DOM FIGURE 2 Comparison of AZD1656 and PF-04991532 (+)/(-) enantiomers with substrate challenge on gene expression in mouse hepatocytes.

A-D. Comparison of (+) and (-) enantiomers of PF-04991532 and AZD1656 on glucokinase activity assayed at 0.5mM glucose (A-B) and gene expression in hepatocytes in MEM with 5mM glucose (C-D). E. Pathway showing xylitol metabolism to fructose 6-P (F6P) and triose phosphates and sites of action of: AZD1656 and PF-04991532 (GKAs); S4048 (inhibitor of G6P hydrolysis); AOA (amino-oxyacetate, inhibitor of oxidation of cytoplasmic NADH). F-N. Hepatocytes from C57BL/6 mice were incubated for either 1h for metabolite analysis (F,G) or 4h for mRNA analysis (H-O) in MEM containing 5mM glucose (5G) and the additions indicated: 10μM AZD1656 (AZD); 10μM PF(+)04991532 (PF); xylitol (2mM, XY); AOA (0.1 mM) or with 25mM glucose without (25G) or with 0.4μM S4048 (25GS). Cell G6P and G3P are expressed as nmol/mg protein and cell ATP as % 5mM glucose control. mRNA levels are normalized to control incubations with 5mM glucose alone. Values are means ± SEM for n=4 (AOA, xylitol) or n=8 (all other conditions) hepatocyte preparations, * P < 0.05 relative to 5mM glucose alone.

A-D. 对比PF-04991532与AZD1656的(+)、(-)对映体对葡糖激酶（glucokinase）活性的影响，检测条件为0.5mM葡萄糖（A-B组）；以及在含5mM葡萄糖的伊格尔最小必需培养基（Minimum Essential Medium, MEM）中培养的肝细胞内的基因表达情况（C-D组）。E. 展示木糖醇（xylitol）代谢生成6-磷酸果糖（fructose 6-P, F6P）与磷酸丙糖的通路，以及各试剂的作用位点：AZD1656与PF-04991532（葡糖激酶激活剂，GKAs）；S4048（6-磷酸葡萄糖水解抑制剂）；AOA（氨基氧乙酸，细胞质NADH氧化抑制剂）。F-N. 从C57BL/6品系小鼠分离的肝细胞，在含5mM葡萄糖的MEM培养基（标注为5G）中分别孵育1小时以进行代谢物检测（F、G组），或孵育4小时以进行mRNA检测（H-O组），同时添加如下试剂：10μM AZD1656（简称AZD）、10μM (+)PF-04991532（简称PF）、2mM木糖醇（XY）、0.1mM AOA；另有25mM葡萄糖组（25G）以及添加0.4μM S4048的25mM葡萄糖组（25GS）。细胞内6-磷酸葡萄糖（G6P）与3-磷酸甘油醛（G3P）的含量以nmol/mg蛋白为单位进行报告，细胞内ATP含量以5mM葡萄糖对照组的百分比形式表示。mRNA水平以仅添加5mM葡萄糖的孵育对照组为参照进行归一化处理。所有数值均为平均值±标准误（standard error of the mean, SEM），其中AOA、木糖醇组的肝细胞制备样本量为n=4，其余所有组别样本量为n=8；*表示与仅添加5mM葡萄糖的对照组相比，P<0.05。