Cathepsin B contributes to autophagy-related 7 (Atg7)-induced nod-like receptor 3 (NLRP3)-dependent proinflammatory response and aggravates lipotoxicity in rat insulinoma cell line

Abstract

Impairment of glucose-stimulated insulin secretion caused by the lipotoxicity of palmitate was found in β-cells. Recent studies have indicated that defects in autophagy contribute to pathogenesis in type 2 diabetes. Here, we report that autophagy-related 7 (Atg7) induced excessive autophagic activation in INS-1(823/13) cells exposed to saturated fatty acids. Atg7-induced cathepsin B (CTSB) overexpression resulted in an unexpected significant increase in proinflammatory chemokine and cytokine production levels of IL-1β, monocyte chemotactic protein-1, IL-6, and TNF-α. Inhibition of receptor-interacting protein did not affect the inflammatory response, ruling out involvement of necrosis. CTSB siRNA suppressed the inflammatory response but did not affect apoptosis significantly, suggesting that CTSB was a molecular linker between autophagy and the proinflammatory response. Blocking caspase-3 suppressed apoptosis but did not affect the inflammatory response, suggesting that CTSB induced inflammatory effects independently of apoptosis. Silencing of Nod-like receptor 3 (NLRP3) completely abolished both IL-1β secretion and the down-regulation effects of Atg7-induced CTSB overexpression on glucose-stimulated insulin secretion impairment, thus identifying the NLRP3 inflammasome as an autophagy-responsive element in the pancreatic INS-1(823/13) cell line. Combined together, our results indicate that CTSB contributed to the Atg7-induced NLRP3-dependent proinflammatory response, resulting in aggravation of lipotoxicity, independently of apoptosis in the pancreatic INS-1(823/13) cell line.

Keywords: Autophagy; Cytokine; Glucose Metabolism; Inflammation; Insulin Resistance; Pancreatic Islets.

FIGURE 1.

0.5 mm PA stimulated proper autophagy activation in INS-1(823/13) cells. A, PA stimulated expression of autophagy activation-associated proteins LC3II/LC3I, p62, lysosome-associated membrane protein (Lamp2), and CTSB. INS-1(823/13) cells treated with 0, 0.25, 0.5, and 1 mm PA were analyzed by immunoblotting. β-Actin was used as a control. B, ratio of LC3II/actin was analyzed in INS-1(823/13) cells treated with 0, 0.25, 0.5, and 1 mm PA for 12 h (**, p < 0.01). C and D, cells stained with LC3 antibody followed by secondary antibody (green) were visualized by confocal microscopy. INS-1(823/13) cells treated with 0.5 mm PA (D) were compared with control (0 mm PA) (C). E, the number of cytoplasmic puncta/cell is shown. Data are expressed as mean ± S.D. (error bars) from three independent experiments (analyzed by t test; **, p < 0.01 compared with control group).

FIGURE 2.

0.5 mm PA induced appropriate INS cell apoptosis as assessed by morphological and FACS analyses. A–L, images of representative fields were captured at a magnification of 40× under fluorescence microscope (Nikon microscope, Japan). INS-1(823/13) cells treated with 0, 0.25, 0.5, and 1 mm PA (from left to right) and stained with DAPI (A–D), Annexin V (AnnV; E–H), and PI (I–L) were observed under a fluorescence microscope. M, the induction of apoptosis was determined by flow cytometric analysis of Annexin V-FITC and PI staining after treatment with 0, 0.25, 0.5, and 1 mm PA (starting from left to right). Cells in the lower right quadrant are Annexin-positive, early apoptotic cells. The cells in the upper right quadrant are Annexin-positive/PI-positive, late apoptotic cells. N, the percentages of INS-1(823/13) cell apoptosis showing a greater but appropriate degree of apoptosis in the 0.5 mm PA group compared with the control and the group treated with 1 mm PA. The data are expressed as the mean ± S.D. (error bars) of three to five independent experiments with each condition performed in duplicate (**, p < 0.01).

FIGURE 3.

Atg7-induced excessive autophagy activation potentiated inflammatory cytokine production in INS-1(823/13) cells exposed to PA. A, Atg7 transfection induced excessive autophagy activation. The Atg7-transfected 0.5 mm PA-treated (Atg7 + 0.5 mm) group showed increased Atg7, LC3II, and CTSB expression compared with control treated with 0.5 mm PA or mock control (Jab-1 + 0.5 mm PA). B, relative levels of LC3II/actin and CTSB/actin were significantly higher in the 0.5 mm PA + Atg7 group compared with both the 0.5 mm PA group and the 0.5 mm PA + Jab-1 group (* and #, p < 0.05) (n = 6). C, relative mRNA expression levels of the proinflammatory cytokines TNF-α, IL-1β, IL-6, and MCP-1 were determined by real time PCR. Data are expressed as the mean ± S.D. (error bars) (**, p < 0.01) (n = 6). The protein levels of proinflammatory cytokines were examined by Western blot of the INS-1(823/13) cells in the 0.5 mm PA group, the 0.5 mm PA + Atg7 group, and the 0.5 mm PA + Jab-1 group. D, representative Western blot showing protein expression levels of Atg7 and LC3II/LC3I of cells that were incubated for 8 h with 0.5 mm PA + Atg7 in the presence of 10 mm 3-methyladenine (3-MA) or 30 μm necrostatin-1 (left panel). Relative mRNA expression levels of the proinflammatory cytokines TNF-α, IL-1β, IL-6, and MCP-1 were determined by real time PCR (right panel). Data are expressed as the mean ± S.D. (error bars) (**, p < 0.01) (n = 6).

FIGURE 4.

Blockage of CTSB suppressed Atg7-induced inflammatory response. A, CTSB expression was completely inhibited by siRNA. The protein expression levels of Atg7 and CTSB and the ratio of LC3II/LC3I were assayed by Western blot analysis using cell-free lysates prepared from the culture. Actin was used as a loading control. Levels of Atg7/actin, LC3II/actin, and CTSB/actin were analyzed in INS-1(823/13) cells treated with 0.5 mm PA, Atg7 + 0.5 mm PA, and (Atg7 + 0.5 mm PA + siCTSB, respectively (*, #, and &, p < 0.05) (n = 6). B, cell apoptosis was determined by FACS in the 0.5 mm PA group, Atg7 + 0.5 mm PA group, and Atg7 + 0.5 mm PA + siCTSB group. There were no significant differences among them, although siRNA caused a lower trend of apoptosis. C, relative levels of mRNA of the proinflammatory cytokines were determined by real time PCR. Data are expressed as the mean ± S.D. (error bars) (**, p < 0.01) (n = 6).

FIGURE 5.

The caspase-3 inhibitor Z-DEVD-fmk suppressed apoptosis but did not affect inflammatory response. A, caspase-3 was completely inhibited by Z-DEVD-fmk (50 μm). The protein expression levels of caspase-3 and CTSB and the ratio of LC3II/LC3I were assayed by Western blot analysis using cell-free lysates prepared from the culture. Actin was used as a loading control. Levels of LC3II/actin, cleaved caspase-3 (Cl-Cas3)/actin, and CTSB/actin were analyzed in INS-1(823/13) cells treated with 0.5 mm PA, Atg7 + 0.5 mm PA, and Atg7 + 0.5 mm PA + Z-DEVD-fmk, respectively (* and &, p < 0.05) (n = 6). B, INS-1(823/13) cell apoptosis under three different treatments was determined by FACS (**, p < 0.01) (n = 6). C, relative levels of mRNA of the proinflammatory cytokines of the three groups were determined by real time PCR. Data are expressed as the mean ± S.D. (error bars) (**, p < 0.01) (n = 6).

FIGURE 6.

NLRP3 knockdown blocked Atg7-induced proinflammatory response. A, INS-1(823/13) cells were stably transfected with siRNA that targets NLRP3, and protein levels of NLRP3 knockdown cells were analyzed by Western blot analysis. NLRP3 or non-target control (siControl) knockdown cells were treated with 0.5 mm PA and transfected with Atg7. Secreted IL-1β (B) and activated caspase-1 (C) were examined by Western blot analysis. Taken together, these data reveal that excessive autophagy activity induced caspase-1 activation and cytokine secretion through an NLRP3-dependent pathway.

FIGURE 7.

Atg7-induced excessive autophagy activation induced severe impairment of GSIS function. After equilibration at 3.3 mm glucose, INS-1(823/13) cells were stimulated with high glucose (16.7 mm) for 1 h. A, the glucose-stimulated insulin secretion in INS-1(823/13) cells was measured by RIA, and then the insulin secretion index was calculated (insulin release at high glucose/insulin release at basal glucose) (*, p < 0.05; **, p < 0.01) (n = 6). B, the insulin secretion in INS-1(823/13) cells in response to 3.0 and 16.7 mm glucose stimulation was examined (*, p < 0.05; **, p < 0.01) (n = 6). C, total insulin content in INS-1(823/13) cells was extracted by the acid/ethanol method and detected using an RIA kit. Data are expressed as the mean ± S.D. (error bars) (*, p < 0.05) (n = 6).