Regulation of glucose transporter GLUT-4 and hexokinase II gene transcription by insulin and epinephrine

Jared P. Jones and G. Lynis Dohm

Department of Biochemistry, East Carolina University School of Medicine, Greenville, North Carolina 27858

    ABSTRACT
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Abstract
Introduction
Methods
Results
Discussion
References

Transport of glucose across the plasma membrane by GLUT-4 and subsequent phosphorylation of glucose by hexokinase II (HKII) constitute the first two steps of glucose utilization in skeletal muscle. This study was undertaken to determine whether epinephrine and/or insulin regulates in vivo GLUT-4 and HKII gene transcription in rat skeletal muscle. In the first experiment, adrenodemedullated male rats were fasted 24 h and killed in the control condition or after being infused for 1.5 h with epinephrine (30 µg/ml at 1.68 ml/h). In the second experiment, male rats were fasted 24 h and killed after being infused for 2.5 h at 1.68 ml/h with saline or glucose (625 mg/ml) or insulin (39.9 µg/ml) plus glucose (625 mg/ml). Nuclei were isolated from pooled quadriceps, tibialis anterior, and gastrocnemius muscles. Transcriptional run-on analysis indicated that epinephrine infusion decreased GLUT-4 and increased HKII transcription compared with fasted controls. Both glucose and insulin plus glucose infusion induced increases in GLUT-4 and HKII transcription of twofold and three- to fourfold, respectively, compared with saline-infused rats. In conclusion, epinephrine and insulin may regulate GLUT-4 and HKII genes at the level of transcription in rat skeletal muscle.

gene expression; counterregulatory hormones; skeletal muscle

    INTRODUCTION
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Abstract
Introduction
Methods
Results
Discussion
References

COUNTERREGULATORY HORMONE control of glucose disposal in skeletal muscle is vital to maintaining euglycemia. In response to insulin binding to its receptor on skeletal muscle, a signal cascade is initiated that culminates in the recruitment of GLUT-4 protein to the plasma membrane of skeletal muscle (9). GLUT-4 protein is a facilitative glucose transporter found in adipose, heart, and skeletal muscle that, on activation in the plasma membrane, allows glucose to move down its concentration gradient into the cell (17, 18, 22). This concentration gradient is sustained, in part, by hexokinase II (HKII), which phosphorylates glucose, thereby maintaining low intracellular concentrations of free glucose. Transport of glucose across the plasma membrane represents the rate-limiting step in glucose utilization (12, 31).

Previous research suggests that insulin and epinephrine may regulate GLUT-4 and HKII gene transcription in rat skeletal muscle. Garvey et al. (13) demonstrated that streptozotocin (STZ)-induced diabetes decreased GLUT-4 mRNA and GLUT-4 protein in rat skeletal muscle. Our laboratory demonstrated that STZinduced decreases in GLUT-4 mRNA and protein in skeletal muscle could be attributed, in part, to decreased transcription of the GLUT-4 gene (23). Subsequent injection of insulin into the STZ-diabetic animals restored both GLUT-4 mRNA and protein to control values (13). Under euglycemic, hyperinsulinemic conditions, HKII mRNA levels and protein activity in rat skeletal muscle are increased (28). In rat skeletal muscle, it is not known whether insulin-induced increases in GLUT-4 and HKII mRNA and protein are transcriptionally mediated.

Neufer and Dohm (24) previously reported that exercise induces a transient increase in GLUT-4 gene transcription. Exercise also increases HKII gene transcription (25), with corresponding changes in mRNA and protein levels (26). The effects of exercise on GLUT-4 and HKII transcription may be due to epinephrine-induced increases in muscle adenosine 3',5'-cyclic monophosphate (cAMP) concentrations. The purpose of this study was to determine whether epinephrine and/or insulin regulates in vivo GLUT-4 and HKII gene transcription in rat skeletal muscle. We demonstrate that infusion of epinephrine decreases GLUT-4 gene transcription, whereas infusion of insulin increases GLUT-4 and HKII gene transcription in rat skeletal muscle.

    METHODS
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Abstract
Introduction
Methods
Results
Discussion
References

Materials. Radiolabeled UTP and dATP were obtained from Du Pont-New England Nuclear. DNA polymerase I (Klenow fragment) and restriction enzymes were purchased from Promega (Madison, WI). RNasin [ribonuclease (RNase) inhibitor] and nonradiolabeled nucleotides CTP, GTP, ATP, and TTP were obtained from Pharmacia (Uppsala, Sweden). Insulin (Humulin R) was purchased from Eli Lilly (Indianapolis, IN), and TRIzol reagent was purchased from GIBCO-BRL (Gaithersburg, MD). Unless mentioned, all other reagents were of molecular biology grade and were obtained from Sigma Chemical (St. Louis, MO), Fisher Scientific (Springfield, NJ), or Pharmacia.

Animals. Male Sprague-Dawley rats (Harlan Sprague Dawley, Indianapolis, IN) were housed in individual cages and provided food and water ad libitum. Room temperature (20-22°C) and lighting (12:12-h light-dark cycle) were controlled. Rats used in the epinephrine experiment were adrenodemedullated 3 wk before the experiment. Three to five days before the experiment, rats were anesthetized, and jugular catheters were surgically implanted for infusion of epinephrine, saline, glucose, and insulin-glucose solutions.

Experimental design. In the first experiment, adrenodemedullated rats (381 ± 5 g) were fasted for 24 h and killed in the control condition or after being infused for 1.5 h with physiological saline containing epinephrine (30 µg/ml), somatostatin (0.3 mg · kg-1 · h-1), and ascorbic acid (25 µg/ml). A sham-infused control group was infused with physiological saline plus somatostatin (1.05 mg · kg-1 · h-1) and ascorbic acid (25 µg/ml). The infusion rate used in all experiments was 1.68 ml/h. Immediately before the 1.5-h period of infusion, animals were infused with somatostatin for 10 min to lower plasma insulin to baseline levels. After 1.5 h of infusion, rats were anesthetized by intravenous injection of pentobarbital sodium (4.8 mg/100 g body wt), and ~500 mg of mixed gastrocnemius muscle were harvested and quick-frozen to the temperature of liquid nitrogen for RNA isolation (Northern analysis) and cAMP determination. Quadriceps, tibialis anterior, and gastrocnemius muscles were then harvested, minced, and pooled for the isolation of nuclei. Blood for glucose and insulin assays was collected from the descending aorta. Plasma, nuclei, and muscle samples were stored at -70°C until analyzed.

In the second experiment, rats (336 ± 2 g) were fasted for 24 h and killed after being infused for 2.5 h with physiological saline or glucose (625 mg/ml) + 0.1% bovine serum albumin or insulin (39.9 µg/ml) + glucose (625 mg/ml) + 0.1% bovine serum albumin. After 2.5 h of infusion, rats were rapidly anesthetized, and tissues and blood were collected as described above.

RNA isolation and Northern analysis. RNA was isolated from mixed gastrocnemius muscle using TRIzol (GIBCO-BRL) reagent according to the manufacturer's instructions. Briefly, 100 mg of powdered muscle were homogenized in 1 ml of TRIzol. Samples were centrifuged at 12,000 g for 10 min at 4°C; the supernatant was transferred to a new tube and incubated at room temperature for 5 min. To each sample, 200 µl of chloroform were then added, vortexed vigorously for 30 s, and incubated at room temperature for 5 min. Samples were centrifuged for 15 min (12,000 g) at 4°C, and the top aqueous layer was transferred to a fresh tube. RNA was precipitated by the addition of an equal volume of isopropanol and incubated at room temperature for 10 min. Samples were centrifuged for 10 min (12,000 g) at 4°C. RNA pellets were then washed with 4 M LiCl, followed by a 70% ethanol wash. RNA pellets were resuspended in 100 µl of diethyl pyrocarbonate-treated water. RNA (20 µg/sample) was size-fractionated on a 1.25% agarose, 2 M formaldehyde gel and then electrotransferred to Hybond N+ membrane. Blots were probed with random primed [alpha -32P]dATP-labeled cDNA probes (11) for GLUT-4 and 18S ribosomal RNA. Northern blots were visualized by phosphorimaging and quantitated using Imagequant software (Molecular Dynamics, Sunnyvale, CA).

Nuclear isolation and transcriptional run-on analysis. Nuclei were isolated from pooled quadriceps, tibialis anterior, and gastrocnemius muscles according to the method of Zahradka et al. (33) with certain modifications (24). Transcriptional run-on analysis was performed using techniques described by Cornelius et al. (8) with certain modifications (24). Briefly, radiolabeled RNA transcripts were isolated using TRIzol reagent (GIBCO-BRL) as described in RNA isolation and Northern analysis, without the 4 M LiCl wash. RNA pellets were resuspended in 1.0 ml of HYBRISOL I (Oncor, Gaithersburg, MD), heated for 10 min at 65°C, and then triturated to ensure denaturation of the RNA. The concentration (counts · min-1 · µl-1) of 32P-labeled RNA was determined by liquid scintillation spectrometry. Hybridization was done on a Hybond N+ membrane to which 2 µg of GLUT-4, HKII, and beta -actin cDNA and 0.1 µg of genomic DNA were crosslinked. Genomic DNA and the cDNAs of interest were denatured in 0.1 M NaOH for 30 min at 37°C, neutralized in 10× standard sodium phosphate EDTA (SSPE) (1× SSPE = 0.15 M NaCL, 0.01 M NaHPO4, and 1.0 mM EDTA, pH 7.4), and applied to Hybond N+ membrane by use of a slot-blot apparatus (Mini-fold II, Schleicher and Schuell, Keene, NH). Each membrane was trimmed and placed in a bag with 1 ml of sample. All membranes were prehybridized overnight in 1.0 ml of HYBRISOL I at 47°C. Hybridization was done for 3 days at 47°C, after which filters were rinsed for 30 min at 50°C in 2× standard saline citrate (SSC), 30 min at 37°C in 2× SSC containing 10 µg/ml of RNase A, and for 30 min at 55°C in 0.1× SSC and 0.1% sodium dodecyl sulfate (SDS). After drying, membranes were placed on a phosphorimager screen for 3 days. All bands were visualized by phosphorimaging and quantitated using Imagequant software (Molecular Dynamics). All transcripts were normalized to genomic DNA.

RNase protection assay. A pGEM7 plasmid containing a Bgl II-Xmn I fragment from cDNAHKII was used to generate a 32P-labeled antisense RNA. Ten micrograms of RNA per sample were hybridized to HKII-labeled probes for 18 h at 45°C in 20 µl of buffer containing 80% deionized formamide, 0.4 M NaCl, 40 mM piperazine-N,N'-bis(2-ethanesulfonic acid) (pH 6.4), and 1 mM EDTA (pH 8.0). After hybridization, 200 µl of RNase digestion buffer consisting of 10 mM tris(hydroxymethyl)aminomethane · HCl (pH 7.5), 5 mM EDTA (pH 8.0), 200 mM Na acetate (pH 7.5), and a 1:1,000 dilution of RNase A/T1 mixture (Ambion, Austin, TX) were added to each sample, followed by incubation at 37°C for 30 min. Digestion was terminated by the addition of 10 µl of proteinase K (10 µg/ml) and 20 µl of 10% SDS and incubation at 37°C for 15 min. RNA was extracted using equal volumes of a phenol-chloroform-isoamyl alcohol cocktail (25:24:1) and precipitated by adding 1 µl of tRNA (10 mg/ml) and 625 µl of 95% ethanol. A 6% polyacrylamide gel containing 7 M urea was used to size-fractionate the RNA. Results were visualized by phosphorimaging and quantitated using Imagequant software (Molecular Dynamics).

Analyses. A Beckman Glucose II analyzer (Fullerton, CA) or an enzymatic (glucose oxidase/o-dianisidine) colorimetric assay (Sigma Chemical) was used to determine blood glucose concentrations (mM). Insulin concentrations (ng/ml) were determined using a radioimmunoassay kit (Linco Research, St. Louis, MO). Muscle cAMP concentrations were determined by the method of Gilman (15). To determine significant differences between treatment means, a Student's t-test or one-way analysis of variance and post hoc Student-Newman-Keuls tests were performed.

    RESULTS
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Abstract
Introduction
Methods
Results
Discussion
References

The first experiment was designed to determine whether GLUT-4 and/or HKII gene transcription rates were altered in rat skeletal muscle by intravenous infusion of epinephrine for a period of 1.5 h. The biological effects of epinephrine are propagated by an increase in intracellular cAMP levels. To maximize the difference in muscle cAMP between control and epinephrine-infused rats, animals were adrenodemedullated to eliminate endogenous epinephrine secretion. Compared with fasted controls, infusion of epinephrine induced an ~8-fold increase in gastrocnemius cAMP concentrations and a 3.5-fold rise in blood glucose levels (Table 1). In an effort to minimize plasma insulin concentrations, rats were fasted for 24 h before the experiment and somatostatin was added to the infusate. Nevertheless, in response to hyperglycemia, plasma insulin concentrations increased 2.5-fold in epinephrine-infused rats. Nuclear run-on analysis indicated that there were no differences in GLUT-4 and HKII gene transcription between sham-infused and resting controls (data not shown). Infusion of epinephrine, however, decreased GLUT-4 gene transcription to 40% of fasted controls and increased HKII gene transcription to 205% of fasted controls (Fig. 1).

                              
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Table 1.   Gastrocnemius cAMP, plasma glucose, plasma insulin, and gastrocnemius GLUT-4 and HKII mRNA levels in control and epinephrine-infused rats


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Fig. 1.   Transcriptional run-on analysis and determination of relative levels of GLUT-4 and hexokinase II (HKII) transcription from mixed skeletal muscle of control (Con) and epinephrine (Epi)-infused rats. A: phosphorimage showing hybridization of in vitro transcribed RNA to cDNA probes (2 µg) of interest or to rat skeletal muscle genomic DNA (0.1 µg). Plasmid DNA (pGEM, Promega, 2 µg/lane) served as a negative control. Hybridized transcripts were quantitated by Imagequant software. B and C: summary of values for GLUT-4 (B) and HKII (C) transcription (means ± SE; n = 5-7/group). * Significantly (P < 0.05) different from control.

In the first experiment, rats infused with epinephrine were hyperglycemic (Table 1). Previous research suggests that hyperglycemia may decrease GLUT-4 mRNA and protein levels (3, 32); therefore, we could not determine with certainty whether the observed changes in the transcription rate of the GLUT-4 gene were due to the effects of epinephrine and/or glucose. To determine whether hyperglycemia was attenuating GLUT-4 gene transcription, we infused additional rats with glucose to match glucose values of epinephrine-infused rats (Table 2). Nuclear run-on analysis revealed that GLUT-4 gene transcription in epinephrine-infused rats was 67% that of glucose-infused rats (Table 2), suggesting that epinephrine, not glucose, is decreasing transcription.

                              
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Table 2.   cAMP (white muscle), GLUT-4 mRNA (white muscle), plasma glucose, and insulin levels and mixed muscle GLUT-4 gene transcription rates in glucose- and epinephrine-infused rats

We have previously reported that STZ-induced diabetes in rats attenuates GLUT-4 gene transcription in red quadriceps muscle (24). In this study, we tested the in vivo effect of insulin on GLUT-4 and HKII gene transcription in skeletal muscle of healthy animals. In preliminary experiments using an infusion rate of 1.68 ml/h, the concentrations of insulin and glucose needed to obtain hyperinsulinemia and euglycemia after 2.5 h of infusion were determined to be 39.9 µg/ml and 625 mg/ml, respectively. Rats infused with glucose were hyperglycemic, whereas rats infused with saline and insulin plus glucose were euglycemic (Table 3). Compared with saline-infused rats, insulin concentrations were significantly (P < 0.05) higher in both glucose-infused and insulin plus glucose-infused rats (Table 3). There were no differences in gastrocnemius cAMP concentrations (Table 3) among rats infused with saline, glucose, or insulin plus glucose. Nuclear run-on analysis indicated that both glucose and insulin plus glucose increased GLUT-4 gene transcription (~2-fold) compared with saline-infused rats (Fig. 2). Similarly, glucose and insulin plus glucose increased HKII gene transcription about three- to fourfold compared with saline-infused rats (Fig. 2). Interestingly, both GLUT-4 and HKII gene transcription rates were unchanged when insulin concentrations were increased from 3 to 377 ng/ml (Table 3). These data suggest that, under these experimental conditions, the maximum effects of insulin on GLUT-4 and HKII transcription may be reached at physiological concentrations of insulin.

                              
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Table 3.   Gastrocnemius cAMP, plasma glucose and insulin, and gastrocnemius GLUT-4 and HKII mRNA levels in saline-, glucose-, and insulin + glucose-infused rats


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Fig. 2.   Transcriptional run-on analysis and determination of relative levels of GLUT-4 and HKII transcription from mixed skeletal muscle of saline- (Sal), glucose- (Glu), and insulin + glucose (I+G)-infused rats. A: phosphorimage showing hybridization of in vitro transcribed RNA to cDNA probes (2 µg) of interest or to rat skeletal muscle genomic DNA (0.1 µg). Hybridized transcripts were quantitated by Imagequant software. B and C: summary of values for GLUT-4 and HKII transcription (means ± SE; n = 5-6/group). * Significantly (P < 0.05) different from saline-infused rats.

Transcription of the beta -actin gene remained unchanged in all experiments (Figs. 1 and 2) and served as an internal control for comparing results between the epinephrine infusion experiments. In this study, there were no changes in GLUT-4 or HKII mRNA levels with either epinephrine or insulin infusion (Tables 1 and 3). This may be attributed to the short time frame of epinephrine and insulin infusion.

    DISCUSSION
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Abstract
Introduction
Methods
Results
Discussion
References

The first two steps of insulin-stimulated glucose disposal in skeletal muscle entail transport of glucose across the plasma membrane by GLUT-4 and subsequent phosphorylation of glucose by HKII. Glycemic control appears to be largely dependent on glucose disposal in skeletal muscle, as ~75% of a glucose load is deposited in skeletal muscle in response to the insulin-stimulated translocation of GLUT-4 to the plasma membrane (10). The importance of GLUT-4 protein in regulating glucose disposal and insulin sensitivity is indicated by research involving transgenic mice that overexpress GLUT-4 protein. Gibbs et al. (14) demonstrated that overexpression of GLUT-4 protein in skeletal muscle of genetically diabetic (db/db) mice returned plasma glucose levels to normal and improved insulin sensitivity. Their results suggest that increased expression of GLUT-4 may be a viable strategy for improving glycemic control and insulin sensitivity in certain forms of diabetes.

Cellular utilization of glucose in skeletal muscle involves the coupling of transport with phosphorylation. Recently, Chang et al. (5) reported that overexpression of HKII in skeletal muscle of transgenic mice resulted in increased basal and insulin-mediated muscle glucose uptake. Because glucose disposal in skeletal muscle is regulated by counterregulatory hormones, an understanding of whether insulin and/or epinephrine regulates in vivo GLUT-4 and HKII gene transcription becomes increasingly important.

Results from this study indicate that insulin increased transcription of the GLUT-4 gene twofold (Fig. 2), whereas epinephrine decreased GLUT-4 gene transcription by 60% (Fig. 1). These results may explain previous findings indicating that GLUT-4 mRNA and protein levels are altered by insulin and epinephrine. Fourteen days of STZ-induced diabetes decreased GLUT-4 mRNA by 35% and GLUT-4 protein by ~50% in rat skeletal muscle (13). The reduction in GLUT-4 mRNA and protein may be explained by a decrease in GLUT-4 transcription (24). Subsequent injection of insulin into STZ-diabetic rats for 7 days restored both GLUT-4 mRNA and protein to control values (13). In humans, insulin infusion increased GLUT-4 mRNA in skeletal muscle by 35% (1), but protein levels decreased (1, 14) or were unchanged (20). In contrast, other studies have reported that insulin infusion in rats has no effect on GLUT-4 mRNA levels in skeletal muscle (4, 28). Nevertheless, this study suggests that an increase in GLUT-4 transcription could account for part of the insulin-induced increase in GLUT-4 mRNA and protein levels observed in humans and STZ-diabetic rats.

Neufer and Dohm (24) previously reported that exercise induces a transient increase in GLUT-4 gene transcription, an effect that may be due to increased concentrations of plasma epinephrine. Recently, Kuo et al. (19) demonstrated that administration of a nonselective beta -receptor antagonist, propranolol, during exercise blocked the exercise-induced increase in GLUT-4 mRNA and protein. They concluded that the activation of the beta -adrenergic system is intimately involved in the increased expression of GLUT-4 protein with exercise (19). Although exercise was not a component of the present investigation, our results reveal that epinephrine infusion decreased GLUT-4 gene transcription by 60%, suggesting that an exercise-induced increase in GLUT-4 transcription may not be mediated through the beta -adrenergic system.

Cellular glucose utilization involves the coupling of transport with phosphorylation; therefore, HKII activity is intimately involved in glucose metabolism. In an experiment involving L6 cells in which glucose transport was not rate limiting, insulin-induced increases in HKII transcription, mRNA, and protein synthesis correlated with increased glucose utilization (27). In this study, insulin infusion stimulated a three- to fourfold increase in HKII gene transcription (Fig. 2). This in vivo response corresponds to the 3.8-fold increase in HKII gene transcription observed in L6 cells treated with insulin (30). Similarly, in that study, HKII mRNA and protein activity were also augmented in response to insulin treatment (30). Under euglycemic hyperinsulinemic conditions, HKII mRNA levels and protein activity in skeletal muscle of humans (21) and rats (28) are increased. This study suggests that, in conditions of increased insulin, a rise in muscle HKII mRNA could be explained, in part, by an increase in HKII gene transcription.

Epinephrine infusion increased the rate of HKII gene transcription about threefold in the skeletal muscle of these rats (Fig. 1). A similar response in HKII transcription was observed in skeletal muscle of exercised rats (25) and in L6 cells treated with cAMP analogs or catecholamines (27). In this study, hyperglycemia in the epinephrine-infused rats induced insulin secretion such that insulin values were ~2.5-fold higher than in fasted controls (Table 1). Results from our insulin infusion experiment clearly demonstrate that insulin stimulates HKII gene transcription (Fig. 2). As such, the effects of epinephrine on in vivo HKII gene transcription cannot be determined from this particular experiment.

We recognize that the exact individual contributions of epinephrine, insulin, and glucose on GLUT-4 and HKII gene transcription cannot be elucidated from this study. Indeed, there are other metabolic scenarios that suggest complex regulation of these two genes. For example, insulin levels decrease and catecholamine levels rise with prolonged fasting. However, Charron and Khan (6) demonstrated that fasting increases GLUT-4 mRNA and protein in rat skeletal muscle. We have also shown that after 3 days of fasting, GLUT-4 transcription and mRNA levels in white skeletal muscle are increased (23). Further research is needed to identify other regulators of the GLUT-4 gene that would explain this paradox.

In summary, epinephrine decreases GLUT-4 gene transcription, and insulin increases GLUT-4 and HKII gene transcription in rat skeletal muscle. We speculate that previously reported insulin-induced increases in skeletal muscle GLUT-4 and HKII mRNA and protein levels may be transcriptionally mediated.

    ACKNOWLEDGEMENTS

We acknowledge the expert technical assistance of Edward B. Tapscott, Gregory Boyd, Mary Beth Brinn, and Celeste Brown. The cDNA probes used in this study were as follows: GLUT-4, a 2.8 kb EcoR I fragment encoding the 3T3-L1 homolog of the adipose/muscle (insulin-responsive) glucose transporter protein (16); HKII, a 2.8 kb EcoR I fragment obtained from Dr. D. K. Granner, Vanderbilt University School of Medicine, Nashville, TN (2); and beta -actin, a 1.9 kb Hind III fragment obtained from Dr. D. W. Cleveland, The Johns Hopkins University, Baltimore, MD (7). The HKII riboprobe was also obtained from Dr. D. K. Granner (29).

    FOOTNOTES

This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-38416.

Address for reprint requests: J. P. Jones, East Carolina Univ. School of Medicine, Dept. of Biochemistry, Greenville, NC 27858.

Received 26 March 1997; accepted in final form 19 June 1997.

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Abstract
Introduction
Methods
Results
Discussion
References

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