ApoA-I protects pancreatic β-cells from cholesterol-induced mitochondrial damage and restores their ability to secrete insulin

BACKGROUND: High cholesterol levels in pancreatic β-cells cause oxidative stress and decrease insulin secretion. β-cells can internalize apo (apolipoprotein) A-I, which increases insulin secretion. This study asks whether internalization of apoA-I improves β-cell insulin secretion by reducing oxidative stress.
METHODS: Ins-1E cells were cholesterol-loaded by incubation with cholesterol-methyl-β-cyclodextrin. Insulin secretion in the presence of 2.8 or 25 mmol/L glucose was quantified by radioimmunoassay. Internalization of fluorescently labeled apoA-I by β-cells was monitored by flow cytometry. The effects of apoA-I internalization on β-cell gene expression were evaluated by RNA sequencing. ApoA-I-binding partners on the β-cell surface were identified by mass spectrometry. Mitochondrial oxidative stress was quantified in β-cells and isolated islets with MitoSOX and confocal microscopy.
RESULTS: An F 1 -ATPase β-subunit on the β-cell surface was identified as the main apoA-I-binding part..., Identification of the F1-ATPase β-subunit on the Ins-1E cell surface
Tosylactivated Dynabeads (Thermo Fisher Scientific) were used to identify apoA-I binding partners on the Ins-1E cell surface. Briefly, apoA-I (100 µg) was dissolved in borate buffer (0.1 M, pH 9.5) containing ammonium sulphate (1.2 M) and incubated at 37 °C overnight with Dynabeads (5 mg, 165 µL). The beads with bound apoA-I were collected using a magnet and coupling efficiency was determined by measuring the concentration of unbound apoA-I in the supernatant. The beads with bound apoA-I were resuspended in PBS (pH 7.4, 250 µL) with 0.1% (w/v) BSA and incubated for 5 min at room temperature on a rotary shaker. The beads were collected and resuspended in PBS (250 µL) with 0.5 % (w/v) BSA, then incubated for a further 1 h at 37 °C to reduce non-specific binding. The beads were collected again and resuspended in PBS (pH 7.4, 250 µL) without BSA.
Ins-1E cells were seeded in a 12-well plate, washed with KRBH buffer, then ma..., , # The anti-oxidant function of apolipoprotein A-I rescues pancreatic β-cells from cholesterol-induced mitochondrial dysfunction

[https://doi.org/10.5061/dryad.83bk3j9vd](https://doi.org/10.5061/dryad.83bk3j9vd)

The deposited data contains two files. The raw file can be viewed using Xcalibur ThermoScientific software and contains the MSMS profile of the sample. OpenChrom ([www.openchrome.net)](https://www.openchrom.net/) can be used to view *.raw files. This file is named APOA1_KRBH_point5.raw.

The PDF and CSV are the search result file obtained using the Mascot algorithm. These files are named:

Rat_specific_database_search_20220803_F002928_output.pdf Rat_specific_database_search_20220803_F002928_output.csv.

Mascot Daemon was used to search a rat-specific database downloaded from Swiss-Prot on 1st Feb, 2021 and specific taxonomy of Rat was applied. The following parameters were used:

• Semi-tryptic

• MS/MS ion search

• Peptide mass tolerance 5ppm

• Fragment mass tolerance 0.05Da...,