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

<div><p>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 <i>Got1</i>), a key enzyme in the MA shuttle, which generates cytosolic glutamate, and the vesicular glutamate transporters (VGLUT1, VGLUT2, and VGLUT3, gene symbol <i>Slc17a7</i>, <i>Slc17a6</i>, and <i>Slc17a8</i>, respectively), which participate in glutamate transport into secretory vesicles. <i>Got1</i> 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 <i>Slc17a7</i> KO mice exhibited impaired IIIS from pancreatic islets, β-cell specific <i>Slc17a7</i> KO mice showed no significant impairment in IIIS, as assessed by pancreas perfusion experiment. Single <i>Slc17a7</i> KO β-cell lines also retained IIIS, probably due to compensatory upregulation of <i>Slc17a6</i>. Interestingly, triple KO of <i>Slc17a7</i>, <i>Slc17a6</i>, and <i>Slc17a8</i> diminished IIIS, which was rescued by exogenously introduced wild-type <i>Slc17a7</i> or <i>Slc17a6</i> 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.</p></div

Characterization of <i>Slc17a7</i>, <i>Slc17a6</i>, and <i>Slc17a8</i> triple KO β-cell lines.

<p>(A) Insulin secretory response in <i>Slc17a7</i>, <i>Slc17a6</i>, and <i>Slc17a8</i> triple KO (TKO) cell lines. WT and TKO cell lines were stimulated with glucose and incretin (GLP-1 or GIP) (n = 4). Insulin secretion was normalized by cellular insulin content and the data are presented as fold-change relative to the amount of insulin secretion at 16.7 mM glucose. (B, C) Rescue of the VGLUT1 (B) or VGLUT2 (C) activity by introducing WT <i>Slc17a7</i> or <i>Slc17a6</i> into triple KO cell line, respectively. The cell line V39 was transfected with <i>INS1</i> (control) or <i>INS1</i> and rescue construct and stimulated with glucose and GLP-1 (n = 4). C-peptide secretion was normalized by cellular C-peptide content and the data are presented as fold-change relative to the amount of C-peptide secretion at 16.7 mM glucose. (D) The effect of dimethyl glutamate (dmGlu) on insulin secretion. The cell line V39 was stimulated with glucose and dmGlu (n = 4). Insulin secretion was normalized by cellular insulin content. The data are expressed as means ± SEM. Representative results are shown. Similar results were found in 3 independent experiments. Dunnett's method was used for evaluation of statistical significance vs. WT in (A) and vs. 16.7 mM glucose in (D). Welch’s t-test was used for evaluation of statistical significance vs. control in (B) and (C). **p < 0.01; ***p < 0.001.</p

Establishment and characterization of <i>Slc17a7</i> single KO β-cell lines.

<p>(A) Mutations in <i>Slc17a7</i> exon 2 in <i>Slc17a7</i> single KO cell lines induced by the CRISPR/Cas9 nickase system. WT sequence is shown with target sites of sgRNAs. Protospacer adjacent motif (PAM) and mutations are shown in red. Allele 2 of both KO cell lines were not detected by PCR probably due to large deletions. (B) mRNA expression levels of <i>Slc17a6</i> in <i>Slc17a7</i> KO cell lines. The mRNA expression levels of KO cell lines are presented as fold increase relative to those of WT (n = 4). (C) Insulin secretory response in <i>Slc17a7</i> single KO cell lines. Cells were stimulated with glucose and GLP-1 (n = 4). Insulin secretion was normalized by cellular insulin content and the data are presented as fold-change relative to the amount of insulin secretion at 16.7 mM glucose. The data are expressed as means ± SEM. Representative results are shown (B and C). Similar results were found in 3 independent experiments. Welch’s t-test with Bonferroni correction was used for evaluation of statistical significance vs. WT in (B). Dunnett's method was used for evaluation of statistical significance vs. WT in (C). **p < 0.01; ***p < 0.001.</p

Immunofluorescence staining of <i>Slc17a7</i> single KO and <i>Slc17a7</i>, <i>Slc17a6</i>, and <i>Slc17a8</i> triple KO β-cell lines.

<p>Green, Insulin; red, VGLUT1 (A) or VGLUT2 (B); blue, 4',6-diamidino-2-phenylindole (DAPI). Scale bars, 50 μm.</p

Establishment and characterization of <i>Got1</i> KO β-cell lines.

<p>(A) Mutations in <i>Got1</i> exon 1 in two <i>Got1</i> KO cell lines induced by the CRISPR/Cas9 nickase system. WT sequence is shown with target sites of sgRNAs. Protospacer adjacent motif (PAM) and mutations are shown in red. (B) Absence of AST1 protein in <i>Got1</i> KO cell lines revealed by western blotting. (C) Cytosolic glutamate content in <i>Got1</i> KO cell line. WT MIN6-K8 or <i>Got1</i> KO (A64) cell lines were stimulated with [U-¹³C]-glucose and ¹³C-enriched glutamate isotopomers M+2 to M+5 (two to five substitutions of ¹²C by ¹³C) were quantified by mass spectrometry (n = 3). (D) Insulin secretory response in <i>Got1</i> KO cell lines. The cell lines were stimulated with glucose and incretin (GLP-1 or GIP) (n = 4). Insulin secretion was normalized by cellular insulin content and presented as fold-change relative to the amount of insulin secretion at 16.7 mM glucose. (E) Rescue of the AST1 activity by introducing WT <i>Got1</i> into <i>Got1</i> KO cell line. The <i>Got1</i> KO (A60) cell line was transfected with <i>INS1</i> along with <i>Got1</i> or empty construct and stimulated with glucose and GLP-1 (n = 4). C-peptide secretion was normalized by cellular C-peptide content and the data are presented as fold-change relative to the amount of C-peptide secretion at 16.7 mM glucose. (F) The effect of dimethyl glutamate (dmGlu) on insulin secretion. The <i>Got1</i> KO (A60) cell line was stimulated with glucose and dmGlu (n = 4). Insulin secretion was normalized by cellular insulin content. The data are expressed as means ± SEM. Representative results are shown (C, D, E, and F). Similar results were found in 3 independent experiments. Welch’s t-test was used for evaluation of statistical significance vs. 2.8 mM glucose in (C) and vs. control in (E). Dunnett's method was used for evaluation of statistical significance vs. WT in (D) and vs. 16.7 mM glucose in (F). *p < 0.05; ***p < 0.001; n.s., not significant.</p