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Effect of cell concentration on the metabolism of normal and diabetic leucocytes in vitro
Effect of Cell Concentration on the Metabolism of Normal and Diabetic Leucocytes in Vitro By VIcGo ESMANK The glucose uptake, lactic acid and pyruvic acid production, glycogen synthesis, oxygen uptake and carbon dioxide production were studied in leucocytes from 54 nondiabetic and 30 diabetic subjects. In normal leucocytes the magnitude of these parameters, except the CO2 production, was influenced by the concentration of leucocytes used in the individual experiments, and decreased with increasing cell concentration. In diabetic leucocytes a similar dependency was found for the oxygen uptake and
E
pyruvic acid production, but not for the glucose uptake, lactic acid production and glycogen synthesis, leading to the situation that these parameters were lower than in normal leucocytes at low cell concentrations, while the reverse was true at high leucocyte concentrations. The lower glucose uptake, lactic acid production and glycogen synthesis of diabetic leucocytes are not thought to be secondary to a defective transport mechanism for glucose.
INFORMATION about the intermediary metabolism of the insulin-deficient animal has been gained from investigations of isolated tissues from several species. 1 Obvious difficulties in securing material has largely prevented the confirmation of these results in tissues from diabetic man. During the last decade, however. methods have been developed, which permit the study of at least 1 human tissue, the leucocytes, which can be suspended in an artificial medium and thus at a cellular level-without interference from connective tissue, membranes and blood vessels-are able to supply information about the intermediary metabolism in diabetes mellitus, and thereby perhaps form a link to our vast knowledge of insulin-deprived animal tissue. Previous investigations have shown a lower uptake of glucose by lrucocytes from patients with a moderately severe or severe diabetes than by leucocytes from normal subjects, whereas the lactic acid production has been found to be reduced or unaltered.“J The glucose uptake of leucocytes from patients with milder diabetes did not differ from the normal subject.a The results of earlier investigations have suggested that the oxygen uptake of leucocytes is inhibited in experiments in which high concentrations of lencocytes suspended in plasma have been used,“~i whereas this “crowding effect” did not occur with cells suspended in buffer.8-10 Recently a crowding effect on both oxygen consumption and lactic acid production has been demonstrated in leucocytes incubated in serum.” The present report provides evidence of the presence of a crowding effect on glucose metabolism and respiration of leucocytes incubated in buffer and, in ~~~ .~-... -._ XTENSIVE
From the Second Depurtment Aarhus, Denmark. Rexxived for publication
of Internal
NOD. 11,
Medicine,
.4afhu.s Univertity
Medical
School,
1963. :354 ~l~v>\sur.~s~r I’or.. 13, No. ii (APRIL), I!334
CELL CONCENTRATION
EFFECT
ON METABOLISM
OF LEUCOCYTES
355
addition, that this effect is different in normal and diabetic leucocytes, to diametrically opposite interpretations of the results of experiments out with high and low leucocyte concentrations. MATERIAL Fifty-four
nondiabetic
subjects
were
leading carried
AND METHODS
studied,
twenty-seven
were
laboratory personnel, the other were patients in the ward suffering not known to be associated with abnormal leucocyte metabolism. The diabetic group consisted of 30 patients. Eight were under
medical from
students
various
40 years
or
diseases
of age,
ten
were 40-59 years, and 12 were more than 60 years old. Nineteen patients had never received insulin and were nearly all newly diagnosed cases. In the remainder insulin was withdrawn 36-72 hours before the investigation. As judged by blood sugar, glucoseuria, ketoneuria, and insulin requirement 13 had severe diabetes, 8 had moderate, and 9 mild diabetes, which in only 2 patients, however, could be controlled without insulin or oral drugs. The subjects were fasting and had rested in bed for 30 minutes before the investigation.12 The leucocytes were isolated from EDTA (ethylenediaminetetra-acetate) stabilized venous blood by increasing the rouleaux formation of the erythrocytes by means of dextran (fraction TDR-205-11-B-1, intrinsic viscosity 0.4, Pharmacia Ltd.) in a final concentration of 1 per cent. After 30 minutes the sedimentation of the erythrocytes was completed and the supernatant plasma containing the leucocytes, thrombocytes, and the remaining erythrocytes were centrifuged at 125-175 G for 4 minutes, followed by resuspension of the leucocytes in buffer and renewed centrifugation for 2 minutes. The leucocytes were finally suspended in a buffer containing NaCl 91.8 mM, KC1 4.7 mM, CaCla 2.5 mM, KH,PO, 1.2 mM, MgSO, 1.2 mM, Na-acetate acid 0.06 mM, glucose 16.7 mM, and gelatine 1 per phere at 37 C. pH was 7.43. The whole isolation trifugations, was carried out in a warm room at 37 C. By this method the plasma, dextran, and EDTA
24.4 mM, NaHCO, 25.0 mM, ascorbic cent. Under a 5 per cent COa-atmosprocedure, except the two short cenwere diluted
at least
2,000
times
and
the final leucocyte suspension contained 1 thrombocyte per 3-4 leucocytes, and %3 erythrocytes per leucocyte. The leucocytes were counted in duplicate on each of two pipettings. The differential count was made on 260 cells. In each of 50 consecutive experiments with normal and diabetic leucocytes the final suspensions contained 85.2 per cent -t 0.6 (s.e.m.) and 86.8 per cent + 0.6 polymorphonuclear leucocytes, respectively. dioxide production ( ACO,), glucose uptake The oxygen uptake ( AO, ), carbon ( Aglucose), lactic acid production ( Alactic acid), pyruvic acid production ( Apyruvic acid), and glycogen synthesis ( Aglycogen) were measured on 3 ml. aliquots incubated at 37 C. for 2 hours in specially designed reaction vessel@ attached to a Summerson differential manometer.14 minutes with a mixture vapour at 37 C. Glucose, lactic
acid
Before incubation of 95 per cent 0, and
pyruvic
acid
the suspensions and 5 per cent
were
determined
glycogen was degraded as previously described.rsJs The experiments were carried out with cell concentrations
were equilibrated for 30 CO, saturated with water by
enzymatic
of 8-40
methods
x lOs/ml.
and
In 2 and
5 patients, respectively, normal and diabetic leucocytes were investigated at two different cell concentrations. The results were calculated as ~mole/lOla leucocytes/hour. Leukocytes incubated in buffer ordinarily clump. The addition of gelatine and sodiumacetate completely prevented this phenomenon, as judged by microscopic examination and by the presence of a constant leucocyte concentration throughout incubation. In 16 experiments the average percentage difference betwen leucocyte counts before and after incubation was $0.4 per cent + 0.78. As evaluated by other parameters: the occurrence of a constant respiration with time, a potassium uptake (31 ,nEq. K/l010 leucocytes/h),
356 and of a barely to be intact.13
VICGO ESMANN
demonstrable
leakage
of alkaline
phosphatase,
the leukocytes
seemed
RESULTS
When the results were computed as arithmetical means no difference was found between normal and diabetic leucocytes; when, however, the results were calculated as a logarithmic function of the leucocyte concentration used in the actual experiments (fig. l), a difference betwen normal and diabetic leucocytes became apparent, as a crowding effect for Aglucose, Alactic acid, and Aglycogen, amounting to a decrease of 50 per cent, was present for NON-DIABETICS t
..
DIABETICS ---__
301 ---+------
,+&
.
1
+
3.2 ’
Fig. 1.-See
--__
--__.
opposite page.
-
CLO-GL.“l#/ ml
CELL CONCENTFIATION EFFECT ON METABOLISM
OF LEUCOCYTES
357
nondiabetic, but not for diabetic, leucocytes. No difference was found between the crowding effect on normal and diabetic cells with respect to Apyruvic acid and A02. The leucocyte concentration did not influence ACOs, either in normal or in diabetic cells. It should be noted that for diabetic leucocytes the levels of the regression lines for Aglucose, Alactic acid, and in principle also for Aglycogen, are such that they intersect the corresponding regression lines for normal leucocytes approximately at the center, leading to the circumstance that these parameters are lower in diabetic leucocytes in experiments performed with low leucocyte concentrations, and accounting, furthermore, for the lack of difference between the arithmetical means of the parameters. Figure 2 visualizes the differences and similiarities between normal and diabetic leucocytes obtained by interpolating on the regression lines in figure 1 at a leucocyte concentration of 107/ml. It is seen that diabetic leucocytes have a lower glucose uptake, lactic acid production and glycogen synthesis than normal leucocytes, whereas no differences are present in pyruvic acid production and respiration. The differences in glucose metabolism have been ascertained by actual experiments carried out using a fixed leucocytes concentration of 107/m1.13 DISCUSSION The experiments have shown the presence of a crowding effect when leucocytes are incubated in buffer; not only as demonstrated earlier on AO, and Alactic acid,l’ but also on Aglucose, Aglycogen and Apyruvic acid. There is thus no reason to uphold the hypothesis postulated by Hartmann1o that the crowding effect is a phenomenon which occurs only on the incubation of leucocytes in plasma. The cause of the crowding effect is unknown, but it has been demonstrated that it is not due to limiting concentrations of glucose or inorganic phosphate, nor can the high concentrations of lactic acid (app. 80 mg./lOO ml.) produced during incubation with high concentrations of leucocytes be held responsible.13
Fig. l.-The correlation (log y = a + hx) between leucocyte concentration and log A glucose, log A lactic acid, log A glycogen, log A pyruvic acid, log A O2 and log A COz for leucocytes from nondiabetic (ND) and diabetic (D) subjects. The regression lines and standard deviation from the regression lines (%. iog,) is
shown. The correlation coefficients r, their p-value, and the differences between the corresponding regression coefficients were: Glucose: rs,, = -0.52, p < 0.005; r, = -0.09, p < 0.70; b. - b, = 0.0081, p < 0.10 (t = 1.93, to,o5 = 1.99). Lactic acid: rSD =“-0.48, p < 0.001; rD = -0.08, p < 0.70; b ND- b, = 0.0083, p < 0.025. Glycogen: rND= -0.37, p < 0.01; rl, = +0.22, p < 0.30; b SD - b, = 0.0194, p < 0.01. Pyruvic acid: r,, = -0.81, p < 0.001; r,, = -0.69, p < 0.001; b - b, = 0.0083, p < 0.20. Oxygen: rKD = -$24, p < 0.005; rD = -0.40, p < 0.02; b ND - b, = 0.0029, p < 0.40. Carbon dioxide: rsU = -0.01, rD = +0.17, p < 0.40 b XI) - b, = 0.0034, p < 0.60.
VIGGO ESMANN
bb0
Glucose Lacllc
I 720
1200
acid
1 730
I Glycogen
Pyruvlc
ac Id Oxygen
Carbon
do-oxide pmol 500
100
l
Normals
/lO”Leuc./h 1000
0
Diabetics
Fig. B.-Values for carbohydrate metabolism and respiration of nondiabetic and diabetic leucoyctes obtained by interpolation on the regression lines of figure 1 at a leucocyte concentration of 105/ml. and expressed in pmole/lO1O leucocytes/h (geometric means).
The presence of a difference in the crowding effect between normal and diabetic leucocytes makes it difficult to evaluate the results of earlier investigations carried out with different cell concentrations;“-* and suggests that experiments with leucocytes should be carried out at low and virtually constant concentrations. Leucocytes at a concentration of 107/ml. have a high glucose uptake as compared to rat liver slices,l” rat diaphragm,” and rat epididymal fat,l* the values being approximately 14 mg., 7 mg., 2 mg., and 0.4 mg. glucose/Gm. wet weight/hour, respectively. The respiration is low, with QoZ = app.2, where the corresponding value for liver,l” diaphragm,20 and adipose tissuelR are Qo~ = app. 10. A characteristic of leucocytes, by which they differ from the majority of other normal tissues, is that these cells have a high aerobic glycolysis. The demonstration of the presence of a glycogen synthesis (2.4 mg./Gm. wet weight/hour) is contrary to the findings of McKinney et a1.,2r a fact which can probably be referred to different experimental conditions. The amount of glycogen synthesized is somewhat higher than that found by Rafaelsen22 in rat diaphragm (1.4 mg. glycogen/Gm. wet weight/hour). Despite large standard deviation from the regression line of the correlation between leucocyte concentration and log aC02, the probability that RQ at a leucocyte concentration of 107/ml. exceeds 0.5 - 0.8 is <0.05. Under the present experimental circumstances, where a large amount of glucose is catabolized, the finding of a low RQ suggests the occurrence of a CO2 fixation. This process has been demonstrated to occur in leucocytes from rabbit and guinea pig exudates .23,2* A stimulatory effect of CO2 on the oxygen con-
CELL
CONCENTRATION
EFFECT
ON METABOLISM
OF LEUCOCYTES
359
sumption of human leucocytes have been shown by Bicz,25 who suggested that the effect was due to a fixation of CO2 to p-enolpyruvate. It has been shown that Aglucose, Alactic acid, and Aglycogen are reduced in diabetic leucocytes. This reduction is of an extent comparable with the increase in these parameters which occurs when diabetics are treated with insulin.28 It has also been shown that insulin treatment is capable of restoring the reduced glycogen content of diabetic leucocytes to normal levels.15 The fact that the stimulatory effect of insulin in viva is not demonstrable within minutes, but has first been demonstrated after 24 hours of insulin treatment, together with the fact that the presence of a specific effect of insulin on leucocytes in vitro is questionable, suggest that in leucocytes the decreased carbohydrate metabolism of diabetic leucocytes cannot be caused by a defect in the mechanism of the transport of glucose. ACKNOWLEDGMENT I gratefully acknowledge the financial support from the Danish State Research Foundation, Aarhus University Research Foundation, and the Rheinholdt W. Jorck and Wife’s Foundation. Pharmacia Ltd., Sweden, generously supplied the high molecular dextran. I am indebted to Mrs. Ruth Malmkjaer for her technical assistance. REFERENCES 1. Krahl, M. E.: The Action of Insulin on Cells. New York, Academic Press, 1961. 2. Martin, S. P., McKinney, G. R., Green, R., and Becker, C.: The influence of glucose, fructose and insulin on the metabolism of leucocytes of healthy and diabetic subjects. J. Clin. Invest. 32: 1171, 1953. 3. Dumm, M.: Glucose utilization and lactate production by leucocytes of patients with diabetes mellitus. Proc. Sot. Exp. Biol. Med. 95:571, 1957. 4. -: Effect of tolbutamide on glucose uptake by leukocytes of patients with diabetes mellitus. J. Appl. Physiol. 14: 1023, 1959. 5. Barron, E. G., and Harrop, G. A., Jr.: Studies on cell metabolism. V. The metabolism of leucocytes. J. Biol. Chem. 84:89, 1929. 6. Soffer, J. L., and Wintrobe, M. M.: The metabolism of leucocytes from normal and leukaemic blood. J. Clin. Invest. 11:661, 1932. 7. Bird, R. M., Clemens, J. A., and Becker, L. M.: The metabolism of leucocytes taken from peripheral blood of leukaemic patients. Cancer 4: 1009, 1951. 8. Ponder, E., and McLeod, J.: The effect
of haemolytic substances on white cell respiration. J. Gen. Physiol. 20:267, 1936. 9. Marinelarena, R.: The Effects of Various Chemical Substances and Bacteria on the Glycolytic and Respiratory Activities of Leucocytes. Diss., Univ. Michigan, 1950. 10. Hartmann, J. D.: Effect of cell concentration on oxygen consumption of leucocytes under varying conditions. Proc. Sot. Exp. Biol. Med. 79:3, 1952. 11. Luganova, I. S., and Seits, I. F.: 0 kolichestvennoi kharakteristike dykhaniya i glikoliza v belykh krovyanykh kletkakh cheloveka. Biull. Exp. Biol. Med. 12:57, 1958. 12. Bisset, S. K., and Alexander, W. D.: Effect of muscular activity prior to venepuncture on the respiration of leucocytes in uitru. Nature 181:909, 1958. 13. Esmann, V.: Carbohydrate Metabolism and Respiration in Leucocytes from Normal and Diabetic Subjects. Aarhus (Denmark), University Press, 1962. 14. Summerson, W. N.: Combination manometer. J. Biol. Chem. 131:579, 1939.
360
. * VIC(‘0
15. Esmann, V.: The glycogen content of leucocytes from diabetic and nondiabetic patients. Stand. J. Clin. Lab. Invest. 13: 134, 1961. 16. Renold, A. E., Teng, C. T., Nesbett, F. B., and Hastings, A. B.: Studies on carbohydrate metabolism in rat liver slices. II. The effect of fasting and of hormonal deficiencies. J. Biol. Chem. 204:533, 1953. 17. Rafaelsen, 0. J., and Lundbaek, K.: Action of carbutamide on isolated diaphragm of alloxan-diabetic rats. Metabolism 8:757, 1959. 18. Jungas, R. L., and Ball, E. G.: Studies on the metabolism of adipose tissue. V. The effect of a growth hormone preparation and insulin on the oxygen glucose uptake, and consumption, lactic acid production. J. Biol. Chem. 235: 1894, 1960. 19. Elliott, K. A. C., Greig, M. E., and Benoy, M. P.: The metabolism of lactic and pyruvic acids in normal and tumour tissue. II. Rat liver, brain and testis. Biochem. J. 31:1003, 1937. 20. Stadie, W. S., and Zapp, J. A.: The effect of insulin upon the synthesis of
ESMANN
glycogen by rat diaphragm in u&o. J. Biol. Chem. 170:55, 1947. 21
McKinney,
G. R.,
dles, R. W., tory
and
man
glycolytic
leucocytes
Rafaelsen,
0.
diabetic
J.:
metabolism
oitro.
Action
of
the
isolated
corporation
III.
W.,
Metabolic
metabolism
Carbon
dioxide
ide tension
Fed. Proc.
Influenw
tion.
Cancer
26. Esmann. cocytes.
V.:
M.
19:42,
Effect
Diabetes
1960. diox-
of nor-
human leucocytes.
on endogenous Res.
L.: with
of carbon
on the respiration
mal and leukaemic I.
Biophys.
associated
The influence
in-
polymorpho-
a~ld Karnowsky, pathways
dia-
1959.
R., and Ljung-
in the rabbit
phagocytosis. 25. Bicz, W.:
rat
8: 195,
nucIear leucocyte. Biochim. Acta 49:593, 1961. 24. Evans,
anti-
carbohydrate
Carbohydrate
in leucocytes.
Appl.
of oral
the
Metabolism
L.:
of huJ.
on
23. Noble, E. P.. Stjemholm, dahl,
Run-
Respira-
195253.
drugs
phragm.
S. P., R.:
activities
in
Physiol. 5:335, 22
Martin,
and Green,
20:184,
respira1960.
of insulin 12:fi45,
1963.
Viggo Esmann, M.D., Senior Physician, Department of Medicine C, Copenhagen County Hospital, Glostmp, Denmark.
on le11-
E
pyruvic acid production, but not for the glucose uptake, lactic acid production and glycogen synthesis, leading to the situation that these parameters were lower than in normal leucocytes at low cell concentrations, while the reverse was true at high leucocyte concentrations. The lower glucose uptake, lactic acid production and glycogen synthesis of diabetic leucocytes are not thought to be secondary to a defective transport mechanism for glucose.
INFORMATION about the intermediary metabolism of the insulin-deficient animal has been gained from investigations of isolated tissues from several species. 1 Obvious difficulties in securing material has largely prevented the confirmation of these results in tissues from diabetic man. During the last decade, however. methods have been developed, which permit the study of at least 1 human tissue, the leucocytes, which can be suspended in an artificial medium and thus at a cellular level-without interference from connective tissue, membranes and blood vessels-are able to supply information about the intermediary metabolism in diabetes mellitus, and thereby perhaps form a link to our vast knowledge of insulin-deprived animal tissue. Previous investigations have shown a lower uptake of glucose by lrucocytes from patients with a moderately severe or severe diabetes than by leucocytes from normal subjects, whereas the lactic acid production has been found to be reduced or unaltered.“J The glucose uptake of leucocytes from patients with milder diabetes did not differ from the normal subject.a The results of earlier investigations have suggested that the oxygen uptake of leucocytes is inhibited in experiments in which high concentrations of lencocytes suspended in plasma have been used,“~i whereas this “crowding effect” did not occur with cells suspended in buffer.8-10 Recently a crowding effect on both oxygen consumption and lactic acid production has been demonstrated in leucocytes incubated in serum.” The present report provides evidence of the presence of a crowding effect on glucose metabolism and respiration of leucocytes incubated in buffer and, in ~~~ .~-... -._ XTENSIVE
From the Second Depurtment Aarhus, Denmark. Rexxived for publication
of Internal
NOD. 11,
Medicine,
.4afhu.s Univertity
Medical
School,
1963. :354 ~l~v>\sur.~s~r I’or.. 13, No. ii (APRIL), I!334
CELL CONCENTRATION
EFFECT
ON METABOLISM
OF LEUCOCYTES
355
addition, that this effect is different in normal and diabetic leucocytes, to diametrically opposite interpretations of the results of experiments out with high and low leucocyte concentrations. MATERIAL Fifty-four
nondiabetic
subjects
were
leading carried
AND METHODS
studied,
twenty-seven
were
laboratory personnel, the other were patients in the ward suffering not known to be associated with abnormal leucocyte metabolism. The diabetic group consisted of 30 patients. Eight were under
medical from
students
various
40 years
or
diseases
of age,
ten
were 40-59 years, and 12 were more than 60 years old. Nineteen patients had never received insulin and were nearly all newly diagnosed cases. In the remainder insulin was withdrawn 36-72 hours before the investigation. As judged by blood sugar, glucoseuria, ketoneuria, and insulin requirement 13 had severe diabetes, 8 had moderate, and 9 mild diabetes, which in only 2 patients, however, could be controlled without insulin or oral drugs. The subjects were fasting and had rested in bed for 30 minutes before the investigation.12 The leucocytes were isolated from EDTA (ethylenediaminetetra-acetate) stabilized venous blood by increasing the rouleaux formation of the erythrocytes by means of dextran (fraction TDR-205-11-B-1, intrinsic viscosity 0.4, Pharmacia Ltd.) in a final concentration of 1 per cent. After 30 minutes the sedimentation of the erythrocytes was completed and the supernatant plasma containing the leucocytes, thrombocytes, and the remaining erythrocytes were centrifuged at 125-175 G for 4 minutes, followed by resuspension of the leucocytes in buffer and renewed centrifugation for 2 minutes. The leucocytes were finally suspended in a buffer containing NaCl 91.8 mM, KC1 4.7 mM, CaCla 2.5 mM, KH,PO, 1.2 mM, MgSO, 1.2 mM, Na-acetate acid 0.06 mM, glucose 16.7 mM, and gelatine 1 per phere at 37 C. pH was 7.43. The whole isolation trifugations, was carried out in a warm room at 37 C. By this method the plasma, dextran, and EDTA
24.4 mM, NaHCO, 25.0 mM, ascorbic cent. Under a 5 per cent COa-atmosprocedure, except the two short cenwere diluted
at least
2,000
times
and
the final leucocyte suspension contained 1 thrombocyte per 3-4 leucocytes, and %3 erythrocytes per leucocyte. The leucocytes were counted in duplicate on each of two pipettings. The differential count was made on 260 cells. In each of 50 consecutive experiments with normal and diabetic leucocytes the final suspensions contained 85.2 per cent -t 0.6 (s.e.m.) and 86.8 per cent + 0.6 polymorphonuclear leucocytes, respectively. dioxide production ( ACO,), glucose uptake The oxygen uptake ( AO, ), carbon ( Aglucose), lactic acid production ( Alactic acid), pyruvic acid production ( Apyruvic acid), and glycogen synthesis ( Aglycogen) were measured on 3 ml. aliquots incubated at 37 C. for 2 hours in specially designed reaction vessel@ attached to a Summerson differential manometer.14 minutes with a mixture vapour at 37 C. Glucose, lactic
acid
Before incubation of 95 per cent 0, and
pyruvic
acid
the suspensions and 5 per cent
were
determined
glycogen was degraded as previously described.rsJs The experiments were carried out with cell concentrations
were equilibrated for 30 CO, saturated with water by
enzymatic
of 8-40
methods
x lOs/ml.
and
In 2 and
5 patients, respectively, normal and diabetic leucocytes were investigated at two different cell concentrations. The results were calculated as ~mole/lOla leucocytes/hour. Leukocytes incubated in buffer ordinarily clump. The addition of gelatine and sodiumacetate completely prevented this phenomenon, as judged by microscopic examination and by the presence of a constant leucocyte concentration throughout incubation. In 16 experiments the average percentage difference betwen leucocyte counts before and after incubation was $0.4 per cent + 0.78. As evaluated by other parameters: the occurrence of a constant respiration with time, a potassium uptake (31 ,nEq. K/l010 leucocytes/h),
356 and of a barely to be intact.13
VICGO ESMANN
demonstrable
leakage
of alkaline
phosphatase,
the leukocytes
seemed
RESULTS
When the results were computed as arithmetical means no difference was found between normal and diabetic leucocytes; when, however, the results were calculated as a logarithmic function of the leucocyte concentration used in the actual experiments (fig. l), a difference betwen normal and diabetic leucocytes became apparent, as a crowding effect for Aglucose, Alactic acid, and Aglycogen, amounting to a decrease of 50 per cent, was present for NON-DIABETICS t
..
DIABETICS ---__
301 ---+------
,+&
.
1
+
3.2 ’
Fig. 1.-See
--__
--__.
opposite page.
-
CLO-GL.“l#/ ml
CELL CONCENTFIATION EFFECT ON METABOLISM
OF LEUCOCYTES
357
nondiabetic, but not for diabetic, leucocytes. No difference was found between the crowding effect on normal and diabetic cells with respect to Apyruvic acid and A02. The leucocyte concentration did not influence ACOs, either in normal or in diabetic cells. It should be noted that for diabetic leucocytes the levels of the regression lines for Aglucose, Alactic acid, and in principle also for Aglycogen, are such that they intersect the corresponding regression lines for normal leucocytes approximately at the center, leading to the circumstance that these parameters are lower in diabetic leucocytes in experiments performed with low leucocyte concentrations, and accounting, furthermore, for the lack of difference between the arithmetical means of the parameters. Figure 2 visualizes the differences and similiarities between normal and diabetic leucocytes obtained by interpolating on the regression lines in figure 1 at a leucocyte concentration of 107/ml. It is seen that diabetic leucocytes have a lower glucose uptake, lactic acid production and glycogen synthesis than normal leucocytes, whereas no differences are present in pyruvic acid production and respiration. The differences in glucose metabolism have been ascertained by actual experiments carried out using a fixed leucocytes concentration of 107/m1.13 DISCUSSION The experiments have shown the presence of a crowding effect when leucocytes are incubated in buffer; not only as demonstrated earlier on AO, and Alactic acid,l’ but also on Aglucose, Aglycogen and Apyruvic acid. There is thus no reason to uphold the hypothesis postulated by Hartmann1o that the crowding effect is a phenomenon which occurs only on the incubation of leucocytes in plasma. The cause of the crowding effect is unknown, but it has been demonstrated that it is not due to limiting concentrations of glucose or inorganic phosphate, nor can the high concentrations of lactic acid (app. 80 mg./lOO ml.) produced during incubation with high concentrations of leucocytes be held responsible.13
Fig. l.-The correlation (log y = a + hx) between leucocyte concentration and log A glucose, log A lactic acid, log A glycogen, log A pyruvic acid, log A O2 and log A COz for leucocytes from nondiabetic (ND) and diabetic (D) subjects. The regression lines and standard deviation from the regression lines (%. iog,) is
shown. The correlation coefficients r, their p-value, and the differences between the corresponding regression coefficients were: Glucose: rs,, = -0.52, p < 0.005; r, = -0.09, p < 0.70; b. - b, = 0.0081, p < 0.10 (t = 1.93, to,o5 = 1.99). Lactic acid: rSD =“-0.48, p < 0.001; rD = -0.08, p < 0.70; b ND- b, = 0.0083, p < 0.025. Glycogen: rND= -0.37, p < 0.01; rl, = +0.22, p < 0.30; b SD - b, = 0.0194, p < 0.01. Pyruvic acid: r,, = -0.81, p < 0.001; r,, = -0.69, p < 0.001; b - b, = 0.0083, p < 0.20. Oxygen: rKD = -$24, p < 0.005; rD = -0.40, p < 0.02; b ND - b, = 0.0029, p < 0.40. Carbon dioxide: rsU = -0.01, rD = +0.17, p < 0.40 b XI) - b, = 0.0034, p < 0.60.
VIGGO ESMANN
bb0
Glucose Lacllc
I 720
1200
acid
1 730
I Glycogen
Pyruvlc
ac Id Oxygen
Carbon
do-oxide pmol 500
100
l
Normals
/lO”Leuc./h 1000
0
Diabetics
Fig. B.-Values for carbohydrate metabolism and respiration of nondiabetic and diabetic leucoyctes obtained by interpolation on the regression lines of figure 1 at a leucocyte concentration of 105/ml. and expressed in pmole/lO1O leucocytes/h (geometric means).
The presence of a difference in the crowding effect between normal and diabetic leucocytes makes it difficult to evaluate the results of earlier investigations carried out with different cell concentrations;“-* and suggests that experiments with leucocytes should be carried out at low and virtually constant concentrations. Leucocytes at a concentration of 107/ml. have a high glucose uptake as compared to rat liver slices,l” rat diaphragm,” and rat epididymal fat,l* the values being approximately 14 mg., 7 mg., 2 mg., and 0.4 mg. glucose/Gm. wet weight/hour, respectively. The respiration is low, with QoZ = app.2, where the corresponding value for liver,l” diaphragm,20 and adipose tissuelR are Qo~ = app. 10. A characteristic of leucocytes, by which they differ from the majority of other normal tissues, is that these cells have a high aerobic glycolysis. The demonstration of the presence of a glycogen synthesis (2.4 mg./Gm. wet weight/hour) is contrary to the findings of McKinney et a1.,2r a fact which can probably be referred to different experimental conditions. The amount of glycogen synthesized is somewhat higher than that found by Rafaelsen22 in rat diaphragm (1.4 mg. glycogen/Gm. wet weight/hour). Despite large standard deviation from the regression line of the correlation between leucocyte concentration and log aC02, the probability that RQ at a leucocyte concentration of 107/ml. exceeds 0.5 - 0.8 is <0.05. Under the present experimental circumstances, where a large amount of glucose is catabolized, the finding of a low RQ suggests the occurrence of a CO2 fixation. This process has been demonstrated to occur in leucocytes from rabbit and guinea pig exudates .23,2* A stimulatory effect of CO2 on the oxygen con-
CELL
CONCENTRATION
EFFECT
ON METABOLISM
OF LEUCOCYTES
359
sumption of human leucocytes have been shown by Bicz,25 who suggested that the effect was due to a fixation of CO2 to p-enolpyruvate. It has been shown that Aglucose, Alactic acid, and Aglycogen are reduced in diabetic leucocytes. This reduction is of an extent comparable with the increase in these parameters which occurs when diabetics are treated with insulin.28 It has also been shown that insulin treatment is capable of restoring the reduced glycogen content of diabetic leucocytes to normal levels.15 The fact that the stimulatory effect of insulin in viva is not demonstrable within minutes, but has first been demonstrated after 24 hours of insulin treatment, together with the fact that the presence of a specific effect of insulin on leucocytes in vitro is questionable, suggest that in leucocytes the decreased carbohydrate metabolism of diabetic leucocytes cannot be caused by a defect in the mechanism of the transport of glucose. ACKNOWLEDGMENT I gratefully acknowledge the financial support from the Danish State Research Foundation, Aarhus University Research Foundation, and the Rheinholdt W. Jorck and Wife’s Foundation. Pharmacia Ltd., Sweden, generously supplied the high molecular dextran. I am indebted to Mrs. Ruth Malmkjaer for her technical assistance. REFERENCES 1. Krahl, M. E.: The Action of Insulin on Cells. New York, Academic Press, 1961. 2. Martin, S. P., McKinney, G. R., Green, R., and Becker, C.: The influence of glucose, fructose and insulin on the metabolism of leucocytes of healthy and diabetic subjects. J. Clin. Invest. 32: 1171, 1953. 3. Dumm, M.: Glucose utilization and lactate production by leucocytes of patients with diabetes mellitus. Proc. Sot. Exp. Biol. Med. 95:571, 1957. 4. -: Effect of tolbutamide on glucose uptake by leukocytes of patients with diabetes mellitus. J. Appl. Physiol. 14: 1023, 1959. 5. Barron, E. G., and Harrop, G. A., Jr.: Studies on cell metabolism. V. The metabolism of leucocytes. J. Biol. Chem. 84:89, 1929. 6. Soffer, J. L., and Wintrobe, M. M.: The metabolism of leucocytes from normal and leukaemic blood. J. Clin. Invest. 11:661, 1932. 7. Bird, R. M., Clemens, J. A., and Becker, L. M.: The metabolism of leucocytes taken from peripheral blood of leukaemic patients. Cancer 4: 1009, 1951. 8. Ponder, E., and McLeod, J.: The effect
of haemolytic substances on white cell respiration. J. Gen. Physiol. 20:267, 1936. 9. Marinelarena, R.: The Effects of Various Chemical Substances and Bacteria on the Glycolytic and Respiratory Activities of Leucocytes. Diss., Univ. Michigan, 1950. 10. Hartmann, J. D.: Effect of cell concentration on oxygen consumption of leucocytes under varying conditions. Proc. Sot. Exp. Biol. Med. 79:3, 1952. 11. Luganova, I. S., and Seits, I. F.: 0 kolichestvennoi kharakteristike dykhaniya i glikoliza v belykh krovyanykh kletkakh cheloveka. Biull. Exp. Biol. Med. 12:57, 1958. 12. Bisset, S. K., and Alexander, W. D.: Effect of muscular activity prior to venepuncture on the respiration of leucocytes in uitru. Nature 181:909, 1958. 13. Esmann, V.: Carbohydrate Metabolism and Respiration in Leucocytes from Normal and Diabetic Subjects. Aarhus (Denmark), University Press, 1962. 14. Summerson, W. N.: Combination manometer. J. Biol. Chem. 131:579, 1939.
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15. Esmann, V.: The glycogen content of leucocytes from diabetic and nondiabetic patients. Stand. J. Clin. Lab. Invest. 13: 134, 1961. 16. Renold, A. E., Teng, C. T., Nesbett, F. B., and Hastings, A. B.: Studies on carbohydrate metabolism in rat liver slices. II. The effect of fasting and of hormonal deficiencies. J. Biol. Chem. 204:533, 1953. 17. Rafaelsen, 0. J., and Lundbaek, K.: Action of carbutamide on isolated diaphragm of alloxan-diabetic rats. Metabolism 8:757, 1959. 18. Jungas, R. L., and Ball, E. G.: Studies on the metabolism of adipose tissue. V. The effect of a growth hormone preparation and insulin on the oxygen glucose uptake, and consumption, lactic acid production. J. Biol. Chem. 235: 1894, 1960. 19. Elliott, K. A. C., Greig, M. E., and Benoy, M. P.: The metabolism of lactic and pyruvic acids in normal and tumour tissue. II. Rat liver, brain and testis. Biochem. J. 31:1003, 1937. 20. Stadie, W. S., and Zapp, J. A.: The effect of insulin upon the synthesis of
ESMANN
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Viggo Esmann, M.D., Senior Physician, Department of Medicine C, Copenhagen County Hospital, Glostmp, Denmark.
on le11-