Metformin Enhances B Cell Function and Antibody Responses of Elderly Individuals With Type-2 Diabetes Mellitus

Abstract

Our previous work has shown that young and elderly patients with Type-2 Diabetes Mellitus (T2DM) treated with Metformin have optimal B cell function and serum antibodies specific for the seasonal influenza vaccine. In this paper, we have evaluated B cell function and the metabolic requirements of B cell antibody responses in elderly T2DM patients (E_T2DM) taking or not Metformin, and compared to those of healthy elderly (E_H) and healthy young (Y_H) individuals. Results show that Metformin significantly increases in vivo B cell function, measured by influenza vaccine-specific serum antibodies, in E_T2DM patients to the levels observed in E_H and more importantly in Y_H individuals. Metformin also decreases the frequencies of pro-inflammatory B cell subsets, as well as intrinsic inflammation and metabolic requirements of peripheral B cells from E_T2DM. This hyper-metabolic phenotype of B cells from E_T2DM is needed to support intrinsic inflammation, measured by the expression of transcripts for markers of the senescence-associated secretory phenotype (SASP), and the secretion of autoimmune antibodies. Importantly, B cell function in E_T2DM patients taking Metformin is not only increased as compared to that in E_T2DM patients not taking Metformin, but is comparable to B cell function measured in Y_H individuals. These results altogether strongly support the anti-aging effects of Metformin on humoral immunity.

Keywords: B cells; Type-2 Diabetes Mellitus; aging; autoimmunity; inflammation.

FIGURE 1

Metformin significantly enhances the serum H1N1-specific antibody response of E_T2DM patients to the levels observed in E_H and more importantly in Y_H individuals. Serum samples were collected from the 4 groups of individuals and evaluated by H1N1-specific HAI. Results show the reciprocal of the titers 4 weeks after vaccination. Mean comparisons between groups were performed by one-way ANOVA. *p < 0.05.

FIGURE 2

Metformin decreases the frequencies of pro-inflammatory B cell subsets in the blood of E_T2DM patients. (A) Gating strategies and a representative contour plot from one individual/group. In this representative plot, naïve (IgD + CD27−) are in the lower right quadrant, IgG memory (IgD + CD27+) are in the upper right quadrant, swIg (IgD-CD27+) are in the upper left quadrant and DN (IgD-CD27-) are in the lower left quadrant. (B) Frequencies of the 4 B cell subsets in the 4 groups of individuals. Mean comparisons between groups were performed by one-way ANOVA. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

FIGURE 3

Metformin decreases mRNA expression of SASP markers in ex vivo-isolated B cells from E_T2DM patients. B cells were resuspended in TRIzol, then the RNA was extracted and the expression of SASP markers detected by qPCR to evaluate expression of RNA for the pro-inflammatory cytokines TNF-α (A) and IL-6 (B), and for the cell cycle regulator p16^INK4 (C). qPCR values are measures of RNA expression of target genes, relative to the housekeeping gene GAPDH, calculated as 2^−ΔCts. Mean comparisons between groups were performed by one-way ANOVA. *p < 0.05, ***p < 0.001, ****p < 0.0001.

FIGURE 4

Metformin decreases glucose uptake in ex vivo-isolated B cells from E_T2DM patients. Glucose uptake was measured by flow cytometry and the glucose fluorescent analog 2-NBDG. (A) MFI (mean fluorescence intensity) of 2-NBDG staining in B cells from the 4 groups of individuals. (B) qPCR values of Glut1 mRNA expression, relative to GAPDH, calculated as 2^−ΔCts in the same B cells in (A). Mean comparisons between groups were performed by one-way ANOVA. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

FIGURE 5

Metformin decreases OCR and ECAR in ex vivo-isolated B cells from E_T2DM patients. B cells were seeded into the wells of an extracellular flux analyzer at the concentration of 2 × 10⁵/well in triplicate and run in a mitostress test. (A) OCR results (left) and maximal respiration results (right) are shown. Mean comparisons between groups were performed by one-way ANOVA. *p < 0.05, ***p < 0.001. (B) ECAR results.

FIGURE 6

Metformin decreases autoimmune antibody secretion in CpG-stimulated B cells from E_T2DM patients. (A) Results show OD values at 1:200 sample dilution. (B) Correlations of anti-dsDNA IgG (OD values) with 2-NBDG MFI. Pearson’s regression coefficients and p values are indicated at the bottom of the figure.