- Email: [email protected]
Sucrose-Diet Feeding Induces Gene Expression of Heat Shock Protein in Rat Brain under Stress
Biochemical and Biophysical Research Communications 274, 355–358 (2000) doi:10.1006/bbrc.2000.3108, available online at http://www.idealibrary.com on
Sucrose-Diet Feeding Induces Gene Expression of Heat Shock Protein in Rat Brain under Stress Haruaki Kageyama,* Eiji Suzuki,† Takayuki Kashiwa,* Masao Kanazawa,‡ Toshimasa Osaka,* Shuichi Kimura,* Yoshio Namba,* and Shuji Inoue* ,1 *Division of Geriatric Health and Nutrition, National Institute of Health and Nutrition, 1-23-1 Toyama Shinjuku-ku, Tokyo, 162-8636, Japan; †Department of Psychiatry, Kitasato University School of Medicine, Kanagawa, 228-8520, Japan; and ‡Third Department of Internal Medicine, Tokyo Medical College, Tokyo, 160-8402, Japan
Received June 20, 2000
Stress-induced hyperphagia is enhanced in the presence of sweets, particularly sucrose, which may act to attenuate stress. Recently, it was also reported that heat shock protein (HSP) may be involved in the defense against stress. To explore whether sucrose alters gene expression of HSP under stress, we determined the HSP mRNA levels in the hypothalamus, cerebellum, and cerebral cortex after restraint stress in sucrose-diet-fed rats. Competitive RT-PCR revealed that gene expressions of HSP27 in the cerebral cortex and cerebellum and of HSP70 in the cerebral cortex, hypothalamus, and cerebellum were induced by restraint stress under a sucrose-diet-fed condition. However, restraint stress by itself or sucrose diet alone did not induce expression of HSP27 or HSP70 mRNA in any of the three anatomical parts. It is suggested that sucrose facilitates the gene expression of HSP27 and HSP70 in brain after restraint stress, which may attenuate stress. © 2000 Academic Press Key Words: sucrose diet; heat shock protein; gene expression; brain; restraint stress; competitive RT-PCR.
Humans and rodents can induce to overfeed under physiological stress, and this is termed stress-induced hyperphagia. Overeating is observed during mild-tail pinch in rodents (1), and this pinch facilitates hyperphagia in the presence of a sucrose diet (2). These findings suggest that sucrose feeding may attenuate stress. Since the brain is vulnerable to a stressful environment, it induces several gene expressions and/or proteins related to protection to the stressor, or defenses to stress. The brain is the most important site where glucose is utilized. Thus, if a massive amount of glucose is consumed in the brain during stress, sucrose To whom correspondence should be addressed. Fax: ⫹81-3-32059536. E-mail: [email protected]. 1
may play an important role in counteracting stress. However, the protective function of sucrose against stress in the brain remains unknown. Heat shock protein (HSP) was initially identified as a protein induced by heat (3). It allowed denatured polypeptides to reacquire their native conformation, and thus it can prevent cellular damage during or after thermal stress (4). Studies on HSP have presumed that overexpression of HSP gene is induced not only by thermal stress but also by environmental stress (5). Several subtypes have been known in HSP family. HSP70 is one of the most well-studied HSPs. This protein functions to help the cell in the repair process after environmental stress such as heat, UV irradiation or oxidative stress (6) as well as molecular chaperones. HSP27 is one of the small heat shock proteins, which are abundant in brain, particularly glial cells. Small HSPs may act as molecular chaperones (7), or protect against toxic chemicals or stress (8 –11). In this study, to explore the possibility that sweets attenuate stress, we examined whether a sucrose diet alters HSPs mRNA expression in rat brain after restraint stress. MATERIALS AND METHODS Animals. Male Wistar rats weighing 240 –280 g (Japan SCL Inc., Hamamatsu, Japan) were kept in individual cages on a rotating 12-h light– dark cycle with an ambient temperature of 24 ⫾ 1°C and given free access to food and water. A sucrose-diet-fed group (n ⫽ 10) ingested a purified experimental diet (Oriental Food Co., Tokyo, Japan) that contained (as percent of calories) 60% sucrose, 11% corn oil, and 29% animal protein. A chow-diet-fed group (n ⫽ 10) was fed standard rat chow (Oriental Food Co.) which contained 60% vegetable starch, 11% corn oil, and 29% animal protein. Sucrose and chowdiet feedings were given for one week. Restraint model. Both the conscious sucrose-diet-fed group (n ⫽ 5) and the chow-diet-fed group (n ⫽ 5) were restrained in a dorsal position on restrainers secured at the head and extremities with a clip for 60 min. At the end of the 60-min stress exposure, the animals were killed by decapitation. Trunk blood was collected for subse-
355
0006-291X/00 $35.00 Copyright © 2000 by Academic Press All rights of reproduction in any form reserved.
Vol. 274, No. 2, 2000
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS TABLE 1
Plasma Glucose and Insulin in Sucrose-Diet-Fed and Chow-Diet-Fed Rats
Insulin (ng/ml) Glucose (mM)
Chow
Sucrose
Chow ⫹ stress
Sucrose ⫹ stress
1.09 ⫾ 0.36
1.10 ⫾ 0.23
0.53 ⫾ 0.14
6.4 ⫾ 0.1
6.4 ⫾ 0.1
10.0 ⫾ 0.5
a,b
0.62 ⫾ 0.15 9.8 ⫾ 0.8 a,b
Note. Data represent the mean ⫾ standard error (SEM). Symbols indicate statistical significance by one-way ANOVA at P ⬍ 0.05; a, versus chow diet; b, versus sucrose diet.
quent assay, and then the cerebral cortex, cerebellum and hypothalamus were removed and stored at ⫺80°C until assay. RNA preparation. Total RNA was isolated from cerebral cortex, cerebellum and hypothalamus using the TRIzol reagent (Life Technologies, Inc., Rockville, MD). RNA was quantified spectrophotometrically and confirmed by ethidium bromide staining of 18S and 28S ribosomal RNA. Primer design and generation of competitor. Gene specific primers were designed to amplify target regions of the HSP70, HSP27, and -actin genes. The competitor for -actin gene was generated to insert a 36 bp fragment at the 388 nt position (Accession Nos. X00351, J00074, M10278). The competitor for -actin gene size was 171 bp. The competitor for the HSP70 gene was generated to insert a 40 bp fragment at the 370 nt position (Accession No. L16764). The competitor size for the HSP70 gene was 252 bp. The competitor for the HSP27 gene was generated to insert a 40 bp fragment at the 170 nt position (Accession No. M86389). The competitor size for the HSP27 gene was 352 bp. These competitors were provided by Mitsubishi Kagaku BCL Inc. (Tokyo, Japan). Reverse transcription-competitive PCR. First strand cDNA was synthesized in a total volume of 20 l of reaction solution containing 4 g of total RNA, reverse transcriptase buffer (Life Technologies, Inc., Rockville, MD), 2 A/ml random primer (TaKaRa, Shiga, Japan), 0.5 mmol/liter each deoxyribonucleoside triphosphate (dNTP) (TaKaRa), 1.23 U/l ribonuclease inhibitor (TaKaRa) and 0.8 U/l AMV reverse transcriptase (Life Technologies, Inc). Samples were incubated at 42°C for 60 min and the reactions were terminated at 95°C for 5 min; the volume of reaction solution was then up to 40 l. A series of competitive PCRs set up using 10-fold dilution of DNA competitor (2 l) with a constant amount (2 l) of the first-strand cDNA was performed in 25 l of PCR mixtures containing 0.2 mmol/l of each dNTP (TaKaRa), Taq DNA polymerase buffer (Boehringer Mannheim, GmbH, Germany), 0.25 pmol/liter of each primer set and 0.012 unit/l Taq DNA polymerase (Roche Diagnostics Corporation). The sense -actin primer was 5⬘-labelled with TAMRA fluorescent dye, whereas the sense HSP27 and HSP70 primers were 5⬘-labelled with FITC fluorescent dye. After denaturing at 94°C for 180 s, the PCR mixtures were subjected to 35 cycles of PCR amplification with a cycle profile including denaturation for 30 s at 94°C, annealing for 30 s at 55°C and extension for 1 min at 72°C, then chase reaction for 3 min at 72°C and holding at 4°C. The PCR products were electrophoresed on 6% denaturated polyacrylamide gel and quantitatively analyzed on a fluorescent image analyzer, FMBIO II Multi-View (TaKaRa). The concentration of the target mRNA was determined at the competition equivalent point as described in detail elsewhere (12). Measurements. Plasma glucose levels were determined by glucose oxidase method (Glucose B-test, Wako Pure Pharmaceutical Co., Osaka, Japan). Immunoreactive insulin (IRI) concentrations
FIG. 1. HSP27 and HSP70 mRNA levels in cerebral cortex of standard chow-fed group (chow), sucrose-diet-fed group (sucrose), standard chow-fed group with restraint stress (chow ⫹ stress) and sucrose-diet-fed group with restraint stress (sucrose ⫹ stress). Means ⫾ SEM of five animals per group. a, P ⬍ 0.05 versus standard chow-fed group; b, P ⬍ 0.05 versus sucrose-diet-fed group; c, P ⬍ 0.05 versus standard chow-fed group with restraint stress.
were determined by a radioimmunoassay kit (Dia Sorin, Inc., Stillwater, NM). Statistics. Data are expressed as means ⫾ standard error (SEM). Statistical analyses were performed with the Stat View J-4.5 of the Macintosh system. Statistical significance was assessed by one-way ANOVA, and P ⬍ 0.05 was accepted as a significant difference.
RESULTS Table 1 shows the characteristic profiles of two dietfed rats. There were no differences in plasma glucose or insulin between the chow-diet- and sucrose-diet-fed animals. Restraint stress significantly increased the plasma glucose in both rat groups (P ⬍ 0.005). Plasma insulin decreased in both groups, but the decreases were not significant. The expressions of HSP27 and HSP70 in the cerebral cortex were not induced in sucrose-diet-fed rats without restraint stress. The expression of HSP70 mRNA increased with restraint stress in this tissue, but that of HSP27 mRNA did not. In chow-diet-fed rats, on the other hand, neither the expression of HSP70 nor HSP27 mRNA was altered (Fig. 1). The expression of HSP70 mRNA in the cerebellum was significantly suppressed in sucrose-diet-fed rats
FIG. 2. HSP27 and HSP70 mRNA levels in cerebellum of standard chow-fed group (Chow), sucrose-diet-fed group (sucrose), standard chow-fed group with restraint stress (chow ⫹ stress) and sucrose-diet-fed group with restraint stress (sucrose ⫹ stress). Means ⫾ SEM of five animals per group. a, P ⬍ 0.05 versus standard chow-fed group; b, P ⬍ 0.05 versus sucrose-diet-fed group.
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FIG. 3. HSP27 and HSP70 mRNA levels in hypothalamus of standard chow-fed group (Chow), sucrose-diet-fed group (sucrose), standard chow-fed group with restraint stress (chow ⫹ stress) and sucrose-diet-fed group with restraint stress (sucrose ⫹ stress). Means ⫾ SEM of five animals per group. a, P ⬍ 0.05 versus standard chow-fed group; b, P ⬍ 0.05 versus sucrose-diet-fed group.
compared to that in chow-diet-fed rats without restraint stress (P ⬍ 0.05, Fig. 2). The expressions of HSP70 and HSP27 were significantly enhanced by restraint stress in the cerebellum of sucrose diet fed rats (P ⬍ 0.05, Fig. 2), while they were not enhanced by restraint stress in chow-diet-fed rats (Fig. 2). The expressions of HSP27 and HSP70 mRNA in the hypothalamus were not induced in sucrose-diet-fed rats without restraint stress. These expressions were significantly induced by restraint stress in the hypothalamus of sucrose-diet-fed rats (P ⬍ 0.05, Fig. 3); however, no expressions were induced in all three tissues in chow-diet-fed rats.
an increase in expression of HSP70 in brain by restraint stress using Northern blotting was not observed (14). Although the gene expression in the whole brain was measured in that study, we separately evaluated the induction of both HSP27 and HSP70 mRNA expression in the hypothalamus, cerebellum and cerebral cortex by stress using competitive RT-PCR, which is more sensitive than Northern blotting. We also failed to demonstrate an increase in the expression of HSP27 and HSP70 mRNA in the hypothalamus, cerebellum or cerebral cortex after restraint stress in chowdiet-fed rats, which is consistent with their results. HSP is known to play a role as a molecular chaperone; however, the significance of reaction to stress remains unclear. Fukudo et al. (15) suggested that an increase in HSP by stress plays a protective role against stress in a vulnerable organ such as brain. In this context, our findings that the sucrose diet induced the gene expression of HSPs in the brain by restraint stress imply that sucrose may have the potential to attenuate stress. In summary, sucrose-diet feeding induced the expression of HSP70 and HSP27 mRNA by restraint stress in the hypothalamus, cerebellum and cerebral cortex. We suggest that a sucrose diet acts as an inducer which helps to induce expression of HSP mRNA in brain under certain stress, which may lead to an amelioration of that stress. ACKNOWLEDGMENT
DISCUSSION In the present study we found that gene expressions of HSP27 in the hypothalamus and cerebellum, and that of HSP70 in the cerebral cortex, hypothalamus and cerebellum were induced by restraint stress in sucrose-diet-fed rats. On the other hand, we failed to demonstrate an increase in the expressions of HSP27 and HSP70 mRNA in any brain area examined after restraint stress in chow-diet-fed rats. Plasma glucose was increased by stress. However, an increase in plasma glucose by stress did not alter expression of HSP27 and HSP70 mRNA since those were not altered by restraint stress in chow-diet-fed rats. The sucrose diet by itself did not induce the gene expression since HSP27 and HSP70 mRNA did not increase in sucrose-diet-fed rats without restraint stress. Restraint stress by itself did not induce the gene expression since HSP27 and HSP70 did not increase by restraint stress in chow-diet-fed rats. Thus, our findings suggest that sucrose diet plays a role as an inducer that helps to induce expression of HSP27 and HSP70 mRNA in the cerebral cortex, hypothalamus and cerebellum under restraint stress. It was reported that HSP70 and HSP27 mRNA in aortic tissues and HSP70 mRNA in adrenal gland were highly induced after restraint stress (13, 14); however,
This study was supported, in part, by a grant of Agriculture and Livestock Industries Corporation (Japan).
REFERENCES
357
1. Rowland, N. E., and Antelman, S. M. (1976) Stress-induced hyperphagia and obesity in rats: A possible model for understanding human obesity. Science 191, 310 –312. 2. Fullerton, D. T., Getto, C. J., Swift, W. J., and Carlson, I. H. (1985) Sugar, opioids and binge eating. Brain Res. Bull. 14, 673– 680. 3. Tissieres, A., Mitchell, H. K., and Tracy, U. M. (1974) Protein synthesis in salivary glands of Drosophila melanogaster: Relation to chromosome puffs. J. Mol. Biol. 84, 389 –398. 4. Morimoto, R. I., Tissieres, A., and Georgopoulous, C. (1990) The stress response, function of the proteins, and perspectives. In Stress Proteins in Biology and Medicine (Morimoto, R. I., Tissieres, A., and Georgopoulous, C., Eds.), pp. 1–37, Cold Spring Harbor Press, NY. 5. Putnak, J. R., and Schlesinger, J. J. (1990) Protection of mice against yellow fever virus encephalitis by immunization with a vaccinia virus recombinant encoding the yellow fever virus nonstructural proteins, NS1, NS2a and NS2b. J. Gen. Virol. 71, 1697–1702. 6. Schlesinger, M. J. (1990) Heat shock proteins. J. Biol. Chem. 265, 12111–12114. 7. Jakob, U., Gaestel, M., Engel, K., and Buchner, J. (1993) Small heat shock proteins are molecular chaperones. J. Biol. Chem. 268, 1517–1520.
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8. Ciocca, D. R., Fuqua, S. A., Lock-Lim, S., Toft, D. O., Welch, W. J., and McGuire, W. L. (1992) Response of human breast cancer cells to heat shock and chemotherapeutic drugs. Cancer Res. 52, 3648 –3654. 9. Oesterreich, S., Weng, C. N., Qiu, M., Hilsenbeck, S. G., Osborne, C. K., and Fuqua, S. A. (1993) The small heat shock protein hsp27 is correlated with growth and drug resistance in human breast cancer cell lines. Cancer Res. 53, 4443– 4448. 10. Huot, J., Lambert, H., Lavoie, J. N., Guimond, A., Houle, F., and Landry, J. (1995) Characterization of 45-kDa/54-kDa HSP27 kinase, a stress-sensitive kinase which may activate the phosphorylation-dependent protective function of mammalian 27-kDa heat-shock protein HSP27. Eur. J. Biochem. 227, 416 – 427. 11. Mairesse, N., Bernaert, D., Del Bino, G., Horman, S., Mosselmans, R., Robaye, B., and Galand, P. (1998) Expression of HSP27 results in increased sensitivity to tumor necrosis factor, etoposide, and H2O2 in an oxidative stress-resistant cell line. J. Cell Physiol. 177, 606 – 617.
12. Auboeuf, D., and Vidal, H. (1997) The use of the reverse transcription-competitive polymerase chain reaction to investigate the in vivo regulation of gene expression in small tissue samples. Anal. Biochem. 245, 141–148. 13. Blake, M. J., Udelsman, R., Feulner, G. J., Norton, D. D., and Holbrook, N. J. (1991) Stress-induced heat shock protein 70 expression in adrenal cortex: An adrenocorticotropic hormonesensitive; age-dependent response. Proc. Natl. Acad. Sci. USA 88, 9873–9877. 14. Udelsman, R., Blake, M. J., Stagg, C. A., Li, D. G., Putney, D. J., and Holbrook, N. J. (1993) Vascular heat shock protein expression in response to stress. Endocrine and autonomic regulation of this age-dependent response. J. Clin. Invest. 91, 465– 473. 15. Fukudo, S., Abe, K., Hongo, M., Utsumi, A., and Itoyama, Y. (1997) Brain– gut induction of heat shock protein (HSP) 70 mRNA by psychophysiological stress in rats. Brain Res. 757, 146 –148.
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Sucrose-Diet Feeding Induces Gene Expression of Heat Shock Protein in Rat Brain under Stress Haruaki Kageyama,* Eiji Suzuki,† Takayuki Kashiwa,* Masao Kanazawa,‡ Toshimasa Osaka,* Shuichi Kimura,* Yoshio Namba,* and Shuji Inoue* ,1 *Division of Geriatric Health and Nutrition, National Institute of Health and Nutrition, 1-23-1 Toyama Shinjuku-ku, Tokyo, 162-8636, Japan; †Department of Psychiatry, Kitasato University School of Medicine, Kanagawa, 228-8520, Japan; and ‡Third Department of Internal Medicine, Tokyo Medical College, Tokyo, 160-8402, Japan
Received June 20, 2000
Stress-induced hyperphagia is enhanced in the presence of sweets, particularly sucrose, which may act to attenuate stress. Recently, it was also reported that heat shock protein (HSP) may be involved in the defense against stress. To explore whether sucrose alters gene expression of HSP under stress, we determined the HSP mRNA levels in the hypothalamus, cerebellum, and cerebral cortex after restraint stress in sucrose-diet-fed rats. Competitive RT-PCR revealed that gene expressions of HSP27 in the cerebral cortex and cerebellum and of HSP70 in the cerebral cortex, hypothalamus, and cerebellum were induced by restraint stress under a sucrose-diet-fed condition. However, restraint stress by itself or sucrose diet alone did not induce expression of HSP27 or HSP70 mRNA in any of the three anatomical parts. It is suggested that sucrose facilitates the gene expression of HSP27 and HSP70 in brain after restraint stress, which may attenuate stress. © 2000 Academic Press Key Words: sucrose diet; heat shock protein; gene expression; brain; restraint stress; competitive RT-PCR.
Humans and rodents can induce to overfeed under physiological stress, and this is termed stress-induced hyperphagia. Overeating is observed during mild-tail pinch in rodents (1), and this pinch facilitates hyperphagia in the presence of a sucrose diet (2). These findings suggest that sucrose feeding may attenuate stress. Since the brain is vulnerable to a stressful environment, it induces several gene expressions and/or proteins related to protection to the stressor, or defenses to stress. The brain is the most important site where glucose is utilized. Thus, if a massive amount of glucose is consumed in the brain during stress, sucrose To whom correspondence should be addressed. Fax: ⫹81-3-32059536. E-mail: [email protected]. 1
may play an important role in counteracting stress. However, the protective function of sucrose against stress in the brain remains unknown. Heat shock protein (HSP) was initially identified as a protein induced by heat (3). It allowed denatured polypeptides to reacquire their native conformation, and thus it can prevent cellular damage during or after thermal stress (4). Studies on HSP have presumed that overexpression of HSP gene is induced not only by thermal stress but also by environmental stress (5). Several subtypes have been known in HSP family. HSP70 is one of the most well-studied HSPs. This protein functions to help the cell in the repair process after environmental stress such as heat, UV irradiation or oxidative stress (6) as well as molecular chaperones. HSP27 is one of the small heat shock proteins, which are abundant in brain, particularly glial cells. Small HSPs may act as molecular chaperones (7), or protect against toxic chemicals or stress (8 –11). In this study, to explore the possibility that sweets attenuate stress, we examined whether a sucrose diet alters HSPs mRNA expression in rat brain after restraint stress. MATERIALS AND METHODS Animals. Male Wistar rats weighing 240 –280 g (Japan SCL Inc., Hamamatsu, Japan) were kept in individual cages on a rotating 12-h light– dark cycle with an ambient temperature of 24 ⫾ 1°C and given free access to food and water. A sucrose-diet-fed group (n ⫽ 10) ingested a purified experimental diet (Oriental Food Co., Tokyo, Japan) that contained (as percent of calories) 60% sucrose, 11% corn oil, and 29% animal protein. A chow-diet-fed group (n ⫽ 10) was fed standard rat chow (Oriental Food Co.) which contained 60% vegetable starch, 11% corn oil, and 29% animal protein. Sucrose and chowdiet feedings were given for one week. Restraint model. Both the conscious sucrose-diet-fed group (n ⫽ 5) and the chow-diet-fed group (n ⫽ 5) were restrained in a dorsal position on restrainers secured at the head and extremities with a clip for 60 min. At the end of the 60-min stress exposure, the animals were killed by decapitation. Trunk blood was collected for subse-
355
0006-291X/00 $35.00 Copyright © 2000 by Academic Press All rights of reproduction in any form reserved.
Vol. 274, No. 2, 2000
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS TABLE 1
Plasma Glucose and Insulin in Sucrose-Diet-Fed and Chow-Diet-Fed Rats
Insulin (ng/ml) Glucose (mM)
Chow
Sucrose
Chow ⫹ stress
Sucrose ⫹ stress
1.09 ⫾ 0.36
1.10 ⫾ 0.23
0.53 ⫾ 0.14
6.4 ⫾ 0.1
6.4 ⫾ 0.1
10.0 ⫾ 0.5
a,b
0.62 ⫾ 0.15 9.8 ⫾ 0.8 a,b
Note. Data represent the mean ⫾ standard error (SEM). Symbols indicate statistical significance by one-way ANOVA at P ⬍ 0.05; a, versus chow diet; b, versus sucrose diet.
quent assay, and then the cerebral cortex, cerebellum and hypothalamus were removed and stored at ⫺80°C until assay. RNA preparation. Total RNA was isolated from cerebral cortex, cerebellum and hypothalamus using the TRIzol reagent (Life Technologies, Inc., Rockville, MD). RNA was quantified spectrophotometrically and confirmed by ethidium bromide staining of 18S and 28S ribosomal RNA. Primer design and generation of competitor. Gene specific primers were designed to amplify target regions of the HSP70, HSP27, and -actin genes. The competitor for -actin gene was generated to insert a 36 bp fragment at the 388 nt position (Accession Nos. X00351, J00074, M10278). The competitor for -actin gene size was 171 bp. The competitor for the HSP70 gene was generated to insert a 40 bp fragment at the 370 nt position (Accession No. L16764). The competitor size for the HSP70 gene was 252 bp. The competitor for the HSP27 gene was generated to insert a 40 bp fragment at the 170 nt position (Accession No. M86389). The competitor size for the HSP27 gene was 352 bp. These competitors were provided by Mitsubishi Kagaku BCL Inc. (Tokyo, Japan). Reverse transcription-competitive PCR. First strand cDNA was synthesized in a total volume of 20 l of reaction solution containing 4 g of total RNA, reverse transcriptase buffer (Life Technologies, Inc., Rockville, MD), 2 A/ml random primer (TaKaRa, Shiga, Japan), 0.5 mmol/liter each deoxyribonucleoside triphosphate (dNTP) (TaKaRa), 1.23 U/l ribonuclease inhibitor (TaKaRa) and 0.8 U/l AMV reverse transcriptase (Life Technologies, Inc). Samples were incubated at 42°C for 60 min and the reactions were terminated at 95°C for 5 min; the volume of reaction solution was then up to 40 l. A series of competitive PCRs set up using 10-fold dilution of DNA competitor (2 l) with a constant amount (2 l) of the first-strand cDNA was performed in 25 l of PCR mixtures containing 0.2 mmol/l of each dNTP (TaKaRa), Taq DNA polymerase buffer (Boehringer Mannheim, GmbH, Germany), 0.25 pmol/liter of each primer set and 0.012 unit/l Taq DNA polymerase (Roche Diagnostics Corporation). The sense -actin primer was 5⬘-labelled with TAMRA fluorescent dye, whereas the sense HSP27 and HSP70 primers were 5⬘-labelled with FITC fluorescent dye. After denaturing at 94°C for 180 s, the PCR mixtures were subjected to 35 cycles of PCR amplification with a cycle profile including denaturation for 30 s at 94°C, annealing for 30 s at 55°C and extension for 1 min at 72°C, then chase reaction for 3 min at 72°C and holding at 4°C. The PCR products were electrophoresed on 6% denaturated polyacrylamide gel and quantitatively analyzed on a fluorescent image analyzer, FMBIO II Multi-View (TaKaRa). The concentration of the target mRNA was determined at the competition equivalent point as described in detail elsewhere (12). Measurements. Plasma glucose levels were determined by glucose oxidase method (Glucose B-test, Wako Pure Pharmaceutical Co., Osaka, Japan). Immunoreactive insulin (IRI) concentrations
FIG. 1. HSP27 and HSP70 mRNA levels in cerebral cortex of standard chow-fed group (chow), sucrose-diet-fed group (sucrose), standard chow-fed group with restraint stress (chow ⫹ stress) and sucrose-diet-fed group with restraint stress (sucrose ⫹ stress). Means ⫾ SEM of five animals per group. a, P ⬍ 0.05 versus standard chow-fed group; b, P ⬍ 0.05 versus sucrose-diet-fed group; c, P ⬍ 0.05 versus standard chow-fed group with restraint stress.
were determined by a radioimmunoassay kit (Dia Sorin, Inc., Stillwater, NM). Statistics. Data are expressed as means ⫾ standard error (SEM). Statistical analyses were performed with the Stat View J-4.5 of the Macintosh system. Statistical significance was assessed by one-way ANOVA, and P ⬍ 0.05 was accepted as a significant difference.
RESULTS Table 1 shows the characteristic profiles of two dietfed rats. There were no differences in plasma glucose or insulin between the chow-diet- and sucrose-diet-fed animals. Restraint stress significantly increased the plasma glucose in both rat groups (P ⬍ 0.005). Plasma insulin decreased in both groups, but the decreases were not significant. The expressions of HSP27 and HSP70 in the cerebral cortex were not induced in sucrose-diet-fed rats without restraint stress. The expression of HSP70 mRNA increased with restraint stress in this tissue, but that of HSP27 mRNA did not. In chow-diet-fed rats, on the other hand, neither the expression of HSP70 nor HSP27 mRNA was altered (Fig. 1). The expression of HSP70 mRNA in the cerebellum was significantly suppressed in sucrose-diet-fed rats
FIG. 2. HSP27 and HSP70 mRNA levels in cerebellum of standard chow-fed group (Chow), sucrose-diet-fed group (sucrose), standard chow-fed group with restraint stress (chow ⫹ stress) and sucrose-diet-fed group with restraint stress (sucrose ⫹ stress). Means ⫾ SEM of five animals per group. a, P ⬍ 0.05 versus standard chow-fed group; b, P ⬍ 0.05 versus sucrose-diet-fed group.
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FIG. 3. HSP27 and HSP70 mRNA levels in hypothalamus of standard chow-fed group (Chow), sucrose-diet-fed group (sucrose), standard chow-fed group with restraint stress (chow ⫹ stress) and sucrose-diet-fed group with restraint stress (sucrose ⫹ stress). Means ⫾ SEM of five animals per group. a, P ⬍ 0.05 versus standard chow-fed group; b, P ⬍ 0.05 versus sucrose-diet-fed group.
compared to that in chow-diet-fed rats without restraint stress (P ⬍ 0.05, Fig. 2). The expressions of HSP70 and HSP27 were significantly enhanced by restraint stress in the cerebellum of sucrose diet fed rats (P ⬍ 0.05, Fig. 2), while they were not enhanced by restraint stress in chow-diet-fed rats (Fig. 2). The expressions of HSP27 and HSP70 mRNA in the hypothalamus were not induced in sucrose-diet-fed rats without restraint stress. These expressions were significantly induced by restraint stress in the hypothalamus of sucrose-diet-fed rats (P ⬍ 0.05, Fig. 3); however, no expressions were induced in all three tissues in chow-diet-fed rats.
an increase in expression of HSP70 in brain by restraint stress using Northern blotting was not observed (14). Although the gene expression in the whole brain was measured in that study, we separately evaluated the induction of both HSP27 and HSP70 mRNA expression in the hypothalamus, cerebellum and cerebral cortex by stress using competitive RT-PCR, which is more sensitive than Northern blotting. We also failed to demonstrate an increase in the expression of HSP27 and HSP70 mRNA in the hypothalamus, cerebellum or cerebral cortex after restraint stress in chowdiet-fed rats, which is consistent with their results. HSP is known to play a role as a molecular chaperone; however, the significance of reaction to stress remains unclear. Fukudo et al. (15) suggested that an increase in HSP by stress plays a protective role against stress in a vulnerable organ such as brain. In this context, our findings that the sucrose diet induced the gene expression of HSPs in the brain by restraint stress imply that sucrose may have the potential to attenuate stress. In summary, sucrose-diet feeding induced the expression of HSP70 and HSP27 mRNA by restraint stress in the hypothalamus, cerebellum and cerebral cortex. We suggest that a sucrose diet acts as an inducer which helps to induce expression of HSP mRNA in brain under certain stress, which may lead to an amelioration of that stress. ACKNOWLEDGMENT
DISCUSSION In the present study we found that gene expressions of HSP27 in the hypothalamus and cerebellum, and that of HSP70 in the cerebral cortex, hypothalamus and cerebellum were induced by restraint stress in sucrose-diet-fed rats. On the other hand, we failed to demonstrate an increase in the expressions of HSP27 and HSP70 mRNA in any brain area examined after restraint stress in chow-diet-fed rats. Plasma glucose was increased by stress. However, an increase in plasma glucose by stress did not alter expression of HSP27 and HSP70 mRNA since those were not altered by restraint stress in chow-diet-fed rats. The sucrose diet by itself did not induce the gene expression since HSP27 and HSP70 mRNA did not increase in sucrose-diet-fed rats without restraint stress. Restraint stress by itself did not induce the gene expression since HSP27 and HSP70 did not increase by restraint stress in chow-diet-fed rats. Thus, our findings suggest that sucrose diet plays a role as an inducer that helps to induce expression of HSP27 and HSP70 mRNA in the cerebral cortex, hypothalamus and cerebellum under restraint stress. It was reported that HSP70 and HSP27 mRNA in aortic tissues and HSP70 mRNA in adrenal gland were highly induced after restraint stress (13, 14); however,
This study was supported, in part, by a grant of Agriculture and Livestock Industries Corporation (Japan).
REFERENCES
357
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