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

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

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>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

A Novel Diphenylthiosemicarbazide Is a Potential Insulin Secretagogue for Anti-Diabetic Agent

<div><p>Insulin secretagogues are used for treatment of type 2 diabetes. We attempted to discover novel small molecules to stimulate insulin secretion by using in silico similarity search using sulfonylureas as query, followed by measurement of insulin secretion. Among 38 compounds selected by in silico similarity search, we found three diphenylsemicarbazides and one quinolone that stimulate insulin secretion. We focused on compound 8 (C8), which had the strongest insulin-secreting effect. Based on the structure-activity relationship of C8-derivatives, we identified diphenylthiosemicarbazide (DSC) 108 as the most potent secretagogue. DSC108 increased the intracellular Ca²⁺ level in MIN6-K8 cells. Competitive inhibition experiment and electrophysiological analysis revealed sulfonylurea receptor 1 (SUR1) to be the target of DSC108 and that this diphenylthiosemicarbazide directly inhibits ATP-sensitive K⁺ (K_ATP) channels. Pharmacokinetic analysis showed that DSC108 has a short half-life in vivo. Oral administration of DSC108 significantly suppressed the rises in blood glucose levels after glucose load in wild-type mice and improved glucose tolerance in the Goto-Kakizaki (GK) rat, a model of type 2 diabetes with impaired insulin secretion. Our data indicate that DSC108 is a novel insulin secretagogue, and is a lead compound for development of a new anti-diabetic agent.</p></div

Chemical structures of DSC108 and its sodium-salt form (DSC108-Na).

<p>Chemical structures of DSC108 and its sodium-salt form (DSC108-Na).</p

Effects of DSC108 and glibenclamide on the membrane potential of pancreatic β-cells.

<p>Membrane potential recording of isolated β-cells was performed by the patch-clamp method in the current clamp mode. (A) Representative membrane potential changes after treatment with 30 μM DSC108 or 1 μM glibenclamide. (B) Summarized data of membrane potential changes after treatment with 30 μM DSC108 (n = 7) or 1 μM glibenclamide (n = 4). Values are expressed as mean ± SEM. **P < 0.01 vs. pretreatment (Student paired <i>t</i> test).</p

Improvement of glucose tolerance in GK rats by DSC108-Na.

<p>(A) Insulinotropic effect of DSC108-Na in vivo. 30 mg/kg of DSC108-Na was orally administered to GK rats and plasma insulin concentrations were measured. Data are expressed as mean ± SEM (n = 6 for each group). *P < 0.05 (paired <i>t</i> test). (B) Changes in blood glucose levels after oral glucose load following administration of DSC108-Na in GK rats (left). Vehicle or 100 mg/kg of DSC108-Na was orally administered to rats at -20 min, and glucose was orally loaded at 0 min. AUC of glucose is represented in bar graphs (right). Data are expressed as mean ± SEM (n = 6 for each group). Arrow and arrowhead indicate the administration of compound and glucose, respectively. *P < 0.05 (paired <i>t</i> test).</p

Insulin secretory properties of DSC108.

<p>(A) Effect of C8 and DSC108 on insulin secretion from MIN6-K8 cells. Cells were stimulated by each concentration of compound in the presence of 11.2 mM glucose for 30 min. Data are fold-increase in insulin secretion relative to vehicle. Values are expressed as mean ± SEM (n = 3 for each compound). *P < 0.05, **P < 0.01 (Student unpaired <i>t</i> test). (B) Effect of DSC108 at 10 μM on the dynamics of insulin secretion from mouse perfused pancreata in the presence of 2.8 mM glucose. Values are expressed as mean ± SEM (n = 3).</p