Figure 1.

Effects of hyperosmotic culture conditions and drugs combinations on cell growth and metabolic phenotype. Experimental results for the shaken flask batch cultures for control conditions (filled squares, C), high osmolarity conditions induced with 200 mM D-mannitol (filled circles, M), control conditions with the addition of 8 μM alpha-lipoic acid (ALA) and 300 μM calcium hydroxycitrate (HCA) (empty triangles, D), and high osmolarity conditions with the addition of 8 μM ALA and 300 μM HCA (empty circles, D + M). (A,B) Batch cell concentration and cell growth rate were determined. Cell growth rates were calculated from the maximum growth rate phase in each condition. Cell growths were highly inhibited under the high osmolarity condition and/or with the drugs combinations compared to the control condition. This is highlighted by the cell-specific growth rate, which was higher in the control condition. (C) Viable cell density. Cell viabilities were flat and quite similar for all conditions, except for the drugs combination condition where cell viability fell suddenly after 70 h of culture and reached 43% at the end of the culture. (D) Metabolic ratios of lactate per mole of glucose consumed (Y_LAC/GLC, grey bars) and glutamate per mole of glutamine consumed (Y_GLU/GLN, grey bars) were set. Lactate yield was higher in the D, M, and D + M conditions compared to control. Glutamate yield was higher in the D condition but relatively close to control for the M and D + M culture conditions. (E) Specific maximum metabolic rates for each metabolite. Glucose consumption rate was higher under high osmolarity. Glutamine consumption was higher in drugs and/or mannitol compared to control. The same was true for the lactate production rate, which was found to be much more pronounced in drugs and/or mannitol. Glutamate production rate was higher in the D and D + M conditions, which are markers of reduced glutaminolytic rates.