Plasma apolipoprotein concentrations in familial apolipoprotein A-I and A-II deficiency (Tangier disease)

Familial apolipoprotein A-I and A-II deficiency (Tangier disease) is characterized by cholesterol ester deposition in histiocytes, decreased plasma cholesterol and low density lipoprotein cholesterol (C-LDL), and a striking deficiency of high density lipoproteins (HDL). We measured plasma lipid, lipoprotein cholesterol, and plasma apolipoprotein (apo) A-I, A-II, B, C-I, C-II, C-III, D, and E concentrations in 7 Tangier homozygotes, 2 obligate heterozygotes, and 50 normal subjects. Heterozygotes had modest reductions in high density lipoprotein cholesterol (C-HDL), plasma apoA-I, and apoA-II levels. Mean concentrations (±SD) of plasma C-HDL and apolipoproteins A-I, A-II, B, C-I, C-II, C-III, D, and E in mg% in normals were: 50 ± 14, 134 ± 24, 68 ± 18, 98 ± 20, 7 ± 2, 3.7 ± 2, 13 ± 5, 10 ± 4, and 10 ± 4, respectively; and in homozygotes were: 1 ± 1, 1.3 ± 0.7, 4.8 ± 2.5, 82.6 ± 18, 4.1 ± 1.7, 2.3 ± 0.9, 6.5 ± 3.8, 2.2 ± 0.5, and 5.4 ± 3.1, respectively. Homozygotes had C-HDL, apoA-I and apoA-II levels which were 2%, 1%, and 7% (p < .001) of normal, respectively, and mean levels of apolipoproteins B, C-I, C-II, C-III, D, and E which were 84%, 59%, 62%, 50%, 22%, and 54% of normal, respectively. There was heterogeneity of these latter apolipoprotein concentrations among homozygotes. Mean apoC-I, apoC-III, apoD, and apoE levels were significantly less than normal (pp < .05) in homozygotes. These data indicate that homozygotes have variable but generally decreased apoC and apoE levels, a deficiency of apoD, and a striking reduction in plasma apoA-I and apoA-II concentrations.     

High-density apolipoprotein A-I and A-II kinetics in relation to regional adiposity

High-density lipoprotein (HDL) cholesterol and apolipoprotein (apo) A-I concentrations decrease with increasing central adiposity. The present study investigated possible mechanisms for these effects by examining the relationship between body mass index, regional adiposity, and HDL apo A-I and A-II metabolism. Fifteen sedentary men and 10 male endurance athletes aged 22 to 44 served as subjects. HDL apo A-I and A-II metabolism was examined using 125I-labeled autologous HDL. Chest and thigh skinfold thickness and the ratio of chest to thigh skinfold thickness were used as indices of regional adiposity. The relationship of adiposity to HDL metabolism was examined using correlational and multiple regression analysis. In both subject groups, the fractional catabolic rate of apo A-I and A-II increased with increasing chest skinfold thickness and chest to thigh skinfold ratio (.43 < r2 < .66). This effect was partially independent of triglyceride or HDL cholesterol concentrations. Apo A-I and A-II fractional catabolic rates increased with increasing body mass index only in the sedentary men. Concentrations and synthetic rates (mg.d-1.kg-1) of apo A-I and A-II were not consistently related to body mass index or regional adiposity. Peripheral adiposity assessed by thigh skinfold thickness was not correlated with any parameter of apo metabolism. We conclude that HDL apo A-I and A-II catabolism increases with increasing central adiposity.     

Tissue-specific expression and developmental regulation of the rat apolipoprotein B gene.

Expression of the apolipoprotein B (apoB) gene was examined in a variety of fetal, neonatal, and adult rat tissues by probing RNA blots with a cloned rat apoB cDNA. Among 10 adult male tissues surveyed, small intestine had the highest concentration of apoB mRNA. Its abundance in liver and adrenal gland was 40% and 0.5%, respectively, of that in small bowel, while none was detected in colon, kidney, testes, spleen, lung, heart, or brain. ApoB mRNA is as abundant in 18-day fetal liver as at any subsequent period of hepatic development. In contrast, the concentration of apoB mRNA remains low in fetal intestine until the last (21st) day of gestation, when it increases sharply to levels that are several-fold higher than in the liver. ApoB mRNA levels in fetal membranes harvested during this late gestational period were 10 times greater than in fetal liver. Since the major lipoprotein species in 19-day fetal plasma is low density lipoprotein, these observations suggest that fetal liver, and particularly its functional homologue, the yolk sac, are the principal sites of fetal lipoprotein synthesis at this stage of development. A 20-fold increase in placental apoB mRNA concentrations during the last 48 hr of pregnancy (to a level that is 50% of that encountered in fetal membrane RNA) suggests a specific role for this organ in maternal-fetal lipid transport immediately prior to parturition. Pulse-labeling experiments using 21-day fetal tissue slices showed that the liver synthesizes both apoB-100 (B-PI) and apoB-48 (B-PIII) albeit in somewhat different ratios than the adult organ. Fetal intestine produces almost exclusively the smaller apoB species, while fetal membranes and placenta synthesize only the larger peptide. The postnatal pattern of apoB mRNA accumulation is similar in liver and intestine. Profound decreases were observed during the late suckling and weaning periods, followed by an increase to adult levels. These final concentrations were similar to those encountered at birth. Analysis of these developmental changes offers an opportunity to generate testable hypotheses about the factors that modulate apoB synthesis.     

Regulation of Na,K-ATPase gene expression by thyroid hormone in rat cardiocytes.

Synthesis and activity of the enzymatic equivalent of the sodium pump, Na,K-ATPase, are regulated by thyroid hormone in responsive tissues. The purpose of this study was to determine whether triiodothyronine (T3) regulates the level of the messenger RNA (mRNA) coding for Na,K-ATPase alpha- and beta-subunits in the heart. The expression of Na,K-ATPase mRNAs in in vitro myocardial cells was directly assayed by Northern and slot blot hybridization using Na,K-ATPase alpha- and beta-isoform-specific cDNA probes. Exposure of cultured neonatal rat cardiocytes to 10(-8) M T3 resulted in 1) threefold to fourfold increase in alpha 1- and beta 1-mRNA accumulation, with a maximum elevation at 48 hours, 2) sevenfold increase in alpha 2-mRNA accumulation with a peak elevation at 72 hours, and 3) transient threefold increase in alpha 3-mRNA within the first 24 hours followed by a deinduction thereafter. The increase in alpha 1-mRNA accumulation by T3 occurred over the physiological T3 concentration range with an EC50 of 5 x 10(-10) M. This was associated with a twofold increase in alpha 1-subunit protein accumulation and an increase in Na,K-ATPase transport activity. The half-life of alpha 1-mRNA analyzed by actinomycin D chase was less than 3 hours and was not affected by T3. Transfection experiments with the luciferase reporter gene revealed that thyroid hormone response sequences are located within the 5'-flanking regions of each alpha-isoform gene.(ABSTRACT TRUNCATED AT 250 WORDS)     

Regulation of prolactin receptor gene expression by thyroid hormone status in the rat.

The nature and tissue distribution of prolactin receptor (PRL-R) mRNA in both male and female rats was studied. A single mRNA species of 2.2 kb was identified in the liver, kidney, adrenal, prostate, lactating mammary gland and ovary but not in the male lung, heart, skeletal muscle, thymus, adipose tissue or brain. There were distinct and contrasting sex differences in abundance of PRL-R mRNA in some tissues: liver (female much greater than male), kidney and adrenal (male much greater than female). A mRNA species of 4 kb was occasionally detected in the male adrenal and female liver. Given previous reports on the effects of thyroid status on PRL binding, the effects of thyroxine (T4), propylthiouracil (PTU) or combined treatment on PRL-R mRNA were assessed. In the male rat, PTU treatment markedly increased (three- to fourfold) PRL-R mRNA in the liver but decreased it (approximately 50%) in the kidney. These changes were reflected in similar changes in lactogenic binding activity. T4 or PTU treatment increased PRL-R mRNA in the prostate, with no obvious changes in binding. No major changes were seen in adrenal glands. In the female rat, PTU had little effect on PRL-R mRNA in any tissue, although binding of 125I-labelled lactogen was decreased in both the liver and kidney. There was an unexpected threefold rise in PRL-R mRNA in the female kidney following combined T4 and PTU treatment. Overall, there was a quite close correlation between the effects of thyroid status on PRL-R mRNA levels and specific lactogenic binding to membranes prepared from the same tissue samples. These studies provide data on the tissue distribution and size of PRL-R mRNA in rats and suggest a novel and complex tissue- and sex-dependent regulation by thyroid hormone.     

Differential expression genes analyzed by cDNA array in the regulation of rat hepatic fibrogenesis.

PURPOSE: To analyze the gene expression pattern in rat hepatic fibrogenesis and further assess the role of some key genes during the pathological process. METHODS: Hepatic fibrosis was induced by intraperitoneal injection of dimethylnitrosamine or carbon tetrachloride (CCl(4)) injection subcutaneously in rats, and identification of the hepatic fibrosis related genes with cDNA microarray was performed. After some key genes up-regulated during the development of hepatic fibrosis were screened and confirmed, their effects on the function of the activated rat hepatic stellate cells (HSC) were assessed using the small interfering RNA (siRNA) technique. RESULTS: Using an Atlas rat cDNA array, a number of differentially expressed genes in fibrotic liver tissues were identified compared with non-diseased control. A total of 15 genes predominantly associated with the mitogen-activated protein kinase (MAPK) signal transduction pathway were upregulated in the fibrotic liver. Immunohistochemical study revealed that the expressions of both extracellular signal-regulated kinases (ERK) and ribosomal protein S6 kinase (RSK), two of the key genes in the MAPK pathway, were remarkably induced, which was closely correlated to that of collagen types I and III during the development of hepatic fibrosis. Transfection of siRNA targeting ERK1 mRNA (siERK1) into HSC led to a 66% and 72% reduction of ERK1 mRNA and protein expression, respectively. Furthermore, siERK1 exerted the inhibition of the proliferation of HSC, accompanied by the induction of HSC apoptosis and reduction of collagen types I and III. In addition, siERK1 abolished the effect of platelet-derived growth factor-BB on the proliferation of HSC. CONCLUSIONS: The present study provided strong evidence for the participation of the MAPK pathway in the pathogenesis of hepatic fibrosis. Selective targeting of ERK1 inhibitors to HSC might present as a novel strategy for the treatment of hepatic fibrosis.     

Insulin regulation of rat growth hormone gene expression

Insulin has been shown previously to inhibit basal and glucocorticoid- or T3-stimulated rat GH (rGH) synthesis, secretion, and mRNA levels in cultured rat pituitary tumor cells (GH3 cells) or pituitaries. The effects of insulin on rGH gene expression in GH3 cells were examined in greater detail in the current studies. Cells were deinduced for 5 days in medium devoid of steroids, T3, and insulin. Cells were then treated for 48 h with insulin (5 X 10(-9) M), dexamethasone (Dex; 10(-6) M), T3 (10(-8) M), insulin plus Dex, or insulin plus T3. When media and hormones were not replaced daily the results were similar to those obtained previously. Insulin decreased both basal and glucocorticoid-stimulated rGH mRNA levels to approximately 70% of control levels, as measured by cytoplasmic dot hybridization. By contrast, when media and hormones were replaced daily, rGH mRNA levels increased by 1.5 to 7-fold in response to insulin in the absence or presence of Dex or T3, measured by both cytoplasmic dot hybridization and RNA (Northern) blotting. Dex increased rGH mRNA levels under both sets of conditions, verifying the specific nature of the insulin influence. Maximum rGH gene expression was achieved after a 48-h exposure to insulin. The observed insulin effects were probably mediated through insulin rather than insulin-like growth factor I or II receptors, since the concentration of insulin employed was near the Kd of the hormone for its receptor measured in the same cells. These results suggest that insulin is capable of regulating rGH gene expression. The action of insulin can be either positive or negative and is influenced by the metabolic state of the cell.     

Immunohistochemical localization and mRNA expression of apolipoprotein A-I in rat spinal cord

Apolipoproteins in the cerebrospinal fluid (CSF) play important roles in lipid metabolism in the central nervous system. Although it has been demonstrated that apo E is synthesized in the neuron, the synthesis of apo A-I has only been determined in fish and chicken. It was demonstrated that apo A-I concentrations in the CSF were increased in poliovirus-infected macaques, however, the origin of the CSF apo A-I was not determined. The present immunohistochemical study provided evidence that apo A-I was localized within the nerve cell body of the rat spinal cord. In situ hybridization also showed that apo A-I mRNA was predominantly expressed in the neurons. As a further experiment, we compared apo A-I levels in the spinal cord from control rats and rats with experimental allergic encephalomyelitis (EAE), which was induced by sensitization with myelin basic protein. Although no significant changes in serum apo A-I levels were observed, apo A-I levels in the spinal cord were significantly elevated in EAE rats. Furthermore, apo A-I in the spinal cord of rats with EAE was not seen in the nerve cell body, but at the interstitium, particularly in lesions where inflammation had occurred. The current study clearly demonstrated that apo A-I is synthesized in the neurons of the rat spinal cord and the synthesis was suppressed in EAE rats.     

Promoter-specific regulation of the brain-derived neurotropic factor gene by thyroid hormone in the developing rat cerebellum.

Thyroid hormone (TH) plays a critical role in normal cerebellar development. However, the molecular mechanisms of TH action in the developing cerebellum are not fully understood. This action could be exerted in part through brain-derived neurotropic factor (BDNF), as cerebellar BDNF messenger RNA (mRNA) expression is lower, and replacement of BDNF partially reverses the abnormal neurogenesis in the hypothyroid rat. The rat BDNF gene consists of four noncoding exons (exons I-IV), each of which is linked to a different promoter, and a protein-coding exon (exon V). To study promoter-specific regulation of the BDNF gene by TH, ribonuclease protection assay of each exon mRNA was performed using total developing rat cerebellar RNA. During cerebellar development, all exon mRNAs were detected, but with different expression patterns; among noncoding exon mRNAs, exon II mRNA was the most abundant. Daily TH replacement induced a 3-fold increase in exon II mRNA on postnatal day (P) 15. On P30, exon II mRNA was still much greater in the TH-replaced animal. Exon I mRNA was detected on P2 and P7. However, in contrast to exon II mRNA, TH treatment suppressed the expression of exon I mRNA on P2. Exon III and IV mRNAs were not detected on P2 and P7, but small amounts were observed starting on P15 in TH-replaced animals. They were not detected by P30 in hypothyroid animals. In contrast, in the cerebral cortex, although all exons are differentially regulated during development, the expression of each mRNA was not significantly altered by TH. These results indicate that TH regulates BDNF gene expression in a promoter-, developmental stage-, and brain region-specific manner, which may play an important role in region- and stage-specific regulation of brain development by TH.     

Human apolipoprotein A-I gene expression increases high density lipoprotein and suppresses atherosclerosis in the apolipoprotein E-deficient mouse.

Atherosclerosis is a complex disease with both genetic and environmental determinants. Apolipoprotein (Apo) E-deficient mice have been created that are highly susceptible to atherosclerosis. In order to assess the role of human apolipoprotein (hApo) A-I and high density lipoprotein (HDL) in atherosclerosis susceptibility, transgenic mice overexpressing the hApo A-I gene were crossed with Apo E-deficient mice. Apo E-/-, hApo A-I mice with two-fold elevation in HDL cholesterol have markedly diminished atherosclerosis with less fibroproliferative lesions by 8 months of age. A strong reciprocal relationship between HDL cholesterol levels and atherosclerosis was found with HDL levels accounting for 78% of the observed variance in mean lesion area. The effect of HDL on atherosclerosis resistance was independent of non-HDL cholesterol.     

In vivo metabolism of apolipoprotein E within the HDL subpopulations LpE,LpE:A-I,LpE:A-II and LpE:A-I:A-II

High-density lipoproteins can be separated into distinct particles based on their apolipoprotein content. In the present study, the in vivo metabolism of apoE within the apoE-containing HDL particles LpE, LpE:A-I, LpE:A-II and LpE:A-I:A-II was assessed in control subjects and in patients with abetalipoproteinemia (ABL), in whom HDL are the sole plasma lipoproteins. The metabolism of apoE within these HDL subspecies was investigated in three separate studies which differed by donor or recipient status: (1) particles purified from normolipidemic plasma and reassociated with 125I or 131I-labeled apoE injected into normolipidemic subjects (study 1); (2) particles purified from ABL plasma injected into normolipidemic subjects (study 2); and (3) particles purified from ABL plasma injected into ABL subjects (study 3). The plasma residence times (RT, hours) in study 1 were 14.3+/-2.9, 11.3+/-3.4, and 9.1+/-1.2 for apoE within LpE:A-I:A-II, LpE:A-II and LpE:A-I, respectively, while those in study 2 were 10.1+/-2.2, 9.7+/-2.4, 7.9+/-1.0 and 7.3+/-0.8 for apoE within LpE:A-I:A-II, LpE:A-II, LpE:A-I and LpE, respectively. In study 3, RTs for apoE within LpE:A-I:A-II and LpE were 8.7+/-0.9 and 6.8+/-0.9, respectively. In comparison, RT for apoA-I on LpA-I:A-II has been reported to be 124.1+/-5.5 h and that for apoA-I on LpA-I 105.8+/-6.2 h. Thus, apoE within the different apoE-containing HDL particles was metabolized rapidly and at a similar rate in control and ABL subjects. The plasma RT of apoE was longest when injected on LpE:A-I:A-II particles and shortest when injected on LpE. In summary, our data show that: (1) the plasma RT of apoE within HDL is approximately ten times shorter than that of apoA-I within HDL, and (2) apoE within HDL is metabolized at a slower rate when apoproteins A-I and A-II are present (LpE:A-I:A-II RT>LpE:A-II>LpE:A-I>LpE). These differences were related to the lipid and apolipoprotein composition of the HDL subspecies, and, in control subjects, to the transfer of apoE from HDL subspecies to apoB-containing lipoproteins as well.     

Differential regulation of thyroid hormone receptor messenger ribonucleic acid levels by thyrotropin-releasing hormone

In addition to its well known actions in stimulating TSH and PRL synthesis and secretion, TRH has been shown to decrease the concentration of thyroid hormone receptors (TRs) in GH4C1 cells as measured by nuclear thyroid hormone (T3) binding. In the present study we have investigated the effects of TRH on the levels of mRNA encoding the different forms of TR, TR beta-1, TR beta-2, and TR alpha-1 as well as that of the non-T3-binding variant, c-erbA alpha-2. GH3 cells were incubated with 100 nM TRH in the presence or absence of 1 nM T3 for 48 h, and mRNA levels were determined by Northern blot analysis. Results revealed that there is differential regulation of the individual TRs by TRH at the pretranslational level. The mRNA for the pituitary-specific form of TR, TR beta-2, was down-regulated by 60% by TRH in GH3 cells, while that of its alternative splice product, TR beta-1, was unchanged. A modest change was observed in TR alpha-1 mRNA levels, which were down-regulated by 20%; there was no change in c-erbA alpha-2 mRNA levels. Levels of nuclear T3 binding were assessed under the same conditions, and 100 nM TRH was found to decrease binding by 40% from 0.78 to 0.46 fmol/micrograms DNA. A similar change in nuclear T3 binding was seen after incubation with 1 nM T3. The effect of TRH on the GH mRNA response to T3 was investigated. In the absence of TRH there was a 4-fold induction of GH mRNA after incubation with 1 nM T3. In the presence of 100 nM TRH, no significant induction in GH mRNA by T3 was seen, indicating that T3 responsiveness as well as receptor concentration are diminished by TRH under these conditions.     

Metabolism of HDL apolipoprotein A-I and A-II in Type 1 (insulin-dependent) diabetes mellitus

Summary Concentrations of HDL cholesterol and apolipoprotein A-I are commonly increased in Type 1 (insul-independent) diabetes mellitus but the mechanisms whereby diabetes influences HDL metabolism have not been studied. We investigated the metabolism of HDL apoproteins A-I and II in normolipidaemic Type 1 diabetic men (n=17, HbA₁ 6.4–11.9%) without microalbuminuria but with a wide range of HDL cholesterol (0.85–2.10 mmol/l) and in nondiabetic men (n=18) matched for body mass index and the range of HDL cholesterol. Input rates and fractional catabolic rates for apolipoproteins A-I and II were determined following injection of ¹²⁵I-apolipoprotein A-I and ¹³¹I-apolipoprotein A-II tracers. Additional multicompartmental analysis was performed using a model to describe the kinetics of HDL particles containing only apolipoprotein A-I (Lp A-I) and apolipoprotein A-I and apolipoprotein A-II (Lp A-I/ A-II). No gross differences from normal subjects were observed in the mean levels of lipids, lipoproteins, apoproteins and the lipolytic enzymes in the diabetic men as a result of the selection process. Furthermore, the relationship between apolipoprotein A kinetics and plasma HDL cholesterol levels appeared to be preserved in the diabetic group. However, some normal interrelationships were disrupted in the diabetic men. Firstly, the rate of apolipoprotein A-II synthesis was 22% lower than in control subjects (p<0.05). Modelling indicated that this was due to decreased input of Lp A-I/A-II particles whereas the input of Lp A-I particles was similar in the two groups. Secondly, there was no correlation between VLDL triglyceride and HDL cholesterol or VLDL triglyceride and the fractional catabolic rate of apolipoproteins A-I and A-II in diabetic men in contrast to that seen in control subjects. We conclude that there is a disruption in the normal association between VLDL and HDL metabolism in Type 1 diabetic men and postulate that the observed differences may be due to the therapeutic use of exogenous insulin.