Cellular expression of retinal dehydrogenase types 1 and 2: effects of vitamin A status on testis mRNA

We examined expression of retinal dehydrogenase (RALDH) types 1 and 2 in liver and lung, and the effect of vitamin A status on testis expression by in situ hybridization. Liver expressed RALDH1 and RALDH2 only in stellate cells and hepatocytes, respectively. Lung expressed RALDH1 and RALDH2 throughout the epithelia of the airways, from the principal bronchi to the respiratory bronchiole. Vitamin A-sufficient rats expressed RALDH1 in spermatocytes, with less intense expression in spermatogonia and spermatids, and expressed RALDH2 in interstitial cells, spermatogonia, and spermatocytes. Neither Sertoli nor peritubular cells showed detectable RALDH1 or RALDH2 mRNA. Vitamin A deficiency produced a sevenfold increase in RALDH1 and a 70-fold decrease in RALDH2 mRNA in testis. In each case, the net change reflected extensive loss of germ cells, increased intensity of expression in residual germ cells, and expression in Sertoli and peritubular cells. Low-dose RA relatively early during vitamin A depletion supported spermatogenesis and affected expression of both RALDHs, but did not reinstate "vitamin A normal" expression patterns. These results show that: RALDH1 and RALDH2 have distinct mRNA expression patterns in multiple cell types in three vitamin A target tissues; RALDH expression occurs in cell types that express cellular retinol-binding protein and retinol dehydrogenase isozymes (except stellate cells, for which retinol dehydrogenase expression remains unknown); vitamin A deficiency and RA supplementation affects the loci and intensity of RALDH mRNAs in testis; and low-dose RA does not substitute completely for retinol. Overall, these data provide insight into the unique functions of RALDH1 and RALDH2 in retinoid metabolism.     

Roles of vitamin A status and retinoids in glucose and fatty acid metabolism

The rising prevalence of metabolic diseases, such as obesity and diabetes, has become a public health concern. Vitamin A (VA, retinol) is an essential micronutrient for a variety of physiological processes, such as tissue differentiation, immunity, and vision. However, its role in glucose and lipid metabolism has not been clearly defined. VA activities are mediated by the metabolite of retinol catabolism, retinoic acid, which activates the retinoic acid receptor and retinoid X receptor (RXR). Since RXR is an obligate heterodimeric partner for many nuclear receptors involved in metabolism, it is reasonable to assume that VA status and retinoids contribute to glucose and lipid homeostasis. To date, the impacts of VA and retinoids on energy metabolism in animals and humans have been demonstrated in some basic and clinical investigations. This review summarizes the effects of VA status and retinoid treatments on metabolism of the liver, adipocytes, pancreatic β-cells, and skeletal muscle. It proposes a mechanism by which the dietary and hormonal signals converge on the promoter of sterol regulatory element-binding protein 1c gene to induce its expression, and in turn, the expression of lipogenic genes in hepatocytes. Future research projects relevant to the VA's roles in metabolic diseases are also discussed.     

Lecithin:retinol acyltransferase from mouse and rat liver. CDNA cloning and liver-specific regulation by dietary vitamin a and retinoic acid

Lecithin:retinol acyltransferase (LRAT), present in microsomes, catalyzes the transfer of the sn-1 fatty acid of phosphatidylcholine to retinol bound to a cellular retinol-binding protein. In the present study we have cloned mouse and rat liver LRAT cDNA and tested the hypothesis that LRAT mRNA, like LRAT activity, is regulated physiologically in a liver-specific manner. The nucleotide sequences of mouse and rat liver LRAT cDNA each encode a 231-amino acid protein with 94% similarity between these species, and approximately 80% similarity to a cDNA for LRAT from human retinal pigment epithelium. Expression of rat LRAT cDNA in HEK293T cells resulted in functional retinol esterification and storage. RNA from several rat tissues hybridized with liver LRAT cDNA. However, LRAT mRNA was virtually absent from the liver of vitamin A-deficient animals, while being unaffected in intestine and testis. LRAT mRNA was rapidly induced by retinoic acid (RA) in liver of vitamin A-deficient mice and rats (P < 0.01). LRAT mRNA and enzymatic activity were well correlated in the same livers of rats treated with exogenous RA (r = 0.895, P < 0.0001), and in a dietary study that encompassed a broad range of vitamin A exposure (r = 0.799, P < 0.0001). Liver total retinol of <100 nmol/g was associated with low LRAT expression (<33% of control).We propose that RA, derived exogenously or from metabolism, serves as an important signal of vitamin A status. The constitutive expression of liver LRAT during retinoid sufficiency would serve to divert retinol into storage pools, while the curtailment of LRAT expression in retinoid deficiency would maintain retinol for secretion and delivery to peripheral tissues.     

Vitamin neurotoxicity

Vitamins contain reactive functional groups necessary to their established roles as coenzymes and reducing agents. Their reactive potential may produce injury if vitamin concentration, distribution, or metabolism is altered. However, identification of vitamin toxicity has been difficult. The only well-established human vitamin neurotoxic effects are those due to hypervitaminosis A (pseudotumor cerebri) and pyridoxine (sensory neuropathy). In each case, the neurological effects of vitamin deficiency and vitamin excess are similar. Closely related to the neurological symptoms of hypervitaminosis A are symptoms including headache, pseudotumor cerebri, and embryotoxic effects reported in patients given vitamin A analogs or retinoids. Most tissues contain retinoic acid (RA) and vitamin D receptors, members of a steroid receptor superfamily known to regulate development and gene expression. Vitamin D3 effects on central nervous system (CNS) gene expression are predictable, in addition to the indirect effects owing to its influence on calcium and phosphorus homeostasis. Folates and thiamine cause seizures and excitation when administered in high dosage directly into the brain or cerebrospinal fluid (CSF) of experimental animals but have rarely been reported to cause human neurotoxicity, although fatal reactions to i.v. thiamine are well known. Ascorbic acid influences CNS function after peripheral administration and influences brain cell differentiation and 2-deoxyglucose accumulation by cultured glial cells. Biotin influences gene expression in animals that are not vitamin-deficient and alters astrocyte glucose utilization. The multiple enzymes and binding proteins involved in regeneration of retinal vitamin A illustrate the complexity of vitamin processing in the body. Vitamin A toxicity is also a good general model of vitamin neurotoxicity, because it shows the importance of the ratio of vitamin and vitamin-binding proteins in producing vitamin toxicity and of CNS permeability barriers. Because vitamin A and analogs enter the CNS better than most vitamins, and because retinoids have many effects on enzyme activity and gene expression, Vitamin A neurotoxicity is more likely than that of most, perhaps all other vitamins. Megadose vitamin therapy may cause injury that is confused with disease symptoms. High vitamin intake is more hazardous to peripheral organs than to the nervous system, because CNS vitamin entry is restricted. Vitamin administration into the brain or CSF, recommended in certain disease states, is hazardous and best avoided. The lack of controlled trials prevents us from defining the lowest human neurotoxic dose of any vitamin. Large differences in individual susceptibility to vitamin neurotoxicity probably exist, and ordinary vitamin doses may harm occasional patients with genetic disorders.(ABSTRACT TRUNCATED AT 400 WORDS)     

Gene Expression Profile Analysis of Type 2 Diabetic Mouse Liver

Liver plays a key role in glucose metabolism and homeostasis, and impaired hepatic glucose metabolism contributes to the development of type 2 diabetes. However, the precise gene expression profile of diabetic liver and its association with diabetes and related diseases are yet to be further elucidated. In this study, we detected the gene expression profile by high-throughput sequencing in 9-week-old normal and type 2 diabetic db/db mouse liver. Totally 12132 genes were detected, and 2627 genes were significantly changed in diabetic mouse liver. Biological process analysis showed that the upregulated genes in diabetic mouse liver were mainly enriched in metabolic processes. Surprisingly, the downregulated genes in diabetic mouse liver were mainly enriched in immune-related processes, although all the altered genes were still mainly enriched in metabolic processes. Similarly, KEGG pathway analysis showed that metabolic pathways were the major pathways altered in diabetic mouse liver, and downregulated genes were enriched in immune and cancer pathways. Analysis of the key enzyme genes in fatty acid and glucose metabolism showed that some key enzyme genes were significantly increased and none of the detected key enzyme genes were decreased. In addition, FunDo analysis showed that liver cancer and hepatitis were most likely to be associated with diabetes. Taken together, this study provides the digital gene expression profile of diabetic mouse liver, and demonstrates the main diabetes-associated hepatic biological processes, pathways, key enzyme genes in fatty acid and glucose metabolism and potential hepatic diseases.     

Mechanisms involved in the intestinal absorption of dietary vitamin A and provitamin A carotenoids

Vitamin A is an essential nutrient for humans and is converted to the visual chromophore, 11-cis-retinal, and to the hormone, retinoic acid. Vitamin A in animal-derived foods is found as long chain acyl esters of retinol and these are digested to free fatty acids and retinol before uptake by the intestinal mucosal cell. The retinol is then reesterified to retinyl esters for incorporation into chlylomicrons and absorbed via the lymphatics or effluxed into the portal circulation facilitated by the lipid transporter, ABCA1. Provitamin A carotenoids such as β-carotene are found in plant-derived foods. These and other carotenoids are transported into the mucosal cell by scavenger receptor class B type I (SR-BI). Provitamin A carotenoids are partly converted to retinol by oxygenase and reductase enzymes and the retinol so produced is available for absorption via the two pathways described above. The efficiency of vitamin A and carotenoid intestinal absorption is determined by the regulation of a number of proteins involved in the process. Polymorphisms in genes for these proteins lead to individual variability in the metabolism and transport of vitamin A and carotenoids. This article is part of a Special Issue entitled Retinoid and Lipid Metabolism.     

Abcb11 deficiency induces cholestasis coupled to impaired β-fatty acid oxidation in mice

The bile salt export pump (BSEP) is an ATP-binding cassette transporter that serves as the primary system for removing bile salts from the liver. In humans, deficiency of BSEP, which is encoded by the ABCB11 gene, causes severe progressive cholestatic liver disease from early infancy. In previous studies of Abcb11 deficiency in mice generated on a mixed genetic background, the animals did not recapitulate the human disease. We reasoned that ABCB11 deficiency may cause unique changes in hepatic metabolism that are predictive of liver injury. To test this possibility, we first determined that Abcb11 knock-out (KO) C57BL/6J mice recapitulate human deficiency. Before the onset of cholestasis, Abcb11 KO mice have altered hepatic lipid metabolism coupled with reduced expression of genes important in mitochondrial fatty acid oxidation. This was associated with increased serum free-fatty acids, reduced total white adipose, and marked impairment of long-chain fatty acid β-oxidation. Importantly, metabolomic analysis confirmed that Abcb11 KO mice have impaired mitochondrial fatty acid β-oxidation with the elevated fatty acid metabolites phenylpropionylglycine and phenylacetylglycine. These metabolic changes precede cholestasis but may be of relevance to cholestatic disease progression because altered fatty acid metabolism can enhance reactive oxygen species that might exacerbate cholestatic liver damage.     

Vitamin A deficiency in mice alters host and gut microbial metabolism leading to altered energy homeostasis

Vitamin A deficiency (A−) is a worldwide public health problem. To better understand how vitamin A status influences gut microbiota and host metabolism, we systematically analyzed urine, cecum, serum and liver samples from vitamin A sufficient (A+) and deficient (A−) mice using ¹H NMR-based metabolomics, quantitative (q)PCR and 16S rRNA gene sequencing coupled with multivariate data analysis. The microbiota in the cecum of A− mice showed compositional as well as functional shifts compared to the microbiota from A+ mice. Targeted ¹H NMR analyses revealed significant changes in microbial metabolite concentrations including higher butyrate and hippurate and decreased acetate and 4-hydroxyphenylacetate in A+ relative to A− mice. Bacterial butyrate-producing genes including butyryl-CoA:acetate CoA-transferase and butyrate kinase were significantly higher in bacteria from A+ versus bacteria from A− mice. A− mice had disturbances in multiple metabolic pathways including alterations in energy (hyperglycemia, glycogenesis, TCA cycle and lipoprotein biosynthesis), amino acid and nucleic acid metabolism. A− mice had hyperglycemia, liver dysfunction, changes in bacterial metabolism and altered gut microbial communities. Moreover, integrative analyses indicated a strong correlation between gut microbiota and host energy metabolism pathways in the liver. Vitamin A regulates host and bacterial metabolism, and the result includes alterations in energy homeostasis.     

Iron and zinc status in rats with diet-induced marginal deficiency of vitamin A and/or copper

The hypothesis was tested that there are interactions of marginal copper and vitamin A deficiency regarding iron and zinc status. Copper restriction (1 vs 5 mg Cu/kg diet) significantly lowered copper concentrations in plasma and tissues of rats and reduced blood hemoglobin, hematocrit, and iron concentrations in tibia and femur, but raised iron concentrations in liver. Vitamin A restriction (0 vs 4000 IU vitamin A/kg diet) reduced plasma retinol concentrations and induced a fall of blood hemoglobin and hematocrit. Neither copper nor vitamin A restriction for up to 42 d affected feed intake and body wt gain. There were no interrelated effects of vitamin A and copper deficiency on iron status. Copper deficiency slightly depressed liver, spleen, and kidney zinc concentrations. Vitamin A deficiency lowered zinc concentrations in heart, but only when the diets were deficient in copper.     

Vitamin A deficiency modulates iron metabolism via ineffective erythropoiesis

Vitamin A modulates inflammatory status, iron metabolism and erythropoiesis. Given that these factors modulate the expression of the hormone hepcidin (Hamp), we investigated the effect of vitamin A deficiency on molecular biomarkers of iron metabolism, the inflammatory response and the erythropoietic system. Five groups of male Wistar rats were treated: control (AIN-93G), the vitamin A-deficient (VAD) diet, the iron-deficient (FeD) diet, the vitamin A- and iron-deficient (VAFeD) diet or the diet with 12 mg atRA/kg diet replacing all-trans-retinyl palmitate by all-trans retinoic acid (atRA). Vitamin A deficiency reduced serum iron and transferrin saturation levels, increased spleen iron concentrations, reduced hepatic Hamp and kidney erythropoietin messenger RNA (mRNA) levels and up-regulated hepatic and spleen heme oxygenase-1 gene expression while reducing the liver HO-1 specific activity compared with the control. The FeD and VAFeD rats exhibited lower levels of serum iron and transferrin saturation, lower iron concentrations in tissues and lower hepatic Hamp mRNA levels compared with the control. The treatment with atRA resulted in lower serum iron and transferrin concentrations, an increased iron concentration in the liver, a decreased iron concentration in the spleen and in the gut, and decreased hepatic Hamp mRNA levels. In summary, these findings suggest that vitamin A deficiency leads to ineffective erythropoiesis by the down-regulation of renal erythropoietin expression in the kidney, resulting in erythrocyte malformation and the consequent accumulation of the heme group in the spleen. Vitamin A deficiency indirectly modulates systemic iron homeostasis by enhancing erythrophagocytosis of undifferentiated erythrocytes.     

Effect of excess and deficiency of vitamin A on the utilization of FFA by liver and skeletal muscle.

Fatty acid metabolism in liver and skeletal muscle has been studied in rats treated with high doses of vitamin A and in those made vitamin A-deficient. Ingestion of 30,000 IU of vitamin A for two days resulted in increased incorporation of palmitate-1-14C into triglycerides but not into phospholipids. Accumulation of hepatic triglycerides was observed in vitamin A-fed rats. Deficiency of vitamin A did not cause any change in the triglyceride or phospholipid content of the liver. The rate of hepatic fatty acid oxidation and ketogenesis was markedly increased in vitamin A-fed rats. The experimental evidence indicated that vitamin A may have a stimulatory effect on these processes apart from that exerted by the high plasma FFA level in vitamin A-fed rats. Oxidation of palmitate-1-14C into C32 by skeletal muscle (latissimus dorsi) was also increased as a result of vitamin A administration. Vitamin A deficiency did not cause any change in fatty acid oxidation by liver and skeletal muscle. Hepatic palmitoyl-CoA synthetase activity was decreased in vitamin A-deficient rats. The results presented suggest that vitamin A may be required for the uptake and utilization of fatty acids by liver and akeletal muscle.     

Disruption of the lecithin:retinol acyltransferase gene makes mice more susceptible to vitamin A deficiency

Lecithin:retinol acyltransferase (LRAT) catalyzes the esterification of retinol (vitamin A) in the liver and in some extrahepatic tissues, including the lung. We produced an LRAT gene knock-out mouse strain and assessed whether LRAT-/- mice were more susceptible to vitamin A deficiency than wild type (WT) mice. After maintenance on a vitamin A-deficient diet for 6 weeks, the serum retinol level was 1.34 +/- 0.32 microM in WT mice versus 0.13 +/- 0.06 microM in LRAT-/- mice (p < 0.05). In liver, lung, eye, kidney, brain, tongue, adipose tissue, skeletal muscle, and pancreas, the retinol levels ranged from 0.05 pmol/mg (muscle and tongue) to 17.35 +/- 2.66 pmol/mg (liver) in WT mice. In contrast, retinol was not detectable (<0.007 pmol/mg) in most tissues from LRAT-/- mice after maintenance on a vitamin A-deficient diet for 6 weeks. Cyp26A1 mRNA was not detected in hepatic tissue samples from LRAT-/- mice but was detected in WT mice fed the vitamin A-deficient diet. These data indicate that LRAT-/- mice are much more susceptible to vitamin A deficiency and should be an excellent animal model of vitamin A deficiency. In addition, the retinol levels in serum rapidly increased in the LRAT-/- mice upon re-addition of vitamin A to the diet, indicating that serum retinol levels in LRAT-/- mice can be conveniently modulated by the quantitative manipulation of dietary retinol.     

Transfer of Vitamins E and A from yolk to embryo during development of the king penguin (Aptenodytes patagonicus).

Since the yolk lipids of the king penguin (Aptenodytes patagonicus) are rich in n-3 fatty acids, which are potentially susceptible to peroxidative damage, the yolk contents and yolk-to-embryo transfer of antioxidants and lipid-soluble vitamins were investigated under conditions of natural incubation in the wild. The concentration of vitamin E in the unincubated egg was 155 microg/g wet yolk, of which 88% was alpha-tocopherol and the rest was gamma-tocopherol. Vitamin A (2.9 microg/g) was present in the yolk entirely as retinol; no retinyl esters were detected. Throughout the latter half of the incubation period, vitamins E and A were taken up from the yolk into the yolk sac membrane (YSM) and later accumulated in the liver, with vitamin A being transferred in advance of vitamin E. In the YSM, vitamin A was present almost entirely as retinyl ester, indicating that the free retinol of the yolk is rapidly esterified following uptake. Retinyl esters were also the predominant form in the liver. The retinyl esters of the liver and YSM displayed different fatty acid profiles. At hatching, the brain contained relatively little vitamin E (4.7 microg/g) compared to the much higher concentration in the liver (482.9 microg/g) at this stage. Ascorbic acid was not detected in the yolk but was present at a high concentration in the brain at day 27 (404.6 microg/g), decreasing to less than half this value by the time of hatching. This report is the first to delineate the yolk-to-embryo transfer of lipid-soluble vitamins for a free-living avian species. The yolk fatty acids of the king penguin provide an extreme example of potential oxidative susceptibility, forming a basis for comparative studies on embryonic antioxidant requirements among species of birds whose yolk lipids differ in their degree of unsaturation.     

Vitamin A and microsomal membranes: the effect of retinol deficiency on lipid microviscosity and phospholipid turnover in rat liver microsomes

Studies with the use of the fluorescent probe pyrene revealed that vitamin A deficiency in maturing male rats results in the increased microviscosity of liver lipids. This effect seems to be due to changes in the lipid composition of microsomal membranes (increased cholesterol/phospholipid ratio and lowered polyunsaturated fatty acid content) as well as to the low level of retinol. Analysis of microsomal phospholipids labeled with [3H]palmitate and [14C]glycerol revealed that vitamin A deficiency accelerates the turnover of the glycerol skeleton but sharply decelerates that of fatty acid residues. It is concluded that the observed effect of retinol on the structural and functional properties of biological membranes is due to its ability to control the microviscosity and turnover of membrane lipids.     

Composition of α-tocopherol and fatty acids in porcine tissues after dietary supplementation with vitamin E and different fat sources

The present study evaluated the effect of increasing supplementation of all-rac-α-tocopheryl acetate and dietary fatty acid composition during a four week period after weaning on porcine tissue composition of α-tocopherol stereoisomers and fatty acids, and on hepatic expression of genes involved in transfer of α-tocopherol, and oxidation and metabolism of fatty acids. From day 28 to 56 of age, pigs were provided 5% of tallow, fish oil or sunflower oil and 85, 150, or 300 mg/kg of all-rac-α-tocopheryl acetate. Samples of liver, heart, and adipose tissue were obtained from littermates at day 56. Tissue fatty acid composition was highly influenced by dietary fat sources. Dietary fatty acid composition (P<0.001) and vitamin E supplementation (P<0.001) influenced the α-tocopherol stereoisomer composition in liver, i.e. less proportion of the RRR-α-tocopherol was observed in pigs provided fish oil and the highest dose of vitamin E in comparison with other dietary treatments. In addition, the stereoisomer composition of α-tocopherol in heart, and adipose tissue was influenced by dietary treatments. Expression of genes in liver involved in the regulation of FA conversion, SCD (P=0.002) and D6D (P=0.04) were lower in pigs fed fish oil compared to other treatments, whereas the fatty acid oxidation, as indicated by the expression of PPAR-α, was higher when sunflower and fish oil was provided (P=0.03). Expression of α-TTP in liver was higher in pigs fed fish oil (P=0.01). Vitamin E supplementation did not influence significantly the hepatic gene expression.     

Regulation of CYP26 (cytochrome P450RAI) mRNA expression and retinoic acid metabolism by retinoids and dietary vitamin A in liver of mice and rats.

Retinoic acid (RA), through nuclear retinoid receptors, regulates the expression of numerous genes. However, little is known of the biochemical mechanisms that regulate RA concentration in vivo. CYP26 (P450RAI), a novel cytochrome P450, is expressed during embryonic development, induced by all-trans RA, and capable of catalyzing the oxidation of [3H]RA to polar retinoids including 4-oxo-RA. Here we report that CYP26 expression in adult liver is regulated by all-trans RA and dietary vitamin A, and is correlated with the metabolism of all-trans RA to polar metabolites. In normal mouse and rat liver, CYP26 mRNA was barely detectable; however, after acute treatment with all-trans RA CYP26 mRNA and RA metabolism by liver microsomes were significantly induced. Aqueous-soluble RA metabolites were detected, but their formation was not induced. The expression of retinoid receptors, RAR-gamma and RXR-alpha, was not changed after RA treatment in vivo. In a model of chronic vitamin A ingestion during aging, CYP26 mRNA expression, determined by Northern blot and RT-PCR analysis, increased progressively with dietary vitamin A (P<0.0001; marginal < control < supplemented) and age (P<0.003). The relative expression of CYP26 mRNA was positively correlated with liver total retinol (log10), ranging from undetectable CYP26 expression at liver retinol concentrations below approximately 20 nmol/g to a three- to fourfold elevation at concentrations >10,000 nmol/g (r=0.90, P<0.0001). We conclude that CYP26 expression and RA metabolism are regulated in adult liver not only acutely by RA administration, as may be relevant to retinoid therapy, but under chronic dietary conditions relevant to vitamin A nutrition in humans.