by isolated adipocytes. REFERENCES J. H., WEERASINGHE, L. C. C.. BASSETT, J. M., AND RANDLE, P. J. (1972) Biochem. J. 126, 525-532. FINK, Ii., CLINE, R. E., AND FINK, R. M. (1963) Anal. Chem. 35, 389. GARRATT. C. J., HARRISON, D. M., AND WICKS, M. P. (1972) Biochem. J. 126, 123. DOLE, V. P., AND MEINERTZ, H. (1960) J. Biol. Chem. 235, 2575. BRAY, G. A. (1960) Biochemidry 1, 279. PATTERSON, M. S., AND GREEN, R. C. (1965) And. Chem. 37, 854.
1. ASHCROFT,
2. 3. 4.
5. 6.
S.