Effects of a supplementation during exercise and recovery.

The present study was designed to investigate whether a protein hydrolysate enriched in branched chain amino acids and antioxidants, trace and mineral elements, and vitamins would affect performance and fatigue. Eighteen sportsmen underwent testing before and after 28 days supplementation with either treatment in protein hydrolysate or placebo. Testing included exhaustive aerobic and anaerobic exercises with determination of blood lactate concentration through exercise and recovery and antioxidant status, but also measurements of maximal oxidative capacity (V. (max)) and citrate synthase activity (CS) from a resting muscle biopsy. Protein hydrolysate resulted in a significant decrease in fatigue indices, without affecting performances. A significant increase in enzymatic antioxidant and a decrease in oxidative damage were observed at rest after treatment but not with a placebo. Decrease in maximal blood lactate and improvement of blood lactate removal were only observed after protein hydrolysate treatment. Furthermore, CS increased significantly, whereas no change was observed in V. (max). In conclusion, this protein hydrolysate treatment induced adaptations that may promote a decrease in fatigue during exercises, potentially explained by changes in parameters used to represent oxidative damage and antioxidant status at rest and changes in lactate metabolism.

Deficiency of the cysteine protease cathepsin S impairs microvessel growth.

During angiogenesis, microvascular endothelial cells (ECs) secrete proteinases that permit penetration of the vascular basement membrane as well as the interstitial extracellular matrix. This study tested the hypothesis that cathepsin S (Cat S) contributes to angiogenesis. Treatment of cultured ECs with inflammatory cytokines or angiogenic factors stimulated the expression of Cat S, whereas inhibition of Cat S activity reduced microtubule formation by impairing cell invasion. ECs from Cat S-deficient mice showed reduced collagenolytic activity and impaired invasion of collagens type I and IV. Cat S-deficient mice displayed defective microvessel development during wound repair. This abnormal angiogenesis occurred despite normal vascular endothelial growth factor and basic fibroblast growth factor levels, implying an essential role for extracellular matrix degradation by Cat S during microvessel formation. These results demonstrate a novel function of endothelium-derived Cat S in angiogenesis.

Preferential pharmacological inhibition of macrophage ACAT increases plaque formation in mouse and rabbit models of atherogenesis.

The cholesteryl ester, foam cell-enriched vulnerable plaque is a principle pharmacological target for reducing athero-thrombosis. Acyl CoA:cholesterol Acyl Transferase (ACAT) catalyzes the esterification of free cholesterol in intestine, liver, adrenal and macrophages, leading in the latter cells to intracellular cholesteryl ester accumulation and foam cell formation in the arterial intima. Previous studies suggested the existence of several isoforms of ACAT with different tissue distribution and this has largely been confirmed by molecular cloning of ACAT-1 and ACAT-2. We developed a series of ACAT inhibitors that preferentially inhibited macrophage ACAT relative to hepatic or intestinal ACAT based on in vitro assays and ex vivo bioavailability studies. Four of these compounds were tested in three models of atherosclerosis at oral doses shown to give sufficient bioavailable monocyte/macrophage ACAT inhibitory activity. In fat-fed C57BL/6 mice, chow fed apo E-/- mice and KHC rabbits, the various ACAT inhibitors had either no effect or increased indices of atherosclerotic foam cell formation. Direct and indirect measurements suggest that the increase in plaque formation may have been related to inhibition of macrophage ACAT possibly leading to cytotoxic effects due to augmented free cholesterol. These results suggest that pharmacological inhibition of macrophage ACAT may not reduce, but actually aggravate, foam cell formation and progression.

Thiazolidinedione- and tumor necrosis factor alpha-induced downregulation of peroxisome proliferator-activated receptor gamma mRNA in differentiated 3T3-L1 adipocytes.

Thiazolidinediones (TZDs) are antidiabetic insulin-sensitizing agents that bind to peroxisome proliferator-activated receptor gamma (PPARgamma) and have potent adipogenic effects on 3T3-L1 preadipocytes. In fully differentiated 3T3-L1 adipocytes, TZDs markedly decreased PPARgamma mRNA levels without reducing the expression of genes that are positively regulated by PPARgamma, such as adipocyte lipid-binding protein 2 (aP2) or lipoprotein lipase-(LPL). PPARgamma mRNA levels were also downregulated by tumor necrosis factor alpha (TNFalpha), an antiadipogenic cytokine. We propose that the downregulation of PPARgamma is not the common denominator of the metabolic effects of TZDs and TNFalpha on mature adipocytes.

Sterol regulatory element-binding protein-1 is regulated by glucose at the transcriptional level.

In vivo studies suggest that sterol regulatory element-binding protein (SREBP)-1 plays a key role in the up-regulation of lipogenic genes in the livers of animals that have consumed excess amounts of carbohydrates. In light of this, we sought to use an established mouse hepatocyte cell line, H2-35, to further define the mechanism by which glucose regulates nuclear SREBP-1 levels. First, we show that these cells transcribe high levels of SREBP-1c that are increased 4-fold upon differentiation from a prehepatocyte to a hepatocyte phenotype, making them an ideal cell culture model for the study of SREBP-1c induction. Second, we demonstrate that the presence of precursor and mature forms of SREBP-1 protein are positively regulated by medium glucose concentrations ranging from 5. 5 to 25 mm and are also regulated by insulin, with the amount of insulin in the fetal bovine serum being sufficient for maximal stimulation of SREBP-1 expression. Third, we show that the increase in SREBP-1 protein is due to an increase in SREBP-1 mRNA. Reporter gene analysis of the SREBP-1c promoter demonstrated a glucose-dependent induction of transcription. In contrast, expression of a fixed amount of the precursor form of SREBP-1c protein showed that glucose does not influence its cleavage. Fourth, we demonstrate that the glucose induction of SREBP could not be reproduced by fructose, xylose, or galactose nor by glucose analogs 2-deoxy glucose and 3-O-methyl glucopyranose. These data provide strong evidence for the induction of SREBP-1c mRNA by glucose leading to increased mature protein in the nucleus, thus providing a potential mechanism for the up-regulation of lipogenic genes by glucose in vivo.

Absence of ACAT-1 attenuates atherosclerosis but causes dry eye and cutaneous xanthomatosis in mice with congenital hyperlipidemia.

Acyl-CoA:cholesterol acyltransferase (ACAT) catalyzes esterification of cellular cholesterol. To investigate the role of ACAT-1 in atherosclerosis, we have generated ACAT-1 null (ACAT-1-/-) mice. ACAT activities were present in the liver and intestine but were completely absent in adrenal, testes, ovaries, and peritoneal macrophages in our ACAT-1-/- mice. The ACAT-1-/- mice had decreased openings of the eyes because of atrophy of the meibomian glands, a modified form of sebaceous glands normally expressing high ACAT activities. This phenotype is similar to dry eye syndrome in humans. To determine the role of ACAT-1 in atherogenesis, we crossed the ACAT-1-/- mice with mice lacking apolipoprotein (apo) E or the low density lipoprotein receptor (LDLR), hyperlipidemic models susceptible to atherosclerosis. High fat feeding resulted in extensive cutaneous xanthomatosis with loss of hair in both ACAT-1-/-:apo E-/- and ACAT-1-/-:LDLR-/- mice. Free cholesterol content was significantly increased in their skin. Aortic fatty streak lesion size as well as cholesteryl ester content were moderately reduced in both double mutant mice compared with their respective controls. These results indicate that the local inhibition of ACAT activity in tissue macrophages is protective against cholesteryl ester accumulation but causes cutaneous xanthomatosis in mice that lack apo E or LDLR.

Embryonic lethality and defective neural tube closure in mice lacking squalene synthase.

Squalene synthase (SS) catalyzes the reductive head-to-head condensation of two molecules of farnesyl diphosphate to form squalene, the first specific intermediate in the cholesterol biosynthetic pathway. We used gene targeting to knock out the mouse SS gene. The mice heterozygous for the mutation (SS+/-) were apparently normal. SS+/- mice showed 60% reduction in the hepatic mRNA levels of SS compared with SS+/+ mice. Consistently, the SS enzymatic activities were reduced by 50% in the liver and testis. Nevertheless, the hepatic cholesterol synthesis was not different between SS+/- and SS+/+ mice, and plasma lipoprotein profiles were not different irrespective of the presence of the low density lipoprotein receptor, indicating that SS is not a rate-limiting enzyme in the cholesterol biosynthetic pathway. The mice homozygous for the disrupted SS gene (SS-/-) were embryonic lethal around midgestation. E9.5-10.5 SS-/- embryos exhibited severe growth retardation and defective neural tube closure. The lethal phenotype was not rescued by supplementing the dams either with dietary squalene or cholesterol. We speculate that cholesterol is required for the development, particularly of the nervous system, and that the chorioallantoic circulatory system is not mature enough to supply the rapidly growing embryos with maternal cholesterol at this developmental stage.