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Mitochondrial dysfunction and adipogenic reduction by prohibitin silencing in 3T3-L1 cells.

Liu D, Lin Y, Kang T, Huang B, Xu W, Garcia-Barrio M, Olatinwo M, Matthews R, Chen YE, Thompson WE - PLoS ONE (2012)

Bottom Line: However, little is known about the effect of mitochondrial PHBs in adipogenesis.Knockdown of PHB1 or PHB2 by oligonucleotide siRNA significantly reduced the expression of adipogenic markers, the accumulation of lipids and the phosphorylation of extracellular signal-regulated kinases.Our results suggest that PHBs are critical mediators in promoting 3T3-L1 adipocyte differentiation and may be the potential targets for obesity therapies.

View Article: PubMed Central - PubMed

Affiliation: Cardiovascular Research Institute, Morehouse School of Medicine, Atlanta, Georgia, United States of America. dliu@msm.edu

ABSTRACT
Increase in mitochondrial biogenesis has been shown to accompany brown and white adipose cell differentiation. Prohibitins (PHBs), comprised of two evolutionarily conserved proteins, prohibitin-1 (PHB1) and prohibitin-2 (PHB2), are present in a high molecular-weight complex in the inner membrane of mitochondria. However, little is known about the effect of mitochondrial PHBs in adipogenesis. In the present study, we demonstrate that the levels of both PHB1 and PHB2 are significantly increased during adipogenesis of 3T3-L1 preadipocytes, especially in mitochondria. Knockdown of PHB1 or PHB2 by oligonucleotide siRNA significantly reduced the expression of adipogenic markers, the accumulation of lipids and the phosphorylation of extracellular signal-regulated kinases. In addition, fragmentation of mitochondrial reticulum, loss of mitochondrial cristae, reduction of mitochondrial content, impairment of mitochondrial complex I activity and excessive production of ROS were observed upon PHB-silencing in 3T3-L1 cells. Our results suggest that PHBs are critical mediators in promoting 3T3-L1 adipocyte differentiation and may be the potential targets for obesity therapies.

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Silencing of PHBs reduced the adipogenesis in 3T3-L1 cells.A, B and C. 3T3-L1 preadipocytes were transfected with the siControl or siRNAs targeting PHB1 (siPHB1-1, siPHB1-2 and siPHB1-3) or PHB2 (siPHB2-1, siPHB2-2 and siPHB2-3), respectively. A and B. Cells were harvested for total RNA isolation and quantitative real-time PCR to determine the mRNA expression levels of PHB1 (A) and PHB2 (B) three days post transfection. 18S rRNA was used as an internal control. ** p<0.01 compared to siControl. C. Cells were harvested to determine the protein expression levels of PHB1 and PHB2 with Immunoblotting three days post transfection. β-actin was used as a loading control. D, E and F. 3T3-L1 cells were treated with adipogenic inducers (day 0) three days post transfection of siRNAs targeting PHB1or PHB2. siControl was used as the control for siPHBs. D. The levels of C/EBPβ at day1, and the levels of PHB1, PHB2, PPARγ and aP2 at day 7 were determined using immunoblotting. HSP90 was used as a loading control. E. The cells were stained with Oil Red O dye at day 7. The quantification of accumulated lipid was performed by readings on a spectrophotometer at 510 nm for cell-released dye. **p<0.01 compared to siControl. F. Phospho-ERK (p-ERK) in siRNA transfected 3T3-L1 cells was detected using immunoblotting at the indicated times of treatment with adipogenic inducer cocktail. ERK was used as a loading control.
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pone-0034315-g002: Silencing of PHBs reduced the adipogenesis in 3T3-L1 cells.A, B and C. 3T3-L1 preadipocytes were transfected with the siControl or siRNAs targeting PHB1 (siPHB1-1, siPHB1-2 and siPHB1-3) or PHB2 (siPHB2-1, siPHB2-2 and siPHB2-3), respectively. A and B. Cells were harvested for total RNA isolation and quantitative real-time PCR to determine the mRNA expression levels of PHB1 (A) and PHB2 (B) three days post transfection. 18S rRNA was used as an internal control. ** p<0.01 compared to siControl. C. Cells were harvested to determine the protein expression levels of PHB1 and PHB2 with Immunoblotting three days post transfection. β-actin was used as a loading control. D, E and F. 3T3-L1 cells were treated with adipogenic inducers (day 0) three days post transfection of siRNAs targeting PHB1or PHB2. siControl was used as the control for siPHBs. D. The levels of C/EBPβ at day1, and the levels of PHB1, PHB2, PPARγ and aP2 at day 7 were determined using immunoblotting. HSP90 was used as a loading control. E. The cells were stained with Oil Red O dye at day 7. The quantification of accumulated lipid was performed by readings on a spectrophotometer at 510 nm for cell-released dye. **p<0.01 compared to siControl. F. Phospho-ERK (p-ERK) in siRNA transfected 3T3-L1 cells was detected using immunoblotting at the indicated times of treatment with adipogenic inducer cocktail. ERK was used as a loading control.

Mentions: To investigate the possible roles of increasing PHBs during adipogenesis, we screened siRNA for effective knockdown of PHBs expression in this model. Three different siRNA oligonucleotides, siPHB1-1, siPHB1-2 and siPHB1-3, were used to target PHB1; while siPHB2-1, siPHB2-2 and siPHB2-3 were used to target PHB2. Commercially available universal siControl was used as a control. Three days after the transfection of siRNA in 3T3-L1 cells, the mRNA levels of PHB1 were markedly reduced by siPHB1-1 (68%), siPHB1-2 (56%) or siPHB1-3 (25%), whereas the mRNA levels of PHB2 were not significantly changed (Fig. 2A). Similarly, the mRNA levels of PHB2 were decreased by siPHB2-1 (64%), siPHB2-2 (38%) or siPHB2-3 (68%), whereas mRNA levels of PHB1 were virtually identical (Fig. 2B). Interestingly, the protein levels of both PHB1 and PHB2 could be down-regulated by either siPHB1 or siPHB2 transfection (Fig. 2C). These data suggest that, in mouse 3T3-L1 preadipocytes, PHB1 and PHB2 are present in an interdependent complex and are consistent with previous findings in yeast, C. elegans, MEFs, human HeLa cells and MCF-7 cells [12], [14], [16], [17], [18]. Our earlier studies showed that PHB silencing could increase cellular sensitivity to apoptosis [20]. In the current study, we did not see the induction of cleaved caspase-3 in 3T3-L1 cells by simply silencing PHB1 or PHB2 without an apoptotic inducer (data not shown), thus consistent with the observation in PHB2-deficient MEFs [18]. For all of the following experiments, siPHB1-1 and siPHB2-3 were used to knockdown PHB1 and PHB2, respectively.


Mitochondrial dysfunction and adipogenic reduction by prohibitin silencing in 3T3-L1 cells.

Liu D, Lin Y, Kang T, Huang B, Xu W, Garcia-Barrio M, Olatinwo M, Matthews R, Chen YE, Thompson WE - PLoS ONE (2012)

Silencing of PHBs reduced the adipogenesis in 3T3-L1 cells.A, B and C. 3T3-L1 preadipocytes were transfected with the siControl or siRNAs targeting PHB1 (siPHB1-1, siPHB1-2 and siPHB1-3) or PHB2 (siPHB2-1, siPHB2-2 and siPHB2-3), respectively. A and B. Cells were harvested for total RNA isolation and quantitative real-time PCR to determine the mRNA expression levels of PHB1 (A) and PHB2 (B) three days post transfection. 18S rRNA was used as an internal control. ** p<0.01 compared to siControl. C. Cells were harvested to determine the protein expression levels of PHB1 and PHB2 with Immunoblotting three days post transfection. β-actin was used as a loading control. D, E and F. 3T3-L1 cells were treated with adipogenic inducers (day 0) three days post transfection of siRNAs targeting PHB1or PHB2. siControl was used as the control for siPHBs. D. The levels of C/EBPβ at day1, and the levels of PHB1, PHB2, PPARγ and aP2 at day 7 were determined using immunoblotting. HSP90 was used as a loading control. E. The cells were stained with Oil Red O dye at day 7. The quantification of accumulated lipid was performed by readings on a spectrophotometer at 510 nm for cell-released dye. **p<0.01 compared to siControl. F. Phospho-ERK (p-ERK) in siRNA transfected 3T3-L1 cells was detected using immunoblotting at the indicated times of treatment with adipogenic inducer cocktail. ERK was used as a loading control.
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pone-0034315-g002: Silencing of PHBs reduced the adipogenesis in 3T3-L1 cells.A, B and C. 3T3-L1 preadipocytes were transfected with the siControl or siRNAs targeting PHB1 (siPHB1-1, siPHB1-2 and siPHB1-3) or PHB2 (siPHB2-1, siPHB2-2 and siPHB2-3), respectively. A and B. Cells were harvested for total RNA isolation and quantitative real-time PCR to determine the mRNA expression levels of PHB1 (A) and PHB2 (B) three days post transfection. 18S rRNA was used as an internal control. ** p<0.01 compared to siControl. C. Cells were harvested to determine the protein expression levels of PHB1 and PHB2 with Immunoblotting three days post transfection. β-actin was used as a loading control. D, E and F. 3T3-L1 cells were treated with adipogenic inducers (day 0) three days post transfection of siRNAs targeting PHB1or PHB2. siControl was used as the control for siPHBs. D. The levels of C/EBPβ at day1, and the levels of PHB1, PHB2, PPARγ and aP2 at day 7 were determined using immunoblotting. HSP90 was used as a loading control. E. The cells were stained with Oil Red O dye at day 7. The quantification of accumulated lipid was performed by readings on a spectrophotometer at 510 nm for cell-released dye. **p<0.01 compared to siControl. F. Phospho-ERK (p-ERK) in siRNA transfected 3T3-L1 cells was detected using immunoblotting at the indicated times of treatment with adipogenic inducer cocktail. ERK was used as a loading control.
Mentions: To investigate the possible roles of increasing PHBs during adipogenesis, we screened siRNA for effective knockdown of PHBs expression in this model. Three different siRNA oligonucleotides, siPHB1-1, siPHB1-2 and siPHB1-3, were used to target PHB1; while siPHB2-1, siPHB2-2 and siPHB2-3 were used to target PHB2. Commercially available universal siControl was used as a control. Three days after the transfection of siRNA in 3T3-L1 cells, the mRNA levels of PHB1 were markedly reduced by siPHB1-1 (68%), siPHB1-2 (56%) or siPHB1-3 (25%), whereas the mRNA levels of PHB2 were not significantly changed (Fig. 2A). Similarly, the mRNA levels of PHB2 were decreased by siPHB2-1 (64%), siPHB2-2 (38%) or siPHB2-3 (68%), whereas mRNA levels of PHB1 were virtually identical (Fig. 2B). Interestingly, the protein levels of both PHB1 and PHB2 could be down-regulated by either siPHB1 or siPHB2 transfection (Fig. 2C). These data suggest that, in mouse 3T3-L1 preadipocytes, PHB1 and PHB2 are present in an interdependent complex and are consistent with previous findings in yeast, C. elegans, MEFs, human HeLa cells and MCF-7 cells [12], [14], [16], [17], [18]. Our earlier studies showed that PHB silencing could increase cellular sensitivity to apoptosis [20]. In the current study, we did not see the induction of cleaved caspase-3 in 3T3-L1 cells by simply silencing PHB1 or PHB2 without an apoptotic inducer (data not shown), thus consistent with the observation in PHB2-deficient MEFs [18]. For all of the following experiments, siPHB1-1 and siPHB2-3 were used to knockdown PHB1 and PHB2, respectively.

Bottom Line: However, little is known about the effect of mitochondrial PHBs in adipogenesis.Knockdown of PHB1 or PHB2 by oligonucleotide siRNA significantly reduced the expression of adipogenic markers, the accumulation of lipids and the phosphorylation of extracellular signal-regulated kinases.Our results suggest that PHBs are critical mediators in promoting 3T3-L1 adipocyte differentiation and may be the potential targets for obesity therapies.

View Article: PubMed Central - PubMed

Affiliation: Cardiovascular Research Institute, Morehouse School of Medicine, Atlanta, Georgia, United States of America. dliu@msm.edu

ABSTRACT
Increase in mitochondrial biogenesis has been shown to accompany brown and white adipose cell differentiation. Prohibitins (PHBs), comprised of two evolutionarily conserved proteins, prohibitin-1 (PHB1) and prohibitin-2 (PHB2), are present in a high molecular-weight complex in the inner membrane of mitochondria. However, little is known about the effect of mitochondrial PHBs in adipogenesis. In the present study, we demonstrate that the levels of both PHB1 and PHB2 are significantly increased during adipogenesis of 3T3-L1 preadipocytes, especially in mitochondria. Knockdown of PHB1 or PHB2 by oligonucleotide siRNA significantly reduced the expression of adipogenic markers, the accumulation of lipids and the phosphorylation of extracellular signal-regulated kinases. In addition, fragmentation of mitochondrial reticulum, loss of mitochondrial cristae, reduction of mitochondrial content, impairment of mitochondrial complex I activity and excessive production of ROS were observed upon PHB-silencing in 3T3-L1 cells. Our results suggest that PHBs are critical mediators in promoting 3T3-L1 adipocyte differentiation and may be the potential targets for obesity therapies.

Show MeSH
Related in: MedlinePlus