Management of giant cell arteritis: Recommendations of the French Study Group for Large Vessel Vasculitis (GEFA).

PURPOSE: Management of giant cell arteritis (GCA, Horton's disease) involves many uncertainties. This work was undertaken to establish French recommendations for GCA management. METHODS: Recommendations were developed by a multidisciplinary panel of 33 physicians, members of the French Study Group for Large Vessel Vasculitis (Groupe d'étude français des artérites des gros vaisseaux [GEFA]). The topics to be addressed, selected from proposals by group members, were assigned to subgroups to summarize the available literature and draft recommendations. Following an iterative consensus-seeking process that yielded consensus recommendations, the degree of agreement among panel members was evaluated with a 5-point Likert scale. A recommendation was approved when ≥ 80% of the voters agreed or strongly agreed. RESULTS: The 15 retained topics resulted in 31 consensus recommendations focusing on GCA nomenclature and classification, the role of temporal artery biopsy and medical imaging in the diagnosis, indications and search modalities for involvement of the aorta and its branches, the glucocorticoid regimen to prescribe, treatment of complicated GCA, indications for use of immunosuppressants or targeted biologic therapies, adjunctive treatment measures, and management of relapse and recurrence. CONCLUSIONS: The recommendations, which will be updated regularly, are intended to guide and harmonize the standards of GCA management.

[Vascular involvement in Cogan's syndrome. A case report]. / Atteinte vasculaire et syndrome de Cogan. Une observation.

Vascular manifestations of Cogan's syndrome are rarely reported. We report the case of a young woman followed for typical Cogan's disease. Serious vascular involvement was found only during work-up for arterial hypertension. This case highlights potentially asymptomatic nature of extensive vasculitis affecting large and medium-sized vessels in Cogan's disease. Careful screening is required to prevent life-threatening complications.

Glycine-induced neurotoxicity in organotypic hippocampal slice cultures.

The role of the neutral amino acid glycine in excitotoxic neuronal injury is unclear. Glycine coactivates glutamate N-methyl-D-aspartate (NMDA) receptors by binding to a distinct recognition site on the NR1 subunit. Purely excitatory glycine receptors composed of NR1 and NR3/NR4 NMDA receptor subunits have recently been described, raising the possibility of excitotoxic effects mediated by glycine alone. We have previously shown that exposure to high concentrations of glycine causes extensive neurotoxicity in organotypic hippocampal slice cultures by activation of NMDA receptors. In the present study, we investigated further properties of in vitro glycine-mediated toxicity. Agonists of the glycine recognition site of NMDA receptors (D-serine and D-alanine) did not have any toxic effect in hippocampal cultures, whereas competitive blockade of the glycine site by 7-chlorokynurenic acid was neuroprotective. Stimulation (taurine, beta-alanine) or inhibition (strychnine) of the inhibitory strychnine-sensitive glycine receptors did not produce any neurotoxicity. The toxic effects of high-dose glycine were comparable in extent to those produced by the excitatory amino acid glutamate in our model. When combined with sublethal hypoxia/hypoglycemia, the threshold of glycine toxicity was decreased to less than 1 mM, which corresponds to the range of concentrations of excitatory amino acids measured during in vivo cerebral ischemia. Taken together, these results further support the assumption of an active role of glycine in excitotoxic neuronal injury.

Pathophysiology of apolipoprotein E deficiency in mice: relevance to apo E-related disorders in humans.

Apolipoprotein E (apo E) deficiency (or its abnormalities in humans) is associated with a series of pathological conditions including dyslipidemia, atherosclerosis, Alzheimer's disease, and shorter life span. The purpose of this study was to characterize these conditions in apo E-deficient C57BL/6J mice and relate them to human disorders. Deletion of apo E gene in mice is associated with changes in lipoprotein metabolism [plasma total cholesterol (TC) (>+400%), HDL cholesterol (-80%), HDL/TC, and HDL/LDL ratios (-93% and -96%, respectively), esterification rate in apo B-depleted plasma (+100%), plasma triglyceride (+200%), hepatic HMG-CoA reductase activity (-50%), hepatic cholesterol content (+30%)], decreased plasma homocyst(e)ine and glucose levels, and severe atherosclerosis and cutaneous xanthomatosis. Hepatic and lipoprotein lipase activities, hepatic LDL receptor function, and organ antioxidant capacity remain unchanged. Several histological/immunohistological stainings failed to detect potential markers for neurodegenerative disease in the brain of 37-wk-old male apo E-KO mice. Apo E-KO mice may have normal growth and development, but advanced atherosclerosis and xanthomatosis may indirectly reduce their life span. Apo E plays a crucial role in regulation of lipid metabolism and atherogenesis without affecting lipase activities, endogenous antioxidant capacity, or appearance of neurodegenerative markers in 37-wk-old male mice.

Potency of select statin drugs in a new mouse model of hyperlipidemia and atherosclerosis.

Poloxamer-407 (P-407) is a nonionic surfactant that induces atheroma formation in the aortas of C57BL/6 mice with long-term (14 weeks) administration. The objectives of the present study were to determine the mechanism(s) responsible for the induction of hypercholesterolemia as well as to determine whether this animal model may be of potential use in rank ordering the efficacy (lipid lowering) of various statin drugs. The effect of long-term (16 weeks) administration of P-407 on the catalytic activities of rate-limiting enzymes of cholesterol biosynthesis [HMG-CoA reductase (HMGR)] and catabolism [microsomal cholesterol 7alpha-hydroxylase (C7alphaH) and mitochondrial sterol 27 hydroxylase (S27H)] was assessed in C57BL/6 mice. Effects of P-407 on these enzymes were compared in mice fed an atheroma-inducing diet (high-cholesterol, supplemented with cholic acid) and animals maintained on a basal diet and injected with saline (controls) after 16 weeks. The mean value for the activities of C7alphaH in P-407-injected mice was 24.3+/-3.8 pmol min(-1) mg(-1) and was significantly (P<0.05) less than the mean value determined for sham-injected control animals (37.0+/-14.3 pmol min(-1) mg(-1)). In contrast, the mean values for the catalytic activities of S27H and HMGR did not change with P-407 administration. Neither C7alphaH nor S27H activity in mice fed the high-cholesterol diet differed from values for control animals, whereas the mean HMGR activity was drastically reduced (-94%, P<0.05). The hypercholesterolemic effect of P-407 is not due to altered cholesterol biosynthesis, but is mediated by reduced cholesterol catabolism due to decreased activity of the rate limiting enzyme (C7alphaH) in the classic bile acid synthetic pathway. Plasma triglyceride lowering resulting from the oral administration of equal doses of various statin drugs appeared, in general, to be positively correlated with their relative aqueous solubility and paralleled the efficacy of these agents to lower low-density-lipoprotein-associated cholesterol (LDL-C) in humans. The plasma triglyceride lowering effect of the five statin drugs tested produced the following rank order; pravastatin sodium (-44%)>atorvastatin calcium (-36%)>simvastatin (-33%)>lovastatin (-25%)>fluvastatin sodium (-19%). While reductions in plasma total cholesterol following administration of the statin drugs was not as profound as that observed with triglycerides, the relative rank order or trend was preserved. The percent reduction in plasma triglycerides in the present model appears to be a useful parameter with which to predict the relative reduction in plasma LDL-C expected for these agents in humans.

Macrophage 3-hydroxy-3-methylglutaryl coenzyme a reductase activity in sitosterolemia: effects of increased cellular cholesterol and sitosterol concentrations.

Sitosterolemia is a rare, recessively inherited disease characterized clinically by accelerated atherosclerosis and xanthomas and biochemically by hyperabsorption and retention of sitosterol and other plant sterols in tissues. Decreased cholesterol biosynthesis and inhibition of 3-hydroxy-3-methylgluratyl coenzyme A (HMG-CoA) reductase and other enzymes in the biosynthetic pathway have been associated with enhanced low-density lipoprotein (LDL) receptor function. We examined the effects of cholesterol and sitosterol on sterol concentrations and composition and HMG-CoA reductase activity in monocyte-derived macrophages (MDM) from 12 control and 3 homozygous sitosterolemic subjects. The cells were cultured up to 7 days in media devoid of plant sterols, but containing increasing amounts of serum cholesterol. Before culture, MDM from the homozygous sitosterolemic subjects contained 22% more total sterols than cells from control subjects. Plant sterols and stanols represented 15.6% of MDM total sterols in sitosterolemic cells, but only 3.8% in control cells. After 7 days of culture in 10% delipidated serum (DLS) (20 microg/mL cholesterol, no sitosterol), all plant sterols were eliminated so that cells from both phenotypes contained only cholesterol. When DLS was replaced with fetal bovine serum (FBS) (300 micromL cholesterol), with and without addition of 200 microg/mL LDL, cholesterol levels in MDM from sitosterolemic subjects increased 108% (P <.05) compared with a 65% increase (P <.04) in control MDM cultured similarly. MDM HMG-CoA reductase activity from the 3 sitosterolemic subjects, which was significantly lower than controls at baseline (24 +/- 3 v 60 +/- 10 pmol/mg/min, P <.05), was not downregulated by increased cellular cholesterol levels, as observed in control cells. Control MDM were also cultured in medium that contained 10% DLS and was supplemented with 100 microg/mL cholesterol or sitosterol dissolved in ethanol or the ethanol vehicle alone. In contrast to cellular cholesterol accumulation, which significantly downregulated HMG-CoA reductase activity (-53%, P <.05), the increase in cellular sitosterol up to 25.1% of total sterols did not change MDM HMG-CoA reductase activity. Evidence of a normal HMG-CoA reductase protein in sitosterolemic cells, which was not derepressed upon removal of cellular sitosterol, and the failure of cellular sitosterol to inhibit normal HMG-CoA reductase activity argue against feedback inhibition by sitosterol as a mechanism for low reductase activity in this disease. The larger accumulation of sterols and inadequate downregulation of HMG-CoA reductase in MDM may be mechanisms for foam cell formation and explain, in part, the increased risk of atherosclerosis in sitosterolemia.

Plant stanol fatty acid esters inhibit cholesterol absorption and hepatic hydroxymethyl glutaryl coenzyme A reductase activity to reduce plasma levels in rabbits.

The aim of this study was to study the inhibitory effect of dietary stanols (campestanol and sitostanol) fatty acid esters (SE) on intestinal cholesterol absorption. New Zealand white rabbits were fed regular chow alone or enriched with 0.2% cholesterol, 0.33% SE + cholesterol, 0.66% SE + cholesterol, 1.2% SE + cholesterol, 2.4% SE + cholesterol, and 1.2% SE alone. After 2 weeks, plasma cholesterol levels increased 3.6 times in the cholesterol group and did not decrease after addition of 0.33% or 0.66% SE to the cholesterol-enriched diets. However, after addition of 1.2% SE to the cholesterol diet, plasma cholesterol concentration decreased 50% (P <.001), but it did not decrease further after doubling of SE to 2.4%. Percent cholesterol absorption measured by the plasma dual-isotope ratio method was 73.0% +/- 8.1 % in the cholesterol group, which was similar to untreated baseline control. The percent absorption of cholesterol did not decrease significantly after addition of 0.33% or 0.66% SE to the cholesterol diet but decreased 43.8% (P <.001) in the 1.2% SE + cholesterol group, a finding similar to those in rabbits fed 1.2% SE alone. Increasing SE to 2.4% in the cholesterol diet did not further decrease absorption. Hepatic hydroxymethyl glutaryl coenzyme A (HMG-CoA) reductase activity reflecting cholesterol synthesis and low-density lipoprotein receptor-mediated binding unexpectedly decreased 67% (P <.01) and 57% (P <.05) in rabbits fed 1.2% SE alone. Increasing dietary SE intake to 1.2% reduced cholesterol absorption and plasma levels. Dietary SE intake below 1.2% was ineffective and above 2.4% did not further decrease percent absorption or plasma cholesterol levels. These results support the hypothesis that dietary SEs competitively displace cholesterol from intestinal micelles to reduce cholesterol absorption and decrease plasma cholesterol levels.

Cholecystectomy prevents expansion of the bile acid pool and inhibition of cholesterol 7alpha-hydroxylase in rabbits fed cholesterol.

To study the effect of cholecystectomy on the regulation of classic and alternative bile acid syntheses, gallbladder-intact (n = 20) and cholecystectomized (n = 20) New Zealand White rabbits were fed either chow or chow with 2% cholesterol (3 g/day). After 10 days, bile fistulas were constructed in half of each rabbit group to recover and measure the bile acid pool and biliary bile acid flux. After cholesterol feeding, the bile acid pool size increased from 268 +/- 55 to 444 +/- 77 mg (P < 0.01) with a 2-fold rise in the biliary bile acid flux in intact rabbits but did not expand the bile acid pool (270 +/- 77 vs. 276 +/- 62 mg), nor did the biliary bile acid flux increase in cholecystectomized rabbits. Ileal apical sodium-dependent bile acid transporter protein increased 46% from 93 +/- 6 to 136 +/- 23 units/mg (P < 0.01) in the intact rabbits but did not change in cholecystectomized rabbits (104 +/- 14 vs. 99 +/- 19 units/mg) after cholesterol feeding. Cholesterol 7alpha-hydroxylase activity was inhibited 59% (P < 0.001) while cholesterol 27-hydroxylase activity rose 83% (P < 0.05) after cholesterol feeding in the intact rabbits but neither enzyme activity changed significantly in cholesterol-fed cholecystectomized rabbits. Fecal bile acid outputs reflecting bile acid synthesis increased significantly in the intact but not in the cholecystectomized rabbits fed cholesterol. Removal of the gallbladder prevented expansion of the bile acid pool after cholesterol feeding as seen in intact rabbits because ileal bile acid transport did not increase. As a result, cholesterol 7alpha-hydroxylase was not inhibited.

Impaired biliary lipid secretion in obese Zucker rats: leptin promotes hepatic cholesterol clearance.

Human obesity is associated with elevated plasma leptin levels. Obesity is also an important risk factor for cholesterol gallstones, which form as a result of cholesterol hypersecretion into bile. Because leptin levels are correlated with gallstone prevalence, we explored the effects of acute leptin administration on biliary cholesterol secretion using lean (FA/-) and obese (fa/fa) Zucker rats. Zucker (fa/fa) rats become obese and hyperleptinemic due to homozygosity for a missense mutation in the leptin receptor, which diminishes but does not completely eliminate responsiveness to leptin. Rats were infused intravenously for 12 h with saline or pharmacological doses of recombinant murine leptin (5 microg x kg(-1) x min(-1)) sufficient to elevate plasma leptin concentrations to 500 ng/ml compared with basal levels of 3 and 70 ng/ml in lean and obese rats, respectively. Obesity was associated with a marked impairment in biliary cholesterol secretion. In biles of obese compared with lean rats, bile salt hydrophobicity was decreased whereas phosphatidylcholine hydrophobicity was increased. High-dose leptin partially normalized cholesterol secretion in obese rats without altering lipid compositions, implying that both chronic effects of obesity and relative resistance to leptin contributed to impaired biliary cholesterol elimination. In lean rats, acute leptin administration increased biliary cholesterol secretion rates. Without affecting hepatic cholesterol contents, leptin downregulated hepatic activity of 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase, upregulated activities of both sterol 27-hydroxylase and cholesterol 7alpha-hydroxylase, and lowered plasma very low-density lipoprotein cholesterol concentrations. Increased biliary cholesterol secretion in the setting of decreased cholesterol biosynthesis and increased catabolism to bile salts suggests that leptin promotes elimination of plasma cholesterol.

Hepatic cholesterol and bile acid synthesis, low-density lipoprotein receptor function, and plasma and fecal sterol levels in mice: effects of apolipoprotein E deficiency and probucol or phytosterol treatment.

We compared hepatic cholesterol metabolism in apolipoprotein (apo) E-knockout (KO) mice with their wild-type counterparts. We also investigated the effects of treatment with phytosterols or probucol on the activity of hepatic 3-hydroxy-3-methyl-glutaryl coenzyme A (HMG-CoA) reductase (cholesterol synthesis), cholesterol 7 alpha-hydroxylase and sterol 27-hydroxylase (bile acid synthesis), and low-density lipoprotein (LDL) receptor function in this animal model of atherogenesis. These findings were then related to treatment-induced changes in plasma, hepatic, and fecal sterol concentrations. Mouse liver membranes have binding sites similar to LDL receptors; the receptor-mediated binding represents 80% of total binding and is LDL concentration-dependent. These binding sites have higher affinity for apo E-containing particles than apo B only-containing particles. Deletion of apo E gene was associated with several-fold increases in plasma cholesterol levels, 1.5-fold increase in hepatic cholesterol concentrations, 50% decrease in HMG-CoA reductase activity, 30% increase in cholesterol 7 alpha-hydroxylase and 25% decrease in LDL receptor function. Treatment of apo E-KO mice with either probucol or phytosterols significantly reduced plasma cholesterol levels. Phytosterols significantly increased the activity of hepatic HMG-CoA reductase, and probucol significantly increased cholesterol 7 alpha-hydroxylase activity. Neither treatment significantly altered hepatic LDL receptor function. Phytosterols, but not probucol, significantly increased fecal sterol excretion and decreased hepatic cholesterol concentrations. Plasma cholesterol lowering effects of phytosterols and probucol are due to different mechanisms: stimulation of cholesterol catabolism via increased bile acid synthesis by probucol and decreased cholesterol absorption by phytosterols. In the absence of apo E, hepatic LDL receptors could not be upregulated and did not contribute to the cholesterol lowering effects of either agent.

Mechanisms for cholesterol homeostasis in rat jejunal mucosa: effects of cholesterol, sitosterol, and lovastatin.

The effects of feeding cholesterol, sitosterol, and lovastatin on cholesterol absorption, biosynthesis, esterification, and LDL receptor function were examined in the rat jejunal mucosa. Cholesterol absorption was measured by the dual-isotope plasma ratio method; the rate-limiting enzyme of cholesterol biosynthesis, 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase, was measured as total and expressed enzyme activities (in the absence and presence of a phosphatase inhibitor, NaF, respectively); mucosal total and esterified cholesterol concentrations were determined by gas-liquid chromatography; LDL receptor function was assayed as receptor-mediated binding of (125)I-labeled LDL to mucosal membranes. Feeding 2% sitosterol or 0.04% lovastatin for 1 week significantly (P < 0.01) decreased the amounts of cholesterol absorbed per day (-85% and -63%, respectively). In contrast, feeding 2% cholesterol for 1 week increased the amounts of absorbed cholesterol 27-fold, even though the percent absorption significantly decreased. With all three treatments, there was a coordinate regulation of total HMG-CoA reductase activity and receptor-mediated LDL binding. Cholesterol feeding downregulated both total jejunal HMG-CoA reductase activity (P < 0.05) and receptor-mediated LDL binding (P < 0.01), whereas lovastatin- and sitosterol-supplemented diets significantly upregulated both of these parameters. In the control, cholesterol-fed, and sitosterol-fed animals, about half of the total jejunal HMG-CoA reductase activity was expressed (in functional dephosphorylated form). However, in the lovastatin-treated rats with 4-fold stimulation of HMG-CoA reductase, only 23% of the total enzyme activity was expressed. Changes in total HMG-CoA reductase activity and receptor-mediated LDL binding in all tested groups occurred with no change in total concentrations of mucosal cholesterol, and only cholesterol-fed animals had increased mucosal esterified cholesterol concentrations. Thus, in response to various fluxes of dietary or newly formed cholesterol, HMG-CoA reductase and receptor-mediated LDL binding are coordinately regulated to maintain constant cellular cholesterol concentrations in the jejunum.

Is there a relationship between 3-hydroxy-3-methylglutaryl coenzyme a reductase activity and forebrain pathology in the PKU mouse?

Previous reports have suggested that elevated levels of phenylalanine inhibit cholesterol synthesis. The goals of this study were to investigate if perturbations in cholesterol synthesis exist in the PAH(enu2) genetic mouse model for phenylketonuria (PKU), and if so, initiate studies determining if they might underlie the white matter pathology that exists in PKU forebrain. Gross sections and electron microscopy showed that select tracts were hypomyelinated in adult PKU mouse forebrain but not hindbrain. The activity of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR), the rate controlling enzyme in the cholesterol biosynthetic pathway, was examined in isolated microsomes from forebrain, hindbrain, and liver to assess if perturbations in cholesterol biosynthesis were occurring. HMGR activity was normal in unaffected PKU hindbrain and was increased 2-4-fold in PKU liver compared to control. HMGR activity in the forebrain, however, was decreased by 30%. Because normal numbers of MBP-expressing glia (oligodendrocytes) were present, but the number of glia expressing HMGR was reduced by 40% in the hypomyelinated tracts, the decreased HMGR activity seemed to result from a down-regulation of HMGR expression in affected oligodendrocytes. Exposure of an oligodendrocyte-like glioma cell line to physiologically relevant elevated levels of Phe resulted in a 30% decrease in cholesterol synthesis, a 28% decrease in microsomal HMGR activity, and a 28% decrease in HMGR protein levels. Measurement of HMGR activity after addition of exogenous Phe to control brain microsomes revealed that Phe is a noncompetitive inhibitor of HMGR; physiologically relevant elevated levels of exogenous Phe inhibited HMGR activity by 30%. Taken together, these data suggest that HMGR is moderately inhibited in the PKU mouse. Unlike other cell types in the body, a subset of oligodendrocytes in the forebrain seems to be unable to overcome this inhibition. We speculate that this may be the cause of the observed pathology in PKU brain.

Apolipoprotein E regulates dietary cholesterol absorption and biliary cholesterol excretion: studies in C57BL/6 apolipoprotein E knockout mice.

The present study examined the role of apolipoprotein E (apoE) in the regulation of dietary cholesterol absorption and biliary cholesterol excretion. Increasing dietary cholesterol from 0.02% to 0.5% in C57BL/6 wild-type mice decreased the percentage of dietary cholesterol that is absorbed by 25%, and this decrease was associated with a 2-fold increase in gallbladder biliary cholesterol concentration. In contrast, increasing dietary cholesterol from 0. 02% to 0.5% in C57BL/6 apoE knockout mice produced no significant suppression of the percentage dietary cholesterol absorption and increased gallbladder biliary cholesterol concentration only 16%. Whereas in wild-type mice, the increase in dietary cholesterol increased the hepatic excretion of biliary cholesterol 4-fold, there was only a 2-fold increase in apoE knockout mice. On both the low- and the high-cholesterol diets, whole liver and isolated hepatocyte cholesterol content was higher in the apoE knockout mice. These results suggest that, in response to dietary cholesterol, apoE may play a critical role in decreasing the percentage absorption of dietary cholesterol and increasing biliary cholesterol excretion. These observations suggest a mechanism whereby the absence of apoE contributes to the propensity for tissue cholesterol deposition and accelerated atherogenesis in apoE knockout mice.

Ileal bile acid transport regulates bile acid pool, synthesis, and plasma cholesterol levels differently in cholesterol-fed rats and rabbits.

We investigated the effect of ileal bile acid transport on the regulation of classic and alternative bile acid synthesis in cholesterol-fed rats and rabbits. Bile acid pool sizes, fecal bile acid outputs (synthesis rates), and the activities of cholesterol 7alpha-hydroxylase (classic bile acid synthesis) and cholesterol 27-hydroxylase (alternative bile acid synthesis) were related to ileal bile acid transporter expression (ileal apical sodium-dependent bile acid transporter, ASBT). Plasma cholesterol levels rose 2.1-times in rats (98 +/- 19 mg/dl) and 31-times (986 +/- 188 mg/dl) in rabbits. The bile acid pool size remained constant (55 +/- 17 mg vs. 61 +/- 18 mg) in rats but doubled (254 +/- 46 to 533 +/- 53 mg) in rabbits. ASBT protein expression did not change in rats but rose 31% (P < 0.05) in rabbits. Fecal bile acid outputs that reflected bile acid synthesis increased 2- and 2.4-times (P < 0.05) in cholesterol-fed rats and rabbits, respectively. Cholesterol 7alpha-hydroxylase activity rose 33% (24 +/- 2.4 vs. 18 +/- 1.6 pmol/mg/min, P < 0.01) and mRNA levels increased 50% (P < 0.01) in rats but decreased 68% and 79%, respectively, in cholesterol-fed rabbits. Cholesterol 27-hydroxylase activity remained unchanged in rats but rose 62% (P < 0.05) in rabbits. Classic bile acid synthesis (cholesterol 7alpha-hydroxylase) was inhibited in rabbits because an enlarged bile acid pool developed from enhanced ileal bile acid transport. In contrast, in rats, cholesterol 7alpha-hydroxylase was stimulated but the bile acid pool did not enlarge because ASBT did not change. Therefore, although bile acid synthesis was increased via different pathways in rats and rabbits, enhanced ileal bile acid transport was critical for enlarging the bile acid pool size that exerted feedback regulation on cholesterol 7alpha-hydroxylase in rabbits.

Comparative regulation of hepatic sterol 27-hydroxylase and cholesterol 7alpha-hydroxylase activities in the rat, guinea pig, and rabbit: effects of cholesterol and bile acids.

The regulation of the classic and alternative bile acid synthetic pathways by key hepatic enzyme activities (microsomal cholesterol 7alpha-hydroxylase and mitochondrial sterol 27-hydroxylase, respectively) was examined in bile acid depletion and replacement and cholesterol-feeding experiments with rats, guinea pigs, and rabbits. The bile acid pool was depleted by creating a bile fistula (BF) and collecting bile for 2 to 5 days, and it was replaced by intraduodenal infusion of the major biliary bile acids (taurocholic acid [TCA], glycochenodeoxycholic acid [GCDCA], and glycocholic acid [GCA] in the rat, guinea pig, and rabbit, respectively) at rates equivalent to the measured hepatic flux of the bile acids. To study the effects of cholesterol, the animals were fed for 7 days on a basal diet with and without 2% cholesterol. Cholesterol 7alpha-hydroxylase and sterol 27-hydroxylase activities, measured by isotope incorporation assays, were related to bile acid output and composition and hepatic cholesterol concentrations. Intraduodenal infusion of bile acids increased the output of the tested bile acids, but did not significantly change hepatic cholesterol concentrations and had no effect on sterol 27-hydroxylase activity. Neither bile acid depletion nor replacement affected sterol 27-hydroxylase activity when three different substrates (cholesterol, 5beta-cholestane-3alpha,7alpha-diol, and 5beta-cholestane-3alpha,7alpha,12alpha-triol) were tested. In contrast, feeding 2% cholesterol increased hepatic cholesterol concentrations in rats, guinea pigs, and rabbits threefold, twofold, and eightfold, respectively, and increased hepatic mitochondrial sterol 27-hydroxylase activity (conversion of cholesterol to 27-hydroxycholesterol) in all three animal models. The stimulation and feedback inhibition of cholesterol 7alpha-hydroxylase activity by bile acid depletion and replacement were observed in all three animal models, whereas the effect of cholesterol feeding was species-dependent (cholesterol 7alpha-hydroxylase activity increased in the rat, did not change in the guinea pig, and was inhibited in the rabbit). Thus, in contrast to sterol 27-hydroxylase, which was upregulated by cholesterol but not affected by bile acid depletion and replacement in all three animal models, cholesterol 7alpha-hydroxylase activity was controlled consistently and inversely by the hepatic flux of bile acids, but was species-dependent in its response to a 1-week feeding with 2% cholesterol.

Renal effects of low to moderate doses of dopamine in newborn piglets.

PURPOSE: Administration of dopamine to adult animal and human subjects results in increased renal blood flow, and it may also enhance the glomerular filtration rate. However, renal hemodynamic effects of exogenous dopamine in the neonate are unclear. In this study, we examined the renal actions of low to moderate doses of exogenous dopamine in newborn piglets. METHODS: The animals were anesthetized, catheterized for vascular access and urine collection, and assigned randomly to a control group or treatment groups receiving dopamine infusion at 2, 5, or 10 microg/kg/min. Data were collected at baseline, during dopamine infusion, and 1 hour after cessation of infusion. Mean arterial blood pressure (MAP) and heart rate (HR) were monitored. Glomerular filtration rate (GFR), cardiac index (CI), and renal blood flow (RBF) were determined. Fractional excretion of sodium (FENa) was calculated. RESULTS: Dopamine did not alter renal blood flow nor did it significantly alter CI in spite of a modest increase in heart rate and mean arterial blood pressure. There was a statistically significant increase in GFR at 10 microg/kg/min and in FENa at all doses. CONCLUSIONS: Low doses of dopamine produce significant natriuresis probably by direct action on renal tubules and at moderate doses via, both, increase in GFR and a direct tubular effect. Low and moderate doses of dopamine do not increase RBF as seen in adult animals, possibly because of immaturity of dopaminergic receptors in newborn piglets.

Histologic, hematologic, and biochemical characteristics of apo E-deficient mice: effects of dietary cholesterol and phytosterols.

In this study, we examined the effects of a "Western-type" diet containing 9% (w/w) fat and 0.15% (w/w) cholesterol, in the presence or absence of 2% (w/w) phytosterol mixture over an 18-week period in apolipoprotein E-deficient mice. Addition of phytosterols to the high cholesterol diet was associated with normalization of the depressed hepatic 3-hydroxy-3-methylglutaryl-coenzyme A reductase activity (from 22.3+/-6.3 to 55.4+/-19.9 pmol/mg protein/minutes, p < 0.05). This finding was associated with a significant decrease in plasma and hepatic cholesterol concentrations compared with animals fed the high cholesterol diet without phytosterols (33.3+/-5.0 versus 19.2+/-6.2 pmol/mg protein, p < 0.05). The activities of cholesterol 7alpha-hydroxylase and sterol 27-hydroxylase were comparable between the two groups of mice. Urinalyses and hematologic data were comparable between the two groups except for significantly lower platelet counts in the phytosterol-treated animals (681.6+/-118.9 versus 857.1+/-185.4 x10(9)/L, p < 0.05). The phytosterol-treated animals had significantly (p < 0.05) less fragile erythrocytes when exposed to 0.08, 0.07, or 0.05 M NaCl compared with cholesterol-fed mice. The consumption of the Western-type diet was associated with the development of xanthomatous skin lesions in 33% of the cholesterol-fed animals, but in none of the phytosterol-treated animals. Histologic examination revealed oil red O-negative vacuolation in liver and kidney parenchymal cells of the cholesterol-fed group, but not in the phytosterol-treated mice. Arrested spermatogenesis and atrophy of seminiferous tubules were observed, to a variable extent, in both groups of animals. We conclude that addition of the phytosterol mixture (2% w/w) to a Western-type diet in apolipoprotein E-deficient mice significantly decreases plasma and hepatic cholesterol concentrations, increases hepatic 3-hydroxy-3-methylglutaryl-coenzyme A reductase activity, and prevents cutaneous xanthomatosis and vacuolation in the parenchymal cells of kidneys and livers.

Increasing dietary cholesterol induces different regulation of classic and alternative bile acid synthesis.

We investigated the effect of increasing dietary cholesterol on bile acid pool sizes and the regulation of the two bile acid synthetic pathways (classic, via cholesterol 7alpha-hydroxylase, and alternative, via sterol 27-hydroxylase) in New Zealand white rabbits fed 3 g cholesterol/per day for up to 15 days. Feeding cholesterol for one day increased hepatic cholesterol 75% and cholesterol 7alpha-hydroxylase activity 1.6 times without significant change of bile acid pool size or sterol 27-hydroxylase activity. After three days of cholesterol feeding, the bile acid pool size increased 83% (P < 0.01), and further feeding produced 10%-20% increments, whereas cholesterol 7alpha-hydroxylase activity declined progressively to 60% below baseline. In contrast, sterol 27-hydroxylase activity rose 58% after three days of cholesterol feeding and remained elevated with continued intake. Bile drainage depleted the bile acid pool and stimulated downregulated cholesterol 7alpha-hydroxylase activity but did not affect sterol 27-hydroxylase activity. Thus, increasing hepatic cholesterol does not directly inhibit cholesterol 7alpha-hydroxylase and initially favors enzyme induction, whereas increased bile acid pool is the most powerful inhibitor of cholesterol 7alpha-hydroxylase. Sterol 27-hydroxylase is insensitive to the bile acid flux but is upregulated by increasing hepatic cholesterol.

Regulation of rat hepatic 3beta-hydroxysterol delta7-reductase: substrate specificity, competitive and non-competitive inhibition, and phosphorylation/dephosphorylation.

The mechanism for the catalytic reduction of the double bond at C-7, 8 in 7-dehydrocholesterol by 3beta-hydroxysterol Delta7-reductase was investigated by testing structurally related sterols as substrates and potential inhibitors. The hepatic smooth endoplasmic reticulum was identified as the site of enzyme activity. All putative substrates contained 27 carbons, but differed from 7-dehydrocholesterol by the addition of either an ethyl substituent at C-24 (7-dehydrositosterol), a double bond at C-22 with a methyl substituent at C-24 (ergosterol), epimerization of the hydroxyl from the 3beta- to 3alpha-configuration (7-dehydroepicholesterol), or a saturated double bond at C-5,6 (lathosterol). Two non-steroidal compounds that inhibit 3beta-hydroxysterol Delta7-reductase in vivo (AY 9944 and BM 15.766) were also tested. Ergosterol, 7-dehydrositosterol, and 7-dehydroepicholesterol were reduced at C-7, 8 to form brassicasterol, sitosterol, and epicholesterol, respectively, but 75% less efficiently than 7-dehydrocholesterol. Increasing concentrations of these sterols competitively inhibited 3beta-hydroxysterol Delta7-reductase activity. The double bond at C-7,8 in lathosterol was not reduced. AY 9944 and BM 15.766 inhibited 3beta-hydroxysterol Delta7-reductase activity non-competitively. 3beta-Hydroxysterol-Delta7-reductase activity declined after microsomes were exposed to alkaline phosphatase, and enzyme activity was increased by phosphorylation with Mg2+, and ATP. These results demonstrate that the reduction of the double bond at C-7,8 requires binding of the enzyme protein with the B-ring of the sterol substrate that contains a double bond at C-5,6. The reaction is hindered by substituents located on the apolar side-chain and epimerization of the hydroxyl group in ring A to a 3alpha-configuration. 3beta-Hydroxysterol Delta7-reductase exists in two forms: an active phosphorylated form and an inactive dephosphorylated form.