Deficient Rat Liver Research Articles

Molecular metabolic fingerprinting approach to investigate the effects of borneol on metabolic alterations in the liver of nitric oxide deficient hypertensive rats

Hypertension is one of the major risk factor that underlie a wide range of cardiovascular irregularities which causes functional and metabolic alterations in vascular system and major organs. Nitric oxide is the central regulator of the vascular system and its deficiency leads to increased blood pressure and metabolic alterations in liver. Fourier transform infrared spectroscopy (FTIR) is a vibrational spectroscopic technique that uses infrared radiation to vibrate molecular bonds with in the sample that absorbs it and different samples contain diverse configurations of molecular bonds. Both wavenumber and area of the vibrational spectra can be used to explore the qualitative and quantitative constituent of macromolecules. In this study, we intended to evaluate the protective role of borneol, a natural terpene on liver metabolism in a nitric oxide deficient model of hypertension through interpretation of FTIR spectral information. Results demonstrate that FTIR can successfully indicate the molecular changes that occur in all groups. The over all findings demonstrate that in nitric oxide deficient animal model of hypertension, the liver metabolic program is altered through increasing the structural modification in proteins and triglycerides, and quantitative alteration in proteins, lipids, and glycogen. All the above mentioned modifications were protected by borneol in liver and showed its ability to exert a novel defensive action on hepatic metabolism.

Zonal expression of hepatocytic marker enzymes during liver repopulation

Hepatocytes are metabolically specialised cells displaying distinctive gene expression patterns within the liver lobule. Here, we investigate whether pre-cultured adult rat hepatocytes adopt periportal and pericentral enzyme expression following their transplantation into the regenerating rat liver. Isolated primary rat hepatocytes, representing a mixture of both periportal and pericentral origin, lost expression of carbamoyl phosphate synthetase I (CPS I) and cytochrome P450 subtype 2B1 (CYP2B1) in culture as shown by immunofluorescence and Western blot analysis. Accordingly, urea synthesis and CYP2B1 enzyme activity decreased. Hepatocytes from DPPIV (CD26) wild type rats were cultured for 4 and 7 days, and then transplanted into the livers of CD26 deficient rats following prior treatment with retrorsine and partial hepatectomy to drive selective donor cell proliferation. CD26 positive donor cells engrafted in the periportal regions and grew in clusters expanding into the parenchyma as time proceeded. Ten weeks after transplantation, cells derived from donors surrounding the portal veins expressed CPS I, but not CYP2B1. The reverse was true for CD26 positive cells in close proximity to the central veins displaying immunoreactivity to CYP2B1, but no longer to CPS I. Hepatocytes lose their specific marker enzyme expression in culture. After transplantation, donor hepatocytes proliferate in the host parenchyma whilst acquiring the position-specific enzyme expression of the surrounding periportal and pericentral host hepatocytes. These results indicate the high degree of plasticity of gene expression in hepatocytes subjected to a change in microenvironment.

Feeding Period of Selenium-Deficient Diet and Response of Redox Relating Minerals

Dynamics of redox relating biotrace elements, selenium (Se), iron (Fe), and zinc (Zn) in liver, kidney, and spleen of selenium deficient Wistar male rats in a series of feeding period (from 0 to 8 weeks) were studied using instrumental neutron activation analysis (INAA). Aspartate aminotransferase (AST), alanine aminotransferase (ALT), and blood urea nitrogen (BUN) for the plasma fraction of the rat bloods and the concentration of vitamin C and vitamin E in the liver homogenates were measured. The initial purpose of this study was to find Fe and Zn as sensitive indices of the tissue oxidative stress levels. However, the relationships among the biotrace elements and the oxidative stress/injury were much complicated. Control group, which was fed Se-deficient diet with Na2SeO4 (0.1 mg selenium/l) in drinking water, showed strange response of Se and Zn contents in the kidney and showed high BUN. Supplementation of inorganic Se by biased Se source may serve as another source of a stress especially in the kidney. The Fe and Zn contents in the liver and kidney look sensitive to the Se-deficiency and/or relating oxidative stresses. Short term exposure to the Se-deficiency appeared to consume Fe and Zn in the liver and kidney. In contrast, long term or chronic exposure to Se-deficiency appeared to accumulate Fe and Zn in liver and kidney.

Time dependent study to evaluate the efficacy of zinc on hepatic marker enzymes and elemental profile in serum and liver of protein deficient rats

This study was designed to determine the time dependent protective effects of zinc sulfate on the serum and liver marker enzymes along with elemental profile in protein deficient Sprague Dawley (S.D.) female rats. Zinc sulfate in the dose of 227 mg/l in drinking water was administered to normal control as well as protein deficient rats for a total duration of 8 weeks. The effects of different treatments were studied on enzymes like alkaline phosphatase (ALP), aspartate aminotransferases (AST) and alanine aminotransferases (ALT) in rat serum at different time intervals of 1, 2, 4 and 8 weeks and in the rat liver at the end of study. The status of different essential elements in liver was also studied. The serum ALP activity got significantly depressed when estimated at the intervals of 4 and 8 weeks. Activity of serum ALT was significantly increased after 4 weeks interval in protein deficient rats and the increasing trend continued upto 8 weeks of protein deficiency. On the other hand, activity of AST showed a significant increase just after 2 weeks and activity continued to be increased up to 8 weeks. Moreover activities of all the hepato marker enzymes showed a significant increase in liver of protein deficient rats. Interestingly, supplementation of Zn to protein deficient rats helped in regulating the altered activities of ALP, AST and ALT both in serum and liver. However, zinc treatment alone to normal rats did not indicate any significant change in the activities of all the enzymes in liver as well serum except at the interval of 2 weeks where a marginal increase in the activity of AST was seen. It has also been observed that concentrations of zinc, copper, iron and selenium were found to be decreased significantly in protein deficient animals. However, the levels of these elements came back to within normal limits when zinc was administered to protein deficient rats.

Fatty Acid Δ5-Desaturase mRNA Is Regulated by Dietary Vitamin A and Exogenous Retinoic Acid in Liver of Adult Rats

Δ5-Desaturase (D5D) catalyzes the Δ5,6 desaturation of dietary essential fatty acids of the n-6 and n-3 series. By subtraction hybridization of vitamin A (VA)-deficient and control rat liver cDNA libraries, we isolated a 106-bp cDNA fragment that proved to be homologous to human liver D5D cDNA and used it as a probe to analyze rat D5D mRNA and clone the rat full-length cDNA. Δ5-Desaturase mRNA was threefold more abundant in liver from VA-deficient rats than in liver from VA-sufficient rats and was expressed dose dependently when dietary VA was varied (VA marginal > control > VA supplemented). Treatment of VA-deficient rats with all-trans-retinoic acid lowered the level of expression of D5D mRNA toward that of VA-sufficient rats. The 3413-bp full-length D5D cDNA cloned from rat liver contains an open reading frame of 447 amino acid residues sharing 92% similarity with its human counterpart. Expression of this cDNA in HEK293T cells incubated with dihomo-γ-linolenic acid (20:3, n-6) resulted in a significantly increased ratio of the product, arachidonic acid (20:4, n-6), to substrate in cell lipid extracts. Δ5-Desaturase mRNA is expressed in relatively high abundance in rat adrenal gland and mammary tissue and moderately in liver, kidney, lung, spleen, thymus, brain, and eye. The regulation of D5D by VA could be important for growth and development, and reproduction, as well as in the control of inflammation.

Vitamin B6 deficiency affects antioxidant defences in rat liver and heart.

We have evaluated the effects of a diet containing normal amounts of lipids and a marginal content of vitamin B6 on lipid peroxidation. Pyridoxal phosphate concentrations of plasma and liver indicated that an initial deficiency state was reached. Vitamin B6 deficiency led to peroxidative stress: TBARS production was higher in the liver (+18.6%) and even more in the heart (+61%) of deficient rats as compared with controls. Furthermore, significant stimulation of glutathione-dependent enzymes occurred in both heart and liver of deficient rats: glutathione peroxidase activity increased in heart (+144%) and liver (+505%); glutathione reductase increased in heart (+54.9%) and liver (+15.5%). No difference in the total glutathione content of the organs of the two groups was observed. The reduced glutathione/oxidized glutathione ratio was significantly lower in deficient rats. Although the activity of glutathione-dependent enzymes was significantly greater in deficient rats than in controls, this stimulation was only partially able to counteract the peroxidative damage due to vitamin B6 deficiency.

Cis-acting elements are required for selenium regulation of glutathione peroxidase-1 mRNA levels.

Classical glutathione peroxidase (GPX1) mRNA levels can decrease to less than 10% in selenium (Se)-deficient rat liver. The cis-acting nucleic acid sequence requirements for Se regulation of GPX1 mRNA levels were studied by transfecting Chinese hamster ovary (CHO) cells with GPX1 DNA constructs in which specific regions of the GPX1 gene were mutated, deleted, or replaced by comparable regions from unregulated genes such as phospholipid hydroperoxide glutathione peroxidase (GPX4). For each construct, stable transfectants were pooled two weeks after transfection, divided into Se-deficient (2 nM Se) or Se-adequate (200 nM Se) medium, and grown for an additional four days. On day of harvest, Se-deficient GPX1 and GPX4 activities averaged 13 +/- 2% and 15 +/- 2% of Se adequate levels, confirming that cellular Se status was dramatically altered by Se supplementation. RNA was isolated from replicate plates of cells and transfected mRNA levels were specifically determined by RNase protection assay. Analysis of chimeric GPX1/GPX4 constructs showed that the GPX4 3'-UTR can completely replace the GPX1 3'-UTR in Se regulation of GPX1 mRNA. We did not find any GPX1 coding regions that could be replaced by the corresponding GPX4 coding regions without diminishing or eliminating Se regulation of the transfected GPX1 mRNA. Further analysis of the GPX1 coding region demonstrated that the GPX1 Sec codon (UGA) and the GPX1 intron sequences are required for full Se regulation of transfected GPX1 mRNA levels. Mutations that moved the GPX1 Sec codon to three different positions within the GPX1 coding region suggest that the mechanism for Se regulation of GPX1 mRNA requires a Sec codon within exon 1. Lastly, we found that addition of the GPX1 3'-UTR to beta-globin mRNA can convey significant Se regulation to beta-globin mRNA levels when a UGA codon is placed within exon 1. We conclude that Se regulation of GPX1 mRNA requires a functional selenocysteine insertion sequence (SECIS) in the 3'-UTR and a Sec codon followed by an intron.

Neoplastic phenotype of gap-junctional intercellular communication-deficient WB rat liver epithelial cells and its reversal by forced expression of connexin 32.

Gap-junctional intercellular communication (GJIC) is involved in cellular growth control and is often reduced in neoplastic cells. In this study, four GJIC-deficient rat liver epithelial cell lines (WB-aB1, WB-bA2, WB-cD6, and WB-dA2) were examined for altered growth and tumorigenicity in comparison with their GJIC-competent parental cell line, WB-F344. WB-aB1 cells were also forced to express connexin 32 (Cx32) by transduction with a Cx32 cDNA retroviral expression vector to help determine whether the restoration of GJIC could reverse their neoplastic phenotype. WB-aB1 and WB-bA2 cells had faster population doubling times (PDTs) and higher saturation densities (SDs) than did WB-F344 cells. In contrast, the growth of WB-cD6 and WB-dA2 cells was not significantly different from that of WB-F344 cells. WB-aB1 and WB-bA2 cells formed tumors in male F344 rats, but WB-cD6 and WB-dA2 cells did not. After transduction of WB-aB1 cells with Cx32, four stable clones (WB-a/32-3, -8, -9, and -10) were isolated that had GJIC levels of 5.2%, 44.5%, 69.8%, and 90.5%, respectively. The growth of poorly coupled clones 3 and 8 was similar to that of parental WB-aB1 cells, but the growth of well-coupled clones 9 and 10 was similar to that of WB-F344 cells. The tumorigenicity of WB-a/32-9 and WB-a/32-10 cells was also significantly lower than that of WB-aB1 cells. Our results suggest that reduced GJIC contributes to neoplastic transformation of WB cells, that additional changes are necessary, and that restoration of GJIC by forced Cx32 protein expression can suppress the neoplastic phenotype of these cells.

Developmental changes in alpha-tocopherol, especially during infantile period.

Developmental changes in expression of alpha-TTP after birth were investigated using rats with respect to plasma changes of tocopherols. The expression of alpha-TTP in the neonatal rat liver, which was very low immediately after birth, increased steadily during the two weeks of life before weanling and reached the adult level at 4 weeks. During the suckling period, the plasma ratio of alpha-tocopherol to gamma-tocopherol linearly increased, because plasma alpha-tocopherol was low immediately after birth, increased rapidly during the period, while gamma-tocopherol remained unchanged. The increase in the ratio seemed to correlate with the developmental expression of alpha-TTP in the liver during this period. The ratio also reached the adult level after 4 weeks. The expression of alpha-TTP was investigated using primary cultured rat hepatocytes. The expression of alpha-TTP was found to be extremely low after 20 h of culture. The decrease in alpha-TTP expression was exacerbated by adding epidermal growth factor to the culture medium and was inhibited by adding dexamethasone. These observations suggest that expression of alpha-TTP may be affected by the state of hepatic differentiation. The Western and Northern blot analysis of alpha-TTP in vitamin E deficient and enriched rats were compared to control rats. The decrease of mRNA of alpha-TTP observed in alpha-Toc-enriched rat liver and the increase of mRNA observed in the vitamin E deficient rat liver indicate the synthesis of alpha-TTP, although alpha-TTP itself was not altered.

Complement C6 and C2 biosynthesis in syngeneic PVG/c- and PVG/c+ rat strains.

A PVG rat with total deficiency of C6 and partial deficiency of C2 (PVG/c-), and a syngeneic control strain (PVG/c+), were used to study the production of extrahepatically synthesized complement. Livers of complement deficient rats were transplanted in sufficient rats (Tx-L). The C6 and C2 levels in Tx-L rats declined within 2 days to 25% and 30%, respectively, and remained stable for more than 6 weeks. To investigate the contribution of C6 synthesis by the liver, C6 sufficient livers were grafted in deficient rats (Tx + 1). After an initial increase, with maximum C6 levels of 119% at 10 days following transplantation, the C6 levels decreased gradually and C6 was no longer detectable 28 days after transplantation. This decline in C6 levels was dependent on antibody production against C6. No significant change in the C3, C4, factor H and factor B levels was observed. Expression of C6 mRNA in the grafted PVG/c+ sufficient liver was comparable to the expression of C6 mRNA in control PVG/c+ livers while C6 mRNA expression in the transplanted PVG/ c- liver and the control PVG/c- liver was lower. In conclusion, it was demonstrated in vivo that not only C6 but also C2 is synthesized extrahepatically in PVG/c rats.

Iron accumulation in tissues of magnesium-deficient rats with dietary iron overload.

The mineral imbalances in magnesium-deficient rats with dietary iron overload were studied. Forty-four male Wistar rats were divided into six groups and fed six diets, two by three, fully crossed: magnesium adequate or deficient, and iron deficient, adequate, or excess. The concentrations of iron, magnesium, calcium, and phosphorus in tissues of the rats were measured. The results were as follows: (1) The excess iron intake reinforced the iron accumulation in liver and spleen of magnesium deficient rats; (2) The saturation of iron binding capacity was enormously elevated in the magnesium deficient rats fed excess iron; and (3) Dietary iron deprivation diminished the degree of calcium deposition in kidney of magnesium deficient rats. These results suggest that magnesium-deprived rats have abnormal iron metabolism losing homeostatic regulation of plasma iron, and magnesium deficient rats with dietary iron overload may be used as an experimental hemochromatosis model.

Localisation of ferritin, transferrin receptor & transferrin in the normal, rion loaded & iron deficient rat liver: [formula omitted]. Dept of Medicine, University of Western Australia, Nedlands, 6009

Localisation of ferritin, transferrin receptor & transferrin in the normal, rion loaded & iron deficient rat liver: [formula omitted]. Dept of Medicine, University of Western Australia, Nedlands, 6009

Suppression of the constitutive microsomal cytochrome P450 2C11 in male rat liver during dietary vitamin A deficiency

The effect of dietary vitamin A deficiency on hepatic microsomal cytochrome P450 (P450) and associated oxidase activities was examined in the male rat. Intake of a deficient diet by weanling rats over 10 weeks led to a pronounced decline in hepatic and serum vitamin A concentrations to levels that were beneath the limits of detection. These changes occurred concurrently with a decrease in total microsomal P450 to 77% of vitamin A adequate control. Measurement of microsomal androstenedione metabolism revealed respective decreases in 16α- and 6β-hydroxylation pathways to 61 and 71% of adequate control; 7α-hydroxylation was not significantly decreased. Immunoquantitation of the principal catalyst of steroid 16α-hydroxylation, the androgen-dependent P450 2C11, indicated a significant decrease in the microsomal content of the enzyme to 78% of control (13.7 ± 0.9 ng/μg protein in deficient rat liver versus 17.5 ± 0.5 in adequate control; P < 0.005). Serum testosterone appeared lower in vitamin A deficient male rats, but did not attain statistical significance. Administration of a diet containing excess vitamin A (500IU/g) to rats for 10 weeks produced marked increases in hepatic vitamin A stores, but did not increase P450 2C11 activities. Thus, the expression and function of P450 2C11 is not related directly to hepatic vitamin A levels. The trend toward lower circulating androgen levels in male rats maintained on the deficient diet for 10 weeks may have a role in P450 2C11 down regulation, but other regulatory factors may also be disrupted in these animals.

Increased malic enzyme activity in selenium-deficient rat liver

The activity of cytosolic malic enzyme was significantly higher (61%) in the liver of rats fed a selenium-deficient diet compared with the controls. Activities of two other NADPH-generating enzymes, glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase, were also increased but to a lesser extent (13%). Glutathione reductase, an NADPH-consuming enzyme involved in the metabolism of glutathione, was higher (26%) in the liver of selenium deficient rats, possibly as a result of an increase in glutathione synthesis and enzymic processes that maintain glutathione in the reduced state. It is concluded that an increased demand for NADPH in selenium deficiency, possibly resulting from increased glutathione metabolism, may induce NADPH-generating enzymes, especially malic enzyme. ( J. Nutr. Biochem. 5:314–316, 1994.)

Alteration of hepatic DNA synthesis in rats treated with N-nitrosodimethylamine during early stages of copper deficiency.

The main aim of this research was to clarify that nutritional dietary copper may participate in the protective action against hepatocarcinogenesis in rats by N-nitrosodimethylamine (NDMA). The copper concentrations in serum and liver from 2 to 8d after rats were first fed a copper deficient diet (copper, 0.6 ppm) decreased significantly compared to those of pair-fed rats (copper in a control diet, 7 ppm). The subcellular distribution of copper in the liver at 5d after feeding of a copper deficient diet began was measured and the copper concentrations in soluble and nuclear fractions decreased at a similar rate in copper deficient rats treated with or without NDMA, compared to those of pair-fed rats. The incorporation of [3H]thymidine into rat liver DNA at 48 h after treatment with NDMA markedly increased under the experimental conditions used. By giving rats a copper deficient diet for a few days the increased incorporation of [3H]thymidine into liver DNA of rats treated with NDMA was enhanced compared to that of pair-fed rats treated with NDMA. The activity of thymidine kinase in liver of copper deficient rats treated with NDMA was also found to increase significantly compared to that of pair-fed rats treated with NDMA.

Tissue iron content in riboflavin and pyridoxine deficient rats

The effect of riboflavin and (or) pyridoxine deficiency and repletion on tissue iron content was studied in rats. The iron content in liver, spleen, and kidney and plasma iron concentration of riboflavin deficient (RD) rats was lower, but hematocrit was not. In pyridoxine deficient (PD) rats versus control rats, the iron content in liver was significantly higher but not in spleen and kidney. In PD rats hematocrit was lower but plasma iron concentration was not. Although combined riboflavin and pyridoxine deficient (CD) rats had lower iron content in liver and spleen compared with control rats, these values were intermediate between those of RD rats and PD rats. After RD and PD rats were repleted, the iron content in liver, spleen, and kidney returned to that of control rats, and the hematological indices were improved significantly. These results suggest that riboflavin and pyridoxine deficiency may impair the absorption and utilization of iron and may result in altered tissue iron content.

Chemotatic activity for human PMN generated during ethanol metabolism by rat hepatocytes: Role of glutathione and glutathione peroxidase

Infiltration of the liver by polymorphonuclear leukocytes is a characteristic feature of alcoholic hepatitis. We have previously shown that liver cytosol metabolizing ethanol generates chemoattractant activity for polymorphonuclear leukocytes and that hydroxyl radical scavengers inhibit this process. To investigate the possibility that endogenous glutathione and glutathione peroxidase also inhibit this process, we evaluated chemoattractant activity production by glutathione and glutathione peroxidase deficient rat liver cytosol during ethanol metabolism. Incubation of cytosol deficient in both glutathione and glutathione peroxidase with 10 mM ethanol for 1 hour resulted in a 500-fold increase in chemoattractant activity when compared to cytosol with normal glutathione and glutathione peroxidase content. These findings provide further evidence for a role of oxygen free radicals in the generation of chemotactic activity and they also suggest that the ethanol-induced decrease in hepatic glutathione and glutathione peroxidase reported by others may have a significant potentiating effect on the recruitment of pro-inflammatory cells into the liver.

Determination of nucleotide sequence of cDNA coding rat glutathione peroxidase and diminished expression of the mRNA in selenium deficient rat liver

The cDNA for rat glutathione peroxidase mRNA was isolated from liver cDNA library in λgt11 by cross-hybridization using the mouse cDNA, and it's nucleotide sequence was determined. The selenocysteine which constitutes an active center of this enzyme was encoded by TGA, a nonsense codon in general, as was the cases with mouse and human glutathione peroxidase. Northern blot analysis elucidated that the mRNA for glutathione peroxidase was markedly diminished in selenium deficient rat liver as compared with that of normal rat livers. The result suggested that the de novo synthesis of the mRNA would be regulated by selenium.

Abnormal Molecules of Mitochondrial Aspartate Aminotransferase in the Liver of Vitamin B-6–Deficient Rats May Be Produced in the Mitochondrial Matrix

The distribution of mitochondrial aspartate aminotransferase (AspATm) in liver cells was studied in rats fed pyridoxine-deficient and control diets. Mitochondrial aminotransferase activity was found mainly in the matrix fraction, with smaller amounts in the outer membranes, intermembrane space and cytosol. The precursor of the enzyme was detected in the liver cytosol of both vitamin B-6--deficient and control rats, and its amount was similar in the two groups. When pyridoxal phosphate was added to the assay system, the ratio of enzyme activity to antigenic activity (E/A) of mitochondrial aspartate aminotransferase in the cytosol of both vitamin B-6--deficient and control rats was about 70% of that in the matrix of control rats. On the other hand, the E/A of the matrix enzyme in deficient rats was 53% of that of controls. From these results we concluded that pyridoxal phosphate is not necessary for translocation of mitochondrial aspartate aminotransferase into mitochondrial matrix and that abnormal molecules of the enzyme may be formed in the matrix of vitamin B-6--deficient rat liver.

Effect of biotin deficiency and supplementation on lipid metabolism in rats: saturated fatty acids

The effects of biotin deficiency and supplementation upon saturated fatty acids in serum, liver, cerebrum, and cerebellum of rats were investigated. Serum total fatty acids were reduced in deficient animals to 29% of normal. The percentage composition of some odd-chain fatty acids was increased in the serum and liver of deficient rats. The relative composition of most saturated fatty acids with carbon-chain lengths ≥ 22 were increased in serum and liver of deficient rats. In contrast, there were no differences in the brain saturated fatty acids of deficient animals compared to those of normal and supplemented animals. These results may indicate that alterations in saturated fatty acids do not play a major role in the neurologic abnormalities in human biotin or inherited multiple-carboxylase deficiencies.