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, 35 (20), 2694-2700

Metformin Improves in Vivo and in Vitro B Cell Function in Individuals With Obesity and Type-2 Diabetes

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Metformin Improves in Vivo and in Vitro B Cell Function in Individuals With Obesity and Type-2 Diabetes

Alain Diaz et al. Vaccine.

Abstract

Metformin (MET), the first-line medication for Type-2 Diabetes (T2D), has been shown to reduce chronic inflammation indirectly through reduction of hyperglycemia, or directly acting as anti-inflammatory drug. The effects of MET on B lymphocytes is uncharacterized. In the present study, we measured in vivo and in vitro influenza vaccine responses in 2 groups of T2D patients: recently diagnosed but not taking anti-diabetic drugs, and patients taking MET. Results show that B cell function and vaccine responses, hampered by obesity and T2D, are recovered by MET. Moreover, MET used in vitro to stimulate B cells from recently diagnosed T2D patients is also able to reduce B cell-intrinsic inflammation and increase antibody responses, similar to what we have seen in B cells from patients taking MET, who show increased responses to the influenza vaccine in vivo. These results are the first to show an effect of MET on B cells.

Keywords: B cells; Inflammation; Metformin.

Conflict of interest statement

Disclosures

Conflicts of interest: none.

Figures

Fig. 1
Fig. 1
The use of MET by T2D patients ameliorates their in vivo and in vitro antibody response to the influenza vaccine. (A) Participants (all obese T2D) were recruited during the 2011–2012, 2012–2013 and 2013–2014 influenza vaccine seasons. Results are expressed as reciprocal of the titer after vaccination at t7. Mean comparisons between groups were performed by Student’s t test (two-tailed). *p < 0.05. (B) PBMC isolated from the blood of the same participants in Fig. 1A before and after vaccination were stimulated with the influenza vaccine for 5 days. Results show fold-increase in AID mRNA expression [qPCR values (2−ΔΔCt) of AID mRNA normalized to GAPDH] after vaccination calculated as follows: qPCR values after vaccination/qPCR values before vaccination. *p < 0.05.
Fig. 2
Fig. 2
T2D patients on MET have increased switched memory (SM) B cells and less late/exhausted memory (LM) B cells. Participants (all obese T2D) are the same as in Fig. 1. One hundred μl of blood were stained to measure switched memory (SM, IgD−CD27+), IgM memory (IgD+CD27+), naïve (IgD+CD27−), late/exhausted memory (LM, IgD−CD27−) B cells. One hundred μl from the same individuals were also stained to measure transitional B cells (CD24brightCD38bright). Top. Representative dot plots, one for a MET-naïve patient and one for a MET-treated patient, are shown. Gates are set based on isotype controls. Bottom. Results are expressed as percentages of CD19 + B cells. *p < 0.05. **p < 0.01.
Fig. 3
Fig. 3
MET in vitro increases the expression of AID mRNA. Participants (all obese T2D) are the same as in Fig. 1. B cells from T2D patients taking MET and those not taking MET were stimulated with CpG for 5 days. The mRNA was extracted, RT reactions performed in the presence of random primers for AID and GAPDH (control) evaluation. Then qPCR were performed. B cells from patients not taking MET were also pre-incubated with MET, before CpG stimulation, for 60 min. Results show qPCR values (2−ΔΔCt) of AID (normalized to GAPDH) RNA expression. *p < 0.05. **p < 0.01.
Fig. 4
Fig. 4
The use of MET in vitro decreases the expression of TNF-α, miR-155 and miR-16 in unstimulated B cells from recently diagnosed T2D patients. Participants (all obese T2D) are the same as in Fig. 1. B cells from T2D patients taking MET and those not taking MET were resuspended in Trizol, RNA was extracted, RT reactions performed in the presence of random primers for TNF-α (A), BAFF (D) and GAPDH (control). B cells from patients not taking MET were also pre-incubated with MET, before Trizol, for 60 min. Results show qPCR values (2−ΔΔCt) of TNF-a and BAFF (normalized to GAPDH). For miRs evaluation, B cells were resuspended in Trizol, RNA was extracted, RT reactions performed in the presence of specific primers for miR-155 (B), miR-16 (C) or U6 (control) evaluation. Then qPCR were performed. B cells from patients not taking MET were also pre-incubated with MET, before Trizol, for 60 min. Results show qPCR values (2−ΔΔCt) of miR-155 and miR-16 (normalized to U6). **p < 0.01. ***p < 0.001. ****p < 0.0001.
Fig. 5
Fig. 5
MET in vitro increases the phosphorylation of AMPK and of its upstream activator p85-PI3K in B cells from recently diagnosed T2D patients. Participants (all obese T2D) are among those in Fig. 1. B cells from T2D patients taking MET and those not taking MET were stimulated with CpG for 30 min. B cells from the patient not taking MET were also pre-incubated with MET, before the stimulation with CpG, for 60 min. Cytoplasmic protein extracts were prepared and analyzed in Western blot. Total UBC9 (or total AMPK) was used as loading control. Results are representative of 4 independent experiments done on individuals with sufficient numbers of B cells to perform protein extraction and Western blot experiments. (A) A representative Western blot is shown. Densitometric analyses (arbitrary units) of p-p85-PI3K (normalized to UBC9) (B) and p-AMPK (normalized to total AMPK) (C) expression are shown and referred to the value in patients taking MET in vivo (taken as 1). ****p< 0.0001.

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