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Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, Indianapolis, IN 46202;
* Department of Materials Science and Engineering, Nagoya Institute of Technology, Nagoya 466-8555, Japan;
Department of Biochemistry, Faculty of Medicine, Mie University, Mie 514-8507, Japan; and
** Department of Health Science, Faculty of Psychological and Physical Sciences, Aichi Gakuin University, Aichi 470-0195, Japan
2To whom correspondence should be addressed. E-mail: raharris{at}iupui.edu.
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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-keto acid dehydrogenase complex (BCKDC) in female but not male rats. We attempted to determine the role played by ovarian hormones in this difference in enzyme regulation. A series of experiments examined the effects of the 4-d estrous cycle, ovariectomy, and replacement of female sex steroids on the catabolism of BCAAs. A proestrous decrease in the activity state of the complex corresponded to an increase in the plasma 17ß-estradiol level. Withdrawal of gonadal steroids by ovariectomy resulted in an increase in the activity state of BCKDC and a decrease in the activity of the branched-chain -keto acid dehydrogenase kinase (BDK). However, 17ß-estradiol reversed these effects, resulting in an increase in the BDK activity, thereby decreasing the activity of the complex. Progesterone administration was ineffective. The changes in the percentage of active BCKDC caused by 17ß-estradiol withdrawal and replacement resulted from changes in the amount of BDK protein associated with the complex and therefore its activity. Thus, the marked diurnal variation in the activity state of BCKDC exhibited by female rats involves estrogenic control of BDK activity. We hypothesize that the 17ß-estradiol–controlled feeding pattern produces these variations in BCKDC activity. This may function in female rats to conserve essential amino acids for protein synthesis.
KEY WORDS: • branched-chain -keto acid dehydrogenase complex • kinase • 17ß-estradiol • liver
The BCAAs (leucine, isoleucine, and valine) are essential for protein synthesis, neurotransmitter synthesis, and branched-chain fatty acid synthesis (1). BCAAs, however, cannot be synthesized in the body and must therefore be continuously supplied by diet for maintenance of body protein. Therefore, the catabolism must be tightly regulated under various nutritional and hormonal conditions to avoid a deficiency.
The most important regulatory enzyme in the oxidation pathways of BCAAs is the branched-chain -keto acid dehydrogenase complex (BCKDC),3 which catalyzes the regulated step of BCAA catabolism (2), i.e., the oxidative decarboxylation of branched-chain -keto acids (BCKAs) derived from BCAAs. Under conditions in which BCAAs and their BCKAs are present in excess, dephosphorylation of BCKDC occurs, activating the complex (1,3). In response to changing needs for BCKAs, and therefore BCAAs, under conditions such as dietary insufficiency, the complex is maintained in its inactive, phosphorylated state to conserve these essential amino acids (1,3). BCKDC activity is regulated by the phosphorylation of 2 serine residues of the E1-subunit of BCKDC, catalyzed by a complex-specific kinase (BDK) (4–6). We reported that only the kinase that is bound to the BCKDC and not free BDK is involved in the regulation of BCKDC activity (7). However, no studies on the regulatory factors or mechanisms that control the binding affinity of BDK for the BCKDC have been reported.
The regulation of the activity of BCKDC through kinase-mediated phosphorylation was studied extensively in rat tissues using different nutritional and hormonal stimuli (8–12). We hypothesized that proper regulation of the activity state of BCKDC by BDK is critically important for maintenance of body protein. For example, low-protein feeding (8) and administration of thyroid hormone (11) resulted in a decrease in the hepatic enzyme activity of the complex, due to an upregulation of BDK expression. High-protein feeding (8), starvation (10), and treatment of rats with glucocorticoids (9) resulted in a downregulation of BDK expression. An interesting feature of the regulatory system of BCAA catabolism is a diurnal variation in BDK activity exhibited in female, but not male rats (13). In the middle of the dark period and early in the light period, most of the BCKDC is in the dephosphorylated and active form in both males and females (13). However, by the end of the light period, most of the complex is in its phosphorylated, inactive form in females, but not males, due to an increase in BDK activity (13). Male rats, in contrast, maintain high BCKDC activity throughout the day (13). Because the change in the kinase activity between morning and evening occurs specifically in females, we suggested an involvement of ovarian hormones in the regulatory mechanism.
Diurnal rhythms are also seen in feeding behavior. Most of the food intake (80%) occurs during the dark phase (14). Levels of plasma estradiol are correlated with food intake during many physiologic states, whereas levels of other hypothalamic-pituitary-gonadal hormones are not (15–18). In addition to the daily rhythm of food intake in female rats, an estrous cycle–induced rhythm is also present (19,20). Food intake, feeding activity, and individual indices of meal size and meal number vary with circulating estradiol titers (21–23).
Considering the gender difference in BCAA oxidation in rat liver, a diurnal rhythm in feeding behavior, and the effect of sex-linked hormones on feeding pattern, we hypothesized that female sex steroids may affect the activity of the key enzyme of BCAA catabolism.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Animals. Female Sprague-Dawley rats aged 8 wk (CLEA Japan) were housed in individual cages. Rats consumed tap water and diet (CE2, CLEA Japan) ad libitum. The room was maintained at 24°C with a 12-h light:dark cycle (lights off from 1700 to 0500 h). Rats were adapted to the housing conditions for 1 wk before testing or surgery. The effects of estrous cycle and female sex hormones on the enzymes were examined in individual experiments. The body weight of all rats from the hormone treatment study was measured between 1600 and 1700 h. All of the rats for both experiments were anesthetized by i.p. injection of sodium pentobarbital (50 mg/kg body weight) between 1600 and 1700 h. Livers were removed rapidly from anesthetized rats, immediately freeze clamped, and stored at –80°C until analysis. All procedures involving animals were approved by the Experimental Animal Care Committee of Nagoya Institute of Technology.
Vaginal cellular differentiation analysis. Estrous cycles (proestrus, estrus, metestrus, and diestrus, n = 3) were monitored by daily vaginal cytology smears, taken between 1600 and 1700 h. Cells were identified as leukocytes, nucleated, or cornified epithelial cells. The cell populations ranging from entirely leukocytes (indicating a proestrous stage) to entirely cornified (indicating a diestrous stage) were analyzed for the determination of each stage. Livers were then removed as described above and used for the assay of BCKDC.
Ovariectomy. Ovariectomized (n = 24) and sham-operated (n = 6) female rats were prepared as described previously (13). Rats were anesthetized by i.p. injection of sodium pentobarbital (50 mg/kg body weight) and bilaterally ovariectomized or sham operated. After the surgery, a mixture of 50 IU penicillin and 50 µg of streptomycin was injected i.p. in saline for 2 d.
Hormone treatment. Groups of ovariectomized rats were randomly divided into 4 groups of similar body weight. Eight days after the surgery, rats were administered intrascapular subcutaneous injections twice over a 3-d interval with vehicle (peanut oil, 1 mL/kg body weight, Sigma-Aldrich) and steroid. Steroid (Sigma-Aldrich) doses were as follows: 17ß-estradiol (20 µg/kg body weight; E), progesterone (5 mg/kg body weight; P) and both hormones at the same doses (P + E) (27). Groups of sham-operated control (C) and ovariectomized (O) rats received peanut oil alone. Two days after the second steroid or vehicle treatment, rats were killed as described above.
Determination of uterine weight and blood 17ß-estradiol. Efficacy of the ovariectomies and sex steroid treatments was confirmed by uterine weight and plasma 17ß-estradiol. Uteri were carefully trimmed with respect to fat and connective tissue prior to measurement of their weight. Plasma 17ß-estradiol was measured using a 17ß-estradiol immunoassay kit (Cayman Chemical).
Enzyme assays.
Extraction and assay of the liver BCKDC was performed as described previously (28). Briefly, livers were homogenized in buffer containing 30 g/L Triton X-100 to extract BCKDC from the mitochondrial matrix space. Cell debris was removed by centrifugation at 20,000 x g for 5 min and BCKDC precipitated from the resulting supernatant by the addition of 90 g/L polyethylene glycol (average molecular weight 6000). The activities of the BCKDC in the active form (actual activity) and totally dephosphorylated form (total activity) were determined spectrophotometrically by measuring the rate of NADH production resulting from the conversion of -ketoisovalerate to isobutyryl CoA (28). Dephosphorylation of the complex for measurement of the total activity was performed using broad specificity phosphoprotein phosphatase (24). One unit of the complex catalyzed the formation of 1 µmol NADH/min. The activity state was calculated as the ratio of actual activity (activity of the active form of the enzyme in vivo) to total activity. The assay of BDK was performed by measuring ATP-mediated inactivation of BCKDC as described previously (13). Kinase activity is expressed as the first-order rate constant of BCKDC inactivation.
Extraction of the enzymes. Extraction of the enzymes from rat liver was performed as described previously (7). Dithiothreitol was added as a solid to the buffer just before use throughout the procedures. The protein concentration in the extract was determined by the method of Bradford using the Bio-Rad protein assay dye reagent. Proteins were separated by SDS-PAGE.
Immunoprecipitation.
Antiserum raised against the E1 (+ß) component of the BCKDC was used for the immunoprecipitation of BDK associated with the complex in the liver crude extracts (8 mg of protein). The amount of kinase was determined by immunoblot analysis with antiserum raised against BDK as described below. Protein A-agarose was purchased from Upstate Biotechnology and used as described previously (7).
Immunoblot analysis.
Proteins in the liver extracts (50 µg of protein) or immunoprecipitates were separated by SDS-PAGE according to the method of Laemmli (29). The separated polypeptides were transferred onto polyvinylidene difluoride (PVDF) membranes (Millipore) by the semidry method. The membrane was blocked with 30 g/L bovine serum albumin in TBST (20 mmol/L Tris-HCl, pH 7.5, 0.5 mol/L NaCl and 0.05% (v:v) Tween-20). The blots were then incubated with the primary antibody in blocking buffer, washed 3 times with TBST, and further incubated with the secondary antibody, [125I]anti-rabbit immunoglobulin (Amersham Biosciences), in blocking buffer. The radioactivities associated with the protein bands on the membrane were analyzed by a laser image analyzer (Fuji BAS1000, Fuji Film). Equivalent protein loading for immunoprecipitated proteins was verified by reprobing with E1 (+ß) antiserum (7). The data were expressed on the basis of the relative amount of E1ß protein.
Northern blot analysis. Total RNA was isolated from the frozen powdered liver using TRIzol reagent (Life Technologies) following the manufacturer’s instructions. Poly(A)+ RNA was separated from total RNA using oligo(dT)-cellulose (Takara) and quantified by measuring the absorbance at 260 nm. The ratios of absorbance at 260 nm to that at 280 nm were consistently 1.7–1.9. Poly(A)+ RNA (2 µg) from each sample were resolved in a 10 g/L agarose-formaldehyde gel and then transferred to a Hybond N+ nylon membrane by capillary flow of 20X SSC solution. 32P-Labeled cDNA probes for both BDK and ß-actin were prepared by a Rediprime Labeling System (Amersham Biosciences). Hybridization was performed at 68°C using QuikHyb (Stratagene). The distribution of radioactivity on the membrane was analyzed with a BAS1000 imaging analyzer.
Statistical analysis. Results are expressed as means ± SE. Statistical analysis was performed using one-way ANOVA and Fisher’s paired least-significant difference test. Differences with P < 0.05 were considered significant.
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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, E1ß and E2) were not affected by ovariectomy or steroid treatments (results not shown). Estradiol increases the protein level of hepatic BDK associated with the complex. The level of kinase protein bound to the complex was decreased by ovariectomy (P < 0.0001; Fig. 3). Treatment with 17ß-estradiol increased the level of kinase protein associated with the complex (P < 0.0001), whereas treatment with progesterone had no effect (Fig. 3). The level of protein associated with the complex was correlated directly with BDK activity (r = 0.997, P < 0.001) and inversely with the activity state of BCKDC (r = –0.989, P < 0.005) (Table 2).
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Our study began with the discovery of a marked diurnal variation in the activity state of the hepatic BCKDC in female rats (13). Regardless of gender, most of the complex was in its dephosphorylated active form in the middle of the dark period and early in the light period, and this form of the complex predominated in male rats at the end of the light period (13). In contrast, most of the complex in female rats became phosphorylated and inactive by the end of light period (13).
Through the 4-d estrous cycle, the activity state of BCKDC was at the lowest level during the proestrous phase when 17ß-estradiol levels were 
2 times higher than other phases (21). It is important to mention here that previous studies clearly revealed that estrogen inhibits appetite, resulting in low food intake (15,30,32). Therefore, during the proestrous phase, when estrogen level is highest and also the complex activity is lowest (Fig. 1), food intake decreases. Several factors or mechanisms might potentially determine the complex activity through the 4-d estrous cycle because this study showed an inverse relationship between the activity state of BCKDC and the plasma 17ß-estradiol concentration only in the proestrous and estrous phases. This estrous cycle–induced variation of the activity state of BCKDC, however, convinced us of the existence of female-specific regulation of the complex by estrogens.
Estrogens influence the diurnal rhythm in both estrogen binding activity (33) and the expression of cytoplasmic estrogen receptors (34). In addition, the most important mechanisms for the regulation of BCKDC involve increased expression of BDK. For instance, low-protein feeding (8) and administration of thyroid hormone (11), which are examples of long-term control mechanisms of the activity of the complex, result in upregulation of BDK expression. Accordingly, our first hypothesis based on the literature was that BDK expression might be modulated by 17ß-estradiol. In this study, however, we demonstrated a direct stimulatory effect of 17ß-estradiol not on BDK expression, but rather on BDK activity (Table 2). By stimulating BDK activity, 17ß-estradiol could decrease the proportion of BCKDC in the active, dephosphorylated state (Table 2) without reduction of the E1, E1ß and E2 components of the complex (results not shown). Moreover, the increase in BDK activity (and decrease in BCKDC activity), that occurred exclusively in 17ß-estradiol–injected ovariectomized rats, was accompanied by a marked change in the amount of BDK protein associated with the complex (Fig. 3), but with no change in total kinase protein (results not shown). We therefore propose that the change in the amount of BDK bound to the complex could be the key feature of the regulatory mechanism that determines the diurnal rhythm of BCKDC activity. It is likely that the amount of BDK protein associated with the complex plays an important role, especially in the short-term control of complex activity.
Why do only female rats have lower BCKDC activity in the evening? To address this issue, we focused on their feeding behavior. Gender differences in feeding pattern in normal male and female rats are well recognized. Renvyle and colleagues (14) reported intersex differences related to light-phase eating. Except for the slightly lower food intakes among females, dark-phase food intake does not differ between sexes, whereas female rats eat less of their food during the light phase relative to males (14). Renvyle et al. (14) reported that the overall mean food intake is 2 g in the dark for each 1 g eaten in the light by male rats, i.e., male rats eat 65% of their daily food intake during the dark cycle. However, the food intake of females is much more dominated by dark-phase feeding, with a ratio of 4:1 (14). We suggest that this phenomenon could explain a profound diurnal rhythm in the activity state of BCKDC in female rats. The homeostasis of food intake is maintained in both male and female rats via different mechanisms. Daily changes are clearly seen only in female rats because estrogens modulate food intake via the hypothalamus (17,35). Accordingly, our final hypothesis is that the 17ß-estradiol–controlled feeding pattern might produce the variations in BCKDC activity. This possibility could help explain the importance of the catabolism of BCAAs; tight control of BCKDC activity is important for conserving as well as disposing of BCAAs (1–3). BCAAs are toxic in excess as evidenced by maple syrup urine disease, which explains why animals have such an efficient oxidative mechanism for their disposal. However, liver BCKDC should dispose of only those BCAAs in excess of requirements for protein synthesis. Because BCAAs are essential amino acids, it is equally important to have measures to shut off BCAAs disposal, when animals eat less, to ensure their continuous availability for protein synthesis and growth.
More work is clearly required to understand the underlying regulatory mechanism of the oxidation of BCAAs in female rats. It is especially necessary to determine the interrelations among 17ß-estradiol, feeding behavior, and BCAA catabolism. In this regard, the antiobesity effect of estrogens is of interest (32). Ovariectomy of female rats increases body weight and the weight of subcutaneous and peritoneal fat, with increased food intake (32). As would be expected, these changes are fully reversed by subcutaneous replacement of estradiol (32). Most interesting, the difference in body weight and fat accumulation in ovariectomized rats was abolished by matching daily energy intake (32). Our present study demonstrated that 17ß-estradiol inhibits body weight gain and increases the amount of BDK protein associated with the complex in ovariectomized rats allowed free access to food. The pair-feeding method reported by Ouchi et al. (32) might be useful for investigation of factors regulating the diurnal rhythm of BCKDC activity in female rats and the change in BDK binding to the complex.
We conclude that the more slowly growing female rats have the hepatic BCKDC under tighter control as a part of a mechanism designed to conserve BCAAs. The detailed mechanism by which the changes in the binding affinity of BDK for the complex are induced remains to be defined.
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FOOTNOTES |
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3 Abbreviations used: BCKA, branched-chain -keto acid; BCKDC, branched-chain -keto acid dehydrogenase complex; BDK, branched-chain -keto acid dehydrogenase kinase; C, sham-operated control; E, 17ß-estradiol; E1, the -subunit of branched-chain -keto acid decarboxylase; E1ß, the ß-subunit of branched-chain -keto acid decarboxylase; E2, dihydrolipoyl transacylase; EIA, enzyme immunoassay; O, ovariectomized; P, progesterone; TBST, 20 mmol/L Tris-HCl, pH 7.5, 0.5 mol/L NaCl and 0.05% (v:v) Tween-20.
Manuscript received 10 May 2004. Initial review completed 8 June 2004. Revision accepted 21 July 2004.
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LITERATURE CITED |
|---|
|
TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
|---|
1. Harris, R. A., Joshi, M. & Jeoung, N. H. (2004) Mechanisms responsible for regulation of branched-chain amino acid catabolism. Biochem. Biophys. Res. Commun. 313:391-396.[Medline]
2. Harper, A. E., Miller, R. H. & Block, K. P. (1984) Branched-chain amino acid metabolism. Annu. Rev. Nutr. 4:409-454.[Medline]
3. Harris, R. A., Kobayashi, R., Murakami, T. & Shimomura, Y. (2001) Regulation of branched-chain alpha-keto acid dehydrogenase kinase expression in rat liver. J. Nutr. 131:841S-845S.
4. Shimomura, Y., Nanaumi, N., Suzuki, M., Popov, K. M. & Harris, R. A. (1990) Purification and partial characterization of branched-chain alpha-ketoacid dehydrogenase kinase from rat liver and rat heart. Arch. Biochem. Biophys. 283:293-299.[Medline]
5. Popov, K. M., Zhao, Y., Shimomura, Y., Kuntz, M. J. & Harris, R. A. (1992) Branched-chain alpha-ketoacid dehydrogenase kinase. Molecular cloning, expression, and sequence similarity with histidine protein kinases. J. Biol. Chem. 267:13127-13130.
6. Harris, R. A., Popov, K. M., Zhao, Y., Kedishvili, N. Y., Shimomura, Y. & Crabb, D. W. (1995) A new family of protein kinases—the mitochondrial protein kinases. Adv. Enzyme Regul. 35:147-162.[Medline]
7. Obayashi, M., Sato, Y., Harris, R. A. & Shimomura, Y. (2001) Regulation of the activity of branched-chain 2-oxo acid dehydrogenase (BCODH) complex by binding BCODH kinase. FEBS Lett. 491:50-54.[Medline]
8. Popov, K. M., Zhao, Y., Shimomura, Y., Jaskiewicz, J., Kedishvili, N. Y., Irwin, J., Goodwin, G. W. & Harris, R. A. (1995) Dietary control and tissue specific expression of branched-chain alpha-ketoacid dehydrogenase kinase. Arch. Biochem. Biophys. 316:148-154.[Medline]
9. Huang, Y. S. & Chuang, D. T. (1999) Down-regulation of rat mitochondrial branched-chain 2-oxoacid dehydrogenase kinase gene expression by glucocorticoids. Biochem. J. 339:503-510.
10. Kobayashi, R., Shimomura, Y., Murakami, T., Nakai, N., Otsuka, M., Arakawa, N., Shimizu, K. & Harris, R. A. (1999) Hepatic branched-chain alpha-keto acid dehydrogenase complex in female rats: activation by exercise and starvation. J. Nutr. Sci. Vitaminol. 45:303-309.
11. Kobayashi, R., Shimomura, Y., Otsuka, M., Popov, K. M. & Harris, R. A. (2000) Experimental hyperthyroidism causes inactivation of the branched-chain alpha-ketoacid dehydrogenase complex in rat liver. Arch. Biochem. Biophys. 375:55-61.[Medline]
12. Kobayashi, R., Murakami, T., Obayashi, M., Nakai, N., Jaskiewicz, J., Fujiwara, Y., Shimomura, Y. & Harris, R. A. (2002) Clofibric acid stimulates branched-chain amino acid catabolism by three mechanisms. Arch. Biochem. Biophys. 407:231-240.[Medline]
13. Kobayashi, R., Shimomura, Y., Murakami, T., Nakai, N., Fujitsuka, N., Otsuka, M., Arakawa, N., Popov, K. M. & Harris, R. A. (1997) Gender difference in regulation of branched-chain amino acid catabolism. Biochem. J. 327:449-453.
14. Laviano, A., Meguid, M. M., Gleason, J. R., Yang, Z. J. & Renvyle, T. (1996) Comparison of long-term feeding pattern between male and female Fischer 344 rats: influence of estrous cycle. Am. J. Physiol. 270:R413-R419.[Medline]
15. Wade, G. N. (1972) Gonadal hormones and behavioral regulation of body weight. Physiol. Behav. 8:523-534.[Medline]
16. Tarttelin, M. F. & Gorski, R. A. (1973) The effects of ovarian steroids on food and water intake and body weight in the female rat. Acta Endocrinol. 72:551-568.
17. Ganesan, R. (1994) The aversive and hypophagic effects of estradiol. Physiol. Behav. 55:279-285.[Medline]
18. Flanagan-Cato, L. M., King, J. F., Blechman, J. G. & O’Brien, M. P. (1998) Estrogen reduces cholecystokinin-induced c-Fos expression in the rat brain. Neuroendocrinology 67:384-391.[Medline]
19. Drewett, R. F. (1974) The meal patterns of the oestrous cycle and their motivational significance. Q. J. Exp. Psychol. 26:489-494.[Medline]
20. Blaustein, J. D. & Wade, G. N. (1976) Ovarian influences on the meal patterns of female rats. Physiol. Behav. 17:201-208.[Medline]
21. Butcher, R. L., Collins, W. E. & Fugo, N. W. (1974) Plasma concentration of LH, FSH, prolactin, progesterone and estradiol-17ß throughout the 4-day estrous cycle of the rat. Endocrinology 94:1704-1708.
22. Cecchini, D. J., Chattoraj, S. C., Fanous, A. S., Panda, S. K., Brennan, T. F. & Edelin, K. C. (1983) Radioimmunoassay of 2-hydroxyestrone in plasma during the estrous cycle of the rat: interrelationships with estradiol, progesterone, and the gonadotropins. Endocrinology 112:1122-1126.
23. Lu, J. K., LaPolt, P. S., Nass, T. E., Matt, D. W. & Judd, H. L. (1985) Relation of circulating estradiol and progesterone to gonadotropin secretion and estrous cyclicity in aging female rats. Endocrinology 116:1953-1959.
24. Harris, R. A., Paxton, R. & Parker, R. A. (1982) Activation of the branched-chain alpha-ketoacid dehydrogenase complex by a broad specificity protein phosphatase. Biochem. Biophys. Res. Commun. 107:1497-1503.[Medline]
25. Shimomura, Y., Paxton, R., Ozawa, T. & Harris, R. A. (1987) Purification of branched chain alpha-ketoacid dehydrogenase complex from rat liver. Anal. Biochem. 163:74-78.[Medline]
26. Shimomura, Y., Nanaumi, N., Suzuki, M. & Harris, R. A. (1991) Immunochemical identification of branched-chain 2-oxo acid dehydrogenase kinase. FEBS Lett. 288:95-97.[Medline]
27. Cagen, L. M. & Baer, P. G. (1987) Effects of gonadectomy and steroid treatment on renal prostaglandin 9-ketoreductase activity in the rat. Life Sci. 40:95-100.[Medline]
28. Nakai, N., Kobayashi, R., Popov, K. M., Harris, R. A. & Shimomura, Y. (2000) Determination of branched-chain alpha-keto acid dehydrogenase activity state and branched-chain alpha-keto acid dehydrogenase kinase activity and protein in mammalian tissues. Methods Enzymol. 324:48-62.[Medline]
29. Laemmli, U. K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (Lond.) 227:680-685.[Medline]
30. Geary, N. & Asarian, L. (1999) Cyclic estradiol treatment normalizes body weight and test meal size in ovariectomized rats. Physiol. Behav. 67:141-147.[Medline]
31. Varma, M., Chai, J. K., Meguid, M. M., Laviano, A., Gleason, J. R., Yang, Z. J. & Blaha, V. (1999) Effect of estradiol and progesterone on daily rhythm in food intake and feeding patterns in Fischer rats. Physiol. Behav. 68:99-107.[Medline]
32. Liang, Y. Q., Akishita, M., Kim, S., Ako, J., Hashimoto, M., Iijima, K., Ohike, Y., Watanabe, T., Sudoh, N., Toba, K., Yoshizumi, M. & Ouchi, Y. (2002) Estrogen receptor beta is involved in the anorectic action of estrogen. Int. J. Obes. Relat. Metab. Disord. 26:1103-1109.[Medline]
33. Nagle, C. A., Cardinali, D. P. & Rosner, J. M. (1974) Diurnal rhythms in tissue radioactivity uptake after 3H-estradiol and 3H-testosterone administration to castrated rats. Steroids Lipids Res. 5:107-112.[Medline]
34. Roy, E. J. & Wilson, M. A. (1981) Diurnal rhythm of cytoplasmic estrogen receptors in the rat brain in the absence of circulating estrogens. Science (Washington, DC) 213:1525-1527.
35. Butera, P. C., Xiong, M., Davis, R. J. & Platania, S. P. (1996) Central implants of dilute estradiol enhance the satiety effect of CCK-8. Behav. Neurosci. 110:823-830.[Medline]
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