Hepatocyte nuclear factor-1alpha is an essential regulator of bile acid and plasma cholesterol metabolism.

Maturity-onset diabetes of the young type 3 (MODY3) is caused by haploinsufficiency of hepatocyte nuclear factor-1alpha (encoded by TCF1). Tcf1-/- mice have type 2 diabetes, dwarfism, renal Fanconi syndrome, hepatic dysfunction and hypercholestrolemia. Here we explore the molecular basis for the hypercholesterolemia using oligonucleotide microchip expression analysis. We demonstrate that Tcf1-/- mice have a defect in bile acid transport, increased bile acid and liver cholesterol synthesis, and impaired HDL metabolism. Tcf1-/- liver has decreased expression of the basolateral membrane bile acid transporters Slc10a1, Slc21a3 and Slc21a5, leading to impaired portal bile acid uptake and elevated plasma bile acid concentrations. In intestine and kidneys, Tcf1-/- mice lack expression of the ileal bile acid transporter (Slc10a2), resulting in increased fecal and urinary bile acid excretion. The Tcf1 protein (also known as HNF-1alpha) also regulates transcription of the gene (Nr1h4) encoding the farnesoid X receptor-1 (Fxr-1), thereby leading to reduced expression of small heterodimer partner-1 (Shp-1) and repression of Cyp7a1, the rate-limiting enzyme in the classic bile acid biosynthesis pathway. In addition, hepatocyte bile acid storage protein is absent from Tcf1-/- mice. Increased plasma cholesterol of Tcf1-/- mice resides predominantly in large, buoyant, high-density lipoprotein (HDL) particles. This is most likely due to reduced activity of the HDL-catabolic enzyme hepatic lipase (Lipc) and increased expression of HDL-cholesterol esterifying enzyme lecithin:cholesterol acyl transferase (Lcat). Our studies demonstrate that Tcf1, in addition to being an important regulator of insulin secretion, is an essential transcriptional regulator of bile acid and HDL-cholesterol metabolism.

Lipoprotein lipase enhances binding of lipoproteins to heparan sulfate on cell surfaces and extracellular matrix.

Lipoprotein lipase enhances binding at 4 degrees C of human plasma lipoproteins (chylomicrons, VLDL, intermediate density lipoprotein, LDL, and HDL3) to cultured fibroblasts and hepG-2 cells and to extracellular matrix. Heparinase treatment of cells and matrix reduces the lipoprotein lipase enhanced binding by 90-95%. Lipoprotein lipase causes only a minimal effect on the binding of lipoproteins to heparan sulfate deficient mutant Chinese hamster ovary cells while it promotes binding to wild type cells that is abolished after heparinase treatment. With 125I-LDL, lipoprotein lipase also enhances uptake and proteolytic degradation at 37 degrees C by normal human skin fibroblasts but has no effect in heparinase-treated normal cells or in LDL receptor-negative fibroblasts. These observations prove that lipoprotein lipase causes, predominantly, binding of lipoproteins to heparan sulfate at cell surfaces and in extracellular matrix rather than to receptors. This interaction brings the lipoproteins into close proximity with cell surfaces and may promote metabolic events that occur at the cell surface, including facilitated transfer to cellular receptors.

Lipolysis exposes unreactive endogenous apolipoprotein E-3 in human and rat plasma very low density lipoprotein.

Endogenous apolipoprotein E in VLDL is poorly expressed in receptor binding processes. Yet catabolism of VLDL-remnants by cellular receptors depends on functional apo E molecules. To better understand remnant catabolism phenomena, we determined the metabolism of VLDL and post-lipolysis VLDL by cultured cells. Partial lipolysis was achieved by incubation of VLDL with lipoprotein lipase in vitro (human) or recirculation (rat) in supradiaphragmatic animals. Lipolyzed VLDL exhibit metabolic activities 2-20-fold higher than control VLDL, that are saturable and dependent on the presence of LDL receptors. The ligand responsible for receptor interaction of lipolyzed VLDL (apo E or apo B-100) and its source (endogenous or transferred) was studied with monoclonal antibodies and with lipoproteins from E-3/3 and E-2/2 subjects. The data unequivocally proved that lipolysis causes exposure of unreactive endogenous apo E-3 at the VLDL surface, possibly by a change of conformation of the protein. Apo B-100 becomes biologically expressed only in lipolyzed VLDL-III. Lipolyzed VLDL, however, is less reactive to exogenous apo E-3 than control VLDL indicating that endogenous and exogenous apo E are oriented differently in VLDL. It is proposed that VLDL delivers triglycerides to tissues when apo E is unreactive but becomes a remnant after the protein becomes exposed and directs the particles from lipoprotein lipase sites to cellular receptors.

Effects of exogenous apo E-3 and of cholesterol-enriched meals on the cellular metabolism of human chylomicrons and their remnants.

The effects of exogenous apo E-3 and of cholesterol-enriched meals on the binding, cell association and proteolytic degradation of human chylomicrons and their remnants were determined in cultured human skin fibroblasts. Chylomicrons were prepared from plasma of normolipemic humans 4 h after a fat meal with normal or high cholesterol content. Remnants were obtained after incubation of chylomicrons with lipoprotein lipase in vitro. Cellular metabolism of chylomicrons was minimal, less than 10% that of LDL. Exogenous apo E-2 enhanced chylomicron metabolism by 3-4-fold. The cellular metabolism of remnants was 2.5-3.5-fold higher as compared to intact chylomicrons but their response to exogenous apo E-3 was considerably lower. The cellular metabolism of chylomicrons and chylomicron remnants obtained from subjects eating cholesterol-enriched fat meal was the highest either without or with added exogenous apo E-3. Yet, even in the preparation that exhibits the highest metabolic activity (apo E-3 enriched remnants from cholesterol-enriched meals) the absolute proteolytic degradation was about two-thirds that of LDL. We conclude that although LDL-receptors take up and degrade chylomicron remnants, the rate of catabolism of remnants by this route can not explain the rapid and complete remnant removal process as observed in vivo.

Significance of high HbA1 levels in normal glucose tolerance.

The significance of high hemoglobin A1 (HbA1) levels (greater than or equal to 8.0%) found in 12.1% of 648 individuals with normal glucose tolerance constituting a part of a representative population sample was examined. Measurement error in HbA1 and/or glucose-tolerance levels was precluded by HbA1 remaining in the same range over 3.5 yr in 89.7% of 29 individuals with initially high and 68.1% of 22 individuals with initially low (less than 6.5%) HbA1. Rate of deterioration to glucose intolerance (6.9%) in the high group during that period resembled the rate (11.8%) in a control group (n = 279). Fasting plasma glucose significantly accounted for only 2.4% of total HbA1-population variance. No correlation of HbA1 was found with other correlates of glucose tolerance or with daily caloric intake and physical activity. A small but significant increment in HbA1 was associated with smoking (7.1 vs. 6.8%, P less than .01) and with clinically overt atherosclerosis (7.3 vs. 6.9%, P less than .01). We conclude that factors unrelated to glucose metabolism are the main determinants of HbA1 level in normal glucose tolerance and play an important role in diabetes as well. These factors have bearing on evaluation of diabetic control by HbA1 and possibly on risk for diabetic complications.

Binding to heparan sulfate is a major event during catabolism of lipoprotein lipase by HepG2 and other cell cultures.

Lipoprotein lipase (LPL) is rapidly and efficiently cleared from the circulation by the liver through an as yet unclear mechanism. In the present study, we determined the nature of LPL interactions with the liver parenchimal cell line HepG2 as compared to other cells in culture. Binding, cell association and degradation of 125I-labelled bovine milk LPL by HepG2 cells, normal and low density lipoprotein (LDL) receptor-negative human fibroblasts and Chinese hamster ovary (CHO) cells show similar values irrespective of source and origin. LPL metabolism in HepG2 cells was characterized by a high capacity to degrade the enzyme, an extremely high sensitivity to heparin and was inhibited by 60%-70% after treatment of the cells with sodium chlorate and heparinase (but not chondroitinase). These findings suggested an important role for heparan sulfate in the process of cell interaction and metabolism of LPL. To further clarify the role of heparan sulfate in determining the LPL-cell interactions, we compared the metabolism of LPL in wild type and mutant heparan sulfate-deficient CHO cells. Heparan sulfate-deficient CHO cells show a low capacity to bind and degrade LPL, about 10%-20% that of the wild type cells. In another set of experiments, we sought to determine whether LPL interactions with HepG2 cells are affected by triglyceride-rich lipoproteins. The results clearly show that whereas unlabeled LPL dramatically enhanced the metabolism of radioiodinated very low density lipoprotein (VLDL), unlabeled VLDL had no effect on radioiodinated LPL metabolism in these cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Efficacy and safety of a combination fluvastatin-bezafibrate treatment for familial hypercholesterolemia: comparative analysis with a fluvastatin-cholestyramine combination.

PURPOSE: Familial hypercholesterolemia (FH) carries a markedly increased risk for coronary artery disease (CAD). Reduction of plasma low-density lipoprotein cholesterol (LDL-C) levels to the normal range may prevent premature atherosclerosis and usually requires a combination of cholesterol-lowering drugs. The major objective of this study is to compare two different drug combinations for the treatment of heterozygous FH. PATIENTS AND METHODS: The current investigation is a short-term, double-blind study comparing the efficacy and safety of fluvastatin when combined with cholestyramine (group 1) or with bezafibrate (group 2) in 38 patients with heterozygous FH. RESULTS: After 6 weeks of combination treatment, in comparison to a drug-free baseline (patients receiving single-blind placebo during the lead-in period of an earlier study, ie, before ever receiving fluvastatin), the combination of 40 mg/d of fluvastatin with 400 mg/d of bezafibrate in group 2 reduced plasma LDL-C levels by 35% as compared with 32% in group 1, and reduced the LDL-C/high-density cholesterol (HDL-C) ratio by 46%, compared to 37% in group 1 (a non-significant difference for both comparisons). When compared to an intermittent 6-week open-label administration of 40 mg fluvastatin monotherapy, the addition of cholestyramine or bezafibrate each reduced LDL-C by an additional 13% (P < 0.01 for both regimens). CONCLUSIONS: Fluvastatin-bezafibrate is superior to a fluvastatin-cholestyramine combination for lowering serum triglycerides and elevating HDL-C serum levels in patients in conjunction with a significant lowering of LDL-C/HDL-C ratios, and may be an effective synergistic therapy for heterozygous FH. No episodes of myositis were seen in this short-term study, a finding that is in agreement with most of the reported studies on statin-fibrate combinations reviewed here.

Platelet parameters and aggregation in essential and reactive thrombocytosis.

Platelet characteristics were assessed in 15 patients with essential thrombocytosis (ET), 89 patients with reactive thrombocytosis (RT), and 23 normal controls. A platelet volume distribution width (PDW) greater than or equal to 10.5 was found in 50%, 21%, and 14% of the three groups, respectively (P = 0.01 between patients with ET and patients with RT; P = 0.02 between patients with RT and controls), reflecting an excess of extreme values at both ends of the distribution. Compared with controls, the increase in platelet number in patients with RT was about twofold throughout the platelet volume range, whereas ET was characterized by a fivefold increase in small platelets less than 7.5 fL and threefold increase in larger size platelets. Mean platelet volume (MPV) was significantly lower in patients with ET versus patients with RT and in patients with RT versus controls (mean +/- SD 7.5 +/- 1.2 vs. 8.8 +/- 0.1 and 10.2 +/- 1.8 fL, respectively, P less than 0.01). Rate of in vitro platelet aggregation greater than or equal to 50% was significantly lower in patients with ET versus patients with RT and in patients with RT versus controls (0%, 23%, and 45%, respectively, P less than 0.01). Aggregation rate was positively correlated with MPV (r = 0.54; P less than 0.0001). Aggregation rate in patients with ET was significantly lower (P = 0.01) than expected from their reduced MPV alone. Despite these group differences, the overlap of individual platelet characteristics between the three groups precludes their usefulness for diagnostic purposes.

Memory deficits and cholinergic impairments in apolipoprotein E-deficient mice.

Apolipoprotein E-deficient mice provide a useful system for studying the role of apolipoprotein E (apoE) in the function of distinct neuronal systems. In the present study we focused on the cholinergic system of these mice. This was pursued by measurements of specific biochemical, physiological and cognitive parameters. Morris Water Maze tasks revealed impairments in working memory but not in reference memory of the apoE-deficient mice. Measurements of brain choline acetyltransferase activities revealed them to be markedly lower in the hippocampus and frontal cortex of the apoE-deficient mice than in the corresponding brain areas of the controls, but unaltered in other brain areas. In addition, hypothermia induced by the centrally acting muscarinic agonist, oxotremorine, was reduced in the apoE-deficient mice as compared to controls. These results show that apoE-deficient mice have cholinergic deficits and highlight the importance of this mouse model for studying the interactions between apoE and the cholinergic nervous system.

Phosphorylation of tau in apolipoprotein E-deficient mice.

It has been suggested that the deleterious effects of the allele E4 of apolipoprotein E (apoE) in Alzheimer's disease (AD) are related to its inability to interact with the microtubule associated protein tau and to thereby prevent its hyperphosphorylation. In the present study we investigated the effects of apoE on tau phosphorylation by immunoblot analysis of the levels and extents of phosphorylation of tau of apoE-deficient mice. This revealed that mAb AT8, which is directed against a phosphorylated tau epitope, labels tau of the apoE-deficient mice more intensely than that of control mice and that the opposite occurs with mAb Tau1, which is directed against dephosphorylated tau epitopes. mAb ALZ50 also labeled the tau enriched preparations of the apoE-deficient mice more intensely than those of the controls, whereas the extents of their labeling by the phosphorylation insensitive anti-tau mAb 134 were similar. These results suggest that tau of apoE-deficient mice is hyperphosphorylated.

Derangement in stress response of apolipoprotein E-deficient mice.

Apolipoprotein E (apoE) is associated with familial and sporadic Alzheimer's disease (AD). Stress has been identified as a putative risk factor of AD. Thus, in the present study we examined the susceptibility of apoE-deficient mice to stress. The results obtained revealed that the elevation of corticosterone levels in apoE-deficient mice following restraint stress is markedly lower than in controls, and that these mice differ in their behavioral pain response to noxious stimuli in both stress and non-stress conditions. These findings suggest an interplay between apoE and the response to stressful stimuli and provide a model for elucidating the relationship between apoE and susceptibility to stress.

[Screening and treatment of hyperlipidemias in Israel].

Hyperlipidemias are a major risk factor for cardiovascular disease. Screening and treatment guidelines for the metabolic disorders associated with them have been adopted and published by the Israel Atherosclerosis Society. We examined the frequency of plasma lipid determinations and treatment of hyperlipidemia by a sample of family practitioners, primary physicians, cardiologists and internists working in ambulatory, primary medical care who filled out questionnaires for consecutive adult clinic patients. Of 2015 patients, 1029 (51%) were reported to be free of cardiovascular disease or its risk factors, 606 (30%) had at least 1 risk factor, 171 (8%) had peripheral or cerebrovascular atherosclerotic disease, 131 (7%) had had a myocardial infarction and 78 (4%) had undergone coronary angioplasty or bypass surgery. Although the frequency of determinations of total HDL and LDL cholesterol increased from those without a risk factor to those after bypass surgery, the overall rates were lower than expected in all groups. With regard to risk factors, in those with a family history of cardiovascular disease lipid determinations were done most frequently, approaching the frequency in the bypass group. Combined hypercholesterolemia and hypertriglyceridemia was the most common metabolic aberration in all categories. The rate of drug therapy increased from those with no risk factor to those after bypass surgery. Therapy with resin was reported in 3-14%, fibric acid derivatives in 29-53% and statins in 33-57% of the various patient groups. Again, the drug therapy profile of the group with a positive family history was similar to that of the bypass group.(ABSTRACT TRUNCATED AT 250 WORDS)

[Hypercalcemia following rhabdomyolysis-induced acute renal failure].

Hypercalcemia is an uncommon clinical feature during recovery from acute renal failure. Moderate hypercalcemia developed during recovery of kidney function in a 24-year-old man with acute renal failure following polytrauma-induced rhabdomyolysis (myoglobinuria). He presented with multiple hematomas of the back, chest, abdomen and upper and lower limbs. He was asymptomatic during the hypercalcemic phase, when Ca reached 13.2 mg and PTH levels (immunoradiometric assay) were low, 8.7 muug/ml (normal 10-65).

Mechanisms of inhibition by apolipoprotein C of apolipoprotein E-dependent cellular metabolism of human triglyceride-rich lipoproteins through the low density lipoprotein receptor pathway.

The mechanism of inhibition by apolipoprotein C of the uptake and degradation of triglyceride-rich lipoproteins from human plasma via the low density lipoprotein (LDL) receptor pathway was investigated in cultured human skin fibroblasts. Very low density lipoprotein (VLDL) density subfractions and intermediate density lipoprotein (IDL) with or without added exogenous recombinant apolipoprotein E-3 were used. Total and individual (C-I, C-II, C-III-1, and C-III-2) apoC molecules effectively inhibited apoE-3-mediated cell metabolism of the lipoproteins through the LDL receptor, with apoC-I being most effective. When the incubation was carried out with different amounts of exogenous apoE-3 and exogenous apoC, it was shown that the ratio of apoE-3 to apoC determined the uptake and degradation of VLDL. Excess apoE-3 overcame, at least in part, the inhibition by apoC. ApoC, in contrast, did not affect LDL metabolism. Neither apoA-I nor apoA-II, two apoproteins that do not readily associate with VLDL, had any effect on VLDL cell metabolism. The inhibition of VLDL and IDL metabolism cannot be fully explained by interference of association of exogenous apoE-3 with or displacement of endogenous apoE from the lipoproteins. IDL is a lipoprotein that contains both apoB-100 and apoE. By using monoclonal antibodies 4G3 and 1D7, which specifically block cell interaction by apoB-100 and apoE, respectively, it was possible to assess the effects of apoC on either apoprotein. ApoC dramatically depressed the interaction of IDL with the fibroblast receptor through apoE, but had only a moderate effect on apoB-100. The study thus demonstrates that apoC inhibits predominantly the apoE-3-dependent interaction of triglyceride-rich lipoproteins with the LDL receptor in cultured fibroblasts and that the mechanism of inhibition reflects association of apoC with the lipoproteins and specific concentration-dependent effects on apoE-3 at the lipoprotein surface.

Remnant particles and their metabolism.

The data described in this chapter demonstrate that the metabolic control of processes responsible for the formation, uptake and clearance of remnant particles is considerably more complex than previously believed. It now appears that several interacting reactions are involved in the process, and evidence is accumulating that defects in any one of these reactions may severely affect the optimal metabolic cascade. Proper exposure of receptor-binding domains in apoE and perhaps apoB-100 molecules is mandatory. Lipoprotein lipase-induced triglyceride hydrolysis is essential and responsible for the formation of remnant particles from secreted triglyceride-rich lipoproteins. The existence of apoE molecules that exhibit normal function is important but perhaps not always essential. Sequestration in the liver through lipoprotein lipase and/or apoE-mediated binding to heparan sulphate ('bridging' effect) appears to play an exceedingly important role during the early phase of the remnant clearance process. The 'bridging' is responsible not only for sequestration in the liver but also for enhanced uptake and lysosomal degradation of the particles. At this stage, association with the remnants of newly secreted, liver-derived apoE molecules may occur and add to the affinity of the particles towards receptors, especially if the new apoE molecules are inserted in a favourable conformational configuration. A role for the hepatic lipase has been suggested but is yet to be proved. Finally, it should be emphasized that remnants are cleared from the plasma predominantly, if not exclusively, following interaction with cellular receptors. Although the LDL receptor avidly internalizes remnant particles and is apparently active in species with a low LDL concentration (e.g. mice and rats), a second specialized and specific receptor or receptors must exist. Whether the LRP is the only remnant receptor or other, as yet unidentified, receptor proteins are also present, remains to be established. Data published in the last few years have begun to elucidate the interactions and consequences of the many reactions and proteins that are involved with the metabolism of remnant lipoproteins. More is to be learned, including the association of remnants in processes that lead to initiation/progression of atherosclerosis.

The role of native apolipoprotein B-containing lipoproteins in atherosclerosis: cellular mechanisms.

Apolipoprotein B-containing lipoproteins play a central role in the pathogenesis of atherosclerosis. The processes that determine the atherogenicity of these lipoproteins, however, remain unclear. A new development that involves the interactions of lipoproteins, enzymes, apolipoproteins, and cell-surface heparan sulphate may explain, at least in part, the enhanced atherogenicity of these lipoproteins. In addition, the development of specific transgenic apolipoproteins, and apolipoprotein-deficient mice models that develop atherosclerosis, provide new tools for studies on the role of native apolipoprotein-B containing lipoproteins in atheroma formation.

Abnormal composition of hypertriglyceridemic very low density lipoprotein determines abnormal cell metabolism.

The metabolism of very low density lipoprotein (VLDL) from normolipemic (NTG) subjects, hypertriglyceridemic (HTG) subjects, and hypertriglyceridemic subjects treated with bezafibrate (BZ) was studied in cultured human skin fibroblasts. The binding, cell association, and proteolytic degradation of 125I-labeled lipoproteins and the capacity to regulate cellular sterol synthesis was determined with and without maximal stimulation of the lipoprotein by exogenous recombinant or plasmatic apolipoprotein (apo) E-3. The VLDL was separated into three density subfractions: I, II, and III. Multiple differences between HTG and NTG lipoproteins were found, which all reverted toward normal with therapy. Even in the presence of an optimal concentration of apo E-3, HTG-VLDL demonstrated 100% to 200% higher metabolic activities, indicating a better association or a better biological expression of apo E-3 at the surface of the lipoprotein. There was a strong and linear relationship between the cholesterol ester/protein ratios of the different VLDLs and their proteolytic degradations by the cells (r = 0.95). Thus, the composition/structure alterations of VLDL appear to determine their apo E-3-dependent cellular catabolism. In addition, HTG-VLDLs not enriched with apo E-3 exhibited a capacity to down-regulate cellular sterol synthesis independently of their uptake and degradation by the cells. This abnormality appeared to reflect the ability of the VLDL to donate cholesterol to the cells and was not observed in receptor-negative cells. Thus, HTG-VLDL is much more capable than NTG-VLDL of introducing cholesterol to cells by at least two mechanisms: 1) accelerated uptake and degradation and 2) direct transfer of cholesterol to the cells. Both processes are potentially atherogenic and are reversible when triglyceride-lowering therapy is instituted.