Endocrinol Metab.  2024 Apr;39(2):353-363. 10.3803/EnM.2023.1809.

Docosahexanoic Acid Attenuates Palmitate-Induced Apoptosis by Autophagy Upregulation via GPR120/mTOR Axis in Insulin-Secreting Cells

• Hong SW
• Lee J
• Moon SJ
• Kwon H
• Park SE
• Rhee EJ
• Lee WY

• KMID: 2554640
• DOI: http://doi.org/10.3803/EnM.2023.1809

Abstract

Background
Polyunsaturated fatty acids (PUFAs) reportedly have protective effects on pancreatic β-cells; however, the underlying mechanisms are unknown.
Methods
To investigate the cellular mechanism of PUFA-induced cell protection, mouse insulinoma 6 (MIN6) cells were cultured with palmitic acid (PA) and/or docosahexaenoic acid (DHA), and alterations in cellular signaling and apoptosis were examined.
Results
DHA treatment remarkably repressed caspase-3 cleavage and terminal deoxynucleotidyl transferase-mediated UTP nick end labeling (TUNEL)-positive red dot signals in PA-treated MIN6 cells, with upregulation of autophagy, an increase in microtubule- associated protein 1-light chain 3 (LC3)-II, autophagy-related 5 (Atg5), and decreased p62. Upstream factors involved in autophagy regulation (Beclin-1, unc51 like autophagy activating kinase 1 [ULK1], phosphorylated mammalian target of rapamycin [mTOR], and protein kinase B) were also altered by DHA treatment. DHA specifically induced phosphorylation on S2448 in mTOR; however, phosphorylation on S2481 decreased. The role of G protein-coupled receptor 120 (GPR120) in the effect of DHA was demonstrated using a GPR120 agonist and antagonist. Additional treatment with AH7614, a GPR120 antagonist, significantly attenuated DHA-induced autophagy and protection. Taken together, DHA-induced autophagy activation with protection against PA-induced apoptosis mediated by the GPR120/mTOR axis.
Conclusion
These findings indicate that DHA has therapeutic effects on PA-induced pancreatic β-cells, and that the cellular mechanism of β-cell protection by DHA may be a new research target with potential pharmacotherapeutic implications in β-cell protection.

Keyword

Docosahexaenoic acids; Autophagy; Insulin-secreting cells; G-protein-coupled receptor

Figure

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  Fig. 1. Docosahexaenoic acid (DHA) prevents palmitic acid (PA)induced apoptosis in insulin-secreting cells. (A, B) Mouse insulinoma 6 (MIN6) cells were incubated with 40 μM DHA in the presence or absence of 500 μM PA for 24 hours. Whole cell lysates were extracted and cleaved caspase-3 (c-Casp3) expression, an apoptosis marker, was measured by immunoblotting analysis. The ratio to total caspase-3 (t-Casp3)/β-actin was described (n=3). (C, D) After treatment with DHA and/or PA, MIN6 cells were fixed for terminal deoxynucleotidyl transferase-mediated UTP nick end labeling (TUNEL) staining assay to observe apoptosis. Apoptotic cells were visualized with red dot signals (white arrows). The nuclei were indicated by 4’,6-diamidino2-phenylindole (DAPI). Scale bars=20 µm. The number of TUNEL-positive cells was counted and described as a ratio to 102 cells (n=3–4). VEH, vehicle; CON, control. aP<0.01, bP<0.001 compared with the VEH group; cP<0.05, dP<0.01 compared with the PA group.

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  Fig. 2. Docosahexaenoic acid (DHA) upregulates autophagy dose-dependently in insulin-secreting cells. (A) Mouse insulinoma 6 (MIN6) cells exposed to DHA at 0, 10, 20, 30, and 40 μM for 24 hours. Expression of autophagy-related factors including light chain 3 (LC3) I/II, p62, autophagy-related 5 (Atg5) was measured by immunoblotting analysis. (B) MIN6 cells were treated with 40 μM DHA or vehicle for 24 hours, and analyzed by immunofluorescence to morphologically observe the LC3 expression level. LC3 was detected with green fluorescence (white arrowheads), and nuclei were indicated by 4’,6-diamidino2-phenylindole (DAPI). Scale bars=20 µm.

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  Fig. 3. Docosahexaenoic acid (DHA) upregulates autophagy in a palmitate-treated manner in mouse insulinoma 6 (MIN6) cells. (A, B) Light chain 3 (LC3) I, II, p62, and autophagy-related 5 (Atg5) expression were measured in MIN6 cells after incubation with and without 500 µM palmitate and/or 40 µM DHA for 24 hours. The ratio to β-actin was identified (n=3–4). (C) LC3 and Atg5 expression was detected using double immunofluorescence. LC3 and Atg5 are respectively visualized by green and red fluorescence, and LC3 and Atg5 co-localization is indicated with white arrowheads. Nuclei were marked by 4’,6-diamidino2-phenylindole (DAPI). Scale bar=20 μm. VEH, vehicle; PA, palmitic acid. aP<0.05, bP<0.01 compared with the VEH group; cP<0.01 compared with the PA group.

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  Fig. 4. Docosahexaenoic acid (DHA) affects protein kinase B (Akt)/mammalian target of rapamycin (mTOR)/unc51 like autophagy activating kinase 1 (ULK1) axis. (A, B, C) Mouse insulinoma 6 (MIN6) cells were incubated with 40 μM DHA in the presence or absence of 500 μM palmitate for 24 hours. Whole cell lysates were extracted and analyzed by immunoblotting with antibodies specific for ULK1, Beclin-1, mTOR phosphorylated on S2448 or S2481, or total mTOR (t-mTOR), phosphorylated Akt (p-Akt), total Akt (t-Akt), and β-actin. (D) The ratio to β-actin was described. All values are expressed as the mean±standard error of the mean (n=3–4). VEH, vehicle; PA, palmitic acid. aP<0.05, bP<0.01 compared with the VEH group; cP<0.05, dP<0.01, eP<0.001 compared with the PA group.

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  Fig. 5. Inhibition of G protein-coupled receptor 120 (GPR120) strictly blocks the docosahexaenoic acid (DHA) effect in mouse insulinoma 6 (MIN6) cells. MIN6 cells were pretreated with 20 μM AH7614 or dimethyl sulfoxide (DMSO) for 2 hours, and then incubated with palmitate/DHA for 24 hours. (A) Whole cell lysates (WCLs) were extracted and analyzed by immunoblotting with antibodies specific for phosphorylated protein kinase B (p-Akt), total Akt (t-Akt), mammalian target of rapamycin (mTOR) phosphorylated on S2448 or S2481, or total mTOR (t-mTOR), unc51 like autophagy activating kinase 1 (ULK1), Beclin-1 light chain 3 (LC3) II/I, and β-actin. (B) The ratio of each factor’s band intensity to β-actin was described. (C) Cleaved caspase-3 (c-Casp3) and total caspase-3 (t-Casp3) were measured by immunoblotting analysis, and the level of c-Casp3 was normalized to t-Casp3/β-actin. All values are expressed as the mean±standard error of the mean (n=3). VEH (V), vehicle; PA (P), palmitic acid; NS, non-significant. aP<0.01 compared with the VEH group of DMSO set; bP<0.05 compared with the PA group of DMSO set; cP<0.01 compared with VEH group of AH7614 set.

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  Fig. 6. Autophagy activation by G protein-coupled receptor 120 (GPR120) agonist via protein kinase B (Akt)/mammalian target of rapamycin (mTOR) axis. Mouse insulinoma 6 (MIN6) cells were incubated with or without 500 μM palmitate and/or 50 μM GSK137647 for 24 hours. (A, B) Whole cell lysates were extracted and analyzed by immunoblotting with antibodies specific for phosphorylated Akt (p-Akt), total Akt (t-Akt), mTOR phosphorylated on S2448 or S2481, or total mTOR (t-mTOR), unc51 like autophagy activating kinase 1 (ULK1), Beclin-1 light chain 3 (LC3) I/II, and β-actin. All values are expressed as the mean±standard error of the mean (n=3–4). VEH, vehicle; PA, palmitic acid. aP<0.05, bP<0.001 compared with the VEH group; cP<0.05, dP<0.01, eP<0.001 compared with the PA group.

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