Bidirectional regulation of uncoupling protein-3 and GLUT-4 mRNA in skeletal muscle by cold

Baozhen Lin, Sean Coughlin, and Paul F. Pilch

Department of Biochemistry, Boston University School of Medicine, Boston, Massachusetts 01228

    ABSTRACT
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Abstract
Introduction
Procedures
Results
Discussion
References

To elucidate the possible role of the mitochondrial uncoupling protein (UCP)-3 in skeletal muscle as a regulator of adaptive thermogenesis and energy balance, we examined the modulation by cold exposure (5°C) of UCP-3 and glucose transporter isoform GLUT-4 mRNAs in male Sprague-Dawley rats. In skeletal muscle, UCP-3 and GLUT-4 mRNAs increased two- to threefold between 6 and 24 h of cold exposure and then decreased to 50% of the control value after 6 days in the cold. In contrast, skeletal muscle UCP-2 mRNA showed a small increase on day 3 and returned to normal after 6 days. The bidirectional regulation of UCP-3 and GLUT-4 mRNAs in skeletal muscle by cold suggests that UCP-3 may be a major mediator of acute adaptive thermogenesis but then is downregulated, along with GLUT-4, in the chronic state to preserve energy. In contrast, cold exposure caused only transient changes of UCP-2 and GLUT-4 mRNA in heart. These data are consistent with the necessity of the heart to continuously expend energy to maintain blood circulation, regardless of environmental conditions.

uncoupling protein; thermogenesis; energy balance; glucose utilization; heart

    INTRODUCTION
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Abstract
Introduction
Procedures
Results
Discussion
References

ADAPTIVE THERMOGENESIS, defined as a major component of energy expenditure, is regulated by environmental stimuli such as cold exposure or chronic dietary excess (16, 36). This process has been implicated in the regulation of body temperature, body weight, and metabolism. In rodents, brown adipose tissue (BAT) plays a major role in thermogenesis in response to cold via the action of the BAT-specific mitochondrial uncoupling protein (UCP), UCP-1 (16), as has been known for some time. UCP-1 is thought to mediate transport of fatty acid across the inner mitochondrial membrane, and the fatty acids then become protonated and diffuse back into the mitochondrial matrix. By this mechanism, UCP-1 promotes dissipation of the proton and electrochemical gradients across the inner mitochondrial membrane (12, 18). This futile cycle leads to the uncoupling of oxidation from respiration and to the generation of heat without synthesis of ATP (18, 26, 33). Recently, two new uncoupling proteins have been cloned, UCP-2 (11, 13, 31) and UCP-3 (5, 31, 39). In the rat, UCP-2 mRNA is expressed in a wide variety of tissues, whereas UCP-3 mRNA is predominantly expressed in skeletal muscle but can be detected at low levels in fat and heart (14, 31, 39). Because skeletal muscle is the largest organ in the body by mass, it would seem reasonable that UCP-3 would play a role in energy expenditure there, and, in fact, UCP-3 has been shown to be upregulated by thyroid hormone and by nutritional state (4, 14, 29, 31, 32). Curiously, fasting upregulates UCP-3 mRNA expression, whereas refeeding downregulates it (4, 32). Another environmental condition that might be expected to regulate UCP-3 is cold exposure, but there are conflicting reports stating, on the one hand, no effect of this condition on UCP-3 mRNA expression (4) and, on the other hand, cold-dependent UCP-3 mRNA downregulation (29).

In addition to its probable role in energy balance via UCP-3, skeletal muscle utilizes the major nutrient glucose in a tightly regulated insulin-dependent process. In response to this hormone, the muscle (and fat)-specific glucose transporter, GLUT-4, is translocated to the cell surface where it functions to clear glucose from the blood for immediate energy use or for storage as muscle glycogen. The transporter translocation process is being extensively studied (reviewed in Ref. 20), and it has been shown that at least two other "nutritional" molecules, the transferrin receptor and the insulin-like growth factor II receptor, also traffic to the cell surface in an insulin-dependent manner, although they do not exhibit an identical subcellular distribution with GLUT-4 (9, 21, 22). Interestingly, two recently described novel proteins are coresidents with intracellular GLUT-4 vesicles: an aminopeptidase of unknown physiological function (23, 25) and sortilin (30), a presumed vesicular sorting protein (34). The aminopeptidase (insulin-responsive aminopeptidase; IRAP) is the only other known protein that traffics in an insulin-dependent manner identical to that of GLUT-4 (19, 23, 25), whereas the trafficking of sortilin is still unclear (30). Studying the modulation of IRAP and sortilin expression by condition, such as cold (and denervation, see DISCUSSION), may give clues about the nature of their membrane vesicle environment and its regulation.

To elucidate the relationship of nutritional uptake (i.e., glucose) to the regulation of energy coupling and to elucidate the possible role of UCP-3 in skeletal muscle as a regulator of adaptive thermogenesis and energy balance, we examined the effect of cold exposure on changes in muscle UCP-3 and GLUT-4 mRNAs as well as the expression of some of GLUT-4's coresident proteins. We report here that there is bidirectional and similar regulation of UCP-3 and GLUT-4 by cold exposure, and we explain the discrepant results in the literature concerning the former protein. On the other hand, other proteins coresident with GLUT-4 do not show any change in response to cold. Thus the regulation of insulin-sensitive glucose transport or the expression of coresident protein is not tightly linked to GLUT-4 expression.

    EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results
Discussion
References

Animals. Male Sprague-Dawley rats (150-175 g, 6-7 wk old) were purchased from Taconic Breeding Laboratory (New York, NY). For cold-exposure experiments, rats were either kept at 22°C or exposed individually to cold (5°C) for the indicated time. The animals had free access to water and standard rat chow (Charles River Laboratories). To obtain the different muscle fiber types, rats were killed by CO2 asphyxiation, and then individual muscle fiber groups were dissected, namely flexor digitorum superficialis, extensor digitorum lateralis, soleus, tibialis cranialis, extensor digitorum longus (EDL), peroneus longus, and flexor digitorum profundus (15). These surgical procedures were approved by the Animal Ethical Committee of Boston University School of Medicine. On the day of the study, specific tissues were taken, frozen in liquid nitrogen, and kept at -80°C until extraction of RNA and cellular membrane protein was performed.

Molecular cloning of rat UCP-3 and UCP-2 cDNA. Human UCP-3 cDNA (GenBank accession no. AF001787) was a gift of Dr. Brad Lowell (Beth Israel Hospital, Boston, MA) (39). The DNA insert of human UCP-3 was excised, labeled with [alpha -32P]dCTP (NEN Life Science Products) by random priming, and used to screen 5'-stretch cDNA library from skeletal muscle [primed with oligo(dT)+random primers, respectively] of adult male Sprague-Dawley rats (Clontech). Six 150-mm agar plates, each containing 50,000 phage plaques, were transferred to nylon filter disks (Du Pont-NEN) and hybridized to probe as described in the manufacturer's instructions. Positive plaques were picked and rescreened until a single plaque was obtained. Lambda cDNA clones were purified by Nucleobond AX (The Nest Group) and directly sequenced using lambda gt11 primers and synthetic oligonucleotide primers corresponding to the gene specific sequence. We verified that the sequence of our clone was identical to that published for rat UCP-3 (GenBank accession nos. U92069 and AB006614). The rat UCP-2 cDNA clone was isolated by PCR from a rat leg skeletal muscle cDNA library [primed with oligo(dT)+random primers, respectively, made by our laboratory]. The primers used are in positions corresponding to rat UCP-2 (GenBank accession no. AB006613), nucleotides 122-145 (primer 1) and 660-639 (primer 2).

RNA isolation and analysis. Total RNA was isolated from rat tissues as described by Chomczynski and Sacchi (6). Tissues were homogenized in solution D (4 M guanidinium thiocyanate, 25 mM sodium citrate, pH 7.0, 0.5% Sarkosyl, and 0.1 M 2-mercaptoethanol). The lysate was extracted with acidic phenol-chloroform and then subjected to an isopropanol precipitation at -20°C. Poly(A)+ RNA was selected using an oligo(dT) cellulose (type 3, Collaborative Biomedical Products) according to the manufacturer's instructions. RNA was quantitated by ultraviolet spectrophotometry. For Northern blot analysis, 40 µg of total RNA or 5 µg of poly(A)+ mRNA were separated on formaldehyde agarose gels and transferred to Gene Screen Plus (Du Pont-NEN) by capillary transfer. After ultraviolet cross-linking, filters were prehybridized, hybridized, and subjected to analysis as described previously (8). In general, hybridization was carried out overnight at 42°C in hybridization solution containing a cDNA probe with a specific radioactivity of 106-107 counts · min-1 · ml-1. For rehybridization, the probe was stripped from membranes by washing the membrane in 100°C 0.1× SSC (1× SSC is 0.15 M NaCl and 0.015 M sodium citrate, pH 7.0)-0.1% SDS two times, each wash being for 10 min. The cDNAs utilized in these studies were rat UCP-3, rat UCP-2, GLUT-4 (GenBank accession no. M23383), IRAP (GenBank accession no. U32990), and sortilin (GenBank accession no. AF023621). All probes were labeled by random priming using the Klenow fragment of DNA polymerase (Promega) and [alpha -32P]dCTP (NEN Life Science Products).

RT-PCR. Total RNA (1 µg) was reverse transcribed into cDNA by using random primer and Superscriptase II (GIBCO). PCR amplification was performed on one quarter of the RT reaction using an Expand Long Template PCR system (Boehringer Mannheim). Parallel amplifications (20, 25, and 30 cycles) of the same cDNA were used to determine the optimum number of cycles. After 25 and 30 cycles, a readily detectable signal in the linear range was obtained.

Preparation of total cellular membranes and Western blot analyses. The total cellular membrane was isolated from muscle as described by Coderre et al. (7). Briefly, muscle tissue was homogenized in a buffer containing 20 mM HEPES, 250 mM sucrose, 2 mM EDTA, 1 µM leupeptin, 1 µM pepstatin, and 1 µM aprotinin A, pH 7.4, at 4°C. The homogenate was centrifuged at 2,000 g for 10 min at 4°C. The supernatant was saved and centrifuged again at 9,000 g for 20 min. The resulting supernatant was then centrifuged at 180,000 g for 90 min at 4°C. The resulting cellular membrane-enriched pellet was resuspended in PBS-1% Triton X-100, and the protein content was determined with a bicinchoninic acid kit (BCA protein assay kit, Pierce) according to the manufacturer's instructions. The membrane protein was then subjected to SDS-PAGE (28) and transferred to an Immobilon membrane. The membranes were incubated with either a mouse monoclonal anti-GLUT-4 antibody (1F8) (17) or a mouse monoclonal anti-transferrin receptor antibody (Zymed Laboratory). The antigen-antibody complexes were visualized with peroxidase-conjugated secondary antibodies (Sigma) and an enhanced chemiluminescent substrate kit (Pierce).

    RESULTS
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Abstract
Introduction
Procedures
Results
Discussion
References

As described in EXPERIMENTAL PROCEDURES, we cloned rat UCP-3 cDNA (GenBank accession no. AF035943) and obtained the exact sequence published by Matsuda et al. (31) (GenBank accession no. AB006614) during the course of our studies. The rat UCP-2 cDNA clone was isolated by PCR from a rat hindlimb skeletal muscle cDNA library with primers designed from rat UCP-2 cDNA sequence (GenBank accession no. AB006613). To further characterize UCP-3 and UCP-2 mRNA expression, their tissue distribution in rats was examined by Northern blot analyses as shown in Fig. 1. As shown in Fig. 1A and as expected, UCP-3 message produces a very strong signal in muscle and weak signals in white fat and heart. UCP-2 mRNA is highly expressed in lung, fat, and heart (Fig. 1). With longer exposure, UCP-2 mRNA is detected in muscle and kidney as well (data not shown). GLUT-4 mRNA shows the same tissue distribution as UCP-3, but the magnitude of the signal varies from that of UCP-3 in that heart and fat both show strong GLUT-4 expression, as is well known (2). On the other hand, the two proteins showing complete (IRAP) or substantial (sortilin) intracellular localization with GLUT-4 exhibit much wider tissue distribution, although both are expressed to a moderate or substantial degree in muscle, heart, and fat. In Fig. 1B, the distribution of UCP-3 and GLUT-4 mRNA in muscle fiber types is shown. UCP-3 message is most strongly expressed in the soleus muscle (lane 3), a red muscle rich in mitochondria, and is expressed to a much lesser degree in EDL (lane 5). Similarly, GLUT-4 mRNA is also most strongly expressed in this type of muscle fiber.


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Fig. 1.   Expression patterns of uncoupling protein (UCP)-3 by Northern blot. A: poly(A)+ RNA from male Sprague-Dawley rat tissues was isolated as described in EXPERIMENTAL PROCEDURES. After electrophoresis in a 1% formaldehyde agarose gel and transfer to a nylon membrane, RNA was hybridized with specific probes. Each lane contained 5 µg of poly(A)+ pooled from 2 rats. As a control, UCP-2 and major components of glucose transporter isoform GLUT-4-containing vesicles [GLUT-4, insulin-responsive aminopeptidase (IRAP), and sortilin] were also analyzed on same blot. B: total RNA was isolated from different muscle fiber groups (40 µg each) and analyzed by Northern blot. Each muscle fiber group from 2 rats was pooled. Lane 1, flexor digitorum superficialis; lane 2, extensor digitorum lateralis; lane 3, soleus; lane 4, tibialis cranialis; lane 5, extensor digitorum longus (EDL); lane 6, peroneus longus; lane 7, flexor digitorum profundus. Blot was hybridized with a UCP-3 probe and then stripped and reprobed with GLUT-4 as a control.

The effect of cold exposure on this same group of mRNAs is shown in Fig. 2. In Fig. 2A, it can be seen that UCP-3 message expression rises after 6 h of cold exposure to a maximum at about day 1. It then decreases to 50% of the control value after 6 days. GLUT-4 mRNA shows a similar bidirectional regulation in response to cold as does UCP-3, although GLUT-4 mRNA shows its highest expression at 3 h, which may be caused by the combined stimulation of cold and shivering (muscle contraction). However, UCP-2 mRNA shows a small, delayed increase and peaks at day 3 in skeletal muscle in response to cold. We also measured the effects of cold on the mRNA for two proteins, IRAP and sortilin, that normally reside with GLUT-4 in intracellular membranes in the absence of insulin (19, 25, 30). These mRNAs show essentially no change due to cold exposure. The consistency of mRNA loading was verified by rRNA staining (data not shown) as well as the fact that neither sortilin nor IRAP mRNA showed any marked changes (Fig. 2A). Figure 2B shows that the bidirectional pattern of UCP-3 mRNA expression due to cold exposure is true for two specific muscles, the EDL and the soleus, as well as for the remaining muscle fibers after dissection of the former two muscle types. UCP-3 message expression is maximal at 1 day of cold exposure for all the muscles examined. Thus the effect of cold is a general one on most or all muscle fiber types.


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Fig. 2.   Time course of stimulation of UCP-3 in skeletal muscle by cold exposure. A: male rats were either kept at 22°C or exposed individually to cold (5°C) for indicated time during which they had free access to chow and water. Total RNA (40 µg) was isolated from leg skeletal muscle and was analyzed by Northern blot. For each time point, 2 rats were used. Each lane contains total RNA from a single rat. A Northern blot was probed successively for UCP-3, UCP-2, GLUT-4, IRAP, and sortilin. Quantitative analysis of radioactivity was analyzed in an InstantIMAGER (Packard). Left: representative blots. Right: quantitative mRNA levels (average from 2 rats). B: total RNA was isolated from EDL, soleus, and remaining muscles (muscle after dissection of EDL and soleus) of cold-exposed rats for indicated time points. Total RNA (40 µg) was analyzed by Northern blot. For each time point, 2 rats were used. At each time, EDL, soleus, and remaining muscles were taken from a single rat. A representative blot is shown. d, Day(s).

We also examined the effect of cold on cardiac muscle gene expression, as shown in Fig. 3. The level of UCP-3 mRNA is very low in this tissue, so RT-PCR was used for more sensitive detection of UCP-3 expression. There is little if any change of cardiac UCP-3 mRNA during 1 day of cold exposure. As a control for the PCR analysis, we measured IRAP mRNA expression by both Northern blotting and PCR. Again, the data show little change due to cold, although there may be a slight (30%) increase at the 3-h time point. On the other hand, GLUT-4 mRNA expression does transiently increase at 3 and 6 h of cold exposure but then returns to basal after 24 h, the same pattern as observed for UCP-2 mRNA. As a control, sortilin mRNA in heart again shows little or no change on cold exposure.


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Fig. 3.   Time course of stimulation of UCP-3 in heart by cold exposure. Total RNA (40 µg) was isolated from hearts of cold-exposed rats for indicated time points and analyzed by Northern blot where indicated. As shown at right, top 2 panels (UCP-3 and IRAP) were analyzed by RT-PCR (30 cycles). IRAP mRNA was detected by both methods as a control. Total RNA (1 µg) was used for RT, and one quarter of RT reaction was used for PCR. For each time point, 2 rats were analyzed. Each lane contains RNA from a single rat. A representative blot is shown.

Figures 1-3 show mRNA expression, but it is the proteins that are mediating the actual metabolic changes. Unfortunately, we do not yet have anti-UCP-3 antisera, but, in Fig. 4, we measured GLUT-4 and transferrin receptor protein expression in skeletal muscle as a function of cold exposure. As expected from Fig. 1, GLUT-4 protein in skeletal muscle decreases with time (i.e., 3 and 6 days) in the cold, whereas the transferrin receptor protein is unchanged.


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Fig. 4.   Western blot analysis of GLUT-4 expression in skeletal muscle by cold exposure. Total membrane protein was isolated from skeletal muscle of cold-exposed rats for indicated time points, and Western blots were performed as described in EXPERIMENTAL PROCEDURES. Protein (100 µg) was analyzed by SDS-PAGE and stained for GLUT-4 and transferrin receptor by using peroxidase-conjugated secondary antibodies and enhanced chemiluminescent substrate kit. For each time point, 2 rats were used. Each lane contains protein from a single rat. Consistency of protein loading was verified by Coomassie staining of gel. A representative blot is shown.

    DISCUSSION
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Abstract
Introduction
Procedures
Results
Discussion
References

Our intention in the present study was to determine whether a condition known to affect energy utilization and thermogenesis, namely cold exposure, would regulate in a similar or identical matter two genes potentially involved in these processes, UCP-3 and GLUT-4, that are expressed in the same tissues in a similar but nonidentical fashion. In skeletal muscle, where the greatest mass of both these proteins is found, their expression in response to cold was very similar. In the first day of cold exposure, their expression increased, particularly that of UCP-3, presumably a thermoregulatory response necessary to warm the muscle to the initial shock of cold. Muscle contraction is known to stimulate GLUT-4 gene expression (35). Thus the rapid increase of GLUT-4 mRNA level at 3 h of cold exposure is most probably due to the activation of shivering thermogenesis in the skeletal muscles. Then, during cold acclimation, the mechanism of heat production progressively changes from shivering to nonshivering thermogenesis (24). As the animals adapt to prolonged cold exposure, they downregulate both UCP-3 and GLUT-4 mRNA levels as well as GLUT-4 protein level to preserve energy, presumably consistent with their altered metabolic state. The fact that UCP-1 knockout animals are unable to tolerate cold (10) and our own data suggest that UCP-3 expression in muscle is normally tied more closely to the regulation of nutrient utilization (e.g., glucose metabolism) than it is to thermoregulation in its chronic functional state. If thermoregulation, i.e., heat production, were the primary role of muscle UCP-3, then prolonged cold would more likely upregulate expression of this gene. Thus the downregulation of UCP-3 mRNA expression in muscle under prolonged cold exposure provides further evidence that the physiological functions of UCPs are very important in regulating energy balance (27). Our data also clarify the conflicting data in the literature, in which one group concluded there was no change in UCP-3 mRNA after 2 days of cold exposure (4), whereas another group showed a substantial decrease after 10 days of cold exposure (29). Our data indicate that both studies are correct, but they missed the interesting time point at 1 day of cold exposure, at which time UCP-3 mRNA expression shows a dramatic but transient increase.

The observed similar regulation of UCP-3 and GLUT-4 mRNAs due to chronic cold is in good agreement with previous reports that glucose uptake, synthesis, and translocation of GLUT-4 were elevated in L6 and 3T3-L1 cells by treatment of chemical uncouplers such as 2,4-dinitrophenol (1, 38). Together with our finding, it is tempting to speculate that uncoupler as well as UCP-3, abundantly expressed in skeletal muscle, may also contribute to regulation of glucose metabolism. We also demonstrate that UCP-2 mRNA shows a small increase at day 3 of cold exposure in skeletal muscle. This stimulatory effect of cold exposure on skeletal muscle UCP-2 mRNA in the rat was also found after 2 days of cold exposure by Boss et al. (3), but a higher induced level in soleus or no change in tibialis anterior and gastrocnemius muscles of UCP-2 mRNA was shown in that paper. This discrepancy between our and their results might be due to differences in muscle fibers or in the duration of cold exposure. Taken together, our data indicate that skeletal muscle is the major site of thermogenesis in response to acute cold exposure via the action of UCP-3. On the other hand, all of the genes we examined showed no change or transient change (e.g., returning to basal after 24 h) in cardiac muscle (Fig. 3), a result in good agreement with the necessary function of heart, an organ in which one would not expect major changes, particularly increases, in UCP expression that could result in lower energy efficiency, a likely pathological condition.

Another issue the current study addresses is the possible role of GLUT-4 levels on the expression of its companion proteins that reside in its intracellular compartments. It is not yet clear whether the membrane compartments harboring GLUT-4 represent something unique or a more general trafficking compartment (20), and, if the former situation applies, one might expect common regulation of its constituents. Thus, whereas acute and prolonged cold exposure alters GLUT-4 mRNA and protein expression, it is without substantial effect on the expression of sortilin and IRAP mRNAs (Fig. 2) and transferrin receptor protein (Fig. 4), and these proteins partially [transferrin receptor (9), sortilin (30)] or completely [IRAP (19, 25)] colocalize with GLUT-4 in intracellular vesicles. These data complement similar data on muscle denervation (40) in which GLUT-4 expression also substantially decreases without any change in the expression of IRAP and the transferrin receptor. On the other hand, the tissue distributions of sortilin and IRAP are much wider than that of GLUT-4, so they are not specific to the insulin-regulated vesicles. Furthermore, it was shown that IRAP is also present in the preadipocyte as well as myoblast (unpublished data). Therefore, IRAP may be a ubiquitous marker for a specialized regulated secretory vesicle as well as GLUT-4-containing vesicles. Together, these results tend to support the hypothesis that changes in GLUT-4 expression itself are not essential to its membrane environment or trafficking pattern (see also Ref. 37, in which the same conclusion applies to GLUT-4 overexpression on adipocytes).

    ACKNOWLEDGEMENTS

We are grateful to Dr. K. V. Kandror for advice and discussion and to Dr. Brad Lowell for providing us with the human UCP-3 cDNA.

    FOOTNOTES

This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-30425, DK-36424 (both to P. F. Pilch), and DK-49147 (to N. Ruderman).

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. F. Pilch, Dept. of Biochemistry, Boston Univ. School of Medicine, 715 Albany St., Boston, MA 02118.

Received 22 January 1998; accepted in final form 12 May 1998.

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Abstract
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Discussion
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Am J Physiol Endocrinol Metab 275(3):E386-E391
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