Figure 3: Saturating MDH1 and GPD1/GPD1L drives lactate production.

(A) Transfecting H-Ras (G12V) into non-transformed 3T3 cells results in detectable H-Ras (G12V) protein expression.

(B) Non-transformed 3T3 cells are contact inhibited when grown to confluency, but H-Ras 3T3 cells are no longer sensitive to contact inhibition. n=3 replicates per group.

(C and D) Overexpressing MDH1 (C) and GPD1 (D) in H-Ras 3T3 cells increases MDH1 and GPD1/GPD1L activities, respectively. Control cells overexpressed GFP instead of a dehydrogenase. MDH1 activities was determined by the fraction of [2-²H] malate normalized to the fraction of [1-²H] GAP. GPD1/GPD1L activities was determined by the fraction of [1,2-²H] G3P normalized to the fraction of [1-²H] DHAP and [4-²H] NADH. n=3 replicates per group. p values were determined by using a two-tailed Student’s t test.

(E) Lactate-excretion rates decrease when MDH1 or GPD1 is overexpressed in H-Ras 3T3 cells. Control cells overexpressed GFP instead of a dehydrogenase. n=3 replicates per experimental group. p values were determined by using a one-way ANOVA followed by Dunnett’s test.

(F) Relative glycolytic activity in H-Ras 3T3 cells as a function of 2DG concentration. Glycolytic activity was determined by the fraction of M+1 GAP during [4-²H] glucose labeling. n=3 replicates per 2DG concentration.

(G) Relative fluxes of MDH1, GPD1/GPD1L, and LDH as a function of glycolytic activity in H-Ras 3T3 cells. Glycolytic activities were derived from (F). MDH1 activity was determined by the fraction of [2-²H] malate normalized to the fraction of [1-²H] GAP. GPD1/GPD1L activity was determined by the fraction of [1,2-²H] G3P normalized to the fraction of [1-²H] DHAP and [4-²H] NADH. LDH activity was determined by the amount of lactate excreted over time. Data were fit with a logistic function. The R² value is the coefficient of determination. n = 3 replicates per experimental group.

(H) First derivatives of MDH1, GPD1/GPD1L, and LDH activities in H-Ras 3T3 cells. Calculations were based on the fitted equations shown in (G).

(I) Relative glycolytic activity in non-transformed proliferating 3T3 cells as a function of 2DG concentration. Glycolytic activity was determined by the fraction of M+1 GAP during [4-²H] glucose labeling. n=3 replicates per 2DG concentration.

(J) Relative fluxes of MDH1, GPD1/GPD1L, and LDH as a function of glycolytic activity in non-transformed proliferating 3T3 cells. Glycolytic activities were derived from (I). MDH1 activity was determined by the fraction of [2-²H] malate normalized to the fraction of [1-²H] GAP. GPD1/GPD1L activity was determined by the fraction of [1,2-²H] G3P normalized to the fraction of [1-²H] DHAP and [4-²H] NADH. LDH activity was determined by the amount of lactate excreted over time. Data were fit with a logistic function. The R² value is the coefficient of determination. n=3 replicates per experimental group.

Error bars denote standard error. *p≤0.05, **p≤0.001, ***p≤0.001, n.s.>0.05.

Ctrl, control; OE, overexpression; KD, knockdown; Lac, lactate; Glc, glucose; GAP, glyceraldehyde 3-phosphate; G3P, glycerol 3-phosphate; DHAP, dihydroxyacetone phosphate.