(Received for publication, August 15, 1995; and in revised form, October 24, 1995)
From the
The effect of increased expression of glycogen phosphorylase on
glucose metabolism in human muscle was examined in primary cultured
fibers transduced with recombinant adenovirus AdCMV-MGP encoding muscle
glycogen phosphorylase. Increments of 20-fold in total enzyme activity
and of 14-fold of the active form of the enzyme were associated with a
30% reduction in basal glycogen levels. Total glycogen synthase
activity was doubled in AdCMV-MGP-transduced cells even though the
activity ratio was decreased. Incubation with forskolin, which
inactivated glycogen synthase and activated glycogen phosphorylase,
induced greater net glycogenolysis in engineered cells. In unstimulated
fibers, lactate production was three times higher in AdCMV-MGP fibers
as compared with controls, despite similar rates of glycogenolysis. In
transduced fibers incubated with 2-deoxyglucose, the level of
2-deoxyglucose 6-phosphate was about 8-fold elevated over the control
even though hexokinase activity was unmodified in AdCMV-MGP fibers.
Overexpression of glycogen phosphorylase also led to enhancement of
[U-C]glucose incorporation into glycogen,
lactate, and lipid. Accordingly, determination of lipid cell content
revealed that engineered cells were accumulating lipids. Furthermore,
CO
formation from
[U-
C]glucose was 1.6-fold higher, whereas
CO
formation from
[6-
C]glucose was unmodified, in AdCMV-MGP
fibers. Our data show that in human skeletal muscle cells in culture,
the increase in glycogen phosphorylase activity is able to up-regulate
glycogen synthase activity indicating the enhancement of glycogen
turnover. We suggest that the increase in glycogen phosphorylase and,
thereby, in glycogen metabolism, is sufficient to enhance glucose
uptake in the muscle cell. Glucose taken up by engineered muscle cells
is essentially disposed of through nonoxidative metabolism and
converted into lactate and lipid.
Glycogen phosphorylase is the rate-limiting enzyme of glycogen
breakdown. In muscle, glycogen serves mainly to provide glucose for
energy production during exercise, although it is also consumed in the
resting state. During exercise muscle glycogenolysis is triggered by
the dual control of contractile activity and epinephrine(1) .
These stimuli result in the release of Ca and an
increase in cyclic AMP, respectively, which in turn lead to the
phosphorylation and activation of glycogen phosphorylase by
phosphorylase kinase(2) .
During exercise, glucose uptake and metabolism are greatly increased in muscle despite low physiological concentrations of insulin. Indeed, it has been shown that insulin is not required to mediate glucose uptake during contractions (3, 4) and that contractile activity augments glucose uptake by muscle even in severely insulin-deficient diabetic rats(5) . Furthermore, in exercised muscle, glucose uptake and disposal are enhanced independently of insulin(6) . Insulin sensitivity of glucose uptake and glycogen synthesis are increased in exercised muscle, in normal humans and insulin-deficient type I diabetic patients (7, 8) . The mechanism by which exercise increases basal and insulin-stimulated muscle glucose uptake remains to be elucidated. The system accounting for such effects appears to be located at a post-receptor level, because exercise does not affect the amount of insulin receptors or insulin-stimulated kinase activity(9, 10) . Breakdown of glycogen stores (11, 12, 13) or the activation of glycogen synthase (9) have been suggested as possible mediators of this phenomenon. On the other hand, even though these studies are consistent with the fact that glucose uptake is limited by glucose metabolism, other studies suggest that it is glucose transport that limits glucose uptake(14, 15) .
We have previously shown that adenoviruses constitute a very efficient vehicle to deliver DNA into nondividing fused C2C12 myotubes(16) . In this study, we have used adenoviruses bearing the rabbit muscle glycogen phosphorylase cDNA to increase the expression of the enzyme in human myotubes in culture. This approach has allowed us to evaluate the contribution of phosphorylase to the regulation of glucose metabolism in human muscle fibers and the repercussion of the stimulation of the glycogenolytic process. Our data show that human muscle fibers overexpressing glycogen phosphorylase show a higher capacity for glucose uptake and metabolism through nonoxidative glycolysis and lipid synthesis. These results may be related to the physiological mechanism by which muscle glucose disposal is increased during and after exercise.
Figure 1: Glycogenolysis in unstimulated and forskolin-challenged human fibers. Noninfected (squares) or AdCMV-MGP-transduced fibers (circles) were incubated in the absence (open symbols) or the presence (closed symbols) of 100 µM forskolin for the indicated times. Glycogen content was measured as described under ``Materials and Methods.'' The results are the means ± S.E. of seven independent experiments performed in triplicate. prot, protein.
As expected, in AdCMV-MGP fibers total glycogen phosphorylase activity (measured in the presence of the activator AMP) was clearly higher (20-fold over the control) (Table 1), as was phosphorylase a activity (assayed without AMP), which increased 14 times. Therefore, in transduced cells, the -AMP/+AMP activity ratio was significantly lower, suggesting that a high proportion of the exogenous glycogen phosphorylase was kept inactive in the basal state (Fig. 2). After incubation of the cells with forskolin, a time-dependent activation of phosphorylase could be observed in both control and transduced cells (Fig. 2). A 1.3-fold increment in the -AMP/+AMP activity ratio was obtained in control cells, which was already maximal after 20 min of incubation and had disappeared after 120 min. In contrast, an increment of about 2-fold was detected in AdCMV-MGP cells, which led to activity ratio values (around 0.85) equivalent to those measured in control cells after forskolin challenge, indicating that almost all of the phosphorylase was in the active form after forskolin treatment.
Figure 2: Time-dependent effect of forskolin on glycogen phosphorylase activity ratio. Noninfected (squares) or AdCMV-MGP-transduced fibers (circles) were incubated in the absence (open symbols) or the presence (closed symbols) of 100 µM forskolin for the indicated times. Glycogen phosphorylase (GP) activity was determined as described under ``Materials and Methods.'' The results are the means ± S.E. of seven independent experiments performed in duplicate.
In AdCMV-MGP cells, total glycogen synthase activity (assayed in the presence of 10 mM glucose 6-P) was significantly higher than that of control cells (about 2-fold increment) (Table 1). In contrast, the active form (assayed without glucose 6-P) was only slightly increased. Thus, the -Glc6P/+Glc6P activity ratio was lower in transduced cells (Fig. 3). Treatment of the cells with forskolin induced a time-dependent decrease in the -Glc6P/+Glc6P activity ratio in control and AdCMV-MGP fibers. The maximal inactivation was reached between 40 and 60 min and was of similar magnitude (about 50%) for both type of cells. By 120 min, the activity ratio had returned to basal values in control and transduced fibers.
Figure 3: Time-dependent effect of forskolin on glycogen synthase activity ratio. Noninfected (squares) or AdCMV-MGP-transduced fibers (circles) were incubated in the absence (open symbols) or the presence (closed symbols) of 100 µM forskolin for the indicated times. Glycogen synthase (GS) activity was measured as described under ``Materials and Methods.'' The results are the means ± S.E. of four independent experiments performed in duplicate. G6P, glucose 6-P.
We examined whether glycogenolysis resulted in changes in lactate formation. Although control cells showed a very small accumulation of lactate in the medium during a 120-min incubation, cells overexpressing myophosphorylase demonstrated a time-dependent increase in lactate concentration. As shown in Fig. 4, lactate production (calculated as the difference in lactate concentration) in 2 h was 9.55 µmol/mg protein in transduced cells and only 2.27 µmol/mg protein in control cells. It should be mentioned that during this period of time, cellular glycogen content decreased by 0.21 µmol of glucose/mg of protein in transduced cells, and a similar decrease (0.18 µmol of glucose/mg of protein) was observed in control cells (Fig. 1). Therefore, data suggested that the increase in lactate is larger than can be accounted for net glycogen breakdown, suggesting an increase in glucose metabolism.
Figure 4:
Time course of lactate production in human
muscle fibers in culture. Human fibers nontransduced () or
transduced with AdCMV-MGP (
) viruses preincubated as described
under ``Materials and Methods'' were incubated with 10 mM glucose. Lactate accumulated in the medium was measured at the
times indicated. The results are the means ± S.E. of four
independent experiments performed in triplicate. prot.,
protein.
Examination of the CO
production revealed
that in cells that had been exposed to AdCMV-MGP viruses,
CO
release was elevated 1.6-fold over the
controls.
CO
production from
[U-
C]glucose mainly reflects CO
derived from the decarboxylation of glucose in the pentose
phosphate pathway and in the pyruvate dehydrogenase step in the pathway
of fatty acid synthesis. Because lipid synthesis was stimulated in
these cells, we used [6-
C]glucose to determine
whether the overexpression of myophosphorylase also stimulated glucose
oxidation. We found that there was no difference in the
CO
production from
[6-
C]glucose in AdCMV-MGP-transduced myofibers
compared with control cells. Therefore, the observed increase in
CO
released from
[U-
C]glucose was essentially due to lipid
synthesis.
Figure 5:
Histochemical staining of lipid content.
Representative phase contrast micrographs (100) of Oil Red O
stained cultured human fibers untransduced (A) or transduced
with AdCMV-MGP (B) 7 days after
infection.
A remarkable consequence of the overexpression of
phosphorylase is an enhancement of glucose uptake and consumption by
the muscle cell, probably secondary to increased turnover and
utilization of glycogen stores. Consistent with this interpretation, we
found that engineered cells exhibited an enhancement in the production
of lactate that could not be explained solely by net depletion of
glycogen. Although depletion of glycogen was similar in control and
AdCMV-MGP cells, the rate of net lactate production was much higher in
engineered cells, as was the incorporation of radioactivity from C-glucose into lactate. Because the incorporation of
C-glucose into glycogen was also increased in engineered
cells, one possible explanation for the increase in lactate production
is that glycogen turnover is stimulated, leading to an increase in
glucose conversion into glycogen and its subsequent degradation to
lactate. Moreover, AdCMV-MGP-transduced cells showed an enhanced
accumulation of the phosphorylated metabolite of 2-deoxyglucose, a
glucose analog that is not metabolized beyond its 6-phosphorylated
form. The increase in 2-deoxyglucose 6-P concentration found in
engineered cells seems to reflect their elevated capacity to transport
glucose, because hexokinase activity is unchanged by overexpression of
phosphorylase. Our results may be relevant to the increase in glucose
uptake and metabolism observed during exercise. It has been long
hypothesized that exercise stimulation of glucose transport is related
to the lowering of muscle glycogen stores. In vivo, exercise
activates glycogen phosphorylase by a dual control of epinephrine and
contractile activity, and both stimulating events have been associated
to the enhancement of glucose uptake. In isolated muscles it has been
observed that a bout of glycogen-depleting exercise increases basal
glucose transport(4, 31) . In addition, in rats
injected with epinephrine, which activates phosphorylase and reduces
glycogen stores independent of exercise, basal glucose transport
activity is increased (13) . We demonstrate that cultured human
muscle fibers with higher levels of glycogen phosphorylase activity
show an enhanced capacity for glucose uptake and consumption, despite
having a relatively unaltered rate of glycogen depletion and unmodified
hexokinase activity.
Controversy exists regarding whether it is
glucose transport or its intracellular metabolism that limits glucose
utilization by muscle tissue(14, 32) . Our data
provide evidence that it is the intracellular glucose utilization that
limits the rate of glucose uptake. We show that in AdCMV-MGP-transduced
cells, along with the increase in glucose uptake, there is a
stimulation of [U-C]glucose conversion into
lactate, lipids, and glycogen. We propose that the influx of glucose
triggered by the overexpression of phosphorylase increases glucose
disposal to lactate and lipid. Accordingly, we found that the
concentration of the intermediate metabolite glucose 6-P is increased
in engineered cells (as described under ``Results''). The
reverse situation has been observed in muscle fibers from
myophosphorylase-deficient patients (McArdle's disease) in which
the levels of intermediate glycolytic metabolites such as glucose 6-P
are depleted(33) . Moreover, our results suggest that the
moderate increase in
CO
observed from
[U-
C]glucose is due to the pentose pathway or
the pyruvate dehydrogenase step rather than to the oxidation of glucose
in the Krebs cycle. Therefore, the glucose taken up by AdCMV-MGP muscle
cells appears to be essentially consumed through nonoxidative
metabolism.
Our data also show that overexpression of glycogen phosphorylase induces an increase in total glycogen synthase activity even though glycogen synthase mRNA levels appear to be unmodified (data not shown). Even though total glycogen synthase activity is increased, there is only a small increase in the level of the active form, and thus the activity ratio is decreased. These results might be related to previous observations showing that exercise increases the total activity of both glycogen synthase and glycogen phosphorylase(34, 35) , conferring to the muscle cells enhanced capacity for glycogen depletion and resynthesis. Additionally, Westergaard and colleagues (36) demonstrated in athletes an elevation of total glycogen synthase activity together with a decrease in the activity ratio, which is accompanied by no difference in immunoreactive glycogen synthase. As in this study, it is suggested that post-translational modifications of the enzyme and not regulation of gene expression seem to account for modulation of glycogen synthase activity in muscle. The fact that an increase in glycogen phosphorylase activity results in an increase in the activity of glycogen synthase suggests the presence of a local control of glycogen turnover. It seems that a compensatory mechanism exists that tends to equilibrate the rates of glycogenolysis and glycogenesis to maintain glycogen turnover and net glycogen content. The simultaneous existence of both reactions or glycogen cycling has been clearly demonstrated in liver(37) . Furthermore, in liver, glycogen turnover is greater in the fed state than in the fasted state (38) , and thus it has been suggested that glycogen concentration may exert a regulatory effect on glycogen turnover. We found that in AdCMV-MGP-treated muscle cells, there is a compensatory mechanism that up-regulates glycogen synthase activity following the increase in phosphorylase, despite a moderate decrease in glycogen content. Therefore, our data suggest the involvement of local undetermined regulatory factors.
It is concluded that in human skeletal muscle cells, the increase in glycogen phosphorylase activity is sufficient to up-regulate glycogen metabolism and to drive the uptake of glucose. Therefore, increased intracellular metabolism of glucose may be the primary event in the induction of a higher capacity to take up glucose by skeletal muscle. Our finding may be related to the exercise-induced enhancement in muscle glucose transport. Additionally, we show that the increase in glucose utilization is associated with increased production of lactate and accumulation of lipid. In summary, it is shown that muscle cells respond to the increase in the glycogenolytic capacity by increasing glucose uptake and consumption.