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. 2010 Nov 26;285(48):37170-7.
doi: 10.1074/jbc.M110.157396. Epub 2010 Sep 14.

Hepatic overexpression of a constitutively active form of liver glycogen synthase improves glucose homeostasis

Susana Ros  1 , Delia Zafra, Jordi Valles-Ortega, Mar García-Rocha, Stephen Forrow, Jorge Domínguez, Joaquim Calbó, Joan J Guinovart
Affiliations

Hepatic overexpression of a constitutively active form of liver glycogen synthase improves glucose homeostasis

Susana Ros et al. J Biol Chem. .

Abstract

In this study, we tested the efficacy of increasing liver glycogen synthase to improve blood glucose homeostasis. The overexpression of wild-type liver glycogen synthase in rats had no effect on blood glucose homeostasis in either the fed or the fasted state. In contrast, the expression of a constitutively active mutant form of the enzyme caused a significant lowering of blood glucose in the former but not the latter state. Moreover, it markedly enhanced the clearance of blood glucose when fasted rats were challenged with a glucose load. Hepatic glycogen stores in rats overexpressing the activated mutant form of liver glycogen synthase were enhanced in the fed state and in response to an oral glucose load but showed a net decline during fasting. In order to test whether these effects were maintained during long term activation of liver glycogen synthase, we generated liver-specific transgenic mice expressing the constitutively active LGS form. These mice also showed an enhanced capacity to store glycogen in the fed state and an improved glucose tolerance when challenged with a glucose load. Thus, we conclude that the activation of liver glycogen synthase improves glucose tolerance in the fed state without compromising glycogenolysis in the postabsorptive state. On the basis of these findings, we propose that the activation of liver glycogen synthase may provide a potential strategy for improvement of glucose tolerance in the postprandial state.

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Figures

FIGURE 1.
FIGURE 1.
Effects of the overexpression of WT LGS or activated mutant LGS on hepatic GS activity in rat liver. A, RT-PCR analysis of LGS mRNA levels in livers of rats overexpressing β-gal, WT LGS, or a constitutively active mutant LGS form (mutant LGS). B, total GS activity (units (U)/g of liver) of liver homogenates from fasted (white bars) or fed (black bars) rats overexpressing β-gal, WT LGS, or mutant LGS. C, GS activity (units/g of liver) calculated in the absence of glucose 6-phosphate. In all cases (A–C), data represent the mean ± S.E. (error bars) of the following: seven fasted and seven fed β-gal-overexpressing rats; five fasted and six fed WT LGS-overexpressing rats; and five fasted and five fed mutant LGS-overexpressing rats. * or **, statistical difference for comparisons with β-gal group at the same metabolic state with p < 0.05 or p < 0.005, respectively. D, representative Western blot analysis of three liver extracts of fasted or fed rats with antibodies against LGS or GAPDH as a load control. In all cases, 20 μg of protein were analyzed per lane.
FIGURE 2.
FIGURE 2.
Effects of the overexpression of WT LGS and activated mutant LGS on glycogen content in rat liver and muscle. A, liver glycogen content (mg/g of liver) measured in rats overexpressing β-gal, WT LGS, or mutant LGS. B, muscle glycogen content (mg/g of muscle) determined in the same animals. In all cases, data represent the mean ± S.E. (error bars) of the following: seven fasted and seven fed β-gal-overexpressing rats; five fasted and six fed WT LGS-overexpressing rats; and five fasted and five fed mutant LGS-overexpressing rats. * or **, statistical difference for comparisons with β-gal group at the same metabolic state with p < 0.05 or p < 0.005, respectively.
FIGURE 3.
FIGURE 3.
Effects of activated mutant LGS overexpression on ultracellular structure as shown by electron microscopy analysis of rat liver sections. Cellular ultrastructure analysis by electron microscopy of liver biopsies from the rats overexpressing β-gal or the constitutively active mutant form of LGS, fasted for 18 h or fed ad libitum. Scale bar, 5 μm (fed rats) or 1 μm (fasted rats).
FIGURE 4.
FIGURE 4.
Effects of the overexpression of WT LGS and activated mutant LGS on glucokinase, GLUT2, glycogen phosphorylase, and phosphoenolpyruvate carboxykinase expression levels in rat liver. A, RT-PCR analysis of liver GK, GLUT2, GP, and PEPCK mRNA levels in livers of fed and fasted rats overexpressing β-gal; data are relative to the fed β-gal group. B, RT-PCR analysis in fasted overexpressing β-gal, WT LGS, or mutant LGS rats; data are relative to the fasted β-gal group of rats. C, RT-PCR analysis in fed overexpressing β-gal, WT LGS, or mutant LGS rats; data are relative to the fed β-gal group of rats. In all cases, relative expression levels were calculated with the 2ΔΔCt method using 18 S rRNA as endogenous control, and data represent the mean ± S.E. (error bars) of the following: seven fasted and seven fed β-gal-overexpressing rats; five fasted and six fed WT LGS-overexpressing rats; and five fasted and five fed mutant LGS-overexpressing rats. In A the asterisk indicates significant difference, with p < 0.05.
FIGURE 5.
FIGURE 5.
Intraperitoneal glucose tolerance test. A, rats overexpressing β-gal, WT LGS, or activated mutant LGS were fasted for 18 h before receiving an intraperitoneal glucose bolus of 2 g/kg body weight. Tail vein blood samples were taken, and glucose concentrations were measured at the times indicated after the glucose bolus. The area under the curve was measured for each experimental group (AUC, inset). B, liver glycogen content (mg/g of liver) determined at the starting point (β-gal-fasted) and at the end point of the IPGTT (180 min). The inset shows a lower scale graph. In all cases, data are mean ± S.E. (error bars) for seven fasted β-gal-overexpressing rats and nine β-gal-, six WT LGS-, and five mutant LGS-overexpressing animals from the IPGTT. C, liver samples taken after the IPGTT were processed for immunofluorescence analysis with an antibody against LGS. Representative confocal microscopy images of liver sections from rats overexpressing β-gal, WT LGS, or activated mutant LGS. Laser intensity was adjusted so that the endogenous LGS signal (β-gal-overexpressing animals) was hardly observable. Lower right panel, magnification of the mutant LGS image (area inside the box) to show the aggregated, peripheral distribution of LGS. Scale bar, 20 μm. In A, the single or double asterisks indicate those time points at which blood glucose concentrations were significantly lower in rats overexpressing mutant LGS than in rats overexpressing β-gal, with p < 0.05 or p < 0.005 respectively; in B the double asterisk denotes statistical difference for comparisons with the β-gal IPGTT group with p < 0.005, and the ampersand denotes statistical difference between the fasted β-gal group and the β-gal IPGTT group, with p < 0.005.
FIGURE 6.
FIGURE 6.
Characterization of transgenic mice expressing activated mutant LGS in liver. A, representative Western blot analysis of liver extracts of wild type and activated mutant LGS transgenic mice (two samples of each) with antibodies against LGS or actin as a load control. In all cases, 20 μg of protein were analyzed per lane. B, GS activity ratio (−glucose 6-phosphate/+glucose 6-phosphate (−Glc-6-P/+Glc-6-P)) of liver homogenates from wild type (n = 8) and mutant LGS transgenic (n = 4) mice. Data represent the mean ± S.E. (error bars). C, liver glycogen content (mg/g of liver) measured in fasted (white bars) or fed (black bars) wild type and mutant LGS transgenic mice. Data represent the mean ± S.E. of six fasted and four fed wild type mice and five fasted and four fed mutant LGS transgenic mice. D, wild type (n = 9) and mutant LGS transgenic (n = 6) mice were fasted for 18 h before receiving an intraperitoneal glucose bolus of 2 g/kg body weight. Tail vein blood samples were taken, and glucose concentrations were measured at the times indicated after the glucose bolus. The area under the curve was measured for each experimental group (AUC, inset). Data represent the mean ± S.E. The single or double asterisks denote statistical difference for comparisons with WT group at the same metabolic state with p < 0.05 or p < 0.005, respectively. The double ampersand denotes statistical difference (p < 0.005) between the fasted and fed states for each group of mice.
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