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Brain isoform glycogen phosphorylase as a novel hepatic progenitor cell marker.

Huang YW, Chiu CC, Liang JD, Chiou LL, Huang GT, Yu MJ, Lee HS - PLoS ONE (2015)

Bottom Line: Immunoblotting results show that GPBB was expressed in two liver progenitor cell lines Lig-8 and WB-F344.Short hairpin RNA (shRNA)-mediated GPBB knockdown followed by glucose deprivation test shows that GPBB aids in liver progenitor cell survival under low glucose conditions.Furthermore, shRNA-mediated GPBB knockdown followed by SB-induced cell differentiation shows that reducing GPBB expression delayed liver progenitor cell differentiation.

View Article: PubMed Central - PubMed

Affiliation: Institute of Biotechnology, National Taiwan University, Taipei, Taiwan; Division of Medical Devices and Cosmetics, Food and Drug Administration, Ministry of Health and Welfare, Taipei, Taiwan.

ABSTRACT
An appropriate liver-specific progenitor cell marker is a stepping stone in liver regenerative medicine. Here, we report brain isoform glycogen phosphorylase (GPBB) as a novel liver progenitor cell marker. GPBB was identified in a protein complex precipitated by a monoclonal antibody Ligab generated from a rat liver progenitor cell line Lig-8. Immunoblotting results show that GPBB was expressed in two liver progenitor cell lines Lig-8 and WB-F344. The levels of GPBB expression decreased in the WB-F344 cells under sodium butyrate (SB)-induced cell differentiation, consistent with roles of GPBB as a liver progenitor cell marker. Short hairpin RNA (shRNA)-mediated GPBB knockdown followed by glucose deprivation test shows that GPBB aids in liver progenitor cell survival under low glucose conditions. Furthermore, shRNA-mediated GPBB knockdown followed by SB-induced cell differentiation shows that reducing GPBB expression delayed liver progenitor cell differentiation. We conclude that GPBB is a novel liver progenitor cell marker, which facilitates liver progenitor cell survival under low glucose conditions and cell differentiation.

No MeSH data available.


Related in: MedlinePlus

Survival of GPBB knockdown WB-F344 and Lig-8 cells under low glucose conditions.Control or lentivirus expressing shRNA3119 or shRNA3759 targeting GPBB was used to infect Lig-8 (A) and WB-F344 (B) cells. After puromycin selection, stably knockdown cells were cultured in mediums with various levels of glucose: high (4.5 g/L), low (1.0 g/L) or zero glucose/pyruvate for 24 hours. All cells in the supernatant and those attached to the plate were harvested and mixed. The nuclei were stained with propidium iodide before flow cytometry analysis. Cells with nucleus staining intensity less than those of the G0/G1 phase cells were considered apoptotic. Values are mean ± SEM. * P < 0.05 and ** P < 0.01, Student’s test against values of the control knockdown (shControl).
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pone.0122528.g008: Survival of GPBB knockdown WB-F344 and Lig-8 cells under low glucose conditions.Control or lentivirus expressing shRNA3119 or shRNA3759 targeting GPBB was used to infect Lig-8 (A) and WB-F344 (B) cells. After puromycin selection, stably knockdown cells were cultured in mediums with various levels of glucose: high (4.5 g/L), low (1.0 g/L) or zero glucose/pyruvate for 24 hours. All cells in the supernatant and those attached to the plate were harvested and mixed. The nuclei were stained with propidium iodide before flow cytometry analysis. Cells with nucleus staining intensity less than those of the G0/G1 phase cells were considered apoptotic. Values are mean ± SEM. * P < 0.05 and ** P < 0.01, Student’s test against values of the control knockdown (shControl).

Mentions: Given GP a key enzyme in glycogenolysis, we examined whether GPBB plays a role in liver progenitor cell survival in low glucose medium. Control and GPBB knockdown cells were cultured in medium containing various levels of glucose concentrations followed by flow cytometry analysis for apoptosis. As shown in Fig. 8A, the percentages of the cells undergoing apoptosis were similar (~ 10%) for the control and GPBB knockdown Lig-8 cells in the medium containing 4.5 g/L glucose. When the glucose levels were reduced to 1.0 g/L, the percentages of apoptotic cells significantly increased (19.31% ± 3.77% by shRNA3119 and 20.17% ± 3.70% by shRNA3759 vs. 10.81% ± 1.73% by shControl). The percentages of apoptotic cells increased even higher when glucose and pyruvate were both deprived from the medium (21.72% ± 4.24% by shRNA3119 and 24.05% ± 2.41% by shRNA3759 vs. 14.41% ± 1.03% by shControl). These results suggest that the liver progenitor cells depend on GPBB for survival under low glucose conditions. Similar results were observed in GPBB knockdown WB-F344 cells under low glucose conditions. The percentages of apoptotic cells were about 4% in medium containing 4.5 g/L glucose (Fig. 8B). In medium containing 1.0 g/L glucose, the apoptotic percentage increased to 10.94% ± 0.52% by shRNA3119 and 10.60% ± 0.06% by shRNA3759 compared to 2.36% ± 0.77% by shControl. When glucose and pyruvate were deprived, more than 70% of the GPBB knockdown WB-F344 cells underwent apoptosis (79.78% ± 1.07% by shRNA3119 and 72.45% ± 3.86% by shRNA3759 vs. 49.93% ± 4.47% by shControl). Note that the apoptotic percentage of the control cells was reduced, from 18.03% in high glucose medium (4.5 g/L) to 2.36% in low glucose medium (1.0 g/L).


Brain isoform glycogen phosphorylase as a novel hepatic progenitor cell marker.

Huang YW, Chiu CC, Liang JD, Chiou LL, Huang GT, Yu MJ, Lee HS - PLoS ONE (2015)

Survival of GPBB knockdown WB-F344 and Lig-8 cells under low glucose conditions.Control or lentivirus expressing shRNA3119 or shRNA3759 targeting GPBB was used to infect Lig-8 (A) and WB-F344 (B) cells. After puromycin selection, stably knockdown cells were cultured in mediums with various levels of glucose: high (4.5 g/L), low (1.0 g/L) or zero glucose/pyruvate for 24 hours. All cells in the supernatant and those attached to the plate were harvested and mixed. The nuclei were stained with propidium iodide before flow cytometry analysis. Cells with nucleus staining intensity less than those of the G0/G1 phase cells were considered apoptotic. Values are mean ± SEM. * P < 0.05 and ** P < 0.01, Student’s test against values of the control knockdown (shControl).
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pone.0122528.g008: Survival of GPBB knockdown WB-F344 and Lig-8 cells under low glucose conditions.Control or lentivirus expressing shRNA3119 or shRNA3759 targeting GPBB was used to infect Lig-8 (A) and WB-F344 (B) cells. After puromycin selection, stably knockdown cells were cultured in mediums with various levels of glucose: high (4.5 g/L), low (1.0 g/L) or zero glucose/pyruvate for 24 hours. All cells in the supernatant and those attached to the plate were harvested and mixed. The nuclei were stained with propidium iodide before flow cytometry analysis. Cells with nucleus staining intensity less than those of the G0/G1 phase cells were considered apoptotic. Values are mean ± SEM. * P < 0.05 and ** P < 0.01, Student’s test against values of the control knockdown (shControl).
Mentions: Given GP a key enzyme in glycogenolysis, we examined whether GPBB plays a role in liver progenitor cell survival in low glucose medium. Control and GPBB knockdown cells were cultured in medium containing various levels of glucose concentrations followed by flow cytometry analysis for apoptosis. As shown in Fig. 8A, the percentages of the cells undergoing apoptosis were similar (~ 10%) for the control and GPBB knockdown Lig-8 cells in the medium containing 4.5 g/L glucose. When the glucose levels were reduced to 1.0 g/L, the percentages of apoptotic cells significantly increased (19.31% ± 3.77% by shRNA3119 and 20.17% ± 3.70% by shRNA3759 vs. 10.81% ± 1.73% by shControl). The percentages of apoptotic cells increased even higher when glucose and pyruvate were both deprived from the medium (21.72% ± 4.24% by shRNA3119 and 24.05% ± 2.41% by shRNA3759 vs. 14.41% ± 1.03% by shControl). These results suggest that the liver progenitor cells depend on GPBB for survival under low glucose conditions. Similar results were observed in GPBB knockdown WB-F344 cells under low glucose conditions. The percentages of apoptotic cells were about 4% in medium containing 4.5 g/L glucose (Fig. 8B). In medium containing 1.0 g/L glucose, the apoptotic percentage increased to 10.94% ± 0.52% by shRNA3119 and 10.60% ± 0.06% by shRNA3759 compared to 2.36% ± 0.77% by shControl. When glucose and pyruvate were deprived, more than 70% of the GPBB knockdown WB-F344 cells underwent apoptosis (79.78% ± 1.07% by shRNA3119 and 72.45% ± 3.86% by shRNA3759 vs. 49.93% ± 4.47% by shControl). Note that the apoptotic percentage of the control cells was reduced, from 18.03% in high glucose medium (4.5 g/L) to 2.36% in low glucose medium (1.0 g/L).

Bottom Line: Immunoblotting results show that GPBB was expressed in two liver progenitor cell lines Lig-8 and WB-F344.Short hairpin RNA (shRNA)-mediated GPBB knockdown followed by glucose deprivation test shows that GPBB aids in liver progenitor cell survival under low glucose conditions.Furthermore, shRNA-mediated GPBB knockdown followed by SB-induced cell differentiation shows that reducing GPBB expression delayed liver progenitor cell differentiation.

View Article: PubMed Central - PubMed

Affiliation: Institute of Biotechnology, National Taiwan University, Taipei, Taiwan; Division of Medical Devices and Cosmetics, Food and Drug Administration, Ministry of Health and Welfare, Taipei, Taiwan.

ABSTRACT
An appropriate liver-specific progenitor cell marker is a stepping stone in liver regenerative medicine. Here, we report brain isoform glycogen phosphorylase (GPBB) as a novel liver progenitor cell marker. GPBB was identified in a protein complex precipitated by a monoclonal antibody Ligab generated from a rat liver progenitor cell line Lig-8. Immunoblotting results show that GPBB was expressed in two liver progenitor cell lines Lig-8 and WB-F344. The levels of GPBB expression decreased in the WB-F344 cells under sodium butyrate (SB)-induced cell differentiation, consistent with roles of GPBB as a liver progenitor cell marker. Short hairpin RNA (shRNA)-mediated GPBB knockdown followed by glucose deprivation test shows that GPBB aids in liver progenitor cell survival under low glucose conditions. Furthermore, shRNA-mediated GPBB knockdown followed by SB-induced cell differentiation shows that reducing GPBB expression delayed liver progenitor cell differentiation. We conclude that GPBB is a novel liver progenitor cell marker, which facilitates liver progenitor cell survival under low glucose conditions and cell differentiation.

No MeSH data available.


Related in: MedlinePlus