Lifelong benefits on myocardial infarction mortality: 40-year follow-up of the randomized Oslo diet and antismoking study.

BACKGROUND: The effects of saturated fat on atherosclerotic vascular disease are currently debated. OBJECTIVES: In the Oslo cardiovascular study initiated in 1972/1973, a 5-year randomized intervention was conducted in healthy middle-aged men at high risk of coronary heart disease to compare the effects on coronary heart disease incidence of diet and antismoking advice versus control (no intervention). A significant reduction (47%) in first myocardial infarction incidence was observed. We have followed mortality up to 40 years to establish whether a lifelong benefit on mortality risk of myocardial infarction could be observed. METHODS: In the present study, a total of 16 203 men (63% of those invited), aged 40-49 years, participated in a screening examination. Overall, 1232 men with total serum cholesterol levels of 6.9-8.9 mmol L(-1) (80% smokers) were included in the study. The dietary intervention consisted of mainly decreasing the intake of saturated fats and increasing fish and vegetable products, as well as weight reduction in overweight subjects. Smokers were advised to stop smoking. Cox regression analysis was used for statistical analyses. RESULTS: The intervention group showed a sustained reduced risk of death at first myocardial infarction (hazard ratio 0.71, 95% confidence interval 0.51-1.00; P = 0.049), compared to control subjects up to 40 years. During follow-up, the beneficial effect developed gradually but proportionally up to about 15 years after randomization. Later, the curves were parallel. All-cause mortality decreased in the period 8-20 years after randomization, but not thereafter. CONCLUSIONS: Receiving advice about a healthy lifestyle led to a long-term reduced risk of coronary mortality during the following 40 years. Our results suggest that systematically providing effective counselling for a healthy lifestyle for 5 years can lead to lifelong benefits.

Obesity and associated factors in a Palestinian West Bank village population.

OBJECTIVE: To describe body mass index (BMI), waist circumference and waist-hip ratio in a Palestinian West Bank village population, and to assess the associations of these variables to blood pressure and serum lipids. DESIGN: Cross-sectional study. SETTING: Community-based study in a prototypic semi-rural Palestinian village in the central West Bank. SUBJECTS: All individuals aged 30-65 y in the study village were invited for the study and 500 (85%) participated. MAIN OUTCOME MEASURES: BMI > or = 30 was used as the measure of obesity. RESULTS: The prevalence of obesity was 37.5% among women and 18.8% among men. The prevalence of abdominal obesity was 62.5% among women and 14.8% among men. BMI seemed to be the more important correlate of blood pressure whereas waist-hip ratio seemed to be the more important correlate of serum triglycerides, compared to the other obesity measures. CONCLUSIONS: The prevalence of obesity in the study population was very high compared to most other countries in the world, particularly among women. SPONSORSHIP: The study was funded by the Norwegian Universities' Committee for Development Research (NUFU). LCM Stene was supported by a grant from the Throne Holst Foundation.

Nuclear deviation in hepatic parenchymal cells on sinusoidal surfaces in Arctic animals.

In normal rat and human, most of the nuclei of hepatic parenchymal cells are centrally located in the cytoplasm. However, it is reported that the nuclei of hepatic parenchymal cells are situated at a deviated position on sinusoidal surfaces under pathological situations such as chronic hepatitis, hepatocellular carcinoma, adenomatous hyperplasia, or regeneration. During a study on the mechanism of extreme vitamin A-accumulation in hepatic stellate cells of arctic animals including polar bears, arctic foxes, bearded seals, and glaucous gulls, we noticed that these arctic animals displayed the nuclear deviation in hepatic parenchymal cells on sinusoidal surfaces. In this study, we assessed the frequency of hepatic parenchymal cells showing the nuclear deviation on the sinusoidal surfaces in arctic animals. A significantly higher frequency of the nuclear deviation in hepatic parenchymal cells was seen in polar bears (89.8+/-3.4%), arctic foxes (68.6+/-10.5%), bearded seals (63.6+/-8.4%), and glaucous gulls (24.2+/-5.8%), as compared to that of control rat liver (9.8+/-3.5%). However, no pathological abnormality such as fibrosis or necrosis was observed in hepatic parenchymal and nonparenchymal cells of arctic animals, and there were no differences in the intralobular distribution of parenchymal cells displaying the nuclear deviation in the livers from either arctic animals and control rats. The hepatic sinusoidal littoral cells such as stellate cells or extracellular matrix components in the perisinusoidal spaces may influence the nuclear positioning and hence the polarity and intrinsic physiological function of parenchymal cells.

Relationships between plasma levels of organochlorines, retinol and thyroid hormones from polar bears (Ursus maritimus) at Svalbard.

Associations were determined between retinol and the thyroid hormones thyroxine (T4) and triiodothyronine (T3), respectively, and the organochlorine contaminants (OCs) polychlorinated biphenyls (PCBs), 1, 1-dichloro-2,2-bis-(4-chlorophenyl)ethylene (DDE), hexachlorobenzene (HCB), and hexachlorocyclohexanes (HCHs) in blood plasma from polar bears (Ursus maritimus) caught at Svalbard. The blood samples were collected from free-ranging polar bears of different age and sex in 1991-1994. The retinol concentration and the ratio of total T4 (TT4) to free T4(FT4) (TT4/FT4 ratio) decreased linearly with increasing concentrations of PCBs and HCB. Retinol was also negatively associated with HCHs, while the TT4/FT4 ratio was positively associated with DDE. The concentrations of retinol and thyroid hormones were significantly higher in females than in males. However, the TT4/FT4 and TT3/FT3 ratios were significantly higher in males than in females. The concentrations of thyroid hormones were negatively correlated with age in male bears, while in females, thyroid hormones did not change with age. The OCs were found to explain 12, 30, and 7% of the variation of retinol concentrations and the TT4/FT4 and TT3/FT3 ratios, respectively, after correcting for age and sex. The potential consequence of these associations for the individual and the population is unknown.

Food consumption patterns in a Palestinian West Bank population.

OBJECTIVE: To describe the food consumption patterns in relation to wealth status and age groups in a Palestinian West Bank village population. DESIGN: Community-based cross-sectional survey of both households and individuals. A list recall method was used at the household level. At the individual level, a short food-frequency questionnaire was used in addition to a 24-h recall without estimates of portion sizes. SETTING: A Palestinian semi-rural village in the central West Bank. SUBJECTS: All households and all men and women aged 30-65 y in the study village were invited. All 368 households and 85% (n=500) of eligible individuals participated. RESULTS: The mean energy consumption from 25 selected food items on household level was about 13.8 MJ (3300 kcal)/consumption unit/d (a consumption unit corresponds to the expected energy requirement for an adult male). The proportion of dietary energy from fat and the consumption of most animal products was highest among the wealthiest households, and the opposite trend was seen for the consumption of wheat flour and lentils. There seems to be an ongoing trend of increasing consumption of processed products rich in sugar among the younger age groups. CONCLUSION: Shortage of dietary energy on the household level did not seem to be a problem in this population, even among the poorest. Differences in food consumption patterns between the poor and the wealthy, including a higher percentage of energy from fat among the wealthy, may be to the disadvantage of the wealthy with respect to some diet-related chronic diseases. SPONSORSHIP: The Norwegian Universities' Committee for Development Research (NUFU).

Retinoylation of proteins in rat hepatocytes following uptake of chylomicron remnant retinyl ester.

Several proteins may covalently bind retinoic acid, a process called retinoylation. Recently, we have demonstrated that proteins were retinoylated in vivo in liver, kidney and lung. In order to gain further knowledge about the mechanism of this process, we studied retinoylation in rat hepatocytes administered vitamin A as [3H]retinyl esters in chylomicron remnants. This resembles the normal physiological uptake of vitamin A. After 24 h incubation, about 0.0017 mol [3H]retinoid was covalently bound per mol protein. Citral, an inhibitor of the oxidation of retinol to retinoic acid, reduced retinoylation about 40%, indicating that oxidation of retinol to retinoic acid is necessary for a large fraction of the observed covalent modification of proteins. When cells were incubated with physiological concentrations of [3H]retinol or [3H]retinoic acid dissolved in ethanol, much less retinoid was covalently bound per mol protein compared with cells incubated with chylomicron remnant. Saturation of the retinoylation was apparent with retinoic acid around the physiological concentration. Retinoylated proteins were also analysed by SDS-PAGE. In general, the same protein bands were labelled with both [3H]retinol and [3H]retinoic acid, although the intensity of the bands varied. Major bands had an apparent molecular weight of about 16, 35, 50 and 120 kDa. In a parallel experiment in which liver stellate cells were incubated with [3H]retinol, major retinoylated protein bands were about 35, 60 and 65 kDa. Thus, different proteins appear to be retinoylated in hepatocytes and liver stellate cells, suggesting that protein retinoylation is a cell specific phenomenon. These results demonstrate that retinoids presented to hepatocytes as chylomicron remnant retinyl esters are covalently linked to proteins. We therefore suggest that retinoylation of proteins represents a minor but significant pathway whereby cells metabolize vitamin A.

Lovastatin inhibits G1/S transition of normal human B-lymphocytes independent of apoptosis.

Lovastatin is a potent inhibitor of protein prenylation, and it has been reported to have pleiotropic cellular effects. In the present study we have elucidated the effects of lovastatin on cell cycle progression and apoptosis of normal human B-lymphocytes. When added to B-lymphocytes stimulated with anti-immunoglobulin (anti-mu) and SAC, lovastatin (20 microM) inhibited the cells in the late G1 phase of the cell cycle. Thus, no early activation parameters such as Ca(2+) flux or MYC induction were affected by lovastatin, whereas progression of cells into the second cell cycle as well as DNA synthesis was markedly reduced. We therefore examined the effects of lovastatin on components of the cell cycle machinery responsible for regulating the G1/S transition. We demonstrated that pRB phosphorylation, cdk2 activity needed for this phosphorylation, and the levels of cyclin A, D, and E were inhibited after 24 h of lovastatin treatment, while the levels of p27(Kip1) were elevated. There was no effect on p21(Cip1), cyclin D2, cdk4, and cdk6. These data are consistent with the cells being inhibited by lovastatin between 24 and 32 h into G1. Lovastatin added to stimulated B-cells in late G1 still inhibited the DNA synthesis by 60%, but at this point only minor effects were noted on the cell cycle machinery. We therefore looked for induced apoptosis as an explanation for reduced S-phase entry of the cells. However, despite the ability to enhance the apoptosis of unstimulated B-cells from 48 to 61% as judged by the TUNEL method, lovastatin only marginally affected apoptosis when administered to stimulated B-cells. Thus, it appears that accelerated apoptosis cannot account for the effect of lovastatin on cell cycle progression.

Retinyl esters are hydrolyzed in early endosomes of J774 macrophages.

The aim of the current study was to identify the subcellular compartment(s) responsible for the hydrolysis of chylomicron remnant-retinyl esters, in J774.1 cells. The cells were incubated with medium containing chylomicron remnant [(3)H]retinyl ester. Subcellular fractionation was used to separate early endosomes from late endosomes and lysosomes. About 26% and 80% of the total [(3)H]retinyl esters taken up by the J774 cells were hydrolyzed after 10 min and 60 min of chase, respectively. In the early endosomes, there was a 4-fold increase of radioactivity (nearly all radioactivity associated with retinyl esters) during the first 10 min of chase. The radioactivity in early endosomes was reduced by 43% from 10 min to 60 min and remained stable from 60 to 180 min of chase. From 10 to 60 min the amount of retinol in early endosomes increased from 44% to 82%, indicating an efficient hydrolysis of retinyl esters. Less than 10% and 5% of the total cell-associated radioactivity was found in the late endosomes and lysosomes during the entire chase period. In the chase medium, 84% of the total amount of retinoid released during 180 min was present already after 10 min. The percentage of retinol in the medium increased from 25% to 82% during incubation from 10 to 180 min. These data suggest that retinyl esters are endocytosed together with the chylomicron remnant particle and hydrolyzed in the early endosomes in this cell model.-Hagen, E., A. M. Myhre, T. E. Tjelle, T. Berg, and K. R. Norum. Retinyl esters are hydrolyzed in early endosomes of J774 macrophages.

Uptake of vitamin A in macrophages from physiologic transport proteins: role of retinol-binding protein and chylomicron remnants.

Vitamin A plays an important role in reducing infectious disease morbidity and mortality by enhancing immunity, an effect that is partly mediated by macrophages. Thus, knowing how these cells take up vitamin A is important. The results in the present study demonstrate that J774 macrophages efficiently take up chylomicron remnant retinyl esters and retinol-binding protein (retinol-RBP) bound retinol by specific and saturable mechanisms. The binding of (125)I-RBP to plasma membrane vesicles demonstrated that the macrophage receptor had a similar binding affinity, as was discovered previously for other cells. The B(max) for the macrophages was smaller than the values reported for placenta, bone marrow, and kidney, but larger than that reported for liver. The J774 cells also bound and took up [(3)H]retinol-RBP. Approximately 50 to 60% of the uptake may compete with excess unlabeled retinol-RBP and approximately 30 to 40% with excess transtyrethin. Following the uptake of [(3)H]retinol-RBP, an extensive esterification occurred: After 5 hours of incubation, 77.8 +/- 3.9% (SD; n = 3) of the cellular radioactivity was recovered as retinyl esters. The J774 cells also demonstrated saturable binding of chylomicron remnant [(3)H]retinyl esters, and a continuous uptake at 37 degrees C followed by an extensive hydrolysis of the retinyl esters. Binding could be inhibited by approximately 50% by excess unlabeled low density lipoprotein (LDL). In addition, lipoprotein lipase increased the binding of chylomicron remnant [(3)H]retinyl esters by approximately 30% and the uptake of chylomicron remnant [(3)H]retinyl ester by more than 300%. Furthermore, because sodium chlorate reduced binding with 40% and uptake with 55%, the results suggest that proteoglycans are involved in the uptake. Thus, the results suggest that both LDL receptor and LDL-related protein are involved in the uptake of chylomicron remnant [(3)H]retinyl ester in macrophages.

Malignant epithelial tumours in the upper digestive tract: a dietary and socio-medical case-control and survival study.

OBJECTIVE: The aim of the present study was to elucidate the influence of social, dietary and environmental factors on the incidence of malignant epithelial tumours in the upper digestive tract and on the prognosis of patients with these cancers. DESIGN: A population-based case-control study was carried out, and the patients in the study were included in a survival analysis. SETTING: The study was carried out at the Department of Otorhinolaryngology at Ullevål University Hospital, Oslo, Norway. SUBJECTS: In the case-control study, 84 patients and 89 controls were included. Only the patients were included in the survival analysis. RESULTS: Smoking showed the highest odds ratio (OR) for morbidity (OR = 29). The patients had in general a lower social status, and a higher alcohol intake (OR = 6.6). For both beta-carotene and vitamin C, the ORs decreased with increasing intake (OR = 0.2 and 0.3, respectively). Increased ORs were associated with low values for haemoglobin, iron, TIBC, folic acid, magnesium and especially for albumin (OR = 14), and with high values for ferritin, vitamin B12 and thiocyanate (a marker for smoking). Stage of the disease was an important prognostic factor. The relative risk (RR) of dying for disseminated vs localised tumours being 3.2. A poorer prognosis was linked to higher age, to smoking vs no smoking (RR = 2.3), and to lower levels of haemoglobin, albumin, magnesium and thiocyanate. CONCLUSIONS: Strong beer, liquor, consumption of milk and table fat, low social status and smoking seemed to have a negative impact on both disease and survival. Fruit and vegetables might, however, reduce the risk. Whereas low serum albumin, iron and magnesium indicated a high OR for cancer, vitamin C and beta-carotene had the opposite implication. No significant implications on survival could be detected in blood chemistry beyond the stage of disease.

Binding of perlecan to transthyretin in vitro.

Transthyretin is one of two specific proteins involved in the transport of thyroid hormones in plasma; it possesses two binding sites for serum retinol-binding protein. In the present study we demonstrate that transthyretin also interacts in vitro with [35S]sulphate-labelled material from the medium of HepG2 cells. By using the same strategy as for purifying serum retinol-binding protein, [35S]sulphate-labelled medium was specifically eluted from a transthyretin-affinity column. Ion-exchange chromatography showed that the material was highly polyanionic, and its size and alkali susceptibility suggested that it was a proteoglycan. Structural analyses with chondroitinase ABC lyase and nitrous acid revealed that approx. 20% was chondroitin sulphate and 80% heparan sulphate. Immunoprecipitation showed that the [35S]sulphate-labelled material contained perlecan. Further analysis by binding studies revealed specific and saturable binding of 125I-transthyretin to perlecan-enriched Matrigel. Because inhibition of sulphation by treating HepG2 cells with sodium chlorate increased the affinity of the perlecan for transthyretin, and [3H]heparin was not retained by the transthyretin affinity column, the binding is probably mediated by the core protein and is not a protein-glycosaminoglycan interaction. Because perlecan is released from transthyretin in water, the binding might be due to hydrophobic interactions.

Secretion of N-(4-hydroxyphenyl) retinamide-retinol-binding protein from liver parenchymal cells: evidence for reduced affinity of the complex for transthyretin.

The synthetic retinoid 4-HPR has been shown to markedly lower the plasma concentration of both retinol and RBP in rats and humans. We have studied the effect of 4-HPR on the secretion of retinol-RBP from liver cells in vivo and in vitro. In rats maintained with a normal diet, a vitamin A-deficient diet or a normal diet supplemented with 4-HPR, chylomicrons [3H]retinyl esters were rapidly cleared from the plasma. The secretion of chylomicron-derived [3H]retinol from tissues to the circulation, however, was different. In control rats, the lymph-derived [3H]retinol peaked after about 2 hr, whereas 4-HPR treatment effectively reduced this peak of [3H]retinol. Our results suggest that 4-HPR inhibits secretion of retinol-RBP from the liver. Therefore, we decided to study the effect of 4-HPR on the secretion of RBP using the human hepatoma cell line HepG2. Retinol and 4-HPR were found to induce the secretion of RBP. The medium from cells treated with 4-HPR was immunoprecipitated with antibodies against human RBP. HPLC analysis of the precipitated RBP revealed the presence of 4-HPR. When the medium from cells incubated with either 4-HPR or retinol was applied to a TTR affinity column, we found that RBP from cells incubated with 4-HPR had a considerably reduced affinity for TTR. We conclude that 4-HPR binds RBP and thereby induces secretion of RBP in HepG2 cells, and that the secreted 4-HPR-RBP complex has a reduced affinity for TTR. This observation may explain the 4-HPR-induced reduction of plasma retinol and RBP observed in in vivo studies.

Storage of vitamin A in extrahepatic stellate cells in normal rats.

In mammals, vitamin A is primarily stored as retinyl esters in hepatic stellate cells under normal dietary intake of the vitamin. Previously, extrahepatic vitamin A-storing stellate cells have only been identified in animals maintained on a vitamin A-rich diet, and it has not been known whether these cells play a role in normal vitamin A metabolism. The purpose of this study was, to quantify the stellate cell lipid droplet area in hepatic and extrahepatic stellate cells in control rats and in rats fed excess vitamin A. The stellate cells were identified by the gold chloride staining technique, specific autofluorescence of retinyl ester, and by electron microscopy. The stellate cell lipid droplet area was then quantitated by the use of morphometric quantitation. We demonstrated that lipid droplet-containing stellate cells were identified in liver, lung, kidney, and intestine, in normal as well as vitamin A-fed rats. The area of lipid droplets in liver, lung, and intestine stellate cells of normal rats was 0.2, 0.3, and 0.04 mm2 per cm2 tissue, respectively. When the rats were administered excess vitamin A, the hepatic, lung, and intestinal stellate cell lipid droplet area increased about 10-fold, 2-fold, and 40-fold, respectively. Thus the present study shows that extrahepatic stellate cells in lung and intestine of normal rats contain lipid droplets, and that these lipid droplets increase in area when high doses of vitamin A are fed to the animals. These data suggest that not only liver stellate cells but also extrahepatic stellate cells play an important role in vitamin A storage in normal as well as vitamin A-fed animals.

Retinyl ester storage is altered in liver stellate cells and in HL60 cells transfected with cellular retinol-binding protein type I.

It is suggested that cellular retinol-binding proteins are important for intracellular metabolism of retinol. Retinol bound to cellular retinol-binding proteins may be esterified with long chain fatty acids by the enzyme lecithin: retinol acyltransferase or may be oxidized to retinoic acid metabolites used in the mechanism of action of vitamin A. The aim of this present report was to determine whether altered levels of cellular retinol-binding protein type I influenced retinol storage and activation. Two different cell types have been examined after transfection with vectors producing sense or antisense mRNA for cellular retinol-binding protein type I. When HL60 cells were transfected with the expression vector for sense cellular retinol-binding protein type I high amounts of cellular retinol-binding protein type I mRNA and protein were produced. We observed that HL60 cells esterified less retinol than control cells without cellular retinol-binding protein type I. Cellular retinol-binding protein type I had, however, no effects on the proliferation or differentiation of HL60 cells by retinoids. Liver stellate cells transfected with the vector for sense cellular retinol-binding protein type I esterified more retinol than cells transfected with the expression vector for antisense cellular retinol-binding protein type I, while retinol esterification in control cells was intermediate. In conclusion, our data show that cellular retinol-binding protein type I influences retinol esterification both in liver stellate cells and in HL60 cells.

Retinyl ester storage is normal in transgenic mice with enhanced expression of cellular retinol-binding protein type I.

This report describes the production and characterization of transgenic mice with high expression of human cellular retinol-binding protein type I [hCRBP(I)]. In initial experiments, overexpression of hCRBP(I) was driven by the strong promoter SR(alpha), but no transgenic offspring were produced. When we used the less efficient mouse metallothionein I promoter fused to the hCRBP(I) cDNA for microinjection, we obtained 12% transgenic offspring. Two of these transgenic mice (409/1 and 401/2) expressed mRNA and immunoreactive hCRBP(I) in several organs. Both lines had relatively high contents of hCRBP(I) in intestine, testis and epididymis. On the other hand, only 401/2 transgenic mice had high contents of hCRBP(I) in kidney. Effects on storage of vitamin A were studied by measuring the concentration of retinyl esters in different organs. The concentrations of retinyl esters in liver, lung and kidney did not significantly differ between transgenic and control mice, and the concentration of total retinol in plasma was within the normal range in transgenic mice. Furthermore, feeding mice a diet with high or low concentrations of vitamin A for 2 wks resulted in no marked differences in the concentrations of retinyl esters in liver, kidney, lung, intestine and testis in transgenic mice compared with control mice. Therefore, in spite of high expression of hCRBP(I) in several organs, the transgenic mice had normal storage of retinyl esters in all organs studied. The present in vivo study indicates that the CRBP(I) content alone does not control retinyl ester storage.

Retinoylation of proteins in rat liver, kidney, and lung in vivo.

Retinoylation (retinoic acylation) is a posttranslational modification of proteins occurring in a variety of cell types in vitro. This study was done to examine whether retinoylation occurs in vivo. We found that in retinol-deficient rats, radiolabeled retinol or retinoic acid was incorporated into the liver, kidney, and lung in a form that was not removed by extraction with CHCl3:CH3OH. About 98% of the radiolabeled retinoid was acid-soluble after digestion with proteinase K indicating that it was covalently bound to protein. About 50% of the retinoid covalently bound to liver and kidney protein was removed by mild hydrolysis with CH3OH-KOH. Methyl retinoate, all-trans-retinoic acid, and polar metabolites of retinoic acid accounted for essentially all of the retinoids released. We conclude that retinoylation of protein occurs in vivo primarily via the formation of an ester bond.