Pancreatic beta cell mass and alpha cell mass.

<p>(<b>a</b>) Insulin/glucagon (upper), insulin/Pdx1 (middle), and insulin/FoxO1 (bottom) double immunostaining. Representative images of islets are shown. Scale bars, 100 μm. (<b>b</b>) Beta cell mass. Alloxan treatment markedly decreased beta cell mass. The beta cell mass was two-fold higher in liraglutide-treated mice than that in vehicle-treated mice on day 30. (<b>c</b>) Alpha cell mass. Alpha cell mass did not change by alloxan treatment. Liraglutide did not affect alpha cell mass. White bars represent vehicle-treated group (Veh) (n = 4–9), and black bars represent liraglutide-treated group (Lir) (n = 4–9). *<i>p</i> < 0.05</p

Evaluation of beta cell neogenesis.

<p>Double immunostaining for insulin (green) and YFP (red) indicates that pancreatic beta cells were specifically labeled by YFP. Scale bars, 100 μm. Quantification of YFP-labeled beta cells shows that the labeling index did not differ before and after liraglutide treatment. Veh, vehicle-treated group; Lir, liraglutide-treated group. Data are means ± SEM of five mice in each group. NS, difference not significant.</p

Oral glucose tolerance test (OGTT).

<p>Blood glucose and serum insulin levels during 1.5 g/kg OGTT on day 15 (<b>a</b>) and day 30 (<b>b</b>) are shown. Glucose tolerance was significantly improved by liraglutide treatment. Although insulin response was not detected in vehicle treated group, liraglutide improved the response. Insulinogenic index was significantly higher in liraglutide-group than vehicle-group on day 30. White circles and bars represent vehicle-treated group (Veh) (n = 5–6), and black circles and bars represent liraglutide-treated group (Lir) (n = 5–6). *<i>p</i> < 0.05</p

Oxidative stress assessed by 4-HNE staining.

<p>Photographs are representative images of islets stained for insulin (green) and 4-HNE (red). Scale bars, 100 μm. Quantification of 4-HNE-positive cells in islet cells is shown below the photographs. Alloxan treatment induced oxidative stress in pancreatic islets. Liraglutide treatment significantly decreased 4-HNE-positive cells in islet cells. Veh, vehicle-treated group; Lir, liraglutide-treated group. Data are means ± SEM of 4–5 mice in each group. *<i>p</i> < 0.05, **<i>p</i> < 0.01</p

Proliferation and apoptosis of pancreatic beta cells.

<p>(<b>a</b>) Quantification of proliferating beta cells. Photographs are representative images of islets stained for insulin (green) and Ki67 (red). White arrows indicate Ki67- and insulin double-positive cells. Scale bars, 100 μm. The number of Ki67-positive beta cells was significantly increased in liraglutide-group compared to vehicle-group on day 30. (<b>b</b>) Quantification of apoptotic beta cells. Photographs are representative images of islets with insulin and TUNEL staining. White arrows indicate TUNEL- and insulin double-positive cells. Scale bars, 100 μm. The number of TUNEL-positive beta cells was significantly decreased in liraglutide-group compared to vehicle-group. White bars represent vehicle-treated group (Veh) (n = 4–6), and black bars represent liraglutide-treated group (Lir) (n = 4–6). NS, difference not significant. *<i>p</i> < 0.05</p

Metabolic parameters in alloxan-diabetic mice treated with vehicle or liraglutide for 30 days.

<p>(<b>a</b>) Schematic representation of the study. (<b>b</b>) Body weight. There is no difference between two groups. (<b>c</b>) Food intake. Liraglutide slightly decreased food intake compared with vehicle. (<b>d</b>) Blood glucose. Blood glucose levels were significantly lower in liraglutide-treated group than in vehicle-treated group after day 13. (<b>e</b>) Serum insulin. Serum insulin levels were significantly higher in liraglutide-treated group than in vehicle-treated group on day 30. White circles and bars represent vehicle-treated group (Veh) (n = 9–13), and black circles and bars represent liraglutide-treated group (Lir) (n = 10–15). *<i>p</i> < 0.05, **<i>p</i> < 0.01</p

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>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

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

Decreased Warfarin Clearance with the CYP2C9 R150H (*8) Polymorphism

The cytochrome P450 (CYP) 2C9 R150H (*8) allele occurs commonly in African Americans and is associated with lower warfarin dose requirements. We examined whether the CYP2C9*8 allele impacts warfarin clearance through a pharmacokinetic study in warfarin-treated African American patients and an in vitro kinetic study of S-warfarin 7-hydroxylation using cDNA-expressed CYP2C9 enzymes. We observed a 30% reduction in the unbound oral clearance of S-warfarin and 25% lower R- to S-warfarin plasma concentration in patients with the CYP2C9*8 allele (n=12) compared to CYP2C9*1 homozygotes (n=26). Consistent with these findings, the in-vitro intrinsic clearance of S-warfarin was 30% lower with the cDNA-expressed R150H protein compared to the wild-type protein. These data show that the R150H variant of the CYP2C9*8 allele reduces S-warfarin clearance, thus providing clinical and experimental evidence to explain lower warfarin dose requirements with the CYP2C9*8 allele