Essential roles of aspartate aminotransferase 1 and vesicular glutamate transporters in β-cell glutamate signaling for incretin-induced insulin secretion

Abstract

Incretins (GLP-1 and GIP) potentiate insulin secretion through cAMP signaling in pancreatic β-cells in a glucose-dependent manner. We recently proposed a mechanistic model of incretin-induced insulin secretion (IIIS) that requires two critical processes: 1) generation of cytosolic glutamate through the malate-aspartate (MA) shuttle in glucose metabolism and 2) glutamate transport into insulin granules by cAMP signaling to promote insulin granule exocytosis. To directly prove the model, we have established and characterized CRISPR/Cas9-engineered clonal mouse β-cell lines deficient for the genes critical in these two processes: aspartate aminotransferase 1 (AST1, gene symbol Got1), a key enzyme in the MA shuttle, which generates cytosolic glutamate, and the vesicular glutamate transporters (VGLUT1, VGLUT2, and VGLUT3, gene symbol Slc17a7, Slc17a6, and Slc17a8, respectively), which participate in glutamate transport into secretory vesicles. Got1 knockout (KO) β-cell lines were defective in cytosolic glutamate production from glucose and showed impaired IIIS. Unexpectedly, different from the previous finding that global Slc17a7 KO mice exhibited impaired IIIS from pancreatic islets, β-cell specific Slc17a7 KO mice showed no significant impairment in IIIS, as assessed by pancreas perfusion experiment. Single Slc17a7 KO β-cell lines also retained IIIS, probably due to compensatory upregulation of Slc17a6. Interestingly, triple KO of Slc17a7, Slc17a6, and Slc17a8 diminished IIIS, which was rescued by exogenously introduced wild-type Slc17a7 or Slc17a6 genes. The present study provides direct evidence for the essential roles of AST1 and VGLUTs in β-cell glutamate signaling for IIIS and also shows the usefulness of the CRISPR/Cas9 system for studying β-cells by simultaneous disruption of multiple genes.

Inhibition of SNAT5 Induces Incretin-Responsive State From Incretin-Unresponsive State in Pancreatic β-Cells: Study of β-Cell Spheroid Clusters as a Model

“…Cell culture was performed as described previously (21) using DMEM (25 mmol/L glucose and 4 mmol/L glutamine; Sigma-Aldrich). Functional experiments were performed on these cells at 80% of confluency.…”

“…Immunostaining of b-cell lines was performed as described previously (21,22) using anti-SNAT5 antibody (Abcam, Cambridge, U.K.), anti-insulin antibody (DAKO, Agilent, Santa Clara, CA), and anti-Ki67 antibody (DAKO). Immunostaining of mouse pancreata was performed by the polymerbased detection method using anti-SNAT5 antibody (Santa Cruz Biotechnology, Dallas, TX) and N-Histofine Simple Stain Mouse MAX PO (Nichirei Biosciences, Tokyo, Japan) according to the manufacturers' instructions.…”

“…Western blotting was performed as described previously (21) using anti-SNAT5 antibody (Abcam) and anti-actin antibody (formerly Calbiochem, Merck KGaA, Darmstadt, Germany).…”

“…The latter glutamate isotopomers are produced through the MA shuttle (20). To clarify the role of the MA shuttle in IIIS and cAMP production directly, we used aspartate aminotransferase 1 (AST1, gene symbol Got1), a critical enzyme in the shuttle, knockout (KO) clonal b-cells, which we recently established (21). GLP-1-and 8-Br-cAMP-AM-induced insulin secretions were significantly reduced in Got1-KO-SC compared with those in K8-SC ( Fig.…”

Glucagon-like Peptide-1 (GLP-1)

“…Another recent report suggests that incretin-stimulation of insulin secretion requires incorporation of glutamate in the insulin granules. In murine pancreatic Min6-K8 cells, prevention of cytosolic glutamate production by KO of a key component of the malate-aspartate shuttle or triple KO of the vesicular glutamate transporters Slc17a6, Slc17a7, and Slc17a8 impaired GLP-1-induced insulin secretion [515]. GLP-1 also lowers blood glucose in patients with type 1 diabetes, e.g.…”