MolMet Figure 5: Functional characterization of glucose metabolism and adaptive response to substrate challenge in hepatocytes from P446L mice.
A-D) GKRP and GK immunoactivity and enzyme activity after 24h-culture of hepatocytes isolated from male P446L mice (PP and LL genotypes, aged 8-12 wk). (C) Representative immunoblots: protein loading GK 30 μg; GKRP 10 μg (446PP); 35 μg (446LL); D. Glucokinase activity munits/mg protein (n=4-5).
E-F) Glucose phosphorylation ([2-3H]glucose, E) and glycolysis ([2-3H]glucose, F) is similar at 5mM glucose but lower by LL genotype at high glucose, (n=5,5).
G) Metabolic scheme showing metabolism of xylitol and fructose bypass the GK reaction and raises glycerol 3-P (G3P); whereas pharmacological activation of GK with PF-04991532 (PF) or inhibition of glucose 6-phosphatase (G6pc) with S4048 (S4) raises glucose 6-P (G6P). Note that fructose at micromolar concentrations also causes activation of GK via elevation in fructose 1-P and thereby elevation in G6P. However at millimolar concentrations the increase in triose phosphates (G3P) is consequent to flux through ketohexokinase and aldolase B reactions.
H-K) Hepatocyte incubations with glucose (15mM or 25mM) -/+ the GK activator PF-04991532 (PF, 10μM) or inhibitor of glucose 6-phosphatase (S4048, 1 μM) or xylitol (Xyl, 2mM) or fructose (Fru, 10mM) were for 1h for determination of cell ATP (H), glucose 6-P (I), glycerol 3-P (J) expressed as nmol/mg protein or ATP normalized to respective 5mM (K), n=5,5.
L-S) Hepatocyte incubations for RNA extraction and RT-qPCR analysis were for 4h. (L) Basal mRNA levels of genes indicated normalized to Gpd2 mRNA. (M-S) mRNA levels of the genes indicated normalised to the respective 5mM glucose control, n=3,4; # P<0.05 LL vs PP; * P<0.05 relative 5mM glucose control.
MICA: Exploring a new perspective on the mechanism of action of Glucokinase Activators in liver, a preclinical study
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