Long-term supplementation with CLA-FFA or CLA-triacylglycerol reduces BFM in healthy overweight adults.

After 12 mo in a randomized, double-blind, placebo-controlled trial of conjugated linoleic acid (CLA) supplementation (2 groups received CLA as part of a triglyceride or as the free fatty acid, and 1 group received olive oil as placebo), 134 of the 157 participants who concluded the study were included in an open study for another 12 mo. The goals of the extension study were to evaluate the safety [with clinical chemistry analyses and reported adverse events (AEs)] and assess the effects of CLA on body composition [body fat mass (BFM), lean body mass (LBM), bone mineral mass (BMM)], body weight, and BMI. All subjects were supplemented with 3.4g CLA/d in the triglyceride form. Circulating lipoprotein(a) and thrombocytes increased in all groups. There was no change in fasting blood glucose. Aspartate amino transferase, but not alanine amino transferase, increased significantly. Plasma total cholesterol and LDL cholesterol were reduced, whereas HDL cholesterol and triglycerides were unchanged. The AE rate decreased compared with the first 12 mo of the study. Body weight and BFM were reduced in the subjects administered the placebo during the initial 12 mo study (-1.6 +/- 3.2 and -1.7 +/- 2.8 kg, respectively). No fat or body weight changes occurred in the 2 groups given CLA during the initial 12 mo. LBM and BMM were not affected in any of the groups. Changes in body composition were not related to diet and/or training. In conclusion, this study shows that CLA supplementation for 24 mo in healthy, overweight adults was well tolerated. It confirms also that CLA decreases BFM in overweight humans, and may help maintain initial reductions in BFM and weight in the long term.

Long-term supplementation with conjugated linoleic acid (CLA) reduces body fat mass (BFM) and increases or maintains lean body mass (LBM). However, the regional effect of CLA was not studied. The study aimed to evaluate the effect of CLA per region and safety in healthy, overweight and obese adults. A total of 118 subjects (BMI: 28-32 kg/m 2 ) were included in a double blind, placebo-controlled trial. Subjects were randomised into two groups supplemented with either 3·4 g/d CLA or placebo for 6 months. CLA significantly decreased BFM at month 3 (D ¼ 20·9 %, P¼ 0·016) and at month 6 (D ¼ 2 3·4 %, P¼ 0·043) compared with placebo. The reduction in fat mass was located mostly in the legs (D ¼ 2 0·8 kg, P, 0·001), and in women (D ¼ 21·3 kg, P¼ 0·046) with BMI . 30 kg/m 2 (D ¼ 21·9 kg, P¼ 0·011), compared with placebo. The waist -hip ratio decreased significantly (P¼ 0·043) compared with placebo. LBM increased (D ¼ þ0·5 kg, P¼ 0·049) within the CLA group. Bone mineral content was not affected (P¼ 0·70). All changes were independent of diet and physical exercise. Safety parameters including blood lipids, inflammatory and diabetogenic markers remained within the normal range. Adverse events did not differ between the groups. It is concluded that supplementation with CLA in healthy, overweight and obese adults decreases BFM in specific regions and is well tolerated.

FYVE zinc finger domains, which are conserved in multiple proteins from yeast to man, interact specifically with the membrane lipid phosphatidylinositol 3-phosphate (PtdIns(3)P). Here we have investigated the structural requirements for the interaction of the FYVE finger of the early endosome antigen EEA1 with PtdIns(3)P and early endosomes. The binding of the FYVE finger to PtdIns(3)P is Zn 2؉ -dependent, and Zn 2؉could not be replaced by any other bivalent cations tested. By surface plasmon resonance, the wild-type FYVE finger was found to bind to PtdIns(3)P with an apparent K D of about 50 nM and a 1:1 stoichiometry. Mutagenesis of cysteines involved in Zn 2؉ coordination, basic residues thought to be directly involved in ligand binding and other conserved residues, resulted in a 6-to >100-fold decreased affinity for PtdIns(3)P. A mutation in the putative PtdIns(3)P-binding pocket, R1375A, may prove particularly informative, because it led to a strongly decreased affinity for PtdIns(3)P without affecting the FYVE three-dimensional structure, as measured by fluorescence spectroscopy. Whereas the C terminus of EEA1 localizes to early endosomes when expressed in mammalian cells, all the FYVE mutants with reduced affinity for PtdIns(3)P were found to be largely cytosolic. Furthermore, whereas expression of the wild-type EEA1 C terminus interferes with early endosome morphology, the point mutants were without detectable effect. These results support recently proposed models for the ligand binding of the FYVE domain and indicate that PtdIns(3)P binding is crucial for the localization and function of EEA1.Phosphatidylinositol 3-kinases are important regulators of vital cellular processes, including endocytic membrane trafficking, signal transduction, apoptosis, and cytoskeletal organization (1-3). The recent discovery of the conserved FYVE zinc finger (for Fab1p, YOTB, Vac1p, EEA1) 1 (4) as a phosphatidylinositol 3-phosphate (PtdIns (3)P) -specific domain has shed light on the function of this phosphatidylinositol 3-kinase product in cellular processes (5-8). FYVE finger proteins include the membrane trafficking regulators EEA1, Hrs, Vac1p, Vps27p, and Fab1p, the signal transducer SARA, the putative cytoskeletal regulator Fgd1, as well as a number of proteins with unknown function (5, 9 -15).The determination of the crystal structure of the ligand-free FYVE domain of the yeast vacuolar sorting protein Vps27p at high resolution has enabled the modeling of the FYVE-PtdIns (3)P interaction (16). According to this model, the basic residues present in the ␤1 strand ( Fig.

Neutral red is a lysosomal probe and a biological pH indicator. In aqueous solutions, the protonated (NRH) and neutral (NR) forms of monomeric neutral red exhibit distinct absorption maxima (535 and 450 nm, respectively) but have the same fluorescence with a maximum at 637 nm and a quantum yield of 0.02. The similarity of the fluorescence spectra at acidic and basic pH suggests deprotonation of cationic species in the first singlet excited state. The NR fluorescence strongly depends on the solvent polarity as shown by addition of increasing amounts of water to pure dioxane, which gradually shifts the fluorescence maximum from 540 nm in pure dioxane to 637 nm in water. The fluorescence quantum yield increases from 0.17 in dioxane to 0.3 upon addition of 7% water and then decreases, reaching 0.02 in pure water. Immediately after incubation of human skin fibroblasts with neutral red, excitation with 435 nm light produces a fluorescence whose maximum is recorded at 575 nm. This fluorescence is located in the perinuclear region and originates from large fluorescent intracytoplasmic spots, suggesting staining of the endoplasmic reticulum-Golgi complex. At longer times, this fluorescence is shifted to 606 nm, suggesting slow diffusion of the lysosomotropic dye toward the more hydrated and acidic interior of lysosomes. Addition of a lysosomotropic detergent to cells previously incubated with neutral red shifts the fluorescence to the blue. Thus, in complex biological systems, this probe cannot be a good pH indicator but is a very sensitive probe of lysosomal microenvironments.