1 Exercise Physiology and Metabolism Laboratory, Department of Kinesiology and Health Education, University of Texas at Austin, Austin, Texas 78712; and 2 Division of Geriatrics and Gerontology, Washington University of Medicine, St. Louis, Missouri 63110
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ABSTRACT |
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We examined the
effects of amylin on
3-O-methyl-D-glucose
(3-O-MG) transport in perfused rat
hindlimb muscle under hyperinsulinemic (350 µU/ml, 2,100 pmol/l)
conditions. Amylin at 100 nmol/l concentration inhibited
3-O-MG transport relative to control
in all three basic muscle fiber types. Transport decreased in
slow-twitch oxidative (from 5.65 ± 1.13 to 3.46 ± 0.71 µmol · g1 · h
1),
fast-twitch oxidative (from 6.84 ± 0.90 to 4.84 ± 0.76 µmol · g
1 · h
1),
and fast-twitch glycolytic (from 1.27 ± 0.20 to 0.60 ± 0.05 µmol · g
1 · h
1)
muscle. Amylin inhibition of insulin-stimulated glucose transport in
skeletal muscle was accompanied by a 433 ± 72% increase in intracellular glucose 6-phosphate
(G-6-P) despite the absence of
extracellular glucose. The source of hexose units for the formation and
maintenance of G-6-P was likely
glycogen. Amylin increased glycogenolysis, increased lactate formation,
and decreased glycogen synthase activity. Furthermore, the kinetics of
glycogen synthase suggest that this enzyme may control intracellular
G-6-P concentration. Despite the large
increase in G-6-P, no detectable
increase in uridine
diphosphate-N-acetylhexosamines
occurred, suggesting that the proposed glucosamine pathway may not be
involved in transport inhibition. However, decreases in uridine
diphosphate hexoses were detected. Therefore, uridine or
hexosamine-based metabolites may be involved in amylin action.
glucosamine; glycogen synthase; glucose 6-phosphate; lactate; uridine diphosphate-N-acetylhexosamine; uridine diphosphate hexose
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INTRODUCTION |
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AMYLIN is a 37-amino acid peptide that is cosecreted
with insulin from pancreatic -cells (3, 13). It inhibits
insulin-mediated glucose uptake and disposal into glycogen in skeletal
muscle and raises the blood lactate level (2, 6, 18, 22, 24, 28, 29).
The mechanism of this decrease in glucose uptake from the blood into
muscle is not completely understood (4, 6, 15, 22, 23, 29, 30) but may
occur secondarily to an inhibition of glycogen synthesis and
stimulation of glycogenolysis, resulting in an increase in
intracellular glucose 6-phosphate (G-6-P) concentration (2, 16, 22,
29). An increase in intracellular
G-6-P has been postulated to decrease
net glucose uptake via feedback inhibition of hexokinase (2, 22, 27).
A glucose analog commonly used to study in vitro glucose metabolism, 3-O-methyl-D-glucose (3-O-MG), is transported across the sarcolemma but not phosphorylated by hexokinase. Therefore, accumulation of this glucose analog under a short duration and a large concentration gradient approximates unidirectional glucose transport across the sarcolemma. Zierath et al. (30) demonstrated an amylin-mediated inhibition of insulin-stimulated 3-O-MG transport in human muscle strips without changes in glycogen and lactate concentration. However, several other investigators have not been able to confirm these observations in isolated muscle preparations (23, 29). Decreased 3-O-MG transport without changes in glycogen or lactate concentrations would suggest that amylin inhibits glucose transport without the involvement of glycolytic intermediates (30). This is an important observation, because the intermediate metabolites G-6-P and products of the glucosamine pathway [uridine diphosphate (UDP)-N-acetylhexosamines] have been implicated in inhibition of insulin-stimulated glucose transport (5, 8, 26). The present study was undertaken to further examine whether amylin inhibits skeletal muscle 3-O-MG transport and whether this inhibition is associated with metabolic intermediates. To this end, we used the nonrecirculating rat hindlimb perfusion model to investigate the effect of amylin on insulin-stimulated 3-O-MG transport. We found that a pharmacological dose of amylin inhibited insulin-stimulated 3-O-MG transport and that inhibition was associated with an increase in intracellular G-6-P but not UDP-N-acetylhexosamines.
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METHODS |
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Animal care. Eighteen Sprague-Dawley rats (Harlan Sprague Dawley, Indianapolis, IN) 3-4 wk of age were purchased and housed three to a cage and allowed access to food and water ad libitum. The temperature of the animal room was maintained at 23°C with a 12:12-h light-dark cycle. All procedures used in this study were approved by the University of Texas Animal Care and Use Committee.
Perfusion and surgical procedure.
Rats weighing 174.5 ± 1.74 g were fasted for 4-6 h, which
resulted in muscle glycogen concentrations averaging 30.0 ± 1.7 µmol/g for soleus, 31.3 ± 1.6 µmol/g for red gastrocnemius, and 28.1 ± 1.5 µmol/g for white gastrocnemius. Rats were
weight-matched into control and amylin groups, and each pair was
perfused consecutively to minimize the effects of fasting duration on
glycogen concentration and glucose transport rates. The rats were
anesthetized with an intraperitoneal injection of pentobarbital sodium
(65 mg/kg); hindlimbs were surgically isolated, and catheters were
placed in the descending aorta and vena cava as previously described (14). Immediately before catheterization, the soleus (slow-twitch oxidative, SO) and red (fast-twitch oxidative, FO) and white
(fast-twitch glycolytic, FG) portions of the gastrocnemius and
quadriceps muscles were surgically separated, removed from the left
leg, and freeze-clamped with tongs cooled in liquid nitrogen. After the
removal of the preperfusion muscle samples, animals were euthanized
with an intracardiac injection of pentobarbital as the hindlimbs were
being washed out with 25 ml of Krebs-Henseleit buffer (KHB). Catheters
were placed in line with a nonrecirculating perfusion system that
provided a flow rate of 5 ml/min. Perfusate flow and system pressure
were constantly monitored to ensure uniformity among experimental
groups. All perfusates consisted of KHB, pH 7.4, 4% dialyzed fatty
acid-free bovine serum albumin (ICN 105033), 30% (vol/vol) blood bank
time-expired human erythrocytes, and 200 µmol/l pyruvate. The
perfusate was continuously gassed (mixture 95%
O2-5%
CO2), and its temperature was
maintained at 37°C. The right hindlimb was perfused for 20 min with
a perfusion medium containing 1 mmol/l glucose and 2,100 pmol/l (350 µU/ml) insulin (Lilly CP-210) with or without a pharmacological concentration (100 nmol/l) of rat amylin (H-9475; Bachem, King of
Prussia, PA). This high concentration of amylin was selected because in
a prior study we found that a near-maximal dose was necessary for all
attributed effects of amylin to be easily measurable (2). Amylin was
added to the perfusate immediately before each perfusion as a solution
of 0.1 mg/ml protein content dissolved in 1.0% gelatin. Rats not
receiving amylin were perfused with added vehicle. After the initial 20 min of perfusion, the hindlimbs were perfused for an additional 12 min
with a perfusate that also included 2 mmol/l mannitol, 10 mmol/l
3-O-MG, 0.1 µCi/ml
[U-14C]mannitol, and
0.3 µCi/ml
3-O-methyl-D-[3H]glucose
(3-O-[3H]MG)
but not glucose. At the end of the perfusion, the same muscles were
removed from the right leg as had been removed from the left leg for
postperfusion measurements. Muscles were stored at 80°C for
further analysis.
Blood and tissue assays. 3-O-MG transport was determined by incorporation of 3-O-[3H]MG into muscle tissue. Freeze-clamped muscles from the perfused hindlimbs were sectioned and weighed frozen. A 60- to 100-mg piece of each muscle or muscle section was first dissolved in 1 ml of 1 mol/l KOH by incubating it for 20 min at 65°C, then mixed, and finally incubated for an additional 10 min at 65°C. An equal volume of 1 mol/l HCl was then added to the sample and mixed. The neutralized sample was counted for 3H and 14C dpm. 3-O-MG transport was calculated as 3-O-MG retained in the tissue after adjustment for extracellular space determined by [14C]mannitol retained in the tissue. Transport was expressed as micromoles 3-O-MG per gram per hour.
Muscle glycogen content was determined after complete enzymatic degradation to glucose with amyloglucosidase (20). A well-mixed aliquot of the digested muscle from the transport sample was transferred and incubated overnight in 0.3 mol/l sodium acetate buffer, pH 4.8, that contained 5 mg/ml amyloglucosidase (Boehringer Mannheim, Mannheim, Germany). Glycogen was expressed as glucose units determined by the spectrophotometric Trender reaction (model 315, Sigma, St. Louis, MO). To best approximate the in vivo sensitivity of glycogen synthase to allosteric activation by G-6-P (25), glycogen synthase activity was determined at 30°C in the presence of 7 mmol/l ATP, 100 µmol/l UDP-1-glucose, and varying concentrations of G-6-P as described by Bloch et al. (1). Muscle samples were prepared for analysis by homogenization in 5 volumes of 60% glycerol, 50 mmol/l KF, and 20 mmol/l EDTA at pH 7.0 and were further diluted with 8 volumes of 50 mmol/l KF and 20 mmol/l EDTA. In addition to ATP, G-6-P, and UDP-glucose, the reaction buffer contained 50 mmol/l MOPS buffer, 25 mmol/l KF, 20 mmol/l EDTA, 10 mmol/l KH2PO4, and 10 mg/ml glycogen at pH 6.9. An 8-point dose-response curve was performed on four FO muscle samples from each treatment group, and nonlinear regression analysis was used to distinguish changes in sensitivity from changes in responsiveness under these conditions. Responsiveness was defined as the maximal obtainable G-6-P-stimulated reaction rate at physiological substrate and inhibitory nucleotide concentration. Both sensitivity and responsiveness can vary considerably depending on these concentrations (25). Sensitivity was defined as the concentration of G-6-P at which half-maximal reaction rate occurred. To analyze nucleotide-linked hexoses and hexosamines, frozen muscle samples (~180 mg) were homogenized at 4°C in 3 vol of 0.3 mol/l perchloric acid and centrifuged at 2,500 g for 10 min. The perchloric acid was then extracted from the supernatant with 2 volumes of trioctylamine-1,1,2-trichlorotrifluoroethane (1:4; vol/vol), and the aqueous phase was stored atStatistics. Individual fiber type effects were analyzed by paired Student t-tests; overall effects and differences among fiber types were determined by two-way ANOVA. Glycogen synthase dose-response curves were analyzed using a repeated-measure ANOVA to determine whether differences occurred. The sensitivity and responsiveness differences were determined by Student's t-tests. A P value of <0.05 for the alpha error was considered significant.
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RESULTS |
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Amylin inhibited insulin-stimulated muscle 3-O-MG transport when all muscles were analyzed collectively by ANOVA (P < 0.01, Fig. 1). Transport was also inhibited in all three fiber types, and absolute transport rates in micromoles per gram per hour were fiber type dependent, although the relative inhibition of transport among fiber types was similar (32% SO, 30% FO, and 47% FG).
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Muscle glycogen decreased in response to amylin when all muscles were
analyzed collectively (P < 0.01, Table 1). The concentrations of glycogen
for amylin and control rats before perfusion were 29.8 ± 1.4 and
29.8 ± 1.2 µmol/g, respectively. Amylin decreased glycogen 13.9 ± 2.7% from initial values, whereas changes in control glycogen
(1.5 ± 3.2%) did not differ from initial values. No significant fiber type differences occurred.
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Glycogen synthase activity decreased significantly after amylin
treatment. Furthermore, this decrease in activity was due to a decrease
in both sensitivity and responsiveness to
G-6-P (Fig.
2). After amylin treatment, the
responsiveness of glycogen synthase declined 80% (229.2 ± 38.0 vs.
45.3 ± 3.8 nmol
G-6-P · g1 · min
1),
and the sensitivity declined 44% (5.91 ± 0.32 vs. 3.33 ± 0.35 mmol G-6-P/l) compared with the
control. Both the changes in sensitivity and responsiveness were highly
significant (P < 0.01).
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Muscle lactate increased in response to amylin when all muscles were analyzed collectively (P < 0.001, Fig. 3). Compared with control, amylin increased lactate by 76% in FO muscle and 82% in FG muscle, and this effect did not differ between fiber types.
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Amylin significantly increased G-6-P concentration by 547% in FO and 319% in FG muscle (P < 0.001, Fig. 4). No significant difference between FO and FG muscle occurred. Although G-6-P increased three to five times control levels with amylin treatment, no difference was detected in the UDP-N-acetylhexosamines between control and amylin-treated muscles (Fig. 5). The concentration of UDP-hexoses, however, decreased by 36% in FO and 41% in FG muscle with amylin treatment.
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DISCUSSION |
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In the present study we demonstrated that a pharmacological concentration of amylin induced inhibition of insulin-stimulated 3-O-MG transport, stimulation of glycogenolysis, and an increase in the glycolytic intermediates G-6-P and lactate with no change in UDP-N-acetylhexosamines. The relationship between glycogenolysis and the elevation in G-6-P and lactate after amylin exposure has been demonstrated before, both in vivo and in vitro under conditions in which extracellular glucose was provided (2, 4, 16, 22, 28, 29). However, in this study we evaluated the effects of amylin on intracellular G-6-P concentration during conditions in which no glucose substrate was present.
Our finding that amylin reduced the rate of insulin-stimulated glucose transport in skeletal muscle is in agreement with the findings of Zierath et al. (30). However, our results extend these previous findings by demonstrating that the effects of amylin are fiber type independent. Furthermore, we observed that the decrease in insulin-stimulated 3-O-MG transport on amylin exposure was coupled with increases in glycogenolysis and lactate production. This finding is in contrast to the findings of Zierath et al. (30), in which neither glycogenolysis nor lactate production was observed. This is an important distinction between studies, because the results of Zierath et al. suggest that amylin must inhibit transport independently of metabolically driven inhibition, although these investigators did not measure intracellular G-6-P levels. One possible explanation for the differences in results between the two studies is the difference in muscle preparations used. Zierath et al. used an isolated human muscle preparation, whereas we used the rat hindlimb prefusion technique. Thus it is possible that the effect of amylin on human muscle differs from that on rat muscle, or that the human isolated muscle preparation lacks the sensitivity to detect small changes in muscle glycogen and lactate concentrations. However, regardless of the mechanism, both studies clearly demonstrated that amylin inhibits skeletal muscle 3-O-MG transport.
An inhibition of muscle glucose transport by amylin has not always been observed (23, 29). The reason for these disparate findings may involve different experimental models and duration of preexposure to amylin before measurements of glucose transport. In demonstrating the inhibition of insulin-stimulated glucose transport by amylin, Zierath et al. (30) exposed muscle to amylin for 45 min before transport was measured. In the present study, glucose transport was determined after a prior amylin exposure of 20 min. In contrast, Pittner et al. (23) could not detect any effect of amylin on glucose transport. They measured the efflux of 3-O-MG from preloaded isolated rat soleus muscles immediately after exposure to amylin. Also, Young et al. (29), using the isolated soleus preparation, did not observe a decrease in glucose transport after amylin exposure. However, it was unclear as to how long their muscles were exposed to amylin before measurement of glucose transport. Thus, it may take an extended period of time for amylin to induce 3-O-MG transport inhibition, and this temporal response may be subject to the experimental model and conditions being used. For example, the temporal response to amylin could be faster when amylin is administered through hyperperfused capillaries, as occurs during hindlimb perfusion, as opposed to administration during an isolated muscle incubation.
Although extended exposure to amylin appears to result in 3-O-MG transport inhibition, Pittner et al. (23) clearly demonstrated that acute 2-deoxyglucose uptake inhibition can occur in the absence of 3-O-MG transport inhibition. They concluded that hexokinase inhibition occurs after amylin exposure and subsequent to a rise in G-6-P. The large rise in G-6-P seen in our study supports their findings in that hexokinase inhibition would likely occur under these conditions. Also, Young et al. (29) demonstrated that intramuscular G-6-P rises after amylin exposure, whereas glucose uptake and disposal into glycogen are reduced. Their findings led them to conclude that amylin inhibits glucose uptake through feedback inhibition of hexokinase (29).
It needs to be considered that transport and uptake mechanisms are not mutually exclusive but complementary. Acute inhibition of hexokinase would protect the cell from excess glycolytic flux, and inhibition of transport could be advantageous in protecting intracellular compartments from accumulating high glucose concentrations. Both mechanisms could conceivably occur with different temporal relationships to amylin exposure. Compared with the inhibition of hexokinase, it may take an extended rise in G-6-P before transport inhibition occurs.
The source and fate of G-6-P in our experiments may give some insight into how glucose transport is being inhibited, although it is unlikely that G-6-P affects transport directly (17). We therefore investigated the mechanism of amylin-induced G-6-P accumulation and the possibility that it may increase the production of the glucosamine pathway metabolites and lead to a hormone-induced glucose toxicity similar to what has been observed during hyperglycemia (26).
Amylin caused a sharp rise in intracellular G-6-P that corresponded to an increase in glycogenolysis. The rise in G-6-P was disproportional to the increase in muscle lactate, suggesting that glycolysis may be partially inhibited under these conditions. This is in agreement with earlier findings from our laboratory, which investigated this phenomenon in more depth by use of a selective antagonist (2). In addition, amylin caused a substantial inhibition of glycogen synthase activity by reducing both its sensitivity and responsiveness to G-6-P. These changes in glycogen synthase are likely due to phosphorylation and not changes in enzyme concentration (16, 25). Evaluation of the dose-response curve for glycogen synthase indicates that it would normally prevent large fluxes in intracellular G-6-P. This observation supports the theory of Shulman and Rothman (27), who proposed that glycogen synthase activity controls G-6-P concentration rather than G-6-P controlling the rate of glycogen synthase. Thus our observations suggest that amylin may control the concentration of G-6-P indirectly through an effect on glycogen synthase rather than just a mass action effect from increased glycogenolysis. This coordination of glycogen synthase inhibition and glycogenolysis activation in the presence of amylin is important, because it prevents the development of a futile cycle between glycogenolysis and glycogen synthesis.
It has been proposed that increases in the glucosamine biosynthetic pathway may lead to inhibition of glucose transport (7, 26). Fructose 6-phosphate is the substrate for the rate-limiting enzyme glutamine fructose-6-phosphate aminotransferase of this pathway. Fructose 6-phosphate in turn is formed from G-6-P by an equilibrium reaction. Because we have produced a 433% increase in G-6-P by treating the muscles with amylin, an increase in hexosamine metabolites should occur if this pathway is involved in the amylin-induced inhibition of glucose transport. The increase in concentration of UDP-N-acetylhexosamines has been used as an indicator of increased flux through the glucosamine pathway, and increases in these metabolites during chronic hyperglycemia and glucosamine infusion have been correlated to decreased rates of glucose uptake (26).
We could not detect any change in UDP-N-acetylhexosamines. This suggests that the glucose transport inhibition demonstrated in this study is not due to a buildup of glucosamine pathway metabolites. Several studies have shown that glucose toxicity characterized by increases in UDP-N-acetylhexosamines takes several hours to develop in glucose clamp studies and that G-6-P is actually lower than control levels during inhibition of glucose transport (9, 10), whereas, in our study, the G-6-P concentration was greatly elevated. Interestingly, the UDP-hexoses decreased with amylin treatment. UDP-hexoses have been shown to decrease when UDP-N-acetylhexosamines increase (26). This would suggest that a decrease in UDP-hexoses, or an increase in the ratio of UDP-N-acetylhexosamines to UDP-hexoses, could be involved in amylin-mediated inhibition of glucose transport. Thus amylin-induced glucose transport inhibition appears to work differently from chronic hyperglycemia. In addition, the decrease in UDP-hexoses without increases in UDP-N-acetylhexosamines might suggest that the latter is not directly responsible for glucose transport inhibition when this pathway is activated. Clearly, more studies are needed to determine the role of the glucosamine pathway in both chronic and acute transport inhibition.
In conclusion, three important aspects of amylin action have been revealed in this study. First, amylin can inhibit insulin-stimulated glucose transport, and thus glucose uptake, without the involvement of hexokinase. This brings into question whether decreases in glucose uptake, which occur concurrently with sustained increases in G-6-P, are necessarily the result of hexokinase inhibition. Second, an increase in the proposed end point of the glucosamine pathway is not involved in glucose transport inhibition, although it is possible that other aspects of this pathway may be involved. Third, amylin can elevate intracellular G-6-P in the absence of extracellular glucose. This appears to be due to activation of glycogenolysis accompanied by restrictions in glycolysis and glycogen synthesis. Further research will be required to determine the exact mechanism by which amylin inhibits insulin-stimulated glucose transport in skeletal muscle.
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ACKNOWLEDGEMENTS |
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We are grateful to Joe Hancock for excellent technical assistance.
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FOOTNOTES |
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This work was supported by a research grant from Pfizer, Inc.
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: J. L. Ivy, Bellmont Hall, Rm. 222, Dept. of Kinesiology and Health Education, Univ. of Texas at Austin, Austin, TX 78712.
Received 17 February 1998; accepted in final form 11 June 1998.
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