Effects of nutrients and insulin on transcriptional and post-transcriptional regulation of glucose-6-phosphate dehydrogenase synthesis in rat liver

Biochimica et Biophysica Acta, 1006 (1989) 104-110 Elsevier

104

BBALIP 53256

Effects of nutrients and insulin on transcriptional and post-transcriptional regulation of glucose-6-phosphate dehydrogenase synthesis in rat liver Akihiko K a t s u r a d a i, N o b u k o Iritani i, Hitomi F u k u d a ~, Y o h k o M a t s u m u r a z, Tamio Noguchi 2 and Takehiko T a n a k a ~I Te~ukcopama6akuln College. 2 Department of Nutrition and Pl~ysiologicalChemistry, Osaka University Medical School, Osaka (Japan)

(Received 16 May 1989)

Key words: Transcriptional control; Insulin; Olucose-6-phosphate dehydrogena~; eDNA: (Rat liver)

The transaiptional and post-transcriptional regulation of glucose-6-phosphate dehydrogenase induction of rat liver was InvestJOated using a eDNA cloned in our laboratory. By feeding a carbohydrate/protein diet to fasted rats, the mRNA eoneoneratlon and enzyme Induction el glueme.6-phosphate debydrogenase (EC 1,1.1.49) reached maximal levels about 104old thou In the f m , d rats at 16 h and 72 It, respectively, whereas the transcriptional rate was increased about 3-fold In 6 It. in the proteIn fed (without carbohydrate) group, both the mRNA concentration and enzyme induction were Ineremed to about 60~ of the levels in the carbohydrate/protein fed group and in the group fed on a carbohydrate diet (without protein) to 30.-40~. Further, dietary fat significantly r ¢ ~ the transcriptional rate, mRNA concentration and enzyme induction to less than half, suggesting that dietary fat primarily reduced transcription. Thus, dietary nutflents appear to he Invelved In the steps preceding the translation. On the other hand, in diabetic rats, the transcriptional rate was alpifleonfly deaemed as compared to the normal level and restored by Insulin-treatment in 4 h. The mLNA ¢oneentrathm was very low In diabetic r a ~ at~ was restored to the normal level by insulin treatment in 8 h, and was half ratoeed by fructose feeding. However, the enzyme induction of giucose-6-phosphate dehydrogenase was scarcely restored by fmete~ unless aeemapankd by insulIn treatment. Thus, it is suggested that insulin is involved in translation as well as In trans~ption, Further, the Insulin-dependent increase of 8lucose-6-phosphate dehydrogenase mRNA was blocked by eydohexhuhle, suggesting that synthesis of a peptide is required.

Introduction The induction of hepatic iipogenic enzymes is markedly increased in animals fed a fat-free/highcarbohydrate diet. The eDNA of malic enzyme (EC I.I.IA0), which is one of the lipogenic enzymes and a NADPH donor for fatty acid synthesis, was previously cloned in our laboratory [1]. We have investigated the transcriptional and post-transcriptional regulation of malic enzyme using eDNA in rat liver [1,2]. Carbohydrate feeding (even without protein feeding) markedly i ~ the concentration of malic enzyme mRNA, rather than the transcriptional rate. However, enzyme induction was not increa,_sedmuch without protein feeding,

Ommpondence: N. IritanL Tezukayama Gakuin College, 4-Cho,

Jtmmidai, Sahi, Osaka,590.01,Japan.

On the other hand, glucose-6-phosphate dehydrogenase, which is one of the enzymes in the pentose phosphate pathway, also provides NADPH for fatty acid synthesis. A number of studies on the nutritional and hormonal regulation of glucose-6-phosphate dehydrogenase in the enzyme level have been reported [3-10]. Kletzien et al. [11] reported that a high-carbohydrate diet increased the amount of glucose-6-phosphate dehydrogenase mRNA, which paralleled the increase in enzyme activity. However, the transcriptional and posttranscriptional regulation of glucose-6-phosphate dehydrogenase is not very clear. In the present experiment, the effects of dietary carbohydrate, protein and fat on the transcriptional rate, mRNA concentration and enzyme induction of glucose-6-phosphate dehydrogenase in rat liver have been investigated following cDNA cloning. In addition, as insulin is thought to be the main inducer of lipogenic enzymes under physiological condition [7-22], insulin action on the transcription of glu-

0005-2760/~9/~03.50 © 1989 Elsevier Science Publishers B.V. (Biomedical Division)

105 cose-6-phosphate dehydrogenase has been investigated. We previously found that a high-fructose diet, even in diabetic rats, increased malic enzyme mRNA aetivhy, but to a lesser extent than insulin treatment did [12]. Therefore, the effect of fructose on glucose-6-phosphate dehydrogenase induction was also investigated in diabetic rats. Materials and Methods

Chemicals [a-32p]dATP (3000 Ci/mmol), [a-32p]dCTP (3000 Ci/mmol) and [7-32p]ATP (3000 Ci/mmol) were purchased from New England Nuclear. [a-32p]UTP was purchased from ICN. Nylon filter was purchased from Amersham (Hybond N) and New England Nuclear (Colony Plaque Screen). Reverse transcriptase, restriction enzymes, synthetic EcoRI linker and nucleotides were purchased from Takara Shuzo Co. (Japan). Sequenase was obtained from United States Biochemical Corporation. fl-Actin eDNA was purchased from Wako Pure Chemicals (Japan). Animals Male Wistar rats (Shizuoka Animals, Japan), 5 weeks old, were fasted for 2 days and then refed for 3 days with four kinds of diet: (I) a 67% carbohydrate and 18% protein diet, (II) an 85% carbohydrate diet without protein, (IlI) an 85% protein diet without carbohydrate, and (IV) a 57% carbohydrate, 18% protein and 10% fat diet (w/w). Sucrose, casein and corn oil were used for carbohydrate, protein and fat, respectively. Each diet contained the common components of 9.9?0 cellulose, 5?0 salts, 0.1% choline chloride and vitamins [23]. The animals were allowed to take water ad libitum and were fed essentially the isocalorie diet for body weight. They were kept under an automatic lighting schedule from 0800 h to 2000 h at 24* C. Male Wistar rats, 6 weeks old, were made diabetic by intravenous injection of streptozotocin (6 rag/100 g) after starvation for 20 h [24]. Blood glucose was assayed 3 days after streptozotoein treatment [25] and rats with blood glucose levels of over 300 mg/dl were used for experiments. The diabetic rats were given a high-glucose diet ad libitum. The composition of the high-glucose diet was the same as diet (I). For insulin treatment, animals were subcutaneously injected with Lente insulin and intraperitoneally with Actrapid insulin as described in the figures and tables. Some animals were intraperitoneally injected with cyeloheximide (1 mg/100 g) just after the insulin injection and killed 6 h after the insulin injection. For fructose feeding, animals were given 1 ml of 20% fructose orally at the beginning and shifted to a high-fructose diet. Fructose was substituted for glucose in the diet. An aliquot of each liver was quickly removed and homogenized with 3 vols of 0.25 M sucrose

to measure the activity of glucose-6-phosphate dehydrogenase in 105000 x g supernatant [26]. Liver nuclei were isolated as described below. Another aliquot of the liver was immediately frozen to measure the concentration of mRNA.

Preparation of RNA RNA was isolated from the tissues of the experimental animals with guanidine tldocyanate as described by Chirgwin et al. [27]. Poly(A)+RNA was isolated by affinity chromatography of oligo(dT)-ceUulose [28]. cDNA synthesis and library construction Poly (A)+RNA (1 /~g) from rat liver was used to prepare eDNA in a reaction employing reverse transcriptase. The second strand was synthesized as described by Gubler and Hoffman [29]. The doublestranded eDNA was then treated with EcoRl methylase [30] and blunt ended with a large fragment of DNA polymerase I. EcoRI linkers were ligated to the cDNA with T4 polynucleotide ligase [30]. The eDNA was then digested with EcoRI and then excess linkers were removed by 1% agarose gel electrophoresis. The resulting eDNA fragment was ligated to EcoRI-cleaved Agtl0 arms. The eDNA packaged into phage particles using Gigapack Gold (Vector Cloning System) according to the supplier's protocol. Recombinant DNA was used to transform Escherichia colt (C600 hfl) as described [31]. Screening of the ~ gtl 0 library A synthetic 50-mer probe, identical to the eDNA for the human glucose-6-phosphate dehydrogenase (nucleic acid residues 1102-1151) was prepared [32]. The sequence of the probe is as follows: 5 '-TCCAGCTCCGACTCCTCGGGGTTGAAGAACATGCCCGGCTTCTTGGTCAT-3'. The 5'-terminal of the probe was labeled with [732P]ATP using T4 polynucleotide kinase by the method of Maniatis et al. [30]. Recombinant clones were fixed onto colony plaque screens (New England Nuclear) according to the supplier's protocol. The filters were prehybridized for at least 3 h at 42°C in 50 mM Tris-HCl (pH 7.5), 6 x SSC (1 x SSC: 0.15 M NaCl, 0.015 M sodium citrate), 5 × Denhardt's solution, I% SDS and 100/~g/ml of denatured salmon sperm DNA. The Denhardt's solution contained 0.02% Ficoll, 0.02% polyvinylpyrrolidone and 0.02% bovine serum albumin. The 32p-labeled probe was hybridized to the filters for 24 h at 37°C in the same solution. The filters were washed twice at 42°C in 1 x SSC and 0.170 SDS for 20 min per wash. The filters were exposed for various lengths of time at - 7 0 °C to Kodak X-Omat AR film with Du pont Lighting-Plus intensifying screens. DNA from the clones that had hybridized with the probe was isolated from plate lysates as described by Manfioletti et al. [33] and subcloned in phage M13mp9 [34] for

106 sequence analysis by dideoxy chain termination using Sequenase [35,361.

Dot blot hybridization assay Cloned glucose-6-phosphate dehydrogenase cDNA was subdoned into the EcoRl site of pUCI8 [37]. The cDNA was isolated by the alkaline lysis method [30]. The cDNA was labeled by a mulfiprimer DNA labeling system kit (Ama,~un) using [3zP]dATP. To measure the mRNA concentration of glucose-6-phosphate dehydrogenase, the RNA (20 ttS) was denatured with formamide at 65°C for 15 rain, spotted on a nylon filter and then was radiated with ultraviolet light for 5 min. To measure the mRNA concentration of p-actin, 5 pg of the RNA was spotted on the filter. The filter was prehybridized at 50°C for at least 4 h in a reaction mixture of 50 mM Tris-HCI (pH 7.5), 6 × SSC, 5 × Denhardt's solution, 1% SDS, 50% formamide and 100 ~$/ml of denatured salmon sperm DNA (medium A). The hybridization was carried out for 36 h at 42°C in medium A with ~2P.labeled cloned glucose-6-phosphate dehydrogenase eDNA insert. The nitrocellulose filter was then washed with 0.1 x SSC and 0.1% SDS at room temperature, and three times at 55°C. The filters were exposed for various lengths of time at - 70 ° C to Kodak X-Omat AR film with Du pont Lighting-Plus intensify. ing screens. Relative densities of the hybridization signals were determined by scanning the autoradiograms at 525 rim. Northern blot analysis Northern blot analysis of RNA was performed as described by Gonzales and Kasper [38]. Poly(A) '~RNA (5 or 20~tg) was denatured in 50% formamide, 2.2 M formaldehyde, and 20 mM 3-morpholinepropanesulfohie acid (pH 7.0) at 65°C for 5 rain. The RNA was electrophoresed on a 0.8% aSarose gel containin8 2.2 M formaldehyde. The gel was blotted onto nylon filter as des~bed by Thomas [39]. The filter was radiated with ultraviolet light for 5 rain and then baked at 80 °C for 2 h. Prehybridization, hybridization and autoradiosraphy were carried out using the dot blot hybridization method. Isolation of nuclei and measurement of transcription Isolation of nuclei from liver and subsequent in vitro transcription assay were performed as described by Lamers et al. [40]. Briefly, liver nuclei were isolated and suspended in 50% glycerol, 5 mM MgCI 2, 0.1 mM EDTA, 50 mM Hepes (pH 7.5) and stored at - 8 0 o C. The transcription reaction mixture contained nuclei (20 absorbance units at 260 nm), 25% glycerol, 2.5 mM MgCI 2, 0.05 mM EDTA, 75 mM Hepes (pH 7.5), 100 mM KCI, 4 mM dithiothreitol, 0.5 mM CTP, 0.5 mM GTP, 1.0 mM ATP, creatine kinase (0.04 mg/ml), 8.8 mM creatine phosphate and 100-200 ltCi of [32P]UTP

in a final vol. of 0.2 ml. Incubation was carried out at 30°C for 20 min. The nuclei were then treated with ribonuclease-free deoxyribonuclease I (EC 3.1.21.1) and proteinase K (EC 3.4.21.14), and RNA was extracted with phenol/chloroform (1:1) and precipitated with ethanol. [32p]RNA was hybridized to 3/tg of pG1 or pUC18 bound to a nitrocellulose filter in 0.5 ml of 50 mM Tris (pH 7.5), 6 × SSC, 5 x Denhardt's solution, 0.1% SDS, 50% formamide and 100 Izg/rnl of denatured salmon sperm DNA. About 9000 cpm of [3H]cRNA synthesized from the pG1 cDNA insert [41] was included in each hybridization mixture to assess the hybridization efficiency. The filter was washed at 50°C for 30 rain with 2 x SSC containing 0.1% SDS, and washed at 50°C with 0.2 × SSC containing 0.1% SDS and then rinsed with 0.2 × SSC. Then the filters were treated with 10 /tg/ml ribonuclease A in 2 × SSC at 37 °C for 20 rain and washed with 2 × SSC at 50 °C for 30 rain. The filters were air dried and then autoradiographed using an intensifying screen. To quantitate the amount of RNA bound to each dot of the cDNA, the autoradiographic image of the dot was scanned using a densitometer [42]. Results

Isolation and properties of cDNA clone for rat liver glucose.6.phosphate dehydrogenase The ~,gtl0 cDNA library was screened with a (5'"~2P)-labeled oligonuclcotide probe. From 5.104 unamplified recombinant phages, one positive clone (pG1) was isolated. Then the clone was subcloned into the EeoRI site of pUC18 and M13mp9. The nucleotide sequence was determined by the dideoxytermination method. The cDNA insert has 374 base pairs. The nucleotide sequence data perfectly coincide with the position 1217-1590, containing the termination codon, as was reported by He et al. [43] for glucose-6-phosphate dehydrogenase cDNA of rat liver. The restriction map of the cloned cDNA is shown in Fig. 1. Northern blot-hybridization analyses of poly(A) + RNA of rat tissues revealed a single hybridization band of about 21 S, as shown in Fig. 2. On applying 5/tg of poly(A) + RNA, 0 L..

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1.5 I

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Fig. l. Restrictionmap and sequencestrategyfor glucose-6-pbosphate dehydrogenasecDNA.The blackbar indicatesthe codingregion.

107 Transcriptional rate cO

.~-28 S ~--18 S

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mRNA concentration

ID C

1

2

3

4

5

6

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Fig. 2. Northern blot analysis of poly(A) + RNA in rat tissues. Poly(A) + RNAs were isolated from the pooled tissues of two rats and separated on a 0.8~ agarose gel containing 2.2 M formaldehyde. The RNA was then transferred to a nitrocellulose filter and hybridized with the 32P-labeled eDNA probe. The RNA markers were 18 S (2.0 kb) and 28 S (5.1 kb). Lanes 1, 2, 3, 4, 5 and 6 show RNA bands fol~ muscle, liver, intestine, heart, brain and adipose tissue, respectively. 5 /~g of liver, intestine and adipose tissue RNA were applied on the gel, and 20 ~g of muscle, heart and brain RNA.

the hybridization band was clearly found in liver, adipose tissue and intestine. Although the band was not found in heart, brain and muscle at that time, it was found in these tissues on applying 20 #g of the RNA. Effects of dietary nutrients on transcriptional rate, m R N A concentration and enzyme induction T h e h e p a t i c t r a n s c r i p t i o n a l rates, m R N A c o n c e n t r a t i o n s a n d e n z y m e activities o f g l u e o s e - 6 - p h o s p h a t e deh y d r o g e n a s e in rats r e f e d a 677o c a r b o h y d r a t e / 1 8 % p r o t e i n diet, a n 85% c a r b o h y d r a t e diet ( w i t h o u t p r o t e i n ) o r a n 85% p r o t e i n diet ( w i t h o u t c a r b o h y d r a t e ) w e r e m e a s u r e d . I n a p r e v i o u s r e p o r t [44], we s h o w e d t h a t c h a n g e s in the e n z y m e a c t i v i t y w e r e a c c o m p a n i e d b y p r o p o r t i o n a l c h a n g e s in t h e q u a n t i t y o f i m m u n o c h e m i tally reactive protein during dietary manipulation. Using t h e e D N A c l o n e d as d e s c r i b e d a b o v e , the m R N A c o n c e n t r a t i o n w a s m e a s u r e d b y d o t - b l o t h y b r i d i z a t i o n as-

Enzyme activity

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Fig. 3. Changes in transcriptional rate, mRNA concentration and enzyme induction of glucose-6-phosphate dehydrogenase after feeding the carbohydrate/protein diet to fasted rats. After refeeding the carbohydrate/protein diet to fasted rats, time courses for transcriptional rate, mRNA concentration and enzyme activity of hepatic glucose-6-phosphate dehydrogenase were followed. The animals were killed at the time indicated after the refeeding. The enzyme activities are expressed as mU/mg protein (nmol utilized NADP/nfin per mg at 37 o C). Other results are showr as fold change for the values of fasted rats (at zero time). The experiment was repeated three times and one of the typical results i.~ shown in the figure. Mean 4. S.D. (n = 3). Means with different superscript letters are significantly different at P < 0.05 at least (by one-way analysis of variance).

say. The rate of transcription was also measured by in vitro transcription assay of nuclei isolated from livers. The time courses for transcriptional rate, mRNA con-

TABLE !

Effects of dietary nutrients on enzyme induction, mRNA concentration and transcriptional rate of glucose.6-phosphate dehydrogenase in rat liver Rats were fasted for 2 days and then refed with four kinds of diet: 67~ carbohydrate/18~ protein diet, 85~ carbohydrate diet, 85~ protein diet and 57~ carbohydrate/18~ protein/10~ fat diet. The enzyme activities in the 105000 × g supernatant of liver homogenates were measured after refeeding for 3 days. 1 mU is defined as 1 nmol utifized NADP/min per mg at 37 o C. Data in parentheses show the fold change of the enzyme activity. The animals were killed at 16 h after refceding to measure the transcriptional rate and mRNA concentration. The concentrations of total cellular mRNA were quantified by dot-blot hybridization in duplicate. Relative rates of transcription were measured as described in 'Materials and Methods'. Hybridization efficiency averaged 50~. The results are shown as the fold change for the values of the carbohydrate/protein group. Mean 4- S.D. ( n = 6-9). Significantly different from carbohydrate/protein: * P < 0.001; * * P < 0.01 (by Student's t-test). Dietary group

Enzyme activity (mU/mg)

mRNA concentration (fold change)

Transcriptional rate (fold change)

Carbohydrate/protein Carbohydrate Protein Carbohydrate/protein/fat Fasted

392 +48.1 122 + 19.6 227 + 35.3 122 4. 23.5 47.04-11.8

1.004.0.21 0.394-0.10 0.57 4. 0.12 0.36 4- 0.10 0.11 4-0.04

1.004.0.10 0.324-0.20 0.52 4.0.17 0.53 4- 0.09 0.31 4-0.16

(1.00) *(0.31) * (0.58) * (0.31) *(0.12)

* ** * *

* * * *

108 centration and enzyme induction of glucose-6-phosphate dehydrogenase after refeeding the carbohydrate/ protein diet to fasted rats are shown in Fig. 3. The transcriptional rates reached a maximum level of 3-fold above the fasted level within 6 h and a similar level within 16 h. The mRNA concentration reached a maximal level of ll-fold in 16 h and then slightly decreased. The enzyme level reached a maximum level of 10-fold in 3 dayq. Thus, to compare effects of dietary nutrients on 81ucos~>4~.phosphate dehydrogenase induction, the transcfiptimal rate and mRNA concentration were measured tt 16 h and enzyme induction at 3 days, after refeeding. The results are shown in Table I. By feeding the carbohydrate/protein diet, the transcriptional rates of glucose-6-phosphate dehydrogenase in the liver gene were increased about 3-fold above the levels in the fasted animals. Although no significant itlcreases in the rates were statistically found by feeding diets containing either carbohydrate or protein alone above the fasted level, the rates tended to be increased by protein rather than by carbohydrate. In the carbohydrate/protein diet group, the enzyme and m R N A levels of 81ucose,6-phosphate dehydrogenase were increased about 10.fold above the corresponding levels in the fasted animals. In the carbohydrate diet group, the enzyme induction and m R N A concentration were 30-~)~$ of the levels in the carbohydrate/protein diet groups. In the protein diet group, the enzyme induction and mRNA concentration were about 60~ of those in the carbohydrate/protein group, On feeding fat, enzyme induction and m R N A concentration of $1ucose-6-phosphate dehydrogenase decreased to about one-third of the corresponding control levels in the fat-free diet group, The transcriptional rate was also significantly reduced by feeding fat,

Effects o/in~din.treatmont and fructose.feeding on transcriptional rate, mRNA concentration and enzyme induction The insulin action on 81ucose-6-phosphate dehydro8ChaSe synthesis was investigated using diabetic rats. In the diabetic rats, the transcriptional rate, m R N A concentration and enzyme activity of 81ucose-6-phosphate dehydrogenase were 34~, 13~ and 6~$ of the normal levels, respectively, Thus, changes in the transcriptional rate, m R N A concentration and enzyme induction were examined after injection of insufin, Further, when diabetic rats adapted to a 67~ glucose diet were shifted to a 67% fructose diet, the changes were examined and compared to the changes after the insulin treatment. The time course for the transcriptional rate after insulin treatment is shown in Fig, 4. The transcriptional rate reached the maximal level of 2.7-fold (about normal level) in 6 h. Fructose feeding slightly increased the transcriptional rate, but not significantly (1.00 + 0.10, 0.34 :!: 0.19 and 0.42 + 0.24, in fold change, for normal,

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Fig. 4. Changes in the transcriptional rate of glucose-6-phosphate dehydrogenase in diabetic rat liver after insulin treatment. Diabetic rats fed on a glucose diet were injected with 0.5 U of Actrapid insulin intraperitoneallly and 1.5 U of Lente insulin subcutaneously. Then the animals were killed at the time indicated after insulin treatment. The results are shown as the fold change over the values of diabetic rats (at zero time). The experiment was repeated three times and one typical result is shown in the figure. Mean :t:S.D. (n - 3). Means with different superscript letters are significantlydifferent at P < 0.05 at least (by one-wayanalysisof variance).

diabetic and fructose-fed diabetic rats, respectively). In.~ulin treatment caused a 7-fold increase to normal level in the m R N A concentration in 16 h and a 17-fold increase (also normal) in the enzyme activity in 3 days (Fig. 5). On the other hand, fructose feeding caused a maximum 4-fold increase in the m R N A concentration in 16 h, and in the enzyme activity, in 3 days. Thus, by feeding fructose to diabetic rats, the enzyme activity was not increased to the same extent as the mRNA, in contrast to the levels after the insufin treatment. It was demonstrated that changes in the enzyme activity of glucose-6-phosphate dehydrogenase in diabetic rat liver were accompanied by proportional changes in the quan-

TABLE !I Effects of cycloheximide on glucose.6.phosphate dehydrogenase mRNA concenwa;ion in rat liver

The diabetic rats adapted to a high.glucosediet were injected with 0.5 U of Actrapid insulin intraperitoneally and 1.5 U Lente insulin subcutaneously, and then cycloheximide(1 mg/100 g). The animals were killed 6 h after insulin treatment. Data are normalized to the value for non-treated diabetic rats and are expressed as the fold change. Data in parentheses are the results for p-actin. Mean+ S.D. (n ffi6). Significantly different from non-cycloheximide treatment, * P < 0.001; from non-insulin treatment, * * P < 0.001 (by Student's t-tesO. Treatment None Insulin

Cycloheximide treated (fold change) 0.92 + 0.38 (1.17:1:0.17) 2.38+0.62 * ** (1.15 :t:0.09)

Non-treated (fold change) 1.00:1:0.38 (1.00:!:0.17) 10.7 +1.46 ** (1.04+ 0.26)

109

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Fig. 5. Changes in mRNA concentration and enzyme activity of glucose-6-phosphate dehydrogenase in diabetic rat liver after insulin treatment or fructose feeding. For insuhn treatment, diabetic rats fed on a glucose diet were injected with 0.5 U of Actrapid insulin intraperitoneally and 1.5 U of Lente insulin subcutaneously. For 1, 2 or 3 days of insulin treatment, animals were subcutaneously injected with Lente insulin (1.5 U/rat) every 12 h. For fructose feeding, the diabetic animals were given 200 mg fructose orally and their diet was changed to a high fructose one. Then the animals were killed at the time indicated. The mRNA concentrations are normalized to the values for diabetic rats at zero time. Enzyme activities are expressed as mU/mg protein. Filled and open circles show the levels for asulin treatment and fructose feeding, respectively. Each point is the mean + S.D. (n = 6). Means with different superscript letters in each time course are significantly different at P < 0.05 at least (by one-way analysis of variance). :,A ihc ic~sulin treatment, the mRNA concentration was significantly different from that in fructose feeding (at P < 0.05 at least) at 4, 8, 16, 24, 48 and 72 h, and the enzyme activity was significantly higher at 16, 24, 48 and 72 h.

It appeared that protein was more responsible for increasing glucose-6-phosphate dehydrogenase synthesis than carbohydrate, probably at the transcription. Further, the transcriptional rate of glucose-6-phosphate dehydrogenase was significantly decreased by feeding fat. Thus, dietary nutrients appeared to be primarily involved in transcription of the glucose-6-phosphate dehydrogenase gene. lit has been postulated that in diabetic animals, the concentration of glucolytic metabolites, which are increased as a consequence of a primary action of insulin to induce glucokinase [45-47], are decreased. Even in the diabetic state, the level of fructoldnase is not markedly reduced, despite the low level of liver glucokinase [48], and fructose could be metabolized to yield an appreciable level of glycolytic intermediates. In the diabetic rats, feeding fructose increased the mRNA concentration of glucose-6-phosphate dehydrogenase to 60% of the normal level and increased the enzyme induction to 30%, while fructose feeding increased the transcription rate slightly (but not significantly). Therefore, the glycolytic intermediates may be involved in the stability of glucose-6-phosphate dehydrogenase mRNA. By feeding fructose to diabetic animals, the enzyme induction of glucose-6-phosphate dehydrogenase was not restored to the same extent as the mRNA concentration was. In diabetic animals, glucose-6-phosphate d~hydrogenase induction could be restored to the normal level only with insulin treatment. Further, the transcriptional rate in diabetic rats was decreased, but restored by insulin treatment. Thus, the present results suggest that insulin is involved in the transcription and translation of glucose-6-phosphate dehydrogenase. The insuhn-dependent increases in the transcriptional rate and mRNA concentration were blocked by cyclohexJmide, suggesting that synthesis of a protein is required. Acknowledgement

tity of immunochemically reactive protein during insulin or fructose treatment (data not shown). The insulin-dependent increase in the mRNA concentration of glucose-6-phosphate dehydrogenase was markedly inhibited by cycloheximide injection, as shown in Table II. The mRNA concentration of fl-actin was not changed by insulin treatment or by cycloheximide treatment. Discussion

When fasted rats were fed a carbohydrate/protein diet, a carbohydrate diet (without protein) or a protein diet (without carbohydrate), the transcriptional rates, mRNA concentrations and enzyme induction were about parallel. The levels in the protein diet group were about 60% of those in the carbohydrate/protein diet and in the carbohydrate diet group they were 30-40%.

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