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Hypoxia promotes glycogen accumulation through hypoxia inducible factor (HIF)-mediated induction of glycogen synthase 1.

Pescador N, Villar D, Cifuentes D, Garcia-Rocha M, Ortiz-Barahona A, Vazquez S, Ordoñez A, Cuevas Y, Saez-Morales D, Garcia-Bermejo ML, Landazuri MO, Guinovart J, del Peso L - PLoS ONE (2010)

Bottom Line: When oxygen becomes limiting, cells reduce mitochondrial respiration and increase ATP production through anaerobic fermentation of glucose.Significantly, knockdown of either HIF1alpha or GYS1 attenuated hypoxia-induced glycogen accumulation, while GYS1 overexpression was sufficient to mimic this effect.Altogether, these results indicate that GYS1 regulation by HIF plays a central role in the hypoxic accumulation of glycogen.

View Article: PubMed Central - PubMed

Affiliation: Departamento de Bioquímica, Universidad Autónoma de Madrid, Madrid, Spain.

ABSTRACT
When oxygen becomes limiting, cells reduce mitochondrial respiration and increase ATP production through anaerobic fermentation of glucose. The Hypoxia Inducible Factors (HIFs) play a key role in this metabolic shift by regulating the transcription of key enzymes of glucose metabolism. Here we show that oxygen regulates the expression of the muscle glycogen synthase (GYS1). Hypoxic GYS1 induction requires HIF activity and a Hypoxia Response Element within its promoter. GYS1 gene induction correlated with a significant increase in glycogen synthase activity and glycogen accumulation in cells exposed to hypoxia. Significantly, knockdown of either HIF1alpha or GYS1 attenuated hypoxia-induced glycogen accumulation, while GYS1 overexpression was sufficient to mimic this effect. Altogether, these results indicate that GYS1 regulation by HIF plays a central role in the hypoxic accumulation of glycogen. Importantly, we found that hypoxia also upregulates the expression of UTP:glucose-1-phosphate urydylyltransferase (UGP2) and 1,4-alpha glucan branching enzyme (GBE1), two enzymes involved in the biosynthesis of glycogen. Therefore, hypoxia regulates almost all the enzymes involved in glycogen metabolism in a coordinated fashion, leading to its accumulation. Finally, we demonstrated that abrogation of glycogen synthesis, by knock-down of GYS1 expression, impairs hypoxic preconditioning, suggesting a physiological role for the glycogen accumulated during chronic hypoxia. In summary, our results uncover a novel effect of hypoxia on glucose metabolism, further supporting the central importance of metabolic reprogramming in the cellular adaptation to hypoxia.

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Hypoxia increases glycogen synthase activity.c2c12 myoblasts were incubated at 21% (Nx) or 1% oxygen (Hx) for the indicated periods of time and processed to determine GYS1/pGYS1(phospho-Ser 641) levels (A) and glycogen synthase activity in the absence (B) or presence of 6.6 mM of the allosteric modulator glucose-6-phospate (C). Graphs represent the mean of duplicated measures and error bars the range. (D) The graph represents the ratio of glycogen synthase activity in the absence (I) and presence (T) of glucose-6-phosphate. The experiment was repeated twice with similar results.
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pone-0009644-g004: Hypoxia increases glycogen synthase activity.c2c12 myoblasts were incubated at 21% (Nx) or 1% oxygen (Hx) for the indicated periods of time and processed to determine GYS1/pGYS1(phospho-Ser 641) levels (A) and glycogen synthase activity in the absence (B) or presence of 6.6 mM of the allosteric modulator glucose-6-phospate (C). Graphs represent the mean of duplicated measures and error bars the range. (D) The graph represents the ratio of glycogen synthase activity in the absence (I) and presence (T) of glucose-6-phosphate. The experiment was repeated twice with similar results.

Mentions: The experiments described above demonstrated that hypoxia induced GYS1 transcription in a HIF-dependent manner. Importantly, gene transcription correlates with an increment of muscle glycogen synthase protein (figure 2B and 4A). Since glycogen synthase (from now on GS) activity is subjected to a complex regulation by allosteric modulators and phosphorylation by several kinases, protein levels do not necessarily correlate with activity. Therefore, we next asked whether the increment in GYS1 protein resulted in augmented GS activity. Figure 4B shows that GS activity increased in parallel with protein accumulation (figure 4A). Importantly, in the presence of the allosteric activator glucose-6-phosphate, that drives conversion of GS to its fully active state, samples from hypoxic cells still displayed increased GS activity as compared to control samples (figure 4C). Thus, hypoxia increased the total amount of GS but did not alter the proportion of active/inactive conformations as shown by the I/T ratio (figure 4D). In agreement, hypoxia did not modify the fraction of phosphorylated GYS1 (figure 4A). These results strongly suggest that GYS1 induction by HIF leads to augmented GS activity.


Hypoxia promotes glycogen accumulation through hypoxia inducible factor (HIF)-mediated induction of glycogen synthase 1.

Pescador N, Villar D, Cifuentes D, Garcia-Rocha M, Ortiz-Barahona A, Vazquez S, Ordoñez A, Cuevas Y, Saez-Morales D, Garcia-Bermejo ML, Landazuri MO, Guinovart J, del Peso L - PLoS ONE (2010)

Hypoxia increases glycogen synthase activity.c2c12 myoblasts were incubated at 21% (Nx) or 1% oxygen (Hx) for the indicated periods of time and processed to determine GYS1/pGYS1(phospho-Ser 641) levels (A) and glycogen synthase activity in the absence (B) or presence of 6.6 mM of the allosteric modulator glucose-6-phospate (C). Graphs represent the mean of duplicated measures and error bars the range. (D) The graph represents the ratio of glycogen synthase activity in the absence (I) and presence (T) of glucose-6-phosphate. The experiment was repeated twice with similar results.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2837373&req=5

pone-0009644-g004: Hypoxia increases glycogen synthase activity.c2c12 myoblasts were incubated at 21% (Nx) or 1% oxygen (Hx) for the indicated periods of time and processed to determine GYS1/pGYS1(phospho-Ser 641) levels (A) and glycogen synthase activity in the absence (B) or presence of 6.6 mM of the allosteric modulator glucose-6-phospate (C). Graphs represent the mean of duplicated measures and error bars the range. (D) The graph represents the ratio of glycogen synthase activity in the absence (I) and presence (T) of glucose-6-phosphate. The experiment was repeated twice with similar results.
Mentions: The experiments described above demonstrated that hypoxia induced GYS1 transcription in a HIF-dependent manner. Importantly, gene transcription correlates with an increment of muscle glycogen synthase protein (figure 2B and 4A). Since glycogen synthase (from now on GS) activity is subjected to a complex regulation by allosteric modulators and phosphorylation by several kinases, protein levels do not necessarily correlate with activity. Therefore, we next asked whether the increment in GYS1 protein resulted in augmented GS activity. Figure 4B shows that GS activity increased in parallel with protein accumulation (figure 4A). Importantly, in the presence of the allosteric activator glucose-6-phosphate, that drives conversion of GS to its fully active state, samples from hypoxic cells still displayed increased GS activity as compared to control samples (figure 4C). Thus, hypoxia increased the total amount of GS but did not alter the proportion of active/inactive conformations as shown by the I/T ratio (figure 4D). In agreement, hypoxia did not modify the fraction of phosphorylated GYS1 (figure 4A). These results strongly suggest that GYS1 induction by HIF leads to augmented GS activity.

Bottom Line: When oxygen becomes limiting, cells reduce mitochondrial respiration and increase ATP production through anaerobic fermentation of glucose.Significantly, knockdown of either HIF1alpha or GYS1 attenuated hypoxia-induced glycogen accumulation, while GYS1 overexpression was sufficient to mimic this effect.Altogether, these results indicate that GYS1 regulation by HIF plays a central role in the hypoxic accumulation of glycogen.

View Article: PubMed Central - PubMed

Affiliation: Departamento de Bioquímica, Universidad Autónoma de Madrid, Madrid, Spain.

ABSTRACT
When oxygen becomes limiting, cells reduce mitochondrial respiration and increase ATP production through anaerobic fermentation of glucose. The Hypoxia Inducible Factors (HIFs) play a key role in this metabolic shift by regulating the transcription of key enzymes of glucose metabolism. Here we show that oxygen regulates the expression of the muscle glycogen synthase (GYS1). Hypoxic GYS1 induction requires HIF activity and a Hypoxia Response Element within its promoter. GYS1 gene induction correlated with a significant increase in glycogen synthase activity and glycogen accumulation in cells exposed to hypoxia. Significantly, knockdown of either HIF1alpha or GYS1 attenuated hypoxia-induced glycogen accumulation, while GYS1 overexpression was sufficient to mimic this effect. Altogether, these results indicate that GYS1 regulation by HIF plays a central role in the hypoxic accumulation of glycogen. Importantly, we found that hypoxia also upregulates the expression of UTP:glucose-1-phosphate urydylyltransferase (UGP2) and 1,4-alpha glucan branching enzyme (GBE1), two enzymes involved in the biosynthesis of glycogen. Therefore, hypoxia regulates almost all the enzymes involved in glycogen metabolism in a coordinated fashion, leading to its accumulation. Finally, we demonstrated that abrogation of glycogen synthesis, by knock-down of GYS1 expression, impairs hypoxic preconditioning, suggesting a physiological role for the glycogen accumulated during chronic hypoxia. In summary, our results uncover a novel effect of hypoxia on glucose metabolism, further supporting the central importance of metabolic reprogramming in the cellular adaptation to hypoxia.

Show MeSH
Related in: MedlinePlus