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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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Identification of FTO in cardiomyocytes and cardiac tissue and evidence for its upregulation by leptin.Panel A shows FTO gene expression in cultured rat ventricular myocytes under control conditions (Ctl) and its stimulation by leptin (L) and lack of effect in the presence of the leptin receptor receptor antagonist SHLA (A). Panels B and C show Western blots and their quantitative analyses with identical treatments as shown in panel A. These panels also show suppression of FTO in myocytes transfected with FTO siRNA. Panels D and E demonstrate Western blots and their quantitative assessment of whole hearts from control animals or animals fed a high fat diet (H) for 12 weeks. The results also show suppression of cardiac FTO protein expression in animals treated with an antibody directed against the leptin receptor (LRAb) every two days during the feeding period. Panels F and G show identical treatments as for panels D and E demonstrating inhibition of STAT3 phosphorylation in hearts of animals treated with the LRAb. Quantitative data are presented as mean+SEM. N = 8 for myocyte data in panels A and C and N = 5 for data shown in panels E and G. *P<0.05 from respective control group (or H group in panels E and G); +P<0.05 from respective group in the absence of siFTO (-siFTO).
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pone-0074235-g001: Identification of FTO in cardiomyocytes and cardiac tissue and evidence for its upregulation by leptin.Panel A shows FTO gene expression in cultured rat ventricular myocytes under control conditions (Ctl) and its stimulation by leptin (L) and lack of effect in the presence of the leptin receptor receptor antagonist SHLA (A). Panels B and C show Western blots and their quantitative analyses with identical treatments as shown in panel A. These panels also show suppression of FTO in myocytes transfected with FTO siRNA. Panels D and E demonstrate Western blots and their quantitative assessment of whole hearts from control animals or animals fed a high fat diet (H) for 12 weeks. The results also show suppression of cardiac FTO protein expression in animals treated with an antibody directed against the leptin receptor (LRAb) every two days during the feeding period. Panels F and G show identical treatments as for panels D and E demonstrating inhibition of STAT3 phosphorylation in hearts of animals treated with the LRAb. Quantitative data are presented as mean+SEM. N = 8 for myocyte data in panels A and C and N = 5 for data shown in panels E and G. *P<0.05 from respective control group (or H group in panels E and G); +P<0.05 from respective group in the absence of siFTO (-siFTO).

Mentions: Our first set of experiments was aimed to identify the presence of FTO in cultured myocytes and determine whether this can be modulated by leptin treatment. Figure 1 summarizes our results in which FTO gene expression was determined as well as protein levels assessed by Western blotting. As evident from Figure 1A, 24 h leptin treatment resulted in a three-fold increase in FTO gene expression. Moreover, Western blot analysis (Figure 1B and 1C) showed substantial abundance of FTO in myocytes as well as the ability of 24 h leptin treatment to produce approximately a two-fold elevation in FTO levels. These data also demonstrate that siRNA directed at FTO substantially decreased FTO protein levels (Figure 1B and 1C). Although leptin tended to increase FTO gene and protein expression in siFTO-treated myocytes no significant effect of leptin was seen under this condition. Moreover, all effects of leptin on FTO were, as would be expected, completely abrogated by the highly potent and specific leptin receptor antagonist SHLA.


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)

Identification of FTO in cardiomyocytes and cardiac tissue and evidence for its upregulation by leptin.Panel A shows FTO gene expression in cultured rat ventricular myocytes under control conditions (Ctl) and its stimulation by leptin (L) and lack of effect in the presence of the leptin receptor receptor antagonist SHLA (A). Panels B and C show Western blots and their quantitative analyses with identical treatments as shown in panel A. These panels also show suppression of FTO in myocytes transfected with FTO siRNA. Panels D and E demonstrate Western blots and their quantitative assessment of whole hearts from control animals or animals fed a high fat diet (H) for 12 weeks. The results also show suppression of cardiac FTO protein expression in animals treated with an antibody directed against the leptin receptor (LRAb) every two days during the feeding period. Panels F and G show identical treatments as for panels D and E demonstrating inhibition of STAT3 phosphorylation in hearts of animals treated with the LRAb. Quantitative data are presented as mean+SEM. N = 8 for myocyte data in panels A and C and N = 5 for data shown in panels E and G. *P<0.05 from respective control group (or H group in panels E and G); +P<0.05 from respective group in the absence of siFTO (-siFTO).
© Copyright Policy
Related In: Results  -  Collection

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

pone-0074235-g001: Identification of FTO in cardiomyocytes and cardiac tissue and evidence for its upregulation by leptin.Panel A shows FTO gene expression in cultured rat ventricular myocytes under control conditions (Ctl) and its stimulation by leptin (L) and lack of effect in the presence of the leptin receptor receptor antagonist SHLA (A). Panels B and C show Western blots and their quantitative analyses with identical treatments as shown in panel A. These panels also show suppression of FTO in myocytes transfected with FTO siRNA. Panels D and E demonstrate Western blots and their quantitative assessment of whole hearts from control animals or animals fed a high fat diet (H) for 12 weeks. The results also show suppression of cardiac FTO protein expression in animals treated with an antibody directed against the leptin receptor (LRAb) every two days during the feeding period. Panels F and G show identical treatments as for panels D and E demonstrating inhibition of STAT3 phosphorylation in hearts of animals treated with the LRAb. Quantitative data are presented as mean+SEM. N = 8 for myocyte data in panels A and C and N = 5 for data shown in panels E and G. *P<0.05 from respective control group (or H group in panels E and G); +P<0.05 from respective group in the absence of siFTO (-siFTO).
Mentions: Our first set of experiments was aimed to identify the presence of FTO in cultured myocytes and determine whether this can be modulated by leptin treatment. Figure 1 summarizes our results in which FTO gene expression was determined as well as protein levels assessed by Western blotting. As evident from Figure 1A, 24 h leptin treatment resulted in a three-fold increase in FTO gene expression. Moreover, Western blot analysis (Figure 1B and 1C) showed substantial abundance of FTO in myocytes as well as the ability of 24 h leptin treatment to produce approximately a two-fold elevation in FTO levels. These data also demonstrate that siRNA directed at FTO substantially decreased FTO protein levels (Figure 1B and 1C). Although leptin tended to increase FTO gene and protein expression in siFTO-treated myocytes no significant effect of leptin was seen under this condition. Moreover, all effects of leptin on FTO were, as would be expected, completely abrogated by the highly potent and specific leptin receptor antagonist SHLA.

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