Roux-en-Y gastric bypass reduces plasma cholesterol in diet-induced obese mice by affecting trans-intestinal cholesterol excretion and intestinal cholesterol absorption.

OBJECTIVE: Bariatric surgery appears as the most efficient therapeutic alternative in morbidly obese patients. In addition to its efficiency to decrease body weight, it also improves metabolic complications associated to morbid obesity, including dyslipidemia. Although the cholesterol-lowering effect varies with the bariatric procedures, the underlying molecular mechanisms remain poorly defined. This study aims to assess the consequence of both restrictive (sleeve gastrectomy; SG) and malabsorptive (Roux-en-Y gastric bypass; RYGB) procedures on cholesterol metabolism in mice. SUBJECTS: Ten-week-old C57BL6/J males were fed with a high-fat diet for 8-14 weeks before sleeve or RYGB surgery. RESULTS: SG has a modest and transient effect on plasma cholesterol levels, linked to a reduction in food intake. In contrast, modified RYGB led to a sustained ≈35% reduction in plasma cholesterol concentrations with a drastic increase in fecal cholesterol output. Mechanistically, RYGB exerts a synergystic effect on cholesterol metabolism by inducing the trans-intestinal cholesterol efflux and reducing the intestinal cholesterol absorption. CONCLUSIONS: In mice, RYGB, but not sleeve, strongly favors plasma cholesterol elimination by concomitantly increasing trans-intestinal cholesterol excretion and by decreasing intestinal cholesterol absorption. Our models open new perspective for deciphering the hypocholesterolemic effects of bariatric procedures.

K+ and NH4(+) modulate gill (Na+, K+)-ATPase activity in the blue crab, Callinectes ornatus: fine tuning of ammonia excretion.

To better comprehend the mechanisms of ionic regulation, we investigate the modulation by Na+, K+, NH4(+) and ATP of the (Na+, K+)-ATPase in a microsomal fraction from Callinectes ornatus gills. ATP hydrolysis obeyed Michaelis-Menten kinetics with KM=0.61+/-0.03 mmol L(-1) and maximal rate of V=116.3+/-5.4 U mg(-1). Stimulation by Na+ (V=110.6+/-6.1 U mg(-1); K0.5=6.3+/-0.2 mmol L(-1)), Mg2+ (V=111.0+/-4.7 U mg(-1); K0.5=0.53+/-0.03 mmol L(-1)), NH4(+) (V=173.3+/-6.9 U mg(-1); K0.5=5.4+/-0.2 mmol L(-1)) and K+ (V=116.0+/-4.9 U mg(-1); K0.5=1.5+/-0.1 mmol L(-1)) followed a single saturation curve, although revealing site-site interactions. In the absence of NH4(+), ouabain (K(I)=74.5+/-1.2 micromol L(-1)) and orthovanadate inhibited ATPase activity by up to 87%; the inhibition patterns suggest the presence of F0F1 and K+-ATPases but not Na+-, V- or Ca2+-ATPase as contaminants. (Na+, K+)-ATPase activity was synergistically modulated by K+ and NH4(+). At 10 mmol L(-1) K+, increasing NH4(+) concentrations stimulated maximum activity to V=185.9+/-7.4 U mg(-1). However, at saturating NH4(+) (50 mmol L(-1)), increasing K+ concentrations did not stimulate activity further. Our findings provide evidence that the C. ornatus gill (Na+, K+)-ATPase may be particularly well suited for extremely efficient active NH4(+) excretion. At elevated NH4(+) concentrations, the enzyme is fully active, regardless of hemolymph K+ concentration, and K+ cannot displace NH4(+) from its exclusive binding sites. Further, the binding of NH4(+) to its specific sites induces an increase in enzyme apparent affinity for K+, which may contribute to maintaining K+ transport, assuring that exposure to elevated ammonia concentrations does not lead to a decrease in intracellular potassium levels. This is the first report of modulation by ammonium ions of C. ornatus gill (Na+, K+)-ATPase, and should further our understanding of NH4(+) excretion in benthic crabs.

Influence of homocysteine on matrix metalloproteinase-2: activation and activity.

Increased levels of the physiological amino acid homocysteine (Hcy) are considered a risk factor for vascular disease. Hyperhomocysteinemia causes an intense remodelling of the extracellular matrix in arterial walls, particularly an elastolysis involving metalloproteinases. We investigated the activation of the latent elastolytic metalloproteinase proMMP-2 (72 kDa) by Hcy. Hcy was proved to exert a dual effect, activating proMMP-2 at low molar ratio (MR 10:1) and inhibiting active MMP2 at high molar ratio (MR > 1000:1). Methionine and the disulphide homocystine did not activate nor inhibit MMP-2, showing that the activation as well as the inhibition requires the thiol group to be free. The activation of proMMP-2 by Hcy is in accordance with the "cysteine-switch" mechanism, but occurs without further autoproteolysis of the enzyme molecule. In contrast with Hcy, the other physiological thiol compounds cysteine and reduced glutathione did not activate proMMP-2. These results suggest that the direct activation of proMMP2 by Hcy could be one of the mechanisms involved in the extracellular matrix deterioration in hyperhomocysteinemia-associated arteriosclerosis.

Effects of folate supplementation in hyperhomocysteinemic pigs.

OBJECTIVES: The aim of this study was to evaluate the therapeutic effects of folic acid in the pig model of hyperhomocysteinemia. BACKGROUND: We have previously shown that pigs fed a methionine-rich diet develop hyperhomocysteinemia, arterial lesions and thrombotic events. Elevated homocysteine level is an independent risk factor for atherosclerosis that can be markedly lowered with daily folic acid administration. However, it is not known whether this treatment can prevent arterial lesions. METHODS: Three groups of pigs were studied: 8 control subjects received a standard diet; 8 received a methionine-rich diet for four months; 8 received a methionine-rich diet for 1 month and then the methionine-rich diet + 5 mg/day folic acid for 3 months. At month 4 after hemodynamic investigation, all the pigs were sacrificed. RESULTS: Control animals developed few usual vascular streaks. All the pigs fed a methionine-rich diet without folic acid treatment developed hyperhomocysteinemia (10.3+/-1.3 micromol/liter at basal state, 18.2+/-2.5 micromol/liter at one month and 14.6+/-3.8 micromol/liter at four months), hemodynamic abnormalities and diffuse arterial lesions with smooth muscle cell hyperplasia, endothelial alterations and elastic lamina dislocation. In this group, one pig died of venous thromboembolism and one of myocardial infarction. The pigs fed a methionine-rich diet + folic acid displayed similar arterial lesions and two had thrombotic events (one myocardial infarction and one pulmonary embolism), despite normalization of homocysteine levels (10.9+/-1.3 micromol/liter at basal state, 19.5+/-2.5 micromol/liter at one month and 11.4+/-3.8 micromol/liter at four months). CONCLUSIONS: In the pig model of hyperhomocysteinemia, 5 mg/day folic acid did not prevent arterial lesions or thrombotic events.

[Homocysteine and coronary events in coronary disease patients]. / Homocystéine et événements coronaires chez le coronarien.

The objective of this study was to determine the prognostic value of serum homocysteine levels in patients with coronary heart disease. Homocysteine was assayed in 76 coronary patients with a mean age of 59.2 years hospitalized for myocardial ischaemia or myocardial infarction. Percutaneous transluminal angioplasty was performed in 47 (70%) of these patients during this hospitalization. The mean follow-up for these patients was 22 months (range: 11 to 67 months). In these patients, serum homocysteine levels were not correlated with the usual risk factors of coronary heart disease (age, sex, treated hypercholesterolaemia, smoking, diabetes) except for hypertension. It was strongly correlated with serum creatinine (R = 0.61; p = 0.0001). Eleven patients presented a major event during follow-up (8 deaths, 1 nonfatal myocardial infarction, 1 cardiac transplantation) and 16 underwent a revascularization procedure. The blood homocysteine level does not have any prognostic value for any coronary events. However, it is higher in patients who develop a major event than in those which do not (15.8 +/- 4 mumol/l versus 11.5 +/- 6.6 mumol/l, p = 0.05). Using multivariate analysis, taking into account age, serum creatinine and serum homocysteine, only serum homocysteine was predictive of major event-free survival (p = 0.02).

Association of mild hyperhomocysteinemia with cardiac graft vascular disease.

In non-transplant patients mild hyperhomocysteinemia is an independent risk factor for vascular disease. The aim of this study was to determine whether hyperhomocysteinemia is associated with graft vascular disease. Fasting total plasma homocysteine was assessed in 18 patients with graft vasculopathy and 18 transplanted patients without graft vasculopathy matched for age, sex and the time since transplant. All were on cyclosporin. Graft vasculopathy was defined at coronary angiography as stenoses > or = 25%, or aneurysms. We found that hyperhomocysteinemia ( > or = 15 micromol/l) is common among transplanted heart recipients and significantly more frequent in the patients with graft vasculopathy (17/18 versus 11/18). Accordingly, the mean homocysteinemia was significantly higher in the group with graft vasculopathy (23.6+/-7.8 versus 16.9+/-7.1 micromol/l, P=0.01). The elevation of homocysteine plasma levels in the heart transplant recipients has probably multiple causes. The main cause seems to be renal failure. Additional causes could be azathioprine treatment or genetic polymorphisms. These results suggest that besides the immunological factors, homocysteine can play an additional role in the pathogenesis of graft vascular disease.

Hyperhomocysteinemia induces elastolysis in minipig arteries: structural consequences, arterial site specificity and effect of captopril-hydrochlorothiazide.

Hyperhomocysteinemia is a risk factor for arterial diseases, and the deterioration of the arterial elastic structures is one of the possible mechanisms underlying this epidemiological association. The aim of this paper is to quantitatively characterize such structural alterations and to explore their causes in a previous model of dietary induced mild hyperhomocysteinemia in minipigs. After four months, both a morphodensitometrical analysis of the elastic structure and a biochemical analysis of elastin and elastase activities were performed on the infrarenal abdominal aorta (IRAA) and the proximal left interventricular coronary artery (LIVCA) of control (C), hyperhomocysteinemic (H) and captopril-hydrochlorothiazide (Cp-Htz, 25 + 12.5 mg/d)-treated (H+/-Cp) minipigs (n = 8/group). Hyperhomocysteinemia was found to induce an increase in parietal elastolytic metalloproteinase activities. It resulted in opening and enlargement of fenestrae through the medial elastic laminae and in a decrease in medial elastin content (p < 10(-3)), expressed as well as volume density (%) as weight concentration (microg elastin/mg dry tissue). The thickness of the media and its basic lamellar organization was unchanged. The reduction in volume density was more dramatic in LIVCA (H: 4.7 +/- 0.9 vs C: 8.8 +/- 2.4), where it was evenly distributed within the media, than in IRAA (H: 6.7 +/- 1.1 vs C: 9.3 +/- 1.2), where the deep medial layers were less affected. Cp-Htz partly prevented the hyperhomocysteinemia-induced reduction of the medial elastin content in LIVCA (5.7 +/- 1.2) and IRAA (7.9 +/- 1.4). This effect, occurring in the subintimal layers of the media in both arteries but not in the deeper layers, resulted in a less beneficial effect in LIVCA than in IRAA. This result parallels the moderate beneficial therapeutic effect of ACE inhibitors against coronary atherosclerosis in humans. This paper reports for the first time a quantitative analysis of the arterial site-dependent deterioration of the elastic structure caused by mild hyperhomocysteinemia and the involvement of metalloproteinases in this process. These results confirm that the plaque-independent damage to elastic structure previously described in hyperhomocysteinemic-atherosclerotic minipigs was mainly due to homocysteine. This highlights that the metalloproteinase-related elastolysis and the subsequent structural deterioration is one of the major events underlying the epidemiological association between mild hyperhomocysteinemia and arterial diseases.

Plasma homocysteine as a risk factor for vascular disease. The European Concerted Action Project.

CONTEXT: Elevated plasma homocysteine is a known risk factor for atherosclerotic vascular disease, but the strength of the relationship and the interaction of plasma homocysteine with other risk factors are unclear. OBJECTIVE: To establish the magnitude of the vascular disease risk associated with an increased plasma homocysteine level and to examine interaction effects between elevated plasma homocysteine level and conventional risk factors. DESIGN: Case-control study. SETTING: Nineteen centers in 9 European countries. PATIENTS: A total of 750 cases of atherosclerotic vascular disease (cardiac, cerebral, and peripheral) and 800 controls of both sexes younger than 60 years. MEASUREMENTS: Plasma total homocysteine was measured while subjects were fasting and after a standardized methionine-loading test, which involves the administration of 100 mg of methionine per kilogram and stresses the metabolic pathway responsible for the irreversible degradation of homocysteine. Plasma cobalamin, pyridoxal 5'-phosphate, red blood cell folate, serum cholesterol, smoking, and blood pressure were also measured. RESULTS: The relative risk for vascular disease in the top fifth compared with the bottom four fifths of the control fasting total homocysteine distribution was 2.2 (95% confidence interval, 1.6-2.9). Methionine loading identified an additional 27% of at-risk cases. A dose-response effect was noted between total homocysteine level and risk. The risk was similar to and independent of that of other risk factors, but interaction effects were noted between homocysteine and these risk factors; for both sexes combined, an increased fasting homocysteine level showed a more than multiplicative effect on risk in smokers and in hypertensive subjects. Red blood cell folate, cobalamin, and pyridoxal phosphate, all of which modulate homocysteine metabolism, were inversely related to total homocysteine levels. Compared with nonusers of vitamin supplements, the small number of subjects taking such vitamins appeared to have a substantially lower risk of vascular disease, a proportion of which was attributable to lower plasma homocysteine levels. CONCLUSIONS: An increased plasma total homocysteine level confers an independent risk of vascular disease similar to that of smoking or hyperlipidemia. It powerfully increases the risk associated with smoking and hypertension. It is time to undertake randomized controlled trials of the effect of vitamins that reduce plasma homocysteine levels on vascular disease risk.

Homocysteine induces synthesis of a serine elastase in arterial smooth muscle cells from multi-organ donors.

OBJECTIVES: In heart transplant recipients with diffuse coronary arteriopathy, we have previously demonstrated the prevalence of elevated homocysteinemia, also known as an independent risk factor for myocardial infarction and stroke. In hyperhomocysteinemic mini-pigs we also observed early detectable pathologic changes in the elastic laminae. We hypothesized that homocysteine causes premature breakdown in the arterial elastic fibers by activation of the elastolytic activities. METHODS: We examined the effect of homocysteine on elastase-like production by smooth muscle cells from sub-inguinal arteries of multi-organ donors (23.4 +/- 3.4 yr, n = 8). The freshly isolated cells were incubated for 0-72 h with homocysteine (0-250 microM), in the presence or absence of specific protease inhibitors. RESULTS: Homocysteine was devoid of a direct effect, but after 18 h incubation the elastase-like activities increased by 5-6-fold in the extracellular medium. The enzymes were characterized as serine proteases. Incubation of cells with a nucleic acid synthesis inhibitor (actinomycin D) or a protein synthesis inhibitor (cycloheximide) suppressed the enzyme induction. CONCLUSIONS: This is the first report of serine protease induction by homocysteine in vascular smooth muscle cells. The process may require protein synthesis and account for the early alterations of the arterial elastic structures in heart transplant recipients, and in other hyperhomocysteinemic patients, as well.

Medial elastic structure alterations in atherosclerotic arteries in minipigs: plaque proximity and arterial site specificity.

Using a model of atherosclerosis in minipigs, we analyzed changes in elastic structure within the medial sections of the abdominal aorta and left interventricular coronary artery both in the vicinity of and distal to atheromatous plaques. Twenty-four animals, divided into three groups, were fed either a control diet or a hypercholesterolemic and hyperhomocysteinic atherogenic diet, alone or in association with an antihypertensor, namely isosorbide dinitrate (Risordan). The atherogenic diet, administered for a period of four months, induced in the minipig advanced noncalcified atherosclerotic lesions that were histologically similar to those found in humans. A morphodensitometric analysis of the medial elastic structures was carried out on images obtained from specifically stained transverse arterial sections examined under a light microscope. The volume density of the elastic structures was diminished in the arterial media of the atherosclerotic animals due to opening and widening of the fenestrae in the elastic laminate and increased communication between the interlamellar spaces. Whereas this elastolytic process was uniform and independent of the proximity of atheromatous plaques in the left interventricular coronary artery, it was intensified in the vicinity of the plaques in the abdominal aorta. Overall elastolytic activity was increased in the walls of atheromatous artery in both arterial sites, and metalloproteinases were implied in this increase of activity. We previously reported that treatment with isosorbide dinitrate significantly reduced the moderate systolic hypertension and the increase in transparietal stress observed in the abdominal aorta of atheromatous animals. We report here that isosorbide dinitrate prevented the atherogenic-diet-induced deterioration of the elastic structure in these arteries; complete inhibition of changes to the elastic laminae was evident in areas remote from plaque formation, but only partial inhibition in the vicinity of such plaques. It did not, however, prevent structural damage in the left interventricular coronary artery or modify the increase in parietal elastolytic activity in either of the two arteries. This suggests that damage to the elastic structure in atheromatous arteries is dependent not only on overall elastolytic activity but also on localized factors, possibly related to parietal stresses, affected by the presence of atheromatous plaques.