Glucose phosphorylation is required for insulin-dependent mTOR signalling in the heart - PubMed (original) (raw)

Glucose phosphorylation is required for insulin-dependent mTOR signalling in the heart

Saumya Sharma et al. Cardiovasc Res. 2007.

Abstract

Objective: Insulin regulates both glucose uptake and postnatal cardiac growth. The anabolic effects of insulin are mediated by the mammalian target of rapamycin (mTOR), an evolutionarily conserved kinase which is also a convergence point between nutrient sensing and cell growth. We postulated that mTOR signalling in the heart requires the metabolism of glucose.

Methods: We interrogated the insulin-mediated mTOR signalling pathway in response to different metabolic interventions regulating substrate metabolism in the isolated working rat heart and in isolated cardiomyocytes.

Results: Although insulin enhanced Akt activity, phosphorylation of mTOR and its downstream targets (p70S6K and 4EBP1) required the addition of glucose. Glucose-dependent p70S6K phosphorylation was independent of the hexosamine biosynthetic pathway, the AMP kinase pathway, and the pentose phosphate pathway. However, inhibition of glycolysis downstream of hexokinase markedly enhanced p70S6K phosphorylation. Furthermore, 2-deoxyglucose activated p70S6K suggesting that phosphorylation of glucose is required for carbohydrate-mediated mTOR signalling in the heart. Lastly, we also found enhanced p70S6K phosphorylation in the hearts of diabetic rats.

Conclusion: Phosphorylation of glucose is necessary for insulin-dependent mTOR activity in the heart, suggesting a link between intermediary metabolism and cardiac growth.

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Figures

Figure 1

Figure 1. Immunoblots and densitometry ratios

A. Immunoblots (n=5) of mTOR signalling proteins in isolated working rat hearts perfused glucose (GLU) at 5mM and 15mM, and amino acids (AA) at physiologic plasma concentrations. Densitometry of the ratio of phosphorylated and total p70S6K (n=5). B. Immunoblots (n=5) of phosphorylated and total Akt and GSK 3β in isolated working rat hearts perfused, with GLU (5mM and 15mM), and AA. C. p70S6K signalling in working rat hearts perfused GLU (5mM) after animals were gavaged for 7 days with either vehicle or rapamycin. D. Immunoblot (n=3) of p70S6K signalling in neonatal cardiomyocytes incubated in PBS and insulin (1μg/ml) ± rapamycin (0.1μM), with the addition of glucose (5mM).* p< 0.05 compared to no insulin

Figure 2

Figure 2. Ratoinale for the experimental strategy

Glucose is taken up by glucose transporters (GLUT) and phosphorylated inside the myocyte by hexokinase. Glucose 6-phosphate may and enter a number of pathways. Glycolysis and glucose oxidation result in ATP generation which may inhibit AMP kinase. Fructose 6-phosphate is converted to glucosamine-6-phosphate and enters the hexosamine biosynthetic pathway ultimately leading to O-GlcNacylation of proteins. Glucose 6-phosphate can also enter the pentose phosphate pathway and generate xylulose-5-phophate. In our experiments we utilized several substrates, activators, or inhibitors to assess whether glucose 6-phosphate regulates mTOR activation. These are underlined. Inhibitors or activators are in italics as well. AICAR 5-aminoimidazole-4-carboxamide ribonucleotide. GFAT glutamine:fructose 6-phosphate amidotransferase, HK hexokinase.

Figure 3

Figure 3. Immunoblots

A. Phosphorylation of p70S6K (n=3) in working rat hearts perfused with GLU (5mM), insulin (40μU/ml), Glucosamine (5mM). B. p70S6K signalling in rat hearts perfused (n=6) with GLU (5mM) + insulin (40μU/ml) with the addition of azaserine (10 mM and 20 mM). C. Immunoblot of p70S6K, AMP kinase, ACC phosphorylation in rat hearts perfused with GLU (5mM) + Insulin with the addition of AICAR (0.4mM). D. Immunoblot (n=3) of p70S6K phosphorylation in neonatal cardiomyocytes incubated in Ham's F-10 media + insulin (1μg/ml) and AICAR (1mM) or azaserine (10mM). E. p70S6K phosphorylation in rat hearts perfused with PLC/AcAc + Insulin (40μU/ml) with the addition of xylitol (0.5mM) (n=3).

Figure 4

Figure 4. Immunoblots

A. p70S6K phosphorylation in rat hearts perfused with insulin (40μU/ml) with the addition of pyruvate (0.05mM)/lactate (0.5mM) (n=3). B. Immunoblot (n=3) of p70S6K signalling in neonatal cardiomyocytes incubates in PBS or media ± insulin (1μg/ml) with the addition of AICAR (1mM) or iodoacetate (10mM). C. ACC phosphorylation in the cardiomyocytes exposed to AICAR and iodoacetate. D. Immunoblot (n=3) of p70S6K signalling in neonatal cardiomyocytes incubates in PBS+ insulin (1μg/ml) with the addition of GLU (5 mM and 15mM), 3-O methylglucose (3-O-MG) (5mM and 15mM), or 2-deoxyglucose (2-DG) (5mM and 15mM). E. Phosphorylation of p70S6K (n=3) in rat hearts perfused with PLC/AcAc + insulin (40μU/ml) with the addition of 2-DG (5mM).

Figure 5

Figure 5. Immunoblots and densitometry ratios

A. p70S6K phosphorylation in ZDF rat hearts perfused with insulin (40μU/ml) with the addition of glucose (5 mM) alone or with glucose (5 mM) and sodium oleate (0.4mM) (n=3). B. Densitometry of the ratio of phosphorylated and total p70S6K (n=3) C. Akt phosphorylation in ZDF rat hearts perfused with insulin (40μU/ml) with the addition of glucose (5 mM) alone or with glucose (5 mM) and sodium oleate (0.4mM) (n=3). D. Densitometry of the ratio of phosphorylated and total Akt (n=3). * p<0.01 between ZDF and ZL rat hearts. # p<0.05 between GLU and GLU+OLE

Figure 6

Figure 6. The proposed scheme for glucose 6-phosphate mediated mTOR signalling in the heart

Insulin activates PI3 kinase and Akt, which increases the uptake of glucose (by translocation of GLUT4 to the cell surface). Once it is phosphorylated, glucose induces mTOR activation. Glucosamine is phosphorylated to glucosamine 6-phosphate, which likely participates in mTOR signalling. Glycogen breakdown provides alternative carbohydrate source for mTOR activation. It is unknown whether Akt/PKB directly participates in mTOR signalling in the heart. Abbreviations: GLUT, glucose transporter; RTK, receptor tyrosine kinase; IGF1, insulin-like growth factor 1; PKB, protein kinase B; mTOR, mammalian target of rapamycin.

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