Linagliptin treatment is associated with altered cobalamin (VitB12) homeostasis in mice and humans

Abstract

Linagliptin is a dipeptidyl peptidase-4 (DPP-4) inhibitor used for the treatment of type 2 diabetes, with additional beneficial effects for the kidney. Treatment of mice with linagliptin revealed increased storage of cobalamin (Cbl, Vitamin B12) in organs if a standard Cbl diet (30 µg Cbl/kg chow) is given. In order to translate these findings to humans, we determined methylmalonic acid (MMA), a surrogate marker of functional Cbl homeostasis, in human plasma and urine samples (n = 1092) from baseline and end of trial (6 months after baseline) of the previously completed MARLINA-T2D clinical trial. We found that individuals with medium Cbl levels (MMA between 50 and 270 nmol/L for plasma, 0.4 and 3.5 µmol/mmol creatinine for urine, at baseline and end of trial) exhibited higher MMA values at the end of study in placebo compared with linagliptin. Linagliptin might inhibit the N-terminal degradation of the transcobalamin receptor CD320, which is necessary for uptake of Cbl into endothelial cells. Because we demonstrate that linagliptin led to increased organ levels of Cbl in mice, sustained constant medium MMA levels in humans, and inhibited CD320 processing by DPP-4 in-vitro, we speculate that linagliptin promotes intra-cellular uptake of Cbl by prolonging half-life of CD320.

Figure 1

MS detection of CN and OH cobalamin in different organs from 4 independent mice studies. Box-and-whisker of log OHCbl and CNCbl standardized intensities obtained from experiments listed in Table 1. The inset shows the basic experimental parameters. The number of data points are for Exp#1:88; Exp#2: 12; Exp#3: 36; Exp#4: 96. **Significant differences between linagliptin treatment and placebo. CNCbl, cyanocobalamin; OHCbl, Hydroxycobalamin, lg, natural logarithm; HFD, high fat diet; N/X, 5/6 nephrectomy.

Figure 2

Distribution of MMA values in plasma and urine at V3 and V7 in individuals with medium MMA levels in dependence of treatment. MMA values for plasma (nmol/L, left, A and B) and urine samples (µmol/mmol creatine, right, C and D) for placebo (green) and linagliptin (blue) at V3 (top, A and C), and V7 (bottom, B and D). The number of samples in each group is shown at the top right. Statistically significant differences between placebo and linagliptin are indicated (Welch t-test). Data is shown as a box-and-whisker plot and the corresponding kernel density estimation. The data shows a shift of MMA values at V7 to the right (towards higher MMA values) in the Placebo group. The shift is also depicted in median and mean values shown in Tables 5 and 6, respectively. MMA, methylmalonic acid; n.s., not significant; V3, baseline; V7, end of study.

Figure 3

N-terminal processing of CD320. Substrate-product ratios of tryptic peptides derived from the N-terminal part of CD320 (CD320_36-58: SPLSTPTSAQAAGPSSGSCPPTK and CD320_38-58: LSTPTSAQAAGPSSGSCPPTK). The conversion rate in each group is given as mean + /−  SD and individual data points. Left: The column bars show a time-dependent increase in conversion rate due to DPP-4 incubation time. Right: The ratio between both tryptic peptides is depicted as increase in conversion rate due to DPP-4 and inhibition of increase by linagliptin (***p < 0.0005). DPP-4, dipeptidyl peptidase-4; sCD320, soluble form of CD320.