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        Vitamin B12 deficiency results in the abnormal regulation of serine dehydratase and tyrosine aminotransferase activities correlated with impairment of the adenylyl cyclase system in rat liver
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        Vitamin B12 deficiency results in the abnormal regulation of serine dehydratase and tyrosine aminotransferase activities correlated with impairment of the adenylyl cyclase system in rat liver
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        Vitamin B12 deficiency results in the abnormal regulation of serine dehydratase and tyrosine aminotransferase activities correlated with impairment of the adenylyl cyclase system in rat liver
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Abstract

The aim of the present study was to elucidate the mechanism of the vitamin B12 deficiency-induced changes of the serine dehydratase (SDH) and tyrosine aminotransferase (TAT) activities in the rat liver. When rats were maintained on a vitamin B12-deficient diet, the activities of these two enzymes in the liver were significantly reduced compared with those in the B12-sufficient control rats (SDH 2·8 (sd 0·56) v. 17·5 (sd 6·22) nmol/mg protein per min (n 5); P < 0·05) (TAT 25·2 (sd 5·22) v. 41·3 (sd 8·11) nmol/mg protein per min (n 5); P < 0·05). In the B12-deficient rats, the level of SDH induction in response to the administration of glucagon and dexamethasone was significantly lower than in the B12-sufficient controls. Dexamethasone induced a significant increase in TAT activity in the primary culture of the hepatocytes prepared from the deficient rats, as well as in the cells from the control rats. However, a further increase in TAT activity was not observed in the hepatocytes from the deficient rats, in contrast to the cells from the controls, when glucagon was added simultaneously with dexamethasone. The glucagon-stimulated production of cAMP was significantly reduced in the hepatocytes from the deficient rats relative to the cells from the control rats. Furthermore, the glucagon-stimulated adenylyl cyclase activity in the liver was significantly lower in the deficient rats than in the controls. These results suggest that vitamin B12 deficiency results in decreases in SDH and TAT activities correlated with the impairment of the glucagon signal transduction through the activation of the adenylyl cyclase system in the liver.

Vitamin B12 is taken-up into mammalian cells and converted to its coenzyme forms, methylcobalamin (MeCbl) and 5′-deoxyadenosylcobalamin (AdoCbl)1, 2. MeCbl functions in methionine synthase, which catalyses the synthesis of methionine from homocysteine with 5-methyltetrahydrofolate as a methyl donor, and participates in one-carbon and folate metabolism3, 4. In contrast, AdoCbl is required by methylmalonyl-CoA mutase, which converts l-methylmalonyl-CoA to succinyl-CoA, an intermediate of the TCA cycle, during the final stage in the degradation pathways of some amino acids, odd-numbered fatty acids and cholesterols5. It is known that B12 deficiency causes haematological and neurological abnormalities, hepatic injury and growth retardation in mammals68.

Serine dehydratase (SDH) is the enzyme that catalyses the pyridoxal 5′-phosphate-dependent deamination of serine and threonine to produce pyruvate and 2-oxobutyrate, respectively, in the liver. The expression level of SDH in the liver is dramatically increased under conditions of gluconeogenesis, such as the feeding of a high-protein diet, starvation and diabetic mellitus911. Glucagon, glucocorticoids and insulin play pivotal roles in the regulation of SDH expression12, 13. Tyrosine aminotransferase (TAT) catalyses the rate-limiting step of tyrosine degradation, and the expression of TAT is also regulated by glucagon, glucocorticoids and insulin14, 15.

It has been reported that amino acid metabolism is disordered by B12 deficiency, and certain amino acids, in particular serine and threonine, are abnormally increased in the plasma and excreted into the urine of animals with B12 deficiency1618. Previously, we found that SDH activity in the liver was significantly reduced due to B12 deficiency in rats16, although neither MeCbl nor AdoCbl is required for the enzyme as a cofactor. Thus, it is possible that a decrease in hepatic SDH activity results in the abnormal increase in the plasma and urinary levels of serine and threonine in the B12-deficient rats. However, the mechanisms by which SDH activity in the liver is affected by B12 deficiency have not yet been determined.

In the present study, we examined the effects of vitamin B12 deficiency on the hormonal regulation of SDH and TAT activities in the rat liver. We report here that glucagon signal transduction through activation of the adenylyl cyclase (AC) system is impaired by B12 deficiency, and consequently the activities of SDH and TAT are abnormally regulated in the rat liver under B12-deficient conditions.

Materials and methods

Diets

In the present study, standard and non-protein diets with or without vitamin B12 were used (Table 1). In the non-protein diet, defatted soyabean in the standard diet was substituted by glucose. When vitamin B12 was fed to the rats, cyanocobalamin (CN-Cbl) was added at 25 or 100 μg/kg diet. The concentration of folic acid in the experimental diet was 2 mg/kg diet on the basis of AIN-93VX.

Table 1 Composition of the experimental diets (g/kg)*

* When vitamin B12 was fed to the rats, cyanocobalamin was added at 25 or 100 μg/kg diet.

Defatted soyabeans contained about 50 % crude protein and 50 % carbohydrate.

Mineral mixture (AIN-93G-MX) and vitamin B12-free vitamin mixture (AIN-93G-VX without vitamin B12) were purchased from Clea (Tokyo, Japan).

Animal treatments

All experimental procedures involving laboratory animals were approved by the Animal Care and Use Committee of Osaka Prefecture University. Male weanling Wistar rats (3 weeks old), born to 14-week-old parent rats, which had been fed the standard diet without vitamin B12 for 8 weeks, were used. The parent rats were purchased from Kiwa Laboratory Animals (Wakayama, Japan). The weanling rats were randomly allocated to the B12-deficient and control (B12-sufficient) groups, and individually housed under controlled temperature (22 ± 2°C), humidity (55 ± 10 %) and lighting (from 08.00 to 20.00 hours) conditions. The vitamin B12-deficient and control rats were allowed free access to the standard diet without or with CN-Cbl (25 μg/kg), respectively, and water. After being maintained for 17 weeks, at 20 weeks of age these rats were anaesthetised with diethyl ether, and their livers excised.

In the vitamin B12-supplementation experiment, the B12-deficient rats at 20 weeks old were fed the standard diet supplemented with CN-Cbl at 100 μg/kg for 2 weeks. The livers were obtained from the B12-supplemented rats (22 weeks old) under diethyl ether anaesthesia. All experimental rats were killed at 10.00 hours.

Enzyme assays

The livers were homogenised using a Teflon homogeniser in tri(hydroxymethyl)-aminomethane (Tris)-HCl buffer (0·1 mol/l; pH 7·4) containing KCl (0·15 mol/l) and EDTA (1 mmol/l) for the SDH assay, or in potassium phosphate buffer (50 mmol/l; pH 7·6) containing KCl (0·15 mol/l), EDTA (1 mmol/l) and pyridoxal 5′-phosphate (0·2 mmol/l) for the TAT assay. The homogenate was centrifuged at 100 000 g for 15 min, and the supernatant fraction obtained was used as a crude enzyme solution.

SDH activity was determined with serine as a substrate19. Briefly, the reaction mixture (1 ml) contained potassium phosphate buffer (0·1 mol/l; pH 8·0), KCl (0·15 mol/l), serine (0·2 mol/l), pyridoxal 5′-phosphate (0·4 mmol/l), NADH (0·15 mmol/l), five units of lactate dehydrogenase (from rabbit muscle) and the crude enzyme solution. The reaction was monitored at 37°C by following the decrease in absorbance at 340 nm due to the consumption of NADH.

TAT activity was determined on the basis of previous reports20. The assay depends on the alkali-catalysed conversion of the reaction product, p-hydroxyphenylpyruvate, to p-hydroxybenzaldehyde and oxalate. The reaction mixture contained potassium phosphate buffer (0·1 mol/l; pH 7·6), tyrosine (5·6 mmol/l), α-ketoglutarate (10 mmol/l), pyridoxal phosphate (50 μmol/l), and the crude enzyme solution in a total volume of 0·93 ml, at pH 7·6. The reaction was started by the addition of enzyme and allowed to proceed for 15 min at 37°C, at which time it was stopped by the addition of 0·07 ml of 10 m-KOH. The reaction was read at 331 nm by using a value of 19 900/m per cm for the molar absorbance of p-hydroxybenzaldehyde.

The K m values of SDH for serine and TAT for tyrosine were determined from Lineweaver–Burk plots. The protein concentration was measured according to Bradford21 with bovine serum albumin as a standard.

Measurement of hepatic vitamin B12 content

The liver (about 1 g) was homogenised in four volumes of 10 mm-sodium acetate buffer (pH 4·8) containing 0·2 % (w/v) potassium cyanide, and the homogenate was boiled for 30 min. The boiled suspension was centrifuged at 3000 g for 10 min. The supernatant fraction was used for the vitamin B12 assay by a microbiological method using Lactobacillus leichmannii ATCC 7830 and B12 assay medium (Nissui, Tokyo, Japan). The liver extract was diluted with distilled water to a B12 concentration range of 0·01–0·1 μg/l and used as the assay sample. The turbidity (%T) of a test culture of L. leichmannii ATCC 7830 grown at 37°C for 16–21 h was measured at 660 nm with a spectrophotometer.

Administration of glucagon and dexamethasone in rats fed the non-protein diet

The vitamin B12-deficient or control rats at 20 weeks of age, which had been maintained on the standard diet, were fed the non-protein diet without or with supplementation with CN-Cbl (25 μg/kg), respectively, for 5 d; then the food was removed 12 h before the hormonal administration. Glucagon (Sigma-Aldrich, St Louis, MO, USA) and dexamethasone (Sigma-Aldrich) were suspended in tricaprylin and emulsified with an equal volume of PBS, and the emulsion (0·5 ml), containing 1 mg glucagon and 5 mg dexamethasone, was injected intraperitoneally22. At 1 d after the administration, the livers were excised for the determination of SDH activity.

Primary culture of hepatocytes

Parenchymal hepatocytes were isolated from rats (age 20 weeks) maintained on the standard diet without (vitamin B12-deficient group) or with (control group) supplementation with CN-Cbl, by perfusion of the liver with collagenase according to a previous paper23. The cells were suspended in Dulbecco's modified Eagle's medium supplemented with 1 mm-insulin, seeded in a collagen-coated 60 mm dish at a density of 7 ×  104 cells/cm2, and cultured at 37°C in 5 % CO2–95 % air. After 6 h, the medium was replaced by Dulbecco's modified Eagle's medium containing 0·8 trypsin inhibitor units/ml trypsin inhibitor and aprotinin (0·12 μg/ml). These cells were then cultured for an additional 18 h, and used as the primary cultured hepatocytes for the following experiments.

To study the induction of SDH and TAT, the primary cultured hepatocytes were stimulated with glucagon (0·5 μmol/l) and/or dexamethasone (10 μmol/l) in Dulbecco's modified Eagle's medium containing 0·8 trypsin inhibitor units/ml trypsin inhibitor and aprotinin (0·12 μg/ml) for 24 h. These cells were then harvested and disrupted by repeated freeze-thawing in Tris-HCl buffer (0·1 mol/l; pH 7·4) containing phenylmethylsulfonyl fluoride (1 mmol/l), leupeptin (0·5 μg/ml) and pepstatin (1 μg/ml) for the SDH assay, or in potassium phosphate buffer (20 mmol/l; pH 7·0) containing pyridoxal 5′-phosphate (0·2 mmol/l) for the TAT assay. After centrifugation at 40 000 g for 20 min, the supernatant fraction obtained was used as a crude enzyme solution for the determination of SDH or TAT activity.

To examine the glucagon-induced production of cAMP, the primary cultured hepatocytes were stimulated with glucagon (0·5 μmol/l) in Hank's salt solution at 37°C. After 0, 1, 3 or 6 min incubation, HCl was added to the culture medium at the final concentration of 0·1 mol/l. The amount of cAMP produced during the incubation was determined by measuring the concentration of cAMP in the culture medium using a commercially available kit (cAMP enzyme immunoassay system; Amersham Pharmacia Biotech, Little Chalfont, Bucks, UK) according to the instructions of the manufacturer.

Measurement of adenylyl cyclase activity

The plasma membrane fraction was prepared from the livers of the B12-deficient or control rats (20 weeks old) according to a previously described method24. The AC activity was assayed as described by Salomon et al. 25 with some modifications. Briefly, the AC reaction was performed at 30°C for 10 min in a mixture (0·1 ml) which contained Tris-HCl buffer (25 mmol/l; pH 7·6), [α-32P]ATP (1 mmol/l; 180 MBq), [3H]cAMP (1 mmol/l; 2 MBq), GTP (0·1 mmol/l), MgCl2 (5 mmol/l), dithiothreitol (1 mmol/l), creatine phosphate (20 mmol/l), creatine phosphokinase (100 units/ml), 3-isobutyl-1-methylxanthine (0·5 mmol/l) and the plasma membrane preparation. The reaction was stopped by the addition of an equal volume of 10 % (w/w) SDS solution containing cAMP (2·5 mmol/l) and ATP (10 mmol/l), and heated at 100°C for 10 min. cAMP was isolated by sequential chromatography of the reaction mixture on Dowex 50-X4 and neutral alumina, and its radioactivity was counted for determination of AC activity.

Statistical analyses

The B12 content, and SDH and TAT activities, in the liver were compared among the vitamin B12-deficient, B12-supplemented and control rats by one-way ANOVA, followed by the Scheffé post hoc test. The other data were evaluated by two-way ANOVA, and post hoc analyses were done by the Scheffé post hoc test. These analyses were performed using GB-Stat 5.4 (Dynamic Microsystems, Silver Spring, MD, USA). All data are presented as mean values and standard deviations, and statistical significance was defined as P < 0·05.

Results

Hepatic serine dehydratase and tyrosine aminotransferase activities in vitamin B12-deficient rats

To induce vitamin B12 deficiency, weanling rats (3 weeks old) were fed a standard diet (containing about 20 % protein) without B12 for 17 weeks. At 20 weeks of age, the body weight of the B12-deficient group (305 (sd 24·4) g) was about 50 % of that of the B12-sufficient control groups (613 (sd 36·4) g) (P < 0·05). The hepatic B12 content in the B12-deficient rats was reduced to less than 10 % of that in the control rats (P < 0·05) (Table 2). The B12 deficiency resulted in a significant decrease in SDH activity in the liver, with the activity in the deficient rats being reduced to less than 20 % of that in the control rats. When the B12-deficient rats were fed a B12-supplemented diet (in which CN-Cbl was included at 100 μg/kg) for 2 weeks, SDH activity was increased to a level comparable with that in the control rats, in response to a significant increase in the hepatic B12 content. TAT activity in the liver was also significantly lower in the deficient group relative to the control. Furthermore, the decreased TAT activity in the deficient rats was restored to a normal level by supplementation with B12 for 2 weeks. The K m values of SDH for serine and TAT for tyrosine were not significantly different among the experimental groups (SDH, 115·8 (sd 34·2), 99·0 (sd 24·7) and 126·6 (sd 26·0) mmol/l; TAT, 2·3 (sd 0·33), 2·8 (sd 0·49) and 2·6 (sd 0·30) mmol/l in the control, vitamin B12-deficient and B12-supplemented rats, respectively).

Table 2 Effects of vitamin B12 deficiency on serine dehydratase (SDH) and tyrosine aminotransferase (TAT) activities and vitamin B12 content in liver of the B12-deficient, B12-supplemented and B12-sufficient control groups

(Mean values and standard deviations for five rats)

a,b,c Values within a column with unlike superscript letters are significantly different (P < 0·05).

Abnormalities in the dietary and hormonal regulations of the serine dehydratase activity in the liver under the vitamin B12-deficient conditions

It is well known that SDH activity in the liver is dramatically changed depending on the content of dietary protein11. When the vitamin B12-sufficient control rats, which had been maintained on the standard diet (containing about 20 % protein) for 2 weeks, were fed the non-protein diet for 5 d, a significant decrease in SDH activity was induced in the liver, as expected (Fig. 1). However, in the vitamin B12-deficient rats, feeding of the non-protein diet did not cause any significant effect on the hepatic SDH activity. Thus, the significant difference in SDH activity between the B12-deficient and control rats was lost when these rats received a non-protein diet. When glucagon and dexamethasone were administered by intraperitoneal injection into the B12-sufficient control rats that had been maintained on the non-protein diet for 5 d, a significant increase (over 10-fold) in SDH activity was induced in the liver 24 h after the injection. In response to the injection of glucagon and dexamethasone, an increase in the hepatic SDH activity was also observed in the B12-deficient rats; however, the extent of the increase in the deficient group was significantly lower than that in the control. These results suggest that the dietary and hormonal regulations of SDH activity are disordered in the rat liver under vitamin B12-deficient conditions.

Fig. 1 Induction of serine dehydratase (SDH) in the liver by the administration of glucagon and dexamethasone under vitamin B12-deficient conditions. Hepatic SDH activity was determined in the deficient or control rats just before the feeding of the non-protein diet (□), at 5 d after the feeding of the non-protein diet () or at 24 h after the administration of glucagon and dexamethasone (■). Values are the means of five rats, with their standard deviations represented by vertical bars. a,b,c Mean values with unlike letters are significantly different (P < 0·05).

Effects of vitamin B12 deficiency on the induction of serine dehydratase and tyrosine aminotransferase by glucagon and dexamethasone in primary culture of hepatocytes

Hepatocytes were isolated from the vitamin B12-deficient or control (B12-sufficient) rats (20 weeks old), and cultured in serum-free medium without vitamin B12. To the primary culture of the hepatocytes, glucagon and/or dexamethasone was added, and SDH activity in the cells was determined 24 h after the stimulation (Fig. 2). A significant increase (about 6-fold) in SDH activity was observed in the hepatocytes prepared from the control rats when they were stimulated simultaneously with glucagon and dexamethasone, although neither glucagon nor dexamethasone alone caused any significant effect. These results are consistent with a previous report showing that both glucagon and dexamethasone are required to induce SDH26. However, in the hepatocytes from vitamin B12-deficient rats, even when glucagon and dexamethasone were added at the same time, SDH activity was not significantly enhanced.

Fig. 2 Induction of serine dehydratase (SDH) by glucagon (Glc) and dexamethasone (Dex) in the primary culture of hepatocytes prepared from vitamin B12-deficient rats. The primary cultures of hepatocytes were incubated in the presence or absence of Glc and/or Dex for 24 h, and SDH activity was then determined. Values are the means of five rats, with their standard deviations represented by vertical bars. a,b Mean values with unlike letters are significantly different (P < 0·05).

TAT activity was significantly increased in the primary cultures of the hepatocytes prepared from either the vitamin B12-deficient or control rats when stimulated with dexamethasone (Fig. 3). In the hepatocytes from the control rats, a further increase in TAT activity was induced when glucagon was simultaneously added with dexamethasone, although TAT activity was not increased by the addition of glucagon alone. In contrast, in the cells from the B12-deficient rats, glucagon did not cause any effect on TAT activity even in the presence of dexamethasone.

Fig. 3 Induction of tyrosine aminotransferase (TAT) by glucagon (Glc) and dexamethasone (Dex) in the primary culture of hepatocytes prepared from vitamin B12-deficient rats. The primary cultures of hepatocytes were incubated in the presence or absence of Glc and/or Dex for 24 h, and TAT activity was then determined. Values are the means of four rats, with their standard deviations represented by vertical bars. a,b,c Mean values with unlike letters are significantly different (P < 0·05).

Glucagon-induced cAMP production in the hepatocytes of the vitamin B12-deficient rats

A primary culture of the hepatocytes was prepared from the vitamin B12-deficient rats and incubated with glucagon, and cAMP production was followed during the incubation. As shown in Fig. 4, cAMP was produced in response to the stimulation with glucagon in the hepatocytes from the B12-deficient rats, as well as the hepatocytes from the B12-sufficient control rats. However, the level of cAMP production was significantly lower in the hepatocytes from the deficient rats after 3 and 6 min of the stimulation.

Fig. 4 Glucagon-stimulated cAMP production in the primary culture of hepatocytes from vitamin B12-deficient rats. The primary cultures of hepatocytes prepared from B12-deficient (●) or control (○) rats were incubated with glucagon for 6 min. Values are the means of five rats, with their standard deviations represented by vertical bars. * Mean value is significantly different from that of the primary cultures of hepatocytes prepared from the control rats at the same time point (P < 0·05).

Adenylyl cyclase activity in the liver of the vitamin B12-deficient rats

The plasma membrane fraction was prepared from the liver of the vitamin B12-deficient or control (B12-sufficient) rats, and AC activity in the membrane fraction was compared between the two groups. As shown in Fig. 5, the basal AC activity without any stimulation was slightly lower in the B12-deficient rats compared with the controls, but no significant difference was observed. When the plasma membrane fraction from the control rats was stimulated with glucagon, AC activity was increased about 3-fold compared with that without such stimulation (P < 0·05). However, the stimulation with glucagon did not induce a significant increase in AC activity in the B12-deficient rats. In addition, AC activity obtained after stimulation with forskolin was significantly lower in the B12-deficient rats than in the control rats.

Fig. 5 Effects of vitamin B12 deficiency on adenylyl cyclase (AC) activity in the liver. AC activity in the plasma membrane was determined in the absence (□) or presence of forskolin (100 μmol/l) () or glucagon (1 μmol/l) (■). Values are the means of four rats, with their standard deviations represented by vertical bars. a,b Mean values with unlike letters are significantly different (P < 0·05).

Discussion

In mammalian cells, MeCbl and AdoCbl are required for methionine synthase and methylmalonyl-CoA mutase, respectively, as coenzymes1, 5. It is well known that in mammals with a vitamin B12 deficiency these enzyme activities are significantly lower and the plasma and urinary levels of homocysteine and methylmalonic acid are abnormally elevated5, 27, 28. We have previously reported that SDH activity in the liver is significantly reduced in rats with dietary vitamin B12 deficiency16, although neither MeCbl nor AdoCbl participates in the SDH enzyme reaction. Furthermore, we also found that serine and threonine are abnormally elevated in the plasma and excreted into the urine in the B12-deficient rats. Impairment of folic acid metabolism due to a decrease in the activity of methionine synthase might cause the abnormalities of the conversion of serine and tetrahydrofolate to glycine and 5,10-methylene-tetrahydrofolate, which is catalysed by serine hydroxymethyltransferase. However, it is conceivable that the decrease of SDH activity is largely responsible for the accumulation of serine in plasma.

SDH, which catalyses the conversion of serine and threonine to pyruvate and 2-oxobutyrate, respectively, is thought to be one of the key enzymes involved in gluconeogenesis, and the nutritional and hormonal regulations of the enzyme expression in the liver have been well studied. It is reported that rats fed a high-protein diet show a 100-fold increase of hepatic SDH activity compared with that in rats maintained on a protein-free diet11, 29. In addition, it is also known that glucagon and glucocorticoids play pivotal roles in the induction of SDH, whereas insulin suppresses the enzyme induction12, 13.

SDH activity in the liver was significantly lower in the vitamin B12-deficient rats than in the B12-sufficient controls when the rats were maintained on the standard diet, which contained about 20 % protein (Table 2). In the B12-deficient rats, no significant change in SDH activity occurred even if a non-protein diet was fed, in contrast to the effect in the B12-sufficient control rats, in which the enzyme activity was markedly decreased by the absence of dietary protein (Fig. 1). These results suggest that vitamin B12 deficiency results in the impairment of the nutritional regulation of SDH activity, and that the activity is maintained at a low level in the liver of B12-deficient rats even if the dietary protein level is increased.

The level of SDH induction in response to the administration of glucagon and dexamethasone was significantly lower in the vitamin B12-deficient rats than in the B12-sufficient controls (Fig. 1). Furthermore, in the primary culture of hepatocytes prepared from B12-deficient rats, in contrast to the cells from the control rats, SDH was not induced even when glucagon and dexamethasone were simultaneously added to the medium (Fig. 2). This indicates that the B12 deficiency results in impairment of the hormonal regulation of SDH activity in rat liver.

The activity of TAT, as well as SDH, in the liver was significantly lower in the vitamin B12-deficient rats than in the controls when the rats were maintained on a standard diet that contained about 20 % protein (Table 2). Since TAT is rate limiting for tyrosine degradation14, it is reasonable to postulate that the decrease in TAT activity due to B12 deficiency would result in abnormal tyrosine metabolism. Indeed, in our previous study16, it was observed that tyrosine, together with serine and threonine, increased in the plasma due to vitamin B12 deficiency in rats.

In contrast to SDH, whose induction requires both glucagon and glucocorticoids26, TAT can be induced in primary cultures of hepatocytes by the action of glucocorticoids alone without any need for glucagon, although glucagon is required in addition to glucocorticoids for the maximal induction of TAT23. TAT was induced by dexamethasone in the primary culture of hepatocytes prepared from the vitamin B12-deficient rats at a level comparable with that observed in the cells from the B12-sufficient control rats (Fig. 3), suggesting that B12 deficiency does not have any effect on the function of glucocorticoids in the liver. However, in the hepatocytes from the deficient rats, in contrast to the cells from the control rats, glucagon did not potentiate the induction of TAT by dexamethasone, suggesting that the glucagon signal transduction is impaired under the B12-deficient conditions. It is well known that glucagon regulates the expression of many kinds of enzymes involved in glycolysis, glyconeogenesis and the urea cycle, in addition to SDH and TAT; therefore, energy and amino acid metabolism is thought to be broadly affected by the impairment of glucagon signal transduction in mammals with B12 deficiency. Actually, it has been reported that B12 deficiency results in abnormal increases in many kinds of amino acids, in addition to serine, threonine and tyrosine, in the plasma and urine16, 18, and induces growth retardation16, 28 in mammals; however, the details of the mechanisms underlying these effects remain to be elucidated.

Glucagon stimulates AC in the plasma membrane, increasing the intracellular concentration of cAMP. After activation of cAMP-dependent protein kinase A, this enzyme catalyses the phosphorylation of cAMP response element-binding protein. The binding of cAMP response element-binding protein to the cAMP response element, located upstream of the transcriptional start sites, modulates the expression of SDH and TAT30, 31. The glucagon-stimulated AC consists of three integral proteins in the plasma membrane: the glucagon receptor, the catalytic unit of AC, and the GTP-binding protein (Gs) which links the receptor and the catalytic unit32, 33. The level of glucagon-stimulated cAMP production was significantly reduced in the primary culture of hepatocytes from the vitamin B12-deficient rats compared with the cells from the B12-sufficient controls (Fig. 4), indicating that the glucagon-stimulated AC system is impaired in the liver under B12-deficient conditions. The forskolin-stimulated AC activity in the liver was significantly lower in the B12-deficient rats than in the controls, although no significant difference between the two groups was observed in the basal level of AC activity (Fig. 5). It is known that forskolin, when added at higher concentrations (over 1 μm), directly interacts with the catalytic unit of AC without the participation of activated Gs and stimulates its catalytic activity34. Thus, the reduced responsiveness of AC to forskolin at 100 μm observed in the B12-deficient rats suggests that B12 deficiency results in dysfunction of the catalytic unit of AC in the liver. In addition, in the B12-deficient rats, in contrast to the B12-sufficient controls, a significant increase in AC activity was not induced by treatment with glucagon. This result may indicate that the functional coupling between Gs and the catalytic unit of AC is also disordered in the liver under B12-deficient conditions. The reduction of cAMP production due to the dysfunction of the AC system would cause a decrease in the level of cAMP response element-binding protein phosphorylated by cAMP-dependent protein kinase A, leading to attenuation of the effect mediated through the cAMP response element located upstream of the transcriptional start site of SDH or TAT, and thereby decrease the expression of their SDH and TAT proteins in the B12-deficient rats.

The AC system functionally depends on the maintenance of a suitable membrane environment, and changes in the plasma membrane lipid composition result in alteration of the basal and Gs-mediated stimulation of AC activity32, 33, 35. Phosphatidylcholine has been shown to act as a dominant factor that regulates the function of the catalytic unit of AC and contributes to the functional coupling between Gs and the catalytic unit3638. Since MeCbl acts as a cofactor for methionine synthase in mammalian cells, the synthesis of S-adenosyl-methionine, which is utilised as a methyl donor in the conversion of phosphatidylethanolamine to phosphatidylcholine, would be affected under B12-deficient conditions39. The synthesis of methionine from betaine, which is derived from choline, by betaine-homocysteine methyl transferase is also linked to the synthesis of S-adenosyl-methionine. Indeed, Åkesson et al. 40 reported that in rats fed a B12-deficient diet, a significant decrease in the proportion of phosphatidylcholine in the phospholipids, with a concomitant increase in that of phosphatidylethanolamine, occurred in the liver, even though the diet was supplemented with choline. Therefore, as a hypothesis, a decrease in the proportion of phosphatidylcholine in the plasma membrane might account, at least in part, for the impairment of the glucagon-stimulated AC system in the liver of B12-deficient rats. Previously, Hatta et al. 41 reported that the β-adrenoceptor–Gs–AC system was impaired in rats fed a vitamin B12-deficient diet.

In summary, the data obtained in the present study show that vitamin B12 deficiency results in impairment of the glucagon-stimulated AC system in the rat liver. As a result of the dysfunction of glucagon signalling, the regulation of the activity of SDH and TAT in the liver would be disordered in vitamin B12-deficient rats.

Acknowledgements

The present study was supported in part by Grants-in-Aid for Scientific Research from the Japan Society for the Promotion of Science (16380092 to Y. N. and 17580116 to H. I.).

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