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Identification of fat mass and obesity associated (FTO) protein expression in cardiomyocytes: regulation by leptin and its contribution to leptin-induced hypertrophy.

Gan XT, Zhao G, Huang CX, Rowe AC, Purdham DM, Karmazyn M - PLoS ONE (2013)

Bottom Line: The recently-identified fat mass and obesity-associated (FTO) protein is associated with various physiological functions including energy and body weight regulation.Responses in cardiomyocytes were accompanied by JAK2/STAT3 activation whereas JAK2/STAT3 inhibition abolished these effects.Two other pro-hypertrophic factors, endothelin-1 or angiotensin II had no effect on FTO expression and FTO knockdown did not alter the hypertrophic response to either agent.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology and Pharmacology, The University of Western Ontario, London, Ontario, Canada.

ABSTRACT
The recently-identified fat mass and obesity-associated (FTO) protein is associated with various physiological functions including energy and body weight regulation. Ubiquitously expressed, FTO was identified in heart homogenates although its function is unknown. We studied whether FTO is specifically expressed within the cardiac myocyte and its potential role pertaining to the hypertrophic effect of the adipokine leptin. Most experiments were performed using cultured neonatal rat cardiomyocytes which showed nuclei-specific FTO expression. Leptin significantly increased FTO expression which was associated with myocyte hypertrophy although both events were abrogated by FTO knockdown with siRNA. Administration of a leptin receptor antibody to either normal or obese rats significant reduced myocardial FTO protein expression. Responses in cardiomyocytes were accompanied by JAK2/STAT3 activation whereas JAK2/STAT3 inhibition abolished these effects. Expression of the cut-like homeobox 1(CUX1) transcriptional factor was significantly increased by leptin although this was restricted to the cathepsin L-dependent, proteolytically-derived shorter p110CUX1 isoform whereas the longer p200CUX1 protein was not significantly affected. Cathepsin L expression and activity were both significantly increased by leptin whereas a cathepsin L peptide inhibitor or siRNA specific for CUX1 completely prevented the leptin-induced increase in FTO expression. The cathepsin L peptide inhibitor or siRNA-induced knockdown of either CUX1 or FTO abrogated the hypertrophic response to leptin. Two other pro-hypertrophic factors, endothelin-1 or angiotensin II had no effect on FTO expression and FTO knockdown did not alter the hypertrophic response to either agent. This study demonstrates leptin-induced FTO upregulation in cardiomyocytes via JAK2/STAT3- dependent CUX1 upregulation and suggests an FTO regulatory function of leptin. It also demonstrates for the first time a functional role of FTO in the cardiomyocyte.

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Upregulation of CUX1 by leptin.Panels A and B show that leptin increases CUX1 immunofluorescence intensity in cultured cardiomyocytes. Panels C to E demonstrate representative Western blots (C) and corresponding quantitative data (D, E) documenting increased protein expression of p110 CUX 1 with a non-significant effect on the p200CUX1 isoform. The increase in p110 CUX protein levels was abolished by the cathepsin L inhibitory peptide (CLi). Quantitative data are presented as mean +SEM. N = 7. *P<0.05 from control; +P<0.05 from respective group in the absence of CLi (-CLi). Horizontal bar in bottom right images indicates 200 µm.
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pone-0074235-g005: Upregulation of CUX1 by leptin.Panels A and B show that leptin increases CUX1 immunofluorescence intensity in cultured cardiomyocytes. Panels C to E demonstrate representative Western blots (C) and corresponding quantitative data (D, E) documenting increased protein expression of p110 CUX 1 with a non-significant effect on the p200CUX1 isoform. The increase in p110 CUX protein levels was abolished by the cathepsin L inhibitory peptide (CLi). Quantitative data are presented as mean +SEM. N = 7. *P<0.05 from control; +P<0.05 from respective group in the absence of CLi (-CLi). Horizontal bar in bottom right images indicates 200 µm.

Mentions: As CUX1 has been suggested as a possible regulator of FTO (please see Discussion), we sought to determine the effect of leptin on CUX1 and the potential role of the latter on leptin-induced FTO upregulation in cardiomyocytes. Total CUX1 as determined by immunofluorescence staining was significantly increased by leptin (Figure 5A and 5B). Individual CUX1 isoforms were measured using Western blotting (Figures 5C to 5E) which showed a significant increase in p110CUX1 protein expression with leptin treatment (Figure 5C and 5D). Although p200 CUX1protein levels tended to increase in response to leptin there were no significant differences between treatment groups (Figure 5C and 5E). The leptin-induced increase in p110 CUX1 protein expression was completely supressed by the cathepsin L peptide inhibitor (Figure 5C and 5D). siRNA directed at CUX1 produced a 80% reduction in basal FTO levels and a complete prevention of the ability of leptin to increase FTO expression (Figure 6).


Identification of fat mass and obesity associated (FTO) protein expression in cardiomyocytes: regulation by leptin and its contribution to leptin-induced hypertrophy.

Gan XT, Zhao G, Huang CX, Rowe AC, Purdham DM, Karmazyn M - PLoS ONE (2013)

Upregulation of CUX1 by leptin.Panels A and B show that leptin increases CUX1 immunofluorescence intensity in cultured cardiomyocytes. Panels C to E demonstrate representative Western blots (C) and corresponding quantitative data (D, E) documenting increased protein expression of p110 CUX 1 with a non-significant effect on the p200CUX1 isoform. The increase in p110 CUX protein levels was abolished by the cathepsin L inhibitory peptide (CLi). Quantitative data are presented as mean +SEM. N = 7. *P<0.05 from control; +P<0.05 from respective group in the absence of CLi (-CLi). Horizontal bar in bottom right images indicates 200 µm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3760875&req=5

pone-0074235-g005: Upregulation of CUX1 by leptin.Panels A and B show that leptin increases CUX1 immunofluorescence intensity in cultured cardiomyocytes. Panels C to E demonstrate representative Western blots (C) and corresponding quantitative data (D, E) documenting increased protein expression of p110 CUX 1 with a non-significant effect on the p200CUX1 isoform. The increase in p110 CUX protein levels was abolished by the cathepsin L inhibitory peptide (CLi). Quantitative data are presented as mean +SEM. N = 7. *P<0.05 from control; +P<0.05 from respective group in the absence of CLi (-CLi). Horizontal bar in bottom right images indicates 200 µm.
Mentions: As CUX1 has been suggested as a possible regulator of FTO (please see Discussion), we sought to determine the effect of leptin on CUX1 and the potential role of the latter on leptin-induced FTO upregulation in cardiomyocytes. Total CUX1 as determined by immunofluorescence staining was significantly increased by leptin (Figure 5A and 5B). Individual CUX1 isoforms were measured using Western blotting (Figures 5C to 5E) which showed a significant increase in p110CUX1 protein expression with leptin treatment (Figure 5C and 5D). Although p200 CUX1protein levels tended to increase in response to leptin there were no significant differences between treatment groups (Figure 5C and 5E). The leptin-induced increase in p110 CUX1 protein expression was completely supressed by the cathepsin L peptide inhibitor (Figure 5C and 5D). siRNA directed at CUX1 produced a 80% reduction in basal FTO levels and a complete prevention of the ability of leptin to increase FTO expression (Figure 6).

Bottom Line: The recently-identified fat mass and obesity-associated (FTO) protein is associated with various physiological functions including energy and body weight regulation.Responses in cardiomyocytes were accompanied by JAK2/STAT3 activation whereas JAK2/STAT3 inhibition abolished these effects.Two other pro-hypertrophic factors, endothelin-1 or angiotensin II had no effect on FTO expression and FTO knockdown did not alter the hypertrophic response to either agent.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology and Pharmacology, The University of Western Ontario, London, Ontario, Canada.

ABSTRACT
The recently-identified fat mass and obesity-associated (FTO) protein is associated with various physiological functions including energy and body weight regulation. Ubiquitously expressed, FTO was identified in heart homogenates although its function is unknown. We studied whether FTO is specifically expressed within the cardiac myocyte and its potential role pertaining to the hypertrophic effect of the adipokine leptin. Most experiments were performed using cultured neonatal rat cardiomyocytes which showed nuclei-specific FTO expression. Leptin significantly increased FTO expression which was associated with myocyte hypertrophy although both events were abrogated by FTO knockdown with siRNA. Administration of a leptin receptor antibody to either normal or obese rats significant reduced myocardial FTO protein expression. Responses in cardiomyocytes were accompanied by JAK2/STAT3 activation whereas JAK2/STAT3 inhibition abolished these effects. Expression of the cut-like homeobox 1(CUX1) transcriptional factor was significantly increased by leptin although this was restricted to the cathepsin L-dependent, proteolytically-derived shorter p110CUX1 isoform whereas the longer p200CUX1 protein was not significantly affected. Cathepsin L expression and activity were both significantly increased by leptin whereas a cathepsin L peptide inhibitor or siRNA specific for CUX1 completely prevented the leptin-induced increase in FTO expression. The cathepsin L peptide inhibitor or siRNA-induced knockdown of either CUX1 or FTO abrogated the hypertrophic response to leptin. Two other pro-hypertrophic factors, endothelin-1 or angiotensin II had no effect on FTO expression and FTO knockdown did not alter the hypertrophic response to either agent. This study demonstrates leptin-induced FTO upregulation in cardiomyocytes via JAK2/STAT3- dependent CUX1 upregulation and suggests an FTO regulatory function of leptin. It also demonstrates for the first time a functional role of FTO in the cardiomyocyte.

Show MeSH
Related in: MedlinePlus