Hormonal Regulation of Aldolase B Gene Expression in Rat Primary Cultured Hepatocytes

Hormonal Regulation of Aldolase B Gene Expression in Rat Primary Cultured Hepatocytes

ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Vol. 350, No. 2, February 15, pp. 291–297, 1998 Article No. BB970527 Hormonal Regulation of Aldolase B Gene ...

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ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS

Vol. 350, No. 2, February 15, pp. 291–297, 1998 Article No. BB970527

Hormonal Regulation of Aldolase B Gene Expression in Rat Primary Cultured Hepatocytes Jun-itsu Ito, Takejiro Kuzumaki, Kaoru Otsu, Yoshihito Iuchi, and Kiichi Ishikawa1 Department of Biochemistry, Yamagata University School of Medicine, Iida-Nishi 2-2-2, Yamagata 990-23, Japan

Received September 16, 1997, and in revised form November 13, 1997

Gene expression of aldolase B, an important enzyme for glucose and fructose metabolism, is regulated by hormones. We examined direct effects of major hormones on aldolase B gene expression in rat primary cultured hepatocytes, in comparison with those on the gene expression of phospho(enol)pyruvate carboxykinase (PEPCK), a key enzyme for gluconeogenesis. Insulin, dexamethasone, and high concentration of glucose increased aldolase B mRNA abundance in the hepatocytes. Glucagon strongly suppressed aldolase B gene expression, and this hormone canceled the stimulative effects of insulin, dexamethasone, and high concentration of glucose. Epinephrine and thyroxine slightly reduced aldolase B mRNA abundance, but these hormones did not cancel the stimulative effects of insulin and dexamethasone. To the contrary, expression of PEPCK gene was suppressed by insulin, dexamethasone, and high concentration of glucose, and remarkably induced by glucagon. Glucagon rapidly suppressed aldolase B gene expression at the transcriptional level. Forskolin and dibutyryl cAMP mimicked the suppressive effect of glucagon on aldolase B gene expression. These results suggest that glucagon may be a key regulator of aldolase B gene transcription through a cAMP/protein kinase A-signaling pathway. q 1998 Academic Press Key Words: Aldolase B gene; phospho(enol)pyruvate carboxykinase gene; transcriptional control; primary cultured hepatocyte; hormonal effects; glucagon.

Liver functions as a major site of blood glucose homeostasis. Many hormones are involved in regulation of the glycolytic and gluconeogenetic pathways in the liver. Insulin lowers blood glucose levels by activating key enzymes in the glycolytic pathway in many organs and by suppressing those in the gluconeogenetic path1 To whom correspondence should be addressed. Fax: 0236-285230. E-mail: [email protected]

way in the liver (1). In contrast, glucagon increases blood glucose levels by suppressing glycogen synthesis and glycolysis and by stimulating glycogenolysis and gluconeogenesis. Glucocorticoids, epinephrine, growth hormone, and thyroxine are also known to be involved in regulation of blood glucose level (2–4). Hormonal regulation of glycolysis and gluconeogenesis in the liver can be divided into short-term and longterm regulations. Short-term regulation involves allosteric regulation of enzyme activities through phosphorylation of corresponding enzymes. Glucagon raises the intracellular cAMP level, which in turn activates PKA.2 The phosphorylation of glycogen phosphorylase by PKA accelerates glycogen breakdown. The phosphorylation of pyruvate kinase and 6-phosphofructo2-kinase/fructose 2,6-bisphosphatase by PKA inhibits these enzyme activities and suppresses glycolysis. All changes of these enzyme activities enhance glucose production in the liver. Insulin, on the other hand, opposes the action of glucagon by activating phosphodiesterase and inhibiting PKA (4). Long-term regulation involves quantitative changes of key enzymes by hormonal regulation of gene transcription. The expression of glucokinase and L-type pyruvate kinase genes, which are key enzymes in the glycolytic pathway, is induced by insulin and inhibited by glucagon (5). Conversely, the expression of PEPCK and fructose 1,6-bisphosphatase genes, which are key enzymes in the gluconeogenetic pathway, is induced by glucagon and inhibited by insulin (6, 7). Aldolase B (EC 4.1.2.13) plays an important role in regulation of glucose and fructose metabolism. It catalyzes the conversion between fructose 1,6-bisphosphate and glyceraldehyde 3-phosphate/dihydroxyacetone phosphate in both glycolytic and gluconeogenetic pathways. It also can catalyze the conversion of fruc2 Abbreviations used: PEPCK, phospho(enol)pyruvate carboxykinase; GAPDH, glyceraldehyde phosphate dehydrogenase; CRE, cyclic AMP-responsive element; CREB, CRE- binding protein; PKA, protein kinase A; FBS, fetal bovine serum.

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tose 1-phosphate to glyceraldehyde and dihydroxyacetone phosphate in fructose metabolism (8). Aldolase B gene is transcribed tissue specifically in the liver, kidney, and upper intestine (9, 10). Dietary regulation of aldolase B gene expression is accomplished through the action of hormones (11). Insulin, triiodo-L-thyronine, and cortisone are effective in vivo to increase the mRNA abundance of aldolase B in the liver, and glucagon is effective to reduce it (12, 13). We previously reported that dietary regulation of aldolase B gene expression was mediated by insulin and glucagon levels in the plasma (14). In this paper, we report the qualitative and quantitative analyses of regulatory effects of major hormones on aldolase B gene expression in rat primary cultured hepatocytes. Our results demonstrate that the effect of glucagon is predominant over all other hormones via cAMP/PKA pathway. Possible mechanism for the inhibition of aldolase B gene transcription by glucagon is discussed. MATERIALS AND METHODS Chemicals. Insulin, glucagon, dexamethasone, thyroxine, epinephrine, somatotropin (growth hormone), forskolin, dibutyryl cAMP, and Williams’ medium E were purchased from Sigma Chemical Co. (St. Louis, MO). Collagenase was from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). Type I collagen and 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H-7) were from Funakoshi Chemical Co. (Tokyo, Japan). [a-32P]dCTP (3000 Ci/mmol) and [a-32P]UTP (3000 Ci/mmol) were from Amersham Japan Co. (Tokyo, Japan). Random primer DNA labeling kit was from Takara Shuzo Co. (Kyoto, Japan). Primary hepatocytes culture. Eight-week-old male Wistar rats (180–220 g) were kept in our Laboratory Animal Center and fed pellets of nutritionally balanced Charles River Formula-1 (Oriental East Co., Japan) and water ad libitum. Hepatocytes were isolated from the rat liver by the two-step collagenase perfusion method as described by Seglen et al. (15). Tissue culture plates (60 mm diameter) were treated overnight at 47C with 2.5 ml/plate of 0.3 mg/ml type I collagen. After washing the plates with phosphate-buffered saline, the hepatocytes (2 1 106 cells) in 4 ml of Williams’ medium E (supplemented with 100 nM insulin, 100 nM dexamethasone, 30 mg/ml kanamycin, 25 mg/ml cefazolin, and 10% FBS) were seeded onto each plate. After incubation of the cells at least for 1 h, nonattached hepatocytes were removed and the attached hepatocytes were cultured in the fresh medium containing 1 nM insulin, 1 nM dexamethasone, and 5% FBS for 20 h. After successive incubation for 3 h in the fresh medium containing 5% FBS (without insulin and dexamethasone), the hormones to be tested were added to the medium. When the cells were cultured in a high concentration of glucose, the medium (11 mM glucose) was changed to that containing 33 mM glucose. DNA probes. DNA probes used for Northern blot hybridization and nuclear run-on assay are as follows: rat aldolase B cDNA (0.8 kb PvuII fragment) (16), rat PEPCK cDNA (1.3 kb SalI–SphI fragment) (17), rat GAPDH cDNA (0.81 kb HindIII–BamHI fragment) (18), rat albumin cDNA (0.95 kb HindIII–PstI fragment) (19). All cDNAs were inserted in pBSK (/) plasmids. The DNA fragment was used as a probe in Northern blot hybridization and whole plasmid was used as a probe in nuclear run-on assay. RNA extraction and Northern blot hybridization. Total RNA was extracted from hepatocytes by the acid guanidinium thiocyanate– phenol–chloroform method (20). Each 10 mg of total RNA was elec-

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trophoresed in 1% agarose gel containing 6% formaldehyde and RNA was completely transferred to a Hybond-N/ membrane (Amersham Japan Co., Tokyo, Japan) (21). RNA was fixed to the membrane by UV cross-linking with an ultraviolet cross-linker (Amersham Japan Co.). Probes labeled with 32P were prepared with random primer DNA labeling kit. After prehybridization for 2 h in prehybridization solution (51 SSC, 1% SDS, 11 Denhardt’s solution), the membrane was incubated with a 32P-labeled probe in hybridization solution (0.75 M NaCl, 20 mM Tris–HCl (pH 8.0), 2.5 mM EDTA, 11 Denhardt’s solution, 1% SDS, and 50 mg/ml salmon sperm DNA) at 657C for 16 h. The membrane was washed twice in washing solution (30 mM NaCl, 3 mM sodium citrate (pH 7.0), and 0.1% SDS) at 657C for 30 min. The radioactivity of a hybridized RNA was quantified by Fujix Bioimaging Analyzer BAS 1000 Mac (Fuji Photo Film Co. Ltd., Tokyo, Japan). The amount of RNA sample applied was confirmed by an intensity of ethidium bromide staining of ribosomal RNAs in the same sample. The radioactive counts of hybridized RNAs were equalized on the basis of the amount of ribosomal RNAs. Nuclear run-on assay. Nuclei of primary cultured hepatocytes were isolated and suspended in lysis buffer (50 mM Tris–HCl (pH 8.3), 40% glycerol, 5 mM MgCl2 , and 0.1 mM EDTA) according to the method described by Greenberg and Ziff (22), and stored at 0807C. Nuclei (40– 50 mg of DNA) were incubated in 100 ml of reaction mixture containing 50 mM Tris–HCl (pH 8.0), 5 mM MgCl2 , 150 mM KCl, 2.5 mM dithiothreitol, 1 mM of ATP, GTP, and CTP, and 200 mCi of [a-32P]UTP at 257C for 30 min as described by Danesch et al. (23). RNA labeled with 32 P was purified by the acid guanidinium thiocyanate–phenol–chloroform method (20) and hybridized to the DNA probes immobilized on Hybond-N/ membrane in hybridization solution containing 50% formamide, 51 SSC, 50 mM sodium phosphate (pH 6.5), 0.1% SDS, 11 Denhardt’s solution, and 250 mg/ml of salmon sperm DNA at 657C for 48 h. The membrane was washed twice in washing solution (30 mM NaCl, 3 mM sodium citrate (pH 7.0), and 0.1% SDS) at 657C for 30 min. The radioactivity on a hybridized RNA was quantified by Fujix Bioimaging Analyzer BAS 1000 Mac.

RESULTS

Effects of Hormones on Aldolase B, PEPCK, GAPDH, and Albumin Gene Expression Primary cultured hepatocytes were prepared from the livers of rats fed ad libitum and cultured in the medium containing insulin and dexamethasone as described under Materials and Methods. Prior to each experiment, the cells were cultured for 3 h in the medium without insulin and dexamethasone. The hormones under study were then added to the culture medium, and the mRNA levels of aldolase B, PEPCK, GAPDH, and albumin were determined by Northern blot hybridization (Fig. 1). Insulin increased the mRNA abundance of aldolase B and GAPDH by 1.5- to 2-fold that in the hepatocytes cultured without insulin (control) in a dose-dependent manner. In contrast, insulin suppressed PEPCK mRNA to 10–25% of the control. Dexamethasone caused an increase of aldolase B and albumin mRNAs by 2- to 2.5-fold over the control but suppressed PEPCK mRNA. In contrast to the effects of insulin and dexamethasone, glucagon suppressed the mRNA level of aldolase B to almost 25% of the control, and raised that of PEPCK by more than 35-fold. Epinephrine suppressed aldolase B mRNA to 70–80% of the control and raised that of PEPCK by about 10-fold. With the exception of aldolase B mRNA whose level

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FIG. 1. Effects of hormones on aldolase B, GAPDH, albumin, and PEPCK gene expression. (A) Northern blot hybridization in the hepatocytes cultured with glucagon. Lane 1, cells cultured without glucagon; lane 2–7, cells cultured with various concentrations of glucagon (lane 2, 10010 M; lane 3, 1009 M; lane 4, 1008 M; lane 5, 1007 M; lane 6, 1006 M; lane 7, 1005 M). (B) The dose-dependent changes of mRNA levels of aldolase B (j), GAPDH (h), and albumin (n) caused by hormones. (C) The dose-dependent changes of mRNA level of PEPCK caused by hormones. Primary cultured hepatocytes were isolated and cultured by the procedures described under Materials and Methods. Hepatocytes were cultured with various concentrations of insulin, dexamethasone, glucagon, epinephrine, growth hormone, and thyroxine for 3 or 24 h. Total cellular RNAs were extracted at 3 h (PEPCK) or at 24 h (aldolase B, GAPDH, and albumin) after the addition of hormones to the culture medium and analyzed by Northern blot hybridization. The radioactivity of each specific RNA band was quantified by Fujix Bioimaging Analyzer BAS 1000 Mac. The values represent relative radioactive counts of the bands in comparison with that of the control (no hormone) taken as 1.00. The values represent the mean { SE (n Å 3–7).

decreased slightly by addition of thyroxine, neither growth hormone nor thyroxine exerted any significant effect on other mRNA abundance. Synergistic Effects of Hormones on Aldolase B and PEPCK Gene Expression We examined the synergistic effects of major hormones on the expression of aldolase B and PEPCK

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genes since many hormones act synergistically in vivo. Insulin and dexamethasone exerted their effects additively to increase aldolase B mRNA by about 4-fold. Interestingly, glucagon canceled these effects. The stimulative effect of glucagon on PEPCK mRNA was not canceled by insulin and/or dexamethasone (Fig. 2A). Epinephrine, growth hormone, and thyroxine produced no significant effect on the effects of insulin and dexamethasone on aldolase B and PEPCK gene expres-

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FIG. 3. Effect of glucose concentration on aldolase B and PEPCK gene expression. Hepatocytes were cultured without hormones (C), with 100 nM glucagon (G), with 100 nM insulin (I), or with 100 nM glucagon plus 100 nM insulin (G/I). The medium containing 11 mM glucose was changed to the medium containing 33 mM glucose (high glucose) at the time when hormones were added to the culture medium. Total cellular RNAs were extracted at 3 h (PEPCK) and at 24 h (aldolase B) after the addition of hormones to the culture medium and analyzed by Northern blot hybridization. The values represent relative radioactive counts of the bands in comparison with that of the control (no hormone) taken as 1.00. The values represent the mean { SE (n Å 3).

FIG. 2. Synergistic effects of hormones on aldolase B and PEPCK gene expression. (A) Hepatocytes were treated with 100 nM insulin (I), 100 nM dexamethasone (D), or 100 nM insulin plus 100 nM dexamethasone (I/D) in the presence (underlined) or absence of 100 nM glucagon (G). Glucagon was simultaneously added with insulin or dexamethasone to the culture medium. (B) Hepatocytes were treated with 100 nM insulin (I) or 100 nM dexamethasone (D). 1 mM epinephrine (E), 100 mIU/ml growth hormone (GH), and 100 nM thyroxine (T) were simultaneously added with insulin or dexamethasone to the culture medium. Total cellular RNAs were extracted at 3 h (PEPCK) and at 24 h (aldolase B) after the addition of hormones to the culture medium and analyzed by Northern blot hybridization. The values represent relative radioactive counts of the bands in comparison with that of the control (no hormone) taken as 1.00. The values represent the mean { SE (n Å 3–4).

decreased to about 50% of the control (Fig. 3). Glucagon effectively suppressed aldolase B mRNA and increased PEPCK mRNA even if cells were cultured in the high concentration of glucose. Effect of Glucagon on Aldolase B, Albumin, GAPDH, and PEPCK Transcription Rates Transcription rates were measured by nuclear runon assay with nuclei prepared from the hepatocytes cultured with or without glucagon. When hepatocytes were cultured with 100 nM glucagon, the transcription rate of aldolase B gene decreased to about 75% of that of the hepatocytes cultured without glucagon (control) at 30 min after the addition of glucagon, and to about 30% of the control at 6 h (Fig. 4). The transcription

sion except that epinephrine exerted a stimulative effect on PEPCK gene expression (Fig. 2B). These results suggest that the effect of glucagon predominates over the effects of other hormones on aldolase B and PEPCK gene expression. Effect of Glucose Concentration on Aldolase B and PEPCK Gene Expression All experiments presented in Figs. 1 and 2 were carried out using the medium containing 11 mM glucose. When hepatocytes were cultured in the medium containing 33 mM glucose (high glucose), the aldolase B mRNA increased by about 4-fold while PEPCK mRNA

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FIG. 4. Effect of glucagon on the transcription rates of aldolase B, albumin, GAPDH, and PEPCK genes. Hepatocytes were cultured with or without 100 nM glucagon. At each time point indicated at the top of the figure, the nuclei were isolated and used for the nuclear run-on assay. The labeled RNA was hybridized to the specific cDNAs (indicated at the left of each row) spotted on the membrane and detected by Fujix Bioimaging Analyzer BAS 1000 Mac.

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scription rate of PEPCK gene increased by 18-fold within 30 min and then decreased rapidly. These results indicate that the effect of glucagon on the transcription of aldolase B and PEPCK genes occurs immediately after the addition of glucagon to the culture medium. Effect of cAMP on Aldolase B and PEPCK Gene Expression To clarify the signaling pathways of glucagon leading to down-regulation of aldolase B gene expression and up-regulation of PEPCK gene expression, we examined the effects of dibutyryl cAMP, forskolin, and H-7 (Fig. 6). Similar to glucagon, forskolin (an activator of adenylate cyclase) and dibutyryl cAMP decreased aldolase

FIG. 5. Changes of mRNA levels and transcription rates of aldolase B and PEPCK genes as a function of time after glucagon addition. Hepatocytes were cultured with or without 100 nM glucagon for the time period indicated on the abscissa of each graph. At each time point, total cellular RNAs were extracted for Northern blot hybridization and nuclei were isolated for nuclear run-on assay. The radioactivity of each band was quantified by Fujix Bioimaging Analyzer BAS 1000 Mac. The values represent relative radioactive counts of the bands in comparison with that of the sample at 0 h taken as 1.00. The values in the graphs of nuclear run-on assay were normalized relative to the values of GAPDH. The results are the means of two independent experiments carried out in duplicate. Upper graphs, mRNA levels determined by Northern blot hybridization; lower graphs, transcription levels determined by nuclear run-on assay. (s) Cells cultured without glucagon; (l) cells cultured with 100 nM glucagon.

rate of PEPCK gene increased by about 18-fold within 30 min after the addition of glucagon and then decreased. The transcription rates of albumin and GAPDH did not change appreciably after the addition of glucagon. These results indicate that glucagon exerted the effect on aldolase B and PEPCK gene expression at the transcriptional level. Changes of mRNA Levels and Transcription Rates as a Function of Time after the Addition of Glucagon The changes in mRNA levels and transcription rates of aldolase B and PEPCK genes by glucagon were shown as a function of time (Fig. 5). The mRNA level of aldolase B remained constant for 2 h after the addition of glucagon, and then decreased to about 20% of the control. The transcription rate of aldolase B gene decreased immediately and fell to about 30% at 6 h after the addition of glucagon. The PEPCK mRNA increased by about 35-fold within 3 h after the addition of glucagon and then decreased gradually. The tran-

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FIG. 6. Effects of dibutyryl cAMP, forskolin, and H-7 on (A) aldolase B and (B) PEPCK gene expression. Hepatocytes were cultured for 3 or 24 h with various concentrations of forskolin, dibutyryl cAMP (db-cAMP), and H-7. H-7 was simultaneously added to the culture medium with 100 nM glucagon. Total cellular RNAs were extracted at 3 h (PEPCK) and at 24 h (aldolase B) after the addition of reagents to the culture medium and analyzed by Northern blot hybridization. The values represent relative radioactive counts of the bands in comparison with that of the control (no hormone and no reagent) taken as 1.00. The values in the graph of glucagon / H-7 were normalized relative to the level of mRNA in the cells treated with each concentration of H-7 without glucagon. The values represent the mean { SE (n Å 3).

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B mRNA and increased PEPCK mRNA in a dose-dependent manner. H-7, an isoquinolinesulfonamide derivative which is a potent inhibitor of cyclic nucleotidedependent protein kinases (24), canceled the effect of glucagon on aldolase B and PEPCK gene expression. The results strongly suggest that glucagon exerted its effects on the transcription of aldolase B and PEPCK genes through the cAMP/PKA-signaling pathway. DISCUSSION

The liver is the most important organ for the control of glucose level in the blood, and glucose metabolism in the liver is controlled by many hormones. Hepatocytes store excess glucose as glycogen and supply glucose from glycogen when needed. Glucagon is the major regulator of glycogen metabolism by regulating the activities of glycogen synthetase and glycogen phosphorylase by phosphorylation. When glycogen is depleted, glucose is mainly produced by inducing the gene expression of PEPCK, fructose 1,6-bisphosphatase, and glucose-6phosphatase, which are key enzymes in the gluconeogenetic pathway. Inversely, glucagon suppresses the gene expression of glucokinase, phosphofructokinase, and Ltype pyruvate kinase, which are key enzymes in the glycolytic pathway (5–7). In this experiment, we showed that glucagon acted dominantly over other hormones in regulation of aldolase B and PEPCK gene expression. Glucagon regulated these genes at the transcriptional level. Glucagon shut off the transcription of aldolase B gene and induced that of PEPCK gene immediately after the addition of glucagon. These results suggest that the blood level of glucagon is a fundamental factor to determine the blood glucose level in vivo. We observed that forskolin and dibutyryl cAMP mimicked the effect of glucagon on the gene expression of aldolase B and PEPCK. These results suggest that glucagon suppresses the transcription of aldolase B gene and activates the transcription of PEPCK gene through the activation of PKA which phosphorylates and activates CREB. Two elements homologous to cAMP-responsive element (CRE-1 and CRE-2) are found in the promoter region of PEPCK gene. CRE-1 (094-CTTACGTCAGAG-084), but not CRE-2, is the potent transcriptional activating site recognized by CREB (7, 25, 26). Similarly, we could find two CRE-like sequences in the promoter region (from about 0300 to /100) of aldolase B gene, one in the upstream (CRE 089) and one in the downstream (CRE /13) of transcription start site. Gel retardation assay and DNA footprinting assay (data not shown) showed that some nuclear proteins could bind to both sequences. In addition to the fundamental elements such as TATA box and polypurine (PPu) elements to which ubiquitous transcription factors (i.e., TFIID including TATA-binding factor (TBP) and polypurine factors) bind, aldolase B

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promoter has at least three elements essential for tissue-specific expression (sites A, B, and C) to which many liver-enriched nuclear factors bind. HNF-1, ALFB (27), NF-Y, ALF-C2 (28), and C/EBP activate the transcription of aldolase B gene, and HNF-3, Ryb-a (29), and ALF-C1 (28) suppress its transcription. The binding of these trans-factors synergistically determines the level of aldolase B gene transcription in the liver (31). In addition to these fundamental regulations, many hormones synergistically control the transcription of this gene in the liver. Our preliminary study suggests that CRE /13 and/or CRE 089 sites are the potential candidate(s) for the cis-element of glucagon effect. If this is true, then the mechanism for suppression of aldolase B gene transcription by glucagon can be considered as follows. When glucagon is given to the hepatocytes, CREB is phosphorylated at Ser-133 by PKA activated by binding the increased cAMP (30–33). When the phosphorylated CREB or CREB-related proteins bind to CRE /13 and/or CRE 089 sites, it is possible that basal transcription factor complexes including RNA polymerase II cannot form around the transcription initiation site. This model can explain that the effect of glucagon is predominant over the effects of other hormones for suppression of aldolase B gene transcription. However, this working hypothesis is not verified yet. Further critical examination to certify the involvement of these CRE-like elements must be carried out to elucidate the mechanism for suppression of aldolase B gene transcription by glucagon. ACKNOWLEDGMENTS We thank Drs. D. K. Granner (Vanderbilt University Medical Center, U.S.A.), S. Ruppert (German Cancer Research Center, Germany), and C. Noda (Tokushima University, Japan) for their generous supply of cDNA probes for PEPCK, GAPDH, and albumin, respectively. Thanks are also given to Professor M. Jacobs-Lorena (Case Western Reserve University, U.S.A.) for critical reading of the manuscript.

REFERENCES 1. O’Brien, M. O., and Granner, D. K. (1991) Biochem. J. 278, 609– 619. 2. Sikama, H., and Ui, M. (1975) Am. J. Physiol. 229, 962–966. 3. Vigneri, R., Squatrito, S., Pezzino, V., Filetti, S., Branca, S., and Polosa, P. (1976) Diabetes 25, 167–172. 4. Pilkis, S. J., El-Maghrabi, M. R., and Claus, T. H. (1988) Annu. Rev. Biochem. 57, 755–783. 5. Matsuda, T., Noguchi, T., Yamada, K., Takenaka, M., and Tanaka, T. (1990) J. Biochem. 108, 778–784. 6. Lemaigre, F. P., and Rousseau, G. G. (1994) Biochem. J. 303, 1– 14. 7. Pilkis, S. J., and Granner, D. K. (1992) Annu. Rev. Physiol. 54, 885–909. 8. Adelman, R. C., Spolter, P. D., and Weinhouse, S. (1966) J. Biol. Chem. 241, 5467–5472. 9. Horecker, B. L., Tsolas, O., and Lai, C. Y. (1974) The Enzymes

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10. 11.

12.

13.

14. 15. 16.

17.

18. 19. 20.

(Boyer, P. D., Ed.), Vol. 7, pp. 213–258. Academic Press, New York. Sato, J., Tsutsumi, K., Ishikawa, M., and Ishikawa, K. (1987) Arch. Biochem. Biophys. 254, 116–123. Weber, A., Marie, J., Cottreau, D., Simon, M-P., Besmond, C., Dreyfus, J-C., and Kahn, A. (1984) J. Biol. Chem. 259, 1798– 1802. Vaulont, S., Munnich, A., Marie, J., Reach, G., Pichard, A-L., Simon, M-P., Besmond, C., Barbry, P., and Kahn, A. (1984) Biochem. Biophys. Res. Commun. 125, 135–141. Munnich, A., Besmond, C., Darquy, S., Reach, G., Vaulont, S., Dreyfus, J-C., and Kahn, A. (1985) J. Clin. Invest. 75, 1045– 1052. Gomez, P. F., Ito, K., Huang, Y., Otsu, K., Kuzumaki, T., and Ishikawa, K. (1994) Arch. Biochem. Biophys. 314, 307–314. Seglen, P. O. (1976) Methods Cell Biol. 13, 29–83. Tsutsumi, K., Mukai, T., Tsutsumi, R., Mori, M., Daimon, M., Tanaka, T., Yatsuki, H., Hori, K., and Ishikawa, K. (1984) J. Biol. Chem. 259, 14572–14575. Sasaki, K., Cripe, T. P., Koch, S. R., Andreone, T. L., Petersen, D. D., Beale, E. G., and Granner, D. K. (1984) J. Biol. Chem. 259, 15242–15251. Ruppert, S., Boshart, M., Bosch, F. X., Schmid, W., Fournier, R. E. K., and Schu¨tz, G. (1990) Cell 61, 895–904. Kioussis, D., Hamilton, R., Hanson, R. W., Tilghman, S. M., and Taylor, J. M. (1979) Proc. Natl. Acad. Sci. USA 76, 4370–4374. Chomczynski, P., and Sacchi, N. (1987) Anal. Biochem. 162, 156– 159.

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21. Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual (Ford, N., and Nolan, C., Eds.), 2nd ed., pp. 7.43–7.50, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. 22. Greenberg, M. E., and Ziff, E. B. (1984) Nature 311, 433–438. 23. Danesch, U., Hashimoto, S., Renkawitz, R., and Schu¨tz, G. (1983) J. Biol. Chem. 258, 4750–4753. 24. Hidaka, H., Inagaki, M., Kawamoto, S., and Sasaki, Y. (1984) Biochemistry 23, 5036–5041. 25. Quinn, P. G., Wong, T. W., Magnuson, M. A., Shabb, J. B., and Granner, D. K. (1988) Mol. Cell. Biol. 8, 3467–3475. 26. Park, E. A., Roesler, W. J., Liu, J., Klemm, D. J., Gurney, A. l., Thatcher, J. D., Shuman, J., Friedman, A., and Hanson, R. W. (1990) Mol. Cell. Biol. 10, 6264–6272. 27. Tsutsumi, K., Ito, K., Yabuki, T., and Ishikawa, K. (1993) FEBS Lett. 321, 51–54. 28. Yabuki, T., Ejiri, S., and Tsutsumi, K. (1993) Biochim. Biophys. Acta 1216, 15–19. 29. Ito, K., Tsutsumi, K., Kuzumaki, T., Gomez, P. F., Otsu, K., and Ishikawa, K. (1994) Nucleic Acids Res. 22, 2036–2041. 30. Lamph, W. W., Dwarki, V. J., Ofir, R., Montminy, M. R., and Verma, I. M. (1990) Proc. Natl. Acad. Sci. USA 87, 4320–4324. 31. Ishikawa, K., Ito, K., and Tsutsumi, K. (1993). Yamagata Med. J. 11, 1–13. 32. Gonzalez, G. A., Yamamoto, K. K., Fisher, W. H., Karr, D., Menzel, P., Biggs III, W. B., Vale, W. W., and Montminy, M. R. (1989) Nature 337, 749–752. 33. Gonzalez, G. A., and Montminy, M. R. (1989) Cell 59, 675–680.

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