Clinical trials and animal studies have shown that sodium-glucose co-transporter type 2 (SGLT2) inhibitors improve pancreatic beta cell function. Our study aimed to investigate the effect of dapagliflozin on islet morphology and cell phenotype, and explore the origin and possible reason of the regenerated beta cells. Two diabetic mouse models, db/db mice and pancreatic alpha cell lineage-tracing (glucagon-β-gal) mice whose diabetes was induced by high fat diet combined with streptozotocin, were used. Mice were treated by daily intragastric administration of dapagliflozin (1 mg/kg) or vehicle for 6 weeks. The plasma insulin, glucagon and glucagon-like peptide-1 (GLP-1) were determined by using ELISA. The evaluation of islet morphology and cell phenotype was performed with immunofluorescence. Primary rodent islets and αTC1.9, a mouse alpha cell line, were incubated with dapagliflozin (0.25-25 μmol/L) or vehicle in the presence or absence of GLP-1 receptor antagonist for 24 h in regular or high glucose medium. The expression of specific markers and hormone levels were determined. Treatment with dapagliflozin significantly decreased blood glucose in the two diabetic models and upregulated plasma insulin and GLP-1 levels in db/db mice. The dapagliflozin treatment increased islet and beta cell numbers in the two diabetic mice. The beta cell proliferation as indicated by C-peptide and BrdU double-positive cells was boosted by dapagliflozin. The alpha to beta cell conversion, as evaluated by glucagon and insulin double-positive cells and confirmed by using alpha cell lineage-tracing, was facilitated by dapagliflozin. After the dapagliflozin treatment, some insulin-positive cells were located in the duct compartment or even co-localized with duct cell markers, suggestive of duct-derived beta cell neogenesis. In cultured primary rodent islets and αTC1.9 cells, dapagliflozin upregulated the expression of pancreatic endocrine progenitor and beta cell specific markers (including Pdx1) under high glucose condition. Moreover, dapagliflozin upregulated the expression of Pcsk1 (which encodes prohormone convertase 1/3, an important enzyme for processing proglucagon to GLP-1), and increased GLP-1 content and secretion in αTC1.9 cells. Importantly, the dapagliflozin-induced upregulation of Pdx1 expression was attenuated by GLP-1 receptor antagonist. Except for glucose-lowering effect, dapagliflozin has extra protective effects on beta cells in type 2 diabetes. Dapagliflozin enhances beta cell self-replication, induces alpha to beta cell conversion, and promotes duct-derived beta cell neogenesis. The promoting effects of dapagliflozin on beta cell regeneration may be partially mediated via GLP-1 secreted from alpha cells.

Glucagon-like peptide-1 (GLP-1) and its based agents improve glycemic control. Although their attenuating effect on hepatic glucose output has drawn our attention for decades, the potential mechanisms remain unclear. Cytokine array kit was used to assess cytokine profiles in db/db mice and mouse primary hepatocytes treated with exenatide (exendin-4). Two diabetic mouse models (db/db and Pax6m/+) were treated with a GLP-1 analog exenatide or liraglutide. The expression and secretion of fibroblast growth factor 21 (FGF21) in the livers of diabetic mice, primary mouse and human hepatocytes, and the human hepatic cell line HepG2 treated with or without GLP-1 analog were measured. Blockage of FGF21 with neutralizing antibody or siRNA, or hepatocytes isolated from Fgf21 knockout mice were used, and the expression and activity of key enzymes in gluconeogenesis were analyzed. Serum FGF21 level was evaluated in patients with type 2 diabetes (T2D) receiving exenatide treatment. Utilizing the cytokine array, we identified that FGF21 secretion was upregulated by exenatide (exendin-4). Similarly, FGF21 production in hepatocytes was stimulated by exenatide or liraglutide. FGF21 blockage attenuated the inhibitory effects of the GLP-1 analogs on hepatic glucose output. Similar results were also observed in primary hepatocytes from Fgf21 knockout mice. Furthermore, exenatide treatment increased serum FGF21 level in patients with T2D, particularly in those with better glucose control. We identify that function of GLP-1 in inhibiting hepatic glucose output is mediated via the liver hormone FGF21. Thus, we provide a new extra-pancreatic mechanism by which GLP-1 regulates glucose homeostasis. FUND: National Key Research and Development Program of China, the National Natural Science Foundation of China, the Natural Science Foundation of Beijing and Peking University Medicine Seed Fund for Interdisciplinary Research.

Under extreme conditions or by genetic modification, pancreatic α-cells can regenerate and be converted into β-cells. This regeneration holds substantial promise for cell replacement therapy in diabetic patients. The discovery of clinical therapeutic strategies to promote β-cell regeneration is crucial for translating these findings into clinical applications. In this study, we reported that treatment with REMD 2.59, a human glucagon receptor (GCGR) monoclonal antibody (mAb), lowered blood glucose without inducing hypoglycemia in normoglycemic, streptozotocin-induced type 1 diabetic (T1D) and non-obesity diabetic mice. Moreover, GCGR mAb treatment increased the plasma glucagon and active glucagon-like peptide-1 levels, induced pancreatic ductal ontogenic α-cell neogenesis, and promoted α-cell proliferation. Strikingly, the treatment also increased the β-cell mass in these two T1D models. Using α-cell lineage-tracing mice, we found that the neogenic β-cells were likely derived from α-cell conversion. Therefore, GCGR mAb-induced α- to β-cell conversion might represent a pre-clinical approach for improving diabetes therapy.

Aims/hypothesis: Glucagon receptor (GCGR) antagonism ameliorates hyperglycaemia and promotes beta cell regeneration in mouse models of type 2 diabetes. However, the underlying mechanisms remain unclear. The present study aimed to investigate the mechanism of beta cell regeneration induced by GCGR antagonism in mice. Methods: The db/db mice and high-fat diet (HFD)+streptozotocin (STZ)-induced mice with type 2 diabetes were treated with antagonistic GCGR monoclonal antibody (mAb), and the metabolic variables and islet cell quantification were evaluated. Plasma cytokine array and liver RNA sequencing data were used to screen possible mediators, including fibroblast growth factor 21 (FGF21). ELISA, quantitative RT-PCR and western blot were applied to verify FGF21 change. Blockage of FGF21 signalling by FGF21-neutralising antibody (nAb) was used to clarify whether FGF21 was involved in the effects of GCGR mAb on the expression of beta cell identity-related genes under plasma-conditional culture and hepatocyte co-culture conditions. FGF21 nAb-treated db/db mice, systemic Fgf21-knockout (Fgf21^−/−) diabetic mice and hepatocyte-specific Fgf21-knockout (Fgf21^Hep−/−) diabetic mice were used to reveal the involvement of FGF21 in beta cell regeneration. A BrdU tracing study was used to analyse beta cell proliferation in diabetic mice treated with GCGR mAb. Results: GCGR mAb treatment improved blood glucose control, and increased islet number (db/db 1.6±0.1 vs 0.8±0.1 per mm², p<0.001; HFD+STZ 1.2±0.1 vs 0.5±0.1 per mm², p<0.01) and area (db/db 2.5±0.2 vs 1.2±0.2%, p<0.001; HFD+STZ 1.0±0.1 vs 0.3±0.1%, p<0.01) in diabetic mice. The plasma cytokine array and liver RNA sequencing data showed that FGF21 levels in plasma and liver were upregulated by GCGR antagonism. The GCGR mAb induced upregulation of plasma FGF21 levels (db/db 661.5±40.0 vs 466.2±55.7 pg/ml, p<0.05; HFD+STZ 877.0±106.8 vs 445.5±54.0 pg/ml, p<0.05) and the liver levels of Fgf21 mRNA (db/db 3.2±0.5 vs 1.8±0.1, p<0.05; HFD+STZ 2.0±0.3 vs 1.0±0.2, p<0.05) and protein (db/db 2.0±0.2 vs 1.4±0.1, p<0.05; HFD+STZ 1.6±0.1 vs 1.0±0.1, p<0.01). Exposure to plasma or hepatocytes from the GCGR mAb-treated mice upregulated the mRNA levels of characteristic genes associated with beta cell identity in cultured mouse islets and a beta cell line, and blockage of FGF21 activity by an FGF21 nAb diminished this upregulation. Notably, the effects of increased beta cell number induced by GCGR mAb were attenuated in FGF21 nAb-treated db/db mice, Fgf21^−/− diabetic mice and Fgf21^Hep−/− diabetic mice. Moreover, GCGR mAb treatment enhanced beta cell proliferation in the two groups of diabetic mice, and this effect was weakened in Fgf21^−/− and Fgf21^Hep−/− mice. Conclusions/interpretation: Our findings demonstrate that liver-derived FGF21 is involved in the GCGR antagonism-induced beta cell regeneration in a mouse model of type 2 diabetes. Graphical

Insulin-producing cells (IPCs) derived from human embryonic stem cells (hESCs) hold great potential for cell transplantation therapy in diabetes. Tremendous progress has been made in inducing differentiation of hESCs into IPCs in vitro, of which definitive endoderm (DE) protocol mimicking foetal pancreatic development has been widely used. However, immaturity of the obtained IPCs limits their further applications in treating diabetes. Forkhead box O1 (FoxO1) is involved in the differentiation and functional maintenance of murine pancreatic β cells, but its role in human β cell differentiation is under elucidation. Here, we showed that although FoxO1 expression level remained consistent, cytoplasmic phosphorylated FoxO1 protein level increased during IPC differentiation of hESCs induced by DE protocol. Lentiviral silencing of FoxO1 in pancreatic progenitors upregulated the levels of pancreatic islet differentiation-related genes and improved glucose-stimulated insulin secretion response in their progeny IPCs, whereas overexpression of FoxO1 showed the opposite effects. Notably, treatment with the FoxO1 inhibitor AS1842856 displayed similar effects with FoxO1 knockdown in pancreatic progenitors. These effects were closely associated with the mutually exclusive nucleocytoplasmic shuttling of FoxO1 and Pdx1 in the AS1842856-treated pancreatic progenitors. Our data demonstrated a promising effect of FoxO1 inhibition by the small molecule on gene expression profile during the differentiation, and in turn, on determining IPC maturation via modulating subcellular location of FoxO1 and Pdx1. Therefore, we identify a novel role of FoxO1 inhibition in promoting IPC differentiation of hESCs, which may provide clues for induction of mature β cells from hESCs and clinical applications in regenerative medicine.

Objective: Glucagon receptor (GCGR) blockage improves glycemic control and increases circulating glucagon-like peptide-1 (GLP-1) level in diabetic animals and humans. The elevated GLP-1 has been reported to be involved in the hypoglycemic effect of GCGR blockage. However, the source of this elevation remains to be clarified. Research design and methods: REMD 2.59, a human GCGR monoclonal antibody (mAb), was administrated for 12 weeks in db/db mice and high-fat diet+streptozotocin (HFD/STZ)-induced type 2 diabetic (T2D) mice. Blood glucose, glucose tolerance and plasma GLP-1 were evaluated during the treatment. The gut length, epithelial area, and L-cell number and proliferation were detected after the mice were sacrificed. Cell proliferation and GLP-1 production were measured in mouse L-cell line GLUTag cells, and primary mouse and human enterocytes. Moreover, GLP-1 receptor (GLP-1R) antagonist or protein kinase A (PKA) inhibitor was used in GLUTag cells to determine the involved signaling pathways. Results: Treatment with the GCGR mAb lowered blood glucose level, improved glucose tolerance and elevated plasma GLP-1 level in both db/db and HFD/STZ-induced T2D mice. Besides, the treatment promoted L-cell proliferation and LK-cell expansion, and increased the gut length, epithelial area and L-cell number in these two T2D mice. Similarly, our in vitro study showed that the GCGR mAb promoted L-cell proliferation and increased GLP-1 production in GLUTag cells, and primary mouse and human enterocytes. Furthermore, either GLP-1R antagonist or PKA inhibitor diminished the effects of GCGR mAb on L-cell proliferation and GLP-1 production. Conclusions: The elevated circulating GLP-1 level by GCGR mAb is mainly due to intestinal L-cell proliferation and GLP-1 production, which may be mediated via GLP-1R/PKA signaling pathways. Therefore, GCGR mAb represents a promising strategy to improve glycemic control and restore the impaired GLP-1 production in T2D.