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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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Effect of FTO knockdown on glucose uptake and downstream signaling in 3T3-L1 cells.(A) Cells were transfected with siRNA and then incubated 48 hrs post-transfection or differentiated for one week. Cells were then exposed to 80 µM 2-NBDG in DMEM media lacking glucose and sodium pyruvate for 5 minutes and glucose-associated fluorescence was measured. Glucose uptake was decreased in both naïve (−22%, p = 0.017) and differentiated (−30%, p = 0.0002) 3T3-L1 adipocytes. Y-axis represents Arbitrary Fluorescent Units (±SEM) from two independent experiments run in quintuplicate and analyzed by one-way ANOVA with post-hoc analysis. (B) FTO knockdown decreases AMPk activation. 3T3-L1 cells were transfected with siRNA and then incubated 48 hrs post-transfection or differentiated for one week and subsequently immunoblotted. pAMPk (Thr172) was increased in both naïve (204.4%) and differentiated cells (37.8%) while total AMPk was unchanged. (C) FTO knockdown increases Akt phosphorylation. pAkt (Ser473) was increased in both naïve (69.9%) and differentiated cells (24.9%) while total Akt was unchanged. Blots and densitometry data from a representative of two similar independent experiments.
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pone-0038444-g003: Effect of FTO knockdown on glucose uptake and downstream signaling in 3T3-L1 cells.(A) Cells were transfected with siRNA and then incubated 48 hrs post-transfection or differentiated for one week. Cells were then exposed to 80 µM 2-NBDG in DMEM media lacking glucose and sodium pyruvate for 5 minutes and glucose-associated fluorescence was measured. Glucose uptake was decreased in both naïve (−22%, p = 0.017) and differentiated (−30%, p = 0.0002) 3T3-L1 adipocytes. Y-axis represents Arbitrary Fluorescent Units (±SEM) from two independent experiments run in quintuplicate and analyzed by one-way ANOVA with post-hoc analysis. (B) FTO knockdown decreases AMPk activation. 3T3-L1 cells were transfected with siRNA and then incubated 48 hrs post-transfection or differentiated for one week and subsequently immunoblotted. pAMPk (Thr172) was increased in both naïve (204.4%) and differentiated cells (37.8%) while total AMPk was unchanged. (C) FTO knockdown increases Akt phosphorylation. pAkt (Ser473) was increased in both naïve (69.9%) and differentiated cells (24.9%) while total Akt was unchanged. Blots and densitometry data from a representative of two similar independent experiments.

Mentions: After determining the effect of FTO knockdown on cellular ATP levels, we wanted to examine additional metabolic traits modulated by FTO expression. To determine if FTO downregulation affects glucose uptake in SH-SY5Y neuronal cells and 3T3-L1 adipocytes, cells were treated with FTO siRNA and glucose uptake was determined by measuring uptake of fluorescent 2-NBDG. As shown in Figure 2A, glucose uptake into both naïve and differentiated SH-SY5Y cells was significantly decreased by −27% (FTO siRNA 98040±7898 (Fluorescence units (FU) vs Control siRNA 134220±4648 FU; p = 0.007; N = 5) and −51% (FTO siRNA 65952±7281 FU vs Control siRNA 135202±17647 FU; p = 0.015; N = 5), respectively. Additionally, glucose uptake into naïve and differentiated 3T3-L1 adipocytes was decreased by −22% (siRNA 35616±2501 FU vs Control siRNA 45745±3000 FU; p = 0.017; N = 5) and −30% (siRNA 122764±3634 FU vs Control siRNA 174581±3643 FU; p = 0.0002; N = 5), respectively (Fig 3A).


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)

Effect of FTO knockdown on glucose uptake and downstream signaling in 3T3-L1 cells.(A) Cells were transfected with siRNA and then incubated 48 hrs post-transfection or differentiated for one week. Cells were then exposed to 80 µM 2-NBDG in DMEM media lacking glucose and sodium pyruvate for 5 minutes and glucose-associated fluorescence was measured. Glucose uptake was decreased in both naïve (−22%, p = 0.017) and differentiated (−30%, p = 0.0002) 3T3-L1 adipocytes. Y-axis represents Arbitrary Fluorescent Units (±SEM) from two independent experiments run in quintuplicate and analyzed by one-way ANOVA with post-hoc analysis. (B) FTO knockdown decreases AMPk activation. 3T3-L1 cells were transfected with siRNA and then incubated 48 hrs post-transfection or differentiated for one week and subsequently immunoblotted. pAMPk (Thr172) was increased in both naïve (204.4%) and differentiated cells (37.8%) while total AMPk was unchanged. (C) FTO knockdown increases Akt phosphorylation. pAkt (Ser473) was increased in both naïve (69.9%) and differentiated cells (24.9%) while total Akt was unchanged. Blots and densitometry data from a representative of two similar independent experiments.
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Related In: Results  -  Collection

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pone-0038444-g003: Effect of FTO knockdown on glucose uptake and downstream signaling in 3T3-L1 cells.(A) Cells were transfected with siRNA and then incubated 48 hrs post-transfection or differentiated for one week. Cells were then exposed to 80 µM 2-NBDG in DMEM media lacking glucose and sodium pyruvate for 5 minutes and glucose-associated fluorescence was measured. Glucose uptake was decreased in both naïve (−22%, p = 0.017) and differentiated (−30%, p = 0.0002) 3T3-L1 adipocytes. Y-axis represents Arbitrary Fluorescent Units (±SEM) from two independent experiments run in quintuplicate and analyzed by one-way ANOVA with post-hoc analysis. (B) FTO knockdown decreases AMPk activation. 3T3-L1 cells were transfected with siRNA and then incubated 48 hrs post-transfection or differentiated for one week and subsequently immunoblotted. pAMPk (Thr172) was increased in both naïve (204.4%) and differentiated cells (37.8%) while total AMPk was unchanged. (C) FTO knockdown increases Akt phosphorylation. pAkt (Ser473) was increased in both naïve (69.9%) and differentiated cells (24.9%) while total Akt was unchanged. Blots and densitometry data from a representative of two similar independent experiments.
Mentions: After determining the effect of FTO knockdown on cellular ATP levels, we wanted to examine additional metabolic traits modulated by FTO expression. To determine if FTO downregulation affects glucose uptake in SH-SY5Y neuronal cells and 3T3-L1 adipocytes, cells were treated with FTO siRNA and glucose uptake was determined by measuring uptake of fluorescent 2-NBDG. As shown in Figure 2A, glucose uptake into both naïve and differentiated SH-SY5Y cells was significantly decreased by −27% (FTO siRNA 98040±7898 (Fluorescence units (FU) vs Control siRNA 134220±4648 FU; p = 0.007; N = 5) and −51% (FTO siRNA 65952±7281 FU vs Control siRNA 135202±17647 FU; p = 0.015; N = 5), respectively. Additionally, glucose uptake into naïve and differentiated 3T3-L1 adipocytes was decreased by −22% (siRNA 35616±2501 FU vs Control siRNA 45745±3000 FU; p = 0.017; N = 5) and −30% (siRNA 122764±3634 FU vs Control siRNA 174581±3643 FU; p = 0.0002; N = 5), respectively (Fig 3A).

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