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Effect of glucose on lipolysis and energy metabolism in fat cells
Life Sciences Vol. 9, Part I, pp . 137-150, 1970 . Printed in Great Britain
Pergamon Press
EFFECT OF GLUCOSE ON LIPOLYSIS AND ENERGY METABOLISM IN FAT CELLS R. J. Ho, R. England and H. C. Meng Dept . of Physiology, Vanderbilt University School of Medicine, Nashville, Tennessee 37203 (Received 10 September 1969 ; in final form 14 October 1969)
THE hormone-stimulated release of free fatty acid (FFA) from adipose tissue incubated in vitro has been shown to be reduced in the presence of glucose (1-3).
Differing from the release of FFA, the release of the other
lipolysic product, glycerol, was somewhat increased (4).
This effect was
interpreted to be the result of an increase in esterification due to an increase in the availability of cKrglycerol phosphate derived from glucose metabolism . Therefore, in studying fat mobilisation, glucose was often omitted from the incubation medium .
The requirement of the high energy phosphate compound,
ATP, for the activation of hormone-sensitive lipase was first shown by Rizack (5).
It is now believed that the formation of 3', 5'-cyclic AMP from ATP
appears to be a possible intermediate step in the lipase activation (6, 7) .
This
belief led us to reexamine the role of glucose in the activation of lipase in response to epinephrine that results in lipolysis in fat cells .
In the present
study, the effect of glucose on epinephrine-stimulated FFA and glycerol release were observed under varying conditions .
The results favored the
hypothesis that the activation of hormone-sensitive lipase requires the energy of added substrate if the endogenous energy supply is inadequate . Part of this work has been reported as an abstract (S). Materials and Methods Fasted and fasted-refed albino rats of Sprague-Dawley strain, 137
13 8
GLUCOSE EFFECTS
weighing 150-200 grams, were used .
Vol . 9, No . 3
The isolated fat cells from the epididymal
adipose tissue were prepared according to the method of Rodbell (9).
Krebs-
Ringer bicarbonate buffer containing defatted bovine serum albumin, pH 7 .4, was used throughout .
The incubations of fat cells and measurement of FFA
and glycerol were as those described in the previous publications (10, 11). FFA content in some experiments was measured by a radiochemical assay (12) .
In brief, FFA was converted to 60 Co-FFA complex, and then the radio-
activity of 60 Co in the Co-FFA complex (upper phase) was counted.
This
method was sensitive to as low as 0. 08 nmoles of FFA, and the standard curve was linear up to 200 nmoles per assay.
The sensitivity of this method
can be adjusted by increasing or decreasing the specific activity of 60 Co (N03)Z used .
Results were expressed as nmoles of FFA or glycerol release
per mg of total lipid per 1 or Z hours of incubation.
Adenosine triphosphate
content in fat cells was measured by the luminescence reaction with firefly luciferase (13) .
DNA content was estimated by the method of Ceriotti (14) .
60Co++ was purchased from New England Nuclear Corp ., Boston, Mass . N6 -2 , -0-dibutyryl 3', 5'-adenosine monophosphate cyclic, sodium salt, was supplied by Boehringer Mannheim Corp ., New York, N.Y .
Bovine
serum albumin, fraction V, was purchased from Nutritional Biochemical Corp . , Cleveland, Ohio .
It was defatted in our laboratory and kept as
FFA "free" dry powder . Result s Experiments were performed to search for suitable conditions under which an increase in epinephrine-stimulated release of FFA exerted by glucose could be demonstrated .
Isolated fat cells from either fasted or tasted-refed rats
were prepared in the absence of added glucose .
Fat cells were then distributed
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GLUCOSE EFFECTS
139
and incubated with and without epinephrine (3 .4 x 10 -10 mole/ml) and with and without glucose (2 . 05 mM). Epinephrine stimulated the release of FFA from fat cells of both groups in the absence of glucose .
Upon addition of
glucose, the effect of epinephrine was increased more than two fold using fat cells from fasted rats (Table I) . TABLE I Enhancement of glucose in epinephrine stimulated FFA release from isolated fat cells of fasted rats Exp.
FFA release, nmoles/mg/h -glucose
1
9 .1
P
Glucose* Effect
+glucose 95 .8
10 .5
2
99 . 1 f 8. 7
465 . 0 * 2. 1
3
80 .2
252.3
4
88 .1t5 .6
169.0f7 .1
<0 .01
1 .9
5
67 .7f3 .4
77 .9f4 .3
«.01
1.2
6 161 .7t6 .5 Mean + 5. E.
216.3t7 .4
«.01
1 .3 3. 78 f 1 . 74.4
z0 . 01
4.7 3.1
*Glucose effect : (FFA release, + glucose)/(FFA release,
- glucose)
* The probability of FFA releasenót increased by glucose is <0 . 01 . However, in cells from rats fasted for 72 hours and refed for 48 hours, FFA release showed a 2096 decrease as compared to controls containing no glucose. Fat cells from fasted rats were then used in all following experiments . Epinephrine concentration and the effect of glucose.
In order to
determine whether the above effect of glucose was due simply to an increase in responsiveness of fat cells to epinephrine, or whether an
140
GLUCOSE EFFECTS
Vol. 9, No. 3
increase in epinephrine concentration could bring further increase in FFA release even in the absence of glucose, the epinephrine dose-response relationship in the presence and absence of glucose was studied.
Reat,lts
in Fig. 1 show that the maximum FFA release due to epinephrine in the
L E \ ó E c
W N W
200
100 ~ il
_J W Q W W
0
a
01, 34 3.4
340
EPINEPHRINE (moles/ml X 10 -1( ) Fig. 1
The relationship between the concentration of epinephrine, the release of FFA, and the effect of glucose.
Fat cells from epididymal adipose tissue
of 48 hours fasted rats were incubated with various concentrations of epine phrine from 3.4 x 10 -11 moles/ml to %40 x 10 -9 moles/ml with (~---r) and without (o---.o) glucose (2 . 05 mW .
The results are expressed as nmoles of
FFA release/mg fat cell lipid/br . absence of glucose was less than that in the presence of glucose (2 . 05 mM). At 3 . 4 x 10 -11 moles of epinephrine/ ml, glucose increased the FFA release
GLUCOSE EFFECTS
Vol. 9, No . 3
0 1 2 .5
14 1
10.0 GLUCOSE (mM)
0.5
20.0
Fig. 2 The effect of increasing glucose concentration on epinephrinestimulated lipolysis .
Fat cells were incubated with epinephrine (3 . 4 x 10-10
moles/ml) and various glucose concentrations, as indicated.
FFA (0---*)
and glycerol (i-o) release are expressed as nmoles/mg of lipids/2 hours of incubation .
Each point represents a mean of 4 values f S. E. .
The
differences in glycerol and FFA release between groups with and without glucose are significant (p <0 . 01).
The decrease in FFA release at a 20 mM
glucose concentration was also significant (p <0 . 01) as compared to that observed with 10 mM glucose.
The FFA to albumin molar ratio reached
to 2. 4 at the end of incubation in samples containing no glucose, suggesting the presence of adequate FFA acceptor in the incubation medium . approximately 3 fold as compared to that in he absence of glucose. At 3.4 x
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GLUCOSE EFFECTS
Vol. 9, No. 3
10 -10 moles of epinephrine/ml this effect of glucose was twofold. At 3.4 x 10 -9 moles of epinephrine/ml a 1 .6 fold increase in FFA release by glucose was observed . Effect of increasing gluco se concent ration on the epinephrinestimulated lipolysis .
The stimulatory effect of glucose on both FFA and
glycerol release is deomonstrated, as shown in Fig. 2 .
Fat cells were
incubated with epinephrine, 3.4 x 10 10 mole/ml in the presence of 0, 0. 5, 2 . 5, 10 or 20 mM of glucose. in 3 phases .
1.
The FFA response curve could be recognized
Positive phase.
EFA release was increased when the
glucose concentration was increased ( o to 0. 5 mM, p<0 . O1); 2. Plateau phase .
FFA release was not significantly affected by further increase
in glucose concentration (2 . 5, and 10 mM); 3. Negative phase .
FFA release
was decreased by increasing glucose concentration to 20 mM (p<0 . O1). However, the glycerol reponse curve was increased at each case by increasing the glucose concentration ; the increase in each glucose concentration was greater than that observed without glucose; (p-
This effect of glucose was not found to be significant when
dibutyryl cyclic AMP was used as the lipolytic agent (Table II).
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GLUCOSE EFFECTS
143
TABLE II Glucose-enhanced FFA release from fat cells stimulated by ACTH, caffeine and N6 -2'-0-dibutyryl cyclic AMP
Lipolytic
FFA release, nmole/mg/h
Agents
Glucose effect
P
-glucose
+glucose *
Caffeine, 1 x 10 -3 M
142 f 2. 5
.9 :k14 239
49.0 .01
+68
ACTH, 20 mU/ml
155 t 5 .5
195 t 5 . 3
L0 . 01
+26
Dibutyryl cyclic AMP, 1 x 10 -3 M
231 f 11 . 7
264 t 18 . 1
N. S.
+14
*Glucose 2. 0 mM was used . ** Glucose effect was calculated as follows : t(FFA release, + glucose) -(FFA release, - glucose)/(FFA release, - glucose x 100 Inhibition of glucose-enhance d lipo lysis by non- metabolizable analogs of glucose.
Results in Table III show that glucose-enhanced lipolysis
was inhibited by 3-0-methyl-D-glucose or 2-deoxy-D-glucose .
The
inhibitory effects were 53 and 80% respectively . Effect of epinep hrine on adenosine triphosphate levels in fat cells and the influence of glucose .
The results shown in Table IV indicated that
epinephrine stimulated lipolysis in fat cells of both fed and fasted rats . Epinephrine had little effect on ATP levels in fat cells of fed rats, but decreased the level of ATP in fat cells of fasted rats by 30-50% as
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GLUCOSE EFFECTS
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TABLE III Inhibition of glucose-enhanced lipolysis by 3-0-methyl-D-glucose and 2deoxy-D-glucose .
Expt .
Epinephrine nmole/ml
Additions mm
FFA Release nmole/mg
Substrate Inhibitory effect% effect
99 . 1 f 8 . 7
IA
34
-----
IB
34
3-0-methylglucose, 7 .5
103 3 17
5
IC
34
glucose, 2. 5
475 +25
376
ID
34
glucose, 2. 5 ; + 3-0-methylglucose, 7 .5
277 t 37
178
IIA
-
-----
6 . 5 f 0. 9
--
IIB
34
-----
74 . 3 1 6 . 3
--
IIC
34
glucose, 0. 5
113 . 5 f 5 . 6
39
IID
34
glucose, 0.5+ 2-deoxyglucose, 5.0
IIE
34
2-deoxyglucose, 5 .0
82
2.7
8
53
80
79
*Inhibitory effect was calculated as follows:
PFFA, C)-(FFA, D)/(FFA, C] x 100
compared to fat cells offasted rats incubated in medium containing no epinephrine. Results in Table IV were based on fat cell DNA as reference.
When
fat cells-lipids were used as reference, the level of ATP was 1 . 09 f 0 . 09 x 10 -7 moles/g lipids of fat cells from fasted rats (30 observations) .
In the
presence of epinephrine, this level was decreased to 0. 77 t 0. 06 x 10 -7 moles/g lipid.
An average of 30% decrease in ATP level was observed .
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GLUCOSE EFFECTS
145
TABLE IV Effect of epinephrine on ATP levels in fat cells
Group
Epinephrine nmole/ml
ATP content moles/mg DNA x 10 -7
Diff$ %
FFA release Wmole/mg DNA/h
A
0.34
3.34t0.21 3. 33 t 0 . 56
-0 .3
7 .2 260.0
B##
0.34
1. 92 t 0. 08 0. 95 f 0. 05
-50
2 .0 124. 0
0.34
2. 40 t 0. 13 1. 10 t 0 . 06
-54
21 .0 130.0
-0.34
1. 75 t 0. 09 1. 15 t 0 . 07
--34
2 .4 159 .0
0. 34
3.00*0 .16 1 . 57 t 0 . 09
-48
2 .4 170.0
C##
D
##
E##
ATP levels in fat cells were measured by bioluminescence reaction after 10 minutes incubation with or without epinephrine in the incubation medium . Fat cells were prepared from epididymal adipose tissue of fed rats . ##Fat cells were prepared from epididymal adipose tissue of rat.fasted for 48 hours. $ Diff. %, percent difference, were calculated as follows : (Value, -epi)-(value, + epi)/(value, -epi)]
x100
The results shown in Table V indicate that an increase in ATP levels in fat cells due to the addition of glucose was correlated with the
14 8
GLUCOSE EFFECTS
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TABLE V The effect of glucose on ATP levels in fat cells
Epinephrine nmole/ml
0.34 0 .34
Glucose mm
5 .0
ATP content mole/mg DNA x10~71 . 92 t 0. 08 0. 95 t 0. 05 1. 25 t 0. 07
Diff . %
FFA release Wrnole/mg DNA/h
-50 -35
2 .0 124.0 152 .0
Fat cells were prepared from rats fasted for 48 hours. enhancing action of glucose on epinephrine-stimulated lipolysis. Discussion That glucose can reduce hormone-stimulated FFA release from adipose tissue has been well domumented (1-3).
This effect is accompanied
under certain conditions by accelerated glycerol release showing that lipolysis can in fact be promoted by glucose (4).
To account for this
behavior, it has been postulated that glucose reduces cellular FFA levels by providing p(-glycerolphosphate for re-esterification, thereby simultaneously slowing FFA efflux and relieving inhibition of lipolysis by FFA (4).
The
present study shows that under appropriate conditions glucose can enhance hormone-stimulated-lipolysis without lowering FFA levels . for in this case, glucose
promoted release of both glycerol and FFA.
In free fat cells prepared from fasting rats, this novel action of glucose was more striking than the classical one .
A l ow concentration of
glucose (0 . 5 mM) sufficed to accelerate both FFA release and glycerol release about 2. 5 fold .
Higher levels of glucose (up to 10 mM) increased
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GLUCOSE EFFECTS
14 7
glycerol release another 2. 5 fold while FFA release increased very little if at all.
This second increase in lipolysis was likely brought about by
the same mechanism as the first, certainly not by a reduction in FFA levels . Nevertheless, re-esterification did increase sufficiently to prevent a further marked increase in FFA output ; and, as glucose levels were increased to 20 mM, the re-esterification increment exceeded that of lipolysis, so that FFA output diminished slightly despite a substantial increase in glycerol release .
Although this last pattern of changes resembled the classical
observation (4), it seems illogical to explain the increased glycerol release seen here in terms of decreased FFA inhibition of lipolysis.
It is simpler'in
this case to assume that the entire, continuous increase in lipolysis as a fun ction of gl ucose concentration was due to the same mechanism.
In the above
interpretation, the possibility was ignored that alterations in FFA oxidation might account for the changes in FFA release, for this process is very slow relative to esterification (11) . Regarding the mechanism by which glucose enhanced hormonestimulated FFA release, it appears that glucose acted in one of its usual roles (as energy substrate, reducing substrate or carbon source); for the glycogen-rich adipose cells from fasted-refed rats showed no such requirement, and inhibitors of glucose utilisation prevented the enhancement.
This view
is confirmed and perhaps refined by the correlations between ATP levels and the effects of glucose and epinephrine.
In cells prepared from fasting
rats, ATP levels were lowered still further by addition of epinephrine .
This
suggests that hormone stimulation accelerated ATP utilization and/or impaired ATP synthesis.
Accelerated utilization could have occurred in connection
with activation and esterification of the higher levels of FFA; impaired
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GLUCOSE EFFECTS
Vol. 9, No . 3
synthesis could have occurred from diversion of the limited amounts of carbohydrate to glyceride glycerol .
The glucose enhancement of lipolysis
were correlated with a partial restoration of ATP towards the level seen without hormone.
In cells from fed rats, the higher ATP levels were
comparable with those observed by Denton et al . (15) in adipose tissue of fed rats incubated with glucose and were not affected by epinephrine suggesting that glycogen sufficed for whatever extra metabolic demands accompanied stimulation.
In this case, a glucose requirement for FFA release would not
be expected and was indeed not seen. While correlations of this kind cannot prove that ATP was limiting for lipolysis in cells from fasted rats, the results are consistent with this view .
Moreover, there is no known requirement for glucose carbon or
reducing equivalents in lipolysis; but there is thought to be need for ATP in lipase activation, namely in the synthesis of 3', 5'-cyclic AMP. Several lines of evidence involve this nucleotide in the mediation of hormone effects on adipose tissue lipase (5, 6) .
One might also envision further ATP dependent
steps in lipase activation by analogy with the glycogen phosphorylase system of liver (7) . Summary The ability of glucose to enhance hormone-stimulated lipolysis in isolated fat cells of fasted rats was observed .
Glucose enhanced free
fatty acid (FFA) release stimulated by adrenocorticortrophine (ACTH), epinephrine or caffeine significantly greater than that stimulated by dibutyryl 3', 5'-cyclic AMP.
The enhancement of epinephrine stimulated
FFA release by glucose was inhibited by 2-deoxy-glucose or 3-0-methyl glucose .
Clearly, the traditional theory that glucose enhanced lipolysis
Vol. 9, No . 3
GLUCOSE EFFECTS
149
was a result of removed FFA inhibition cannot explain glucose-enhancement of hormone stimulated release of FFA.
Epinephrine-stimulated lipolysis
correlated with a decrease in ATP content of fat cells from fasted rats that contained little glycogen .
Such a decrease in ATP content was not seen in
fat cells from fed rats after epinephrine.
In fat cells from fasted rats,
glucose-enhanced lipolysis correlated to an increase in ATP content.
This
finding suggests that the activation of lipolysis requires energy ; if the endogenous energy supply is inadequate, the energy of added substrate must be used for such activation processes. Acknowledgements We would like to thank Drs . C. R. Park, O. B. Crofford and M. A. Rizack for discussions and reading the manuscript .
The help of Dr . R. A .
Johnson for the method of ATP measurement is cordially acknowledged . This work was done during the tenure of an Established Investigator ship of the American Heart Association (R. J. Ho) and was supported by grants from the U. S. Public Health Service (HE 04372 and AM-07462). References 1.
D. STEINBERG, M. VAUGHAN, and S. MARGOLIS, J. Biol . Chem . 235, pc 38 (1960) .
2.
A . E. RENOLD, and G. CAHILL, Editors, Handbook of Physidogy, Section 5 : Adipose Tissue, Am . Physiol. Soc. Washington, D. C., (1965) .
3.
H. C. MENG and R. J. HO, Progr. Biochem. Pharmacol. 3, 207 (1967) .
4.
P . R. BALLY, H. KAPELLER, E. R. FROESCH and A LABHART, Ann. N. Y . Acad . Sci ., 131, 143 (1965) .
5.
M.A . RIZACK, J. Biol Chem. , 239, 292 (1964) .
6.
R. W. BUTCHER, R. J. HO, H. C. MENG and E. W. SUTHERLAND, _J . Biol . Chem . , 240, 4515 (1965) .
7.
G. A. ROBISON, R. W. BUTCHER, and E. W. SUTHERLAND, Ann. Rev.
150
GLUCOSE EFFECTS
Vol. 9, No . 3
Biochem. 37, 149 (l968) . 8.
R. J. HO and H. C. MENG, The Physiologist 12, 225 (1969) .
9.
M. RODBELL, J. Biol. Chem . , 239, 375 (1964) . R. J. HO, S. J. HO and H. C. MENG, Metabolism 14, 1010 (1965) .
11 .
R J. HO, B. JEANRENAUD, TH . POSTERNAK, and A. E. RENOLD, Biochem . Biophys. Acta , 144, 78 (1967) .
12 .
R J. HO and H. C. MENG, Anal. Biociem . , in press.
13 .
B. L. STREHLER and J. K. TOTTER in "Method of Biochemical Analysis" Edited by D. Glick, Vol. I, p. 341, Interscience Publisher, N.Y ., 1954 .
14 .
C. KECK, Arch . Biochem . Biophys . 63, 446 (1956) .
15 .
R.M. DENTON, R. E. YORKE and P. J. RANDLE, Biochem. J. 100 407 (1966) .
Pergamon Press
EFFECT OF GLUCOSE ON LIPOLYSIS AND ENERGY METABOLISM IN FAT CELLS R. J. Ho, R. England and H. C. Meng Dept . of Physiology, Vanderbilt University School of Medicine, Nashville, Tennessee 37203 (Received 10 September 1969 ; in final form 14 October 1969)
THE hormone-stimulated release of free fatty acid (FFA) from adipose tissue incubated in vitro has been shown to be reduced in the presence of glucose (1-3).
Differing from the release of FFA, the release of the other
lipolysic product, glycerol, was somewhat increased (4).
This effect was
interpreted to be the result of an increase in esterification due to an increase in the availability of cKrglycerol phosphate derived from glucose metabolism . Therefore, in studying fat mobilisation, glucose was often omitted from the incubation medium .
The requirement of the high energy phosphate compound,
ATP, for the activation of hormone-sensitive lipase was first shown by Rizack (5).
It is now believed that the formation of 3', 5'-cyclic AMP from ATP
appears to be a possible intermediate step in the lipase activation (6, 7) .
This
belief led us to reexamine the role of glucose in the activation of lipase in response to epinephrine that results in lipolysis in fat cells .
In the present
study, the effect of glucose on epinephrine-stimulated FFA and glycerol release were observed under varying conditions .
The results favored the
hypothesis that the activation of hormone-sensitive lipase requires the energy of added substrate if the endogenous energy supply is inadequate . Part of this work has been reported as an abstract (S). Materials and Methods Fasted and fasted-refed albino rats of Sprague-Dawley strain, 137
13 8
GLUCOSE EFFECTS
weighing 150-200 grams, were used .
Vol . 9, No . 3
The isolated fat cells from the epididymal
adipose tissue were prepared according to the method of Rodbell (9).
Krebs-
Ringer bicarbonate buffer containing defatted bovine serum albumin, pH 7 .4, was used throughout .
The incubations of fat cells and measurement of FFA
and glycerol were as those described in the previous publications (10, 11). FFA content in some experiments was measured by a radiochemical assay (12) .
In brief, FFA was converted to 60 Co-FFA complex, and then the radio-
activity of 60 Co in the Co-FFA complex (upper phase) was counted.
This
method was sensitive to as low as 0. 08 nmoles of FFA, and the standard curve was linear up to 200 nmoles per assay.
The sensitivity of this method
can be adjusted by increasing or decreasing the specific activity of 60 Co (N03)Z used .
Results were expressed as nmoles of FFA or glycerol release
per mg of total lipid per 1 or Z hours of incubation.
Adenosine triphosphate
content in fat cells was measured by the luminescence reaction with firefly luciferase (13) .
DNA content was estimated by the method of Ceriotti (14) .
60Co++ was purchased from New England Nuclear Corp ., Boston, Mass . N6 -2 , -0-dibutyryl 3', 5'-adenosine monophosphate cyclic, sodium salt, was supplied by Boehringer Mannheim Corp ., New York, N.Y .
Bovine
serum albumin, fraction V, was purchased from Nutritional Biochemical Corp . , Cleveland, Ohio .
It was defatted in our laboratory and kept as
FFA "free" dry powder . Result s Experiments were performed to search for suitable conditions under which an increase in epinephrine-stimulated release of FFA exerted by glucose could be demonstrated .
Isolated fat cells from either fasted or tasted-refed rats
were prepared in the absence of added glucose .
Fat cells were then distributed
Vol. 9, No. 3
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139
and incubated with and without epinephrine (3 .4 x 10 -10 mole/ml) and with and without glucose (2 . 05 mM). Epinephrine stimulated the release of FFA from fat cells of both groups in the absence of glucose .
Upon addition of
glucose, the effect of epinephrine was increased more than two fold using fat cells from fasted rats (Table I) . TABLE I Enhancement of glucose in epinephrine stimulated FFA release from isolated fat cells of fasted rats Exp.
FFA release, nmoles/mg/h -glucose
1
9 .1
P
Glucose* Effect
+glucose 95 .8
10 .5
2
99 . 1 f 8. 7
465 . 0 * 2. 1
3
80 .2
252.3
4
88 .1t5 .6
169.0f7 .1
<0 .01
1 .9
5
67 .7f3 .4
77 .9f4 .3
«.01
1.2
6 161 .7t6 .5 Mean + 5. E.
216.3t7 .4
«.01
1 .3 3. 78 f 1 . 74.4
z0 . 01
4.7 3.1
*Glucose effect : (FFA release, + glucose)/(FFA release,
- glucose)
* The probability of FFA releasenót increased by glucose is <0 . 01 . However, in cells from rats fasted for 72 hours and refed for 48 hours, FFA release showed a 2096 decrease as compared to controls containing no glucose. Fat cells from fasted rats were then used in all following experiments . Epinephrine concentration and the effect of glucose.
In order to
determine whether the above effect of glucose was due simply to an increase in responsiveness of fat cells to epinephrine, or whether an
140
GLUCOSE EFFECTS
Vol. 9, No. 3
increase in epinephrine concentration could bring further increase in FFA release even in the absence of glucose, the epinephrine dose-response relationship in the presence and absence of glucose was studied.
Reat,lts
in Fig. 1 show that the maximum FFA release due to epinephrine in the
L E \ ó E c
W N W
200
100 ~ il
_J W Q W W
0
a
01, 34 3.4
340
EPINEPHRINE (moles/ml X 10 -1( ) Fig. 1
The relationship between the concentration of epinephrine, the release of FFA, and the effect of glucose.
Fat cells from epididymal adipose tissue
of 48 hours fasted rats were incubated with various concentrations of epine phrine from 3.4 x 10 -11 moles/ml to %40 x 10 -9 moles/ml with (~---r) and without (o---.o) glucose (2 . 05 mW .
The results are expressed as nmoles of
FFA release/mg fat cell lipid/br . absence of glucose was less than that in the presence of glucose (2 . 05 mM). At 3 . 4 x 10 -11 moles of epinephrine/ ml, glucose increased the FFA release
GLUCOSE EFFECTS
Vol. 9, No . 3
0 1 2 .5
14 1
10.0 GLUCOSE (mM)
0.5
20.0
Fig. 2 The effect of increasing glucose concentration on epinephrinestimulated lipolysis .
Fat cells were incubated with epinephrine (3 . 4 x 10-10
moles/ml) and various glucose concentrations, as indicated.
FFA (0---*)
and glycerol (i-o) release are expressed as nmoles/mg of lipids/2 hours of incubation .
Each point represents a mean of 4 values f S. E. .
The
differences in glycerol and FFA release between groups with and without glucose are significant (p <0 . 01).
The decrease in FFA release at a 20 mM
glucose concentration was also significant (p <0 . 01) as compared to that observed with 10 mM glucose.
The FFA to albumin molar ratio reached
to 2. 4 at the end of incubation in samples containing no glucose, suggesting the presence of adequate FFA acceptor in the incubation medium . approximately 3 fold as compared to that in he absence of glucose. At 3.4 x
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GLUCOSE EFFECTS
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10 -10 moles of epinephrine/ml this effect of glucose was twofold. At 3.4 x 10 -9 moles of epinephrine/ml a 1 .6 fold increase in FFA release by glucose was observed . Effect of increasing gluco se concent ration on the epinephrinestimulated lipolysis .
The stimulatory effect of glucose on both FFA and
glycerol release is deomonstrated, as shown in Fig. 2 .
Fat cells were
incubated with epinephrine, 3.4 x 10 10 mole/ml in the presence of 0, 0. 5, 2 . 5, 10 or 20 mM of glucose. in 3 phases .
1.
The FFA response curve could be recognized
Positive phase.
EFA release was increased when the
glucose concentration was increased ( o to 0. 5 mM, p<0 . O1); 2. Plateau phase .
FFA release was not significantly affected by further increase
in glucose concentration (2 . 5, and 10 mM); 3. Negative phase .
FFA release
was decreased by increasing glucose concentration to 20 mM (p<0 . O1). However, the glycerol reponse curve was increased at each case by increasing the glucose concentration ; the increase in each glucose concentration was greater than that observed without glucose; (p-
This effect of glucose was not found to be significant when
dibutyryl cyclic AMP was used as the lipolytic agent (Table II).
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143
TABLE II Glucose-enhanced FFA release from fat cells stimulated by ACTH, caffeine and N6 -2'-0-dibutyryl cyclic AMP
Lipolytic
FFA release, nmole/mg/h
Agents
Glucose effect
P
-glucose
+glucose *
Caffeine, 1 x 10 -3 M
142 f 2. 5
.9 :k14 239
49.0 .01
+68
ACTH, 20 mU/ml
155 t 5 .5
195 t 5 . 3
L0 . 01
+26
Dibutyryl cyclic AMP, 1 x 10 -3 M
231 f 11 . 7
264 t 18 . 1
N. S.
+14
*Glucose 2. 0 mM was used . ** Glucose effect was calculated as follows : t(FFA release, + glucose) -(FFA release, - glucose)/(FFA release, - glucose x 100 Inhibition of glucose-enhance d lipo lysis by non- metabolizable analogs of glucose.
Results in Table III show that glucose-enhanced lipolysis
was inhibited by 3-0-methyl-D-glucose or 2-deoxy-D-glucose .
The
inhibitory effects were 53 and 80% respectively . Effect of epinep hrine on adenosine triphosphate levels in fat cells and the influence of glucose .
The results shown in Table IV indicated that
epinephrine stimulated lipolysis in fat cells of both fed and fasted rats . Epinephrine had little effect on ATP levels in fat cells of fed rats, but decreased the level of ATP in fat cells of fasted rats by 30-50% as
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TABLE III Inhibition of glucose-enhanced lipolysis by 3-0-methyl-D-glucose and 2deoxy-D-glucose .
Expt .
Epinephrine nmole/ml
Additions mm
FFA Release nmole/mg
Substrate Inhibitory effect% effect
99 . 1 f 8 . 7
IA
34
-----
IB
34
3-0-methylglucose, 7 .5
103 3 17
5
IC
34
glucose, 2. 5
475 +25
376
ID
34
glucose, 2. 5 ; + 3-0-methylglucose, 7 .5
277 t 37
178
IIA
-
-----
6 . 5 f 0. 9
--
IIB
34
-----
74 . 3 1 6 . 3
--
IIC
34
glucose, 0. 5
113 . 5 f 5 . 6
39
IID
34
glucose, 0.5+ 2-deoxyglucose, 5.0
IIE
34
2-deoxyglucose, 5 .0
82
2.7
8
53
80
79
*Inhibitory effect was calculated as follows:
PFFA, C)-(FFA, D)/(FFA, C] x 100
compared to fat cells offasted rats incubated in medium containing no epinephrine. Results in Table IV were based on fat cell DNA as reference.
When
fat cells-lipids were used as reference, the level of ATP was 1 . 09 f 0 . 09 x 10 -7 moles/g lipids of fat cells from fasted rats (30 observations) .
In the
presence of epinephrine, this level was decreased to 0. 77 t 0. 06 x 10 -7 moles/g lipid.
An average of 30% decrease in ATP level was observed .
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145
TABLE IV Effect of epinephrine on ATP levels in fat cells
Group
Epinephrine nmole/ml
ATP content moles/mg DNA x 10 -7
Diff$ %
FFA release Wmole/mg DNA/h
A
0.34
3.34t0.21 3. 33 t 0 . 56
-0 .3
7 .2 260.0
B##
0.34
1. 92 t 0. 08 0. 95 f 0. 05
-50
2 .0 124. 0
0.34
2. 40 t 0. 13 1. 10 t 0 . 06
-54
21 .0 130.0
-0.34
1. 75 t 0. 09 1. 15 t 0 . 07
--34
2 .4 159 .0
0. 34
3.00*0 .16 1 . 57 t 0 . 09
-48
2 .4 170.0
C##
D
##
E##
ATP levels in fat cells were measured by bioluminescence reaction after 10 minutes incubation with or without epinephrine in the incubation medium . Fat cells were prepared from epididymal adipose tissue of fed rats . ##Fat cells were prepared from epididymal adipose tissue of rat.fasted for 48 hours. $ Diff. %, percent difference, were calculated as follows : (Value, -epi)-(value, + epi)/(value, -epi)]
x100
The results shown in Table V indicate that an increase in ATP levels in fat cells due to the addition of glucose was correlated with the
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TABLE V The effect of glucose on ATP levels in fat cells
Epinephrine nmole/ml
0.34 0 .34
Glucose mm
5 .0
ATP content mole/mg DNA x10~71 . 92 t 0. 08 0. 95 t 0. 05 1. 25 t 0. 07
Diff . %
FFA release Wrnole/mg DNA/h
-50 -35
2 .0 124.0 152 .0
Fat cells were prepared from rats fasted for 48 hours. enhancing action of glucose on epinephrine-stimulated lipolysis. Discussion That glucose can reduce hormone-stimulated FFA release from adipose tissue has been well domumented (1-3).
This effect is accompanied
under certain conditions by accelerated glycerol release showing that lipolysis can in fact be promoted by glucose (4).
To account for this
behavior, it has been postulated that glucose reduces cellular FFA levels by providing p(-glycerolphosphate for re-esterification, thereby simultaneously slowing FFA efflux and relieving inhibition of lipolysis by FFA (4).
The
present study shows that under appropriate conditions glucose can enhance hormone-stimulated-lipolysis without lowering FFA levels . for in this case, glucose
promoted release of both glycerol and FFA.
In free fat cells prepared from fasting rats, this novel action of glucose was more striking than the classical one .
A l ow concentration of
glucose (0 . 5 mM) sufficed to accelerate both FFA release and glycerol release about 2. 5 fold .
Higher levels of glucose (up to 10 mM) increased
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GLUCOSE EFFECTS
14 7
glycerol release another 2. 5 fold while FFA release increased very little if at all.
This second increase in lipolysis was likely brought about by
the same mechanism as the first, certainly not by a reduction in FFA levels . Nevertheless, re-esterification did increase sufficiently to prevent a further marked increase in FFA output ; and, as glucose levels were increased to 20 mM, the re-esterification increment exceeded that of lipolysis, so that FFA output diminished slightly despite a substantial increase in glycerol release .
Although this last pattern of changes resembled the classical
observation (4), it seems illogical to explain the increased glycerol release seen here in terms of decreased FFA inhibition of lipolysis.
It is simpler'in
this case to assume that the entire, continuous increase in lipolysis as a fun ction of gl ucose concentration was due to the same mechanism.
In the above
interpretation, the possibility was ignored that alterations in FFA oxidation might account for the changes in FFA release, for this process is very slow relative to esterification (11) . Regarding the mechanism by which glucose enhanced hormonestimulated FFA release, it appears that glucose acted in one of its usual roles (as energy substrate, reducing substrate or carbon source); for the glycogen-rich adipose cells from fasted-refed rats showed no such requirement, and inhibitors of glucose utilisation prevented the enhancement.
This view
is confirmed and perhaps refined by the correlations between ATP levels and the effects of glucose and epinephrine.
In cells prepared from fasting
rats, ATP levels were lowered still further by addition of epinephrine .
This
suggests that hormone stimulation accelerated ATP utilization and/or impaired ATP synthesis.
Accelerated utilization could have occurred in connection
with activation and esterification of the higher levels of FFA; impaired
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GLUCOSE EFFECTS
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synthesis could have occurred from diversion of the limited amounts of carbohydrate to glyceride glycerol .
The glucose enhancement of lipolysis
were correlated with a partial restoration of ATP towards the level seen without hormone.
In cells from fed rats, the higher ATP levels were
comparable with those observed by Denton et al . (15) in adipose tissue of fed rats incubated with glucose and were not affected by epinephrine suggesting that glycogen sufficed for whatever extra metabolic demands accompanied stimulation.
In this case, a glucose requirement for FFA release would not
be expected and was indeed not seen. While correlations of this kind cannot prove that ATP was limiting for lipolysis in cells from fasted rats, the results are consistent with this view .
Moreover, there is no known requirement for glucose carbon or
reducing equivalents in lipolysis; but there is thought to be need for ATP in lipase activation, namely in the synthesis of 3', 5'-cyclic AMP. Several lines of evidence involve this nucleotide in the mediation of hormone effects on adipose tissue lipase (5, 6) .
One might also envision further ATP dependent
steps in lipase activation by analogy with the glycogen phosphorylase system of liver (7) . Summary The ability of glucose to enhance hormone-stimulated lipolysis in isolated fat cells of fasted rats was observed .
Glucose enhanced free
fatty acid (FFA) release stimulated by adrenocorticortrophine (ACTH), epinephrine or caffeine significantly greater than that stimulated by dibutyryl 3', 5'-cyclic AMP.
The enhancement of epinephrine stimulated
FFA release by glucose was inhibited by 2-deoxy-glucose or 3-0-methyl glucose .
Clearly, the traditional theory that glucose enhanced lipolysis
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149
was a result of removed FFA inhibition cannot explain glucose-enhancement of hormone stimulated release of FFA.
Epinephrine-stimulated lipolysis
correlated with a decrease in ATP content of fat cells from fasted rats that contained little glycogen .
Such a decrease in ATP content was not seen in
fat cells from fed rats after epinephrine.
In fat cells from fasted rats,
glucose-enhanced lipolysis correlated to an increase in ATP content.
This
finding suggests that the activation of lipolysis requires energy ; if the endogenous energy supply is inadequate, the energy of added substrate must be used for such activation processes. Acknowledgements We would like to thank Drs . C. R. Park, O. B. Crofford and M. A. Rizack for discussions and reading the manuscript .
The help of Dr . R. A .
Johnson for the method of ATP measurement is cordially acknowledged . This work was done during the tenure of an Established Investigator ship of the American Heart Association (R. J. Ho) and was supported by grants from the U. S. Public Health Service (HE 04372 and AM-07462). References 1.
D. STEINBERG, M. VAUGHAN, and S. MARGOLIS, J. Biol . Chem . 235, pc 38 (1960) .
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A . E. RENOLD, and G. CAHILL, Editors, Handbook of Physidogy, Section 5 : Adipose Tissue, Am . Physiol. Soc. Washington, D. C., (1965) .
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H. C. MENG and R. J. HO, Progr. Biochem. Pharmacol. 3, 207 (1967) .
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M.A . RIZACK, J. Biol Chem. , 239, 292 (1964) .
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G. A. ROBISON, R. W. BUTCHER, and E. W. SUTHERLAND, Ann. Rev.
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Biochem. 37, 149 (l968) . 8.
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M. RODBELL, J. Biol. Chem . , 239, 375 (1964) . R. J. HO, S. J. HO and H. C. MENG, Metabolism 14, 1010 (1965) .
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R J. HO, B. JEANRENAUD, TH . POSTERNAK, and A. E. RENOLD, Biochem . Biophys. Acta , 144, 78 (1967) .
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R J. HO and H. C. MENG, Anal. Biociem . , in press.
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B. L. STREHLER and J. K. TOTTER in "Method of Biochemical Analysis" Edited by D. Glick, Vol. I, p. 341, Interscience Publisher, N.Y ., 1954 .
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C. KECK, Arch . Biochem . Biophys . 63, 446 (1956) .
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R.M. DENTON, R. E. YORKE and P. J. RANDLE, Biochem. J. 100 407 (1966) .