Impaired glucose tolerance and predisposition to the fasted state in liver glycogen synthase knock-out mice

Abstract

Conversion to glycogen is a major fate of ingested glucose in the body. A rate-limiting enzyme in the synthesis of glycogen is glycogen synthase encoded by two genes, GYS1, expressed in muscle and other tissues, and GYS2, primarily expressed in liver (liver glycogen synthase). Defects in GYS2 cause the inherited monogenic disease glycogen storage disease 0. We have generated mice with a liver-specific disruption of the Gys2 gene (liver glycogen synthase knock-out (LGSKO) mice), using Lox-P/Cre technology. Conditional mice carrying floxed Gys2 were crossed with mice expressing Cre recombinase under the albumin promoter. The resulting LGSKO mice are viable, develop liver glycogen synthase deficiency, and have a 95% reduction in fed liver glycogen content. They have mild hypoglycemia but dispose glucose less well in a glucose tolerance test. Fed, LGSKO mice also have a reduced capacity for exhaustive exercise compared with mice carrying floxed alleles, but the difference disappears after an overnight fast. Upon fasting, LGSKO mice reach within 4 h decreased blood glucose levels attained by control floxed mice only after 24 h of food deprivation. The LGSKO mice maintain this low blood glucose for at least 24 h. Basal gluconeogenesis is increased in LGSKO mice, and insulin suppression of endogenous glucose production is impaired as assessed by euglycemic-hyperinsulinemic clamp. This observation correlates with an increase in the liver gluconeogenic enzyme phosphoenolpyruvate carboxykinase expression and activity. This mouse model mimics the pathophysiology of glycogen storage disease 0 patients and highlights the importance of liver glycogen stores in whole body glucose homeostasis.

FIGURE 1.

Gene targeting and basic characterization of genetically modified mice. A, partial map of the targeting vector and Gys2 locus containing the LoxP sites flanking the exons 5 and 6 (LGS^Lox) introduced in ES cells by homologous recombination. ES cells containing this modified locus were used to create the transgenic animals. Crossing LGS^(Lox/Lox) mice with alb-CRE^(+/−) mice would excise exons 5 and 6 from the two alleles of Gys2 gene via the action of the CRE recombinase, creating a Gys2 knock-out mouse. B, growth rates of CN (squares), HET (circles), and LGSKO (triangles) male mice (n = 22, 21, and 30, respectively). The mice were weighed weekly, at 8–9 a.m. under random fed conditions from 20 to 150 weeks after birth. There were no statistical differences. C and F, fold change, compared with CN mice, of liver GS protein in liver (C) and muscle GS in muscle (F), normalized by glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as judged by Western blotting (n = 8/genotype). The lower panels show representative examples of the LGS, MGS, and glyceraldehyde-3-phosphate dehydrogenase Western blots (two for each genotype). D and G, total glycogen synthase activity measured in the presence of 7.2 mm glucose-6-P in liver (D) and skeletal muscle (G) total homogenates (n = 8/genotype). E and H, fold change in Gys2 mRNA content in liver (n = 8 per genotype) (E) and Gys1 mRNA in muscle (n = 4 per genotype) (H) compared with CN mice measured with real time PCR. All of the measurements were for 4-month-old male mice. The data are expressed as the means ± S.E. *, p < 0.05 versus CN; #, p < 0.05 versus wild type (WT); $, p < 0.05 versus CRE; +, p < 0.05 versus HET.

FIGURE 2.

Glycogen and triglyceride content in tissues of LGSKO mice. A–C, glycogen content measured in liver (A), skeletal muscle (B), and kidney (C) (n = 8/genotype) under fed conditions of 4-month-old mice. The data are expressed as the means ± S.E. *, p < 0.05 versus CN; #, p < 0.05 versus wild type (WT); $, p < 0.05 versus CRE; +, p < 0.05 versus HET. D and E, glycogen content under random fed (black bars), 6-h fasted (hatched bars), and overnight fasted (white bars) in liver (D) and skeletal muscle (E) of 4-month old mice. *, p < 0.05 versus CN; +, p < 0.05 versus HET; #, p < 0.05 versus fed condition; $, p < 0.05 versus 6 h fasting. F, triglyceride content in liver under random fed conditions of 4-month-old (black bars, n = 8), 7-month-old (hatched bars, n = 14 for CN and n = 16 for LGSKO) and 15-month-old (white bars, n = 8 for CN and n = 10 for LGSKO) mice. *, p < 0.05 versus CN; #, p < 0.05 versus 4-month-old mice; $, p < 0.05 versus 7-month-old mice. The numbers in parentheses indicate the n for the given group. The data are expressed as the means ± S.E.

FIGURE 3.

Glycogen in CN and LGSKO mouse liver sections. A and B, periodic acid-Schiff reagent staining of paraffin-embedded liver sections from random fed CN (A) and LGSKO (B) mice. The sections were counterstained for hematoxylin and eosin. All of the hepatocytes were stained pink in the liver sections from CN. Conversely, LGSKO mice sections only a small amount of cells (about 7%) were stained. The arrows point to those hepatocytes containing glycogen in the LGSKO mice sections. C and D, immunohistochemistry of paraffin-embedded liver sections of CN (C) and LGSKO (D) mice for CRE recombinase and counterstained with hematoxylin. CN mice sections were negative for the staining, whereas LGSKO mice sections were positively stained light brown in almost all the cells. However, some cells, marked by arrows, were not stained, indicating a lack of CRE recombinase protein.

FIGURE 4.

Glucose and insulin tolerance tests and time course of fasting in LGSKO mice. A, GTTs were performed on CN (open symbols) and LGSKO (filled symbols) mice. Glucose (2 mg/g of body weight) was administrated by intraperitoneal injection (circles) or oral gavage (triangles) after overnight (15 h) fasting. B, area under the curve (AUC) of the intraperitoneal injection (IP) or oral gavage (OG) GTT displayed A, expressed as arbitrary units. *, p < 0.05 versus CN with same glucose administration. C, increase in blood lactate (mm) 20 min after intraperitoneal injection of glucose in the GTT of A. D, insulin tolerance test displaying the blood glucose levels of CN (open circles) and LGSKO (filled circles) mice. E, long term fasting of CN (open symbols) and LGSKO (filled symbols) mice. Food was withdrawn between 8 and 9 a.m. Blood glucose levels were measured immediately before and at 2, 4, 6, 12, and 24 h after removing the food. The numbers in parentheses indicates the n for the given group. The data are expressed as the means ± S.E. *, p < 0.05 versus CN.

FIGURE 5.

Whole body glucose metabolism during euglycemic-hyperinsulinemic clamp in LGSKO mice. A and B, blood glucose levels (A) and glucose infusion rate (B) before and during a 120-min euglycemic-hyperinsulinemic clamp of CN (open symbols) and LGSKO (filled symbols) mice. There were no statistical differences at any time between genotypes. C, glucose uptake rate (R_g) in soleus, gastrocnemius, vastus lateralis, diaphragm, heart, white adipose tissue, and brain during the euglycemic-hyperinsulinemic clamp. D–F, endogenous glucose appearance (D, Endo Ra, as total glucose appearance minus glucose infusion), net gluconeogenesis rate (E), and glycogenolysis (as endogenous glucose appearance minus net gluconoegenesis) measured as mg of glucose/kg of mouse/min (F). n = 9–10/genotype for all of the parameters. The data are expressed as the means ± S.E. *, p < 0.05 versus CN; #, p < 0.05 versus time 0.

FIGURE 6.

Gluconeogenesis in LGSKO mice. A, C, and D, fold change of g6pc1 (A), pck1 (C), and PGC-1α (D) mRNA content in liver measured with real time PCR. The fold change is compared with random fed CN mice. B, PEPCK activity measured in 100 kg of liver supernatants. E–G, change of PEPCK1 protein level normalized by glyceraldehyde-3-phosphate dehydrogenase (n = 8) (E), FoxO1 phosphorylated (Thr²⁴) normalized by total FoxO1 content (F), and phosphorylated (Thr¹⁷²) AMPKα normalized by total AMPKα content (G) in liver homogenates. The upper panels show representative Western blot data. The measurements in the lower panels were done in liver of fed (black bars), 6-h fasted (hatched bars), and overnight fasted (white bars) mice. n = 8 for fed mice and n = 5 for 6-h and ON fasted mice. The data are expressed as the means ± S.E. *, p < 0.05 versus CN; +, p < 0.05 versus HET; #, p < 0.05 versus fed; $, p < 0.05 versus 6-h fasted.