Bioscience Reports 3, 675-679 (1983)
Printed in Great Britain
675
Insulin s e n s i t i v i t y of r a t e s of glycolysis and glycogen
synthesis in soleus, stripped soleus, epitrochlearis, and
hemi-diaphragm muscles isolated from sedentary rats
R. A. 3. CHALLISS, 3. ESPINAL, and E. A. NEWSHOLME
Department of Biochemistry, University of Oxford,
South Parks Road, Oxford OXl 3QU, U.K.
(Received 13 July 1983)
The e f f e c t of insulin c o n c e n t r a t i o n s on the rates of
glycolysis and glycogen synthesis in four di fferent in
vitro r a t muscle preparations (i nt act soleus, stripped
s o l e u s , e p i t r o c h l e a r i s , and h e m i - d i a p h r a g m ) w e r e
investigated: the concentrations of insulin that produced
h a l f - m a x i m a l stimulation of the rates of these two
processes in the four muscle preparations were similar about 100 punits/ml.
This is at least 10-fold great er
t h a n t h e c o n c e n t r a t i o n t h a t produced half-maximal
inhibition of lipolysis in isolated adipocytes. Since 100
p u n i t s / m l insulin is outside the normal physiological
range in the rat, it is suggested that, in vivo, insulin
influences
g l u c o s e u t i l i z a t i o n in m u s c l e m a i n l y
indirectly, via changes in the plasma f a t t y acid levels
and the ' g l u c o s e / f a t t y acid cycle'.
Consequently the
v ie w t h a t insulin s t i m u l a t e s g l u c o s e utilization in
m u s c l e m a i n l y by a d i r e c t e f f e c t on m e m b r a n e
transport must be t r e a t e d with caution.
Insulin can a c u t e l y increase the rate of glucose utilization by
muscle in a direct and an indirect manner; directly, it stimulates the
r ate of membrane transport of glucose ( i ) ; indirectly, it inhibits the
r ate of lipolysis in adipose tissue, which lowers the plasma f a t t y acid
c o n c e n t r a t i o n and hence decreases the rate of f a t t y acid oxidation,
which favours glucose utilization. The regulatory relationship between
f a t t y acids and glucose is known as the g l u c o s e / f a t t y acid cycle (2,3).
An i m p o r t a n t question is whether the direct e f f e c t of insulin on
muscle in vivo is quantitatively important.
The sensitivities of the
processes of lipolysis in adipose tissue and glucose utilization in muscle
to insulin in vitro are reported to differ by at least 10-fold: the
co n cen tr at i on of insulin required for half-maximal inhibition of lipolysis
in isolated incubated adipocytes is about 10 punits/ml (~,5) whereas
the concentration of insulin required for half-maximal stimulation of
glucose utilization in isolated muscle preparations (stripped soleus and
perfused hindquarters) from sedentary rats is at least 100 punits/ml
(6,7).
Since the concent r a t i on of insulin in peripheral blood of the
rat varies between 5 and 50 punits/ml in the fasted and fed states
01983
The Biochemical Society
676
CHALLISS ET AL.
r e s p e c t i v e l y (8), these findings suggest that the direct e f f e c t of
insulin on glucose utilization in muscle is not quantitatively important
in control of glucose utilization after a normal meal. However, the
two m u s c l e preparations used to study insulin sensitivity are both
'extreme' preparations: the isolated incubated stripped soleus preparation (7) c o n t a i n s a high proportion of type-I fibres; and in the
perfused hindquarter of the rat (6) insulin must diffuse from the
v a s c u l a r s y s t e m t h r o u g h the interstitial space to reach the cell
membrane, which could decrease the observed sensitivity to insulin.
Consequently, it was decided to study the insulin sensitivity of glucose
utilization of two other small muscles that can be incubated in vitro,
the epitrochlearis, which contains a high proportion of type-II fibres,
and the hemi-diaphragm. The soleus preparation was also included in
this study to compare results with previous studies.
M a t e r i a l s and M e t h o d s
All c h e m i c a l s and e n z y m e s were obtained from sources given
previously (4) except that insulin was obtained from Sigma Chemical
Co., P o o l e , D o r s e t , U.K., and D-EU-t~C]glucose from Amersham
I n t e r n a t i o n a l , Amersham, Bucks, U.K.
Male Wistar rats (weighing
60-80 g, for preparation of intact soteus and hemi-diaphragms, or
150-170 g for preparation of stripped soleus and epitrochlearis) were
obtained from Bantin and Kingman, Hull, Yorkshire, U.K., and were
kept in the Department's animal house with free access to food and
water for at least #S h before use.
Rats were killed by cervical
dislocation, and muscles were dissected and immersed immediately in
Krebs-Ringer bicarbonate of the following composition (in mM)" Na,
132; K, 6; PO~., 1.2; Ca, 1.3; Mg, 1.2; HCO 3, 25; N-2-hydroxypiperazine-N'-2-ethanesulphonic acid, 7; pyruvate, 5; succinate, #; L-glutamate, 5; and D-glucose, 5. The medium was prepared daily and gassed
with O2:CO 2 (95:5).
Defatted albumin was added to a final conc e n t r a t i o n of 1.5% and the pH adjusted to 7.25.
Muscles were
pre-incubated for 30 min in sealed 25-ml siliconized Erlenmeyer flasks
at 37~
and then transferred to other flasks containing identical
Krebs-Ringer bicarbonate medium except for the exclusion of pyruvate,
s u c c i n a t e , and L - g l u t a m a t e and the inclusion of D-[U-t~C]glucose
(0.25 - 0.5 lJCi/ml) and various insulin concentrations.
Flasks were
gassed continuously in the pre-incubation period and for the first 15
min of the second incubation. After 60 rain, muscles were removed,
rapidly blotted, and freeze-clamped.
The rate of glucose oxidation
was measured by adsorption of carbon dioxide in 2 N NaOH a f t e r
acidification of the medium with 5 N H2SO~ and the radioactivity was
measured on barium a c e t a t e discs (9).
An aliquot of the incubation
m e d i u m was d e p r o t e i n i z e d with #% (w/v) perchloric acid (final
c o n c e n t r a t i o n ) and the p r o t e i n removed by centrifugation.
The
supernatant was neutralized with KOH and precipitated KCIO~ removed
by c e n t r i f u g a t i o n .
L a c t a t e was assayed enzymatically (10) and
radiochemically by separation of lactate on an ion-exchange column
(I l ) . The radioactivity incorporated into glycogen was measured a f t e r
precipitation with ethanol (12).
Preliminary experiments established
that, in all four muscle preparations used in this work, the ATP/ADP
and ATP/AMP concentration ratios were maintained throughout the
GLYCOLYSIS AND GLYCOGEN SYNTHESIS IN MUSCLE
677
i n c u b a t i o n period at values similar to those found in the muscle
freeze-clamped either in situ or immediately after dissection and that
the rates of glycolysis and glycogen synthesis were linear for at least
60 rain.
It was considered that all four muscle preparations were
physiologically viable.
Since the half-life of insulin in several tissues is only several hours
(13) (depending on the concentration of insulin), the total period of
incubation never exceeded 90 min to prevent any significant loss of
receptors during the experiment.
Results
The e f f e c t of insulin on glucose utilization has been studied by
measuring the rate of formation of lactate and the rate of conversion
of [tgC]glucose to [tgC]glycogen as described elsewhere (7).
The
e f f e c t s of insulin on the rates of l a c t a t e production in four muscle
preparations are presented in Table 1.
In both soleus preparations,
insulin increased the rate of l a c t a t e production just over two-fold (see
also reference 14) whereas, for both the hemi-diaphragm and epitrochlearis, the maximum stimulation is less than two-fold. However,
for all preparations the concentration of insulin that produced halfmaximal stimulation of the rate of l a c t a t e production was about 100
punits/ml. Similar results were obtained when glycolysis was measured
by following the formation [ t ~ C ] l a c t a t e from [t~C]glucose (Table I),
except that rates of glycolysis were lower at low insulin concentrations.
This finding is probably explained by a contribution from
g l y c o g e n degradation to l a c t a t e production, which is detected only
when l a c t a t e is measured enzymatically. Furthermore, the contribution
m a y be higher at low insulin levels since this hormone inhibits
glycogenolysis. The rates of conversion of [tgC]glucose to [t~C]CO 2
at zero insulin concentration, in epitrochlearis, hemi-diaphragm, and
stripped soleus were 0.5 + 0.03 (6), 2.0 • 0.011 (5), and 1.2 • 0.1z~
(6) and in the presence of 1000 punits of insulin were 0.7 • 0.03 (6),
3.2 + 0.20 (5), and 1.6 +- 0.01 (6) IJmol/h per g wet wt. of muscle
respectively.
Hence, for each muscle the highest rate of glucose
oxidation is less than 25% of the rate of glycolysis so that any
changes in oxidation cannot account for the large changes observed in
rates of lactate formation.
The effects of insulin on the rates of glycogen synthesis in the
four muscle preparations are shown in Table 1. tn all four preparations, the concentration of insulin required to produce half-maximal
stimulation os glycogen synthesis was at least 100 punits/ml. The two
preparations in which some fibres are damaged, hemi-diaphragm and
stripped soleus, exhibited higher rates of glycogen synthesis both under
basal conditions and in the presence of maximal concentrations of
insulin; the percentage stimulation of glycogen synthesis by insulin was
also considerably greater in these preparations. The reason for these
differences is not known.
Discussion
These findings demonstrate that for three very different rat
muscles, soleus, diaphragm, and epitrochlearis~ and for two separate
1.
Effect of insulin concentration on the rates of ]actate formation and glycogen synthesis
in intact and stripped soleus~ epitrochlearis and hemidiaphragm preparations of the rat
0.77 • 0.06
(12)
1.35 • 0 . 3 0
(12)
1.40 -+ 0.35
(12)
1000
10000
50
10
I00
(12)
Radiochemical
Enzymatic
Radiochemical
Epitrochlearis
14.9 • 1.59
(12)
18.5 • 1.18
(12)
1 8 . 2 • 1.18
(12)
9 . 6 • 1.03
(12)
8 . 1 +- 0 . 7 5
(12)
8.2 • 0.90
16.8 • 1.43
13.2 + 2.74
(5)
1 6 . 5 i 1.97
-
(5)
8.5 i 0.55
(4)
7.0 • 0.55
(6)
7.0 • 0.76
7.4 • 0.39
(5)
9.2 i 0.57
(4)
9.1 • 0 . 4 9
(4)
(4)
8.1 • 0.52
(5)
9.5 + 0.29
(4)
9.3 • 0.61
(5)
(4)
0.48
5.6 • 0.25
(5)
5.2 i
4.9 • 0.46
(6)
-
7.3 • 0.39
(5)
6.5 i 0.36
6.4 • 0.30
(6)
Rate of lactate formation (~Jmo2 lactate/hl~er g wet w t . ]
Enzymatic
Stripped soleus
6.0 • 0.45
(12)
5.2 • 0.77
(12)
3.7 • 0.48
(12)
--
1.3 • 0.17
(6)
1.3 • 0.32
(12)
(12)
I.I • 0.17
1.5
1.4
_
+ 0.19
(4)
• 0.16
(5)
0.86 • 0.Ii
(5)
(4)
0.49 • 0.23
(5)
0.63 • 0.13
(6)
0.46 • 0.05
Rate of glycogen sgnthesis (pmol glucos~l unlt/h per g wet wt.)
0.59 + 0.08
Radiochemical
0.56 • 0.05
(6)
0.56 + 0.05
(12)
-
14.1 + 1.19
(12)
17.8 + 1 . 8 2
(12)
17.9 • 1 . 0 0
(12)
-
(12)
9 . 2 • 1.08
(12)
7.4 + 0.78
(12)
6.0 • 1.15
Enzymatic
Intact soleus
1
0
10000
1000
I00
50
10
I
0
Insulin
conch.
(~un[ts
/ml)
10.7 • 0.39
(8)
13.4 i 0.77
(8)
13.6 • 0.62
(8)
-
(8)
8.8 • 0.66
8.8 • 0.53
(8)
-
E~zymatie
_
4.3 + 0.6[
(8)
5.1 -+ 0 . 8 5
(7)
2.4 + 0.45
(8)
[ . 5 +- 0 . 2 8
(8)
-
(8)
1.3 + 0.16
8.0 • 0.65
(7)
9.8 i 0.48
(8)
10.2 • 0 . 6 6
(8)
(8)
5.3 • 0.20
5.2 • 0.29
(8)
Radiochemical
Hemidiaphragm
Lactote was measured in the incubation medium both enzymatically and radiochemically and the rate of glycogen synthesis
was measured radioebemieally ~s described in Materials and Methods.
Results are presented as means i 8.E.M. with number
of separate muscles given in parentheses.
Table
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uo
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0%
"-J
Oo
GLYCOLYSIS AND GLYCOGEN SYNTHESIS IN MUSCLE
679
processes, glycogen synthesis and glucose utilization, the e f l e c t s of
insulin are similar; the concentration of the hormone that produces
hall-maximal stimulation o5 both processes was at least 100 tJunits/ml.
In isolated adipocytes from the rat, the sensitivities o5 two processes,
glucose uptake and lipotysis, to insulin were the same; the concentration that produced half-maximal stimulation was about 10 lmnits/ml
(4).
Since the e f f e c t of insulin on lipolysis in adipose tissue can
modify glucose utilization in muscle, via e f f e c t s of the 'glucose/fatty
acid cycle', the current findings strongly support the view that, in
normal sedentary animals, insulin controls the utilization of glucose in
muscle primarily through this cycle. However, since exercise training
i n c r e a s e s the sensitivity of muscle glycolysis to insulin (the 50%
response is obtained at about 10 lJunits/ml) (7) in such animals insulin
may control glucose utilization in muscle primarily through a direct
e f f e c t on membrane transport. Thus, statements that insulin increases
glucose utilization in muscle in vivo by direct stimulation o5 membrane
transport of glucose should only be made with due caution.
Acknowledgements
We t h a n k Professor R. R. Porter, F.R.S., for his interest and
e n c o u r a g e m e n t and the British Diabetic Association for financial
support.
J. E. was recipient of an MRC Training Scholarship and
R. A. J. C. was a recipient o5 an SERC Training Scholarship.
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