Leptin induction of UCP1 gene expression is dependent on sympathetic innervation

Philip J. Scarpace and Michael Matheny

Geriatric Research, Education and Clinical Center, Department of Veterans Affairs Medical Center, Gainesville 32608; and Department of Pharmacology and Therapeutics, University of Florida College of Medicine, Gainesville, Florida 32610

    ABSTRACT
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Abstract
Introduction
Methods
Results
Discussion
References

We previously demonstrated that leptin increases uncoupling protein 1 (UCP1) and lipoprotein lipase (LPL) gene expression in brown adipose tissue (BAT) of rats. To determine whether the induction of these transcripts is dependent on sympathetic innervation of BAT, we unilaterally surgically denervated interscapular BAT in both pair-fed and leptin (0.9 mg/day by infusion)-treated rats. In pair-fed rats, the level of UCP1 mRNA in the denervated BAT pad was 30-47% less than in the innervated pad. In the intact BAT pad, leptin administration increased UCP1 mRNA levels by nearly 2.5-fold compared with pair-fed rats. In contrast, in the denervated BAT pad, there was no increase in UCP1 gene expression. When LPL mRNA was examined in pair-fed rats, there was no difference between innervated and denervated BAT pads. With leptin administration, LPL gene expression increased by 75% in both the innervated and denervated BAT pads. beta 3-Adrenergic receptor mRNA was unaffected by either denervation or leptin, whereas uncoupling protein 2 mRNA levels were increased in epididymal white adipose tissue (WAT) but not in perirenal WAT. CGP-12177, a specific beta 3-adrenergic receptor agonist, induced nearly a fourfold increase in UCP1 and a twofold increase in LPL gene expression in both the innervated and denervated BAT pads. These data indicate that the leptin induction of UCP1 gene expression in BAT is dependent on sympathetic innervation but that the leptin induction of LPL gene expression is not.

brown adipose tissue; lipoprotein lipase; uncoupling protein 2; beta 3-adrenergic agonist

    INTRODUCTION
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Abstract
Introduction
Methods
Results
Discussion
References

LEPTIN, the product of the ob gene, is one factor involved in body weight maintenance, and this hormone contributes to the regulation of both food intake and energy expenditure (5, 14, 21, 25). The mechanism of increased energy expenditure appears to involve increased thermogenesis in brown adipose tissue (BAT) (25). Leptin increases sympathetic nerve activity and norepinephrine turnover in BAT (7, 15). We previously reported that leptin administration increases energy expenditure, including increases in whole body oxygen consumption and uncoupling protein 1 (UCP1) gene expression in BAT (25).

Thermogenesis in BAT is primarily mediated by sympathetically innervated beta 3-adrenergic receptors (beta 3ARs) (23). The beta 3AR demonstrates downregulation after stimulation, and both cold exposure and beta 3-adrenergic agonist administration diminish beta 3AR mRNA levels (13). This downregulation of beta 3AR mRNA after cold exposure can be prevented by surgical dissection of the sympathetic nerves of interscapular BAT, suggesting that sympathetic nerve activity can regulate beta 3AR gene expression (13).

Another enzyme, the induction of which in BAT is apparently regulated by beta 3ARs, is lipoprotein lipase (LPL) (31). This enzyme is involved in the assimilation of triglycerides into both BAT and white adipose tissue (WAT) by hydrolyzing lipoprotein triglycerides before their importation into adipocytes (31). LPL gene expression has a unique pattern of regulation; mRNA levels are upregulated by beta -adrenergic stimulation in BAT but downregulated in WAT (1, 28). We previously demonstrated that leptin administration increases the gene expression of LPL in BAT but not in WAT (26).

Collectively, these data suggest that the mechanism by which leptin induces both UCP1 and LPL is through sympathetic activation of beta 3ARs. In addition, because cold-induced sympathetic activation of BAT downregulates beta 3AR mRNA levels, leptin may also modulate beta 3AR mRNA levels. To test whether the induction of these transcripts is dependent on sympathetic innervation of BAT, we unilaterally surgically denervated interscapular BAT followed by either a single leptin injection or a 3-day leptin infusion. We examined UCP1, LPL, and beta 3AR gene expression in the innervated and denervated BAT pads as well as leptin, uncoupling protein 2 (UCP2), and LPL mRNA levels in perirenal WAT (PWAT) and epididymal WAT (EWAT) from pair-fed and leptin-treated rats.

    METHODS
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Abstract
Introduction
Methods
Results
Discussion
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Animals. Six-month-old male F-344 × Brown Norway rats were obtained from Harlan Sprague Dawley (Indianapolis, IN). Rats were examined on arrival and remained in quarantine for 1 wk. Animals were cared for in accordance with the principles of the National Research Council Guide for the Care and Use of Laboratory Animals. Rats were housed individually in microisolated cages with a 12:12-h light-dark cycle (light from 0700 to 1900). Ambient temperature was 26°C, which was thermoneutrality for these rats (24).

Surgical denervation. Rats underwent unilateral surgical denervation of the interscapular BAT under pentobarbital anesthesia according to the method of Bartness et al. (2). A transverse incision was made just anterior to the BAT, separating the BAT from the muscles of the scapulae. The BAT was raised to expose the five intercostal nerve bundles entering each pad. On one side, a section of each nerve bundle was removed with scissors. The rats were maintained on a heat pad until recovery from the anesthesia. Experiments were begun on the 4th day after unilateral denervation. In previous studies, denervation was verified in selected rats by assessing norepinephrine levels in the innervated compared with denervated BAT pads (22). In all tested cases the denervation was successful (22).

Leptin administration. Rats were administered either saline or mouse leptin by a single intraperitoneal injection (1 mg leptin/rat), and killed 5 h later, or by mini pump (0.9 mg leptin/day) for 3 days. Osmotic mini pumps (model 1003D, Alzet, Palo Alto, CA), three per rat, were implanted subcutaneously along the back of the rats. In the 3-day experiment, control rats were pair fed to the amount of food consumed by the leptin-treated rats. The latter were allowed access to food ad libitum. Rats were killed on the 3rd day after mini pump implantation.

Chemicals. Mouse leptin was supplied by Amgen (Thousand Oaks, CA). All other chemicals were obtained from Sigma Chemical (St. Louis, MO).

Tissue harvesting. Rats were killed by cervical dislocation under 85 mg/kg pentobarbital anesthetic. Blood samples were collected by heart puncture, and serum was harvested by a 30-min centrifugation in serum separator tubes. The circulatory system was perfused with 20 ml of cold saline, and BAT, EWAT, and PWAT were excised.

Leptin radioimmunoassay. Serum leptin levels were measured with a rat leptin radioimmunoassay kit (Linco Research, St. Charles, MO).

mRNA levels. Total cellular RNA was extracted using a modification of the method of Chomczynski and Sacchi (6). The integrity of the isolated RNA was verified using agarose gels (1%) stained with ethidium bromide. The RNA was quantified by spectrophotometric absorption at 260 nm by use of multiple dilutions of each sample.

The probe to detect leptin mRNA was a 33-mer antisense oligonucleotide (5'-GGTCTGAGGCAGGGAGCAGCTCTTGGAGAAGGC) (28) that was end-labeled using terminal deoxynucleotidyl transferase (Promega, Madison, WI). The oligonucleotide was based on a region of the mRNA downstream from the site of the primary mutation in ob/ob mice (28). We previously demonstrated by Northern analysis that this probe binds to a single mRNA species of 4.1 kb (20). The UCP2 cDNA (IMAGE 389584) was provided by Craig Warden (11), the UCP1 cDNA was provided by Leslie Kozak (17), the beta 3AR cDNA was provided by James Granneman (12), the rat pre pro neuropeptide Y (NPY) cDNA was provided by Janet Allen (University of Glasgow, Glasgow, UK), and mouse LPL and hormone-sensitive lipase cDNA clones were obtained from ATCC (Manassas, VA). The cDNAs were labeled using a randon primer kit (Prime-a-Gene, Promega, Madison, WI). For dot blot analysis, multiple concentrations of the RNA were immobilized on nylon membranes with a dot blot apparatus (Bio-Rad, Richmond, CA). The membranes were baked at 80°C for 2 h. The baked membranes were prehybridized in 10 ml Quikhyb (Stratagene, LaJolla, CA) for 20 min followed by hybridization in the presence of a labeled probe and 100 µg denatured salmon sperm DNA. After hybridization for 2 h at 65°C, the membranes were washed and exposed to a phosphorimaging screen for 48 h. The latent image was scanned using a Phosphor Imager (Molecular Dynamic, Sunnyvale, CA) and analyzed by Image Quant Software (Molecular Dynamics). Intensities per milligram of total cellular RNA were calculated by comparison with internal laboratory standards of WAT or BAT mRNA present on each nylon membrane. In some cases, nylon membranes probed for specific mRNA were stripped and rehybridized with beta -actin.

Statistical analysis. Data were analyzed by one-way or two-way ANOVA. When the main effect was significant, a post hoc test was applied to determine individual differences between means.

    RESULTS
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Abstract
Introduction
Methods
Results
Discussion
References

Leptin infusion. The interscapular BAT on one side was surgically denervated. The contralateral side was sham operated. On the 3rd day after unilateral denervation, leptin was administered by mini pump infusion for 3 days. Leptin administration for 3 days to rats maintained at thermoneutrality raised serum leptin levels by nearly 10-fold (Table 1). Leptin treatment resulted in a 30% decrease in food intake after 1 day and a 56% decrease on the 2nd and 3rd days (Table 2). The pair-fed rats were limited to the daily amount of food consumed by the leptin-treated, ad libitum-fed rats. Both leptin treatment and pair feeding resulted in a significant loss of body weight compared with prerestriction weight levels, but there was no greater loss of body weight in the leptin-administered rats compared with the pair-fed rats (Table 2).

                              
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Table 1.   Serum leptin after a single injection or 3 days of continuous infusion of leptin

                              
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Table 2.   Food consumption and body weight during 3 days of leptin infusion

UCP1 expression. We previously determined that 7-day leptin infusion increased UCP1 and LPL gene expression in BAT. To determine whether these increases were mediated by sympathetic innervation of BAT, UCP1 and LPL gene expression was examined in both the intact and denervated BAT pads. As expected, in the pair-fed rats, the level of UCP1 mRNA in the denervated BAT pad was 30% less than in the innervated pad (Fig. 1). When the denervated pad was compared with the innervated one from the same rats, the decrease in UCP1 mRNA was significant (P = 0.029, paired t-test). In the intact BAT pad, leptin administration increased UCP1 mRNA levels by nearly 2.5-fold compared with pair-fed rats (Fig. 1). In contrast, in the denervated BAT pad, there was no increase in UCP1 gene expression (Fig. 1). When LPL mRNA levels were examined, a different picture emerged. In the pair-fed rats, there was no difference in the levels of LPL mRNA between the innervated and denervated BAT pads (Fig. 2). Furthermore, with leptin administration, LPL gene expression increased in both the innervated and denervated BAT pads (Fig. 2). In contrast to BAT, leptin administration had no effect on LPL mRNA levels in either EWAT or PWAT (Table 3).


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Fig. 1.   Brown adipose tissue (BAT) uncoupling protein 1 (UCP1) mRNA levels in 3-day leptin-treated or pair-fed rats in which BAT had been unilaterally denervated 3 days earlier. Values are means ± SE of 8 rats per group and are expressed in arbitrary units per µg total RNA, with level in intact pair-fed rats set to 100 and SE adjusted proportionally. * P < 0.001 for difference with leptin or with denervation by two-way ANOVA; P = 0.002 for interaction between leptin and denervation; P = 0.001 for difference with leptin in intact BAT.


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Fig. 2.   BAT lipoprotein lipase (LPL) mRNA levels in 3-day leptin-treated or pair-fed rats in which BAT had been unilaterally denervated 3 days earlier. Values are means ± SE of 8 rats per group and are expressed in arbitrary units per µg total RNA, with level in intact pair-fed rats set to 100 and SE adjusted proportionally. * P = 0.0001 for difference with leptin by two-way ANOVA.

                              
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Table 3.   Levels of transcripts after 3-day infusion of leptin

beta 3-Adrenergic agonist administration. To verify that the denervated BAT pad is still capable of stimulation by beta -adrenergic agonists, CGP-12177, a specific beta 3AR agonist, was administered to rats in which the BAT had been unilaterally denervated. A single injection of CGP-12177 induced a nearly fourfold increase in UCP1 gene expression in both the innervated and denervated BAT pads (Fig. 3). Similarly, the beta 3-adrenergic agonist increased LPL gene expression in both the innervated and denervated BAT pads (Fig. 3). In contrast, there was no effect of the beta 3-adrenergic agonist on LPL mRNA levels in EWAT (data not shown). However, as expected, CGP-12177 diminished leptin gene expression in EWAT (100 ± 6.4 vs. 76 ± 7.3 arbitrary units per µg total RNA, P = 0.028 by one-way ANOVA). CGP-12177 administration also downregulated beta 3AR levels in BAT (100 ± 3.3 vs. 83.8 ± 4.1 arbitrary units per µg total RNA, P = 0.008). In contrast, neither denervation nor leptin administration had any effect on beta 3AR mRNA levels in either the denervated or innervated BAT pads (Table 3).


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Fig. 3.   beta 3-Adrenergic receptor agonist CGP-12177 induction of UCP1 (top) and LPL (bottom) mRNA in BAT from 3-day leptin-treated or pair-fed rats in which BAT had been unilaterally denervated. Values are means ± SE of 8 rats per group expressed in arbitrary units per µg total RNA, with level in intact pair-fed rats set to 100 and SE adjusted proportionally. * P = 0.0001 for difference with CGP-12177 by two-way ANOVA; dagger  P = 0.005 for difference with denervation by two-way ANOVA.

Leptin gene expression. We previously reported that 7-day leptin infusion downregulated leptin gene expression in WAT (26). This same phenomenon was observed in the present study after 3 days of leptin infusion. Leptin mRNA levels in both EWAT and PWAT were downregulated by 43 and 33%, respectively, after leptin administration (Fig. 4).


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Fig. 4.   Downregulation of leptin mRNA in epididymal white adipose tissue (EWAT) and perirenal WAT (PWAT) after 3-day leptin administration compared with pair-fed rats. Values are means ± SE of 7-8 rats per group expressed in arbitrary units per µg total RNA, with level in intact pair-fed rats set to 100 and SE adjusted proportionally. * P < 0.05 (EWAT) or P < 0.01 (PWAT) for difference with leptin by one-way ANOVA.

UCP2 expression. In addition to UCP1, another uncoupling protein that may be involved in energy expenditure is UCP2 (11). This transcript is found in a variety of tissues including WAT (11). After leptin administration, UCP2 mRNA levels were increased by 60% in EWAT but not in PWAT (Table 3).

Single leptin injection. Some effects of leptin are mediated by the hypothalamus, whereas others may be the result of a direct action at the target site. Because many of the effects of leptin mediated by the hypothalamus have a slow onset, we reasoned that an acute dose of peripherally administered leptin may act sooner on peripheral target sites that are the result of a direct action compared with those mediated by the hypothalamus. Five hours after a single injection of leptin, various transcript levels were examined in WAT and both innervated and denervated BAT. In both the control and leptin-treated rats, the level of UCP1 was nearly 50% less in the denervated compared with the innervated BAT (P < 0.0004 by two-way ANOVA). In the leptin-treated rats, there was a small (27%) but nonsignificant increase in UCP1 mRNA in innervated BAT (100 ± 10 vs. 127 ± 25 arbitrary units per µg RNA) that was absent in the denervated BAT (52.5 ± 7.0 vs. 54.7 ± 9.9). Furthermore, there were no significant changes in LPL gene expression after a single dose of leptin in either BAT or EWAT (data not shown).

Surprisingly, in EWAT, a tissue in which the effects of leptin may possibly be direct, there was neither a downregulation of leptin gene expression nor an induction of UCP2 gene expression (data not shown). One of the most universal observations after leptin administration is the inhibition of NPY gene expression in the hypothalamus. After the single leptin injection, NPY mRNA levels in the hypothalamus were unchanged. In contrast, after the 3-day leptin infusion, there was a significant (P < 0.001) 35% decrease in NPY mRNA levels (data not shown).

    DISCUSSION
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Abstract
Introduction
Methods
Results
Discussion
References

Leptin contributes to both the negative regulation of food intake and the positive regulation of energy expenditure (14, 21, 25). We previously reported that leptin administration increases thermogenesis in BAT, including increases in oxygen consumption and UCP1 gene expression (25). Thermogenesis in BAT is mediated by norepinephrine activation of sympathetically innervated beta 3ARs (24). The beta 3-adrenergic signal transduction pathway serves both to activate BAT mitochondrial UCP1 and to induce new synthesis of this protein (30). Sympathetic innervation of BAT is necessary to maintain levels of UCP1 (8), and denervation of BAT prevents the cold-induced increase in BAT hyperplasia and UCP1 (9). Activated UCP1 uncouples mitochondria, allowing high rates of substrate oxidation and heat production without phosphorylation of adenosine 5'-diphosphate (16).

Leptin increases sympathetic nerve activity to BAT (15) as well as norepinephrine turnover in BAT (7). These reports, coupled with our previous report indicating a leptin-induced increase in UCP1 mRNA, suggest that leptin increases sympathetic activation of the beta 3-adrenergic pathway, leading to an increase in thermogenesis in BAT. The present study confirms this by demonstrating that surgical denervation of BAT prevents the leptin-induced increase in UCP1 gene expression. This finding is further supported by a recent report indicating that leptin does not increase UCP1 gene expression when applied directly to isolated adipocytes (27). In the present study, the lack of response to leptin in the denervated BAT pad was not due to surgically induced damage, because both the innervated and denervated BAT pads responded equally to the CGP-12177 induction of UCP1 gene expression. In addition, the beta 3-adrenergic agonist, as expected, diminished leptin gene expression in PWAT and EWAT. These latter findings were similar to our previous report (20) and suggest that the denervation of BAT did not disrupt the beta 3-adrenergic regulation of leptin gene expression in WAT.

Along with UCP1, LPL is another gene that is induced by beta -adrenergic stimulation of BAT (28). However, in contrast to the findings with UCP1 gene expression, surgical denervation of BAT did not prevent the leptin-induced increase in LPL mRNA levels. Leptin increased LPL gene expression equally in both the denervated and innervated BAT pads. LPL gene expression is believed to be regulated by beta -adrenergic stimulation in BAT but not in WAT (1). Moreover, the observation that cold exposure, a condition that increases sympathetic activation of BAT, upregulates LPL mRNA (28) suggests that LPL gene expression is regulated by sympathetic nerve activity. In the present study, we confirmed that beta 3-adrenergic administration increases LPL gene expression in BAT but not in WAT and that leptin, independent of sympathetic activation and presumably independent of beta 3ARs, upregulates LPL gene expression. These data suggest that leptin may act directly on BAT or possibly through another intermediate to increase LPL mRNA. One known stimulator of LPL gene expression is insulin. However, in our previous study, insulin levels were unchanged by a 7-day leptin infusion (26), suggesting against a role for insulin in the leptin-induced upregulation of LPL mRNA. A recent report investigated the direct effects of leptin on isolated brown adipocytes (27). Leptin increased the gene expression of LPL but not UCP1 after a 24-h exposure (27). These data corroborate our findings and suggest that the induction of LPL gene expression by leptin, in vivo, is mediated by a direct action on BAT.

In addition to UCP1 and LPL gene expression, the present report examined the effect of leptin administration on another transcript that may be involved in energy balance, UCP2. This uncoupling protein has 59% homology with UCP1 and 73% homology with uncoupling protein 3 (11, 29). Like UCP1, UCP2 can partially uncouple mitochondrial respiration (11). The expression of UCP2, unlike UCP1, is not limited to BAT, and this protein is widely expressed in many tissues, including WAT, heart, and muscle in both rodents and humans (11). Although the biological role of this uncoupling protein is not fully understood, some evidence suggests a role for this protein in energy balance and thermogenesis. In UCP1-deficient mice, UCP2 expression in BAT is upregulated, possibly contributing to the surprising absence of obesity in these mice (10). In another study, cold exposure upregulated the expression of UCP2 in BAT (3). In addition, in rats in which the leptin gene was overexpressed, UCP2 expression was increased in WAT (31). Our previous report indicated that 7-day leptin infusion induced an increase in UCP2 expression in EWAT but not in PWAT or BAT (26). Similar to our previous study, the present study demonstrates that a 3-day leptin infusion increases the expression of UCP2 in EWAT but not in PWAT. These data suggest a potential role for UCP2 in both thermoregulation and energy balance; however, the lack of response in selected tissues in previous studies (11, 30) and in BAT and PWAT in our studies suggests that the role for UCP2 is not clearly defined.

In our previous report, we demonstrated that the leptin-induced reduction in food intake resulted in the expected decrease in leptin gene expression in WAT (26). In addition, there was a leptin-induced downregulation of leptin gene expression independent of food consumption (26). Similarly, in the present report, after 3 days of leptin infusion, leptin mRNA levels were downregulated in PWAT and EWAT compared with pair-fed rats, thus confirming a leptin-induced, food-independent downregulation. Because of the presence of leptin receptors in WAT (19), the downregulation of leptin mRNA may be a result of a direct action of leptin on WAT. We expected that a single dose of leptin would be effective on tissues that were the result of a direct action of leptin compared with those actions that were mediated by the hypothalamus, such as the UCP1 gene expression in BAT. Surprisingly, the single dose of leptin was without effect on any of the transcripts investigated, including UCP1, UCP2, leptin, or NPY gene expression in the hypothalamus. This suggests that both the downregulation of leptin gene expression in WAT and the induction of LPL in BAT may not be due to a direct action of leptin on these tissues or that the response to leptin, whether mediated by central or peripheral leptin receptors, is universally slow.

The present study, as well as other reports, has demonstrated that administration of a beta 3-adrenergic agonist downregulates beta 3AR mRNA in BAT (13, 18). Surprisingly, beta 3AR mRNA levels were unchanged in BAT by either denervation, 3-day leptin infusion, or the 7-day leptin infusion from our previous study (26). Cold exposure, a strong stimulus for sympathetic activation of BAT, downregulates beta 3AR mRNA, and this downregulation was prevented by denervation (13). This suggests that the leptin-induced increase in sympathetic activation of BAT in the present study must be insufficient to downregulate beta 3AR mRNA levels or that any downregulation is being offset by other regulatory agents. Another study reported that leptin did not regulate beta 3AR mRNA in wild type C57BL/6J mice, even though beta 3AR mRNA was upregulated in ob/ob mice (4).

In summary, this study indicates that 3-day leptin infusion increases the gene expression of UCP1 and LPL in BAT, as well as that of UCP2 in EWAT, but does not regulate beta 3AR mRNA levels in BAT. In addition, leptin downregulates its own synthesis in WAT independently of food intake. Furthermore, the leptin-induced increase in UCP1 mRNA in BAT is dependent on sympathetic activation, whereas the increase in LPL gene expression is not. These data suggest that one mechanism by which leptin increases energy expenditure is through increased UCP2 expression in WAT and increased sympathetic activation of UCP1 gene expression in BAT. In addition, leptin upregulates LPL gene expression in BAT, although that mechanism does not involve sympathetic activation.

    ACKNOWLEDGEMENTS

This study was supported by the Medical Research Service of the Department of Veterans Affairs and National Institute on Aging Grant AG-11465.

    FOOTNOTES

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: P. J. Scarpace, Geriatric Research, Education and Clinical Center (182), Dept. of Veterans Affairs Medical Center, Gainesville, FL 32608-1197.

Received 24 February 1998; accepted in final form 22 April 1998.

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Abstract
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Results
Discussion
References

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