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Hepatitis C virus induced a novel apoptosis-like death of pancreatic beta cells through a caspase 3-dependent pathway.

Wang Q, Chen J, Wang Y, Han X, Chen X - PLoS ONE (2012)

Bottom Line: Pancreatic beta cell failure is central to the progression of type 2 diabetes.Our experiments demonstrate that the HCV virion itself is indispensable and has a dose- and time-dependent cytopathic effect on the cells.However, the fact that HCV-infected cells exhibit a dilated, low-density nucleus with intact plasma and nuclear membrane indicates that a novel apoptosis-like death occurs.

View Article: PubMed Central - PubMed

Affiliation: State Key Lab of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, China.

ABSTRACT
Epidemiological and experimental studies have suggested that Hepatitis C virus (HCV) infection is associated with the development of type 2 diabetes. Pancreatic beta cell failure is central to the progression of type 2 diabetes. Using virus infection system, we investigate the influence of HCV infection on the fate of the insulinoma cell line, MIN6. Our experiments demonstrate that the HCV virion itself is indispensable and has a dose- and time-dependent cytopathic effect on the cells. HCV infection inhibits cell proliferation and induces death of MIN6 cells with apoptotic characteristics, including cell surface exposure of phosphatidylserine, decreased mitochondrial membrane potential, activation of caspase 3 and poly (ADP-ribose) polymerase, and DNA fragmentation in the nucleus. However, the fact that HCV-infected cells exhibit a dilated, low-density nucleus with intact plasma and nuclear membrane indicates that a novel apoptosis-like death occurs. HCV infection also causes endoplasmic reticulum (ER) stress. Further, HCV RNA replication was detected in MIN6 cells, although the infection efficiency is very low and no progeny virus particle generates. Taken together, our data suggest that HCV infection induces death of pancreatic beta cells through an ER stress-involved, caspase 3-dependent, special pathway.

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HCV played a direct role in the reduction of cell viability and induction of cell death of beta cells.(A, B) MIN6 cells were mock-infected (a) or infected with 1.0 MOI of the supernatant of HCV-infected Huh7.5.1 (b), ultracentrifugation-purified HCV particles (1.0 MOI) (c), the supernatant of HCV-infected Huh7.5.1 after ultracentrifugation (d), CON1 cell culture medium (e), or the supernatant of HCV-infected Huh7.5.1 after UV radiation treatment (f) at 96 hpi. Light microscopy images (scale bar, 10 µm) (A) were obtained and cell viability (B) of MIN6 cells was determined by MTT assay. (C, D) MIN6 cells were mock-infected or infected with HCV particles (1.0 MOI) with addition of indicated dose of RBV (C) or BILN 2061 (D) at 96 hpi, cell viability was assessed by MTT assay. (E) MIN6 cells were infected with HCV as in Figure 1B. At different time-points, cell number was determined by DNA content staining with the fluorescent dye PI. (F) MIN6 cells were treated as in (A). Cell death induced by the indicated treatment was measured by trypan blue exclusion. (A–F) All activity measurements were done in triplicates. Data represent means + SD of three independent experiments (n = 9). *P<0.05, **P<0.01, compared with respective controls.
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pone-0038522-g002: HCV played a direct role in the reduction of cell viability and induction of cell death of beta cells.(A, B) MIN6 cells were mock-infected (a) or infected with 1.0 MOI of the supernatant of HCV-infected Huh7.5.1 (b), ultracentrifugation-purified HCV particles (1.0 MOI) (c), the supernatant of HCV-infected Huh7.5.1 after ultracentrifugation (d), CON1 cell culture medium (e), or the supernatant of HCV-infected Huh7.5.1 after UV radiation treatment (f) at 96 hpi. Light microscopy images (scale bar, 10 µm) (A) were obtained and cell viability (B) of MIN6 cells was determined by MTT assay. (C, D) MIN6 cells were mock-infected or infected with HCV particles (1.0 MOI) with addition of indicated dose of RBV (C) or BILN 2061 (D) at 96 hpi, cell viability was assessed by MTT assay. (E) MIN6 cells were infected with HCV as in Figure 1B. At different time-points, cell number was determined by DNA content staining with the fluorescent dye PI. (F) MIN6 cells were treated as in (A). Cell death induced by the indicated treatment was measured by trypan blue exclusion. (A–F) All activity measurements were done in triplicates. Data represent means + SD of three independent experiments (n = 9). *P<0.05, **P<0.01, compared with respective controls.

Mentions: To exclude the effects of proinflammatory cytokines from the viral culture supernatants, we analyzed the direct effect of HCV virion. The purified HCV particles induced MIN6 morphologic changes and decreased the number of metabolically active MIN6 cells (Fig. 2Ac and B), whereas virion-depleted culture supernatant had no effect (Fig. 2Ad and B), demonstrating that the virus particles were account for the impairment of cell viability. This was further confirmed by CON1 cell (with HCV subgenomic replicon) culture medium and UV-inactivated HCV supernatants (Fig. 2Ae, f and B). Furthermore, the addition of ribavirin (RBV) and a HCV-specific inhibitor, BILN 2061 could rescue the purified HCV particles induced-decreased cell viability of MIN6 cells in a dose-dependent manner (Fig. 2C and D). RBV (50 µM) and BILN 2061 (5 µM) completely inhibited the cytopathogenic effect of HCV on MIN6 cells. These results supported the hypothesis that HCV played a direct role in the reduction of cell viability of insulinoma beta cell.


Hepatitis C virus induced a novel apoptosis-like death of pancreatic beta cells through a caspase 3-dependent pathway.

Wang Q, Chen J, Wang Y, Han X, Chen X - PLoS ONE (2012)

HCV played a direct role in the reduction of cell viability and induction of cell death of beta cells.(A, B) MIN6 cells were mock-infected (a) or infected with 1.0 MOI of the supernatant of HCV-infected Huh7.5.1 (b), ultracentrifugation-purified HCV particles (1.0 MOI) (c), the supernatant of HCV-infected Huh7.5.1 after ultracentrifugation (d), CON1 cell culture medium (e), or the supernatant of HCV-infected Huh7.5.1 after UV radiation treatment (f) at 96 hpi. Light microscopy images (scale bar, 10 µm) (A) were obtained and cell viability (B) of MIN6 cells was determined by MTT assay. (C, D) MIN6 cells were mock-infected or infected with HCV particles (1.0 MOI) with addition of indicated dose of RBV (C) or BILN 2061 (D) at 96 hpi, cell viability was assessed by MTT assay. (E) MIN6 cells were infected with HCV as in Figure 1B. At different time-points, cell number was determined by DNA content staining with the fluorescent dye PI. (F) MIN6 cells were treated as in (A). Cell death induced by the indicated treatment was measured by trypan blue exclusion. (A–F) All activity measurements were done in triplicates. Data represent means + SD of three independent experiments (n = 9). *P<0.05, **P<0.01, compared with respective controls.
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Related In: Results  -  Collection

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pone-0038522-g002: HCV played a direct role in the reduction of cell viability and induction of cell death of beta cells.(A, B) MIN6 cells were mock-infected (a) or infected with 1.0 MOI of the supernatant of HCV-infected Huh7.5.1 (b), ultracentrifugation-purified HCV particles (1.0 MOI) (c), the supernatant of HCV-infected Huh7.5.1 after ultracentrifugation (d), CON1 cell culture medium (e), or the supernatant of HCV-infected Huh7.5.1 after UV radiation treatment (f) at 96 hpi. Light microscopy images (scale bar, 10 µm) (A) were obtained and cell viability (B) of MIN6 cells was determined by MTT assay. (C, D) MIN6 cells were mock-infected or infected with HCV particles (1.0 MOI) with addition of indicated dose of RBV (C) or BILN 2061 (D) at 96 hpi, cell viability was assessed by MTT assay. (E) MIN6 cells were infected with HCV as in Figure 1B. At different time-points, cell number was determined by DNA content staining with the fluorescent dye PI. (F) MIN6 cells were treated as in (A). Cell death induced by the indicated treatment was measured by trypan blue exclusion. (A–F) All activity measurements were done in triplicates. Data represent means + SD of three independent experiments (n = 9). *P<0.05, **P<0.01, compared with respective controls.
Mentions: To exclude the effects of proinflammatory cytokines from the viral culture supernatants, we analyzed the direct effect of HCV virion. The purified HCV particles induced MIN6 morphologic changes and decreased the number of metabolically active MIN6 cells (Fig. 2Ac and B), whereas virion-depleted culture supernatant had no effect (Fig. 2Ad and B), demonstrating that the virus particles were account for the impairment of cell viability. This was further confirmed by CON1 cell (with HCV subgenomic replicon) culture medium and UV-inactivated HCV supernatants (Fig. 2Ae, f and B). Furthermore, the addition of ribavirin (RBV) and a HCV-specific inhibitor, BILN 2061 could rescue the purified HCV particles induced-decreased cell viability of MIN6 cells in a dose-dependent manner (Fig. 2C and D). RBV (50 µM) and BILN 2061 (5 µM) completely inhibited the cytopathogenic effect of HCV on MIN6 cells. These results supported the hypothesis that HCV played a direct role in the reduction of cell viability of insulinoma beta cell.

Bottom Line: Pancreatic beta cell failure is central to the progression of type 2 diabetes.Our experiments demonstrate that the HCV virion itself is indispensable and has a dose- and time-dependent cytopathic effect on the cells.However, the fact that HCV-infected cells exhibit a dilated, low-density nucleus with intact plasma and nuclear membrane indicates that a novel apoptosis-like death occurs.

View Article: PubMed Central - PubMed

Affiliation: State Key Lab of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, China.

ABSTRACT
Epidemiological and experimental studies have suggested that Hepatitis C virus (HCV) infection is associated with the development of type 2 diabetes. Pancreatic beta cell failure is central to the progression of type 2 diabetes. Using virus infection system, we investigate the influence of HCV infection on the fate of the insulinoma cell line, MIN6. Our experiments demonstrate that the HCV virion itself is indispensable and has a dose- and time-dependent cytopathic effect on the cells. HCV infection inhibits cell proliferation and induces death of MIN6 cells with apoptotic characteristics, including cell surface exposure of phosphatidylserine, decreased mitochondrial membrane potential, activation of caspase 3 and poly (ADP-ribose) polymerase, and DNA fragmentation in the nucleus. However, the fact that HCV-infected cells exhibit a dilated, low-density nucleus with intact plasma and nuclear membrane indicates that a novel apoptosis-like death occurs. HCV infection also causes endoplasmic reticulum (ER) stress. Further, HCV RNA replication was detected in MIN6 cells, although the infection efficiency is very low and no progeny virus particle generates. Taken together, our data suggest that HCV infection induces death of pancreatic beta cells through an ER stress-involved, caspase 3-dependent, special pathway.

Show MeSH
Related in: MedlinePlus