miR-132-3p is a positive regulator of alpha-cell mass and is downregulated in obese hyperglycemic mice

Abstract

Objective: Diabetes is a complex disease implicating several organs and cell types. Within the islets, dysregulation occurs in both alpha- and beta-cells, leading to defects of insulin secretion and increased glucagon secretion. Dysregulation of alpha-cells is associated with transcriptome changes. We hypothesized that microRNAs (miRNAs) which are negative regulators of mRNA stability and translation could be involved in alpha-cell alterations or adaptations during type 2 diabetes.

Methods: miRNA microarray analyses were performed on pure alpha- and beta-cells from high-fat diet fed obese hyperglycemic mice and low-fat diet fed controls. Then, the most regulated miRNA was overexpressed or inhibited in primary culture of mouse and human alpha-cells to determine its molecular and functional impact.

Results: 16 miRNAs were significantly regulated in alpha-cells of obese hyperglycemic mice and 28 in beta-cells. miR-132-3p had the strongest regulation level in alpha-cells, where it was downregulated, while we observed an opposite upregulation in beta-cells. In vitro experiments showed that miR-132-3p, which is inversely regulated by somatostatin and cAMP, is a positive modulator of alpha-cell proliferation and implicated in their resistance to apoptosis. These effects are associated with the regulation of a series of genes, including proliferation and stress markers Mki67 and Bbc3 in mouse and human alpha-cells, potentially involved in miR-132-3p functions.

Conclusions: Downregulation of miR-132-3p in alpha-cells of obese diabetic mice may constitute a compensatory mechanism contributing to keep glucagon-producing cell number constant in diabetes.

Keywords: Alpha-cell; Apoptosis; Glucagon; Proliferation; Type 2 diabetes; miR-132-3p.

Figure 1

miR-132-3p expression in alpha- and beta-cells in LFD and HFD mice. (A) Level of expression in the microarray analyses of miR-132-3p in FACS sorted alpha-cells from LFD mice (n = 5), HFD mice (n = 6), and FACS sorted beta-cells from LFD mice (n = 6) and HFD mice (n = 6). Moderated t-test, Benjamini-Hochberg method * Adj.P.Val. ≤ 0.05 vs LFD; $ Adj.P.Val. ≤ 0.05 vs Alpha-cells. (B) Expression level of miR-132-3p was measured by qPCR in FACS sorted alpha-cells from LFD mice (n = 9) and HFD mice (n = 7). Two-tailed unpaired Student's t test, *P ≤ 0.05 vs LFD.

Figure 2

Regulation of miR-132-3p in primary cultures of mouse alpha-cells. (A) Evaluation by qPCR of the expression of miR-132-3p in primary alpha-cells cultured for 48 h in 1% FBS supplemented DMEM with 0.5% BSA and 5.6 mM glucose (Control), 25 mM glucose and 500 μM palmitate, 25 mM glucose, 500 μM palmitate or 100 nM insulin (n = 4). (B) Evaluation by qPCR of the expression of miR-132-3p in primary alpha-cells cultured in 1% FBS supplemented DMEM with 0.5% BSA and 5.6 mM glucose with control or 10 μM IBMX + 10 μM Forskolin treatment during 48 h (n = 4). (C) Evaluation by qPCR of the expression of miR-132-3p in primary alpha-cells cultured in 1% FBS supplemented DMEM with 0.5% BSA and 5.6 mM glucose with control or 100 nM somatostatin treatment during 48 h (n = 4). One-tailed paired Student's t test,*P ≤ 0.05, **P ≤ 0.01 vs control.

Figure 3

Impact of miR-132-3p on glucagon secretion. (A) 5.6 mM glucose basal and 10 mM arginine stimulated glucagon secretion during 30 min from primary mouse alpha-cells 48 h after transfection with 100 nM of miR-132-3p mimic or inhibitor expressed in % of total glucagon cell content (n = 3) or in fold (stimulated/basal) (insert) (n = 3). (B) 5.6 mM glucose basal and 1 mM glucose stimulated glucagon secretion during 30 min by primary mouse alpha-cells 48 h after transfection with 100 nM of miR-132-3p mimic or inhibitor expressed in % of total glucagon cell content (n = 3) or in fold (stimulated/basal) (insert) (n = 3). Two-way ANOVA,*P ≤ 0.05, ***P ≤ 0.001 vs 5.6 mM glucose basal.

Figure 4

Impact of miR-132-3p on alpha-cell proliferation and apoptosis. (A–B) 48 h BrdU incorporation in primary mouse alpha-cells transfected with 100 nM control mimic or inhibitor (white) or miR-132-3p mimic (dark grey) (n = 8) (A) or inhibitor (light grey) (n = 5) (B). (C–D) TUNEL labeling in primary mouse alpha-cells transfected with 100 nM control mimic or inhibitor (white bars) or miR-132-3p mimic (dark grey) (n = 6) (C) or inhibitor (light grey) (n = 6) (D) and cultured in 1% FBS supplemented DMEM with 0.5% BSA and 5.6 mM glucose for 48 h. (E–F) Active Caspase-3 labeling in primary mouse alpha-cells transfected with 100 nM control mimic or inhibitor (white bars) or miR-132-3p mimic (dark grey) (n = 4) (E) or inhibitor (light grey) (n = 3) (F) and cultured in 1% FBS supplemented DMEM with 0.5% BSA and 5.6 mM glucose for 48 h. Two-tailed paired Student's t test,*P ≤ 0.05, **P ≤ 0.01 vs control.

Figure 5

Impact of miR-132-3p on alpha-cell gene expression. (A,B) Evaluation by qPCR of the expression of genes coding for proteins implicated in glucagon synthesis, secretion and alpha-cell identity in primary mouse alpha-cells transfected with 100 nM control mimic or inhibitor (white bars) or miR-132-3p mimic (dark grey) (A) or inhibitor (light grey) (B) and cultured for 48 h in 10% FBS supplemented DMEM with 5.6 mM glucose (n = 4–9). Multiple t-test with Benjamini, Krieger and Yekutieli method, * FDR ≤ 0.05 vs control. (C,D) Expression of genes coding for cell proliferation markers measured by qPCR in primary mouse alpha-cells transfected with 100 nM control mimic or inhibitor (white bars) or miR-132-3p mimic (dark grey) (C) or inhibitor (light grey) (D) and cultured for 48 h in 10% FBS supplemented DMEM with 5.6 mM glucose. Two-tailed paired Student's t test,*P ≤ 0.05 vs control. (E) Expression of genes coding for cell stress markers measured by qPCR in primary mouse alpha-cells transfected with 100 nM control inhibitor (white bars) or miR-132-3p inhibitor (light grey) and cultured in 1% FBS supplemented DMEM with 0.5% BSA and 5.6 mM glucose. Two-tailed paired Student's t test,*P ≤ 0.05 vs control. (F,G) Potential gene targets of miR-132-3p measured by qPCR in primary mouse alpha-cells transfected with 100 nM control mimic or inhibitor (white bars) or miR-132-3p mimic (dark grey) (F) or inhibitor (light grey) (G) and cultured for 48 h in 10% FBS supplemented DMEM with 5.6 mM glucose. Two-tailed paired Student's t test,*P ≤ 0.05 vs control. (H–I) BTG2 and MKI67 mRNA levels measured by qPCR in human primary alpha-cells transfected with 100 nM control mimic or inhibitor (white bars) or miR-132-3p mimic (dark grey) (H) or inhibitor (light grey) (I) and cultured in PIM-S medium supplemented with 10% human serum for 48 h. One-tailed paired Student's t test,*P ≤ 0.05 vs control. (J) Btg2, Mki67 Bbc3 and Ddit3 mRNA levels measured by qPCR in primary mouse alpha-cells in control condition or treated for 48 h with 100 nM somatostatin in 1% FBS supplemented DMEM with 0.5% BSA and 5.6 mM glucose. One-tailed paired Student's t test,*P.≤0.05 vs control.