課題番号 0１Ａ(追)－７

IAMS Project Report
Principal Investigator: Ilir Mehmeti
Institute of Clinical Biochemistry, Hannover Medical School
Carl-Neuberg-Str. 1, 30625 Hannover / Germany+49 511 532 6525
+49 511 532 6526
Mehmeti.Ilir@mh-hannover.de“Understanding the role of oxidative and ER stress in Pancreatic β cells”

Research summary 2019
Dietary habits can have a major impact on health and may cause obesity and type 2
diabetes mellitus (T2DM). Increased circulating levels of saturated fatty acids (FFAs) andglucose are considered to be major mediators of β-cell dysfunction and death in T2DM.
Although it has been proposed that endoplasmic reticulum (ER) and oxidative stress playa crucial role in gluco/lipotoxicity, their interplay and relative contribution to β-cell
dysfunction and apoptosis has not been fully elucidated. To investigate this issue, we
examined the toxicity of free fatty acid (FFA) compositions mirroring the FFA profilesof various popular edible oils in human EndoC-βH1 β-cells. For this purpose, we madecompositions consisting exclusively of various FFAs in different volumetric percentages
mimicking these oils and additionally mixtures of these compositions. Human EndoCβH1 β-cells were incubated with different oil compositions and the toxicity, lipid droplet
formation, ER-stress, and H2O2 production were analyzed. Compositions with prominentcontent of saturated as well as unsaturated long-chain FFAs showed moderate but
significant toxicity both in human EndoC-βH1 β-cells and rat islets, however, without
further measurable metabolic impairments. On the other hand compositions with highcontent of medium-chain FFAs revealed no toxicity. A composition with 50% of the very
long-chain unsaturated FFA erucic acid caused high toxicity with concomitantperoxisomal H2O2 production. The toxicity of FFAs to human EndoC-βH1 β-cells wasdampened in mixtures of FFA compositions with a significant content of medium-chain
FFAs, but not with a significant proportion of unsaturated FFAs.

Research plan for 2020

Professor Oyadomari’s group have established mouse β-cells lacking a transcription
factor Activating transcription factor 4 (ATF4) by CRISPR/Cas9 gene editing system.
ATF4, a member of the ATF/CREB family, plays a major role in regulating genes thatare involved in amino acid metabolism and redox homeostasis under ER stress condition.
As we obtained the ATF4 knockout MIN6 cells from Professor Oyadomari, we will
characterize the role of ATF4 on mechanism of lipotoxicity to pancreatic β-cells. Thefollowing analysis will be conducted in 2020.

Insulin biosynthesis and secretion•••- Quantification of insulin secretion and also insulin content (RIA)- Quantification of pro-insulin (ELISA and immunohistochemistry)- Oxidative protein folding (Live cell imaging)

ROS and enzymes detection assays
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- General ROS quantification (H2-DCFDA)- Specific superoxide (MitoSOX), hydroxyl radical (HPF, oxyDNA by FACS,Microscopy) and organelle-specific H2O2 quantification (HyPer, Reader,Microscopy)- Quantification of antioxidant enzyme activity (SOD, GPx, and catalase) andglutathione reductase activity (Spectroscopy)- Quantification of Nerf2, and AP1 promoter activity- Quantification of GSH and GSSG

Cell death – Apoptosis detection assays•••••-Initiator caspases (caspase-8, -9 and -12) and effector caspases (caspase-3)
DNA-fragmentation / damage: Propidium iodide / oxyDNA
Cardiolipin peroxidation: 10-N-nonyl acridine orange (NAO)
Mitochondrial: Mitochondrial membrane potential (JC-1)
Release of cytochrome c and SMAC/DIABLO (cell fractionation, WB)