br Materials and methods br Results

Materials and methods
Results
Discussion Our liver-specific Gck (−/−) mice and another line of liver-specific mice [13] exhibited impaired glucose tolerance. Furthermore, both knockout mice showed decreased hepatic glycogen content and reduced Pklr and Fas gene SANT-1 in the liver [11]. However, there were several differences between our mutant mice and theirs. First, their mice showed a significant increase of blood glucose levels in both the fed and fasting states. In contrast, there were no significant difference in blood glucose levels between our Gck (−/−) and Gck (+/+) mice. Second, they reported the presence of hyperinsulinemia (indicative of insulin resistance) in their knockout mice. The insulin levels and insulin tolerance in our Gck (−/−) knockout mice were indistinguishable from those of control mice. Third, glucose-induced insulin secretion in their liver-specific knockout mice during hyperglycemic clamp experiments was markedly decreased; however, insulin secretion was normal after intraperitoneal injection of glucose in our Gck (−/−) mice. Postic et al. used gklox/lox mice as a control of glucokinase knockout mice (gklox/lox + Alb-Cre), but gklox/lox mice themselves exhibited significantly increased blood glucose concentrations when compared to WT mice due to the insertion of the loxP sequences. Although we have no adequate explanation for these differences, the chronic hyperglycemia of gklox/lox mice may have contributed to the different findings of the two studies. A recent study revealed that hepatic glucokinase regulates thermogenesis-related gene expression in BAT [12]. In addition, hepatic glucokinase knockdown in HFD-fed C57BL/6 mice attenuated weight gain with increased expression of Ucp1, Pgc1a, and Dio2 in BAT. However, gene expression was unaffected and body weight was also unchanged in our Gck (−/−) mice. Different experimental conditions (chronic suppression versus acute suppression) or different dietary conditions (normal chow versus HFD) may have contributed to these different findings. In the present study, we generated novel liver-specific glucokinase knockout mice and demonstrated the determinant roles of glucokinase in hepatic glycogen synthesis and glycolysis and lipid synthesis-related gene expression. It has been also reported that hepatic glycogen content regulates nerve-mediated lipolysis in WAT [24]. Further studies of these liver-specific glucokinase knockout mice could be useful to clarify intertissue metabolic communication via glucokinase.
Conflict of interest
Acknowledgments We thank Professor T. Fukuda (Kumamoto University) for technical assistance with preparing hypothalamus. This work was supported by a Grant-in-Aid for Scientific Research (B) (25293212), a grant from the Novo Nordisk Insulin Research Foundation to Kazuya Yamagata, and a Grant-in-Aid for Scientific Research (S) (21220010) to Ken-ichi Yamamura.
Introduction Glucokinase (GK), a member of the hexokinase family, catalyzes the first step in glycolysis, phosphorylation of glucose to glucose-6-phosphate. Glucokinase is responsible for maintaining glucose homeostasis in human body. Mutations that inactivate GK are linked to diabetes, and mutations that activate it are associated with hypoglycemia (Zelent et al., 2011). Glucokinase consists of two domains: the large and small ones (Kamata et al., 2004). According to modern model, in the absence of glucose, GK exists in an open inactive conformation with low affinity for glucose. Glucose binding induces a large-scale structural rearrangement in glucokinase and transfers GK to an active closed conformation with high affinity for glucose (Larion and Miller, 2010, Antoine et al., 2009, Larion et al., 2012). The active site for glucose phosphorylation is in a cleft between two domains. The active closed conformations of GK can be stabilized by binding of glucokinase activator (GKA) (Zelent et al., 2011, Futamura et al., 2006). In closed conformation all known GKAs except for proapoptotic factor BAD (Szlyk et al., 2014) bind to the same allosteric site formed by residues of the small and large domains and two loops that connect them (Kamata et al., 2004, Hinklin et al., 2013, Filipski et al., 2013). GKA binding stabilizes this allosteric site and in turn stabilizes the active conformation and increases the glucokinase activity. The GK allosteric site is located approximately 20Å away from glucose binding site (Kamata et al., 2004). The glucose binding site and the allosteric site of GK are shown in Supplementary (Fig. S1).