Investigation of possible involvement of several genes related to development of hepatocarcinogenesis in rats

A comparative study on the possible involvement of several genes in the susceptibility of chemical carcinogenesis was carried out using carcinogen-resistant DRH rat and -sensitive Donryu and F344 rats. Previously, we observed that the induction of glutathione S-transferase placental form (GST-P) in the liver of Donryu rats by 3'-methyl-4-dimethylaminoazobenzene (3'-Me-DAB) was significantly greater than that of DRH rats. In the present study, we tentatively determined base sequences of the enhancer region including GPE-I and GPE-II (GST-P enhancers I and II) of GST-P genes of DRH, Donryu and F344 rats, but we did not observe any nucleotide polymorphism around these regions. Furthermore, the mRNA levels of silencer binding protein (NFA-1) for the GST-P promoter of rat liver were also similar in the DRH and Donryu rats. Since clonal expansion of putative preneoplastic GST-P-positive foci in the DRH rat liver was significantly suppressed during 3'-Me-DAB administration, we examined whether two opposite growth controlling factors, TGF-alpha and TGF-beta, may participate in such suppression of growth. It was supposed that mannose 6-phosphate/insulin-like growth factor 2 receptor (M6P/IGF2R), at least in part, activates TGF-beta preproprotein. However, we observed that the levels of M6P/IGF2R mRNA in the livers of DRH were not higher than those of Donryu rats after being fed 3'-Me-DAB for 8 weeks. Another important factor in the carcinogenesis is insulin-like growth factor II itself. Although liver tumors induced by 3'-Me-DAB in F344 had high levels of IGF-II mRNA, little IGF-II gene expression existed in normal adult livers of Donryu, F344 and DRH rats. High levels of IGF-II mRNA were detected similarly in the livers of neonates from all these three strains of rats. Finally, we detected a significant increase of AFP (alpha-fetoprotein) mRNA in the livers of Donryu rats around 6 to 8 weeks from the start of 3'-Me-DAB feeding, which is in parallel with detrimental effects of this carcinogen on these rats. A reduced induction of AFP mRNA was observed in DRH rats under the same conditions. Further study will be needed to explain the lower tumor susceptibility in the DRH rat.     

The occurrence of polymorphism of mannose 6-phosphate/insulin-like growth factor 2 receptor gene in laboratory and wild rats

Carcinogen-resistant inbred DRH rat strain was established from closed colony Donryu rats in the presence of 3'-methyl-4-dimethylaminoazobenzene (3'-Me-DAB). Despite using 3'-Me-DAB during the stage of selection, the DRH rats developed normally and did not show any spontaneous tumor at over 1 year of age. In the present study, we examined the polymorphism in mannose 6-phosphate/insulin-like growth factor 2 receptor (M6p/Igf2r) gene and found that the DRH rat showed CCC (Proline)-type polymorphism in exon 48 and the Donryu rat had GCC (Alanine) sequence. Since the DRH rat was developed from the Donryu rat, we examined whether this polymorphism in exon 48 of M6p/Igf2r gene was due to mutation that occurred at the stage of selection in the presence of 3'-Me-DAB, using several other laboratory and wild rats. We detected the presence of polymorphism at the same site of the M6p/Igf2r gene among these rats. It is likely that the polymorphism in exon 48 of the M6p/Igf2r gene is present broadly in rats since ancient times and not due to the mutation during the course of selection unless this site is a hot spot for chemical carcinogens.     

Hepatic fatty acid-binding protein mRNA is regulated by growth hormone.

Hepatic fatty acid-binding protein (FABP) is one of several abundant proteins which may participate in fatty acid uptake and utilization. Using differential hybridization to screen for growth hormone-responsive gene products, a complementary deoxyribonucleic acid (cDNA) was isolated which proved to be a hepatic FABP cDNA fragment. Hypophysectomy caused a 60% reduction in hepatic FABP messenger ribonucleic acid (mRNA) levels in rat liver, and growth hormone administration to hypophysectomized rats resulted in restoration of the expression of hepatic FABP mRNA. Other pituitary hormones did not alter these changes in expression. The response to growth hormone occurred within 4 hours of administration. During development, expression of hepatic FABP mRNA in rat liver was low in late fetal life, with increases to 40% of adult values by day 2 of life. Significant increases to adult levels did not occur until after day 25, when weaning is essentially completed. Alteration of hepatic FABP mRNA expression by growth hormone in rat liver may be important in the complex regulation of fatty acid uptake and metabolism.     

Response of the insulin-like growth factor system to vitamin A depletion and repletion in rats

Vitamin A (VA) and insulin-like growth factors (IGF) are important regulators of a wide range of physiological processes. To investigate the IGF system's involvement in the physiological actions of VA, we examined the effects of VA status on components of the IGF system in rats. Male rats (3-wk-old) fed a VA-deficient diet for 11 wk developed VA deficiency, as confirmed by the depletion of serum retinol and hepatic retinyl palmitate. Rats fed the VA-deficient diet had significantly lower body weight (p < 0.05) and lower serum IGF-I concentrations than the rats fed the control diet. The decreases in serum IGF-I levels were accompanied by approximately 40% lower levels of the IGF-I mRNA in the liver and lungs. With respect to the gene expression of other IGF system components, VA deficiency caused a twofold induction of IGF-I receptor (IGF-IR) mRNA in the heart and a twofold reduction in IGFBP-6 mRNA in the lungs, but did not alter the expression of IGF-II, IGFBP-1, IGFBP-3, IGFBP-4 or IGFBP-5 in all tissues examined. When VA-deficient rats received a single injection of retinoic acid (2 mg/rat), tissue IGF-I and IGF-IR gene expression did not change after 4 or 8 h, while the expression of IGF-II, IGFBP-4, and IGFBP-6 mRNAs in some tissues increased rapidly. These results suggest a possible involvement of the IGF system in mediating the physiological actions of VA, including VA-supported growth, in the rat.     

Effects of Long-Term Oral Administration of Arachidonic Acid and Docosahexaenoic Acid on the Immune Functions of Young Rats

Natural killer (NK) cells have many functional activities, including cytotoxicity and the capacity to produce cytokines and chemokines. NK cell activity is regulated partly by eicosanoids, which are produced from arachidonic acid (ARA) and eicosapentaenoic (EPA) acid. In this study, we investigated the effects of long-term therapy with ARA or docosahexaenoic acid (DHA) on the cytotoxic effects of the NK cells of young rats, which were fed on a nonfish oil diet for two generations. Control oil, ARA (240 mg/kg BW/day) or DHA (240 mg/kg BW/day) were orally administrated to the rats for 13 weeks before determining the cytotoxic activity of NK cells from the spleen against YAC-1 mouse lymphoma cell line, as well as the plasma levels of docosanoids or eicosanoids and inflammatory cytokines. Long-term ARA administration significantly suppressed the cytotoxic activity of NK cells. Moreover, ARA administration significantly increased the plasma levels of ARA, prostaglandin (PG) E₂, and PGD₂. However, DHA administration did not produce any different effects compared with those in the control rats. Furthermore, the inflammatory cytokine levels were not affected by the administration of ARA or DHA. These results suggest that long-term ARA administration has an inhibitory effect on the tumor cytotoxicity of NK cells in rat spleen lymphocytes owing to the enhanced synthesis of PGE₂ and PGD₂ from ARA because of the elevated plasma ARA levels in young rats.     

Developmental increases in rat hepatic microsomal UDP-glucuronosyltransferase activities toward xenoestrogens and decreases during pregnancy

Xenoestrogens, such as bisphenol A and diethylstilbestrol, are glucuronidated by an isoform of UDP-glucuronosyltransferase named UGT2B1 in the livers of adult male rats. In this study, we found that nonylphenol and octylphenol are also conjugated with glucuronic acid by adult rat liver microsomes. Although UDP-glucuronosyltransferase activities toward these xenoestrogens were not detected in the fetal rat liver, a linear increase in enzymatic activities during neonatal development was observed. At 3 weeks after birth, the activities had reached the same level as that of adult rats. The protein and mRNA contents of UGT2B1 also were not detected in the fetal rat liver, but a developmental increase in newborn rat liver was detected by Western and Northern blotting analysis. Additionally, rat hepatic microsomal UDP-glucuronosyltransferase activities toward these xenoestrogens were reduced by about half during pregnancy of mother rats. The results suggest that the reproductive organs of fetal and early-stage neonatal rats, which are sensitive to sex hormones, face a high risk of exposure to free active xenoestrogens.     

大、小鼠吸收和代谢双酚A的差异

目的 探讨相同剂量双酚A(bisphenol A,BPA)经口染毒后,引起大、小鼠血清BPA浓度差异的机制.方法 无特定病原体(specic pathogen free,SPF)级雄性SD大鼠和ICR小鼠各18只,一次性经口给予300 mg/kg的BPA后,在第0.5、1.0、12.0小时时间点各采集6只大、小鼠血液,用荧光-高效液相色谱法(fluorescence-high performance liquid chromatography,FL-HPLC)检测血清中BPA浓度;大、小鼠各6只采用原位小肠吸收模型循环灌流100 ml 0.1 mmol/L BPA灌流液,用FL-HPLC法分别检测第0.5、1.0、2.0小时时间点灌注液中BPA浓度和灌流后2.0 h血清中的浓度;采用逆转录PCR(RT-PCR)方法检测大、小鼠肝脏尿苷二磷酸葡萄糖醛酸转移酶2B1(UDP-glucuronosyltransferase 2B1,UGT2B1)mRNA的表达水平,并采用FL-HPLC法检测UGT2B1的酶活性;大、小鼠各6只禁食24 h后,一次性经口给予300 ms/kg BPA,收集24 h粪便,用FL-HPLC法检测粪便中BPA含量.结果 300 ms/kg BPA经口染毒后第0.5、1.0、12.0小时时间点小鼠血清中BPA浓度分别为(66.57±14.95)、(51.16±16.06)、(22.73±5.00)μg/ml,大鼠血清中BPA浓度分别为(15.63±5.65)、(18.34±5.02)、(7.65±2.58)μg/ml,各时间点小鼠均高于大鼠,差异有统计学意义(F值分别为50.660,17.957,8.420,P值均＜0.05);0～、0.5～、1.0～2.0 h小鼠小肠各时间段吸收速率分别为(10.20±4.20)、(1.49±0.67)、(1.31±0.55)μg·cm-2·min-1均高于大鼠相应时间段吸收率[(1.87 ± 0.69)、(0.47±0.13)、(0.36 ± 0.08)μg·cm-2·min-1],差异均有统计学意义(F值分别为14.954、8.877、11.536,P值均＜0.05).0.1 mmol/L BPA灌肠2 h后小鼠血清中的BPA浓度为(22.64±4.35)μg/ml,高于大鼠的(4.13±0.83)μg/ml,差异有统计学意义(F=74.643,P=0.000);大鼠肝脏中BPA代谢酶UGT2B1 mRNA的表达水平和酶活性明显高于小鼠;300 mg/kg BPA经口染毒24 h后,大鼠经粪便排出的BPA量为(1.50±0.32)mg/g,高于小鼠的(0.57±0.35)ms/g,差异有统计学意义(F=21.215,P=0.001).结论 大、小鼠经口给予300 mg/kg BPA染毒后,由于小鼠肠吸收BPA的能力高于大鼠,而大鼠代谢及排出BPA能力高于小鼠,引起小鼠血清中BPA的浓度明显高于大鼠.     

Chronic vitamin E inadequacy and thermally treated oils affect the synthesis of hepatic metallothionein isoforms

Summary Background: Metallothionein (MT)^# synthesis can be stimulated in many organs not only by various metals such as cadmium, zinc, and copper, but also by many nonmetalic compounds or experimental conditions such as oxidative stress. The latter lead to the hypothesis that MT is induced in response to free radicals formed in tissues and lipid peroxidation. Aims of the study: Whether the relationship between lipid peroxidation amd MT synthesis is a common phenomenon also valid for lipid peroxidation induced by dietary factors such as chronic vitamin E inadequacy and autoxidation products of polyenoic fatty acids derived from thermally oxidized oil was investigated in the presence study. Methods: The relationship between the induction of metallothionein isoforms I and II (MT-I and MT-II) in response to diet-induced lipid peroxidation using a rat model system in which lipid peroxidation was examined in vivo by chronic vitamin E inadequacy or by administration of lipid peroxidation products from a thermally treated polyenoicrich oil with either basal (dietary zinc concentration: 48 mg/kd; experiment 1) or Zn-stimulated MT levels (dietary zinc concentration: 305 mg/kd; experiment 2) was studied. In both experiments, growing male rats were fed diet containing either a fresh or a thermally treated soybean oil with deficient of sufficient amounts of vitamin E (14 and 11 vs. 648 and 560 mg α-tocopherol equivalents per kg diet) over 40 days according to a bifactorial experimental design. Plasma and liver concentrations of tocopherols and hepatic levels of thiobarbituric acid-reacitve substances (TBARS) were measured by high performance liquid chromatography. MT isoform concentrations in rat liver were isolated and quantified by ion-exchange high performance liquid chromatography and atomic absorption spectrometry. Results: Irrespective of the zinc supply, rats receiving inadequate amounts of vitamin E with the diet had markedly lower plasma and liver concentrations of α-tocopherol and total tocopherols than vitamin E-sufficient rats. ANOVA also revealed an interaction between the diet factors vitamin E and oil on tocopherols in plasma and liver of rats from both experiments. In experiment 1, where rats received normal amounts of dietary zinc, ingestion of the thermally treated oil impaired the tocopherol status compared to the treatment with the fresh oil, although this effect was only obvious in the vitamin E-deficient groups. In experiment 2, where rats received excessive amounts of zinc, the thermally treated oil did not contribute to a reduction of the tocopherol status in plasma and liver. In both experiments a significant increase in TBARS level, indicative of lipid peroxidation, was observed in the liver at chronic vitamin E inadequacy, but no effect of the oil was observed. Here, we show that the dietary treatment had some effects on the synthesis of liver metallothionein isoforms. In groups, receiving normal amounts of zinc, there was a significant interaction between the dietary treatments on the levels of MT-I and MT-II in liver. Chronic vitamin E inadequacy which was accompanied by diminisched tocopherol levels in liver induced the synthesis of MT-I and MT-II. When vitamin E inadequacy was combined with the ingestion of a thermally treated polyenoic acid-rich oil hepatic levels of MT-I and MT-II remained low. In experiment 2, where rats were fed the high zinc diet, vitamin E inadequacy caused an increase of hepatic MT-I level just as in experiment 1, although this MT stimulating effect was irrespective of the oil. For MT-II there was a 43% increase in the vitamin E-deficient group fed the fresh oil compared to all the other groups, although this effect was not statistically significant. The liver MT isoform response to stress was similar in rats with basal MT levels and Zn-induced liver MT levels. The failing effect of the thermally treated oil on MT levels which were stimulated by vitamin E deficiency in experiment 2 was possibly due to the low oxidation grade of the thermally treated oil. Conclusion: The present results are strongly indicative of an apparent induction of MT isoform synthesis in response to an impaired antioxidant defence system in the lipid regions of liver cells induced by vitamin E inadequacy. In contrast, thermally treated polyenoic-rich oils with a certain oxidation grade seem to restrain the induction of MT isoform synthesis under the present experimental conditions. Received: 10 January 2000, Accepted: 27 April 2000     

Evaluation of the effects of the flavonoid apigenin on apoptotic pathway gene expression on the colon cancer cell line (HT29)

Apigenin (4',5,7-trihydroxyflavone) is one of the leading components supporting targeted treatment options. We explored the cytotoxic and apoptotic effects of various doses of apigenin administered alone and together with 5-fluorouracil (5-FU)-a chemotherapeutic agent with high cytotoxicity-for different incubation periods, on morphologic, DNA, RNA (messenger RNA [mRNA]), and protein levels on the p53 mutant HT29 human colon adenocarcinoma cell line. Treatment with apigenin alone for a 72-hour incubation at 90-μM dose resulted in an apoptotic percentage of 24.92% (P=.001). A higher percentage (29.13%) was observed after treatment with the same dose of apigenin plus 5-FU for the same incubation period (P=.001). These results were confirmed as mRNA and protein expression levels of caspase-3 increased 2.567-fold and mRNA expression levels of caspase-8 increased 3.689-fold compared with the control group. On the other hand, mRNA expression levels of mammalian target of rapamycin (mTOR) and cyclin D1 (CCND1) decreased by 0.423-fold and 0.231-fold, respectively. To our knowledge this is the first study showing that treatment with apigenin alone results in cell cycle arrest through activation of caspase cascade and stimulation of apoptosis in HT29 cells. It also shows that use of apigenin plus 5-FU further increases this effect. This study draws attention to the probable clinical effectiveness of apigenin plus a chemotherapeutic agent with high cytotoxicity. It also highlights the induction of desirable apoptotic effects by targeting the caspase cascade pathway through administration of reduced doses for shorter incubation periods.     

Dietary sesame seed and its lignan increase both ascorbic acid concentration in some tissues and urinary excretion by stimulating biosynthesis in rats

We previously showed that the intake of sesamin, a major lignan in sesame seed, decreased lipid peroxidation and elevated tocopherol concentration in rat tissues. In this study, we examined the effect of dietary sesame seed and sesamin on the ascorbic acid concentration in rat tissues. Rats (4-wk-old) were fed either a vitamin E-free diet, or a diet containing 50 mg gamma-tocopherol/kg, one containing 2 g sesamin/kg, one containing 50 mg gamma-tocopherol/kg and 2 g sesamin/kg, or one containing 200 g sesame seed/kg for 28 d. The dietary sesamin and sesame seed elevated ascorbic acid concentrations in the liver and kidney, and increased urinary excretion in those Wistar rats. The dietary sesamin also elevated the hepatic mRNA levels of cytochrome P450 (CYP) 2B, and UDP-glucuronosyltransferase (UGT) 1A and 2B. In contrast, neither the sesamin nor the sesame seed affected the liver concentration of ascorbic acid in ODS rats with a hereditary defect in ascorbic acid synthesis, though the dietary sesame seed elevated the UGT1A and 2B mRNA levels in the liver. In addition, the sesame seed elevated the gamma-tocopherol concentration in the various ODS rat tissues and the ascorbic acid concentrations in the kidney, heart and lung, while reducing the thiobarbituric acid reactive substance concentration in the heart and kidney. These results suggest that dietary sesame seed and its lignan stimulate ascorbic acid synthesis as a result of the induction of UGT1A and the 2B-mediated metabolism of sesame lignan in rats. The data of ODS rat studies also suggest that dietary sesame seed enhances antioxidative activity in the tissues by elevating the levels of two antioxidative vitamins, vitamin C and E.     

Effect of tea polyphenols and tea pigments on the inhibition of precancerous liver lesions in rats

The objective of this study was to investigate the inhibitory effect of tea components, tea polyphenols and tea pigments, on precancerous liver lesions in rats. A rat liver precancerous lesion model was established by multiple low-dosage N-nitrosodiethylamine (NDEA) injections, followed by intraperitoneal CCl4 injection and partial hepatectomy (PH). Tea pigments (0.1%) or tea polyphenols (0.1%) were given to Wistar rats in drinking water during the eight weeks of the experiment. The number and area of glutathione S-transferase Pi-positive foci in the rat liver were used as biomarkers of precancerous liver lesions. Western and Northern blot techniques were used to detect rat liver GST-Pi expression at the protein and mRNA levels. At the end of the experiment, tea polyphenols and tea pigments significantly decreased the number and area of GST-Pi-positive foci that were overexpressed in the NDEA-CCl4-PH-treated rats compared with the positive control group. The results also showed that GST-Pi mRNA and protein expression increased significantly in the NDEA-CCl4-PH-treated group, which is consistent with the changing of GST-Pi-positive foci. Tea pigments and tea polyphenols had an inhibitory effect on the overexpression of GST-Pi mRNA and protein in NDEA-CCl4-PH-treated rats. These results suggest that tea pigments and tea polyphenols are effective in preventing the occurrence and progression of precancerous liver lesions in rats.     

β-酪啡肽-7对大鼠生长、相关激素及生长素受体mRNA表达的影响

目的: 探讨β-酪啡肽-7(β-CM-7)对大鼠生长、生长相关激素和生长因子以及生长素受体(GHR)mRNA表达的影响。方法: 将30只生长期雄性SD大鼠随机分为对照组和实验组,分别被灌饲生理盐水和β-CM-7,并记录大鼠日采食量。饲养1个月后检测大鼠的增重情况,应用放射免疫法测定血清中生长相关激素和生长因子的含量,用相对定量RT-PCR法研究两组大鼠肝脏GHR mRNA表达水平的差异。结果: 实验组大鼠与对照组比,增重有提高的趋势,增重率提高8.67%。实验组大鼠的日采食量增加了13.08%(P<0.05)。与对照组相比,实验组大鼠血清中生长激素(GH)和胰岛素显著或极显著升高(P<0.05或P<0.01),三碘甲腺原氨酸(T3)和胰岛素样生长因子-I(IGF-I)有升高趋势,而甲状腺素(T4)有下降趋势。实验组大鼠肝脏的GHR mRNA表达水平显著提高(P<0.05),增加了19.83%。结论: β-CM-7能促进生长期大鼠的生长,增加采食量,影响生长相关激素和生长因子的分泌,并且上调肝脏GHR mRNA表达水平,从而增加生长激素的敏感性。     

The potential beneficial effect of glycine on the carbohydrate moieties of glycoproteins in an experimental model of alcohol-induced hepatotoxicity

Glycine is known to have a protective role against alcohol-induced liver damage. The aim of our study was to evaluate the effect of glycine on liver and brain glycoproteins in alcohol-fed rats. Administering ethanol (7.9 g/kg of body of weight) every day to Wistar rats for 60 days resulted in significantly elevated levels of liver and brain hexosamine, fucose, and sialic acid and significantly reduced levels of total hexoses as compared with those of the control rats. Simultaneous glycine supplementation (0.6 g/kg of body weight) during the last 30 days of the experiment to rats given alcohol normalized the levels of hexosamine, fucose, and sialic acid and elevated the levels of total hexoses in the liver and brain significantly as compared with unsupplemented alcohol-treated rats. Microscopic examination of alcohol-fed rat liver showed inflammatory cell infiltrates and fatty changes, which were reversed on treatment with glycine. Similarly, alcohol-treated rat brain demonstrated edema, which was markedly reduced on treatment with glycine. Thus glycine administration plays a significant role in reducing the toxicity of ethanol.     

The prevention of colon carcinogenesis in rats by dietary cellulose is greater than the promotive effect of dietary lard as assessed by repeated endoscopic observation

We developed a method which we used in the current study to observe the rat colon endoscopically. Our goal was to evaluate the entire course in the development of experimental large bowel tumors through serial observations in the same rat. We compared the effects of dietary lard and cellulose on rat colon tumorigenesis in a 2 x 3 factorial design. Sprague-Dawley rats (n = 90) were divided into 6 diet groups: rats were fed a diet without cellulose that contained 5, 10, and 15 g/100 g lard, or diets containing 15% cellulose diet (15 g cellulose/100 g diet) and the same concentrations of lard. The development of large bowel tumors induced by the administration of 1,2-dimethylhydrazine (25 mg/kg body weight) for 19 wk was examined endoscopically. Tumor induction rates in the 15% cellulose groups were lower than in the 0% cellulose groups (P = 0.008), independent of the lard concentration. These results suggest that the preventive effect of cellulose against large bowel tumorigenesis is greater than the promotive effect of fat under the current experimental conditions.     

L-Carnitine changes the levels of insulin-like growth factors (IGFs) and IGF binding proteins in streptozotocin-induced diabetic rat

This study investigated the effects of L-carnitine on insulin-like growth factor-I/II (IGF-I/II) and insulin-like growth factor binding proteins (IGFBPs) in streptozotocin (STZ)-induced diabetic rats. Each rat in the three L-carnitine-treated groups was injected subcutaneously with L-carnitine, 50 (D50), 100 (D100), or 200 (D200) mg/kg body weight every other day for four weeks, and animals in normal (N) and diabetic (DM) groups received saline by the same method. Diabetic rats had significantly lower carnitine concentrations in serum and liver compared with normal rats. Total carnitine concentrations were increased dose-dependently by carnitine treatment. Total IGF-I in serum from diabetic rats was increased dose-dependently by carnitine treatment, but was statistically significant only in the D200 group. The expression of liver IGF-I mRNA was lower in diabetic rats than in normal rats and increased by L-carnitine treatment. L-Carnitine treatment of diabetic rats had no effect on the levels of IGF-II in serum, liver, and kidney. Although the levels of IGF-II in serum and kidney of diabetic rats were increased in comparison with normal rats, IGF-II mRNA was not expressed in liver. Diabetic rats had markedly lower IGFBP-3 than normal rats did, and IGFBP-3 was increased by L-carnitine treatment. These results demonstrate that L-carnitine treatment of diabetic rats modulates the IGFs/IGFBPs axis. Especially note-worthy is that L-carnitine at a dose of 200 mg/kg/48 h for four weeks was able to restore serum total IGF-I in STZ-induced diabetic rats to nearly normal levels.     

Extremely low activity of methionine synthase in vitamin B-12-deficient rats may be related to effects on coenzyme stabilization rather than to changes in coenzyme induction

Severely vitamin B-12 (B-12)-deficient rats were produced by feeding a B-12-deficient diet. The status of B-12 deficiency was confirmed by an increase in urinary methylmalonate excretion and decreases in liver B-12 concentrations and cobalamin-dependent methionine synthase activity. Rat liver methionine synthase existed almost exclusively as the holoenzyme. In B-12-deficient rats, the level of methionine synthase protein was lower, although the mRNA level was not significantly different from that of control rats. When methylcobalamin, the coenzyme for methionine synthase, was administered to the B-12-deficient rats, growth, liver B-12 concentrations and urinary excretion of methylmalonate were reversed although not always to control (B-12-sufficient) levels in a short period. During this recovery process, methionine synthase activity and its protein level increased, whereas the mRNA level was unaffected. We reported previously that rat apomethionine synthase is very unstable and is stabilized by forming a complex with methylcobalamin. Thus, the extremely low activity of methionine synthase in B-12-deficient rats may be related to effects on "coenzyme stabilization" (stabilization of the enzyme by cobalamin binding) rather than to changes in "coenzyme induction."