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Regulated Expression of 5′-Deleted Mouse GLUT4 Minigenes in Transgenic Mice: Effects of Exercise Training and High-Fat Diet
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS ARTICLE NO.
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Regulated Expression of 5*-Deleted Mouse GLUT4 Minigenes in Transgenic Mice: Effects of Exercise Training and High-Fat Diet Nobuyo Tsunoda,* David W. Cooke,† Shinji Ikemoto,* Kayo Maruyama,* Mayumi Takahashi,* M. Daniel Lane,‡ and Osamu Ezaki*,1 *Division of Clinical Nutrition, National Institute of Health and Nutrition, 1-23-1, Toyama, Shinjuku-ku, Tokyo 162, Japan; and Department of †Pediatrics and Department of ‡Biological Chemistry, The Johns Hopkins University School of Medicine, 725 North Wolfe Street, Baltimore, Maryland 21205
Received September 8, 1997
Fourteen kb murine GLUT4 minigene (Å 07395 GLUT4) contains DNA sequence that confers tissue specific, exercise-induced up-regulation of the GLUT4 gene in skeletal muscle and high-fat diet induceddown-regulation in white adipose tissue. To identify the DNA sequences required for regulated expression, we generated GLUT4 minigene transgenic mice harboring 3237, 2000, 1000, and 442 bp of 5*-flanking region, all exons and introns, and 1 kb of 3 *-flanking sequence of the mouse GLUT4 gene. The 03237-, 02000-, and 01000-GLUT4 constructs were expressed in a tissue-specific manner identical to the endogenous GLUT4. Exercise-induced up-regulation and high-fat diet-induced down-regulation of these constructs also paralleled those of the endogenous GLUT4 gene. In contrast, the 0442 GLUT4 construct was expressed substantially in skeletal muscle (gastrocnemius and quadriceps) and heart, but was only expressed very weakly in white adipose tissue and was not expressed in brown adipose tissue. Furthermore, this 0442 GLUT4 construct failed to respond to exercise or a high-fat diet in either muscle or adipose tissue. These results indicate that brown and white adipocyte-specific enhancer(s) and exercise- and high-fat diet-responsive elements are located between bases 01000 and 0442 of the murine GLUT4 5*-flanking region. q 1997 Academic Press
1 To whom correspondence should be addressed: Osamu Ezaki, M.D., Division of Clinical Nutrition, National Institute of Health and Nutrition, 1-23-1, Toyama, Shinjuku-ku, Tokyo 162, Japan. Fax: 813-3207-3520. E-mail: [email protected]. Abbreviations: GLUT4, muscle/adipocyte insulin-responsive glucose transporter; CAT, chloramphenicol acetyltransferase; MEF2, myocyte enhancer factor 2; LA-PCR, long and accurate polymerase chain reaction; BAT, brown adipose tissue; WAT, white adipose tissue.
GLUT4, the insulin-responsive glucose transporter, which is found in heart, skeletal muscles, and adipose tissues, plays an important role in whole body glucose homeostasis (1). This transporter is responsible for the acute stimulation of glucose uptake by insulin that occurs only in these tissues (2,3). This acute regulation of GLUT4 by insulin involves the rapid translocation of the transporter from an intracellular site to the plasma membrane (4,5). In addition to acute regulation of GLUT4 activity by insulin, expression of the GLUT4 gene is hormonally and metabolically regulated. For example, fasting (6-8), high-fat feeding (9,10), and obesity (11,12) lead to a decrease of GLUT4 mRNA in WAT, while streptozotocin-induced diabetes (13-15) results in a decrease of GLUT4 mRNA in both skeletal muscles and WAT. On the other hand, exercise training (16,17), and T3 administration (18,19) increase GLUT4 mRNA levels in skeletal muscles. Previously, we produced murine GLUT4 minigene transgenic mice that expressed a 1.3 to 2-fold higher level of GLUT4 protein than endogenous GLUT4 in the appropriate tissues: heart and skeletal muscle, BAT, and WAT (20, 21). This GLUT4 minigene contains 7395 bp of 5*-flanking sequence, all exons and introns, and 1 kb of 3*-flanking sequence of the GLUT4 gene, as well as a segment of foreign DNA (281 bp of the CAT gene) inserted in the 3*-untranslated region for transcript identification (20). In these transgenic mice, exercise training increased expression of GLUT4 mRNA derived from the minigene as well as the endogenous gene, and led to a further improvement of glycemic control (21). Furthermore, feeding a high-fat diet decreased expression of GLUT4 mRNA derived from both the minigene and the endogenous gene in WAT, but had no effect on expression of either the minigene or the endogenous gene in muscle (22). These findings
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indicated that the cis-regulatory element(s) controlling exercise-induced up-regulation (in muscle and WAT) and high-fat diet-induced down-regulation (in WAT) of the GLUT4 gene is located within the nucleotide sequence encompassed by the 14 kb of the GLUT4 minigene. In addition, modest increases in GLUT4 expression in muscle prevented the hyperglycemia induced by a high-fat diet (22), showing that pharmacological regulation of GLUT4 expression may be a useful approach in the treatment of diabetes. Olson and Pessin elucidated the location of tissue specific and insulin-responsive elements in the human GLUT4 promoter using the human GLUT4 gene promoter ligated to the CAT reporter gene (23). However, when human DNA is used for producing transgenic mice, species differences might affect transgene expression and regulation. Therefore, we have produced transgenic mice expressing murine GLUT4 minigenes containing 5* deletions of the GLUT4 5*-flanking region. We have investigated the tissue specific expression and the effects of exercise training and high-fat diet feeding on the expression of these GLUT4 minigenes. EXPERIMENTAL PROCEDURES Plasmid Construction
or The Johns Hopkins University School of Medicine Transgenic Mouse Core Facility (Baltimore, MD).
Transgenic Mice Identification of transgenic mice harboring the GLUT4 minigenes and estimation of the minigene copy number were made by Southern blot (25) using the 281 bp CAT tag as probe as described previously (20-22). RNase protection analysis utilized an antisence RNA probe that generates protected fragments of 433 and 399 bp corresponding to the minigene GLUT4 transcript and of 182 bp corresponding to the endogenous GLUT4 transcript (20). RNA transcript was quantified with a BAS2000 image analyzer (Fuji). Exercise training. Mice at 5-7 weeks of age were exercise trained by forced swimming in plastic barrels filled with warm water at 357C as described previously (21). Mice swam for 30-min periods for four times daily separated by 10-min rest periods. Swimming was continued for 3 weeks. Eighteen to 22 hr after the terminal exercise session, gastrocnemius, parametrial (female) and epididymal (male) WAT were isolated by rapid dissection and homogenized in guanidine, and RNA was isolated by centrifugation through a CsCl cushion (26). Mice were allowed free access to laboratory mouse chow (with 11%, 60%, and 29% of total calories from fat, carbohydrate, and protein, respectively) and water. High-fat diet. Beginning at 8 to 14 weeks of age, mice were fed either a high-carbohydrate diet or a high-fat diet. The ingredients of the diets have been described previously (22). Prior to this, all mice were fed laboratory mouse chow. The diets were continued for 3 months, after which the animals were sacrificed and RNA was isolated as described above.
RESULTS
The 5*-deletion GLUT4 minigenes were derived from the 14 kb GLUT4 minigene containing 7395 bp of GLUT4 5*-flanking DNA (24) with a ‘‘tag’’ consisting of 281 bp of foreign DNA (20) and were constructed in several steps as follows. (a) The pGEM-3Z vector (Promega) was digested with EcoRI and HindIII, the ends were filled in by T4 DNA polymerase, and a double stranded DNA oligonucleotide -GGTCGACGGATCCCCCGGGGGATCCGTCGACC-, was ligated into the plasmid, giving rise to pGEM-3ZX, containing the new polylinker site -SalI-BamHI-SmaI-BamHI-SalI-. (b) To make the 03237 GLUT4 minigene, the 14 kb GLUT4 minigene (Å07395) was digested with SalI and BamHI to delete 4158 bps from its 5* region. This insert was blunted and ligated into the SmaI site of pGEM-3ZX. The 02000, 01000, and 0442 GLUT4 minigene constructs were generated by LA-PCR kit Ver. 2 (Takara) using the 14 kb GLUT4 minigene construct as a template, Takara LA Taq polymerase, and the following primers: 5* primer(complementary to sequence in the murine GLUT4 5*-flanking region), 5*-CGCATC-GGATCC(BamHI)-AGGAATTAACCTCAGATCTACA-3* for 02000, 5*-CGCATC-GGATCC(BamHI)GGTTAATAGAAGAGAGCCACCC-3* for 01000, 5*-CGCATC-GGATCC(BamHI)-GGGAACTAAAAATAGCCACTCC-3* for 0442; 3* primer (complementary to sequence in the murine GLUT4 3*-flanking region), 5*-CGCATC-GGATCC(BamHI)-CTGCAGGTCAACGGATCAGTGG-3* for each construct. These primers had 6 bp for efficient restriction enzyme digestion, 6 bp of BamHI site, and 22 bp of sequence complementary to the template. The PCR products were digested with BamHI and subcloned into the BamHI site of pGEM3ZX. Both ends of each construct were sequenced up to 700-800 bps. (c) After large scale propagation of these plasmids harboring the constructs, the plasmids were digested by SalI and the linearized minigene constructs were isolated by agarose gel electrophoresis, followed by electroelution. These minigene constructs were further purified by NACS cartridge (GIBCO BRL) and used for microinjection into the pronucleous of fertilized mouse embryos at either the CHRYSALIS DNX, Inc. Transgenic Animal Sciences (Princeton, NJ)
To identify the DNA sequences responsible for tissuespecific, exercise-induced up-regulation and high-fat diet-induced down-regulation of the mouse GLUT4 gene, several lines of GLUT4 minigene transgenic mice were produced from constructs that contained various lengths of 5*-flanking DNA (Fig. 1). Since expression of the GLUT4 minigene could be affected by the construct copy number and the genomic integration site in the mouse genome, at least two independent founder lines per construct were analyzed in the following experiments, with no substantial differences seen between founder lines, except as noted below for the 0442 construct. The expression pattern in various tissues of the minigene and endogenous GLUT4 from each construct was determined by RNase protection analysis (Fig. 2 and 3). Since the 0442-GLUT4 construct displayed an altered tissue specific expression pattern, results from two independent founder mice lines of the 0442GLUT4 construct are shown (Fig. 2 E and F). Autoradiograms of RNase protection assays from 01000- and 0442-GLUT4 minigene transgenic mice are shown in Fig. 3. Data from heterozygous female transgenic mice are shown. In WAT, the 07395-, 03237, 02000, and 01000-GLUT4 constructs expressed about 50% lower levels in male mice than in female mice, but this difference was not observed in 0442-GLUT4 mice. The 07395-(Å14kb GLUT4) and 03237-GLUT4 constructs
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FIG. 1. (A) Structure of the 14-kb mouse GLUT4 minigene (Å 07395) with a ‘‘tag’’ consisting of 281 bp of foreign DNA (derived from the bacterial CAT gene) inserted at the EcoRI site in the 3* untranslated region. Solid and open boxes indicate the coding and noncoding exon sequences, respectively. Hatched segment indicates the tag sequence. (B) The constructs used to generate the 5*-deleted GLUT4 minigene transgenic mice are shown. The transcription start site of the GLUT4 gene is indicated by the arrow. Nucleotide sequence for each construct is indicated relative to the transcription start site as /1. B, BamHI; E, EcoRI; H, HindIII; Sa, Sal I.
were expressed at substantial levels in skeletal muscles, heart and BAT, but at a relatively low level in WAT. The minigene expression of these constructs in skeletal and heart muscle and BAT was 3 to 5- fold higher than endogenous GLUT4 expression, while in WAT, the minigene expression was only 60 to 80% of the endogenous GLUT4 expression (based on the relative signal from the RNAse protection assay). In contrast, the 02000- and 01000-GLUT4 minigenes were expressed at relatively high levels in all insulin sensitive tissues: skeletal and heart muscle, WAT and BAT. Minigene expression of these constructs was 3 to 5- fold higher than endogenous GLUT4 expression in these tissues. The 0442 GLUT4 minigene was expressed at substantial levels in skeletal muscle (gastrocnemius and quadriceps) and heart, but its expression was very weak in WAT and it was not expressed in BAT. Thus, the 0442 GLUT4 minigene contains a skeletal and heart muscle-specific enhancer(s). A WAT-specific enhancer(s) is present between bases 01000 and 0442, and a weak WAT-specific repressor(s) is located between bases 03237 and 02000. A strong BAT-specific enhancer(s) is present between bases 01000 and 0442. One of the 0442 GLUT4 mice lines (Fig. 2 F) showed significant expression in brain, which does not express endogenous GLUT4. This may be due to its integration site, since the other 0442 GLUT4 mice line (Fig. 2 E) did not show expression in brain, and since aberrant expression of the GLUT4 minigene was occasionally seen in liver, brain, or lung with other minigene constructs.
In each transgenic mouse line, three-weeks of exercise training resulted in a 1.3 to 2.0-fold increase in endogenous GLUT4 mRNA levels in gastrocnemius (Fig. 4). Expression of the 03237-, 02000-, and 01000GLUT4 minigenes was also increased in gastrocnemius, by 1.5 to 2.3-fold. In contrast, exercise training did not increase the expression of the 0442-GLUT4 minigene. These changes of minigene and endogenous GLUT4 mRNA were also observed in WAT. The data of male mice are shown here, but the response was similar in female mice. In comparison with high-carbohydrate feeding, three-months of high-fat feeding resulted in a 50-70% decrease of endogenous GLUT4 mRNA in WAT from each transgenic mouse line (Fig. 5). Since high-fat feeding did not decrease endogenous GLUT4 levels in skeletal muscles, skeletal muscles were not examined. The transgenic mice harboring the 03237, 02000, and 01000-GLUT4 minigenes all displayed 60-85% decreases of minigene GLUT4 mRNA in WAT with highfat feeding, which paralleled that of the endogenous GLUT4 gene. However, expression of the 0442-GLUT4 minigene did not decrease in mice fed a high-fat diet. As previously shown for the 07395-GLUT4 minigene (22), all of these GLUT4 minigene transgenic mice showed improved glycemic control in response to an oral glucose load when fed either a high-carbohydrate or a high-fat diet, consistent with their increased GLUT4 expression in skeletal muscles (data not shown). These data indicated that BAT- and WAT-specific enhancer(s), and exercise-and high-fat diet-responsive elements are located between bases 01000 and 0442 of the murine GLUT4 5*-flanking region. DISCUSSION Previously, we have shown that a 14 kb GLUT4 minigene, which contains 7395 bp of 5*-flanking sequence, all exons and introns, and 1 kb of 3*-flanking sequence of the GLUT4 gene, exhibits tissue-specific expression (21). Furthermore, this 14 kb GLUT4 minigene showed differentiation-induced expression in 3T3-L1 preadipocytes (20), cAMP-induced down-regulation (27), exercise-induced up-regulation (21), and high-fat diet-induced down-regulation (22). The deletion analysis reported here shows that the murine 0442 GLUT4 minigene harboring 442 bp of 5*-flanking DNA possesses a skeletal and heart muscle-specific enhancer, but lacks a BAT and WAT-specific enhancer(s). The 0422 GLUT4 minigene also lacks sequence element(s) responsible for exercise and high-fat diet induced regulation. The 0442 GLUT4 minigene does, however, contain an MEF2 binding site (-CTAAAAATAG-) located at 0437 to 0428, which was necessary for muscle specific expression in C2C12 cells (24, 28). Nevertheless,
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FIG. 2. Tissue-specific expression of GLUT4 mRNA derived from minigene and endogenous GLUT4 in female heterozygous transgenic mice. Five mg of total RNA isolated from gastrocnemius, quadriceps, parametrial WAT, liver, spleen, kidney, heart, lung, intrascapler BAT, and brain from 07395-(A), 03237-(B), 02000-(C), 01000-(D), 0442-(E and F) GLUT4 mice was used to quantitate minigene and endogenous GLUT4 mRNA using RNase protection assay as described under ‘‘Experimental Procedures.’’ Size of the open and solid boxes indicates radioactivity levels of endogenous and minigene GLUT4 protected fragments, respectively. Since minigene GLUT4 protected fragments (433 and 399 bp) have about 2-fold radioactivity greater than that of endogenous GLUT4 protected fragments (182 bp), actual mRNA levels of minigene GLUT4 was about half of its radioactivity in comparison with endogenous GLUT4. The expression levels of minigene and endogenous GLUT4 were expressed as a percentage of that of endogenous GLUT4 from gastrocnemius. Minigene GLUT4 copy numbers are shown in parenthesis. 506
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ever, expression of the murine 0442 GLUT4 construct, though not maximal, was similar in heart and skeletal muscle (Fig. 3). The reason for the difference in expression in heart of the murine 0422 GLUT4 minigene compared to the 0730 human GLUT4-CAT reporter gene is not known at this time. It may be due to sequence elements in the introns and/or 3*-flanking region of the murine GLUT4 gene, or to differences between the human and murine GLUT4 5*-flanking region. The construction of the 5*-deleted GLUT4 minigenes used in this study relied on the recent development of LA-PCR technology (29). Unlike Taq DNA polymerase, LA-polymerase possesses 3* to 5* exonuclease proofreading activity that enables the polymerase to correct nucleotide-misincorporation errors, giving an error rate of only one mutation per 10 kb (30). Sequence
FIG. 3. Expression of minigene and endogenous GLUT4 mRNAs in transgenic mice harboring 01000 GLUT4 minigene (A) and 0442 GLUT4 minigene (B). RNA isolated from the indicated tissues of female heterozygous transgenic mice from 01000 GLUT4 (Fig. 2 D) and 0442 GLUT4 (Fig. 2 E) was subjected to RNase protection analysis as described under ‘‘Experimental Procedures.’’ The 433-bp and 399-bp protected fragment corresponds to GLUT4 minigene message and the 182-bp protected fragment corresponds to endogenous GLUT4. Several bands observed between 399 bp and 182 bp appear to be degraded fragments of the minigene-protected fragment.
in comparison with the larger constructs, two independent founder 0442 GLUT4 mice lines showed a lower expression of the GLUT4 minigene in skeletal muscles and heart, indicating that another element besides MEF2 may be required for maximal expression of GLUT4 in skeletal muscle and heart. These data are consistent with data from the rat GLUT4 gene that showed that MEF2 sequence was essential for myotube-specific expression in C2C12 cells but was not sufficient for its full expression (28). Olson and Pessin reported that 730 bp of the human GLUT4 5*-flanking DNA harboring a MEF2 binding site was sufficient to direct relatively high expression in skeletal muscles but not in heart or WAT (23). How-
FIG. 4. Regulation of minigene and endogenous GLUT4 mRNA in gastrocnemius from male exercise-trained mice. A: Typical autoradiogram. Heterozygous male transgenic mice were divided into two groups, one subjected to the exercise protocol (/) and the other not (0). RNA isolated from gastrocnemius skeletal muscle was subjected to RNase protection assays. The 433-bp and 399-bp protected fragment corresponds to GLUT4 minigene message and the 182-bp protected fragment corresponds to endogenous GLUT4. B: The minigene and endogenous GLUT4 expression levels were quantified by image analyzer and expressed by relative percent to endogenous GLUT4 in gastrocnemius from nonexercised controls. Each data point is mean { standard error of the mean of 2-4 mice (shown in parenthesis).
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quence identity within this proximal 1000 bps upstream of the transcription start site (31), which suggests that hormonal/metabolic regulatory elements are clustered in this region. The present data indicate that BAT- and WAT-specific enhancer(s), and exercise- and high-fat diet-responsive elements are located between bases 01000 and 0442 of the murine GLUT4 5*-flanking region. Olson et al. have shown that this region of the human GLUT4 gene mediates the regulation of GLUT4 expression in streptozotocin-induced diabetes (23). It will be interesting to see if any of these responses are mediated through the same cis-elements or to learn how their trans-acting factors interact. ACKNOWLEDGMENTS We are grateful to M. Koda and M. Kurumiya for excellent technical assistance. This work was supported by research grants from the National Institutes of Health, National Institute of Diabetes and Digestive and Kidney Diseases (Washington, DC), the Japanese Ministry of Health and Welfare, the Japanese Ministry of Education, Science and Culture (Tokyo), and the Japanese Science and Technology Agency (Tokyo).
REFERENCES FIG. 5. Regulation of minigene and endogenous GLUT4 mRNA in white adipose tissues from female high-fat diet fed mice. A: Typical autoradiogram. Heterozygous female transgenic mice were divided into two groups, one subjected to a high-carbohydrate feeding protocol (C) and the other to a high-fat feeding protocol (F). RNA isolated from parametrial WAT was subjected to RNase protection assays. The 433-bp and 399-bp protected fragment corresponds to GLUT4 minigene message and the 182-bp protected fragment corresponds to endogenous GLUT4. B: The minigene and endogenous GLUT4 expression levels were quantified by image analyzer and expressed by relative percent to endogenous GLUT4 in WAT from carbohydrate-fed controls. Each data point is mean { standard error of the mean of 3-4 mice (shown in parenthesis).
analysis of the promoter region of the 0442 GLUT4 minigene identified a single A to G substitution at 0373 bp. While it is possible this PCR introduced mutation caused the loss of exercise and diet-induced regulation in the 0442 GLUT4 minigene, it is much more likely that this loss of regulation is due to the deletion from 01000 to 0442 bp. Furthermore, since all the transgenes produced functional GLUT4 (all transgenic mice had improved glucose tolerance compared to nontransgenic mice), significant errors in the coding region or intron splice sites are unlikely. Given the extremely low error rate of LA-polymerase, it is unlikely that a PCR-introduced mutation in the coding region, introns, or 3*-untranslated region caused a loss of regulation seen in the 0442 construct. Further studies will result in definitive identification of the response element (s). There is substantial identity between the mouse and human GLUT4 gene, with the greatest degree of se-
1. Kahn, B. B. (1996) Diabetes 45, 1644–1654. 2. Garvey, W. T. (1992) Diabetes Care 15, 396–417. 3. Pessin, J. E., and Bell, G. I. (1992) Annu. Rev. Physiol. 54, 911– 930. 4. Suzuki, K., and Kono, T (1980) Proc. Natl. Acad. Sci. USA 77, 2542–2545. 5. Cushman, S. W., and Wardzala, L. J. (1980) J. Biol. Chem. 255, 4758–4762. 6. Charron, M. J., and Kahn, B. B. (1990) J. Biol. Chem. 265, 7994– 8000. 7. Sivitz, W. I., DeSautel, S. L., Kayano, T., Bell, G. I., and Pessin, J. E. (1989) Nature 340, 72–74. 8. Berger, J., Biswas, C., Vicario, P. P., Strout, H. V., Saperstein, R., and Pilch, P. F. (1989) Nature 340, 70–72. 9. Kahn, B. B. (1994) J. Nutr. 124, 1289S–1295S. 10. Leturque, A., Postic, C., Ferre, P., and Girard, J. (1991) Am. J. Physiol. 260, E588–E593. 11. Garvey, W. T., Maianu, L., Huecksteadt, T. P., Birnbaum, M. J., Molina, J. M., and Ciaraldi, T. P. (1991) J. Clin. Invest. 87, 1072– 1081. 12. Pedersen, O., Kahn, C. R., and Kahn, B. B. (1992) J. Clin. Invest. 89, 1964–1973. 13. Napoli, R., Hirshman, M. F., and Horton, E. S. (1995) J. Clin. Invest. 96, 427–437. 14. Kahn, B. B., Rossetti, L., Lodish, H. F., and Charron, M. J. (1991) J. Clin. Invest. 87, 2197–2206. 15. Garvey, W. T., Huecksteadt, T. P., and Birnbaum, M. J. (1989) Science 245, 60–63. 16. Wake, S. A., Sowden, J. A., Storlien, L. H., James, D. E., Clark, P. W., Shine, J., Chisholm, D. J., and Kraegen, E . W. (1991) Diabetes 40, 275–279. 17. Ploug, T., Stallknecht, B. M., Pedersen, O., Kahn, B. B., Ohkuwa , T., Vinten, J., and Galbo, H. (1990) Am. J. Physiol. 259, E778– E786.
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18. Weinstein, S. P., O’Boyle, E., and Haber, R. S. (1994) Diabetes 43, 1185–1189. 19. Shimokawa, T., Kato, M., Shioduka, K., Irie, J., and Ezaki, O. (1997) Biochem. Biophys. Res. Commmun. 235, 790–793. 20. Ezaki, O., Flores-Riveros, J. R., Kaestner, K. H., Gearhart, J., and Lane, M. D. (1993) Proc. Natl. Acad. Sci. USA 90, 3348– 3352. 21. Ikemoto, S., Thompson, K. S., Itakura, H., Lane, M. D., and Ezaki, O. (1995) Proc. Natl. Acad. Sci. USA 92, 865–869. 22. Ikemoto, S., Thompson, K. S., Takahashi, M., Itakura, H., Lane, M. D., and Ezaki, O. (1995) Proc. Natl. Acad. Sci. USA 92, 3096– 3099. 23. Olson, A. L., and Pessin, J. E. (1995) J. Biol. Chem. 270, 23491– 23495.
24. Kaestner, K. H., Christy, R. J., and Lane, M. D. (1990) Proc. Natl. Acad. Sci. USA 87, 251–255. 25. Southern, E. M. (1975) J. Mol. Biol. 98, 503–517. 26. Chirgwin, J. M., Przybyla, A. E., MacDonald, R. J., and Rutter, W. J. (1979) Biochemistry 18, 5294–5299. 27. Kaestner, K. H., Flores-Riveros, J. R., MacLenithan, J. C., Janicot, M., and Lane, M. D. (1991) Proc. Natl. Acad. Sci. USA 88, 1933–1937. 28. Liu, M.-L., Olson, A. L., Edgington, N. P., Moye-Rowley, W. S., and Pessin, J. E. (1994) J. Biol. Chem. 269, 28514–28521. 29. Barnes, W. M. (1994) Proc. Natl. Acad. Sci. USA 91, 2216–2220. 30. Capuano, V., Galleron, N., Pujic, P., Sorokin, A., and Ehrlich, S. D. (1996) Microbiology 142, 3005–3015. 31. Olson, A. L., Edgington, N. P., Moye-Rowley, W. S., and Pessin, J. E. (1995) Endocrinology 136, 1962–1968.
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Regulated Expression of 5*-Deleted Mouse GLUT4 Minigenes in Transgenic Mice: Effects of Exercise Training and High-Fat Diet Nobuyo Tsunoda,* David W. Cooke,† Shinji Ikemoto,* Kayo Maruyama,* Mayumi Takahashi,* M. Daniel Lane,‡ and Osamu Ezaki*,1 *Division of Clinical Nutrition, National Institute of Health and Nutrition, 1-23-1, Toyama, Shinjuku-ku, Tokyo 162, Japan; and Department of †Pediatrics and Department of ‡Biological Chemistry, The Johns Hopkins University School of Medicine, 725 North Wolfe Street, Baltimore, Maryland 21205
Received September 8, 1997
Fourteen kb murine GLUT4 minigene (Å 07395 GLUT4) contains DNA sequence that confers tissue specific, exercise-induced up-regulation of the GLUT4 gene in skeletal muscle and high-fat diet induceddown-regulation in white adipose tissue. To identify the DNA sequences required for regulated expression, we generated GLUT4 minigene transgenic mice harboring 3237, 2000, 1000, and 442 bp of 5*-flanking region, all exons and introns, and 1 kb of 3 *-flanking sequence of the mouse GLUT4 gene. The 03237-, 02000-, and 01000-GLUT4 constructs were expressed in a tissue-specific manner identical to the endogenous GLUT4. Exercise-induced up-regulation and high-fat diet-induced down-regulation of these constructs also paralleled those of the endogenous GLUT4 gene. In contrast, the 0442 GLUT4 construct was expressed substantially in skeletal muscle (gastrocnemius and quadriceps) and heart, but was only expressed very weakly in white adipose tissue and was not expressed in brown adipose tissue. Furthermore, this 0442 GLUT4 construct failed to respond to exercise or a high-fat diet in either muscle or adipose tissue. These results indicate that brown and white adipocyte-specific enhancer(s) and exercise- and high-fat diet-responsive elements are located between bases 01000 and 0442 of the murine GLUT4 5*-flanking region. q 1997 Academic Press
1 To whom correspondence should be addressed: Osamu Ezaki, M.D., Division of Clinical Nutrition, National Institute of Health and Nutrition, 1-23-1, Toyama, Shinjuku-ku, Tokyo 162, Japan. Fax: 813-3207-3520. E-mail: [email protected]. Abbreviations: GLUT4, muscle/adipocyte insulin-responsive glucose transporter; CAT, chloramphenicol acetyltransferase; MEF2, myocyte enhancer factor 2; LA-PCR, long and accurate polymerase chain reaction; BAT, brown adipose tissue; WAT, white adipose tissue.
GLUT4, the insulin-responsive glucose transporter, which is found in heart, skeletal muscles, and adipose tissues, plays an important role in whole body glucose homeostasis (1). This transporter is responsible for the acute stimulation of glucose uptake by insulin that occurs only in these tissues (2,3). This acute regulation of GLUT4 by insulin involves the rapid translocation of the transporter from an intracellular site to the plasma membrane (4,5). In addition to acute regulation of GLUT4 activity by insulin, expression of the GLUT4 gene is hormonally and metabolically regulated. For example, fasting (6-8), high-fat feeding (9,10), and obesity (11,12) lead to a decrease of GLUT4 mRNA in WAT, while streptozotocin-induced diabetes (13-15) results in a decrease of GLUT4 mRNA in both skeletal muscles and WAT. On the other hand, exercise training (16,17), and T3 administration (18,19) increase GLUT4 mRNA levels in skeletal muscles. Previously, we produced murine GLUT4 minigene transgenic mice that expressed a 1.3 to 2-fold higher level of GLUT4 protein than endogenous GLUT4 in the appropriate tissues: heart and skeletal muscle, BAT, and WAT (20, 21). This GLUT4 minigene contains 7395 bp of 5*-flanking sequence, all exons and introns, and 1 kb of 3*-flanking sequence of the GLUT4 gene, as well as a segment of foreign DNA (281 bp of the CAT gene) inserted in the 3*-untranslated region for transcript identification (20). In these transgenic mice, exercise training increased expression of GLUT4 mRNA derived from the minigene as well as the endogenous gene, and led to a further improvement of glycemic control (21). Furthermore, feeding a high-fat diet decreased expression of GLUT4 mRNA derived from both the minigene and the endogenous gene in WAT, but had no effect on expression of either the minigene or the endogenous gene in muscle (22). These findings
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indicated that the cis-regulatory element(s) controlling exercise-induced up-regulation (in muscle and WAT) and high-fat diet-induced down-regulation (in WAT) of the GLUT4 gene is located within the nucleotide sequence encompassed by the 14 kb of the GLUT4 minigene. In addition, modest increases in GLUT4 expression in muscle prevented the hyperglycemia induced by a high-fat diet (22), showing that pharmacological regulation of GLUT4 expression may be a useful approach in the treatment of diabetes. Olson and Pessin elucidated the location of tissue specific and insulin-responsive elements in the human GLUT4 promoter using the human GLUT4 gene promoter ligated to the CAT reporter gene (23). However, when human DNA is used for producing transgenic mice, species differences might affect transgene expression and regulation. Therefore, we have produced transgenic mice expressing murine GLUT4 minigenes containing 5* deletions of the GLUT4 5*-flanking region. We have investigated the tissue specific expression and the effects of exercise training and high-fat diet feeding on the expression of these GLUT4 minigenes. EXPERIMENTAL PROCEDURES Plasmid Construction
or The Johns Hopkins University School of Medicine Transgenic Mouse Core Facility (Baltimore, MD).
Transgenic Mice Identification of transgenic mice harboring the GLUT4 minigenes and estimation of the minigene copy number were made by Southern blot (25) using the 281 bp CAT tag as probe as described previously (20-22). RNase protection analysis utilized an antisence RNA probe that generates protected fragments of 433 and 399 bp corresponding to the minigene GLUT4 transcript and of 182 bp corresponding to the endogenous GLUT4 transcript (20). RNA transcript was quantified with a BAS2000 image analyzer (Fuji). Exercise training. Mice at 5-7 weeks of age were exercise trained by forced swimming in plastic barrels filled with warm water at 357C as described previously (21). Mice swam for 30-min periods for four times daily separated by 10-min rest periods. Swimming was continued for 3 weeks. Eighteen to 22 hr after the terminal exercise session, gastrocnemius, parametrial (female) and epididymal (male) WAT were isolated by rapid dissection and homogenized in guanidine, and RNA was isolated by centrifugation through a CsCl cushion (26). Mice were allowed free access to laboratory mouse chow (with 11%, 60%, and 29% of total calories from fat, carbohydrate, and protein, respectively) and water. High-fat diet. Beginning at 8 to 14 weeks of age, mice were fed either a high-carbohydrate diet or a high-fat diet. The ingredients of the diets have been described previously (22). Prior to this, all mice were fed laboratory mouse chow. The diets were continued for 3 months, after which the animals were sacrificed and RNA was isolated as described above.
RESULTS
The 5*-deletion GLUT4 minigenes were derived from the 14 kb GLUT4 minigene containing 7395 bp of GLUT4 5*-flanking DNA (24) with a ‘‘tag’’ consisting of 281 bp of foreign DNA (20) and were constructed in several steps as follows. (a) The pGEM-3Z vector (Promega) was digested with EcoRI and HindIII, the ends were filled in by T4 DNA polymerase, and a double stranded DNA oligonucleotide -GGTCGACGGATCCCCCGGGGGATCCGTCGACC-, was ligated into the plasmid, giving rise to pGEM-3ZX, containing the new polylinker site -SalI-BamHI-SmaI-BamHI-SalI-. (b) To make the 03237 GLUT4 minigene, the 14 kb GLUT4 minigene (Å07395) was digested with SalI and BamHI to delete 4158 bps from its 5* region. This insert was blunted and ligated into the SmaI site of pGEM-3ZX. The 02000, 01000, and 0442 GLUT4 minigene constructs were generated by LA-PCR kit Ver. 2 (Takara) using the 14 kb GLUT4 minigene construct as a template, Takara LA Taq polymerase, and the following primers: 5* primer(complementary to sequence in the murine GLUT4 5*-flanking region), 5*-CGCATC-GGATCC(BamHI)-AGGAATTAACCTCAGATCTACA-3* for 02000, 5*-CGCATC-GGATCC(BamHI)GGTTAATAGAAGAGAGCCACCC-3* for 01000, 5*-CGCATC-GGATCC(BamHI)-GGGAACTAAAAATAGCCACTCC-3* for 0442; 3* primer (complementary to sequence in the murine GLUT4 3*-flanking region), 5*-CGCATC-GGATCC(BamHI)-CTGCAGGTCAACGGATCAGTGG-3* for each construct. These primers had 6 bp for efficient restriction enzyme digestion, 6 bp of BamHI site, and 22 bp of sequence complementary to the template. The PCR products were digested with BamHI and subcloned into the BamHI site of pGEM3ZX. Both ends of each construct were sequenced up to 700-800 bps. (c) After large scale propagation of these plasmids harboring the constructs, the plasmids were digested by SalI and the linearized minigene constructs were isolated by agarose gel electrophoresis, followed by electroelution. These minigene constructs were further purified by NACS cartridge (GIBCO BRL) and used for microinjection into the pronucleous of fertilized mouse embryos at either the CHRYSALIS DNX, Inc. Transgenic Animal Sciences (Princeton, NJ)
To identify the DNA sequences responsible for tissuespecific, exercise-induced up-regulation and high-fat diet-induced down-regulation of the mouse GLUT4 gene, several lines of GLUT4 minigene transgenic mice were produced from constructs that contained various lengths of 5*-flanking DNA (Fig. 1). Since expression of the GLUT4 minigene could be affected by the construct copy number and the genomic integration site in the mouse genome, at least two independent founder lines per construct were analyzed in the following experiments, with no substantial differences seen between founder lines, except as noted below for the 0442 construct. The expression pattern in various tissues of the minigene and endogenous GLUT4 from each construct was determined by RNase protection analysis (Fig. 2 and 3). Since the 0442-GLUT4 construct displayed an altered tissue specific expression pattern, results from two independent founder mice lines of the 0442GLUT4 construct are shown (Fig. 2 E and F). Autoradiograms of RNase protection assays from 01000- and 0442-GLUT4 minigene transgenic mice are shown in Fig. 3. Data from heterozygous female transgenic mice are shown. In WAT, the 07395-, 03237, 02000, and 01000-GLUT4 constructs expressed about 50% lower levels in male mice than in female mice, but this difference was not observed in 0442-GLUT4 mice. The 07395-(Å14kb GLUT4) and 03237-GLUT4 constructs
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FIG. 1. (A) Structure of the 14-kb mouse GLUT4 minigene (Å 07395) with a ‘‘tag’’ consisting of 281 bp of foreign DNA (derived from the bacterial CAT gene) inserted at the EcoRI site in the 3* untranslated region. Solid and open boxes indicate the coding and noncoding exon sequences, respectively. Hatched segment indicates the tag sequence. (B) The constructs used to generate the 5*-deleted GLUT4 minigene transgenic mice are shown. The transcription start site of the GLUT4 gene is indicated by the arrow. Nucleotide sequence for each construct is indicated relative to the transcription start site as /1. B, BamHI; E, EcoRI; H, HindIII; Sa, Sal I.
were expressed at substantial levels in skeletal muscles, heart and BAT, but at a relatively low level in WAT. The minigene expression of these constructs in skeletal and heart muscle and BAT was 3 to 5- fold higher than endogenous GLUT4 expression, while in WAT, the minigene expression was only 60 to 80% of the endogenous GLUT4 expression (based on the relative signal from the RNAse protection assay). In contrast, the 02000- and 01000-GLUT4 minigenes were expressed at relatively high levels in all insulin sensitive tissues: skeletal and heart muscle, WAT and BAT. Minigene expression of these constructs was 3 to 5- fold higher than endogenous GLUT4 expression in these tissues. The 0442 GLUT4 minigene was expressed at substantial levels in skeletal muscle (gastrocnemius and quadriceps) and heart, but its expression was very weak in WAT and it was not expressed in BAT. Thus, the 0442 GLUT4 minigene contains a skeletal and heart muscle-specific enhancer(s). A WAT-specific enhancer(s) is present between bases 01000 and 0442, and a weak WAT-specific repressor(s) is located between bases 03237 and 02000. A strong BAT-specific enhancer(s) is present between bases 01000 and 0442. One of the 0442 GLUT4 mice lines (Fig. 2 F) showed significant expression in brain, which does not express endogenous GLUT4. This may be due to its integration site, since the other 0442 GLUT4 mice line (Fig. 2 E) did not show expression in brain, and since aberrant expression of the GLUT4 minigene was occasionally seen in liver, brain, or lung with other minigene constructs.
In each transgenic mouse line, three-weeks of exercise training resulted in a 1.3 to 2.0-fold increase in endogenous GLUT4 mRNA levels in gastrocnemius (Fig. 4). Expression of the 03237-, 02000-, and 01000GLUT4 minigenes was also increased in gastrocnemius, by 1.5 to 2.3-fold. In contrast, exercise training did not increase the expression of the 0442-GLUT4 minigene. These changes of minigene and endogenous GLUT4 mRNA were also observed in WAT. The data of male mice are shown here, but the response was similar in female mice. In comparison with high-carbohydrate feeding, three-months of high-fat feeding resulted in a 50-70% decrease of endogenous GLUT4 mRNA in WAT from each transgenic mouse line (Fig. 5). Since high-fat feeding did not decrease endogenous GLUT4 levels in skeletal muscles, skeletal muscles were not examined. The transgenic mice harboring the 03237, 02000, and 01000-GLUT4 minigenes all displayed 60-85% decreases of minigene GLUT4 mRNA in WAT with highfat feeding, which paralleled that of the endogenous GLUT4 gene. However, expression of the 0442-GLUT4 minigene did not decrease in mice fed a high-fat diet. As previously shown for the 07395-GLUT4 minigene (22), all of these GLUT4 minigene transgenic mice showed improved glycemic control in response to an oral glucose load when fed either a high-carbohydrate or a high-fat diet, consistent with their increased GLUT4 expression in skeletal muscles (data not shown). These data indicated that BAT- and WAT-specific enhancer(s), and exercise-and high-fat diet-responsive elements are located between bases 01000 and 0442 of the murine GLUT4 5*-flanking region. DISCUSSION Previously, we have shown that a 14 kb GLUT4 minigene, which contains 7395 bp of 5*-flanking sequence, all exons and introns, and 1 kb of 3*-flanking sequence of the GLUT4 gene, exhibits tissue-specific expression (21). Furthermore, this 14 kb GLUT4 minigene showed differentiation-induced expression in 3T3-L1 preadipocytes (20), cAMP-induced down-regulation (27), exercise-induced up-regulation (21), and high-fat diet-induced down-regulation (22). The deletion analysis reported here shows that the murine 0442 GLUT4 minigene harboring 442 bp of 5*-flanking DNA possesses a skeletal and heart muscle-specific enhancer, but lacks a BAT and WAT-specific enhancer(s). The 0422 GLUT4 minigene also lacks sequence element(s) responsible for exercise and high-fat diet induced regulation. The 0442 GLUT4 minigene does, however, contain an MEF2 binding site (-CTAAAAATAG-) located at 0437 to 0428, which was necessary for muscle specific expression in C2C12 cells (24, 28). Nevertheless,
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FIG. 2. Tissue-specific expression of GLUT4 mRNA derived from minigene and endogenous GLUT4 in female heterozygous transgenic mice. Five mg of total RNA isolated from gastrocnemius, quadriceps, parametrial WAT, liver, spleen, kidney, heart, lung, intrascapler BAT, and brain from 07395-(A), 03237-(B), 02000-(C), 01000-(D), 0442-(E and F) GLUT4 mice was used to quantitate minigene and endogenous GLUT4 mRNA using RNase protection assay as described under ‘‘Experimental Procedures.’’ Size of the open and solid boxes indicates radioactivity levels of endogenous and minigene GLUT4 protected fragments, respectively. Since minigene GLUT4 protected fragments (433 and 399 bp) have about 2-fold radioactivity greater than that of endogenous GLUT4 protected fragments (182 bp), actual mRNA levels of minigene GLUT4 was about half of its radioactivity in comparison with endogenous GLUT4. The expression levels of minigene and endogenous GLUT4 were expressed as a percentage of that of endogenous GLUT4 from gastrocnemius. Minigene GLUT4 copy numbers are shown in parenthesis. 506
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ever, expression of the murine 0442 GLUT4 construct, though not maximal, was similar in heart and skeletal muscle (Fig. 3). The reason for the difference in expression in heart of the murine 0422 GLUT4 minigene compared to the 0730 human GLUT4-CAT reporter gene is not known at this time. It may be due to sequence elements in the introns and/or 3*-flanking region of the murine GLUT4 gene, or to differences between the human and murine GLUT4 5*-flanking region. The construction of the 5*-deleted GLUT4 minigenes used in this study relied on the recent development of LA-PCR technology (29). Unlike Taq DNA polymerase, LA-polymerase possesses 3* to 5* exonuclease proofreading activity that enables the polymerase to correct nucleotide-misincorporation errors, giving an error rate of only one mutation per 10 kb (30). Sequence
FIG. 3. Expression of minigene and endogenous GLUT4 mRNAs in transgenic mice harboring 01000 GLUT4 minigene (A) and 0442 GLUT4 minigene (B). RNA isolated from the indicated tissues of female heterozygous transgenic mice from 01000 GLUT4 (Fig. 2 D) and 0442 GLUT4 (Fig. 2 E) was subjected to RNase protection analysis as described under ‘‘Experimental Procedures.’’ The 433-bp and 399-bp protected fragment corresponds to GLUT4 minigene message and the 182-bp protected fragment corresponds to endogenous GLUT4. Several bands observed between 399 bp and 182 bp appear to be degraded fragments of the minigene-protected fragment.
in comparison with the larger constructs, two independent founder 0442 GLUT4 mice lines showed a lower expression of the GLUT4 minigene in skeletal muscles and heart, indicating that another element besides MEF2 may be required for maximal expression of GLUT4 in skeletal muscle and heart. These data are consistent with data from the rat GLUT4 gene that showed that MEF2 sequence was essential for myotube-specific expression in C2C12 cells but was not sufficient for its full expression (28). Olson and Pessin reported that 730 bp of the human GLUT4 5*-flanking DNA harboring a MEF2 binding site was sufficient to direct relatively high expression in skeletal muscles but not in heart or WAT (23). How-
FIG. 4. Regulation of minigene and endogenous GLUT4 mRNA in gastrocnemius from male exercise-trained mice. A: Typical autoradiogram. Heterozygous male transgenic mice were divided into two groups, one subjected to the exercise protocol (/) and the other not (0). RNA isolated from gastrocnemius skeletal muscle was subjected to RNase protection assays. The 433-bp and 399-bp protected fragment corresponds to GLUT4 minigene message and the 182-bp protected fragment corresponds to endogenous GLUT4. B: The minigene and endogenous GLUT4 expression levels were quantified by image analyzer and expressed by relative percent to endogenous GLUT4 in gastrocnemius from nonexercised controls. Each data point is mean { standard error of the mean of 2-4 mice (shown in parenthesis).
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quence identity within this proximal 1000 bps upstream of the transcription start site (31), which suggests that hormonal/metabolic regulatory elements are clustered in this region. The present data indicate that BAT- and WAT-specific enhancer(s), and exercise- and high-fat diet-responsive elements are located between bases 01000 and 0442 of the murine GLUT4 5*-flanking region. Olson et al. have shown that this region of the human GLUT4 gene mediates the regulation of GLUT4 expression in streptozotocin-induced diabetes (23). It will be interesting to see if any of these responses are mediated through the same cis-elements or to learn how their trans-acting factors interact. ACKNOWLEDGMENTS We are grateful to M. Koda and M. Kurumiya for excellent technical assistance. This work was supported by research grants from the National Institutes of Health, National Institute of Diabetes and Digestive and Kidney Diseases (Washington, DC), the Japanese Ministry of Health and Welfare, the Japanese Ministry of Education, Science and Culture (Tokyo), and the Japanese Science and Technology Agency (Tokyo).
REFERENCES FIG. 5. Regulation of minigene and endogenous GLUT4 mRNA in white adipose tissues from female high-fat diet fed mice. A: Typical autoradiogram. Heterozygous female transgenic mice were divided into two groups, one subjected to a high-carbohydrate feeding protocol (C) and the other to a high-fat feeding protocol (F). RNA isolated from parametrial WAT was subjected to RNase protection assays. The 433-bp and 399-bp protected fragment corresponds to GLUT4 minigene message and the 182-bp protected fragment corresponds to endogenous GLUT4. B: The minigene and endogenous GLUT4 expression levels were quantified by image analyzer and expressed by relative percent to endogenous GLUT4 in WAT from carbohydrate-fed controls. Each data point is mean { standard error of the mean of 3-4 mice (shown in parenthesis).
analysis of the promoter region of the 0442 GLUT4 minigene identified a single A to G substitution at 0373 bp. While it is possible this PCR introduced mutation caused the loss of exercise and diet-induced regulation in the 0442 GLUT4 minigene, it is much more likely that this loss of regulation is due to the deletion from 01000 to 0442 bp. Furthermore, since all the transgenes produced functional GLUT4 (all transgenic mice had improved glucose tolerance compared to nontransgenic mice), significant errors in the coding region or intron splice sites are unlikely. Given the extremely low error rate of LA-polymerase, it is unlikely that a PCR-introduced mutation in the coding region, introns, or 3*-untranslated region caused a loss of regulation seen in the 0442 construct. Further studies will result in definitive identification of the response element (s). There is substantial identity between the mouse and human GLUT4 gene, with the greatest degree of se-
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18. Weinstein, S. P., O’Boyle, E., and Haber, R. S. (1994) Diabetes 43, 1185–1189. 19. Shimokawa, T., Kato, M., Shioduka, K., Irie, J., and Ezaki, O. (1997) Biochem. Biophys. Res. Commmun. 235, 790–793. 20. Ezaki, O., Flores-Riveros, J. R., Kaestner, K. H., Gearhart, J., and Lane, M. D. (1993) Proc. Natl. Acad. Sci. USA 90, 3348– 3352. 21. Ikemoto, S., Thompson, K. S., Itakura, H., Lane, M. D., and Ezaki, O. (1995) Proc. Natl. Acad. Sci. USA 92, 865–869. 22. Ikemoto, S., Thompson, K. S., Takahashi, M., Itakura, H., Lane, M. D., and Ezaki, O. (1995) Proc. Natl. Acad. Sci. USA 92, 3096– 3099. 23. Olson, A. L., and Pessin, J. E. (1995) J. Biol. Chem. 270, 23491– 23495.
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