(Received for publication, June 19, 1995; and in revised form, August 21, 1995)
From the
The role of calmodulin in control of carbohydrate metabolism in
the absence and presence of insulin in isolated mouse soleus muscle was
investigated. The calmodulin antagonist CGS 9343B had no effect on
basal glycogen synthase activity, the contents of high energy
phosphates, glucose-6-P, or glycogen synthesis. However, CGS 9343B
inhibited the basal rates of 2-deoxyglucose uptake and
3-O-methylglucose transport by 30% (p < 0.05) and
40% (p < 0.001), respectively. Insulin activated glycogen
synthase by almost 40% (p < 0.01) and this increase was not
altered in the presence of CGS 9343B. Insulin increased the muscle
content of glucose-6-P (2-fold), as well as glycogen synthesis
(
8-fold), 2-deoxyglucose uptake (
3-fold), and
3-O-methylglucose transport (
2-fold), and these increases
were inhibited by CGS 9343B. In additional experiments on isolated rat
epitrochlearis muscle, it was found that the hypoxia-mediated
activation of 3-O-methylglucose transport was also inhibited
by CGS 9343B. These data demonstrate that: 1) hexose transport, both in
the absence and presence of external stimuli (insulin and hypoxia),
requires functional calmodulin; and 2) insulin-mediated activation of
glycogen synthase does not require functional calmodulin, nor can it be
accounted for by increases in glucose transport or glucose-6-P.
Skeletal muscle is the major site of insulin-stimulated glucose
uptake(1) . Insulin stimulates glucose transport(2) ,
GS ()and glycogen synthesis in skeletal
muscle(3, 4) . A defect in insulin-mediated glycogen
synthesis, i.e. insulin resistance, is considered to underlie
and contribute to the development of noninsulin-dependent diabetes
mellitus(5, 6) . It is therefore important to
elucidate the signaling mechanisms whereby insulin stimulates
glycogenesis in skeletal muscle.
Recent studies by Beitner and
associates (7) indicate that insulin-mediated increases in
binding of hexokinase to mitochondrial membrane and binding of
phosphofructokinase and aldolase to cytoskeletal proteins in skeletal
muscle are blocked by calmodulin antagonists. These results suggest
that calmodulin, generally considered to be a
Ca-transducing protein, is involved in
insulin-mediated activation of glycolysis(8) . Using
nonspecific calmodulin antagonists (TFP and N-(6-aminohexyl)-5-chloro-1-napthalenesulfonamide), a number
of investigators have suggested that calmodulin also plays a role in
insulin-mediated activation of hexose transport/uptake in adipocytes
and skeletal muscle(9, 10, 11, 12) .
Whether calmodulin plays a role in insulin-mediated activation of GS
has, to our knowledge, not been investigated. It has been demonstrated
that calmodulin can be phosphorylated by the insulin-receptor tyrosine
kinase in vitro, and that insulin stimulates calmodulin
phosphorylation in intact hepatocytes(13) . Since activation of
tyrosine kinase activity is a relatively early event in insulin action,
we reasoned that other insulin-sensitive pathways, in addition to
glycolysis, may also require calmodulin. We have therefore assessed the
effects of CGS 9343B, a calmodulin antagonist(14) , on
insulin-mediated activation of GS, glycogen synthesis and hexose
transport in skeletal muscle.
Measurements of phosphorylase, when performed, were on the same
extracts used for GS. Briefly, an aliquot of the 10,000 g supernatant (see (15) ) was diluted (2:1) with ice-cold
buffer consisting of 100 mM MES, 100 mM potassium
fluoride, pH 6.3, and immediately assayed also using the filter paper
technique following the incorporation of
[
C]glucose-1-P into glycogen at 30 °C for 20
min(17) . Phosphorylase a was assayed at 15 mM glucose-1-P (0.14 mCi/mmol) and 3.3 mg/ml glycogen at pH 6.3.
Phosphorylase a+b was assayed in the same manner except that 3.3
mM AMP was included and the glucose-1-P (0.003 mCi/mmol) and
glycogen concentrations were increased to 66 mM and 6.7 mg/ml,
respectively. All enzyme activities were linear with time and extract
volume used (data not shown).
3-OMG transport was
measured essentially as described elsewhere(22) . Paired solei
were incubated at 35 °C in 2 ml of KBB containing 8 mM glucose and 32 mM mannitol. After the initial 30 min
incubation, insulin was added to the designated vials. 30 min later the
muscles were rinsed in the absence of glucose for 10 min at 29 °C
in 2 ml of KBB containing 40 mM mannitol. Thereafter,
transport was measured by incubating the muscles in the absence of
glucose for 15 min at 29 °C in KBB containing 39 mM mannitol (3.9 µCi/mmol, for assessment of extracellular space)
and 1 mM 3-OMG (0.3 mCi/mmol). When insulin or CGS 9343B was
added to the incubation medium, it was also added to the medium used
for the rinse and transport steps. The muscles were blotted, frozen,
and processed for analysis of intracellular 3-OMG as described
previously(21) . Epitrochlearis muscles were also examined as
above, except that for the transport measurement the KBB contained 8
mM 3-OMG and 32 mM mannitol (specific activities for
both were as for the soleus) and the incubation duration was 10 min.
Last, in studies involving hypoxia, epitrochlearis muscles were
incubated for 90 min in KBB continuously gassed with 95% N,
5% CO
, which results in maximal activation of 3-OMG
transport(23) . The rinse procedure and transport measurement
were as above.
CGS 9343B (40 µM) had no significant effects on the muscle contents of high energy phosphates (ATP; control = 4.3 ± 0.1 µmol/g wet wt, CGS 9343B = 4.0 ± 0.2) (phosphocreatine; control = 10.1 ± 0.2, CGS 9343B = 9.6 ± 0.6; n = 7 and p > 0.05 for both variables) or glucose-6-P (Fig. 1). Insulin does not affect ATP or phosphocreatine in this preparation(15) . Because 40 µM CGS 9343B had no effect on the energy status of the muscle, we continued to use this concentration in the experiments described below.
Figure 1:
Glucose-6-P concentrations in isolated
mouse soleus muscle. Muscles were incubated at 35 °C for 90 min in
KBB containing 5 mM glucose ± 40 µM CGS
9343B and frozen in liquid N. Muscles were processed and
analyzed for glucose-6-P as described under ``Experimental
Procedures.'' When insulin was present, it was during the last 30
min of incubation at 20 milliunits/ml. Values are means ± S.E.
for 5-6 muscles per group.**, p < 0.01 (paired
two-tailed t test).
Figure 2:
Glycogen synthesis in isolated mouse
soleus muscle. Muscles were incubated for 90 min as described in legend
to Fig. 1. After 60 min [C]glucose (0.05
mCi/mmol) was added to all vials. When insulin was present (20
milliunits/ml), it was added together with the isotope. Muscles were
frozen, processed, and analyzed for label incorporation into glycogen
as described under ``Experimental Procedures.'' Values are
means ± S.E. for 6 muscles per group.***, p < 0.001
(paired two-tailed t test).
Figure 3: Concentration dependence of CGS 9343B mediated inhibition of glycogen synthesis in isolated mouse soleus muscle. Incubations were as described in the legend to Fig. 2. 100% glycogen synthesis = 2.34 ± 0.21 nmol/mg wet wt/30 min. Values are means of 4-8 muscles per group.
Figure 4: 2-DG uptake in isolated mouse soleus muscle. Muscles were incubated at 35 °C for 80 min in KBB containing 2 mM pyruvate ± 40 µM CGS 9343B, blotted, and frozen. When insulin (20 milliunits/ml) was added, it was after the initial 30 min of incubation. After 60 min, 2-DG (1 mM, 1 mCi/mmol) was added to all vials, and 20 min later the muscles were blotted and frozen. Muscles were processed and analyzed for intracellular 2-DG concentration as described under ``Experimental Procedures.'' Values are means ± S.E. for 4-5 muscles per group. *, p < 0.05; **, p < 0.01 (paired two-tailed t test).
We therefore studied the effect of CGS 9343B on the transport of 3-OMG, a glucose analogue that is transported across the cell membrane but not phosphorylated to any appreciable extent. Insulin stimulated 3-OMG transport 2-fold, and this effect was blocked by CGS 9343B (Fig. 5). Additionally, CGS 9343B decreased the basal rate of 3-OMG transport. These results are similar to those observed with 2-DG.
Figure 5: 3-OMG transport in isolated mouse soleus muscle. Muscles were incubated at 35 °C for 60 min in KBB containing 8 mM glucose ± 40 µM CGS 9343B. When insulin (20 milliunits/ml) was added, it was after the initial 30 min of incubation. After the 60 min incubation, the muscles were rinsed in glucose-free medium for 10 min at 29 °C, followed by measurement of transport activity using 1 mM 3-OMG (0.3 mCi/mmol). Once CGS 9343B or insulin were added, they were also present in the rinse and transport steps. Muscles were processed and analyzed for intracellular 3-OMG as described under ``Experimental Procedures.'' Values are means ± S.E. for 6-10 muscles per group.***, p < 0.001 (paired two-tailed t test).
Because the basal rate of 3-OMG transport in the mouse soleus
was so high, it was difficult to detect a large increase in
insulin-mediated transport. ()Therefore to study the
concentration dependence of the CGS 9343B effect on insulin-mediated
activation of 3-OMG transport, we used the rat epitrochlearis muscle,
in which transport has previously been shown to increase
6-fold in
the presence of insulin(27) . Insulin activated 3-OMG transport
>5-fold and this activation was inhibited by CGS 9343B in a
concentration-dependent manner (Fig. 6). As was the case in
mouse soleus muscle, CGS 9343B also inhibited the basal rate of 3-OMG
transport in the epitrochlearis muscle by
45% (n =
10, p < 0.05 by paired two-tailed t test).
Figure 6: Concentration dependence of CGS 9343B mediated inhibition of 3-OMG transport in isolated rat epitrochlearis muscle. Incubations were as described in the legend to Fig. 5except that the transport step was for 10 min using 8 mM 3-OMG. 100% 3-OMG transport = 0.74 ± 0.08 µmol/ml/10 min. Values are means ± S.E. for 4 muscles per group.
Since CGS 9343B inhibited basal and insulin-mediated activation of
3-OMG transport, we reasoned that the drug may also interfere with the
actions of other activators of hexose transport. Hypoxia potently
stimulates 3-OMG transport in skeletal muscle by a pathway separate to
that of insulin(23) . We therefore studied the effect of CGS
9343B on hypoxia-mediated activation of 3-OMG transport in the
epitrochlearis muscle. Hypoxia increased 3-OMG transport 7-fold (cf. Fig. 7and Fig. 6, wherein basal transport
averages 0.14 µmol/ml/10 min), and CGS 9343B inhibited this
increase by
65%.
Figure 7:
CGS
9343B inhibits hypoxia-mediated activation of 3-OMG transport in
isolated rat epitrochlearis muscle. Muscles were incubated for 90 min
in KBB continuously gassed with 95% N, 5% CO
(40 µM CGS 9343B. Thereafter, muscles were rinsed
and transport was measured as described in the legend to Fig. 6.
Values are means ± S.E. for 6 muscles per group. *, p < 0.05 (paired two-tailed t test).
Figure 8: CGS 9343B slows relaxation in isolated mouse soleus muscle. Original force records showing the slowing of relaxation after tetanic stimulation in one muscle. Similar results were obtained in 3 additional muscles. Stimulation protocol is described under ``Experimental Procedures.'' The superimposed records were obtained after 10 min exposure to 40 µM CGS 9343B and 3 min after washout of the drug. Period of stimulation is shown below the force records (the elevated bar on the left side of abscissa). Observe that while relaxation was slower in the presence of CGS 9343B, the peak tetanic force (which generally occurred at the end of each tetanus) was unaffected by the drug.
These findings suggest that calmodulin is involved in
insulin-mediated activation of glucose transport and glycogen
synthesis, but not in insulin-mediated activation of GS. This
interpretation is valid only to the extent that CGS 9343B, under the
conditions studied, specifically antagonized calmodulin function.
Others have suggested that CGS 9343B is specific for calmodulin, and in
contrast to other antagonists (e.g. TFP), has no effect on
protein kinase C activity (at least up to a concentration of 100
µM)(14) . On the other hand, it was recently
demonstrated that CGS 9343B inhibits membrane currents and prevents
increases in intracellular Ca in rat pheochromocytoma
cells. These results served as a basis for the conclusion that CGS
9343B affects ion fluxes in a manner unrelated to calmodulin
inhibition(28) .
These results warranted further testing on
potential sites of CGS 9343B action in mouse soleus muscle. Because CGS
9343B did not affect GS and phosphorylase fractional activities under
basal conditions, it is unlikely that it had any major effect on
myoplasmic Ca concentrations (an increase in
Ca
would be expected to activate phosphorylase
kinase, resulting in an activation of phosphorylase and inactivation of
GS). The fact that peak tetanic force was not affected by CGS 9343B
indicates that the drug had no significant effect on action potentials,
sarcoplasmic Ca
release, and cross-bridge ability to
produce force. The slowing of relaxation observed with CGS 9343B can be
explained by an effect on phospholamban. This sarcoplasmic reticulum
protein is found in slow muscles(29) , and calmodulin-dependent
phosphorylation of this protein stimulates the sarcoplasmic reticulum
Ca
pumps. Thus calmodulin inhibition would reduce the
rate of sarcoplasmic Ca
uptake and hence slow
relaxation(30, 31) . Last, CGS 9343B did not inhibit
insulin binding, because the drug did not affect the insulin-mediated
activation of GS. Therefore, to the extent tested in previous
studies(14) , as well as in the present study, it is reasonable
to assume that CGS 9343B specifically antagonized calmodulin activity
under the conditions reported herein.
CGS 9343B inhibited
insulin-mediated activation of hexose transport/uptake by 75%, but
insulin-mediated glycogen synthesis by only 40%. Presumably, there was
a redistribution of the glucose entering the muscle such that in the
presence of CGS 9343B relatively more glucose was diverted toward
glycogen synthesis and less toward glycolysis. This can be explained by
a CGS 9343B-mediated inhibition of glycolysis in the presence of
insulin(7, 8) , coupled with a maintained high
activity of GS (present results). These findings indicate that insulin
does not control glucose metabolism by simply accelerating glucose
transport. The high glucose transport rate in the presence of insulin
must be coordinated with activation of glucose metabolizing enzymes (e.g. hexokinase, GS, phosphofructokinase, etc.) to achieve
the glucose metabolizing rates required by the muscle. In this context
it is noteworthy that CGS 9343B inhibited the basal rate of hexose
transport/uptake but had no effect on glycogen synthesis, which may
also be due to a drug-dependent inhibition of glycolysis.
The observation that CGS 9343B inhibited both the basal and insulin-stimulated rate of glucose transport raised the possibility that calmodulin may be involved in other modes of glucose transport activation (e.g. hypoxia and exercise)(23) . This possibility was verified by the finding that CGS 9343B also inhibited hypoxia mediated activation of 3-OMG transport. Because insulin and hypoxia stimulate 3-OMG transport by separate pathways(23) , it is likely that calmodulin is involved in control of hexose transport at a site beyond where the two pathways converge.
The precise step where calmodulin modulates glucose transport is not known. Recently, a photoaffinity labeling technique has been used to demonstrate that insulin-mediated activation of glucose transport in isolated adipocytes and rat soleus muscle is accounted for by an increase in surface accessible Glut-4 transporter proteins(32, 33) . And studies on isolated adipocytes indicate that the surface accessible affinity labeled Glut 4 transporters translocate to the plasma membrane from an intracellular pool upon insulin stimulation(32) . By using Western blotting it has also been shown that insulin and hypoxia stimulate translocation of the Glut 4 transporter to the plasma membrane in skeletal muscle ( (23) and (33) and references within). These observations, coupled with the findings that calmodulin antagonists abolish insulin-mediated translocations of glycolytic enzymes (7, 8) and 3-OMG transport (present study), suggest that calmodulin modulates translocation of glucose transporters.
It may be argued that because CGS 9343B (40
µM) inhibited basal 3-OMG transport, the drug-dependent
inhibition of insulin- and hypoxia-mediated activation of 3-OMG
transport was not due to inhibition of an insulin (or
hypoxia)-dependent process. However, the fact that the CGS 9343B (40
µM) dependent inhibition of 3-OMG transport in the
presence of insulin (0.3 µmol/ml/10 min) and after hypoxia
(
0.5) were greater than the inhibition of basal transport (
0.1)
indicates that CGS 9343B exerted an additional effect on transport in
the presence of insulin or after hypoxia. The same reasoning can also
be applied to the results in the soleus muscle, wherein the inhibition
of insulin-mediated hexose transport/uptake by CGS 9343B was markedly
greater than the inhibition of basal transport/uptake by CGS 9343B (see Fig. 4and Fig. 5).
The next question arises as to how
calmodulin modulates activation of glucose transport. It is generally
believed that an increase in intracellular Ca will
result in an increased binding of Ca
to calmodulin,
thereby inducing a conformational change in the latter, which will
alter the affinity of calmodulin for its site of action (enzyme
activity or structural component)(34) . For this to be the
mechanism of action in the present study would require that hypoxia and
insulin should increase myoplasmic Ca
. The finding
that dantrolene, which inhibits sarcoplasmic reticulum Ca
release, inhibits the hypoxia-mediated activation of 3-OMG
transport in skeletal muscle (23) provides indirect support for
the idea that hypoxia causes an increase in myoplasmic
Ca
. In this context it is noteworthy that 10 min of
cyanide incubation, which inhibits mitochondrial oxygen utilization,
significantly increased myoplasmic free Ca
by 10
± 1 nM (basal value = 28 nM) in
isolated single intact mouse muscle fibers. (
)Whether
insulin increases intracellular Ca
has been debated
(see (12) and references therein). The finding that dantrolene
abolished the insulin-mediated activation of 3-OMG in isolated rat
muscle (12) suggests that insulin also increases myoplasmic
Ca
. However, direct measurements of myoplasmic
Ca
in intact mammalian skeletal muscle after exposure
to insulin have, to our knowledge, not been performed.
Alternatively, insulin may not alter myoplasmic
Ca, but rather stimulate the phosphorylation of
calmodulin. It has recently been demonstrated that insulin increases
the phosphorylation state of calmodulin in isolated hepatocytes, and
that phosphocalmodulin exhibits altered biologic activity in a manner
that can be Ca
-independent(13, 35) .
Which, if any of these explanations is applicable to the present study,
as well as which calmodulin-dependent enzyme/structure is altered by
insulin in skeletal muscle, remains to be determined.
Although CGS 9343B had no effect on GS (presence and absence of insulin), the fact that it abolished the insulin-mediated increase in glucose-6-P allows for assessment of the role of glucose-6-P in insulin-mediated activation of GS. Glucose-6-P stimulates the dephosphorylation of GS by GS phosphatases by allosterically altering the configuration of GS, rendering GS a more suitable substrate for these phosphatases(36) . It has been suggested that an increase in glucose-6-P by insulin, subsequent to activation of glucose transport, stimulates GS activation (i.e. dephosphorylation)(18, 37) . The present findings indicate that neither increases in glucose transport nor glucose-6-P are required for the insulin-mediated activation of GS in mouse soleus. Indeed close examination of the relationship between exogenously added glucose-6-P and GS phosphatase activity in skeletal muscle homogenates ((37) , see Fig. 9) indicates that the insulin-mediated increase in glucose-6-P observed in the present study would be expected to have only a minor effect on GS phosphatase.
In conclusion, these data demonstrate that in isolated skeletal muscle: 1) hexose transport both in the absence and presence of external stimuli (insulin and hypoxia) requires functional calmodulin; and 2) insulin-mediated activation of GS does not require functional calmodulin, nor can it be accounted for by increases in glucose transport or glucose-6-P.