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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 infection induces ER stress in MIN6 cells.(A) mRNA levels of GRP78 and CHOP. MIN6 cells were mock-infected or infected with 1.0 MOI HCV for 24, 48, 72 h. mRNA was analyzed by real-time RT-PCR. The results were normalized with the values obtained from actin in the same sample. Data are expressed as fold-increase relative to the values observed in mock control. The measurements were done in triplicates. Data represent means + SD of three independent experiments (n = 9). *P<0.05, compared with respective controls. (B) Immunoblot analysis of GRP78, CHOP and p-PERK corresponding to (A) probed with the indicated antibodies. Amounts of actin were measured as an internal control to verify equivalent sample loading. Immunoblots are representative of at least three independent experiments.
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pone-0038522-g006: HCV infection induces ER stress in MIN6 cells.(A) mRNA levels of GRP78 and CHOP. MIN6 cells were mock-infected or infected with 1.0 MOI HCV for 24, 48, 72 h. mRNA was analyzed by real-time RT-PCR. The results were normalized with the values obtained from actin in the same sample. Data are expressed as fold-increase relative to the values observed in mock control. The measurements were done in triplicates. Data represent means + SD of three independent experiments (n = 9). *P<0.05, compared with respective controls. (B) Immunoblot analysis of GRP78, CHOP and p-PERK corresponding to (A) probed with the indicated antibodies. Amounts of actin were measured as an internal control to verify equivalent sample loading. Immunoblots are representative of at least three independent experiments.

Mentions: Beta cells are very susceptible to endoplasmic reticulum (ER) stress and chronic ER stress during the development of diabetes could potentially lead to beta cells loss [29]. Therefore, we examined whether HCV infection induced ER stress in MIN6 cells. The 78-kDa glucose-regulated protein (GRP78) is an ER chaperone of which expression is increased upon ER stress. Both the mRNA and protein levels of GRP78 were found to be significantly elevated from 24 to 72 hpi specifically in HCV infected-MIN6 cells as compared to the control (Fig. 6). The expression patterns of another ER stress marker, PKR-like kinase (PERK), were also examined and exhibited an increased phosphorylation status in HCV-infected MIN6 cells (Fig. 6B). These data strongly suggested that the ER stress is an early cell response of MIN6 cells challenged with HCV. HCV infection also mediated an increased expression of C/EBP homologous protein (CHOP) (Fig. 6), which plays an important role as ER stress-induced cell death modulator. Taken together, these results demonstrated that ER stress might play an important role in HCV-induced apoptosis-like death of MIN6 cells.


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 infection induces ER stress in MIN6 cells.(A) mRNA levels of GRP78 and CHOP. MIN6 cells were mock-infected or infected with 1.0 MOI HCV for 24, 48, 72 h. mRNA was analyzed by real-time RT-PCR. The results were normalized with the values obtained from actin in the same sample. Data are expressed as fold-increase relative to the values observed in mock control. The measurements were done in triplicates. Data represent means + SD of three independent experiments (n = 9). *P<0.05, compared with respective controls. (B) Immunoblot analysis of GRP78, CHOP and p-PERK corresponding to (A) probed with the indicated antibodies. Amounts of actin were measured as an internal control to verify equivalent sample loading. Immunoblots are representative of at least three independent experiments.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3366942&req=5

pone-0038522-g006: HCV infection induces ER stress in MIN6 cells.(A) mRNA levels of GRP78 and CHOP. MIN6 cells were mock-infected or infected with 1.0 MOI HCV for 24, 48, 72 h. mRNA was analyzed by real-time RT-PCR. The results were normalized with the values obtained from actin in the same sample. Data are expressed as fold-increase relative to the values observed in mock control. The measurements were done in triplicates. Data represent means + SD of three independent experiments (n = 9). *P<0.05, compared with respective controls. (B) Immunoblot analysis of GRP78, CHOP and p-PERK corresponding to (A) probed with the indicated antibodies. Amounts of actin were measured as an internal control to verify equivalent sample loading. Immunoblots are representative of at least three independent experiments.
Mentions: Beta cells are very susceptible to endoplasmic reticulum (ER) stress and chronic ER stress during the development of diabetes could potentially lead to beta cells loss [29]. Therefore, we examined whether HCV infection induced ER stress in MIN6 cells. The 78-kDa glucose-regulated protein (GRP78) is an ER chaperone of which expression is increased upon ER stress. Both the mRNA and protein levels of GRP78 were found to be significantly elevated from 24 to 72 hpi specifically in HCV infected-MIN6 cells as compared to the control (Fig. 6). The expression patterns of another ER stress marker, PKR-like kinase (PERK), were also examined and exhibited an increased phosphorylation status in HCV-infected MIN6 cells (Fig. 6B). These data strongly suggested that the ER stress is an early cell response of MIN6 cells challenged with HCV. HCV infection also mediated an increased expression of C/EBP homologous protein (CHOP) (Fig. 6), which plays an important role as ER stress-induced cell death modulator. Taken together, these results demonstrated that ER stress might play an important role in HCV-induced apoptosis-like death of MIN6 cells.

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