Aims/hypothesisApolipoprotein A-I (apoA-I), the main protein constituent of HDL, has a central role in the reverse cholesterol-transport pathway, which together with the anti-inflammatory properties of apoA-I/HDL provide cardioprotection. Recent findings of direct stimulation of glucose uptake in muscle by apoA-I/HDL suggest that altered apoA-I and HDL functionality may be a contributing factor to the development of diabetes. We have studied the in vivo effects of short treatments with human apoA-I in a high-fat diet fed mouse model. In addition to native apoA-I, we investigated the effects of the cardioprotective Milano variant (Arg173Cys).MethodsMale C57Bl6 mice on a high-fat diet for 2 weeks that received a single injection of human apoA-I proteins (wild-type and Milano) were analysed for blood glucose and insulin levels during a 3 h incubation followed by glucose tolerance tests. Incorporation of injected human apoA-I protein into HDLs was analysed by native gel electrophoresis.ResultsApoA-I treatment significantly improved insulin secretion and blood glucose clearance in the glucose tolerance test, with an efficiency exceeding that of lean control animals, and led to decreased basal glucose during the 3 h incubation. Notably, the two apoA-I variants triggered insulin secretion and glucose clearance to the same extent.Conclusions/interpretationApoA-I treatment leads to insulin- and non-insulin-dependent effects on glucose homeostasis. The experimental model of short-term (2 weeks) feeding of a high-fat diet to C57Bl6 mice provides a suitable and time-efficient system to unravel the resulting tissue-specific mechanisms of acute apoA-I treatment that lead to improved glucose homeostasis.

Apolipoprotein A-I (apoA-I) is the main protein of high-density lipoprotein (HDL) and a principal mediator of the reverse cholesterol transfer pathway. Variants of apoA-I have been shown to be associated with hereditary amyloidosis. We previously characterized the G26R and L178H variants that both possess decreased stability and increased fibril formation propensity. Here we investigate the Milano variant of apoAI (R173C; apoAI-M), which despite association with low plasma levels of HDL leads to low prevalence of cardiovascular disease in carriers of this mutation. The R173C substitution is located to a region (residues 170 to 178) that contains several fibrillogenic apoA-I variants, including the L178H variant, and therefore we investigated a potential fibrillogenic property of the apoAI-M protein. Despite the fact that apoAI-M shared several features with the L178H variant regarding increased helical content and low degree of ThT binding during prolonged incubation in physiological buffer, our electron microscopy analysis revealed no formation of fibrils. These results suggest that mutations inducing secondary structural changes may be beneficial in cases where fibril formation does not occur.

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Abstract"Host-guest" studies of the B1 domain from Streptococcal protein G have been used previously to establish a thermodynamic scale for the ␤-sheet-forming propensities of the 20 common amino acids. To investigate the contribution of side chain conformational entropy to the relative stabilities of B1 domain mutants, we have determined the dynamics of side chain methyl groups in 10 of the 20 mutants used in a previous study. Deuterium relaxation rates were measured using two-dimensional NMR techniques for 13 CH 2 D groups. Analysis of the relaxation data using the Lipari-Szabo model-free formalism showed that mutations introduced at the guest position caused small but statistically significant changes in the methyl group dynamics. In addition, there was a low level of covariation of the Lipari-Szabo order parameters among the 10 mutants. The variations in conformational free energy estimated from the order parameters were comparable in magnitude to the variations in global stability of the 10 mutants but did not correlate with the global stability of the domain or with the structural properties of the guest amino acids. The data support the view that conformational entropy in the folded state is one of many factors that can influence the folding thermodynamics of proteins.Keywords: ␤-sheet propensity; B1 domain; conformational entropy; dynamics; GB1; NMR; protein G; protein stability Supplemental material: see www.proteinscience.orgThe relative stabilities of folded and unfolded proteins are determined by the interplay of many enthalpic and entropic factors. In this communication we describe an experimental study of the role of conformational entropy within the folded state. It is widely appreciated that folded proteins have substantially reduced conformational entropy relative to the unfolded state, yet retain some residual flexibility. Although the reduction in entropy upon folding is thermodynamically unfavorable, it is compensated by the formation of enthalpically favorable interactions and by a favorable increase in solvent entropy. The native ensemble represents the optimal trade-off between these various factors. Mutation of a protein (or ligand-binding, changes in pH, ionic strength, temperature, pressure, etc.) could potentially affect any of these thermodynamic factors. Therefore, to understand the influence of mutations on protein stability, it would be advantageous to understand, among other things, the effect of the mutations on conformational entropy. Abbreviations and symbols: ASA, accessible surface area; ⌬G conf , conformational free energy; HSQC, heteronuclear single quantum coherence; NMR, nuclear magnetic resonance; R 1 , longitudinal relaxation rate constant; R 1 , transverse relaxation rate constant; S 2 , order parameter; S 2 axis , methyl axis order parameter; S conf , conformational entropy; e , internal correlation time.Article published online ahead of print. Article and publication date are at