DOI: 10.5061/dryad.83bk3j9vd
title: Data from: ApoA-I protects pancreatic β-cells from cholesterol-induced mitochondrial damage and restores their ability to secrete insulin

description: 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 partner. β-cell internalization of apoA-I was time-,
 concentration-, temperature-, cholesterol-, and F 1 -ATPase
 β-subunit-dependent. β-cells with internalized apoA-I (apoA-I + cells) had
 higher cholesterol and cell surface F 1 -ATPase β-subunit levels than
 β-cells without internalized apoA-I (apoA-I − cells). The internalized
 apoA-I colocalized with mitochondria and was associated with reduced
 oxidative stress and increased insulin secretion. The IF 1 (ATPase
 inhibitory factor 1) attenuated apoA-I internalization and increased
 oxidative stress in Ins-1E β-cells and isolated mouse islets.
 Differentially expressed genes in apoA-I + and apoA-I − Ins-1E cells were
 related to protein synthesis, the unfolded protein response, insulin
 secretion, and mitochondrial function. CONCLUSIONS: These results
 establish that β-cells are functionally heterogeneous, and apoA-I restores
 insulin secretion in β-cells with elevated cholesterol levels by improving
 mitochondrial redox balance.
description: <strong>Identification of the
 F<sub>1</sub>-ATPase β-subunit on the Ins-1E cell
 surface</strong> 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 maintained at 4
 °C for 1 h with KRBH with 0.1% (w/v) BSA (200 µL) and Dynabeads (50 µL)
 with bound apoA-I. The supernatant was discarded, and the cells were
 washed gently with ice-cold PBS, then harvested with PBS containing
 EDTA-Na<sub>2</sub> (2 mM) and collected into an Eppendorf
 tube. The Dynabeads and the bound cells were collected with a magnet. The
 supernatant was removed and the beads were washed with PBS (pH 7.4) and
 0.1% (v/v) Tween20, then washed with PBS (pH 7.4). Non-specifically bound
 proteins were removed from the Dynabeads by incubation overnight at room
 temperature with trypsin at protein to enzyme ratio of 100:1. The beads
 were then collected using a magnet, the supernatant was discarded and the
 beads were washed (x3) with PBS. ApoA-I binding partners on the beads were
 eluted with 0.15% (v/v) trifluoro acetic acid, concentrated using C18
 stage tips as previously described and analysed by LC–MS/MS.
 <strong>Mass Spectrometry</strong>
 The eluted peptides were reconstituted in 0.1% (v/v) formic acid
 (10 μL) and resolved by nano-LC using an Ultimate 3000 HPLC and an
 autosampler (Dionex, Amsterdam, The Netherlands). Briefly, the sample (0.5
 µL), was loaded onto a micro C18 pre-column (300 μm×5 mm, Dionex) with
 H<sub>2</sub>O:CH<sub>3</sub>CN (98:2) in 0.1%
 (v/v) trifluoro acetic acid at a flow rate of 10 μL/min. After washing,
 the pre-column was switched (Valco 10 port valve, Dionex) into line with a
 fritless nanocolumn (75 μm×22 cm) containing reverse phase C18 media
 (particle size: 1.9 μm, pore size: 120 Å, Dr. Maisch HPLC GmbH). Peptides
 were eluted using a linear gradient of
 H<sub>2</sub>O:CH<sub>3</sub>CN (98:2 to 64:36) in
 0.1% (v/v) formic acid at a flow rate of 250 nL/min over 90 min. The
 QExactive (Thermo Electron, Bremen, Germany) mass spectrometer was run in
 DDA mode where 2000 V was applied to a low-volume union and the column (45
 °C) was positioned 0.5 cm from the heated capillary (275 °C). A survey
 scan 350–1750 m/z was acquired in the Orbitrap (resolution 70,000 at 200
 m/z) with an accumulation target of 10<sup>6</sup> ions, lock
 mass enabled and up to the 10 most abundant ions (AGC target set to
 10<sup>5</sup>, minimum AGC target set to
 1.5×10<sup>4</sup>) with charge states ≥ +2 and ≤ +6
 sequentially isolated and fragmented. Protein
 dataset-peak lists were generated from raw files using Mascot Daemon
 v2.5.1 (Matrix Science, London, UK, www.matrixscience.com). All MS/MS spectra were searched against the Uniprot database (Feb 2021) of 563,972 sequences for protein identification with the following criteria: (1) taxon, Rat; (2) allowed 1 missed cleavages; (3) variable modifications, oxidation (M), phosphorylation (S,T,Y), Carbomidomethyl (C); (4) peptide tolerance, ±5 ppm; (5) fragment tolerance, ±0.5 Da; (6) peptide charge +2 and +3; and (7) enzyme specificity, semi-tryptic. A decoy database search was also performed. Only proteins that had a significance threshold of p&lt;0.007 (&lt;1% FDR) were recorded.
description: # 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 • 2 missed cleavages • Target FDR of 1% • Modifications for C, M, STY ## Software * Microsoft Word environment/other tabular data software * ThermoScientific Xcalibur software environment