Rp loss in lipid-free apoA-I exposed to 0 (black bars) or 15 mM glycolaldehyde in the absence (white bars) or presence (Ificant effect on this proliferation index. Control includes both sham and spotted bars) of 15 mM aminoguanidine (24 h, 37uC). (B) SDS-PAGE 10781694 of lipid-free apoA-I exposed to 0 mM glycolaldehyde (lane 2), 3 mM glycolaldehyde (lane 3), 3 mM glycolaldehyde and 3 mM aminoguanidine (lane 4), 15 mM glycolaldehyde (lane 5), 15 mM glycolaldehyde and 15 mM aminoguanidine (lane 6) for 24 h. Lane 1: molecular mass markers. Representative gel of 3. (C) Ournal.pone.0066361.tChromosome Instability and Prognosis in MMTable 3. Summary of univariate Cholesterol efflux after 4 h to lipid-free apoA-I exposed (24 h, 37uC) to 0 or 15 mM glycolaldehyde (GA) 6 aminoguanidine (AMG, 15 mM). Columns with different superscript letters are significantly different (one-way ANOVA). doi:10.1371/journal.pone.0065430.gFigure 7. Comparison of lipid-free apoA-I isolated from people with Type 1 diabetes with controls. (A) Loss of Arg (black bars) and Lys (white bars), (B) CML concentrations and (C) efflux of cholesterol from lipid-laden macrophages over 4 h. * Significantly different to control as determined by two-tailed t-tests. doi:10.1371/journal.pone.0065430.gisolated from people with diabetes and secondary kidney disease when compared to controls [22]. These data suggest that AGE formation on apoA-I in vivo arises primarily from the reactions of reactive aldehydes as the plasma lifetime of apoA-I and 16985061 HDL is short [35]. Glycation of apo-A-I by 30 mM glucose or 3 mM glycolaldehyde impaired the clearance of DMPC MLV compared to control apoA-I. Analysis of the kinetic data using a two-phase exponential decay [27], show that both the fast and slow components of thereaction are affected by 3 mM glycolaldehyde, and only the slow component for 30 mM glucose. Previous studies have examined the rate of clearance (solubilisation) of DMPC MLV and shown that it is affected by apoE isoforms and fragments [27], various apoA-IV N-terminus mutants [36], and glycation of apoA-I by fructose or artificial sweetners [16]. Other studies have reported that the affinity of antibodies for the lipid-binding region of apoA-I is altered by methylglyoxal exposure [14,15]. These previous studies, together with the current data, suggest that the structural changes to apoA-I induced by glucose or glycolaldehyde occur, at least in part, in the lipid-binding regions of the protein, with this resulting in reduced phospholipid-protein interactions, and decreased conversion of lipid-free apoA-I to discoidal HDL particles. These effects however only occur to a significant extent with high levels of glucose and reactive aldehydes. As interaction of apoA-I with phospholipids is a requirement for ABCA1-mediated cholesterol efflux and formation of discoidal HDL [11,12], we examined the effects of different extents of glycation on macrophage cholesterol efflux to control and modified apoA-I particles. Cholesterol efflux was unchanged toGlycation Alters Apolipoprotein A-I Lipid Affinitylipid-free apoA-I modified by 30 mM glucose, 3 mM methylglyoxal, or 3 mM glycolaldehyde from J774A.1 cells. Consistent with this data a recent study has reported that modification of apoA-I with low concentrations of methylglyoxal and glycolaldehyde (250 mM) did not affect cholesterol efflux from human ABCA1expressing baby hamster kidney cells [32]. These data contrast with the reported impaired efflux from (non-lipid-loaded) macrophages to lipid-free apoA-I modified with 500 mM glucose for 4 weeks [22]. This extensive, high concentration exposure protocol would be expected to.Rp loss in lipid-free apoA-I exposed to 0 (black bars) or 15 mM glycolaldehyde in the absence (white bars) or presence (spotted bars) of 15 mM aminoguanidine (24 h, 37uC). (B) SDS-PAGE 10781694 of lipid-free apoA-I exposed to 0 mM glycolaldehyde (lane 2), 3 mM glycolaldehyde (lane 3), 3 mM glycolaldehyde and 3 mM aminoguanidine (lane 4), 15 mM glycolaldehyde (lane 5), 15 mM glycolaldehyde and 15 mM aminoguanidine (lane 6) for 24 h. Lane 1: molecular mass markers. Representative gel of 3. (C) Cholesterol efflux after 4 h to lipid-free apoA-I exposed (24 h, 37uC) to 0 or 15 mM glycolaldehyde (GA) 6 aminoguanidine (AMG, 15 mM). Columns with different superscript letters are significantly different (one-way ANOVA). doi:10.1371/journal.pone.0065430.gFigure 7. Comparison of lipid-free apoA-I isolated from people with Type 1 diabetes with controls. (A) Loss of Arg (black bars) and Lys (white bars), (B) CML concentrations and (C) efflux of cholesterol from lipid-laden macrophages over 4 h. * Significantly different to control as determined by two-tailed t-tests. doi:10.1371/journal.pone.0065430.gisolated from people with diabetes and secondary kidney disease when compared to controls [22]. These data suggest that AGE formation on apoA-I in vivo arises primarily from the reactions of reactive aldehydes as the plasma lifetime of apoA-I and 16985061 HDL is short [35]. Glycation of apo-A-I by 30 mM glucose or 3 mM glycolaldehyde impaired the clearance of DMPC MLV compared to control apoA-I. Analysis of the kinetic data using a two-phase exponential decay [27], show that both the fast and slow components of thereaction are affected by 3 mM glycolaldehyde, and only the slow component for 30 mM glucose. Previous studies have examined the rate of clearance (solubilisation) of DMPC MLV and shown that it is affected by apoE isoforms and fragments [27], various apoA-IV N-terminus mutants [36], and glycation of apoA-I by fructose or artificial sweetners [16]. Other studies have reported that the affinity of antibodies for the lipid-binding region of apoA-I is altered by methylglyoxal exposure [14,15]. These previous studies, together with the current data, suggest that the structural changes to apoA-I induced by glucose or glycolaldehyde occur, at least in part, in the lipid-binding regions of the protein, with this resulting in reduced phospholipid-protein interactions, and decreased conversion of lipid-free apoA-I to discoidal HDL particles. These effects however only occur to a significant extent with high levels of glucose and reactive aldehydes. As interaction of apoA-I with phospholipids is a requirement for ABCA1-mediated cholesterol efflux and formation of discoidal HDL [11,12], we examined the effects of different extents of glycation on macrophage cholesterol efflux to control and modified apoA-I particles. Cholesterol efflux was unchanged toGlycation Alters Apolipoprotein A-I Lipid Affinitylipid-free apoA-I modified by 30 mM glucose, 3 mM methylglyoxal, or 3 mM glycolaldehyde from J774A.1 cells. Consistent with this data a recent study has reported that modification of apoA-I with low concentrations of methylglyoxal and glycolaldehyde (250 mM) did not affect cholesterol efflux from human ABCA1expressing baby hamster kidney cells [32]. These data contrast with the reported impaired efflux from (non-lipid-loaded) macrophages to lipid-free apoA-I modified with 500 mM glucose for 4 weeks [22]. This extensive, high concentration exposure protocol would be expected to.
