Diabetes Metab J.  2024 Mar;48(2):231-241. 10.4093/dmj.2022.0366.

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

• Moon S
• Lim JY
• Lee M
• Han Y
• Kim H
• Kwon W
• Jang JY
• Kim MN
• Park KS
• Jung HS

• KMID: 2553594
• DOI: http://doi.org/10.4093/dmj.2022.0366

Abstract

Background
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.

Keyword

Autophagy; Diabetes mellitus; Humans; Insulin; Islets of Langerhans

Figure

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  Fig. 1. Low-dose pancreatic endoplasmic reticulum kinase inhibitor (PERKi) treatment decreased P62 levels in the islets of obese diabetic mice. PERKi GSK2656157 (10 mg/kg/day) was administered for 8 weeks to diabetic mice induced by a high-fat diet and streptozotocin injections, in accordance with the Institutional Animal Care and Use Committee of Seoul National University Hospital (SNU-150327-3-2), as previously described [10]. After euthanasia, pancreas was harvested and preserved in paraffin blocks. Section slides were stained for insulin and P62 in brown, and quantification of the P62 intensity per islet area was calculated. Animal numbers 5–6/group and 10.1 ± 1.8 islets were analyzed in each mouse. (A) immunohistochemical staining for P62 and insulin and (B) P62 staining intensity. (A) Representative images with islets delineated by dashed lines. (B) The lines and error bars on the plot represent mean ± standard error of the mean. One-way analysis of variance (ANOVA) was applied. DM, diabetes mellitus; C, control; V, vehicle.

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  Fig. 2. Low-dose pancreatic endoplasmic reticulum kinase inhibitor (PERKi) enhanced autophagy activity, insulin contents, and insulin secretion in human islets under chronic glucolipotoxicity (GLT). Human islets were isolated from living donors’ pancreas and exposed to GLT (glucose 20 mM and palmitate 0.5 mM) for 29 to 48 hours, with/without GSK2606414. Each treatment group was composed of 25 to 30 islets, resulting in total 120 islets per experiment, by hand-picking of healthy islets estimated by their morphology. The islets were randomly assigned to control, vehicle, and PERKi groups, without blinding to the researchers. (A, C) Western blots and (B) quantitative reverse transcription-polymerase chain reaction of autophagy and unfolded protein response markers. In (C), bafilomycin A (4 nM) was added to the culture medium during the last 5 hours. (D) Islet insulin content, (E) insulin secretion, and (F) insulin secretion adjusted by the islet insulin contents. Because of large inter-participant variation, (B, D, E) are shown on a log-10 scale. In (A, C), one-way analysis of variance (ANOVA) with Bonferroni posttest was used, except for phospho-eukaryotic translation initiation factor 2 alpha (EIF2A) where paired t-test was applied due to limited sample size. For (B), unpaired t-test was used between the control and vehicle. For (D), the Kruskal-Wallis with Dunn’s multiple comparison test was applied. In (E, F), two-way repeated measures ANOVA with Bonferroni posttest was applied. For (B, D, E), logarithmic transformation was performed before the analyses. Experiment numbers 4 to 5. The lines and error bars represent median (interquartile ranges) for (D) and mean ± standard error of the mean (all the others). C, control; V, vehicle; BIP, binding immunoglobulin protein; NS, no significant differences; LC3B, light chain 3B; LG, low glucose; HG, high glucose. aP<0.05 and bP<0.01 between the groups, cP < 0.01 in the paired t-test.

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  Fig. 3. Low-dose pancreatic endoplasmic reticulum kinase inhibitor (PERKi) improved 3-methyladenine (3-MA)-suppressed autophagy and insulin synthesis through autophagy related 7 (ATG7). (A-E) Mouse islets or dispersed cells were treated with 3-MA (0.5 mM) for 24 hours with/without GSK2606414. Autophagy was induced by fetal bovine serum deprivation by further incubation for 5 hours (A, B). Each treatment group was composed of 25 to 30 islets, resulting in total 120 islets from 3 to 4 mice per experiment, by hand-picking of healthy islets estimated by their morphology. The islets were randomly assigned to the groups, without blinding to the researchers. Western blotting of autophagy and unfolded protein response markers was conducted, and representative blots are demonstrated (A, C, D, E). For (B), dispersed islet cells were transduced with BacMam tandem red fluorescent protein (RFP)-green fluorescent protein (GFP)-light chain 3B (LC3B) reagent, incubated overnight, then treated as described above. Before fixation with 4% formaldehyde, live cell nuclei were stained with Hoechst 33342, and GFP or RFP puncta were manually counted in cytoplasm of live cells. About 100 cells were counted in each treatment group by 3 to 6 experiments. Representative pictures are presented: yellow puncta indicating quenching of acid-sensitive GFP by lysosome (in the control), a green punctate (3-MA group) and red puncta indicating autophagolysosome (3-MA with PERKi group) marked by arrows. (F, G, H) Mouse islets were dispersed to single cells, and transfected with 100 nM of siRNA for Atg7 and negative control. Then autophagy was inhibited by 3-MA (0.5 mM) with/without GSK2606414 (20 nM). Each treatment group was composed of 105 cells, resulting in total 3 × 105 cells from 5 to 6 mice per experiment. The cells were randomly assigned to the groups, without blinding to the researchers. After 30 to 48 hours, quantitative reverse transcription-polymerase chain reaction was conducted (Atg7 and insulin 1 [Ins1]), and protein levels were measured by Western blot (ATG7 and tubulin) and enzyme-linked immunosorbent assay (ELISA; insulin contents and secretion). Dot numbers in each group represent experimental numbers. The lines and error bars are median (interquartile ranges) (E) and mean ± standard error of the mean (all the others). One-way analysis of variance (ANOVA) with Bonferroni posttest was applied, except for (B, E) where Kruskal-Wallis with Dunn's multiple comparison test was applied excluding the serum-free control in (E), and for (H) where two-way repeated measures ANOVA with Dunnett's multiple comparisons test was applied. C, control; V, vehicle; NS, no significant differences; EIF2A, eukaryotic translation initiation factor 2 alpha; BIP, binding immunoglobulin protein; LG, low glucose; HG, high glucose; siAtg7, Atg7 siRNA. aP < 0.05, bP < 0.01 in the post hoc analyses.

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  Fig. 4. A working hypothesis of pancreatic endoplasmic reticulum kinase inhibitor (PERKi) action in human β-cells under glucolipotoxicity (GLT). Metabolic stress suppresses microtubule associated protein 1 light chain 3 (LC3) lipidation and low-dose PERKi recovers it, causing enhanced autophagic activity and subsequent improvement in endoplasmic reticulum (ER) function. Especially, ER-phagy would enhance clearance of misfolded proinsulin, which contributes to efficient proinsulin translation/post-translational modification [38]. Observations in this study is shown in red, and inference from literature in black. PE, phosphatidylethanolamine; ATG7, autophagy related 7; P, phosphorylation.

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