Effect of dietary cholesterol and alloxan-diabetes on tissue cholesterol and apolipoprotein E mRNA levels in the rabbit

Alloxan-diabetic rabbits develop hypercholesterolemia and hypertriglyceridemia in response to cholesterol feeding. To determine whether alterations in apolipoprotein composition of plasma lipoproteins were due to changes in apolipoprotein gene expression, we measured the steady state apoE mRNA levels in several tissues from both control and diabetic rabbits. Control rabbits were fed either chow or chow plus 1.5% cholesterol (chow-fed or cholesterol-fed groups) and diabetic rabbits were fed either chow or chow plus 0.5% cholesterol for dietary periods of 5, 21, and 42 days. The tissues examined were liver, small intestine, brain, adrenals, and aorta. ApoE mRNA levels were measured by Northern and dot blot analysis with a human apoE cDNA probe. In control rabbits fed either chow or cholesterol diets for up to 42 days, the steady state apoE mRNA levels remained relatively constant in all of the tissues examined. In contrast, in alloxan-diabetic rabbits fed a 0.5% cholesterol diet, apoE mRNA was reduced in liver, brain, and adrenals (46 +/- 19%, 56 +/- 5%, and 39 +/- 18% of chow-fed control, respectively), but showed little change in the aorta (91 +/- 22% of chow-fed control). Despite a similar increase in plasma cholesterol, the cholesterol content of the liver and adrenals of cholesterol-fed diabetic rabbits were 20% and 50%, respectively, of that of the liver and adrenals in cholesterol-fed control rabbits. The result that apoE mRNA levels and tissue cholesterol content are altered in the diabetic cholesterol-fed rabbit suggests that insulin deficiency in the rabbit may influence apoE gene expression and tissue cholesterol homeostasis.     

Changes in mRNA expression of 3-hydroxy-3-methylglutaryl coenzyme A reductase and cholesterol 7 alpha-hydroxylase in chickens

3-Hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) and cholesterol 7 alpha-hydroxylase (CYP7A1), essential enzymes of cholesterol synthesis and excretion, respectively, were isolated from a chicken liver cDNA library. When their recombinant proteins were overexpressed in HNK293 cells, corresponding enzyme activities were observed. The complete open reading frames of MHGR and CYP7A1 contained (i) 2625 base pairs (bp), predicting a protein of 875 amino acids, and (ii) 1539 bp, predicting a protein of 513 amino acids, respectively. By Northern blot analysis, chicken HMGR mRNA expression was detected in most tissues examined, however, the highest levels were found in liver, brain and ileum. CYP7A1 mRNA was detected only in the liver. Changes in chicken HMGR and CYP7A1 mRNA expression with nutritional state were examined and were shown to respond to certain nutritional treatments, i.e. fast refeeding and cholesterol supplementation. HMGR and CYP7A1 mRNA levels were significantly increased with maturation (i.e. egg producing), when compared to immature chickens. However, these stimulations were not associated with estrogen, although this does enhance triacylglycerol and very low density lipoprotein secretion by the chicken liver. The present study is the first to report the molecular characterization of HMGR and CYP7A1, key enzymes of cholesterol metabolism in avian species.     

Induction of rat liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase mRNA by refeeding and insulin

The effects of fasting/refeeding and untreated or insulin-treated diabetes on the bifunctional enzyme 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase and its mRNA in rat liver were determined. Both enzymatic activities fell to 20% of control values with fasting or streptozotocin-induced diabetes and were coordinately restored to normal within 48 h of refeeding or 24 h of insulin administration. These alterations in enzymatic activities were always mirrored by corresponding changes in amount of enzyme as determined by phosphoenzyme formation and immunoblotting. In contrast, mRNA for 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase did not decrease during starvation or in diabetes, but there was a 3-6-fold increase upon refeeding a high carbohydrate diet to starved rats or insulin treatment of diabetic rats. The decrease of the enzyme in starved or diabetic rats without associated changes in mRNA levels suggests a decrease in the rate of mRNA translation, an increase in enzyme degradation, or both. The rise in enzyme amount and mRNA for 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase with refeeding and insulin treatment suggests an insulin-dependent stimulation of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase gene expression. Northern blots of RNA from heart, brain, kidney, and skeletal muscle probed with restriction fragments of a full-length cDNA from liver showed that only skeletal muscle contained an RNA species that hybridized to any of the probes. Skeletal muscle mRNA for 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase was 2.0 kilobase pairs but in contrast to the liver message (2.2 kilobase pairs) was not regulated by refeeding.     

Identification and expression analysis of rainbow trout <Emphasis Type="Italic">pumilio</Emphasis>-1 and <Emphasis Type="Italic">pumilio</Emphasis>-2

Pumilio is a sequence-specific RNA-binding protein that regulates translation from the relevant mRNA. The PUF-domain, the RNA-binding motif of Pumilio, is highly conserved across species. In the present study, we have identified two pumilio genes (pumilio-1 and pumilio-2) in rainbow trout and analyzed their expression patterns in its tissues. Pumilio-1 mRNA and pumilio-2A mRNA code for typical full length Pumilio proteins that contain a PUF-domain, whereas pumilio-2B mRNA is a splice variant of pumilio-2 and encodes a protein that lacks the PUF-domain. We have also identified a novel 72-bp exon that has not been reported in other animal species but is conserved in fish species. The insertion of this novel exon leads to the expression of an isoform of the Pumilio-2 protein with a slightly altered conformation of the PUF-domain. Pumilio-1 mRNA and pumilio-2A mRNA (irrespective of the presence of the 72-bp exon) are expressed in both the brain and ovaries at high levels, whereas pumilio-2B mRNA is expressed at low levels in all the rainbow trout tissues examined. Western blot analysis also indicates that the full length Pumilio proteins are expressed predominantly in the brain and ovaries. These data suggest that the Pumilio proteins have physiological roles and are involved in regulatory mechanisms in rainbow trout.This work was in part supported by a grant from the Akiyama Foundation to E.I. Nucleotide sequence data for rainbow trout pumilio-1 and pumilio-2 have been deposited in the DDBJ/EMBL/GenBank databases.     

Characterization of the antioxidant status of the heterozygous manganese superoxide dismutase knockout mouse

The antioxidant status of several tissues (liver, kidney, lung, brain, heart, muscle, stomach, and spleen) from heterozygous manganese superoxide dismutase (MnSOD) mutant mice (Sod2-/+) was characterized. The activity of MnSOD was decreased (30 to 80%) in all tissues examined. The levels of mRNA coding for the major antioxidant enzymes (CuZnSOD, catalase, and glutathione peroxidase) were not significantly altered in liver, kidney, heart, lung, or brain in the Sod2-/+ mice. The activities of the enzymes were not altered in any of these tissues, with the exception of a decrease in glutathione peroxidase activity in muscle in the Sod2-/+ mice compared to the Sod2+/+ mice. Thus, there was no up-regulation of the activities of the major antioxidant enzymes to compensate for the decrease in MnSOD activity. Reduced glutathione levels were 30 to 50% lower in the lung, brain, and muscle of the Sod2-/+ mice compared to the wild-type Sod2+/+ mice. In addition, the ratio of GSH/GSSG was decreased approximately 50% in Sod2-/+ muscle, indicating that the decrease in MnSOD activity in the Sod2-/+ mice results in some degree of oxidative stress in this tissue.     

Tissue-specific expression, developmental regulation, and chromosomal mapping of the lecithin: cholesterol acyltransferase gene. Evidence for expression in brain and testes as well as liver

Lecithin:cholesterol acyltransferase (LCAT) catalyzes the esterification of cholesterol in high density lipoproteins, thereby facilitating transport of excess cholesterol from peripheral tissues to liver. We report here studies of the developmental, dietary, and genetic control of LCAT gene expression. In adult male Sprague-Dawley rats fed a standard chow diet LCAT mRNA was most abundant in liver, a major source of the plasma enzyme, but appreciable levels were also present in brain and testes. Since both brain and testes are isolated from blood by tight cellular barriers, undoubtedly greatly reducing the level of plasma-derived LCAT in cerebrospinal fluid and testes, the production of LCAT in these tissues may be important for removal of excess cholesterol. Noteworthy changes in the expression of LCAT mRNA were observed during development of both rodents and humans. On the other hand, LCAT mRNA levels were relatively resistant to dietary challenge or to drugs affecting cholesterol metabolism. Since human epidemiological studies have suggested an association between LCAT levels and variations of high density lipoprotein cholesterol, we examined LCAT gene polymorphisms in a mouse animal model. Mapping of the LCAT gene (Lcat) to mouse Chromosome 8 within 2 centimorgans of the Es-2 locus indicates that it does not correspond to any previously mapped loci affecting high density lipoprotein phenotypes in the mouse.     

Oxidative damage and antioxidant enzyme activities in experimental hypothyroidism

Free radicals are now well known to damage cellular components. To investigate whether age and thyroid level affect peroxidation speed, we examined the levels of malondialdehyde and antioxidant enzyme activities in different age groups of hypothyroid rats. Hypothyroidism was induced in 30- and 60-day-old Wistar Albino rats by the i.p. administration of propylthiouracil (10 mg kg(-1) body weight) for 15 days. While malondialdehyde levels of 30- or 60-day-old hypothyroid rats were increased in liver, they were decreased in the tissues of the heart and thyroid. While glucose-6-phosphate dehydrogenase activity levels did not change in heart, brain and liver tissues of 30-day-old rats, they increased in brain and heart tissues of 60-day-old experimental groups, but decreased in the liver. Catalase activities decreased in the liver and heart of rats with hypothyroidism, but increased in erythrocytes. In control groups while malondialdehyde levels increased in brain, heart and thymus with regard to age, they decreased in plasma. Glucose-6-phosphate dehydrogenase and catalase activities were not affected by age in tissues of the thymus, thyroid and brain, but they were decreased in the heart tissue. The changes in the levels of lipid peroxidation and antioxidant enzyme activities which were determined in different tissues of hypothyroid rats indicate a cause for functional disorder of these tissues. Moreover, there may be changes depending on age at lipid peroxidation and antioxidant enzyme activity levels.     

Differential effects of different statins on endothelin-1 gene expression and endothelial NOS phosphorylation in porcine aortic endothelial cells

It has been reported that 3-hydroxy-3-methylglutaryl CoA (HMG-CoA) reductase inhibitors (statins) produce a variety of cardiovascular protective effects independent of their ability to lower total and low-density lipoprotein cholesterol. Recent studies have also reported that statins produce pleiotropic effects through improved endothelial function, enhanced fibrinolysis, and antithrombotic actions. In the present study, we examined the effects of pitavastatin, pravastatin, atorvastatin, and cerivastatin on endothelin (ET)-1 production in cultured porcine aortic endothelial cells (PAECs). Treatment with cerivastatin but not pitavastatin, pravastatin, or atorvastatin decreased basal and TNF-alpha-stimulated ET-1 release from PAECs in a dose-dependent manner (1-10 microM). Northern blot analysis showed that cerivastatin markedly suppressed prepro ET-1 mRNA expression in both conditions. In addition, these inhibitory effects of cerivastatin on ET-1 release and prepro ET-1 mRNA expression were completely abolished by simultaneous treatment with 200 microM mevalonate. Furthermore, cerivastatin did not have any effects on endothelial nitric oxide synthase (eNOS) protein levels, but induced eNOS phosphorylation at Ser1177. From these findings, it is most likely that cerivastatin suppresses ET-1 production, possibly through an increase in eNOS activity and the subsequent nitric oxide production in PAECs. These findings also suggest that cerivastatin may have beneficial effects on ET-1-related diseases.     

Variation in insulin receptor messenger ribonucleic acid expression in human and rodent tissues

The expression of insulin receptor mRNA was studied in human and rodent tissues by Northern analysis. Human EBV-transformed lymphocytes contained four receptor mRNA species of sufficient length to encode the entire proreceptor: 9.5, 7.9, 7.1, and 5.7 kb. In human fibroblasts, the same four species were observed; however, the 7.9 and 5.7 kb mRNAs were markedly decreased. In mouse liver, rat hepatoma cells, and normal rat brain, kidney, liver, and muscle only two mRNA species (7.4 and 9.6 kb) were detected. Each of these human and rodent mRNAs hybridized equally well with cDNA sequences encoding the binding and kinase domains of the insulin receptor. Several smaller polyadenylated mRNAs (approximately 1.8 to 3.3 kb) were also identified in human cell lines that appeared to separately encode either alpha- or beta-subunit sequences of the receptor. In rats, liver had the highest content of insulin receptor mRNA, followed by kidney, brain, and muscle. The relative amount of the two mRNA species also varied among the rat tissues. The ratio of the 9.6-7.4 kb species was 2.7 in brain but only 1.0 to 1.6 in the other tissues (P less than 0.025). Dexamethasone treatment increased the content of the two insulin receptor mRNAs in rat liver by 2-fold. The half-life of both mRNA species was 70 min in rat hepatoma cells. These findings indicate that insulin receptor gene expression is complex and regulated with differential expression of insulin receptor mRNA and/or alterations in mRNA processing among various tissues.     

Quantitation of apolipoprotein E mRNA in the liver and peripheral tissues of nonhuman primates

A sensitive DNA-excess solution hybridization assay was used to quantitate apo-E mRNA in the liver and peripheral tissues of two nonhuman primates, Macaca fascicularis and Cercopithecus aethiops. When expressed on the basis of total RNA, apo-E mRNA values for M. fascicularis adrenal, brain, testis, and spleen ranged from 17-52% of the liver value. Apo-E mRNA values for mesenteric lymph node, kidney, thymus, and skeletal muscle were 1-5% of the liver value. When expressed on a cellular basis, apo-E mRNA was most abundant in the liver at approximately 1200 molecules/cell. Peripheral tissues showed a continuous range of apo-E mRNA from 1.5 molecules/cell in the thymus up to 350 molecules/cell in the brain. Similar results were obtained with peripheral tissues from C. aethiops in which case apo-E mRNA also was found in skin, lung, skeletal muscle, small intestine, and vascular tissues such as heart, aorta, and brachial artery. Calculation of the total apo-E mRNA/organ showed that most of the apo-E mRNA was present in the liver. However, summation of apo-E mRNA in peripheral tissues indicated that 20-40% of total body apo-E mRNA was extrahepatic. This results indicates that apo-E made in peripheral tissues may play a quantitatively important role in cholesterol metabolism since peripheral tissues have the potential to contribute a significant fraction of plasma apo-E.