Fam3a-mediated prohormone convertase switch in α-cells regulates pancreatic GLP-1 production in an Nr4a2-Foxa2-dependent manner.

ABSTRACT

BACKGROUND: Fam3a has been demonstrated to regulate pancreatic ß-cell function and glucose homeostasis. However, the role and mechanism of Fam3a in regulating α-cell function remains unexplored. METHODS: Glucagon and glucagon-like peptide-1 (GLP-1) levels in pancreas and plasma were measured in global Fam3a knockout (Fam3a-/-) mice. Human islet single-cell RNA sequencing (scRNA-seq) datasets were utilized to analyze gene expression correlations between FAM3A and PCSK1 (encoding PC1/3, which processes proglucagon into GLP-1). Mouse pancreatic α-cell line αTC1.9 cells were transfected with Fam3a siRNA or plasmid for Fam3a knockdown or overexpression to explore the effects of Fam3a on PC1/3 expression and GLP-1 production. The downstream mediator (including Nr4a2) was identified by transcriptomic analysis, and its role was confirmed by Fam3a knockdown or overexpression in αTC1.9 cells. Based on the interacted protein of Nr4a2 and the direct binding to Pcsk1 promoter, the transcription factor Foxa2 was selected for further verification. Nuclear translocation assay and dual-luciferase reporter assay were used to clarify the involvement of Fam3a-Nr4a2-Foxa2 pathway in PC1/3 expression and GLP-1 production. Moreover, α-cell-specific Fam3a knockout (Fam3aα-/-) mice were constructed to evaluate the metabolic variables and hormone levels under normoglycemic, high-fat diet (HFD)-fed and streptozotocin (STZ)-induced diabetic conditions. Exendin 9-39 (Ex9), a GLP-1 receptor antagonist, was used to investigate GLP-1 paracrine effects in Fam3aα-/- mice and in their primary islets. RESULTS: Compared with wild-type mice, pancreatic and plasma active GLP-1 levels were increased in Fam3a-/- mice. Analysis of human islet scRNA-seq datasets showed a significant negative correction between FAM3A and PCSK1 in α-cells. Fam3a knockdown upregulated PC1/3 expression and GLP-1 production in αTC1.9 cells, while Fam3a overexpression displayed inverse effects. Transcriptomic analysis identified Nr4a2 as a key downstream mediator of Fam3a, and Nr4a2 expression in αTC1.9 cells was downregulated and upregulated by Fam3a knockdown and overexpression, respectively. Nr4a2 silencing increased PC1/3 expression, albeit Nr4a2 did not directly bind to Pcsk1 promoter. Instead, Nr4a2 formed a complex with Foxa2 to facilitate Fam3a-mediated Foxa2 nuclear translocation. Foxa2 negatively regulated PC1/3 expression and GLP-1 production. Besides, Foxa2 inhibited the transcriptional activity of Pcsk1 promoter at specific binding sites 10 and 6, and this inhibition was intensified by Nr4a2 in αTC1.9 cells. Compared with Flox/cre littermates, improved glucose tolerance, increased active GLP-1 level in pancreas and plasma, upregulated plasma insulin level in response to glucose, and decreased plasma glucagon level were observed in Fam3aα-/- mice. Primary islets isolated from Fam3aα-/- mice also showed an increase in active GLP-1 and insulin release. In addition, the insulinotropic effect of intra-islet GLP-1 were blocked by Ex9 in Fam3aα-/- mice and in their primary islets. Similarly, HFD-fed Fam3aα-/- mice also exhibited an improved glucose tolerance. Both HFD-fed and STZ-induced diabetic Fam3aα-/- mice showed an increased pancreatic active GLP-1 level, an elevated plasma insulin level and a reduced plasma glucagon level. CONCLUSIONS: Fam3a deficiency in α-cells enhances pancreatic GLP-1 production to improve ß-cell function via paracrine signaling in an Nr4a2-Foxa2-PC1/3-dependent manner. Our study unveils a novel strategy for reprogramming α-cell proglucagon processing output from glucagon to GLP-1 and deepen the understanding of crosstalk between α-cells and ß-cells.