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In vivo pancreatic ?-cell-specific expression of antiaging gene Klotho: a novel approach for preserving ?-cells in type 2 diabetes.

ABSTRACT: Protein expression of an antiaging gene, Klotho, was depleted in pancreatic islets in patients with type 2 diabetes mellitus (T2DM) and in db/db mice, an animal model of T2DM. The objective of this study was to investigate whether in vivo expression of Klotho would preserve pancreatic ?-cell function in db/db mice. We report for the first time that ?-cell-specific expression of Klotho attenuated the development of diabetes in db/db mice. ?-Cell-specific expression of Klotho decreased hyperglycemia and enhanced glucose tolerance. The beneficial effects of Klotho were associated with significant improvements in T2DM-induced decreases in number of ?-cells, insulin storage levels in pancreatic islets, and glucose-stimulated insulin secretion from pancreatic islets, which led to increased blood insulin levels in diabetic mice. In addition, ?-cell-specific expression of Klotho decreased intracellular superoxide levels, oxidative damage, apoptosis, and DNAJC3 (a marker for endoplasmic reticulum stress) in pancreatic islets. Furthermore, ?-cell-specific expression of Klotho increased expression levels of Pdx-1 (insulin transcription factor), PCNA (a marker of cell proliferation), and LC3 (a marker of autophagy) in pancreatic islets in db/db mice. These results reveal that ?-cell-specific expression of Klotho improves ?-cell function and attenuates the development of T2DM. Therefore, in vivo expression of Klotho may offer a novel strategy for protecting ?-cells in T2DM.

SUBMITTER: Lin Y

PROVIDER: S-EPMC4375073 | biostudies-literature | 2015 Apr

REPOSITORIES: biostudies-literature

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In vivo pancreatic β-cell-specific expression of antiaging gene Klotho: a novel approach for preserving β-cells in type 2 diabetes.

Lin Yi Y Sun Zhongjie Z

Diabetes 20141105 4

Protein expression of an antiaging gene, Klotho, was depleted in pancreatic islets in patients with type 2 diabetes mellitus (T2DM) and in db/db mice, an animal model of T2DM. The objective of this study was to investigate whether in vivo expression of Klotho would preserve pancreatic β-cell function in db/db mice. We report for the first time that β-cell-specific expression of Klotho attenuated the development of diabetes in db/db mice. β-Cell-specific expression of Klotho decreased hyperglycem ...[more]

PMID: 25377875

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Project description:Aims/hypothesisPancreatic beta cells are subjected to exogenous damaging factors such as proinflammatory cytokines or excess glucose that can cause accumulation of damage-inducing reactive oxygen species during the pathogenesis of diabetes. We and others have shown that beta cell autophagy can reduce reactive oxygen species to protect against apoptosis. While impaired islet autophagy has been demonstrated in human type 2 diabetes, it is unknown if islet autophagy is perturbed in the pathogenesis of type 1 diabetes. We hypothesised that beta cell autophagy is dysfunctional in type 1 diabetes, and that there is a progressive loss during early diabetes development.MethodsPancreases were collected from chloroquine-injected and non-injected non-obese diabetes-resistant (NOR) and non-obese diabetic (NOD) mice. Age- and BMI-matched pancreas tissue sections from human organ donors (N = 34) were obtained from the Network for Pancreatic Organ Donors with Diabetes (nPOD). Tissue sections were stained with antibodies against proinsulin or insulin (beta cell markers), microtubule-associated protein 1 light chain 3 A/B (LC3A/B; autophagosome marker), lysosomal-associated membrane protein 1 (LAMP1; lysosome marker) and p62 (autophagy adaptor). Images collected on a scanning laser confocal microscope were analysed with CellProfiler and ImageJ. Secondary lysosomes and telolysosomes were assessed in electron micrographs of human pancreatic tissue sections (n = 12), and energy dispersive x-ray analysis was performed to assess distribution of elements (n = 5).ResultsWe observed increased autophagosome numbers in islets of diabetic NOD mice (p = 0.008) and increased p62 in islets of both non-diabetic and diabetic NOD mice (p < 0.001) vs NOR mice. There was also a reduction in LC3-LAMP1 colocalisation in islets of diabetic NOD mice compared with both non-diabetic NOD (p < 0.001) and NOR mice (p < 0.001). Chloroquine elicited accumulation of autophagosomes in the islets of NOR (p = 0.003) and non-diabetic NOD mice (p < 0.001), but not in islets of diabetic NOD mice; and stimulated accumulation of p62 in NOR (p < 0.001), but not in NOD mice. We observed reduced LC3-LAMP1 colocalisation (p < 0.001) in residual beta cells of human donors with type 1 diabetes vs non-diabetic participants. We also observed reduced colocalisation of proinsulin with LAMP1 in donors with type 1 diabetes (p < 0.001). Electron microscopy also revealed accumulation of telolysosomes with nitrogen-dense rings in beta cells of autoantibody-positive donors (p = 0.002).Conclusions/interpretationWe provide evidence of islet macroautophagy/crinophagy impairment in human type 1 diabetes. We also document accumulation of telolysosomes with peripheral nitrogen in beta cells of autoantibody-positive donors, demonstrating altered lysosome content that may be associated with lysosome dysfunction before clinical hyperglycaemia. Similar macroautophagy impairments are present in the NOD mouse model of type 1 diabetes.

Project description:The objective of this study is to investigate whether stem cell delivery of secreted Klotho (SKL), an aging-suppressor protein, attenuates monocrotaline-induced pulmonary vascular dysfunction and remodeling. Overexpression of SKL in mesenchymal stem cells (MSCs) was achieved by transfecting MSCs with lentiviral vectors expressing SKL-green fluorescent protein (GFP). Four groups of rats were treated with monocrotaline, whereas an additional group was given saline (control). Three days later, 4 monocrotaline-treated groups received intravenous delivery of nontransfected MSCs, MSC-GFP, MSC-SKL-GFP, and PBS, respectively. Ex vivo vascular relaxing responses to acetylcholine were diminished in small pulmonary arteries (PAs) in monocrotaline-treated rats, indicating pulmonary vascular endothelial dysfunction. Interestingly, delivery of MSCs overexpressing SKL (MSC-SKL-GFP) abolished monocrotaline-induced pulmonary vascular endothelial dysfunction and PA remodeling. Monocrotaline significantly increased right ventricular systolic blood pressure, which was attenuated significantly by MSC-SKL-GFP, indicating improved PA hypertension. MSC-SKL-GFP also attenuated right ventricular hypertrophy. Nontransfected MSCs slightly, but not significantly, improved PA hypertension and pulmonary vascular endothelial dysfunction. MSC-SKL-GFP attenuated monocrotaline-induced inflammation, as evidenced by decreased macrophage infiltration around PAs. MSC-SKL-GFP increased SKL levels, which rescued the downregulation of SIRT1 (Sirtuin 1) expression and endothelial NO synthase (eNOS) phosphorylation in the lungs of monocrotaline-treated rats. In cultured endothelial cells, SKL abolished monocrotaline-induced downregulation of eNOS activity and NO levels and enhanced cell viability. Therefore, stem cell delivery of SKL is an effective therapeutic strategy for pulmonary vascular endothelial dysfunction and PA remodeling. SKL attenuates monocrotaline-induced PA remodeling and PA smooth muscle cell proliferation, likely by reducing inflammation and restoring SIRT1 levels and eNOS activity.

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In vivo pancreatic ?-cell-specific expression of antiaging gene Klotho: a novel approach for preserving ?-cells in type 2 diabetes.

Publications

In vivo pancreatic β-cell-specific expression of antiaging gene Klotho: a novel approach for preserving β-cells in type 2 diabetes.

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