Autophagy Is Impaired in Neutrophils from Streptozotocin-Induced Diabetic Rats

Abstract

We tested the hypothesis that changes reported on functions of neutrophils from streptozotocin-induced diabetic rats involve autophagy impairment. Wistar rats were rendered diabetic by streptozotocin injection (65 mg/kg, i.v.), and the measurements were carried out 2 weeks afterward. Neutrophils were collected through intraperitoneal cavity lavage after 4 h of i.p. oyster glycogen type 2 injection. Neutrophils cultured with PMA (20 nM) for 1 h were used for analysis of plasma membrane integrity, DNA fragmentation, and mitochondrial depolarization by flow cytometry; expression of Atg5, Atg14, Beclin1, LC3BII, and Rab9 by RT-PCR; the contents of caspase 3, LC3BII/LC3BI, and pS6 by western blotting; ATP content by fluorescence essay; reactive oxygen species production by chemiluminescence (Luminol), and autophagy by immunofluorescence tracking LC3B cleavage. Herein, neutrophils from diabetic rats had high DNA fragmentation, depolarization of mitochondrial membrane, low content of ATP, and high content of cleaved caspase 3 after PMA stimulation. Neutrophils from diabetic rats also had low expression of LC3B, failed to increase the expression of Rab9 and Atg14 induced by PMA stimulation. Neutrophils from diabetic animals also had low cleavage of LC3BI to LC3BII and do not present punctate structures that label autophagosomal membranes after stimulus. The changes of neutrophil function reported in diabetic rats do involve impaired autophagy. The suppression of autophagy in neutrophils from diabetic rats may be associated with the activation of the mTOR signaling as indicated by the high content of pS6.

Keywords: autophagy; cell death and LC3B; diabetes type 1; neutrophil.

Figure 1

Cell integrity, DNA fragmentation, and mitochondrial transmembrane potential in neutrophils from diabetic and control rats at different time points after PMA stimulus. No differences in cell integrity and DNA fragmentation were observed between groups at the different time points. Neutrophils from the diabetic group had depolarization of mitochondrial transmembrane potential before and after 60 min PMA stimulus. Graphs (A) and (B) present the percentage of gated cells. Graph (C) represents the red/green fluorescence ratio of JC1. Analyses were performed in a FACSCalibur flow cytometer. Results are presented as mean ± SEM of six animals per group. (*) indicates p < 0.05 vs the control group before PMA stimulus; (**) indicates p < 0.01 vs the control group before PMA stimulus; (***) indicates p < 0.001 vs the control group before PMA stimulus. (##) indicates p < 0.01 vs the diabetic group before PMA stimulus; (###) indicates p < 0.001 vs the diabetic group before PMA stimulus.

Figure 2

Reactive oxygen species (ROS) production by neutrophils after PMA stimulus. Neutrophils from diabetic rats produced less ROS than the ones from controls. No difference was observed between groups without PMA stimulus. GFX and DPI abolished the production of ROS. Results represent relative chemiluminescence intensity ± SEM from 12 animals per group. (**) indicates p < 0.01.

Figure 3

Content of non-cleaved (35 kDa) and cleaved (17 and 19 kDa) caspase 3. PMA treatment augmented the content of cleaved caspase 3. (A) No differences were observed in the non-cleaved caspase 3. No statistical difference was observed between groups. (B) Neutrophils from diabetic rats without stimulus had higher content of 19 kDa cleaved caspase 3. (A,B) Graphs represent mean OD ± SEM of the bands from six animals per group. (*) indicates p < 0.05.

Figure 4

ATP content in neutrophils before and after PMA stimulus. Neutrophils from diabetic had lower content of ATP. PMA decreased the content of ATP in both groups. Results presented as concentration of ATP (μM) ± SEM from four animals per group. (*) indicates p < 0.05; (***) indicates p < 0.001.

Figure 5

Effect of reactive oxygen species on expression of genes related to autophagy. PMA augmented the expression of Atg14 (A) and Rab9 (D) in the control group and no statistic difference was observed in neutrophils from diabetic rats. LC3B expression was lower in the diabetic group (C). There were no differences in Beclin 1 (B) and Atg5 (E) expression. Results represent the relative expression of the genes ± SEM from 12 animals per group. RPL37a was used as a reference control gene. (*) indicates p < 0.05.

Figure 6

Contents of LC3BI and LC3BII in neutrophils before and after PMA stimulus. Neutrophils from diabetic rats had low cleavage of LC3BI to LC3BII when compared to the control group (A). Graph (B) presents mean OD ± SEM of the bands from six animals per group.

Figure 7

Immunofluorescence of LC3BII/I in neutrophils before and after PMA stimulus. Neutrophils from diabetic rats had low content of LC3BII/I before and after PMA stimulus when compared to the control group (A). Graph (B) presents MIF ± SEM of at least 30 cells per group. Analysis was performed cell by cell. The values were normalized by the control group. Green: LC3BII/I; blue (nucleus): DAPI. (*) indicates p < 0.05.

Figure 8

Contents of pS6 and S6 in neutrophils before and after PMA stimulus. Neutrophils from diabetic rats had high contents of pS6 and S6 when compared to the control group (A). Graphs (B) present mean OD ± SEM of the bands from six animals per group. (*) indicates p < 0.05; (*) indicates p < 0.01.