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(13)C metabolic flux analysis shows that resistin impairs the metabolic response to insulin in L6E9 myotubes.

Guzmán S, Marin S, Miranda A, Selivanov VA, Centelles JJ, Harmancey R, Smih F, Turkieh A, Durocher Y, Zorzano A, Rouet P, Cascante M - BMC Syst Biol (2014)

Bottom Line: Resistin significantly increased glucose uptake and glycolysis, altering pyruvate utilisation by the cell.Resistin prevented the increase in gene expression in pyruvate dehydrogenase-E1 and the sharp decrease in gene expression in cytosolic phosphoenolpyruvate carboxykinase-1 induced by insulin.This impairment cannot be explained by the activity of a single enzyme, but instead due to reorganisation of the whole metabolic flux distribution.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, Faculty of Biology, Universitat de Barcelona, Av Diagonal 643, 08028, Barcelona, Spain. shirleyguzman1@gmail.com.

ABSTRACT

Background: It has been suggested that the adipokine resistin links obesity and insulin resistance, although how resistin acts on muscle metabolism is controversial. We aimed to quantitatively analyse the effects of resistin on the glucose metabolic flux profile and on insulin response in L6E9 myotubes at the metabolic level using a tracer-based metabolomic approach and our in-house developed software, Isodyn.

Results: Resistin significantly increased glucose uptake and glycolysis, altering pyruvate utilisation by the cell. In the presence of resistin, insulin only slightly increased glucose uptake and glycolysis, and did not alter the flux profile around pyruvate induced by resistin. Resistin prevented the increase in gene expression in pyruvate dehydrogenase-E1 and the sharp decrease in gene expression in cytosolic phosphoenolpyruvate carboxykinase-1 induced by insulin.

Conclusions: These data suggest that resistin impairs the metabolic activation of insulin. This impairment cannot be explained by the activity of a single enzyme, but instead due to reorganisation of the whole metabolic flux distribution.

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Metabolic fluxes in L6E9 myotubes not treated and treated with resistin and/or insulin. L6E9 myotubes were treated (resistin+) or not treated (resistin–) with 100 nM resistin for 8 h and then incubated for 6 h with 10 mM glucose, 50%-enriched in [1,2-13C2]-D-glucose in the absence or in the presence of 100 nM insulin. Fluxes were estimated using the software Isodyn. Arrow sizes indicate net fluxes in L6E9 myotubes not treated or treated with resistin that were incubated in the absence of insulin. Colours indicate flux fold-changes in response to insulin. Fluxes plotted are the median values from the 20 best flux sets (Table 3). Grey-coloured fluxes have not been measured. FBP, fructose-1,6-bisphosphate; Hexose-P, glucose-6-phosphate and its isomers; Mal, malate; OAA, oxaloacetate; Pentose-P, ribose-5-phosphate and its isomers; PEP, phosphoenolpyruvate; PYR, pyruvate; Triose-P, glyceraldehyde-3-phosphate and dihydroxyacetone phosphate.
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Figure 1: Metabolic fluxes in L6E9 myotubes not treated and treated with resistin and/or insulin. L6E9 myotubes were treated (resistin+) or not treated (resistin–) with 100 nM resistin for 8 h and then incubated for 6 h with 10 mM glucose, 50%-enriched in [1,2-13C2]-D-glucose in the absence or in the presence of 100 nM insulin. Fluxes were estimated using the software Isodyn. Arrow sizes indicate net fluxes in L6E9 myotubes not treated or treated with resistin that were incubated in the absence of insulin. Colours indicate flux fold-changes in response to insulin. Fluxes plotted are the median values from the 20 best flux sets (Table 3). Grey-coloured fluxes have not been measured. FBP, fructose-1,6-bisphosphate; Hexose-P, glucose-6-phosphate and its isomers; Mal, malate; OAA, oxaloacetate; Pentose-P, ribose-5-phosphate and its isomers; PEP, phosphoenolpyruvate; PYR, pyruvate; Triose-P, glyceraldehyde-3-phosphate and dihydroxyacetone phosphate.

Mentions: After 6 h of incubation, glucose consumption, lactate production, intracellular levels of glycogen and glucose-6-phosphate (G6P) were determined (Table 1), as were the mass-isotopomer distributions in glucose and lactate from the incubation medium, as well as glycogen glucose and ribose isolated from RNA in cell pellets (Table 2). Analyses of the obtained data by Isodyn determined the distribution of the metabolic flux profile of L6E9 myotubes under various incubation conditions (Table 3). Figure 1 summarises the qualitative changes in metabolites and fluxes.


(13)C metabolic flux analysis shows that resistin impairs the metabolic response to insulin in L6E9 myotubes.

Guzmán S, Marin S, Miranda A, Selivanov VA, Centelles JJ, Harmancey R, Smih F, Turkieh A, Durocher Y, Zorzano A, Rouet P, Cascante M - BMC Syst Biol (2014)

Metabolic fluxes in L6E9 myotubes not treated and treated with resistin and/or insulin. L6E9 myotubes were treated (resistin+) or not treated (resistin–) with 100 nM resistin for 8 h and then incubated for 6 h with 10 mM glucose, 50%-enriched in [1,2-13C2]-D-glucose in the absence or in the presence of 100 nM insulin. Fluxes were estimated using the software Isodyn. Arrow sizes indicate net fluxes in L6E9 myotubes not treated or treated with resistin that were incubated in the absence of insulin. Colours indicate flux fold-changes in response to insulin. Fluxes plotted are the median values from the 20 best flux sets (Table 3). Grey-coloured fluxes have not been measured. FBP, fructose-1,6-bisphosphate; Hexose-P, glucose-6-phosphate and its isomers; Mal, malate; OAA, oxaloacetate; Pentose-P, ribose-5-phosphate and its isomers; PEP, phosphoenolpyruvate; PYR, pyruvate; Triose-P, glyceraldehyde-3-phosphate and dihydroxyacetone phosphate.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4363945&req=5

Figure 1: Metabolic fluxes in L6E9 myotubes not treated and treated with resistin and/or insulin. L6E9 myotubes were treated (resistin+) or not treated (resistin–) with 100 nM resistin for 8 h and then incubated for 6 h with 10 mM glucose, 50%-enriched in [1,2-13C2]-D-glucose in the absence or in the presence of 100 nM insulin. Fluxes were estimated using the software Isodyn. Arrow sizes indicate net fluxes in L6E9 myotubes not treated or treated with resistin that were incubated in the absence of insulin. Colours indicate flux fold-changes in response to insulin. Fluxes plotted are the median values from the 20 best flux sets (Table 3). Grey-coloured fluxes have not been measured. FBP, fructose-1,6-bisphosphate; Hexose-P, glucose-6-phosphate and its isomers; Mal, malate; OAA, oxaloacetate; Pentose-P, ribose-5-phosphate and its isomers; PEP, phosphoenolpyruvate; PYR, pyruvate; Triose-P, glyceraldehyde-3-phosphate and dihydroxyacetone phosphate.
Mentions: After 6 h of incubation, glucose consumption, lactate production, intracellular levels of glycogen and glucose-6-phosphate (G6P) were determined (Table 1), as were the mass-isotopomer distributions in glucose and lactate from the incubation medium, as well as glycogen glucose and ribose isolated from RNA in cell pellets (Table 2). Analyses of the obtained data by Isodyn determined the distribution of the metabolic flux profile of L6E9 myotubes under various incubation conditions (Table 3). Figure 1 summarises the qualitative changes in metabolites and fluxes.

Bottom Line: Resistin significantly increased glucose uptake and glycolysis, altering pyruvate utilisation by the cell.Resistin prevented the increase in gene expression in pyruvate dehydrogenase-E1 and the sharp decrease in gene expression in cytosolic phosphoenolpyruvate carboxykinase-1 induced by insulin.This impairment cannot be explained by the activity of a single enzyme, but instead due to reorganisation of the whole metabolic flux distribution.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, Faculty of Biology, Universitat de Barcelona, Av Diagonal 643, 08028, Barcelona, Spain. shirleyguzman1@gmail.com.

ABSTRACT

Background: It has been suggested that the adipokine resistin links obesity and insulin resistance, although how resistin acts on muscle metabolism is controversial. We aimed to quantitatively analyse the effects of resistin on the glucose metabolic flux profile and on insulin response in L6E9 myotubes at the metabolic level using a tracer-based metabolomic approach and our in-house developed software, Isodyn.

Results: Resistin significantly increased glucose uptake and glycolysis, altering pyruvate utilisation by the cell. In the presence of resistin, insulin only slightly increased glucose uptake and glycolysis, and did not alter the flux profile around pyruvate induced by resistin. Resistin prevented the increase in gene expression in pyruvate dehydrogenase-E1 and the sharp decrease in gene expression in cytosolic phosphoenolpyruvate carboxykinase-1 induced by insulin.

Conclusions: These data suggest that resistin impairs the metabolic activation of insulin. This impairment cannot be explained by the activity of a single enzyme, but instead due to reorganisation of the whole metabolic flux distribution.

Show MeSH
Related in: MedlinePlus