Changes in hepatic risk factors, metabolic variables, body composition, and physical fitness in obese children after a one-year weight loss program.

Obesity can cause insulin resistance and cardiovascular and liver disease. The aim of this study was to analyze changes in laboratory values, body composition, and physical fitness before and after a one-year weight loss program with nutritional education, psychological care, and physical exercise. Twenty-two obese children (16 boys, 6 girls; median age 11.9 [range 7-15] years; BMI SDS +2.4 [1.6-3.1]) participated in the program. Outcome measures included liver enzymes, insulin resistance (HOMA), lipids, body composition, physical strength and endurance. All children had an inverse HOMA/body composition correlation; Group 1 (reduced BMI SDS after one year) had lower triglycerides, liver enzymes and improved body composition and fitness (p < 0.05). Group 2 (unchanged or increased BMI SDS) had worse body composition and increased endurance and strength of trunk extension (p < 0.05). Weight loss reduced risk factors for liver disease and improved insulin sensitivity. Body composition proved useful as a non-invasive indicator for insulin sensitivity.

Ligand-dependent structural changes in the V(1) ATPase from Manduca sexta.

The response of V(1) ATPase of the tobacco hornworm Manduca sexta to Mg(2+) and nucleotide binding in the presence of the enhancer methanol has been studied by CuCl(2)-induced disulfide formation, fluorescence spectroscopy, and small-angle X-ray scattering. When the V(1) complex was supplemented with CuCl(2) nucleotide-dependence of A-B-E and A-B-E-D cross-linking products was observed in absence of nucleotides and presence of MgADP+Pi but not when MgAMP.PNP or MgADP were added. A zero-length cross-linking product of subunits D and E was formed, supporting their close proximity in the V(1) complex. The catalytic subunit A was reacted with N-4[4-[7-(dimethylamino)-4-methyl]coumarin-3-yl]maleimide (CM) and spectral shifts and changes in fluorescence intensity were detected upon addition of MgAMP.PNP, -ATP, -ADP+Pi, or -ADP. Differences in the fluorescence emission of these nucleotide-binding states were monitored using the intrinsic tryptophan fluorescence. The structural composition of the V(1) ATPase from M. sexta and conformational alterations in this enzyme due to Mg(2+) and nucleotide binding are discussed on the basis of these and previous observations.

Resolution of the V1 ATPase from Manduca sexta into subcomplexes and visualization of an ATPase-active A3B3EG complex by electron microscopy.

The effect of the ATPase activity of Manduca sexta V(1) ATPase by the amphipathic detergent lauryldimethylamine oxide (LDAO) and the relationship of these activities to the subunit composition of V(1) were studied. The V(1) was highly activated in the presence of 0.04-0.06% LDAO combined with release of the subunits H, C, and F from the enzyme. Increase of LDAO concentration to 0.1-0.2% caused the characterized subcomplexes A(3)B(3)HEGF and A(3)B(3)EG with a remaining ATPase activity of 52 and 65%, respectively. The hydrolytic-active A(3)B(3)EG subcomplex has been visualized by electron microscopy showing six major masses of density in a pseudo-hexagonal arrangement surrounding a seventh mass. The compositions of the various subcomplexes and fragments of V(1) provide an organization of the subunits in the enzyme in the framework of the known three-dimensional reconstruction of the V(1) ATPase from M. sexta (Radermacher, M., Ruiz, T., Wieczorek, H., and Grüber, G. (2001) J. Struct. Biol. 135, 26-37).

Expression, purification, and characterization of subunit E, an essential subunit of the vacuolar ATPase.

A recombinant form of subunit E (Vma4p) from yeast vacuolar ATPases (V-ATPases) has been overexpressed in Escherichia coli, purified to homogeneity, and explored by mass spectrometry. Analysis of the secondary structure of Vma4p by circular dichroism spectroscopy indicated 32% alpha-helix and 23% beta-sheet content. Vma4p formed a hybrid-complex with the nucleotide-binding subunits alpha and beta of the closely related F(1) ATPase of the thermophilic bacterium PS3 (TF(1)). The alpha(3)beta(3)E-hybrid-complex had 56% of the ATPase activity of the native TF(1). By comparison, an alpha(3)beta(3)-formation without Vma4p showed about 24% of total TF(1) ATPase activity. This is the first demonstration of a hydrolytically active hybrid-complex consisting of F(1) and V(1) subunits. The arrangement of subunit E in V(1) has been probed using the recombinant Vma4p, the alpha(3)beta(3)E-hybrid-complex together with V(1) and an A(3)B(3)HEG-subcomplex of the V(1) ATPase from Manduca sexta, respectively, indicating that subunit E is shielded in V(1).