Glucolipotoxicity Suppressed Autophagy and Insulin Contents in Human Islets, and Attenuation of PERK Activity Enhanced Them in an ATG7-Dependent Manner.

BACKGRUOUND: Administration of pancreatic endoplasmic reticulum kinase inhibitor (PERKi) improved insulin secretion and hyperglycemia in obese diabetic mice. In this study, autophagic balance was studied whether to mediate it. METHODS: Human islets were isolated from living patients without diabetes. PERKi GSK2606414 effects were evaluated in the islets under glucolipotoxicity by palmitate. Islet insulin contents and secretion were measured. Autophagic flux was assessed by microtubule associated protein 1 light chain 3 (LC3) conversion, a red fluorescent protein (RFP)-green fluorescent protein (GFP)- LC3 tandem assay, and P62 levels. For mechanical analyses, autophagy was suppressed using 3-methyladenine in mouse islets. Small interfering RNA for an autophagy-related gene autophagy related 7 (Atg7) was transfected to interfere autophagy. RESULTS: PERKi administration to mice decreased diabetes-induced P62 levels in the islets. Glucolipotoxicity significantly increased PERK phosphorylation by 70% and decreased insulin contents by 50% in human islets, and addition of PERKi (40 to 80 nM) recovered both. PERKi also enhanced glucose-stimulated insulin secretion (6-fold). PERKi up-regulated LC3 conversion suppressed by glucolipotoxicity, and down-regulated P62 contents without changes in P62 transcription, indicating enhanced autophagic flux. Increased autophagosome-lysosome fusion by PERKi was visualized in mouse islets, where PERKi enhanced ATG7 bound to LC3. Suppression of Atg7 eliminated PERKi-induced insulin contents and secretion. CONCLUSION: This study provided functional changes of human islets with regard to autophagy under glucolipotoxicity, and suggested modulation of autophagy as an anti-diabetic mechanism of PERKi.

Partial Deletion of Perk Improved High-Fat Diet-Induced Glucose Intolerance in Mice.

Although pancreatic endoplasmic reticulum kinase (PERK) is indispensable to beta cells, low-dose PERK inhibitor improved glucose- stimulated insulin secretion (GSIS) and hyperglycemia in diabetic mice. Current study examined if partial deletion of Perk (Perk+/-) recapitulated the effects of PERK inhibitor, on the contrary to the complete deletion. Perk+/- mice and wild-type controls were fed with a high-fat diet (HFD) for 23 weeks. Glucose tolerance was evaluated along with serum insulin levels and islet morphology. Perk+/- mice on normal chow were comparable to wild-type mice in various metabolic features. HFD-induced obesity was not influenced by Perk reduction; however, HFD-induced glucose intolerance was significantly improved since 15-week HFD. HFD-induced compromises in GSIS were relieved by Perk reduction, accompanied by reductions in phosphorylated PERK and activating transcription factor 4 (ATF4) in the islets. Meanwhile, HFD-induced islet expansion was not significantly affected. In summary, partial deletion of Perk improved glucose tolerance and GSIS impaired by diet-induced obesity, without changes in body weights or islet mass.

A QCM immunosensor employing signal amplification strategies by enlarging the size of nanoparticles using gold or silver staining.

We present quartz crystal microbalance (QCM) immunosensors for the detection of alpha-fetoprotein (AFP) in a human serum immunoassay with high sensitivity. In this study, we employed three types of signal amplification strategies using size enlargement and/or increase in mass of gold and titanium dioxide nanoparticles. Since the basic principle of the QCM sensor is to measure the change in resonance frequency according to the mass change caused by the molecular interactions on the sensor surface, we were able to quantitatively analyze AFP by sandwich immunoassay using gold or titanium dioxide nanoparticles conjugated with anti-AFP detection antibodies and the subsequent three signal amplification techniques in a similar manner. The signal amplification technologies provide the size expansion of gold nanoparticles by gold or silver staining reaction and mass enhancement by photocatalytic silver staining of titanium dioxide nanoparticles. The limit of detections (LODs) of the AFP immunoassay in human serum by the gold and silver staining-mediated signal amplifications for gold nanoparticles were 56 and 87 pg mL-1, respectively, but by the photocatalytic silver staining signal amplification for titanium dioxide nanoparticles was 118 pg mL-1. This means that the signal amplification method through size enhancement by gold staining of gold nanoparticles further improved the detection ability of the QCM immunosensor.

Quartz crystal microbalance cardiac Troponin I immunosensors employing signal amplification with TiO2 nanoparticle photocatalyst.

A sensitive and highly reproducible cardiac troponin I (cTnI) immunoassay in human serum is a challenging research goal for researchers studying biosensors because cTnI can undergo proteolysis and various modifications in blood. Furthermore, the reproducible detection of cTnI at very low concentrations is also required for diagnosing acute myocardial infarction. Here, we present sensitive and highly reproducible quartz crystal microbalance (QCM) immunosensors for the detection of cTnI in human serum. The unique features of this study are the use of a pair of capture antibodies that bind to different epitopes of cTnI, and the use of a signal amplification technique that enlarged the size of the titanium dioxide nanoparticles using photocatalytic silver staining. Since QCM measures changes in the resonance frequency due to the changes in mass occurring on the sensor surface, it is possible to quantitatively analyze cTnI based on the enormous increase in mass using a sandwich immunoassay and subsequent signal amplification by silver staining. The detection limit of the cTnI immunoassay in human serum without photocatalytic silver staining was 307 pg/ml, but 18 pg/ml in photocatalytic silver staining-mediated signal amplification. Thus, amplifying the signal increased the sensitivity and reproducibility of the cTnI immunoassay in human serum.

Sensitive detection of microRNA using QCM biosensors: sandwich hybridization and signal amplification by TiO2 nanoparticles.

MicroRNA-21 (miR-21) is known to act as an important biomarker for cancer, in that its up-regulation is closely related to several types of malignant tumor. Sensitive and accurate detection of miR-21 using a biosensor is highly challenging. In this study, sensitive and selective detection technology for miR-21 molecules using a quartz crystal microbalance (QCM) biosensor was developed. Sandwich hybridization between miR-21 and specially designed probes and a subsequent TiO2 photocatalytic silver enhancement reaction were the driving forces for sensitive detection with high selectivity for miR-21. Using this strategic approach under optimal conditions, the novel QCM biosensor can detect miR-21 with a LOD of 0.87 pM over the entire linear range from 0.1 pM to 10 µM, with a correlation coefficient of 0.988. In addition, the developed QCM biosensor was very effective in the quantification of miR-21 in serum samples, so the proposed miRNA detection method offers great potential for the diagnosis of early disease, such as cancer and vascular diseases, and could be an excellent alternative for biological research and clinical diagnosis.