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Upregulation of glucose metabolism by granulocyte-monocyte colony-stimulating factor
Life Sciences, Printed in the
Vol. 49. U.S.A.
pp.
899-906
Pergamon
UPRE~XJLATI~N OF GLUCOSE METABOLISM COLONY-STIMULATING
Press
BY GRANUL~CYTE-MON~CYTE FACTOR
Agnes Schuler, Zoltan Spolarics’, Charles H. Lang, Gregory J. Bagby, Steve Nelson* and John J. Spitzer Department of Physiology’ and Department of Medicine Section of Pulmonary/Critical-Care Medicine*, Louisiana State University Medical Center, 1901 Perdido Street, 1 New Orleans, Louisiana 70112 (Received
in
final
form
July
16,
1991)
Summary Alterations ,of glucose, metabolism were investigated for 6 hours following an intraarterial’ injection pf murine recombinant granulocyte-monocyte colonystimulating factor (GM-CSF) (30 pg/kg body weight). GM-CSF resulted in a transient elevation of plasma glucose. The rate of whole body glucose appearance, as measured by infusion of [6-‘HIglucose. was increased by about 10 % between 0.5 and 3 hours following GM-CSF injection. In viva glucose utilization of individual tissues was investigated by the tracer 2-deoxyglucose technique. At 30 min. GM-CSF increased glucose utilization by 80-90 % in liver and lung, and 50-60 % jn skin and spleen. At 3 and 6 hours. glucose utilization by these tissues returned toward control levels except for lung. There was a 40-50 % increase in, glucose utilization by skeletal muscle 30 min after GM-CSF which was sustained for 6 hours. Glucose utilization of testis,,ileum and kidney did not change significantly. Plasma concentrations of insulin, glucagon and tumor necrosis factor were not altered in response to GM-CSF. These findings indicate that some of the acute metabolic effects of a short-term ~adminispation of GM-CSF are observed in macrophage-rich tissues, and suggest that GM-CSF may be involved in the metabolic upregulation of immunologically active tissues. Colony-stimulating factors p!ay an important role in the proliferation and differentiation of hemopoetic progenitor ceils (1). Prolonged administration of these factors result in increased numbers of circulating mono- and polymorphonuclear phagocytes. as well as eosinophils (1). Several recent in virro studies have indicated that colony stimulating factors may also be involved in the activation of mature granulocytes and macrophages (2-6). Earlier studies have demonstrated ,tJtat the complex metabolic changes found in sepsis or endotoxemia (7,8) can be only partially simulated by the administration of different cytokjnes such as tumor necrosis factor (TNP),
‘To whom correspondence
and npri’nt requests should be addressed
Copyright
0024-3205191 (c) 1991
$3.00 +.oo Pergamon Press
plc
900
GM-CSF & Glucose Utilization
Vol. 49, No. 12, 1991
interleukins or platelet-activating factor (9-11). This indicates that additional factors may also contribute to mediating the metabolic responses of the host in sepsis and endotoxemia. The aim of the present study was to delineate the short-term effects of GM-CSF on whole body glucose metabolism and on the in vivo glucose utilization of individual tissues. We found that GM-CSF administration resulted in an acute and transient elevation of glucose utilization in macrophage-rich tissues. GM-CSF also increased glucose uptake by skeletal muscle, an effect that was sustained for at least 6 hours. Methods Experimental animals. Male Sprague-Dawley rats (300-340 g, Charles River, Wilmington, MA) were used for the study. The day preceding the experiment catheters were placed in the jugular vein and carotid artery using aseptic surgical procedures. Animals were fasted overnight with free access to water. Experimental protocols. For the determination of in vivo glucose uptake of individual tissues the 2-deoxyglucose tracer technique was used, as described previously (10,12). Murine recombinant GM-CSF (106 IU/mg; a gift from Amgen, Thousand Oaks, CA) was dissolved in 0.9 % saline and injected intraarterially in a dose of 30 p.g/kg body weight. At 0.5, 3 and 6 hours, 2-deoxy-D-[U-14C]glucose (Amersham Corporation, Arlington Heights, 1L) was injected intravenously (13 t.tCi/100 g body weight). The decay of the radioactivity from the plasma and glucose concentrations were monitored for 40 minutes. Animals were sacrificed and the accumulation of the phosphorylated metabolites of 2-deoxyglucose were measured in selected tissues (7). The glucose metabolic rates (Rg) were calculated based on the accumulation of the phosphorylated metabolites in the tissue, the integrated specific activity of the tracer during the 40 rain labeling period and the lump constant as described earlier (7,13). The glucose metabolic clearance rate (MCR) of an individual tissue was calculated by dividing the tissue Rg by the integrated value of plasma glucose concentrations determined during the labeling period. MCR provides an estimate of the avidity of the tissue for glucose independent of the prevailing plasma glucose concentration. Glucose rate of appearance (Ra) was determined by the measurement of glucose specific activity at selected intervals after the primed-constant iv. infusion of [6-3H]glucose (11). Plasma concentrations of glucose, insulin and glucagon were measured from arterial blood samples (11). Mean arterial blood pressure and heart rate were monitored by a pressure transducer attached to the arterial catheter. For the statistical analysis of data, the one-way ANOVA followed by Student-Newman Keuls test, and the Tukey-Kramer comparison of multiple means methods were used (14). Results Glucose Ra was monitored for 6 hours after a bolus injection of GM-CSF. Fig 1 shows that glucose Ra was increased promptly after GM-CSF injection and remained slightly, but significantly elevated for 3 hours. Plasma glucose concentration also tended to be transiently increased. Heart rate was increased throughout the experiment, while no change in the arterial blood pressure was detected (data not shown). Glucose utilization of different tissues was determined by the 2-deoxyglucose tracer technique. Rg of lung, liver, spleen and skin were increased approximately 50-90 % 30 min after
Vol. 49, No. 12, 1991
GM-CSF & Glucose Utilization
901
GM-CSF administration (Fig 2). At 3 and 6 h, the Rg of these tissues returned to normal, except the lung which showed a significantly elevated Rg at 6 hours. Rg in skeletal muscle, assessed from tissue samples of the gastrocnemius, was elevated by approximately 40 % at 30 rain after GM-CSF injection and this increase was sustained for up to 6 hours (Fig 3). No significant change in Rg was observed in the testis, ileum and kidney. Glucose Rate Of A p p e a r a n c e 60
¢°--
E
50
0
E 40 I
I
I
I
I
I
I
I
I
Plasma Glucose C o n c e n t r a t i o n 8
E
I
I
I
I
I
Heart Rate ¢50 -i ¢-. °~
E
-0
4.00 350 300 I
I
I
I
I
I
I
0
1
2
3
4
5
6
TIME (h)
FIG. 1 Effect of GM-CSF on glucose kinetics and heart rate. Glucose appearance rate (Ra), concentration of plasma glucose, and heart rate were measured prior to and for 6 h after GM-CSF injection. Means + S.E. n=9. * indicates significant difference from time-matched saline-injected controls, p<0.05.
902
GM-CSF & Glucose Utilization
200
40
150
30
IO0
20
~=
50
10
.c__
0
E
.E. c
12, 1991
50
250
-6
49, No.
Liver
Lung
E
Vol.
T
0 Skin
Spleen
4-00
RO
o~ 300 ¢v,
6O
200
40
1oo
20
T
0
Sollne
0.Sh
3h
Saline
6h
0.Sh
I
,3h
6h
I
GM-CSF
GM-CSF
Kidney
Ileum 200 250 200
150
150
100
..-..
100
~=
50
{;c
0
50 0
Testis
E
Muscle
5
E
20o
100 :-
,E, c ~" n~
150
8060-
1O0
4050
200 Saline
0.Sh
3h I
GM-CSF
6h
Saline
0.Sh
3h
6h
I
GM-CSF
FIG 2 EffecL of 0M,CSF onth e glucose metabolic rate (Rg) of selected tissues. Rg was determined ~ter 0.5, 3 and 6 hours of'GM-CSF injection as indicated. Open bars representvalues obtained after the injection of saline. Means _+S.E. n=6-16. * indicates significant difference from saline-injected controls, p<0.05.
Vol. 49, No. 12, 1991
GM-CSF & Glucose Utilization
903
The MCR of individual tissues were also calculated to assess the avidity of the various tissues for glucose. Table I indicates that at 30 min MCR was increased in the lung and liver; while in the muscle, MCR was elevated at 3 and 6 h following GM-CSF injection. No consistent changes in MCR were seen for the other tissues examined. TABLE I Effect of GM-CSF on the glucose metabolic clearance rate of individual tissues Metabolic clearance rate Saline
GM-CSF 30 min
6h
3h
Tissues Lung
23 +_ 1.1
32 +_ 1.2 *~
24 +_ 1.9
29 _+ 3.4*
Liver
4 5- 0.2
6 5- 0.6*
5 5- 0.7
5 -+ 0.3
Spleen
40 5- 1.7
47 5- 2.7
41 5- 1.7
45 _+ 3.6
Muscle
12 5- 0.9
12 5- 0.9
17 +_ 1.7 .8'
17 5- 0.9 *&
Plasma glucose 5.6 5- 0.1
7.7 + 0.4 *s
(mM) 5.8 5- 0.3
5,8 5- 0.2
MCR (Ixl plasma/min/g wet weight) was calculated as described in the Materials and Methods. Mean _+ S.E. n=6-16. *, statistical difference from saline; @from GM-CSF at 3h; ~ from GM-CSF at 30 min; s from GM-CSF at 3h and 6h. p< 0.05.
TABLE II Effect of GM-CSF on the plasma concentration of insulin and glucagon (Time after GM-CSF or saline; hours) Hormone; Treatment Insulin
30 min
3h
6h
~U/ml) GM-CSF
28 5- 4
31 +_3
23+3
Saline
24 + 4
24+2
23+_2
Glucagon (pg/ml) GM-CSF
195 5- 27
196 +_ 14
166 + 10
Saline
137 + 13
169 + 10
152 + 5
Mean + S.E. n=7-10.
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GM-CSF & Glucose Utilization
Vol. 49, No.
12, 1991
The changes in glucose metabolism in response to GM-CSF were not accompanied by significant alterations of plasma concentrations of glucagon, insulin (Table II), or the insulin-glucagon ratio (data not shown). The plasma levels of TNF were less than the detection limit (< 40 U/ml) following GM-CSF injection, as measured by the L929 cytotoxicity assay (15) (data not shown). Discussion Several studies indicate that in response to bacterial, endotoxins, TNF, or interleukin-1, GM-CSF is released from different cellular sources (see refs in 1). T-lymphocytes, endothelial cells, macrophages and fibroblasts are the primary cellular sources of plasma GM-CSF. It has been shown that GM-CSF has an important role in the induction of proliferation of bone marrow colonies, and acts in concert with other cytokines in modulating different cellular functions (1,16). To date most studies have focused on the effects of GM-CSF that require several hours or even days to develop. No experimental data are available concerning the acute in vivo metabolic effects of GM-CSF. In the present study we demonstrate an acute effect of GM-CSF on in vivo carbohydrate metabolism. We employed a single injection of the cytokine in a dose resulting in an estimated peak plasma concentration of about 500-1000 U/ml. This dose should provide appropriate tissue concentrations of GM-CSF for biological activity as it has been demonstrated that it is effective at 10-500 U/ml in vitro (2-6). Our findings indicate that the immune-competent tissues are important targets for GM-CSF action. The response resembles the early metabolic effect of bacterial endotoxins (8). Both endotoxin and GM-CSF cause a transient hyperglycemia. However, the transient effect of GM-CSF on glucose utilization by macrophage-rich tissues is in contrast with the sustained effects of endotoxin. On the other hand, the metabolic effect of GM-CSF on skeletal muscle was sustained for at least 6 hours. This finding may indicate a direct action of GM-CSF on this tissue. Alternatively, muscle could be a site for an accumulation of different inflammatory cells following GM-CSF administration, whose increased metabolic activity could contribute to the observed increase in glucose utilization. Earlier we demonstrated somewhat similar metabolic responses 0.5 and 3 after the administration of TNF to that found after GM-CSF (9,17). However, TNF adminisu'ation did not change the glucose utilization of skeletal muscle. This difference in the action of these cytokines, in addition to the fact that an elevation in the plasma concentration of TNF was not detectable in our experiments following GM-CSF, indicates that the metabolic effects of GM-CSF are not simply mediated by TNF. The unaltered plasma concentration of insulin suggests that this hormone is also not responsible for the increased glucose uptake after GM-CSF injection. Administration of GM-CSF resulted in a transient increase in glucose Ra with no significant increase in the plasma glucagon concentrations. An elevated prostaglandin secretion upon interaction of GM-CSF with macrophages (18), if it takes place in the liver, could be a possible mechanism responsible for the increased glucose secretion by hepatocytes (19,20). Elevated plasma glucose concentration is likely to contribute to the early metabolic effect of GM-CSF. However, the Rg and MCR of muscle at 3 hours, or muscle and lung at 6 hours, were also elevated suggesting that the increased glucose uptake after GM-CSF is not merely a consequence of an elevated plasma glucose concentration. We note that other hormones such as catecholamines and glucocorticoids might be involved in mediating the glucose metabolic effects of GM-CSF. However, the selective response of tissues, and the lack of change in plasma insulin concentration do suggest a direct effect of GM-CSF on glucose metabolism of the peripheral tissues.
Vol. 49, No. 12, 1991
GM-CSF & Glucose Utilization
905
From these data we postulate the following sequence of events: GM-CSF induces an increase in glucose Ra which in the early absence of peripheral changes, causes a slight hyperglycemia. This is accompanied by increased glucose uptake matching the elevated glucose Ra. At the same time, the MCR of glucose by some peripheral tissues (lung and liver) increases, while insulin concentrations remain unaltered. Subsequently, glucose MCR of skeletal muscle also increases. The mechanism of the latter enhanced insulin-independent glucose uptake remains to be elucidated. Thus, our findings indicate that GM-CSF may be a potentially important member of the cytokine cascade in eliciting or contributing to the acute and complex metabolic changes in different tissues observed upon endotoxin challenge. The mechanism whereby GM-CSF causes the metabolic changes is not known at this time. Direct effect on the glucose metabolic pathway (glucose trasporters, rate limiting enzymes of glucose metabolism), as well as interaction with known regulatory influences of metabolism (e.g. affecting the release of secondary lipid or protein mediators and/or hormones) could be some of the mechanisms of action of GM-CSF administration. The transient effect of GM-CSF on macrophage-rich tissues indicates that this cytokine alone is not able to completely simulate the metabolic responses of endotoxin, and implies that this agent acts in concert with other cytokines and mediators in regulating metabolic parameters upon immune stimulation. Acknowledgement This work was supported by NIH grant GM-32654 and 38032. GM-CSF was a gift from Amgen. Thanks are due to June T. Bechtel, Jean Carnal and Howard Blakesley for their excellent technical assistance. References 1.
2.
3. 4. 5. 6. 7. 8.
.
10. 11.
N.M. GOUGH and N.A. NICOLA, Colony-Stimulating Factors T.M. Dexter, J.M. Garland, and N.G. Testa (eds), 111-153, Marcel Dekker, Inc., New York and Basel (1990). D. ENGLISH, H.E. BROXMEYER, T.G. GABIG, L.P. AKARD, D.E. WILIAMS and R. HOFFMAN, J. Immunol. 141 2400-2406 (1988). M.S. CAIRO, C. VAN DE VEN, C. TOY, M. MAUSS and L. SENDER, Pediatric. Res. 26 395-399 (1989). M. KLAUSMAN, K.H. PFLUGER, D. KRUMWIEH, F.R. SEILER and K. HAVEMANN, Blut 5._44307-312 (1987). S.W. EDWARDS, C.S. HOLDEN, J.M. HUMPHREYS and C.A. HART, FEBS Lett. 256 62-66 (1989). J.A. HAMILTON, G. VAIRO and S.R. LINGELBACH, J. Cell. Phys. 134 405-412 (1988). K. MIS.SZ.~ROS, C.H. LANG, G.J. BAGBY and J.J. SPITZER, J. Biol. Chem. 262 10965-10970 (1987). J.J. SPITZER, G.J. BAGBY, K. MI~SZAROS and C.H. LANG, Molecular and Cellular Mechanisms of Septic Shock B.L Roth, T.B. Nielsen, A.E. McKee (eds) 145-152, Alan R. Liss, New York. NY (1989). G.J. BAGBY, C.H. LUNG, D.M. HARGROVE, J.J. THOMPSON, L.A. WILSON and J.J. SPITZER, Circ. Shock. 24 111-132 (1988). K. M16.SZAROS, C.H. LANG, G.J. BAGBY and J.J. SPITZER, Biochem. Biophys. Res. Commun. 149 1-6 (1987). C.H. LANG and C. DOBRESCU, Life Sci. 45 2127- 2134 (1989).
906
12. 13. 14. 15. 16. 17. 18. 19. 20.
GM-CSF & Glucose Utilization
Vol. 49, No. 12, 1991
L.M. SOKOLOFF, C. REIVICH, M.H. KENNEDY, C.S. DES ROSIERS, K.D. PETrlGREV, O. SAKURADA and M. SHINIHARA, J. Neurochem. 2._88897-916 (1977). K. MI~SZAROS, C.H. LANG, D.M. HARGROVE and J.J. SPITZER, J. Appl. Physiol. 6..2.71770-1775 (1989). G.W. SNEDECOR and W.G COCHRAN, Statistical Methods (6th ed.) Iowa State University Press, Ames, Iowa (1968). N.B. D'SOUZA, G.J. BAGBY, S. NELSON, C.H. LANG and J.J. SPITZER, Alcohol. Clin. Exp. Res. 13295-298 (1989). C.H. DINARELLO, Lymphokines and the Immune Response S. Cohen (ed) 166-172. CRC Press, Inc., Boca Raton, Florida (1990). Z. SPOLARICS, .~. SCHULER, G.J. BAGBY, C.H. LANG and J.J. SPITZER, J. Leukoc. Biol. 49309-312 (1991). S. HEINDENREICH, J-H. GONG, H. RENZ, A. SCHMIDT M. NAIN and D. GEMSA, J. Immunol. 1431198-1205 (1989). J. KUIPER, Y.B. De RIJKE, F.J. ZIJLSTRA, P.M. Van WAAS and T.J.C. Van BERKEL, Biochem. Biophys. Res. Com. 1571288-1295 (1988). E.B. TURCO and J.A. SPITZER, J. Leukoc. Biol. 4._.88488-494 (1990).
Vol. 49. U.S.A.
pp.
899-906
Pergamon
UPRE~XJLATI~N OF GLUCOSE METABOLISM COLONY-STIMULATING
Press
BY GRANUL~CYTE-MON~CYTE FACTOR
Agnes Schuler, Zoltan Spolarics’, Charles H. Lang, Gregory J. Bagby, Steve Nelson* and John J. Spitzer Department of Physiology’ and Department of Medicine Section of Pulmonary/Critical-Care Medicine*, Louisiana State University Medical Center, 1901 Perdido Street, 1 New Orleans, Louisiana 70112 (Received
in
final
form
July
16,
1991)
Summary Alterations ,of glucose, metabolism were investigated for 6 hours following an intraarterial’ injection pf murine recombinant granulocyte-monocyte colonystimulating factor (GM-CSF) (30 pg/kg body weight). GM-CSF resulted in a transient elevation of plasma glucose. The rate of whole body glucose appearance, as measured by infusion of [6-‘HIglucose. was increased by about 10 % between 0.5 and 3 hours following GM-CSF injection. In viva glucose utilization of individual tissues was investigated by the tracer 2-deoxyglucose technique. At 30 min. GM-CSF increased glucose utilization by 80-90 % in liver and lung, and 50-60 % jn skin and spleen. At 3 and 6 hours. glucose utilization by these tissues returned toward control levels except for lung. There was a 40-50 % increase in, glucose utilization by skeletal muscle 30 min after GM-CSF which was sustained for 6 hours. Glucose utilization of testis,,ileum and kidney did not change significantly. Plasma concentrations of insulin, glucagon and tumor necrosis factor were not altered in response to GM-CSF. These findings indicate that some of the acute metabolic effects of a short-term ~adminispation of GM-CSF are observed in macrophage-rich tissues, and suggest that GM-CSF may be involved in the metabolic upregulation of immunologically active tissues. Colony-stimulating factors p!ay an important role in the proliferation and differentiation of hemopoetic progenitor ceils (1). Prolonged administration of these factors result in increased numbers of circulating mono- and polymorphonuclear phagocytes. as well as eosinophils (1). Several recent in virro studies have indicated that colony stimulating factors may also be involved in the activation of mature granulocytes and macrophages (2-6). Earlier studies have demonstrated ,tJtat the complex metabolic changes found in sepsis or endotoxemia (7,8) can be only partially simulated by the administration of different cytokjnes such as tumor necrosis factor (TNP),
‘To whom correspondence
and npri’nt requests should be addressed
Copyright
0024-3205191 (c) 1991
$3.00 +.oo Pergamon Press
plc
900
GM-CSF & Glucose Utilization
Vol. 49, No. 12, 1991
interleukins or platelet-activating factor (9-11). This indicates that additional factors may also contribute to mediating the metabolic responses of the host in sepsis and endotoxemia. The aim of the present study was to delineate the short-term effects of GM-CSF on whole body glucose metabolism and on the in vivo glucose utilization of individual tissues. We found that GM-CSF administration resulted in an acute and transient elevation of glucose utilization in macrophage-rich tissues. GM-CSF also increased glucose uptake by skeletal muscle, an effect that was sustained for at least 6 hours. Methods Experimental animals. Male Sprague-Dawley rats (300-340 g, Charles River, Wilmington, MA) were used for the study. The day preceding the experiment catheters were placed in the jugular vein and carotid artery using aseptic surgical procedures. Animals were fasted overnight with free access to water. Experimental protocols. For the determination of in vivo glucose uptake of individual tissues the 2-deoxyglucose tracer technique was used, as described previously (10,12). Murine recombinant GM-CSF (106 IU/mg; a gift from Amgen, Thousand Oaks, CA) was dissolved in 0.9 % saline and injected intraarterially in a dose of 30 p.g/kg body weight. At 0.5, 3 and 6 hours, 2-deoxy-D-[U-14C]glucose (Amersham Corporation, Arlington Heights, 1L) was injected intravenously (13 t.tCi/100 g body weight). The decay of the radioactivity from the plasma and glucose concentrations were monitored for 40 minutes. Animals were sacrificed and the accumulation of the phosphorylated metabolites of 2-deoxyglucose were measured in selected tissues (7). The glucose metabolic rates (Rg) were calculated based on the accumulation of the phosphorylated metabolites in the tissue, the integrated specific activity of the tracer during the 40 rain labeling period and the lump constant as described earlier (7,13). The glucose metabolic clearance rate (MCR) of an individual tissue was calculated by dividing the tissue Rg by the integrated value of plasma glucose concentrations determined during the labeling period. MCR provides an estimate of the avidity of the tissue for glucose independent of the prevailing plasma glucose concentration. Glucose rate of appearance (Ra) was determined by the measurement of glucose specific activity at selected intervals after the primed-constant iv. infusion of [6-3H]glucose (11). Plasma concentrations of glucose, insulin and glucagon were measured from arterial blood samples (11). Mean arterial blood pressure and heart rate were monitored by a pressure transducer attached to the arterial catheter. For the statistical analysis of data, the one-way ANOVA followed by Student-Newman Keuls test, and the Tukey-Kramer comparison of multiple means methods were used (14). Results Glucose Ra was monitored for 6 hours after a bolus injection of GM-CSF. Fig 1 shows that glucose Ra was increased promptly after GM-CSF injection and remained slightly, but significantly elevated for 3 hours. Plasma glucose concentration also tended to be transiently increased. Heart rate was increased throughout the experiment, while no change in the arterial blood pressure was detected (data not shown). Glucose utilization of different tissues was determined by the 2-deoxyglucose tracer technique. Rg of lung, liver, spleen and skin were increased approximately 50-90 % 30 min after
Vol. 49, No. 12, 1991
GM-CSF & Glucose Utilization
901
GM-CSF administration (Fig 2). At 3 and 6 h, the Rg of these tissues returned to normal, except the lung which showed a significantly elevated Rg at 6 hours. Rg in skeletal muscle, assessed from tissue samples of the gastrocnemius, was elevated by approximately 40 % at 30 rain after GM-CSF injection and this increase was sustained for up to 6 hours (Fig 3). No significant change in Rg was observed in the testis, ileum and kidney. Glucose Rate Of A p p e a r a n c e 60
¢°--
E
50
0
E 40 I
I
I
I
I
I
I
I
I
Plasma Glucose C o n c e n t r a t i o n 8
E
I
I
I
I
I
Heart Rate ¢50 -i ¢-. °~
E
-0
4.00 350 300 I
I
I
I
I
I
I
0
1
2
3
4
5
6
TIME (h)
FIG. 1 Effect of GM-CSF on glucose kinetics and heart rate. Glucose appearance rate (Ra), concentration of plasma glucose, and heart rate were measured prior to and for 6 h after GM-CSF injection. Means + S.E. n=9. * indicates significant difference from time-matched saline-injected controls, p<0.05.
902
GM-CSF & Glucose Utilization
200
40
150
30
IO0
20
~=
50
10
.c__
0
E
.E. c
12, 1991
50
250
-6
49, No.
Liver
Lung
E
Vol.
T
0 Skin
Spleen
4-00
RO
o~ 300 ¢v,
6O
200
40
1oo
20
T
0
Sollne
0.Sh
3h
Saline
6h
0.Sh
I
,3h
6h
I
GM-CSF
GM-CSF
Kidney
Ileum 200 250 200
150
150
100
..-..
100
~=
50
{;c
0
50 0
Testis
E
Muscle
5
E
20o
100 :-
,E, c ~" n~
150
8060-
1O0
4050
200 Saline
0.Sh
3h I
GM-CSF
6h
Saline
0.Sh
3h
6h
I
GM-CSF
FIG 2 EffecL of 0M,CSF onth e glucose metabolic rate (Rg) of selected tissues. Rg was determined ~ter 0.5, 3 and 6 hours of'GM-CSF injection as indicated. Open bars representvalues obtained after the injection of saline. Means _+S.E. n=6-16. * indicates significant difference from saline-injected controls, p<0.05.
Vol. 49, No. 12, 1991
GM-CSF & Glucose Utilization
903
The MCR of individual tissues were also calculated to assess the avidity of the various tissues for glucose. Table I indicates that at 30 min MCR was increased in the lung and liver; while in the muscle, MCR was elevated at 3 and 6 h following GM-CSF injection. No consistent changes in MCR were seen for the other tissues examined. TABLE I Effect of GM-CSF on the glucose metabolic clearance rate of individual tissues Metabolic clearance rate Saline
GM-CSF 30 min
6h
3h
Tissues Lung
23 +_ 1.1
32 +_ 1.2 *~
24 +_ 1.9
29 _+ 3.4*
Liver
4 5- 0.2
6 5- 0.6*
5 5- 0.7
5 -+ 0.3
Spleen
40 5- 1.7
47 5- 2.7
41 5- 1.7
45 _+ 3.6
Muscle
12 5- 0.9
12 5- 0.9
17 +_ 1.7 .8'
17 5- 0.9 *&
Plasma glucose 5.6 5- 0.1
7.7 + 0.4 *s
(mM) 5.8 5- 0.3
5,8 5- 0.2
MCR (Ixl plasma/min/g wet weight) was calculated as described in the Materials and Methods. Mean _+ S.E. n=6-16. *, statistical difference from saline; @from GM-CSF at 3h; ~ from GM-CSF at 30 min; s from GM-CSF at 3h and 6h. p< 0.05.
TABLE II Effect of GM-CSF on the plasma concentration of insulin and glucagon (Time after GM-CSF or saline; hours) Hormone; Treatment Insulin
30 min
3h
6h
~U/ml) GM-CSF
28 5- 4
31 +_3
23+3
Saline
24 + 4
24+2
23+_2
Glucagon (pg/ml) GM-CSF
195 5- 27
196 +_ 14
166 + 10
Saline
137 + 13
169 + 10
152 + 5
Mean + S.E. n=7-10.
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GM-CSF & Glucose Utilization
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The changes in glucose metabolism in response to GM-CSF were not accompanied by significant alterations of plasma concentrations of glucagon, insulin (Table II), or the insulin-glucagon ratio (data not shown). The plasma levels of TNF were less than the detection limit (< 40 U/ml) following GM-CSF injection, as measured by the L929 cytotoxicity assay (15) (data not shown). Discussion Several studies indicate that in response to bacterial, endotoxins, TNF, or interleukin-1, GM-CSF is released from different cellular sources (see refs in 1). T-lymphocytes, endothelial cells, macrophages and fibroblasts are the primary cellular sources of plasma GM-CSF. It has been shown that GM-CSF has an important role in the induction of proliferation of bone marrow colonies, and acts in concert with other cytokines in modulating different cellular functions (1,16). To date most studies have focused on the effects of GM-CSF that require several hours or even days to develop. No experimental data are available concerning the acute in vivo metabolic effects of GM-CSF. In the present study we demonstrate an acute effect of GM-CSF on in vivo carbohydrate metabolism. We employed a single injection of the cytokine in a dose resulting in an estimated peak plasma concentration of about 500-1000 U/ml. This dose should provide appropriate tissue concentrations of GM-CSF for biological activity as it has been demonstrated that it is effective at 10-500 U/ml in vitro (2-6). Our findings indicate that the immune-competent tissues are important targets for GM-CSF action. The response resembles the early metabolic effect of bacterial endotoxins (8). Both endotoxin and GM-CSF cause a transient hyperglycemia. However, the transient effect of GM-CSF on glucose utilization by macrophage-rich tissues is in contrast with the sustained effects of endotoxin. On the other hand, the metabolic effect of GM-CSF on skeletal muscle was sustained for at least 6 hours. This finding may indicate a direct action of GM-CSF on this tissue. Alternatively, muscle could be a site for an accumulation of different inflammatory cells following GM-CSF administration, whose increased metabolic activity could contribute to the observed increase in glucose utilization. Earlier we demonstrated somewhat similar metabolic responses 0.5 and 3 after the administration of TNF to that found after GM-CSF (9,17). However, TNF adminisu'ation did not change the glucose utilization of skeletal muscle. This difference in the action of these cytokines, in addition to the fact that an elevation in the plasma concentration of TNF was not detectable in our experiments following GM-CSF, indicates that the metabolic effects of GM-CSF are not simply mediated by TNF. The unaltered plasma concentration of insulin suggests that this hormone is also not responsible for the increased glucose uptake after GM-CSF injection. Administration of GM-CSF resulted in a transient increase in glucose Ra with no significant increase in the plasma glucagon concentrations. An elevated prostaglandin secretion upon interaction of GM-CSF with macrophages (18), if it takes place in the liver, could be a possible mechanism responsible for the increased glucose secretion by hepatocytes (19,20). Elevated plasma glucose concentration is likely to contribute to the early metabolic effect of GM-CSF. However, the Rg and MCR of muscle at 3 hours, or muscle and lung at 6 hours, were also elevated suggesting that the increased glucose uptake after GM-CSF is not merely a consequence of an elevated plasma glucose concentration. We note that other hormones such as catecholamines and glucocorticoids might be involved in mediating the glucose metabolic effects of GM-CSF. However, the selective response of tissues, and the lack of change in plasma insulin concentration do suggest a direct effect of GM-CSF on glucose metabolism of the peripheral tissues.
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From these data we postulate the following sequence of events: GM-CSF induces an increase in glucose Ra which in the early absence of peripheral changes, causes a slight hyperglycemia. This is accompanied by increased glucose uptake matching the elevated glucose Ra. At the same time, the MCR of glucose by some peripheral tissues (lung and liver) increases, while insulin concentrations remain unaltered. Subsequently, glucose MCR of skeletal muscle also increases. The mechanism of the latter enhanced insulin-independent glucose uptake remains to be elucidated. Thus, our findings indicate that GM-CSF may be a potentially important member of the cytokine cascade in eliciting or contributing to the acute and complex metabolic changes in different tissues observed upon endotoxin challenge. The mechanism whereby GM-CSF causes the metabolic changes is not known at this time. Direct effect on the glucose metabolic pathway (glucose trasporters, rate limiting enzymes of glucose metabolism), as well as interaction with known regulatory influences of metabolism (e.g. affecting the release of secondary lipid or protein mediators and/or hormones) could be some of the mechanisms of action of GM-CSF administration. The transient effect of GM-CSF on macrophage-rich tissues indicates that this cytokine alone is not able to completely simulate the metabolic responses of endotoxin, and implies that this agent acts in concert with other cytokines and mediators in regulating metabolic parameters upon immune stimulation. Acknowledgement This work was supported by NIH grant GM-32654 and 38032. GM-CSF was a gift from Amgen. Thanks are due to June T. Bechtel, Jean Carnal and Howard Blakesley for their excellent technical assistance. References 1.
2.
3. 4. 5. 6. 7. 8.
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10. 11.
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L.M. SOKOLOFF, C. REIVICH, M.H. KENNEDY, C.S. DES ROSIERS, K.D. PETrlGREV, O. SAKURADA and M. SHINIHARA, J. Neurochem. 2._88897-916 (1977). K. MI~SZAROS, C.H. LANG, D.M. HARGROVE and J.J. SPITZER, J. Appl. Physiol. 6..2.71770-1775 (1989). G.W. SNEDECOR and W.G COCHRAN, Statistical Methods (6th ed.) Iowa State University Press, Ames, Iowa (1968). N.B. D'SOUZA, G.J. BAGBY, S. NELSON, C.H. LANG and J.J. SPITZER, Alcohol. Clin. Exp. Res. 13295-298 (1989). C.H. DINARELLO, Lymphokines and the Immune Response S. Cohen (ed) 166-172. CRC Press, Inc., Boca Raton, Florida (1990). Z. SPOLARICS, .~. SCHULER, G.J. BAGBY, C.H. LANG and J.J. SPITZER, J. Leukoc. Biol. 49309-312 (1991). S. HEINDENREICH, J-H. GONG, H. RENZ, A. SCHMIDT M. NAIN and D. GEMSA, J. Immunol. 1431198-1205 (1989). J. KUIPER, Y.B. De RIJKE, F.J. ZIJLSTRA, P.M. Van WAAS and T.J.C. Van BERKEL, Biochem. Biophys. Res. Com. 1571288-1295 (1988). E.B. TURCO and J.A. SPITZER, J. Leukoc. Biol. 4._.88488-494 (1990).