Figure 4. GlcN effects on glucose- and glutamine-utilizing metabolic pathways.

(a) Schematic diagram of the hexosamine biosynthetic (HBSP, red), glycolytic (blue), pentose phosphate (PPP, green), and glutamine (gln, pink) -derived pathways. GlcN 6-P, the first intermediate in the HBSP, can be generated upon phosphorylation of exogenous GlcN (underlined), or from fructose 6-P + gln. Hexokinase-1 and -2 (HK1, 2), phosphofructokinase (PFK), pyruvate kinase M (PKM), pyruvate dehydrogenase (PDHA), lactate dehydrogenase (LDHA), glucose 6-PO₄ dehydrogenase (G6PD), Glutamine--fructose-6-phosphate aminotransferase (GFAT), tricarboxylic acid cycle (TCA cycle). (b) Schematic diagram of oxidative pathways, the TCA cycle (purple) and oxidative phosphorylation (green), related to glucose or glutamine metabolism. (c) Lactate accumulation in media after 48 hr culture of LG-ESC ± 0.8 mM GlcN. (d) Intracellular pyruvate accumulation after 48 hr culture or LG-ESC ± 0.8 mM GlcN. (e) Extracellular acidification rate (ECAR) by LG-ESC incubated ± 0.8 mM GlcN. (f) Oxygen consumption rate (OCR) by LG-ESC incubated ± 0.8 mM GlcN. (g) Immunoblot of glycolytic enzymes from LG-ESC cultured ± 0.8 mM GlcN. (h) Quantitation of (g). (i) Immunoblot of subunits of mitochondrial respiratory chain enzymes (Complex V ATP synthase (CV-ATP5A), Complex III ubiquinol-cytochrome c reductase (CIII-UQCRC2), Complex IV cyctochrome C oxidase catalytic (CIV-MTCO1), Complex II succinate dehydrogenase iron-sulfur protein (CII-SDHB) and Complex I NADH dehydrogenase (CI-NDUFB8)) from rat heart (positive control) or extracts from LG-ESC cultured ± 0.8 mM GlcN. (j) G6PD activity by LG-ESC grown ± 0.8 mM GlcN for 48 hr or +GlcN only the last hr of culture. Experiments were repeated 2–3 times using triplicate culture dishes. Quantitative data from representative experiments are displayed as the mean ± s.e.m. (N = 3) and were analyzed by Student t-test *P < 0.05, **P < 0.01.