Valproate pretreatment protects pancreatic β-cells from palmitate-induced ER stress and apoptosis by inhibiting glycogen synthase kinase-3β

Abstract

Background: Reduction of pancreatic β-cells mass, major secondary to increased β-cells apoptosis, is increasingly recognized as one of the main contributing factors to the pathogenesis of type 2 diabetes (T2D), and saturated free fatty acid palmitate has been shown to induce endoplasmic reticulum (ER) stress that may contribute to promoting β-cells apoptosis. Recent literature suggests that valproate, a diffusely prescribed drug in the treatment of epilepsy and bipolar disorder, can inhibit glycogen synthase kinase-3β (GSK-3β) activity and has cytoprotective effects in neuronal cells and HepG2 cells. Thus, we hypothesized that valproate may protect INS-1 β-cells from palmitate-induced apoptosis via inhibiting GSK-3β.

Results: Valproate pretreatment remarkable prevented palmitate-mediated cytotoxicity and apoptosis (lipotoxicity) as well as ER distension. Furthermore, palmitate triggered ER stress as evidenced by increased mRNA levels of C/EBP homologous protein (CHOP) and activating transcription factor 4 (ATF4) in a time-dependent fashion. However, valproate not only reduced the mRNA and protein expression of CHOP but also inhibited GSK-3β and caspase-3 activity induced by palmitate, whereas, the mRNA expression of ATF4 was not affected. Interestingly, TDZD-8, a specific GSK-3β inhibitor, also showed the similar effect on lipotoxicity and ER stress as valproate in INS-1 cells. Finally, compared with CHOP knockdown, valproate displayed better cytoprotection against palmitate.

Conclusions: Valproate may protect β-cells from palmitate-induced apoptosis and ER stress via GSK-3β inhibition, independent of ATF4/CHOP pathway. Besides, GSK-3β, rather than CHOP, may be a more promising therapeutic target for T2D.

Figure 1

Palmitate induces INS-1 cells lipotoxicity in a time-concentration dependent fashion. INS-1 cells were treated with 0.25% BSA, 0.5% BSA, 1.0% BSA, 0.25 mM palmitate/0.25% BSA (0.25 mM PA), 0.5 mM PA or 1.0 mM PA in medium without serum. (A) After the treatments for the times indicated on the X-axis, cell viability was assessed by CCK-8 assay. The figure shows representative results of three independent experiments. The given values are mean ± SD of at least five duplicate wells. **P < 0.01; ^§P < 0.001 vs. control (BSA groups). (B-C) After the treatments for 48 h, the cells were stained with Annexin V and PI and assessed by flow cytometry. B shows the percentage of apoptosis. Data are mean ± SD of three independent experiments. ^§P < 0.001 vs. control (BSA groups). C shows the representative result of INS-1 cells apoptosis and necrosis (1% BSA vs. 1 mM PA).

Figure 2

Effect of valproate on cell viability and palmitate-induced cytotoxicity in INS-1 cells. (A) INS-1 cells were exposed to different concentrations of valproate for 24 h or 48 h, then cell viability was measured by CCK-8 assay. The figure shows representative results of three independent experiments. The given values are mean ± SD of at least five duplicate wells. *P < 0.05; **P < 0.01; ^§P, ^βP < 0.001 vs. FBS group. (B) With or without VPA pretreatment for 48 h, INS-1 cells were exposed to 0.25 mM PA for 24 h, then cell viability was assessed by CCK-8 assay. The figure shows representative results of three independent experiments. The given values are mean ± SD of at least five duplicate wells. ^§P < 0.001 vs. 0.25 mM PA.

Figure 3

Valproate pretreatment prevents palmitate-induced apoptosis and ER distension. INS-1 cells were pretreated with or without 1 mM VPA for 48 h before challenged with 0.25 mM PA. (A) After the treatments for 24 h, apoptosis was assessed by staining cells with Hoechst 33342/PI (a-c), meanwhile the morphology of INS-1 cells was also captured (d-f). Apoptosis was characterized by nucleus condensed or fragmented (red arrows) that intensely stained with Hoechst 33342 (blue fluorescence). In each case five to seven microscopic fields were photographed randomly. Scale bars: a-f = 400 × magnification. (B) After the treatments for 24 h or 48 h, the cells were stained with Annexin V and PI, and the percentage of apoptosis was detected by flow cytometry. Data are mean ± SD of three independent experiments. **P < 0.01; ^§P < 0.001. (C) After the treatments for 24 h, (a-c) apoptosis and (d-f) morphology of ER were visualized by electron microscopy. The presence of marked chromatin condensation was considered to be a sign of apoptosis. ER distension is marked with red arrows. Scale bars: a-c = 2 μm; d-f = 1 μm.

Figure 4

Valproate pretreatment protects β-cells from palmitate-induced apoptosis via GSK-3β inhibition. (A) With presence or absence of 1 mM VPA pretreatment, INS-1 cells were exposed to 0.25 mM PA for 48 h. The expressions of phospho-GSK-3β and cleaved caspase-3 were detected by Western blot. GAPDH protein served as loading control. The intensity of protein bands was quantified by densitometry and expressed as fold change compared with control. Data are mean ± SD of three independent experiments. **P < 0.01; ^§P < 0.001. (B) INS-1 cells were pretreated with VPA, LiCl or TDZD-8 before challenged with 0.25 mM PA for the times indicated on the X-axis. After the treatments, cell viability was measured by CCK-8 assay. The figure shows representative results of three independent experiments. The given values are mean ± SD of at least five duplicate wells. ^§P < 0.001 vs. 0.25 mM PA; &P < 0.001 vs. 0.25 mM PA + 1 mM VPA.

Figure 5

Valproate and TDZD-8 pretreatment ameliorate palmitate-induced ER stress by reducing CHOP expression. With presence or absence of 1 mM VPA or 20 μM TDZD-8 pretreatment, INS-1 cells were exposed to 0.25 mM PA for 24 h (A), 0.5 mM PA for the times indicated on the X-axis (B-C), or 0.25 mM PA for 48 h (D). After the treatments, the expressions of ATF4 and CHOP mRNA (A-C) and CHOP protein level (D) were detected by RT-PCR and Western blot, respectively. The intensity of protein bands was quantified by densitometry. Results are presented as fold change compared with control. Data are mean ± SD of three independent experiments. *P < 0.05; **P < 0.01; ^§P < 0.001.

Figure 6

Valproate pretreatment, rather than CHOP knockdown, prevents palmitate-induced INS-1 cells lipotoxicity. (A-B) INS-1 cells were transfected with siCHOP for 48 h, the expressions of CHOP mRNA and protein were detected by RT-PCR and Western blot, respectively. Results are shown as fold change compared with control. Data are mean ± SD of three independent experiments. ^§P < 0.001 vs. control. (C) INS-1 cells were transfected with 50nM CHOP siRNAs for 48 h, then exposed to 0.25 mM PA for 24 h or 48 h. After the treatments, cell viability was assessed by CCK-8 assay. The figure shows representative results of three independent experiments. The given values are mean ± SD of at least five duplicate wells. *P < 0.05; ^§P < 0.001. (D) INS-1 cells were exposed to conditions indicated on the X-axis for 48 h. After the treatments, the percentage of apoptosis was measured by flow cytometry. Data are mean ± SD of three independent experiments. **P < 0.01.