PMC

β-Cell–specific expression of mKL abolished glucose intolerance and increased plasma insulin levels in NOD mice. GTT was performed at 7 days (A) and 14 days (B) after gene delivery. Area under the curve for GTT results (C), ^^P < 0.01. Plasma insulin levels (D). Data are means ± SEM. n = 5 animals/group. *P < 0.05, **P < 0.01, and ***P < 0.001 vs. the ICR/HaJ-PBS group; ^P < 0.05 and ^^^P < 0.001 vs. the NOD-PBS group; ⁺⁺P < 0.01 and ⁺⁺⁺P < 0.001 vs. the NOD-AAV-GFP group.

β-Cell–specific expression of mKL increased insulin storage and attenuated T-cell infiltration and apoptosis in pancreatic islets of NOD mice. A: Representative photomicrograph of Klotho staining (brown color) in cross sections of pancreatic islets. B: Semiquantification of Klotho staining in islets. C: Representative photomicrograph of insulin staining (brown color) in islets. D: Semiquantification of insulin staining in islets. E: Representative images of CD3 staining (brown color, red arrow) in islets. F: The percentage of cells with positive CD3 staining in islets. G: Representative images of apoptotic cells (TUNEL staining, immunofluorescence red color) in islets. H: The percentage of TUNEL-positive cells in islets. Data are mean ± SEM. n = 5 animals/group. *P < 0.05, **P < 0.01, and ***P < 0.001 vs. the ICR/HaJ-PBS group; ^P < 0.05 and ^^P < 0.01 vs. the NOD-PBS group; ⁺P < 0.05 and ⁺⁺P < 0.01 vs. the NOD–AAV-GFP group.

Immunohistochemical analysis of Klotho and insulin expression and apoptosis in pancreatic islets of mice treated with STZ. KL^+/− and WT male mice were injected with STZ or citrate buffer. Animals were killed 5 weeks after the initial injections. A: Representative images of Klotho staining (brown color) in cross sections of mouse pancreatic islets. B: Semiquantification of Klotho staining in pancreatic islets. C: Representative images of insulin staining (brown color) of cross sections of islets. D: Semiquantification of insulin staining in pancreatic islets. E: The percentage of cells with positive insulin staining in pancreatic islets. F: Representative images of TUNEL staining (blue color) in pancreatic islets. Arrows point to apoptotic cells (blue). G: The number of TUNEL-positive apoptotic cells in pancreatic islets. Data = mean ± SEM. n = 4–8 animals/group. **P < 0.05 and ***P < 0.001 vs. the WT group; ⁺⁺P < 0.01 and ⁺⁺⁺P < 0.001 vs. the WT-STZ group.

Effects of the β-cell–specific expression of mKL on insulin storage and apoptosis in pancreatic islets of mice challenged with STZ. 129S1/SvIm male mice were injected with PBS, rAAV-GFP, or rAAV-mKL. One week after gene delivery, these mice were injected with STZ or citrate buffer. Animals were killed 6 weeks after the rAAV injections. A: Representative images of Klotho staining (brown color) in cross sections of mouse pancreatic islets. B: Semiquantification of Klotho staining in pancreatic islets. C: Representative images of insulin staining (brown color) of cross sections of islets. D: Semiquantification of insulin staining in pancreatic islets. E: The percentage of cells with positive insulin staining in pancreatic islets. F: Representative images of TUNEL staining (blue color) in pancreatic islets. Arrows point to apoptotic cells. G: The number of TUNEL-positive apoptotic cells in pancreatic islets. Data = mean ± SEM. n = 4–6 animals/group. **P < 0.01 and ***P < 0.001 vs. the control group; ⁺⁺⁺P < 0.001 vs. the PBS-STZ–treated group.

Half deficiency of Klotho in KL^+/− mice exacerbated the development of T1DM induced by STZ. KL^+/− and WT male mice were injected with STZ or citrate buffer. Blood glucose levels, glucose tolerance, insulin sensitivity, and plasma insulin levels were measured during the 5-week period. Fasting blood glucose levels (A). GTT results at week 2 (B) and week 4 (D) after the initial STZ injections. Area under the curve for GTT results at week 2 (C) and week 4 (E) after the initial STZ injections. Original readings of IST results at week 5 (F) after the initial STZ injections. Normalized blood glucose levels of IST results at week 5 (G) after the initial injections. Plasma insulin levels at week 5 after the injections (H). Urine glucose levels after the injections (I). Data = mean ± SEM. n = 4–8 animals/group. **P < 0.01 and ***P < 0.001 vs. the WT group; ⁺P < 0.05 and ⁺⁺P < 0.01 vs. the WT-STZ group.

Effects of the β-cell–specific expression of mKL on blood glucose levels, glucose tolerance, insulin sensitivity, and plasma insulin levels in STZ-induced diabetic mice. 129S1/SvIm male mice were injected with PBS, rAAV-GFP, or rAAV-mKL, respectively. One week after gene delivery, these mice were injected with STZ or citrate buffer. Blood glucose levels, glucose tolerance, insulin sensitivity, and plasma insulin levels were measured during the 6-week period. Fasting blood glucose levels (A). GTT results at week 3 (B) and week 5 (D) after gene delivery. Area under the curve for GTT results at week 3 (C) and week 5 (E) after gene delivery. Original readings of IST results at week 6 (F) after gene delivery. Normalized blood glucose levels of IST results at week 6 (G) after gene delivery. Plasma insulin levels in mice at week 6 (H) after gene delivery. Urine glucose levels (I). Data are mean ± SEM. n = 4–6 animals/group. *P < 005, **P < 0.01, and ***P < 0.001 vs. the control (treated with PBS-citrate buffer) group; ⁺⁺P < 0.01 and ⁺⁺⁺P < 0.001 vs. the PBS-STZ–treated group.

Expression of mKL increased phosphorylations of FAK and Akt and decreased caspase 3 cleavage in MIN6 β-cells. Transfected cells were seeded on collagen IV–coated six-well plates and then incubated with 1 mmol/L STZ for 24 h. Cells were lysed with Ripa buffer. A: Western blot analysis of phosphorylated FAK (Tyr 397; top panel) and FAK protein (middle panel) in cell lysates. B: Quantification of phosphorylation of FAK. Results were standardized to FAK protein levels and then expressed as fold changes vs. the control group. C: Western blot analysis of phosphorylated Akt (Ser 473; top panel) and Akt protein (bottom panel). D: Quantification of phosphorylation of Akt. Results were standardized to Akt protein levels and then expressed as fold changes vs. the control group. E: Western blot analysis of cleaved caspase 3 in cell lysates. F: Quantification of caspase 3 cleavage. Results were standardized to total caspase 3 and then expressed as fold changes vs. the control group. G: Western blot analysis of integrin β1. H: Quantification of integrin β1. n = 4 wells. *P < 0.05, **P < 0.01, and ***P < 0.001 vs. the control vehicle; ⁺⁺P < 0.01 and ⁺⁺⁺P < 0.001 vs. the control–1 mmol/L STZ group.

Effects of expression of mKL on basal and STZ-induced and TNFα-induced apoptosis, and cell adhesion to collagen IV in MIN6 β-cells. MIN6 β-cells were transfected with plasmid DNA including pmKL, pGFP, or vehicles (transfection agent alone) for 48 h. A: Western blot analysis of Klotho protein expression. B: Quantification of full-length Klotho protein expression. C: Quantification of short-form Klotho protein expression. Results were standardized to β-actin and then expressed as fold changes vs. the control group (transfection reagent alone). n = 4. **P < 0.01 and ***P < 0.001 vs. the control group. D: Apoptotic nuclear change (pointed by arrows, brown color) detected by TUNEL staining in MIN6 β-cells. Transfected MIN6 β-cells were seeded on collagen IV–coated six-well plates and then incubated with or without STZ or TNFα for 24 h. E: The percentage of apoptotic cells. Data are means ± SEM. n = 4 wells. *P < 0.05 and ***P < 0.001 vs. the control group; ⁺P < 0.05 vs. the control-STZ group; ^^^P < 0.001 vs. the control-TNFα group. F: Phase contrast images of transfected MIN6 β-cells cultured in dishes coated with 1% BSA or collagen IV (5 µg/mL) for 3 h. G: Quantification of cell adhesion. n = 4 wells. ***P < 0.001 vs. the control-BSA group; ⁺⁺⁺P < 0.001 vs. the control–collagen IV group.