Secretion of apolipoproteins A-I and B by HepG2 cells: regulation by substrates and metabolic inhibitors.

It was the aim of this study to i) compare the effects of glucose and other hexoses with that of oleate on secretion of apolipoproteins (apos) A-I and B by HepG2 cells, and ii) document the effect of various metabolic inhibitors on the secretion of these apos in the absence or presence of extra glucose/oleate. i) The addition of 10 mM glucose increased secretion of apoA-I and apoB, as measured by enzyme immunoassay, by about 60% when cells were incubated for 48 h in DMEM + 10% fetal calf serum. The addition of extra glucose also increased the mRNA levels for these apos. Increased radioactivity was also found in these apolipoproteins by immunoprecipitation after metabolic labeling with [35S]methionine for 48 h. However, in a pulse-chase experiment (15 min labeling, 2 h chase), glucose was found to increase apoA-I synthesis but not apoB synthesis. More labeled apoB appeared in the medium during the chase because glucose inhibited its intracellular degradation. The effect of glucose on secretion of these apos could be mimicked by fructose and mannose but not by 6-deoxyglucose, showing that the hexoses must enter the cells and be phosphorylated. In contrast, the addition of 0.5 mM oleate had a weak inhibitory effect on secretion of apoA-I whereas it increased the secretion of apoB by more than twofold. The combination of 10 mM glucose and 0.5 mM oleate had no greater effect than glucose alone on apoA-I secretion but increased apoB secretion by fourfold. ii) Inhibiting glycolysis (by glucosamine) lowered secretion of both apoA-I and apoB, while inhibiting lipogenesis (using 8-Br-cyclic AMP or 5-(tetradecyloxy)-2-furancarboxylic acid (TOFA)) did not affect apoA-I secretion but clearly decreased that of apoB. However, the inhibitory effect of TOFA on apoB secretion was much smaller in the presence of 0.5 mM oleate instead of extra glucose. Actinomycin-D and cycloheximide strongly suppressed the stimulatory effect of glucose on secretion of both apolipoproteins. Actinomycin-D also suppressed basal secretion of apoA-I but surprisingly stimulated that of apoB. These observations indicate that in HepG2 cells secretion of apoA-I is strongly dependent on ongoing protein synthesis and can be boosted by glucose, whereas that of apoB is primarily driven by internal (via lipogenesis from glucose) or external supply of fatty acyl-residues.

Effects of stigmastanyl-phosphocholine (Ro 16-6532) and lovastatin on lipid and lipoprotein levels and lipoprotein metabolism in the hamster on different diets.

Previous studies from our laboratory have shown that oral administration of stigmastanyl-phosphocholine (Ro 16-6532) reduces plasma cholesterol levels in experimental animals on diets free of added cholesterol. In the present study, effects of Ro 16-6532 and lovastatin on lipoprotein levels and metabolism were investigated in male golden Syrian hamsters. In hamsters fed a standard diet, Ro 16-6532 (1 mmol/kg/day) lowered cholesterol in all lipoprotein fractions, as well as apoB-100 and apoA-I. In contrast, lovastatin (25 mumol/kg/day) lowered high density lipoprotein (HDL)-cholesterol but had no effect on low density lipoprotein (LDL)-cholesterol or on apoB-100 or apoA-I while triglycerides and very low density lipoprotein (VLDL)-cholesterol increased. In hamsters fed a coconut fat-supplemented diet, Ro 16-6532 reduced all lipoproteins, with a stronger effect on VLDL- and LDL- than on HDL-cholesterol. Also apoB-100 was reduced. Lovastatin (50 mumol/kg/day) reduced LDL-cholesterol, HDL-cholesterol, and apoA-I while triglycerides and VLDL-cholesterol increased. The drop in LDL-cholesterol seen with both drugs in hamsters fed the diet supplemented with coconut fat occurred without any effect on the plasma removal rate of homologous LDL, or on the content of hepatic LDL-receptors. In contrast, the first phase of removal of homologous radioiodinated VLDL from plasma was markedly increased by both compounds, paralleled with an increased uptake of label in the liver and a decreased appearance of labeled apoB-100 in the LDL-fraction. Furthermore, retinyl ester-labeled chylomicrons were also cleared more rapidly in hamsters treated with Ro 16-6532. Hepatic uptake of label from VLDL and chylomicrons was strongly decreased by pre-injection of lactoferrin. In addition, Ro 16-6532 slightly decreased the secretion rate of VLDL in hamsters fed the coconut fat-supplemented diet. Taken together, these results indicate that the reduction of LDL-cholesterol after treatment with Ro 16-6532 and lovastatin observed in the hamster is mainly due to decreased conversion of VLDL into LDL, consequent to an increased hepatic removal of VLDL remnants. Ro 16-6532 also increased the liver uptake of chylomicron remnants. The hepatic uptake system implicated in this remnant removal can be completely blocked by lactoferrin. The nature of this uptake system is still unknown.

Regulation of human apolipoprotein A-I expression in Caco-2 and HepG2 cells by all-trans and 9-cis retinoic acids.

Retinoids are reported to stimulate apolipoprotein (apo) A-I gene promoter activity (Rottman et al. 1991. Mol. Cell. Biol. 11: 3814-3820) and apoA-I protein secretion by monkey hepatocytes (Kaptein et al. 1993. Arterioscler. Thromb. 13: 1505-1514). In this study we have assessed the effects of retinoids on parameters of apoA-I biosynthesis in human cell lines. Caco-2 and HepG2 cells (human intestinal and hepatoma cell lines, respectively, both known to express and secrete apoA-I) were stably transfected with a reporter gene construct containing 1.3 kb of the 5-'flanking region of the human apoA-I gene linked to the firefly luciferase coding region. These cells were incubated for 48 h with 10 microM all-trans retinoic acid (RA) or 9-cis RA. The cells were then assayed for luciferase activity, for apoA-I mRNA level, and for secretion of apoA-I protein in the medium. Secretion of apoB was monitored as well. In Caco-2 cells, all-trans and 9-cis RA increased luciferase activity, mRNA content, and protein secretion by 40% to 80% above control. Strikingly, in HepG2 cells all-trans and 9-cis RA caused a more marked stimulation of luciferase activity (by 100-150%) but a weaker increase of mRNA content and protein secretion (by 25-30%). In contrast, apoB secretion was inhibited by the two retinoids in Caco-2 cells and not changed in HepG2 cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Prolactin lowering activity of the retinoid Ro 14-9706 affecting lactation and pup survival.

The arotinoid Ro 14-9706, though devoid of any teratogenic potential, was found to reduce dose dependently the survival of pups when their mothers were treated with toxic doses during days 6-15 of gestation. The increased mortality was primarily seen during early lactation. When pups derived from treated mothers were nursed by control foster mothers unexposed to the drug, their survival was significantly improved indicating that the increased mortality was not solely due to fetal drug exposure. When pups derived from untreated mothers were fostered by dams that were exposed to the arotinoid during pregnancy, a significant pup mortality (p < 0.01) was observed, suggesting that the nursing behaviour of lactating dams was seriously affected. This impairment could be linked to a prolactin-suppressive activity of the arotinoid during lactation which was also seen during pregnancy. Other pituitary hormones, however, were not affected by the compound. Although the drug induced pronounced structural alterations in mitochondria of adrenocortical cells, visualized by light microscopy as extended vacuolization in the zona fasciculata and reticularis, this pathological finding did not translate into functional impairment of steroidogenesis. Thus, the arotinoid Ro 14-9706 exhibits in rats a prolactin-suppressive activity which affects lactation and subsequently pup survival. This particular endocrinological interference is a new phenomenon and uncommon for retinoids.

Effect of acitretin on the response to an intravenous glucose tolerance test in healthy volunteers.

The effect of the synthetic retinoid acitretin (A) on the disposition of blood glucose and on the serum insulin response following the IV infusion of 139 mmol glucose over 10 min (IGTT) has been investigated in six healthy subjects. The IGTT was performed on Days 1, 10 and 24. On Days 3 to 10 A 50 mg/d was administered. Several parameters of glucose disposition and insulin response (K-values, AUC) were assessed. As a methodological variant, the profiles over time of blood glucose and serum insulin were evaluated by model calculations using the 'minimal model'. Acitretin did not influence any parameter of glucose disposition. The area under the insulin-time curve (baseline corrected) was significantly decreased from 1.20 mU.min.l-1 on Day 1 to 0.89 mU.min.l-1 on Day 10, and was 0.91 mU.min.l-1 on Day 24. The model-derived 'insulin sensitivity' increased from 13.10(-4) l.mU-1.min-1 on Day 1 to 20.10(-4) l.mU-1.min-1 on Day 10 and was 18.10(-4) l.mU-1.min-1 on Day 24. The results suggest that A increased sensitivity to endogenous insulin. It supports a recent report showing greater insulin sensitivity in patients treated with the synthetic retinoid etretinate.

The detection of antibodies to recombinant interferon alfa-2a in human serum.

Three different procedures have been used for detecting antibodies to Roferon-A (recombinant human interferon alfa-2a, rHuIFN alpha-2a) in the serum of patients who received this interferon as part of ongoing clinical trials: an antiviral neutralization bioassay (ANB), the standard method recommended by the World Health Organization (WHO), and the more recently developed radioimmunoassay (RIA) and enzymeimmunoassay (EIA). Although the three tests are based on different principles, the correlation among them was excellent. The assays show differences in sensitivities with the ANB being the least sensitive of the three. The EIA equals the RIA in sensitivity, reproducibility, accuracy and labor and provides the advantage of safety and convenience in the use of non-radioactive materials. Therefore, the EIA has been selected as the most suitable assay for initial screening of the sera of patients receiving Roferon-A for the presence of antibodies to this interferon. EIA positive sera are then tested in the ANB to determine whether or not neutralizing activities are present.

Retardation of spermiation following short-term treatment of rats with theobromine.

Groups of 4-6 Fü-albino rats were examined 24 h after 3 daily doses of 500 mg/kg bw of theobromine administered orally by gavage as well as 1, 2, 4, 6, and 10 weeks after 5 daily doses. Controls received the standard solvent vehicle (SSV) only. A variety of parameters were assessed including body and organ weights, serum clinical chemistry, hematological parameters, epididymal sperm motility and LDH-X fraction in seminal plasma, serum gonadotropins and testosterone, and the morphology of various organs. Testes were perfused with 5% glutaraldehyde and semi-thin sections were evaluated. The most striking morphological observation was a retarded release of late spermatids into the tubular lumen mainly 2 weeks post treatment. This partial disruption of the rigid spermatogenic synchronization was not followed by substantial germ cell death. The other parameters investigated remained relatively normal throughout the study. These observations suggest that theobromine at the dose tested rather selectively interferes with germ cell kinetics. Sertoli cell toxicity could account for these early and subtle effects as well as for the late and severe effects of subchronic exposure of rats to theobromine as reported in the literature.

Lack of effect of tenoxicam on glibornuride kinetics and response.

The pharmacokinetics of glibornuride (25 mg i.v.) and the accompanying insulin and glucose responses were characterized in eight human subjects in the presence and absence of steady-state tenoxicam (20 mg p.o./day for 2 weeks). Tenoxicam affected neither the pharmacokinetic parameters of glibornuride (systemic clearance, volume of distribution and biological half-life) nor the responses of plasma insulin and blood glucose to glibornuride. The single i.v. dose of glibornuride had no detectable effect on the kinetics of tenoxicam.

Comparison of serum 1,25-dihydroxycholecalciferol concentrations in rheumatoid arthritis and osteoarthrosis.

We have previously compared 25-hydroxycholecalciferol levels in the serum of patients with osteoarthrosis and rheumatoid arthritis, finding no significant difference between the circulating levels of this hormone. We have now estimated 1,25-dihydroxycholecalciferol levels on stored sera from the same groups of patients and found no significant difference in the levels of this hormone between the 2 groups. The osteopenia that distinguishes rheumatoid arthritis from osteoarthrosis is not the result of altered levels of systemic 25OHD3 or of 1,25(OH)2D3. Local factors may be more important in its pathogenesis.

Influence of 1,25 dihydroxycholecalciferol on sexual dysfunction and related endocrine parameters in patiens on maintenance hemodialysis.

It has been postulated that hyperparahyroidism is in part responsible for sexual dysfunction in dialyzed patients and that this could improved by 1,25(Oh)2D3. In a single blind study patients on maintenance hemodialysis were treated after a control period with 1,25 (OH)2D3 and with placebo in random order. Sexual performance was assessed with a detailed semistructured psychiatric interview protocol in 14 and several endocrine parameters were analyzed in 15 patients. Under control conditions sexual function was disturbed in 11/14 patients. Plasma testosterone was moderately decreased in men, PRL and PTH levels were distinctly elevated in both sexes. 1,25(OH)2D3 raised serum calcium levels significantly from 9.6 to10.6 mg/100 ml ( P < 0.01) and lowered PTh from 1.39 to 0.82 ng/ml ( P < 0.01). However, no improvement in sexual function (such as libido, frequency of intercourse or masturbation) was found, and apart from a slight rise of testosterone in men and moderate fall of PRl in men and women endocrine parameters remained unchanged. It is concluded that in spite of an improvement of secondary hyperparathyroidism, treatment with 1,25(OH)2D3 for 2 to 4 months was of no value in improving sexual dysfunction in these hemodialysis patients.