Glucose-6-phosphate dehydrogenase (G6PD) is crucial to
NADPH generation and redox homeostasis. We have recently shown that
G6PD deficiency predisposes cells to
oxidant-induced cell death, and it is associated with the impairment of
glutathione regeneration. It remains unclear what other metabolic pathways are affected by
G6PD deficiency and whether the altered metabolism disturbs cellular redox homeostasis and underlies increased susceptibility to
oxidants. In this study, we examined the effects of
diamide on global metabolite profiles of SK-Hep1-derived SK-i-Gi and SK-i-Sc cells, which could inducibly express
short hairpin RNA (
shRNA) against G6PD (Gi) and control
shRNA (Sc), respectively. There was no significant difference in their metabolite profiles under uninduced conditions.
Doxycycline (Dox) addition resulted in over 70% decrease in G6PD activity in SK-i-Gi cells. This was accompanied by relatively minor changes in the metabolome of SK-i-Gi cells. Upon further
diamide treatment, the metabolite profiles of both SK-i-Gi and SK-i-Sc cells changed in a time-dependent manner. A number of metabolic pathways, including those involved in energy metabolism and metabolism of
amino acids and
glutathione, were affected. However, the changes in the metabolite profile of Dox-treated SK-i-Gi cells were distinct from those of control cells (i.e., Dox-treated SK-i-Sc, SK-i-Gi, and SK-i-Sc cells). Cellular
glutathione was depleted, whereas its
disulfide form increased significantly in
diamide, Dox-treated SK-i-Gi cells. Metabolites related to energy metabolism, such as
AMP,
ADP, and
acetylcarnitine, increased to a greater extent in these cells than in
diamide-treated control cells. In contrast,
NAD and
glutathione dropped to lower levels in SK-i-Gi cells than in control cells. The
NAD(+) depletion in SK-i-Gi cells was accompanied by a significant increase in
NAD kinase activity. Targeted analyses revealed that
NADP(+) and
NADPH increased significantly in
diamide, Dox-treated SK-i-Gi cells compared with similarly treated control cells. Our results suggest that
diamide induces oxidation and depletion of
glutathione in SK-i-Gi cells under conditions of G6PD
shRNA induction and subsequently induces conversion of
NAD(+) to
NADP(+) through enhanced
NAD kinase activity. This may represent a compensatory mechanism to restore cellular
NADPH reserve in G6PD-deficient cells. It is accompanied by alteration in pathways of cellular energy metabolism, such as glycolysis and β-oxidation.