Serine Dehydratase mRNA Research Articles

Excess Leucine Intake Induces Serine Dehydratase in Rat Liver

We have hypothesized that rat liver serine dehydratase (SDH) is induced in response to the amount of surplus amino acids from dietary protein. In the present study, we found that excess leucine intake strongly induced SDH activity in the liver but not in the kidney of rats. The increase in activity was accompanied by increases in the levels of SDH mRNA. On the other hand, isoleucine and valine had little effect on SDH induction. These results support our hypothesis and suggest that leucine is a signal for SDH induction.

Enzymatic and biochemical properties of a novel human serine dehydratase isoform

A cDNA clone similar to human serine dehydratase (SDH) is deposited in the GenBank/EMBL databases, but its structural and functional bases remain unknown. Despite the occurrence of mRNA, the expected protein level was found to be low in cultured cells. To learn about physicochemical properties of the protein, we expressed the cDNA in Escherichia coli, and compared the expressed protein with that of a hepatic SDH. The purified protein showed l-serine and l-threonine dehydratase activity, demonstrating to be an isoform of SDH. However, their K m and V max constants were different in a range of two-order. Removal of Pro128 from the hepatic SDH consisting of 328 residues, which is missing in the corresponding position of the isoform consisting of 329 residues, significantly changed the Michaelis constants and K d value for pyridoxal 5′-phosphate, whereas addition of a proline residue to the isoform was without effect. These findings suggest the difference in the structures of the active sites of the two enzymes. Another striking feature was that the expressed level of the isoform in E. coli was 7-fold lower than that of the hepatic SDH. Substitution of Val for Leu287 in the isoform dramatically increased the protein level. The high yield of the mutated isoform was also confirmed by the in vitro transcription and translation experiment. The poor expression of the isoform could be explained by the more stable secondary structure of the mRNA than that of the hepatic SDH mRNA. The present findings may provide a clue as to why the protein level in cultured cells is low.

Inverse correlation between the nitrogen balance and induction of rat liver serine dehydratase (SDH) by dietary protein.

Rats of different ages (3 to 15-wk-old) were fed on a 25% casein diet for one week, and the nitrogen balance and liver serine dehydratase (SDH, EC 4.2.1.13) activity were then determined. The value for nitrogen balance decreased with the age of the rats, while the liver SDH activity increased. A statistical analysis showed clear inverse correlation between the two factors (R(2) = 0.7372, p < 0.01). This result suggests that SDH was induced by response to the amount of surplus amino acids from dietary protein taken beyond the body's requirement. The increase in SDH activity was accompanied by an increase in the level of SDH mRNA. Since the half-life of this mRNA did not change significantly, the induction was mainly controlled at the level of transcription. In addition, the induction seems not to be related to gluconeogenesis, since the mRNA levels of tyrosine aminotransferase (TAT) and phosphoenolpyruvate carboxykinase (PEPCK), other gluconeogenic enzymes, were not changed under these experimental conditions.

Clinical predictors of cognitive function change in schizophrenia

Serine dehydratase mRNA in the livers of rats maintained on laboratory chow (containing 22% protein) under a 12-h light (7:00-19:00)/12-h dark (19:00-7:00) cycle showed a daily oscillation that was maximal at 19:00 and minimal at 7:00 with an amplitude of more than 20-fold. The feeding of high-protein diets (60 and 91% casein) did not affect the oscillation phase. Virtually identical oscillatory phases for the mature serine dehydratase mRNA and its precursor RNAs, and the results of nuclear run-off transcription assays indicated that the oscillation was generated at the level of transcription. Similar oscillation patters were seen in adrenalectomized, starved, or diabetic rats. However, the mRNA oscillation was not precisely reflected in the oscillation of enzyme activity or enzyme protein in both the phase and the amplitude. Rats maintained on laboratory chow under constant light for 2 weeks still showed mRNA oscillation with a peak at 3:00, whereas rats exposed to constant light and fed between 7:00 and 9:00 exhibited an oscillation peak at 3:00-7:00. Under the conditions of a 12-h light/dark cycle and a 2-h restricted feeding between 7:00 and 9:00, a broad peak was seen at 23:00-3:00. These results indicate that the rhythmic expression of serine dehydratase gene is controlled by both meal-responsive and photoresponsive regulators.

Morphological changes induced by extracellular matrix are correlated with maturation of rat small hepatocytes.

Small hepatocytes (SHs), which are known to be hepatic progenitor cells, were isolated from an adult rat liver. SHs in a colony sometimes change their shape from small to large and from flat to rising/piled-up. The aim of the present study is to clarify whether the alteration of cell shape is correlated with the maturation of SHs and whether extracellular matrix (ECM) can induce the morphological changes of SHs. We used liver-enriched transcription factors (LETFs) such as hepatocyte nuclear factor (HNF) 4 alpha, HNF6, CCAAT/enhancer binding proteins (C/EBP) alpha, and C/EBP beta, tryptophan 2,3-dioxygenase (TO), and serine dehydratase (SDH) as markers of hepatic maturation. To enrich the number of SH colonies, the colonies were isolated from dishes and replated. Replated colonies proliferated and the average number of cells per colony was about five times larger at day 9 than at day 1. When the cells were treated with laminin, type IV collagen, a mixture of laminin and type IV collagen, Matrigel or collagen gel (CG), only the cells treated with Matrigel dramatically changed their shape within several days and had reduced growth activity, whereas the cells treated with other ECM did not. HNF4 alpha, HNF6, C/EBP alpha, C/EBP beta, and TO were well expressed in the cells treated with Matrigel. Furthermore, addition of both glucagon and dexamethasone dramatically induced the expression of SDH mRNA and protein in the cells treated with Matrigel. In conclusion, morphological changes of SHs may be correlated with hepatic maturation and basement membrane (BM)-like structure may induce the morphological changes of SHs.

Recovery of mRNA expression of tryptophan 2,3-dioxygenase and serine dehydratase in long-term cultures of primary rat hepatocytes.

Expression of tryptophan 2,3-dioxygenase (TO) and serine dehydratase (SDH) has not previously been maintained or re-induced in long-term cultured hepatocytes. In the present study, we succeeded in inducing expression of TO and SDH mRNAs in adult rat hepatocytes cultured in serum-free L-15 medium supplemented with epidermal growth factor and 2% dimethyl sulfoxide (DMSO). After the start of culture, the expression of TO mRNA rapidly disappeared and at 96 h it was less than 10% of that at isolation. However, after the addition of 2% DMSO from 96 h, the transcript level gradually increased and reached about 40% of that of the isolated cells at day 14. In addition, the expression of TO mRNA was enhanced in cells treated with both 10(-5) M dexamethasone and 10(-7) M glucagon. In contrast, the expression of SDH mRNA decreased very rapidly and we could not detect it after 24 h of culture. Furthermore, 2% DMSO failed to induce it. However, when both 10(-5) M dexamethasone and 10(-7) M glucagon were added to the culture medium at day 9, we observed dramatic induction of SDH mRNA 24 h later. Primary hepatocytes cultured by this method could express and maintain highly differentiated hepatic functions for a long time. Thus, this in vitro system is suitable for the investigation of hepatic functions.

Developmental patterns of diurnal variations in 3-hydroxy-3-methylglutaryl coenzyme A reductase, D-site binding protein (DBP), and serine dehydratase mRNA levels in rat liver

Rats were housed under a 12-hr light (07:00-19:00)/12-hr dark cycle. Hepatic 3-hydroxy-3-methyglutaryl coenzyme A reductase mRNA appeared at 3 weeks of age, and diurnal variation was established at 5 weeks of age. Diurnal variation of the D-Site binding protein (DBP) mRNA was faintly seen 1 week after birth and became evident with age. Serine dehydratase mRNA level was high at 11:00 in the livers of rats at 2–3 days of age, and at 15:00 in those at 1–2 weeks of age. The rhythm became ambiguous at 3–4 weeks of age, but an adult-type rhythm with a peak at 19:00 was established at 5 weeks of age.

Diurnal Rhythms of Rat Liver Serine Dehydratase, D-Site Binding Protein, and 3-Hydroxy-3-methylglutaryl Coenzyme A Reductase mRNA Levels Are Altered by Destruction of the Suprachiasmatic Nucleus of the Hypothalamus

It is generally accepted that a clock generating biological rhythms exists in the suprachiasmatic nuclei (SCN) of the hypothalamus in rodents. We have examined if diurnal variations of rat liver serine dehydratase (SDH), D-site binding protein (DBP), and 3-hydrogy-3-methylglutaryl coenzyme A reductase (HMGR) mRNA levels, each of which exhibits low values during the daytime (06:00-18:00) and high values during the dark period, are controlled by the SCN, The SCN were destructed electrolytically, and, 2 weeks later, rats were sacrificed at 06:00, 12:00, 18:00, and 24:00. Completeness of SCN lesions was assessed by the alteration of spontaneous locomotor activity and histological examination of the brains. The SDH and DBP mRNA levels in SCN-lesioned rats were very low at 06:00 and almost eliminated at 18:00, whereas the HMGR mRNA level at 06:00 was low at 06:00 and slightly increased at 18:00 compared to the control values. When determined at 12:00 and 24:00, the SDH, DBP, and HMGR mRNA levels in sham-operated rats were higher at 24:00 than at 12:00, whereas those in SCN-lesioned rats were higher at 12:00 than at 24:00. The hepatic β-actin and glycine N-methyltransferase mRNA levels showed no significant diurnal variation and were not affected by SCN lesions. These results indicate that the SCN also play a pivotal role in determining rhythmic gene expressions in liver.

Periportal expression of the serine dehydratase gene in rat liver

The mRNA for rat liver serine dehydratase, a gluconeogenic enzyme, exhibits a circadian rhythm with a maximum at the onset of darkness marking the end of the fasting period and a minimum at the onset of light that marks the end of the feeding period, when rats have free access to food and water. In situ hybridization with an antisense cRNA probe revealed that serine dehydratase mRNA was localized in the periportal area of rat liver parenchyma in the evening, whereas it was scarce in the liver in the morning. The predominant localization of serine dehydratase mRNA in the periportal area also occurred in livers of rats that underwent laparotomy, glucagon and dexamethasone administration, and streptozotocin-induced diabetes mellitus, all of which are known to induce serine dehydratase mRNA levels remarkably. Immunostaining revealed that the localization of serine dehydratase protein agreed with that of succinate dehydrogenase, another enzyme known to be predominant in the periportal zone. Thus, the periportal serine dehydratase gene expression strongly supports the idea of metabolic zonation that gluconeogenesis from amino acids occurs preferentially in the periportal parenchyma of rat liver.

Laparotomy Causes a Transient Induction of Rat Liver Serine Dehydratase mRNA

Rat liver serine dehydratase mRNA shows rhythmicity with a high level at the onset of dark (19:00) and a low level at the onset of light (07:00). We have examined the effect of stress (laparotomy) on the rhythm, Upon laparotomy at 09:00 or 17:00, a marked induction of serine dehydratase mRNA occurred 2 h after operation. The elevated mRNA level then decreased and the original mRNA rhythm resumed 2 days later. By contrast, the transcription activator protein DBP mRNA level which shows a similar oscillation was not affected by this treatment. The induction was also seen in adrenalectomized rats that had been treated with hydrocortisone but not with saline or noradrenaline, indicating that glucocorticoids are absolutely necessary for the induction. Pretreatment of rats with phenoxybenzamine, and prazosin prevented the effect of laparotomy, but propranolol and yohimbine had no effect, indicating the necessity of the α1-adrenergic stimulation for the induction. These results suggest that laparotomy releases glucocorticoids and neural noradrenaline that stimulates α1-adrenergic receptors, thereby leading to the serine dehydratase gene expression.

Diurnal Variation of the Serine Dehydratase mRNA Level in Rat Liver

Serine dehydratase mRNA in the livers of rats maintained on laboratory chow (containing 22% protein) under a 12-h light (7:00-19:00)/12-h dark (19:00-7:00) cycle showed a daily oscillation that was maximal at 19:00 and minimal at 7:00 with an amplitude of more than 20-fold. The feeding of high-protein diets (60 and 91% casein) did not affect the oscillation phase. Virtually identical oscillatory phases for the mature serine dehydratase mRNA and its precursor RNAs, and the results of nuclear run-off transcription assays indicated that the oscillation was generated at the level of transcription. Similar oscillation patters were seen in adrenalectomized, starved, or diabetic rats. However, the mRNA oscillation was not precisely reflected in the oscillation of enzyme activity or enzyme protein in both the phase and the amplitude. Rats maintained on laboratory chow under constant light for 2 weeks still showed mRNA oscillation with a peak at 3:00, whereas rats exposed to constant light and fed between 7:00 and 9:00 exhibited an oscillation peak at 3:00-7:00. Under the conditions of a 12-h light/dark cycle and a 2-h restricted feeding between 7:00 and 9:00, a broad peak was seen at 23:00-3:00. These results indicate that the rhythmic expression of serine dehydratase gene is controlled by both meal-responsive and photoresponsive regulators.

Nutritional regulation and tissue-specific expression of the serine dehydratase gene in rat.

The mechanism of dietary regulation and tissue-specific expression of the serine dehydratase gene in rat has been studied. The hepatic serine dehydratase activity and its mRNA showed a parallel increase with increasing protein content in the diet. However, when rats that had been maintained on a high protein diet were fed a protein-free diet, the mRNA level rapidly decreased to 0.5 in 3 h, whereas the enzyme activity gradually fell to a low level over a period of 5 days. With animals maintained on a high protein diet or on a protein-free diet, we examined the sites hypersensitive to DNase I in the 5'-flanking region of serine dehydratase gene in the liver chromatins. A series of DNase I-hypersensitive sites were located within 10.5 kilobase pairs upstream of the transcription start site. The DNA regions at -3050 and -3180 (region II) and -3600 to -3850 (region III) were more susceptible to the nuclease in the expressing than in the nonexpressing liver. A reverse situation obtained at -100 (region I). Kidney contained serine dehydratase mRNA at a level of 5% of liver as determined by Northern blotting. The kidney chromatin was found to be susceptible to DNase I only at region I. No conspicuous DNase I-hypersensitive sites were observed in the relevant regions of chromatins from brain and lung, in which serine dehydratase mRNA was scarcely transcribed. These results suggest that nutritional control and tissue-specific expression of the serine dehydratase gene is closely associated with the alteration of DNase I hypersensitivity at specific sites of the 5'-flanking region of the gene.

Organization and Structure of the 5′ Flanking Region of the Rat Serine Dehydratase Gene1

For study of the mechanisms regulating the induction of serine dehydratase by various hormones in rat liver [Noda et al. (1988) J. Biol. Chem. 263, 14764-14768], we cloned the gene for this enzyme from a rat genomic library. The gene spans about 7.5 kilobases and consists of nine exons and eight introns. The exon-intron boundaries are consistent with the "GT-AG" rule. Southern blot analysis of rat genomic DNA suggested the presence of one copy of serine dehydratase gene per haploid genome. The 5' end of serine dehydratase mRNA is located 148 nucleotides upstream of the initiator methionine codon. ATG, determined by primer extension analysis and S1 nuclease mapping, although an alternative transcription initiation site(s) may be located a few bases downstream. The 5' flanking region of the gene lacks typical TATA and CCAAT sequences, but contains AATAAA and CATT sequences, at -25 to -20 and -54 to -51, respectively. Furthermore, there are five GC box-related sequences. There are three putative glucocorticoid-responsive elements and two copies of the CGTCA motif of the cAMP-responsive element upstream of the promoters. The 5' flanking sequence shows more than 98% homology with that reported by Ogawa et al. [(1988) Proc. Natl. Acad. Sci. U.S.A. 85, 5809-5813], but the first exons have different sequences. Another difference is that a segment of 108 nucleotides is located just upstream of exon 5 in the sequence reported here, but is included as an exon in the sequence of Ogawa et al. The possibility to producing two species of serine dehydratase mRNAs from a single gene by transcription from different sites and alternative splicing is discussed.

Developmental and growth-related regulation of expression of serine dehydratase mRNA in rat liver

In rat liver, serine dehydratase mRNA is undetectable in the late prenatal period, but its level increases rapidly after birth to a transient peak, and then after decrease gradually increases again to a maximum 2 weeks after birth that is slightly higher than that of adult liver. To determine whether mature quiescent hepatocytes proliferate without loss of differentiated functions, we measured the serine dehydratase mRNA contents in regenerating liver and primary cultured hepatocytes from adult rats. Partial hepatectomy resulted in a dramatic decrease in the mRNA content within 24 h and then its recovery within a week. In subconfluent cultures of adult rat hepatocytes that did not grow even in the presence of mitogens, serine dehydratase mRNA was maintained at a high level. However, when the hepatocytes were cultured at low cell density without added mitogens, their serine dehydratase mRNA content decreases to a quarter of that of subconfluent cultures. The possibility that the expression of serine dehydratase mRNA is regulated in G0 G1 transition before entry into the S phase and the relationship of the mRNA with growth are discussed.

Requirements of both glucocorticoids and glucagon as co-inducers for activation of transcription of the serine dehydratase gene in cultured rat hepatocytes.

Induction of translatable mRNA for serine dehydratase (SDH) in primary cultured rat hepatocytes requires both dexamethasone and glucagon or cAMP (Noda, C., Tomomura, M., Nakamura, T., and Ichihara, A. (1986) J. Biochem. (Tokyo) 95, 37-45). This unique hormone requirement was studied further with a cDNA probe complementary to SDH mRNA in primary cultured hepatocytes of adult rats. Dot-blot hybridization analysis of RNA showed that SDH mRNA was induced by dexamethasone and glucagon together, but not by either alone. Insulin or epinephrine caused 40% inhibition of this induction of SDH mRNA. Cycloheximide prevented the induction of SDH mRNA by dexamethasone and glucagon, suggesting that ongoing protein synthesis is required for the induction by glucocorticoids and glucagon. In vitro transcription experiments using nuclei isolated from cultured hepatocytes showed that transcription of the SDH gene was not affected by either dexamethasone or glucagon alone, but markedly enhanced by both hormones together, and that this enhancement was inhibited by insulin or epinephrine. These results indicate that the inhibition of SDH induction by epinephrine or insulin was due to effects of these hormones on the transcriptional rate of the SDH gene.

Isolation and nucleotide sequence of the cDNA for rat liver serine dehydratase mRNA and structures of the 5' and 3' flanking regions of the serine dehydratase gene.

Rat serine dehydratase cDNA clones were isolated from a lambda gt11 cDNA library on the basis of their reactivity with monospecific immunoglobulin to the purified enzyme. Using the cDNA insert from a clone that encoded the serine dehydratase subunit as a probe, additional clones were isolated from the same library by plaque hybridization. Nucleotide sequence analysis of the largest clone obtained showed that it has 1444 base pairs with an open reading frame consisting of 1089 base pairs. The deduced amino acid sequence contained sequences of several portions of the serine dehydratase protein, as determined by Edman degradation. Rat liver serine dehydratase mRNA virtually disappeared from livers of rats fed a protein-free diet for 5 days. Several genomic clones were isolated from two libraries. Determinations of the transcription start site and the structure of the 3' flanking region of the gene indicated that the coded mRNA is 1504 nucleotides long. The 5' promoter region contained a variety of sequences similar to several consensus sequences believed to be important for the regulation of specific gene expression.

Primary structure of rat liver serine dehydratase deduced from the cDNA sequence

The nucleotide sequence of serine dehydratase mRNA of rat liver has been determined from a recombinant cDNA clone, previously cloned in this laboratory, and from a recombinant cDNA clone screened from a primer-extended cDNA library. The sequence of 1322 nucleotides includes the entire protein coding region and noncoding regions on the 3′- and 5′-sides. The deduced polypeptide consists of 327 amino acid residues with a calculated molecular mass of 34462 Da. Comparison of the amino acid sequences of the serine dehydratase polypeptide with those of biosynthetic threonine dehydratase of yeast and biodegradative threonine dehydratase of E. coli revealed various extents of homology. A heptapeptide sequence, Gly-Ser-Phe-Lys-Ile-Arg-Gly, which is the pyridoxal-binding site in the yeast and E. coli threonine dehydratases, was found as a highly conserved sequence.

Molecular cloning of DNA complementary to mRNA of rat liver serine dehydratase

A cDNA clone containing sequences complementary to the mRNA cording for rat hepatic serine dehydratase was isolated to study the multihormonal regulation of this enzyme. Serine dehydratase mRNA was partially purified (50-fold enrichment, 8.2% of the total mRNA activity) from the liver of rats fed high protein diet by polysome immunoadsorption followed by oligo(dT)-cellulose column chromatography. This preparation was used as template for synthesis of cDNA. Double-stranded cDNA sequences were inserted into the plasmid pBR322 and cloned in Esherichia coli DHl. Of 860 transformants screened, 6 clones containing DNA complementary to serine dehydratase mRNA were identified by differential colony hybridization and hybrid-selected translation. The length of serine dehydratase mRNA was estimated to be 1,500 bases by Northern blot analysis. One cloned cDNA comprised about 1,000 base pairs, or 65% of the length of the mRNA. The amount of the mRNA was greatly increased in the liver of rats given high protein diet.

Hormonal control of serine dehydratase mRNA in primary cultures of adult rat hepatocytes.

The mechanism of hormonal induction of serine dehydratase [EC 4.2.1.13, SDH] was studied in primary cultures of adult rat hepatocytes by measuring the rates of syntheses of the enzyme protein and its translatable mRNA. The rate of synthesis of enzyme protein, measured as incorporation of [3H]leucine into the enzyme protein in hepatocytes, was increased 4-5 times by dexamethasone (Dex) plus glucagon. Neither hormone alone increased the rate. The increased rate induced by the two hormones was suppressed by insulin and epinephrine. The decay curves of [3H]leucine-labeled SDH showed that these hormones did not affect the rate of enzyme degradation. The level of translatable mRNA was determined by measuring cell-free synthesis of SDH in a reticulocyte lysate system. Dex plus glucagon increased the level of mRNA of SDH in hepatocytes. Insulin and epinephrine suppressed this increase without changing the rate of mRNA degradation. The level of mRNA changed in parallel with that of the rate of synthesis of the enzyme protein. These results suggest that these hormones regulate transcription of SDH, rather than its translation. After pretreatment of hepatocytes with Dex, further addition of glucagon caused more rapid induction of mRNA of SDH than addition of both hormones together. The effect of glucagon after pretreatment with Dex was inhibited by actinomycin D and alpha-amanitin, suggesting that glucagon does not affect post-transcription, but transcription per se. The requirement for both Dex and glucagon for induction of this enzyme is discussed in comparison with the requirements for either hormone alone for inductions of other gluconeogenic enzymes.