Functional adenosine triphosphate‐sensitive potassium channel is required in high‐carbohydrate diet‐induced increase in β‐cell mass

Abstract Aims/Introduction A high‐carbohydrate diet is known to increase insulin secretion and induce obesity. However, whether or not a high‐carbohydrate diet affects β‐cell mass (BCM) has been little investigated. Materials and Methods Both wild‐type (WT) mice and adenosine triphosphate‐sensitive potassium channel‐deficient (Kir6.2KO) mice were fed normal chow or high‐starch (ST) diets for 22 weeks. BCM and the numbers of islets were analyzed by immunohistochemistry, and gene expression levels in islets were investigated by quantitative real‐time reverse transcription polymerase chain reaction. MIN6‐K8 β‐cells were stimulated in solution containing various concentrations of glucose combined with nifedipine and glimepiride, and gene expression was analyzed. Results Both WT and Kir6.2KO mice fed ST showed hyperinsulinemia and body weight gain. BCM, the number of islets and the expression levels of cyclinD2 messenger ribonucleic acid were increased in WT mice fed ST compared with those in WT mice fed normal chow. In contrast, no significant difference in BCM, the number of islets or the expression levels of cyclinD2 messenger ribonucleic acid were observed between Kir6.2KO mice fed normal chow and those fed ST. Incubation of MIN6‐K8 β‐cells in high‐glucose media or with glimepiride increased cyclinD2 expression, whereas nifedipine attenuated a high‐glucose‐induced increase in cyclinD2 expression. Conclusions These results show that a high‐starch diet increases BCM in an adenosine triphosphate‐sensitive potassium channel‐dependent manner, which is mediated through upregulation of cyclinD2 expression

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

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>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

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

Empagliflozin in Patients with Chronic Kidney Disease

Background The effects of empagliflozin in patients with chronic kidney disease who are at risk for disease progression are not well understood. The EMPA-KIDNEY trial was designed to assess the effects of treatment with empagliflozin in a broad range of such patients. Methods We enrolled patients with chronic kidney disease who had an estimated glomerular filtration rate (eGFR) of at least 20 but less than 45 ml per minute per 1.73 m(2) of body-surface area, or who had an eGFR of at least 45 but less than 90 ml per minute per 1.73 m(2) with a urinary albumin-to-creatinine ratio (with albumin measured in milligrams and creatinine measured in grams) of at least 200. Patients were randomly assigned to receive empagliflozin (10 mg once daily) or matching placebo. The primary outcome was a composite of progression of kidney disease (defined as end-stage kidney disease, a sustained decrease in eGFR to &lt; 10 ml per minute per 1.73 m(2), a sustained decrease in eGFR of &amp; GE;40% from baseline, or death from renal causes) or death from cardiovascular causes. Results A total of 6609 patients underwent randomization. During a median of 2.0 years of follow-up, progression of kidney disease or death from cardiovascular causes occurred in 432 of 3304 patients (13.1%) in the empagliflozin group and in 558 of 3305 patients (16.9%) in the placebo group (hazard ratio, 0.72; 95% confidence interval [CI], 0.64 to 0.82; P &lt; 0.001). Results were consistent among patients with or without diabetes and across subgroups defined according to eGFR ranges. The rate of hospitalization from any cause was lower in the empagliflozin group than in the placebo group (hazard ratio, 0.86; 95% CI, 0.78 to 0.95; P=0.003), but there were no significant between-group differences with respect to the composite outcome of hospitalization for heart failure or death from cardiovascular causes (which occurred in 4.0% in the empagliflozin group and 4.6% in the placebo group) or death from any cause (in 4.5% and 5.1%, respectively). The rates of serious adverse events were similar in the two groups. Conclusions Among a wide range of patients with chronic kidney disease who were at risk for disease progression, empagliflozin therapy led to a lower risk of progression of kidney disease or death from cardiovascular causes than placebo