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. 2015 Jun 5;290(29):18245–18260. doi: 10.1074/jbc.M115.658948

FIGURE 5.

FIGURE 5.

Exploration of the mechanisms by which AMPK affects UGCG activity. A, examination of phosphorylation of UGCG. CHO cells expressing C-terminal FLAG-tagged UGCG were incubated with AICAR or CC for 3 h. The recombinant UGCG was immunoprecipitated from cell lysates by FLAG antibody and then subjected to immunoblot analysis and stained with ProQ Diamond, which detects phosphorylated proteins. B, expression levels of ugcg mRNA in AICAR- or CC-treated cells. The mRNA was extracted from 3T3 cells after treatment with AICAR or CC for 3 h and then quantified as described under “Experimental Procedures.” C, effects of AICAR and CC on intracellular GCase activity. After treatment with AICAR or CC, NBD C6-GlcCer conjugated to BSA was added to cells and incubated, and then NBD C6-ceramide released from GlcCer was measured by TLC. D, effects of metformin and AICAR on intracellular UGCG activity of fibroblasts derived from Gaucher disease patients. Fibroblasts derived from type 1, 2, and 3 Gaucher disease patients were incubated with metformin or AICAR for 8 h and then were incubated with C6-NBD Cer conjugated with BSA for 90 min. Fluorescent sphingolipids were extracted from the cells and then analyzed by TLC. E, quantification of cellular GlcCer levels of fibroblasts derived from a Gaucher disease patient (type 2). Lipids were extracted from fibroblasts treated with metformin (8 mm) or AICAR (1 mm) for 16 h and were then subjected to LC-ESI MS, as described under “Experimental Procedures.” F, sugar nucleotide levels in metformin- or AICAR-treated cells. Sugar nucleotides were extracted from 3T3 cells treated with metformin (8 mm) or AICAR (1 mm) for 8 h and then subjected to ion pair reverse-phase HPLC, as described under “Experimental Procedures.” G, effect of CC on sugar nucleotide levels in AICAR-treated cells. Sugar nucleotides were extracted from 3T3 cells treated with AICAR (1 mm) or AICAR (1 mm) plus CC (20 μm) for 8 h. Bars, means ± S.D. (error bars) of at least three experiments. **, p < 0.01; ***, p < 0.001.