Glycation of human serum albumin impairs binding to the glucagon-like peptide-1 analogue liraglutide

Abstract

The long-acting glucagon-like peptide-1 analogue liraglutide has proven efficiency in the management of type 2 diabetes and also has beneficial effects on cardiovascular diseases. Liraglutide's protracted action highly depends on its capacity to bind to albumin via its palmitic acid part. However, in diabetes, albumin can undergo glycation, resulting in impaired drug binding. Our objective in this study was to assess the impact of human serum albumin (HSA) glycation on liraglutide affinity. Using fluorine labeling of the drug and ¹⁹F NMR, we determined HSA affinity for liraglutide in two glycated albumin models. We either glycated HSA in vitro by incubation with glucose (G25- or G100-HSA) or methylglyoxal (MGO-HSA) or purified in vivo glycated HSA from the plasma of diabetic patients with poor glycemic control. Nonglycated commercial HSA (G0-HSA) and HSA purified from plasma of healthy individuals served as controls. We found that glycation decreases affinity for liraglutide by 7-fold for G100-HSA and by 5-fold for MGO-HSA compared with G0-HSA. A similarly reduced affinity was observed for HSA purified from diabetic individuals compared with HSA from healthy individuals. Our results reveal that glycation significantly impairs HSA affinity to liraglutide and confirm that glycation contributes to liraglutide's variable therapeutic efficiency, depending on diabetes stage. Because diabetes is a progressive disease, the effect of glycated albumin on liraglutide affinity found here is important to consider when diabetes is managed with this drug.

Keywords: albumin; diabetes; glycation; liraglutide; nuclear magnetic resonance (NMR); protein drug interaction; spectroscopy.

Figure 1.

Liraglutide structure (a) and fluorine labeling reaction (b).

Figure 2.

Labeled liraglutide analysis. a, chromatography UV profile indicates that protein is present in fractions 36–42. UV, absorbance at 280 nm; Cond, conductivity; Conc, concentration of NaCl from 0 to 1 m in 50 mm borate buffer, pH 7.5. b, identical 1D ¹H spectra for unlabeled and labeled liraglutide show evidence for no significant structural alteration of the protein due to labeling process (top red spectrum, labeled and ultra-filtrated liraglutide; bottom blue spectrum, unlabeled and dialyzed liraglutide). c, 1D ¹⁹F spectrum of purified labeled liraglutide presents a main peak at −74.333 ppm extending between −74.302 and −74.369 ppm and a degradation peak at −74.472 ppm corresponding to TFA.

Figure 3.

In vitro glycated albumin characterization. a, SDS-PAGE analysis of commercial (A1887)-HSA and G0-, G100-, and MGO-HSA. b, mass-to-charge ratio (m/z) versus the percentage intensity plot for A1887-, G0-, G100-, and MGO-HSA. c, fructosamine levels of G0-, G25- G100-, and MGO-HSA. All data are expressed as means ± S.D. (error bars) of three independent experiments. ***, unpaired t test compared with G0-HSA. G100-HSA, p < 0.0001; MGO-HSA, p = 0.007. ##, unpaired t test comparison between G100- and MGO-HSA, p = 0.003. d, AGE fluorescence measurement on G0-, G25-, G100-, and MGO-HSA. **, unpaired t test compared with G0-HSA: p < 0.01; ***, unpaired t test compared with G0-HSA: p < 0.001; ####, unpaired t test comparison: p < 0.0001.

Figure 4.

NMR experiments for in vitro glycated albumin interaction with liraglutide. a, [HSA]₀/ΔR_2obs is plotted as a function of liraglutide concentration. A linear regression is calculated, and K_d is obtained from the vertical intercept. G0-HSA, K_d = 35 ± 8 μm, n = 3, r² = 0.989 ± 0.009; G25-HSA, K_d = 40 ± 12 μm, n = 3, r² = 0.955 ± 0.037; G100-HSA, K_d = 240 ± 10 μm, n = 3, r² = 0.974 ± 0.028; MGO-HSA, K_d = 173 ± 7 μm, n = 3, r² = 0.976 ± 0.014. b, dissociation constant comparison for non-glycated and in vitro glycated commercial albumin. All data are expressed as means ± S.D. (error bars) of three independent experiments. ***, unpaired t test compared with G0-HSA; p < 0.0001.

Figure 5.

Plasma-extracted albumin analysis. a, SDS-PAGE analysis of albumin sample purified from plasma of patients divided into three groups (ND, D, and D+). b, HbA1c levels (%) of patients divided into three groups (ND, D, and D+). ***, unpaired t test compared with HSA from ND; p < 0.00010 for D and D+. ##, unpaired t test comparison between D and D+; p = 0.0041. c, fructosamine levels of purified albumin from healthy (ND) and diabetic (D and D+) patients. **, unpaired t test compared with HSA from ND; p = 0.0016 and 0.0062, respectively, for D and D+ groups. #, unpaired t test comparison between D and D+ groups; p = 0.0277. d, correlation between percentage HbA1c and albumin fructosamine levels. e, AGE fluorescence measurement on HSA from ND, D, and D+ groups (n = 3); unpaired t test compared with HSA from ND; p = 0.0621 and 0.1282, respectively, for D and D+ groups. f, correlation between percentage HbA1c and albumin-AGE levels. g, levels of FFA bound to purified albumin from healthy (ND) and diabetic (D and D+) patients. h, correlation between levels of FFA bound to purified albumin and albumin fructosamine levels. Error bars, S.D.

Figure 6.

NMR results for plasma-extracted albumin interaction with liraglutide. Shown are albumin-liraglutide K_d values for non-diabetics (ND), diabetics below 11% HbA1c (D), and diabetics above 11% HbA1c (D+). **, unpaired t test comparison between ND and D+ groups; p = 0.0017. #, unpaired t test comparison between D and D+ groups; p = 0.0057. Error bars, S.D.