Insulin synthesis and release activities of cells can be acutely regulated by glucose through its glycolytic and mitochondrial breakdown involving a glucokinase-dependent rate-limiting step. Isolated cell populations are composed of cells with intercellular differences in acute glucose responsiveness that have been attributed to differences in glucokinase (GK) expression and activity. This study first shows that glyceraldehyde can be used as GK-bypassing oxidative substrate and then examines whether the triose can metabolically activate cells with low glucose responsiveness. Glyceraldehyde 1 mM induced a similar cellular ¹⁴CO₂ output and metabolic redox state as glucose 4 mM. Using flow cytometric analysis, glyceraldehyde (0.25-2 mM) was shown to concentration-dependently increase the percent metabolically activated cells at all tested glucose concentrations (2.5 to 20 mM). Its ability to activate cells that are unresponsive to the prevailing glucose level was further illustrated in glucose low-responsive cells that were isolated by flow-sorting. Metabolic activation by glyceraldehyde was associated with an activation of nutrient-driven translational control proteins and an increased protein synthetic response to glucose, however not beyond the maximal rates that are inducible by glucose alone. It is concluded that glucose low-responsive cells can be metabolically activated by the GK-bypassing glyceraldehyde, increasing their acute biosynthetic response to glucose but not their maximal glucose-inducible biosynthetic capacity, which is considered subject to chronic regulation.