Clusterin Protects Lipotoxicity-Induced Apoptosis via Upregulation of Autophagy in Insulin-Secreting Cells

Abstract

Background: There is a great need to discover factors that could protect pancreatic β-cells from apoptosis and thus prevent diabetes mellitus. Clusterin (CLU), a chaperone protein, plays an important role in cell protection in numerous cells and is involved in various cellular mechanisms, including autophagy. In the present study, we investigated the protective role of CLU through autophagy regulation in pancreatic β-cells.

Methods: To identify the protective role of CLU, mouse insulinoma 6 (MIN6) cells were incubated with CLU and/or free fatty acid (FFA) palmitate, and cellular apoptosis and autophagy were examined.

Results: Treatment with CLU remarkably upregulated microtubule-associated protein 1-light chain 3 (LC3)-II conversion in a doseand time-dependent manner with a significant increase in the autophagy-related 3 (Atg3) gene expression level, which is a mediator of LC3-II conversion. Moreover, co-immunoprecipitation and fluorescence microscopy experiments showed that the molecular interaction of LC3 with Atg3 and p62 was markedly increased by CLU. Stimulation of LC3-II conversion by CLU persisted in lipotoxic conditions, and FFA-induced apoptosis and dysfunction were simultaneously improved by CLU treatment. Finally, inhibition of LC3-II conversion by Atg3 gene knockdown markedly attenuated the cytoprotective effect of CLU.

Conclusion: Taken together, these findings suggest that CLU protects pancreatic β-cells against lipotoxicity-induced apoptosis via autophagy stimulation mediated by facilitating LC3-II conversion. Thus, CLU has therapeutic effects on FFA-induced pancreatic β-cell dysfunction.

Keywords: Autophagy; Autophagy related protein 3; Clusterin; Insulin-secreting cells; LC3-II conversion.

Fig. 1

Clusterin (CLU) facilitates light chain 3 (LC3)-II conversion in mouse insulinoma 6 (MIN6) cells in a dose- and time-dependent manner. (A) MIN6 cells in one group were exposed to CLU for 24 hours at concentrations of 0, 1, 10, and 100 μg/L. MIN6 cells in another group were incubated with 10 μg/L CLU for 0, 6, 24, and 48 hours. LC3-I and -II were detected by Western blotting, and the level of LC3-II was normalized to LC3-I and β-actin. All values are expressed as mean±standard error of the mean (n=4–6). (B) LC3 in MIN6 cells was detected by immunolabeling after exposure to 10 μg/L CLU or vehicle (VEH) for 24 hours. The nuclei were visualized by 4′,6-diamidino-2-phenylindole (DAPI). Scale bars=20 μm. ^aP<0.01 and ^bP<0.001 compared to 0 μg/L treated cells; ^cP<0.001 and ^dP<0.05 compared to CLU-exposed cells for 0 hour.

Fig. 2

Clusterin (CLU) remarkably increases light chain 3 (LC3) binding with autophagy-related 3 (Atg3) and p62. Mouse insulinoma 6 (MIN6) cells were treated with 10 μg/L CLU for 24 hours, and protein levels were analyzed from whole lysates. (A) Levels of Atg3, Atg5, and Atg7 were detected by Western blotting and normalized to β-actin. (B, C) Morphological association of LC3 with Atg3 or p62 was analyzed by double immunolabeling using fluorescein isothiocyanate (FITC) and tetramethylrhodamine (TRITC), and co-localized areas are indicated by arrowheads. The nuclei were visualized by 4′,6-diamidino-2-phenylindole (DAPI). Scale bars=20 μm. (D) The number of double-positive cells was counted, and the results are expressed as mean±standard error of the mean per 100 DAPI-positive cells (n=3 in each treatment group). (E) Protein lysates were prepared for co-immunoprecipitation (IP) using LC3. Immunoblotting showed the co-precipitation of Atg3 and p62 with LC3. Rabbit immunoglobulin G (IgG) was used as a negative control. ^aP<0.001 and ^bP<0.05 compared to vehicle (VEH).

Fig. 3

Clusterin (CLU) protects pancreatic β-cells against lipotoxicity-induced apoptosis. Mouse insulinoma 6 (MIN6) cells were incubated with 0.5 mM palmitate (PA) in the presence or absence of 10 μg/L CLU for 24 hours. (A, B) Autophagy-inducing or -related proteins, including light chain 3 (LC3), autophagy-related 3 (Atg3), p62, beclin-1, Unc-51 like autophagy activating kinase (ULK)1, phosphor-mammalian target of rapamycin (mTOR), and β-actin were measured by Western blotting, and the respective ratios of LC3-II, Atg3, p62, and beclin-1 to LC3-I and β-actin were described (n=3–4 in each treatment group). (C, D) Terminal deoxynucleotidyl transferase-mediated UTP nick end labeling (TUNEL) staining was performed to detect apoptosis in MIN6 cells, and TUNEL-positive signals are indicated by red spots. Apoptotic cells are represented by arrowheads that indicate red spots in the nuclei. The nuclei were visualized by 4′,6-diamidino-2-phenylindole (DAPI). Scale bars=20 μm. The number of apoptotic cells was counted and described by a ratio to 100 cells (n=5–6). (E, F) The protein level of cleaved caspase-3 (c-casp3), a marker of apoptosis, was examined by Western blotting, and the level of c-casp3 was normalized to total caspase-3 (t-casp3). All values are expressed as mean±standard error of the mean (n=3–4). ^aP<0.05 and ^bP<0.01 compared to vehicle (VEH); ^cP<0.05 compared to PA.

Fig. 4

Knockdown of the autophagy-related 3 (Atg3) gene attenuates clusterin (CLU)-induced cell protection in pancreatic β-cells. Mouse insulinoma 6 (MIN6) cells were transfected with 10 nM small interfering RNA (siRNA) for the Atg3 gene or si-scramble (Scr), then incubated in 0 or 0.5 mM palmitate (PA) media with or without 10 μg/L CLU for 24 hours. (A–C) The levels of light chain 3 (LC3)-I, II, and Atg3 were analyzed by Western blotting and normalized to the LC3-I and β-actin levels of each sample, respectively. (D, E) Terminal deoxynucleotidyl transferase-mediated UTP nick end labeling (TUNEL) staining was performed to detect apoptosis after treatment, and apoptotic cells are indicated by arrowheads. The nuclei were visualized by 4′,6-diamidino-2-phenylindole (DAPI). Scale bars=20 μm. The number of apoptotic cells was counted and described by a ratio to 100 cells (n=4). (F, G) Cleaved caspase-3 (c-casp3) and total caspase-3 (t-casp3) were measured by Western blotting, and the level of c-casp3 was normalized to t-casp3. All values are expressed as mean±standard error of the mean (n=4). NS, non-significant; NT, non-transfection. ^aP<0.05 and ^bP<0.01 compared to vehicle (VEH) of the si-Scr group; ^cP<0.05, ^dP<0.01, and ^eP<0.001 compared to PA of the si-Scr group.