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Knockdown of the fat mass and obesity gene disrupts cellular energy balance in a cell-type specific manner.

Pitman RT, Fong JT, Billman P, Puri N - PLoS ONE (2012)

Bottom Line: FTO siRNA in SH-SY5Y cells mediated mRNA knockdown (-82%), increased ATP concentrations by up to 46% (P = 0.013) compared to controls, and decreased phosphorylation of AMPk and Akt in SH-SY5Y by -52% and -46% respectively as seen by immunoblotting.Furthermore, glucose uptake was decreased in both SH-SY5Y (-51% p = 0.015) and 3T3-L1 cells (-30%, p = 0.0002).We also show that FTO knockdown decreases NPY mRNA expression in SH-SY5Y cells (-21%) through upregulation of pSTAT3 (118%).

View Article: PubMed Central - PubMed

Affiliation: Department of Biomedical Sciences, University of Illinois College of Medicine, Rockford, Illinois, United States of America.

ABSTRACT
Recent studies suggest that FTO variants strongly correlate with obesity and mainly influence energy intake with little effect on the basal metabolic rate. We suggest that FTO influences eating behavior by modulating intracellular energy levels and downstream signaling mechanisms which control energy intake and metabolism. Since FTO plays a particularly important role in adipocytes and in hypothalamic neurons, SH-SY5Y neuronal cells and 3T3-L1 adipocytes were used to understand how siRNA mediated knockdown of FTO expression alters cellular energy homeostasis. Cellular energy status was evaluated by measuring ATP levels using a luminescence assay and uptake of fluorescent glucose. FTO siRNA in SH-SY5Y cells mediated mRNA knockdown (-82%), increased ATP concentrations by up to 46% (P = 0.013) compared to controls, and decreased phosphorylation of AMPk and Akt in SH-SY5Y by -52% and -46% respectively as seen by immunoblotting. In contrast, FTO siRNA in 3T3-L1 cells decreased ATP concentration by -93% (p<0.0005), and increased AMPk and Akt phosphorylation by 204% and 70%, respectively suggesting that FTO mediates control of energy levels in a cell-type specific manner. Furthermore, glucose uptake was decreased in both SH-SY5Y (-51% p = 0.015) and 3T3-L1 cells (-30%, p = 0.0002). We also show that FTO knockdown decreases NPY mRNA expression in SH-SY5Y cells (-21%) through upregulation of pSTAT3 (118%). These results provide important evidence that FTO-variant linked obesity may be associated with altered metabolic functions through activation of downstream metabolic mediators including AMPk.

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Related in: MedlinePlus

Downregulation of FTO mRNA expression in SH-SY5Y cells and its effects on ATP concentration.(A) SH-SY5Y cells were transfected with siRNA for 24 hours and then incubated for a total of 48 hrs (−81.8%) or 72 hrs (−69.8%) post-transfection. Additionally, SH-SY5Y cells were transfected with siRNA for 48 hrs and then differentiated for 1 week (−59.3%). GAPD mRNA was used as a positive internal control. (B) 3T3-L1 cells were transfected as described above and FTO expression was decreased by −80.3% at 48 hrs post-transfection, 65.3% at 72 hrs post-transfection, and 59.6% after 48 hrs of transfection and 1 week of differentiation. (C, D) FTO knockdown modulates ATP levels in SH-SY5Y and 3T3-L1 cells. Cells were transfected with siRNA and then incubated for 48 hrs post-transfection or differentiated for 1 week. (C) ATP levels were increased in naïve SH-SY5Y cells treated with glucose free DMEM only (46%, p = 0.013), oligomycin (30%, p<0.0001), and differentiated SH-SY5Y cells treated with only glucose-free DMEM (16% p = 0.05), but decreased in differentiated SH-SY5Y cells treated with oligomycin (−16%, p = 0.19). (D) ATP levels were decreased in naïve 3T3-L1 cells treated with glucose free DMEM only (−93%, p = 0.0005), oligomycin (−76%, p = 0.00002), and differentiated 3T3-L1 cells treated with only glucose-free DMEM (−54% p = 0.0068), yet increased in differentiated 3T3-L1 cells treated with oligomycin (179%, p = 0.026). Values indicate percent of control. ATP results are average of 2 independent experiments performed in at least quadruplicate and analyzed by one-way ANOVA with post-hoc analysis with α at 0.05. (E, F) FTO knockdown does alter cell viability of SH-SY5Y and 3T3-L1 cells. Cells were transfected with siRNA, incubated for 48 hrs and viable cells were collected and counted. (E) No difference was found between FTO siRNA treated SH-SY5Y cells and Control siRNA treated cells (p = 0.35). In addition, no difference in the cell viability was detected in FTO siRNA treated SH-SY5Y cells after exposure to oligomycin (p = 0.77). (F) There is no difference in FTO siRNA or Control siRNA treated 3T3-L1 cells (p = 0.35) or in Control siRNA treated 3T3-L1 cells treated with oligomycin compared to FTO siRNA treated cells (p = 0.131). Cell viability results are the result of experiments performed in quadruplicate and analyzed by repeated measures ANOVA with α at 0.05.
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pone-0038444-g001: Downregulation of FTO mRNA expression in SH-SY5Y cells and its effects on ATP concentration.(A) SH-SY5Y cells were transfected with siRNA for 24 hours and then incubated for a total of 48 hrs (−81.8%) or 72 hrs (−69.8%) post-transfection. Additionally, SH-SY5Y cells were transfected with siRNA for 48 hrs and then differentiated for 1 week (−59.3%). GAPD mRNA was used as a positive internal control. (B) 3T3-L1 cells were transfected as described above and FTO expression was decreased by −80.3% at 48 hrs post-transfection, 65.3% at 72 hrs post-transfection, and 59.6% after 48 hrs of transfection and 1 week of differentiation. (C, D) FTO knockdown modulates ATP levels in SH-SY5Y and 3T3-L1 cells. Cells were transfected with siRNA and then incubated for 48 hrs post-transfection or differentiated for 1 week. (C) ATP levels were increased in naïve SH-SY5Y cells treated with glucose free DMEM only (46%, p = 0.013), oligomycin (30%, p<0.0001), and differentiated SH-SY5Y cells treated with only glucose-free DMEM (16% p = 0.05), but decreased in differentiated SH-SY5Y cells treated with oligomycin (−16%, p = 0.19). (D) ATP levels were decreased in naïve 3T3-L1 cells treated with glucose free DMEM only (−93%, p = 0.0005), oligomycin (−76%, p = 0.00002), and differentiated 3T3-L1 cells treated with only glucose-free DMEM (−54% p = 0.0068), yet increased in differentiated 3T3-L1 cells treated with oligomycin (179%, p = 0.026). Values indicate percent of control. ATP results are average of 2 independent experiments performed in at least quadruplicate and analyzed by one-way ANOVA with post-hoc analysis with α at 0.05. (E, F) FTO knockdown does alter cell viability of SH-SY5Y and 3T3-L1 cells. Cells were transfected with siRNA, incubated for 48 hrs and viable cells were collected and counted. (E) No difference was found between FTO siRNA treated SH-SY5Y cells and Control siRNA treated cells (p = 0.35). In addition, no difference in the cell viability was detected in FTO siRNA treated SH-SY5Y cells after exposure to oligomycin (p = 0.77). (F) There is no difference in FTO siRNA or Control siRNA treated 3T3-L1 cells (p = 0.35) or in Control siRNA treated 3T3-L1 cells treated with oligomycin compared to FTO siRNA treated cells (p = 0.131). Cell viability results are the result of experiments performed in quadruplicate and analyzed by repeated measures ANOVA with α at 0.05.

Mentions: To confirm appropriate downregulation of FTO in both naïve and differentiated cells, qPCR was performed at several time points after transfection, and one week after induction of differentiation. Naïve (undifferentiated) SH-SY5Y cells treated with siRNA against FTO exhibited a decrease in FTO mRNA expression by −81.8%, and −69.8% at 48 and 72 hours, respectively (Fig 1A). 3T3-L1 cells exhibited −80.3% and −65.3% decrease in FTO mRNA at 48 and 72 hours after transfection, respectively (Fig 1B). Additionally, sustained knockdown of FTO mRNA was validated after 1 week of differentiation in both SH-SY5Y (−59.3%) and 3T3-L1 cells (−59.6%) (Fig 1A and 1B).


Knockdown of the fat mass and obesity gene disrupts cellular energy balance in a cell-type specific manner.

Pitman RT, Fong JT, Billman P, Puri N - PLoS ONE (2012)

Downregulation of FTO mRNA expression in SH-SY5Y cells and its effects on ATP concentration.(A) SH-SY5Y cells were transfected with siRNA for 24 hours and then incubated for a total of 48 hrs (−81.8%) or 72 hrs (−69.8%) post-transfection. Additionally, SH-SY5Y cells were transfected with siRNA for 48 hrs and then differentiated for 1 week (−59.3%). GAPD mRNA was used as a positive internal control. (B) 3T3-L1 cells were transfected as described above and FTO expression was decreased by −80.3% at 48 hrs post-transfection, 65.3% at 72 hrs post-transfection, and 59.6% after 48 hrs of transfection and 1 week of differentiation. (C, D) FTO knockdown modulates ATP levels in SH-SY5Y and 3T3-L1 cells. Cells were transfected with siRNA and then incubated for 48 hrs post-transfection or differentiated for 1 week. (C) ATP levels were increased in naïve SH-SY5Y cells treated with glucose free DMEM only (46%, p = 0.013), oligomycin (30%, p<0.0001), and differentiated SH-SY5Y cells treated with only glucose-free DMEM (16% p = 0.05), but decreased in differentiated SH-SY5Y cells treated with oligomycin (−16%, p = 0.19). (D) ATP levels were decreased in naïve 3T3-L1 cells treated with glucose free DMEM only (−93%, p = 0.0005), oligomycin (−76%, p = 0.00002), and differentiated 3T3-L1 cells treated with only glucose-free DMEM (−54% p = 0.0068), yet increased in differentiated 3T3-L1 cells treated with oligomycin (179%, p = 0.026). Values indicate percent of control. ATP results are average of 2 independent experiments performed in at least quadruplicate and analyzed by one-way ANOVA with post-hoc analysis with α at 0.05. (E, F) FTO knockdown does alter cell viability of SH-SY5Y and 3T3-L1 cells. Cells were transfected with siRNA, incubated for 48 hrs and viable cells were collected and counted. (E) No difference was found between FTO siRNA treated SH-SY5Y cells and Control siRNA treated cells (p = 0.35). In addition, no difference in the cell viability was detected in FTO siRNA treated SH-SY5Y cells after exposure to oligomycin (p = 0.77). (F) There is no difference in FTO siRNA or Control siRNA treated 3T3-L1 cells (p = 0.35) or in Control siRNA treated 3T3-L1 cells treated with oligomycin compared to FTO siRNA treated cells (p = 0.131). Cell viability results are the result of experiments performed in quadruplicate and analyzed by repeated measures ANOVA with α at 0.05.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3367022&req=5

pone-0038444-g001: Downregulation of FTO mRNA expression in SH-SY5Y cells and its effects on ATP concentration.(A) SH-SY5Y cells were transfected with siRNA for 24 hours and then incubated for a total of 48 hrs (−81.8%) or 72 hrs (−69.8%) post-transfection. Additionally, SH-SY5Y cells were transfected with siRNA for 48 hrs and then differentiated for 1 week (−59.3%). GAPD mRNA was used as a positive internal control. (B) 3T3-L1 cells were transfected as described above and FTO expression was decreased by −80.3% at 48 hrs post-transfection, 65.3% at 72 hrs post-transfection, and 59.6% after 48 hrs of transfection and 1 week of differentiation. (C, D) FTO knockdown modulates ATP levels in SH-SY5Y and 3T3-L1 cells. Cells were transfected with siRNA and then incubated for 48 hrs post-transfection or differentiated for 1 week. (C) ATP levels were increased in naïve SH-SY5Y cells treated with glucose free DMEM only (46%, p = 0.013), oligomycin (30%, p<0.0001), and differentiated SH-SY5Y cells treated with only glucose-free DMEM (16% p = 0.05), but decreased in differentiated SH-SY5Y cells treated with oligomycin (−16%, p = 0.19). (D) ATP levels were decreased in naïve 3T3-L1 cells treated with glucose free DMEM only (−93%, p = 0.0005), oligomycin (−76%, p = 0.00002), and differentiated 3T3-L1 cells treated with only glucose-free DMEM (−54% p = 0.0068), yet increased in differentiated 3T3-L1 cells treated with oligomycin (179%, p = 0.026). Values indicate percent of control. ATP results are average of 2 independent experiments performed in at least quadruplicate and analyzed by one-way ANOVA with post-hoc analysis with α at 0.05. (E, F) FTO knockdown does alter cell viability of SH-SY5Y and 3T3-L1 cells. Cells were transfected with siRNA, incubated for 48 hrs and viable cells were collected and counted. (E) No difference was found between FTO siRNA treated SH-SY5Y cells and Control siRNA treated cells (p = 0.35). In addition, no difference in the cell viability was detected in FTO siRNA treated SH-SY5Y cells after exposure to oligomycin (p = 0.77). (F) There is no difference in FTO siRNA or Control siRNA treated 3T3-L1 cells (p = 0.35) or in Control siRNA treated 3T3-L1 cells treated with oligomycin compared to FTO siRNA treated cells (p = 0.131). Cell viability results are the result of experiments performed in quadruplicate and analyzed by repeated measures ANOVA with α at 0.05.
Mentions: To confirm appropriate downregulation of FTO in both naïve and differentiated cells, qPCR was performed at several time points after transfection, and one week after induction of differentiation. Naïve (undifferentiated) SH-SY5Y cells treated with siRNA against FTO exhibited a decrease in FTO mRNA expression by −81.8%, and −69.8% at 48 and 72 hours, respectively (Fig 1A). 3T3-L1 cells exhibited −80.3% and −65.3% decrease in FTO mRNA at 48 and 72 hours after transfection, respectively (Fig 1B). Additionally, sustained knockdown of FTO mRNA was validated after 1 week of differentiation in both SH-SY5Y (−59.3%) and 3T3-L1 cells (−59.6%) (Fig 1A and 1B).

Bottom Line: FTO siRNA in SH-SY5Y cells mediated mRNA knockdown (-82%), increased ATP concentrations by up to 46% (P = 0.013) compared to controls, and decreased phosphorylation of AMPk and Akt in SH-SY5Y by -52% and -46% respectively as seen by immunoblotting.Furthermore, glucose uptake was decreased in both SH-SY5Y (-51% p = 0.015) and 3T3-L1 cells (-30%, p = 0.0002).We also show that FTO knockdown decreases NPY mRNA expression in SH-SY5Y cells (-21%) through upregulation of pSTAT3 (118%).

View Article: PubMed Central - PubMed

Affiliation: Department of Biomedical Sciences, University of Illinois College of Medicine, Rockford, Illinois, United States of America.

ABSTRACT
Recent studies suggest that FTO variants strongly correlate with obesity and mainly influence energy intake with little effect on the basal metabolic rate. We suggest that FTO influences eating behavior by modulating intracellular energy levels and downstream signaling mechanisms which control energy intake and metabolism. Since FTO plays a particularly important role in adipocytes and in hypothalamic neurons, SH-SY5Y neuronal cells and 3T3-L1 adipocytes were used to understand how siRNA mediated knockdown of FTO expression alters cellular energy homeostasis. Cellular energy status was evaluated by measuring ATP levels using a luminescence assay and uptake of fluorescent glucose. FTO siRNA in SH-SY5Y cells mediated mRNA knockdown (-82%), increased ATP concentrations by up to 46% (P = 0.013) compared to controls, and decreased phosphorylation of AMPk and Akt in SH-SY5Y by -52% and -46% respectively as seen by immunoblotting. In contrast, FTO siRNA in 3T3-L1 cells decreased ATP concentration by -93% (p<0.0005), and increased AMPk and Akt phosphorylation by 204% and 70%, respectively suggesting that FTO mediates control of energy levels in a cell-type specific manner. Furthermore, glucose uptake was decreased in both SH-SY5Y (-51% p = 0.015) and 3T3-L1 cells (-30%, p = 0.0002). We also show that FTO knockdown decreases NPY mRNA expression in SH-SY5Y cells (-21%) through upregulation of pSTAT3 (118%). These results provide important evidence that FTO-variant linked obesity may be associated with altered metabolic functions through activation of downstream metabolic mediators including AMPk.

Show MeSH
Related in: MedlinePlus