Imeglimin, unlike metformin, does not perturb differentiation of human induced pluripotent stem cells towards pancreatic β-like cells and rather enhances gain in β cell identity gene sets

Abstract

Aims/introduction: Metformin treatment for hyperglycemia in pregnancy (HIP) beneficially improves maternal glucose metabolism and reduces perinatal complications. However, metformin could impede pancreatic β cell development via impaired mitochondrial function. A new anti-diabetes drug imeglimin, developed based on metformin, improves mitochondrial function. Here we examine the effect of imeglimin on β cell differentiation using human induced pluripotent stem cell (iPSC)-derived pancreatic islet-like spheroid (SC-islet) models.

Materials and methods: Human iPSCs are differentiated into SC-islets by three-dimensional culture with and without imeglimin or metformin. Differentiation efficiencies of SC-islets were analyzed by flow cytometry, immunostaining, quantitative PCR, and insulin secretion assay. RNA sequencing and oxygen consumption rate were obtained for further characterization of SC-islets. SC-islets were cultured with proinflammatory cytokines, in part mimicking the uterus environment in HIP.

Results: Metformin perturbed SC-islet differentiation while imeglimin did not alter it. Furthermore, imeglimin enhanced the gene expressions of β cell lineage markers. Maintenance of mitochondrial function and optimization of TGF-β and Wnt signaling were considered potential mechanisms for augmented β cell maturation by imeglimin. In the presence of proinflammatory cytokines, imeglimin ameliorated β cell differentiation impaired by cytokines and metformin.

Conclusions: Imeglimin does not perturb differentiation of SC-islet cells and rather enhances gain in β cell identity gene sets in contrast to metformin. This may lead to the improvement of in vitro β cell differentiation protocols.

Keywords: Imeglimin; Metformin; β cell differentiation.

Figure 1

Metformin perturbs SC‐islet differentiation while imeglimin does not alter it. (a) Schematic presentation of the β‐cell differentiation protocol. Imeglimin or metformin was added in the last differentiation stage. (b–d) Immunohistochemical images (b and c) and bright‐field images (d) of SC‐islets treated with imeglimin (Imeg), or metformin (Met), and untreated (Vehicle). Scale bar, 100 μm. (e) The diameters of SC‐islets. n = 62 for Imeg, n = 105 for Met, n = 51 for Vehicle in 3 fields of view. *P < 0.05, **P < 0.01. (f) TBP‐adjusted INS gene expression levels of SC‐islets at the end of differentiation. n = 6 for Imeg, n = 4 for Met, n = 4 for Vehicle. *P < 0.05, **P < 0.01. (g) Quantitative analysis of C‐peptide‐positive β‐like cells in SC‐islets by flow cytometry. n = 4 for Imeg, n = 4 for Met, n = 5 for Vehicle. *P < 0.05, **P < 0.01. (h, i) Insulin secretion in response to 2.8 mm glucose (Low), 25 mm glucose (High), 50 nm exendin‐4 with 25 mm glucose (Ex4) (h), and 40 mm KCl with 25 mm glucose (KCl) (i), adjusted by insulin content of SC‐islets. n = 5 for each group. ^# P < 0.05 compared to low glucose conditions in the same treatment. *P < 0.05, **P < 0.01 compared to the same conditions in Vehicle. (j) Insulin content in 50 spheroids. n = 6 for each group.

Figure 2

Imeglimin differentially changes gene expression patterns of SC‐islets towards maturation. (a) Principal component analysis (PCA) of SC‐islets treated with imeglimin (Imeg) or metformin (Met) and untreated (Vehicle). (b–d) Heatmaps of a subset of mature β‐cell markers (b), immature β‐cell markers (c), and non‐β endocrine cell markers (d) in SC‐islets. (e) KEGG analysis of downregulated (P < 0.05, log₂FC < −0.5), and upregulated (P < 0.05, log₂FC >0.5) in SC‐islets treated with imeglimin compared to those untreated. (f, g) Heatmaps of a subset of cell cycle‐related genes (f), and lipid metabolism‐related genes (g) in SC‐islets.

Figure 3

Imeglimin maintains mitochondrial function, and optimizes TGF‐β/Wnt signaling. (a) Heatmap of a subset of mitochondria‐related genes in SC‐islets treated with imeglimin or metformin and untreated. (b) Seahorse assay measuring mitochondrial respiration in SC‐islets treated with imeglimin (1 mm or 0.1 mm) or metformin (1 mm or 0.1 mm) and untreated. n = 4 for imeglimin and untreated groups, n = 1 for metformin groups (not detected in the other metformin‐treated spheroids). Mean values are shown. Max OCR is shown as a bar graph. (c) A heatmap of a subset of ER stress‐related genes in SC‐islets. (d–g) Heatmaps of a subset of cell signaling‐related genes: NOTCH signal (d), Wnt signal (e), and TGF‐β signal (f). The level of specific gene expressions represented by TPM (g). *P < 0.05, **P < 0.01.

Figure 4

Imeglimin ameliorated β cell differentiation impaired by proinflammatory cytokines and metformin. (a) Schematic presentation of β cell differentiation protocol. Imeglimin or metformin with proinflammatory cytokines were added in the last differentiation stage. (b, c) Immunohistochemical images (b) and bright‐field images (c) of SC‐islets treated with imeglimin (cImeg), metformin (cMet) or not (Cytokine) in the presence of proinflammatory cytokines. Scale bar, 100 μm. (d) The diameters of SC‐islets. n = 87 for cImeg, n = 62 for cMet, n = 88 for Cytokine in 3 fields of view. *P < 0.05, **P < 0.01. (e) TBP‐adjusted INS gene expression levels of SC‐islets treated with imeglimin (cImeg), metformin (cMet), or not in the presence of inflammatory cytokines (Cytokine), along with untreated (Vehicle). n = 5 for cImeg, n = 5 for cMet, n = 6 for Cytokine, n = 4 for Vehicle. *P < 0.05, **P < 0.01. (f) Quantitative analysis of C‐peptide‐positive β‐like cells in SC‐islets by flow cytometry. n = 4 for cImeg, n = 4 for cMet, n = 4 for Cytokine, n = 5 for Vehicle. *P < 0.05, **P < 0.01. (g) C‐peptide secretion in response to 2.8 mm glucose (Low), 25 mm glucose (High), and 40 mm KCl with 25 mm glucose (KCl) of SC‐islets. n = 5 for each group. *P < 0.05, **P < 0.01. For reference, the data for untreated SC‐islets (Vehicle) presented in Figure 1 are given on the left side separated by dotted lines.

Figure 5

Imeglimin preserves iPSC‐derived β‐like cell differentiation impaired by proinflammatory cytokines. (a) PCA of SC‐islets treated with imeglimin (cImeg), metformin (cMeet) or not in presence of inflammatory cytokines (Cytokine), along with untreated (Vehicle). (b–d) Heatmaps of a subset of mature β cell markers (b), immature β cell markers (c), and non‐β endocrine cell markers (d) in SC‐islets. (e) KEGG analysis of downregulated genes (P < 0.05, log₂FC < −0.5) in SC‐islets in Met compared to Cytokine. (f) A heatmap of a subset of ATP dependent K⁺ channel and voltage‐dependent Ca²⁺ channel in SC‐islets. (g, h) Venn's diagrams show common genes between upregulated in cImeg and downregulated in Cytokine (g) and those between upregulated in Cytokine and downregulated in cImeg (h).