1 Department of Molecular Biology and Pharmacology, Washington University School of Medicine, St. Louis, Missouri 63110; 2 Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, Indianapolis, Indiana 46202; and 3 Departments of Pharmacology and Medicine, University of Virginia School of Medicine, Charlottesville, Virginia 22908
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ABSTRACT |
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The effects of transgenic overexpression of glycogen synthase in different types of fast-twitch muscle fibers were investigated in individual fibers from the anterior tibialis muscle. Glycogen synthase was severalfold higher in all transgenic fibers, although the extent of overexpression was twofold greater in type IIB fibers. Effects of the transgene on increasing glycogen and phosphorylase and on decreasing UDP-glucose were also more pronounced in type IIB fibers. However, in any grouping of fibers having equivalent malate dehydrogenase activity (an index of oxidative potential), glycogen was higher in the transgenic fibers. Thus increasing synthase is sufficient to enhance glycogen accumulation in all types of fast-twitch fibers. Effects on glucose transport and glycogen synthesis were investigated in experiments in which diaphragm, extensor digitorum longus (EDL), and soleus muscles were incubated in vitro. Transport was not increased by the transgene in any of the muscles. The transgene increased basal [14C]glucose into glycogen by 2.5-fold in the EDL, which is composed primarily of IIB fibers. The transgene also enhanced insulin-stimulated glycogen synthesis in the diaphragm and soleus muscles, which are composed of oxidative fiber types. We conclude that increasing glycogen synthase activity increases the rate of glycogen synthesis in both oxidative and glycolytic fibers, implying that the control of glycogen accumulation by insulin in skeletal muscle is distributed between the glucose transport and glycogen synthase steps.
muscle fiber type; phosphorylase; uridine diphosphate-glucose; insulin
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INTRODUCTION |
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INSULIN LOWERS BLOOD GLUCOSE by inhibiting hepatic glucose production and by stimulating the uptake and storage of glucose in various insulin-sensitive tissues (4, 46). The synthesis of glycogen, a major storage depot for glucose, is increased in response to insulin in many cell types, and most of the glucose that is removed from the blood after a meal is converted into skeletal muscle glycogen (18, 37). Thus glycogen synthesis in skeletal muscle fibers is of particular relevance to blood glucose homeostasis. Insulin activates both glucose transport and glycogen synthase in skeletal muscle, thereby affecting the initial as well as the final step in the pathway leading from extracellular glucose to glycogen (24). The increase in glucose transport results primarily from the translocation of the glucose transporter GLUT-4 from intracellular compartments to the sarcolemma and transverse-tubular membranes in response to insulin (5, 33). This allows more glucose to enter the muscle fiber, where it is converted to glucose 6-phosphate (G-6-P), glucose 1-phosphate (G-1-P), and uridine diphosphoglucose (UDPG) in a sequence of reactions catalyzed by hexokinase, phosphoglucomutase, and UDPG pyrophosphorylase, respectively. Glycogen synthase catalyzes the final step in which the glucosyl moiety from UDPG is added to preexisting glycogen. Insulin activates glycogen synthase by promoting dephosphorylation of the enzyme (2, 24).
The relative importance of glucose transport and glycogen synthase in controlling the rate of glycogen synthesis has been debated over the years. Recently, much attention has focused on the importance of glucose transporters in determining the rate of glycogen biosynthesis, and a widely held view is that most of the control is at the level of the transporter. For example, studies with transgenic mice overexpressing the glucose transporters GLUT-1 and GLUT-4 have provided compelling evidence that increasing glucose transport is sufficient to increase glycogen synthesis in skeletal muscle (10, 32). The glycogen content of muscles from these transgenic animals was markedly increased, but the activation state of glycogen synthase in the transgenic muscles did not appear different from that in the control muscles. On the basis of these results, it was concluded that glucose transport is strictly rate limiting for glycogen synthesis (32). Mice heterozygous for a disrupted GLUT-4 allele, expressing some 50% of the wild-type level of GLUT-4, also exhibited impaired glycogen synthesis during euglycemic hyperinsulinemic clamps (34). However, the results from animals with genetically modified expression of GLUT-1 or GLUT-4 do not exclude the possibility that activation of glycogen synthase has an important role in the stimulation of glycogen synthesis by insulin. Indeed, our recent finding that glycogen was markedly elevated in muscles of transgenic mice overexpressing glycogen synthase supports the view that increasing glycogen synthase activity is sufficient to promote glycogen accumulation (27).
The heterogeneity of the fibers that comprise skeletal muscle is a complicating factor in all studies of muscle metabolism. Even adjacent fibers in a muscle may have markedly different contractile and metabolic properties (30, 42). A method involving histochemical staining of myosin ATPase activity at different H+ concentrations has been widely used to distinguish three types of fibers, designated types I, IIA, and IIB (1). Fiber type assignments have also been made on the basis of the activities of representative enzymes of glycolytic and oxidative energy metabolism found in different fibers (14, 26, 30, 42). Type I fibers have relatively low levels of glycolytic enzymes and high levels of oxidative enzymes. These fibers also have a slow twitch speed and are much less fatigable than type II fibers, because most of their energy is derived from oxidative metabolism. Type IIB fibers have a fast twitch speed and depend largely on glycogen metabolism to provide the energy to fuel rapid and forceful contractions. These fibers have relatively high levels of glycolytic enzymes and low levels of enzymes of oxidative energy metabolism. Type IIA fibers have a rapid contractile speed and high levels of both glycolytic and oxidative enzymes. One of four myosin heavy-chain isoforms is usually expressed either exclusively or predominantly in a single fiber (35). The ability to detect the different heavy-chain isoforms led to the identification of a third type of fast-twich fiber, designated type IID (or 2X). The oxidative enzyme content of these fibers is higher than that of IIB fibers but generally lower than that of IIA fibers (35).
Single fiber analyses and immunohistochemical studies have demonstrated that the amount of GLUT-4 correlates directly with the levels of enzymes of oxidative energy metabolism, such as malate dehydrogenase (MDH) (19, 28). This finding is consistent with observations that the effect of insulin on glucose transport is highest in muscles composed predominantly of oxidative fiber types (20). Whether glycogen synthase levels correlate with those of GLUT-4 in different fibers is not known, but it is possible that the relative contributions of glucose transport and glycogen synthase to glycogen synthesis could differ among fibers.
The present study extends our previous work (27) on transgenic mice that overexpress glycogen synthase under the control of promoter/enhancer elements of the mouse muscle creatine kinase gene. The transgenic animals overexpress a mutant form of rabbit muscle glycogen synthase having Ser to Ala mutations in two key inactivating phosphorylation sites, sites 2 and 3a (39). We had previously correlated expression of the transgene with elevated glycogen accumulation in various muscles. We now present results from single fiber analyses that allow direct comparison between transgene-stimulated glycogen accumulation and the oxidative capacity of a fiber, as assessed by measurements of MDH activity. In addition, we describe the effects of the transgene on glucose transport and the rates of glycogen synthesis in muscles having different fiber-type compositions.
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METHODS AND MATERIALS |
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Animals
Transgenic mice expressing glycogen synthase (GS) (with mutations in sites 2 and 3a) have been described previously (27). Expression of the transgene was driven by promoter/enhancer elements from the skeletal muscle creatine kinase gene, which favors expression in fast-twitch fibers. Two transgenic lines, GSL3 and GSL30, were used in the experiments presented. Heterozygous male mice were mated to wild-type (C57BL6 × CBA)F1 females. Transgenic pups were identified by using the PCR to amplify chloramphenicol acetyltransferase sequences in tail DNA. All mice were used ~2 mo after birth. At this time the weights of the transgenic mice and the nontransgenic littermates, which were used as controls, were approximately equal (Table 1).
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Experimental Protocols
Single fiber analyses.
Transgenic and age-matched wild-type animals were fed ad libitum and
then anesthetized by subcutaneous injection (1 ml/kg) of a mixture of
ketamine (40 mg/ml), xylazine (10 mg/ml), and acepromazine (1.5 mg/ml).
Hindlimb muscles were exposed and frozen in situ between two stainless
steel blocks (1 cm × 2.5 cm × 2 cm) that had been chilled
in liquid nitrogen. The muscles were freeze-dried at 35°C
and then stored under vacuum at
70°C before use. Single
fibers were manually dissected from the muscle samples (26) and weighed
using a fish pole balance (29). Glucose and G-6-P (13),
G-1-P (29), and the activities of creatine kinase (26) and MDH
(14) were measured in pieces of individual fibers. Phosphorylase
activity and levels of glycogen and UDPG were measured as described by
Henry and Lowry (11).
Incubation of muscles in vitro. All media used for muscle incubations were directly gassed by bubbling with a mixture of 95% O2-5% CO2. Incubations were conducted essentially as described previously (25). Soleus, extensor digitorum longus (EDL), and diaphragm muscles were removed from anesthetized mice and transferred to DMEM. The diaphragm was dissected into two hemidiaphragms, leaving a rib attached to each to avoid the cutting of fibers. Muscles were incubated at 37°C for 45 min to remove endogenous hormones, transferred to Krebs-Henseleit buffer (in mM: 118 NaCl, 4.7 KCl, 2.5 CaCl2, 1.2 potassium phosphate, 1.2 MgSO4, and 25 NaHCO3) plus 5 mM glucose, and incubated as we will now describe.
Measurements of GS activities. Muscles were incubated at 37°C for 30 min either without hormone or with 20 mU/ml insulin and were then frozen in liquid nitrogen. Tissue (~6 mg) was homogenized in 300 µl of a solution containing (in mM) 100 KF, 10 EDTA, 2 EGTA, 5 potassium phosphate, 5 sodium pyrophosphate, 50 Tris · HCl (pH 7.8 at 25°C), and the following protease inhibitors: 1 mM benzamidine, 10 µg/ml leupeptin, 10 µg/ml aprotinin, and 0.1 mM phenylmethylsulfonyl fluoride. Homogenates were centrifuged at 10,000 g for 30 min, and the supernatants were retained for analyses. GS activity was measured both without and with 10 mM G-6-P by the method of Thomas et al. (43). Total activity is defined as that measured in the presence of 10 mM G-6-P and is expressed in terms of extract protein. The activity ratio is the activity measured in the absence of G-6-P divided by the total activity.
Glucose transport activity. 2-Deoxyglucose uptake was measured essentially as described previously (40). Briefly, muscles were incubated at 37°C either without or with 20 mU/ml insulin for 15 min and then transferred to Krebs-Henseleit buffer (10 ml/muscle) containing 0.5 mM 2-deoxy-[1,2-3H]glucose (1 µCi/ml) and 10 mM [1-14C]mannitol (0.1 µCi/ml). After incubations of 15 min without and with insulin, incubations were terminated by washing the muscles in buffer at 4°C. The amount of 2-deoxy-[1,2-3H]glucose retained by the muscles was determined by scintillation counting. The values presented for uptake were corrected for the extracellular space, which was estimated from the amount of [14C]mannitol recovered. Measuring the initial rate of 2-deoxyglucose is presumed to provide a reliable index of glucose transport activity, although the uptake rate is a function of both the transport and phosphorylation of the sugar.
[U-14C]glucose incorporation into glycogen. [14C]glycogen was measured essentially as described previously (41). Muscles were incubated at 37°C in Krebs-Henseleit buffer (10 ml/muscle) containing 5 mM [U-14C]glucose (1 µCi/ml) and increasing concentrations of insulin for 30 min, then rinsed with buffer at 0°C, and frozen in liquid nitrogen. The muscles were weighed, and samples (5-7 mg) were homogenized in 0.4 ml of 30% KOH. The homogenates were heated in a boiling water bath for 20 min. Ethanol was added to a final concentration of 70% to precipitate the glycogen. The samples were centrifuged at 2,000 g for 30 min, and the glycogen pellets were washed three times with 70% ethanol. The amount of [14C]glycogen was determined by scintillation counting.
Materials. Porcine insulin (27 U/mg) was obtained from Eli Lilly. 2-Deoxy-[1,2-3H]glucose, [U-14C]mannitol, and [U-14C]glucose were obtained from New England Nuclear. Most commonly used chemicals and reagents were from Sigma Chemical.
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RESULTS |
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Single Fiber Analyses
In previous work, three lines of transgenic mice overexpressing GS were studied, of which line GSL30 had the highest level of overexpression (27). The transgenic enzyme is present in high levels in anterior tibialis muscles, which are composed of the three types of fast-twitch fiber types (9, 15). To determine whether the transgene affected glycogen accumulation to different extents in these three types of fibers, enzymes and metabolites were measured in single fibers that were manually dissected from freeze-clamped samples of muscles from control and GSL30 animals.Muscle fiber classification.
Creatine kinase and MDH activities were measured to assess differences
between fibers. Type IIB fibers have the lowest levels of MDH activity
of the different types of fibers (14), and in a plot of creatine kinase
vs. MDH activity, the data points from IIB fibers were tightly
clustered and well separated from those derived from the oxidative
fibers (Fig. 1). Type IIA fibers generally have higher levels of enzymes of oxidative metabolism than IID fibers
(9). Consequently, we believe that the fibers with the highest MDH
activities in Fig. 1 are IIA fibers. However, some IID fibers have high
levels of oxidative enzymes; therefore, we have assigned fibers having
an MDH activity higher than 0.25 nmol · min1 · mg
1
to a type IIA\IID category. By this criterion, 53% of the control fibers analyzed were IIB, and 47% were IIA\IID fibers. These
proportions agree well with the fiber type composition of mouse
anterior tibialis muscles estimated by other methods. For example, in a
recent study in which fiber type was assigned on the basis of
reactivity of fibers with isoform-specific myosin heavy-chain
antibodies, the deep region (from which the present fibers were
obtained) of the muscle was found to contain 47% IIB fibers, 24% IIA
fibers, and 27% IID fibers (15). The proportions of IIB and IIA\IID
fibers (50% of each) in transgenic muscles were essentially
the same as in control muscles. Thus the transgene did not
elicit any major change in the overall fiber composition of the muscle.
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Effect of transgenic expression of GS on the glycogen
content of single fibers.
Consistent with other studies of single muscle fibers (14, 26), the
average levels of creatine kinase were approximately twice as high in
IIB fibers as in the oxidative fast-twitch fiber types (Fig. 1).
Because transgenic expression of GS was driven by promoter/enhancer
elements derived from the muscle creatine kinase gene, levels of the
transgene would also be expected to be higher in type IIB fibers. GS
levels were measured by immunoblotting in control and transgenic fibers
(Fig. 2A). As observed in samples of whole muscle (27), GS from single wild-type fibers appeared as
electrophoretically distinguishable species (Fig. 2A), most likely because of differences in phosphorylation, which retards the
electrophoretic mobility of the GS subunit. In fibers from wild-type
muscle, the amount of GS estimated from optical density scanning of the
immunoblot was approximately twice as great in IIA\IID
fibers as in IIB fibers (Fig. 2B). GS in the transgenic IIB and
IIA\IID fibers was much higher than in the respective fibers from wild-type animals, and the level in transgenic IIB fibers
was higher than that in transgenic IIA\IID groups. We
believe that immunoblotting tends to overestimate the expression of the transgenic rabbit protein relative to the endogenous mouse GS, because
the antibodies used were raised against rabbit GS. We previously
observed that the increase due to transgenic expression of GS as
estimated by immunoblotting extracts of whole anterior tibialis muscle
was greater than that predicted from the increase in total synthase
activity in the same extracts (27). Nevertheless, because of the extent
of overexpression, the results indicate that the amount of GS in
transgenic IIB fibers was approximately twice that in the
IIA\IID fibers.
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Increased phosphorylase activities in transgenic fibers.
Glycogen levels in fibers are ultimately determined by the rates of
both synthesis and degradation. Therefore, we investigated the
possibility that the higher levels of glycogen achieved in the IIB
fibers were due to a reduction in phosphorylase, the enzyme that
mediates glycogen degradation. Instead of having less phosphorylase, the transgenic IIB fibers had approximately fourfold higher levels of
the enzyme than the control IIB fibers (Fig.
4). Interestingly, there was a very strong
inverse correlation (r = 0.95 by linear regression)
between MDH and phosphorylase activities in the transgenic fibers.
Thus, in the most oxidative fibers, there was very little difference
between the levels of phosphorylase in control and transgenic fibers
(Fig. 4). There was also a correlation, albeit positive (r
= 0.80), between phosphorylase and glycogen among the different
fibers. Thus the fibers with the most glycogen tended to have the
highest levels of phosphorylase (Fig. 5).
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Effect of the transgene on metabolites in the GS pathway.
To investigate the mechanisms involved in the increase in glycogen
mediated by the transgene, each of the metabolites in the pathway
leading to GS was measured. We had previously found that UDPG, the
immediate precursor to glycogen, was lower in transgenic anterior
tibialis muscles (27). UDPG was also lower in both the transgenic IIB
and IIA\IID fibers than in the corresponding types of
control fibers (Fig. 6). Indeed, at
equivalent MDH activities, the UDPG level in only one transgenic IIB
fiber was found in the control domain, and the UDPG content of all of
the transgenic IIA\IID fibers was lower than in
control fibers of these types. Other precursors in the pathway leading
to glycogen were not decreased in the transgenic fibers (Fig.
7). Glucose levels associated with the
single fibers were modestly higher in the transgenic fibers than in the
control fibers. It should be noted that this measurement includes not
only intracellular glucose but also a fraction of the interstitial
glucose that is recovered with the fibers. G-6-P levels were
~50% higher in both groups of transgenic fibers than in the control
groups. A similar difference in G-6-P levels in samples of
anterior tibialis muscles from control and transgenic mice was observed
previously (27). No difference in G-1-P was observed between
control and transgenic fibers in either the IIB or the
IIA\IID groups.
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Experiments with Muscles Incubated in Vitro
To analyze GS and glucose transport, it was necessary to utilize intact muscle, and experiments were conducted in vitro with diaphragm, EDL, and soleus muscles. These muscles may be maintained in a viable state during short-term incubations, and they have different fiber type compositions, allowing some correlation with data obtained from dissected fibers. On the basis of staining with antibodies against different myosin isoforms, mouse soleus muscles were found to contain approximately equal numbers of type I and IIA fibers (15). The following proportions of fibers were found in EDL muscles: 1% type I, 12% type IIA, 68% type IIB, and 19% type IID fibers (15). In mouse diaphragm the proportions were as follows: 1% type I, 56% type IIA, and 35% of a group containing IIB and IID fibers (31). Although the IIB and IID fibers were not specifically identified by the myosin heavy-chain antibodies used in this study (31), it is likely that very few IIB fibers were present, because almost all fibers in the mouse diaphragm appear to be oxidative types (8).GS activities in muscles incubated in vitro.
Transgenic overexpression of GS driven by the muscle creatine kinase
regulatory gene cassette is highest in the EDL, followed by the
diaphragm and soleus muscles (27). This expression pattern may be seen
by comparing the total GS activities measured after in vitro
incubations of muscles from wild-type animals and two lines of
transgenic mice (Fig. 8).
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Effect of insulin on 2-deoxyglucose uptake in control and transgenic
muscles incubated in vitro.
Glucose transport was assessed by measuring the initial rate of
2-deoxyglucose uptake. The basal rates of uptake were approximately equal in diaphragm, EDL, and soleus muscles from wild-type animals (Fig. 9). The effect of insulin on
increasing glucose transport in these muscles was most pronounced in
the diaphragm, where the hormone produced an eightfold increase in the
initial rate of 2-deoxyglucose uptake (Fig. 9A). Insulin
increased transport activity by ~2-fold and 2.5-fold in the EDL (Fig.
9B) and soleus muscles (Fig. 9C), respectively.
Insulin-stimulated rates of 2-deoxyglucose uptake in muscles from GSL3
and GSL30 mice were not significantly different from the rates in
muscles from wild-type animals, and basal rates of transport were not
higher in any of the transgenic muscles than in wild-type muscles. In
EDL muscles from GSL30 mice, basal 2-deoxyglucose uptake was
significantly less than the basal uptake in wild-type EDL muscles (Fig.
9B).
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Increased glucose incorporation into glycogen in transgenic muscles
incubated in vitro.
To investigate the effect of the transgene on glycogen synthesis,
[U-14C]glucose incorporation into glycogen was
measured in diaphragm, EDL, and soleus muscles from GSL3 animals
incubated with increasing concentrations of insulin (Fig.
10). In all three muscles and at all
insulin concentrations, the mean values for
[14C]glycogen synthesis were higher in the GSL3
muscles than in muscles from wild-type animals, although statistical
significance was not achieved in all cases. The greatest effect of
insulin was observed in diaphragm muscles (Fig. 10A), where
[14C]glycogen synthesis was increased 24-fold
by 2 mU/ml insulin. The difference between the wild-type and transgenic
muscles reached statistical significance at the 20 µU/ml
concentration, where the rate in the transgenic diaphragms was
~2.5-fold higher than in the wild-type muscles. In EDL muscles from
wild-type mice, [14C]glycogen synthesis was
increased fivefold by 2 mU/ml insulin (Fig. 10B). The rates of
[U-14C]glucose incorporation into glycogen
observed either without insulin or with 20 µU/ml insulin were
approximately twofold higher in the GSL3 muscles than in muscles from
wild-type mice. [14C]glycogen synthesis in the
GSL3 soleus was approximately twice that observed in the wild-type
soleus when muscles were incubated with 200 µU/ml insulin (Fig.
10C).
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DISCUSSION |
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An important conclusion from the present study is that increasing glycogen synthase enhances glycogen synthesis in both the glycolytic and oxidative types of fast-twitch fibers. This conclusion is supported by results obtained from microanalytical measurements of glycogen and metabolites in single fibers derived from control and transgenic muscles that had been freeze-clamped in situ and from measurements of the rates of 2-deoxyglucose uptake and glucose incorporation into glycogen in muscles incubated in vitro.
Transgenic overexpression of glycogen synthase was associated with increased glycogen accumulation in both the IIB and the IIA\IID groups of fibers from anterior tibialis muscles (Fig. 3). Although we did not attempt to distinguish between IIA and IID fibers, in any grouping of fibers of approximately equal oxidative capacity, as reflected by MDH activity, glycogen was higher in the transgenic fibers. Because approximately equal numbers of IIA and IID fibers should have been present in the population of fibers that we analyzed (15), it seems likely that the transgene increased glycogen in both of these types of fibers, although to a lesser degree than in type IIB fibers. In addition, the UDPG concentrations were decreased in all groupings of fibers of nearly equal MDH activities, consistent with increased conversion of UDPG to glycogen as a result of increased glycogen synthase (Fig. 6). Expression of the transgene did not increase basal glucose transport or enhance insulin-stimulated glucose transport in diaphragm, EDL, or soleus muscles incubated in vitro (Fig. 9). Therefore, we infer that the increased glycogen synthase activity, rather than an increase in glucose transport, accounted for the increase in glycogen accumulation.
In the present study we also sought to identify fiber-specific differences in glycogen metabolism in response to transgene expression by performing crossover analysis of the precursors in the synthetic pathway leading to glycogen. Crossover plots for the IIB and IIA\IID groups were remarkably similar, and in both, crossover occurred between G-1-P and UDPG (Fig. 7). Thus, with overexpression of glycogen synthase, UDPG pyrophosphorylase became more rate determining for glycogen synthesis in both the IIB and IIA\IID grouping of fibers. The major differences between the two groups were the greater effects of the transgene on increasing glycogen and on decreasing UDPG in the IIB fibers. It seems reasonable to conclude that the higher level of transgenic glycogen synthase observed in the IIB fibers contributed to these differences (Fig. 2).
Increased glucose transport would increase glycogen precursors, including UDPG, and oxidative fibers contain more GLUT-4 than IIB fibers (19). Thus the higher overall capacity for glucose uptake might partly explain why UDPG concentrations were not decreased as much by the transgene in the IIA\IID fibers as in IIB fibers. However, by the same token, the greater glucose transport capacity of IIA\IID fibers would not explain why these fibers accumulated less glycogen than IIB fibers in response to overexpression of glycogen synthase, again indicating that glycogen levels and glucose transport are not always strictly correlated.
As with all transgenic animal models, there is the issue of adaptive changes that accompany expression of the transgene. In our previous analyses of whole muscles from the overexpressing mice (27), we had noted that increased levels of glycogen synthase were accompanied by an increase in the amount of phosphorylase. Another secondary effect resulting from expression of the transgene is the hyperaccumulation of glycogen, which itself has been viewed as a possible regulator of metabolic processes. For example, Danforth (3) described many years ago an inverse correlation between glycogen synthase activity ratio and the amount of glycogen in muscle. It is interesting to speculate that increased glycogen may serve as a signal for increasing phosphorylase. The significant correlation observed between levels of glycogen and phosphorylase activity across fiber types would be consistent with this type of regulation (Fig. 5). One hypothesis is that glycogen upregulates expression of the phosphorylase gene, but in principle glycogen could act either by increasing the synthesis of phosphorylase or by decreasing the rate of phosphorylase degradation. Decreasing the spontaneous contractile activity of myotubes with TTX, which would be expected to increase glycogen stores by reducing energy expenditure of the cells, markedly decreased the rate of phosphorylase degradation (23). Regardless of the mechanism by which phosphorylase is increased in the transgenic fibers, it is unlikely that it is the increase in phosphorylase, which degrades glycogen, that leads to increased glycogen accumulation.
There have also been suggestions that glycogen levels could feed back to inhibit glucose transport (7, 12, 16). In the present study, basal 2-deoxyglucose uptake was significantly lower in EDL muscles from GSL30 mice than in control muscles (Fig. 9). This result was not unexpected, because we had previously found that GLUT-4 levels in the transgenic EDL muscles were ~50% lower than in control muscles (27). Thus our results support the view that accumulated glycogen may decrease glucose transport, at least under certain conditions. However, the existence of a secondary effect of glycogen to inhibit glucose transport would not appear to affect our interpretation that glycogen accumulation in the transgenic animals is due to the increase in glycogen synthase activity, because a decrease in transport would be expected to limit glycogen accumulation by reducing the availability of substrate for glycogen synthesis.
When muscles were incubated in vitro with [14C]glucose, the rate of [14C]glycogen synthesis was higher in the transgenic EDL than in the wild-type EDL (Fig. 10B). This finding provides additional support for the conclusion that elevated glycogen synthase increases glycogen synthesis in IIB fibers, as these fibers account for the large majority of fibers in the EDL. Likewise, the finding of increased [14C]glycogen synthesis in diaphragm muscles (Fig. 10A) strengthens the conclusion that increasing glycogen synthase increases glucose transport in IIA and IID fibers, because the diaphragm is composed primarily of these fiber types. In the presence of insulin, [14C]glycogen synthesis was also increased in the transgenic soleus muscles. However, this increase could have occurred in either the type I or IIA fibers that comprise this muscle. Because single fiber analyses of type I fibers were not performed in the present study, the effect of the transgene in type I fibers remains to be established.
Although we believe that the present results strongly support the conclusion that activation of glycogen synthase by insulin has an important role in the stimulation of glycogen synthesis, we certainly do not mean to imply that glucose transport is unimportant. There is strong evidence that glucose transport is the principal rate-determining step for glucose metabolism under basal conditions, as there appears to be very little free glucose in resting skeletal muscle fibers (45). Under these conditions, increasing glycogen synthase could not increase net glucose uptake by increasing the driving force for the facilitative diffusion of glucose into the muscle fiber. Thus any increase in glycogen synthase would come at the expense of other pathways involved in glucose utilization. This could explain why transgenic overexpression of glycogen synthase did not increase the basal rate of glucose incorporation into glycogen in diaphragms and soleus muscles (Fig. 10, A and C). On the other hand, glucose transport did not appear to be strictly rate limiting for glycogen synthase in EDL muscles even under basal conditions, as the rate of glycogen synthase was increased in response to the increase in glycogen synthase in muscles incubated without insulin (Fig. 10B). In the presence of insulin, more glucose becomes available for glycogen synthase as a result of increased glucose transport, and transgenic overexpression of glycogen synthase increased the rate of [U-14C]glucose incorporation into glycogen in all three muscles (Fig. 10).
Results obtained using transgenic animal models in which either GLUT-1
or GLUT-4 levels were manipulated suggested that the rate of glucose
entry into cells exerted an important influence on glycogen
accumulation (10, 32). Shulman et al. (38) came to similar conclusions
through the application of metabolic control analysis. There is no
contradiction between our results and those just cited, because more
than one step in a multistep metabolic pathway can contribute to
determining the flux (6). Indeed, from a theoretical analysis of
glycogen metabolism by use of metabolic control analysis, Schulz (36)
reached the conclusion that the control of glycogen synthase is
distributed among different steps that assume more or less importance
depending on the physiological conditions. More recently, Jucker and
Shulman (17) analyzed rats during hyperinsulinemic clamp and used
metabolic control analysis to apportion the control of glucose disposal
among glucose transport, glycogen synthase, and glycolysis. The result
was that glucose transport/phosphorylation had a control coefficient of 0.55 and glycogen synthase that of 0.30, meaning that both steps substantially affect glucose disposal. It is interesting to compare these coefficients with the effects of insulin on increasing glucose transport and glycogen synthase activity. In the present experiments, the largest effects of insulin were observed in the diaphragm muscles.
Incubating hemidiaphragms with a maximally effective concentration of
insulin increased the rate of [14C]glucose
incorporation into glycogen by 24-fold (Fig. 10A), an effect
much greater than either the increase (8-fold) in 2-deoxyglucose uptake
(Fig. 9A) or the increase (4-fold) in the
G-6-P/+G-6-P activity ratio of glycogen synthase
(Fig. 8A). These results are consistent with the view that the
stimulation of glycogen synthesis involved increases in both glucose
transport and glycogen synthase.
In conclusion, control of glycogen accumulation by insulin is distributed between transport and synthesis, and increasing either in skeletal muscle leads to increased glycogen accumulation in vivo. The activation of both glucose transport and glycogen synthase by insulin ensures the efficient storage of glucose as glycogen, and thus defects in either may compromise the process of glycogen synthesis.
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ACKNOWLEDGEMENTS |
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This work was supported in part by National Institutes of Health Grants DK-28312 (J. C. Lawrence, Jr.), AR-41189 (J. C. Lawrence, Jr.), and DK-27221 (P. J. Roach).
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: J. C. Lawrence, Jr., Dept. of Pharmacology, Box 448, Health Science Center, University of Virginia, Charlottesville, VA 22908 (E-mail: jcl3p{at}virginia.edu).
Received 4 May 1999; accepted in final form 1 October 1999.
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