Blocking mitochondrial pyruvate import in brown adipocytes induces energy wasting via lipid cycling (2020)
Figure 5

Figure 5: MPC inhibition induces ATP utilization by lipid cycling.

(A,B) Effect of UK5099 on energy expenditure contributed by ATP demand and by mitochondrial proton leak in non-stimulated brown adipocytes. Primary brown adipocytes were pre-treated with vehicle (DMSO) or 100 nM UK5099 for 2 hours. OCR were measured in respirometry media supplemented with 5 mM glucose and 3 mM glutamine in the presence of vehicle or UK5099. Oligomycin A (Oligo; 4 µM), mitochondrial uncoupler FCCP (2 µM) and antimycin A (AA; 4 µM) were injected where indicated. (A) Representative OCR traces averaging 6 technical replicates. (B) Quantification of mitochondrial proton leak and ATP-linked OCR (oligomycin sensitive) as measured in (A) from n=9 individual experiments. Data were normalized to vehicle for each individual experiment. ** p < 0.01 ***, p < 0.001 compared to vehicle by Student´s t test.

(C) To determine the contribution of ATP demand to increased energy expenditure under UK5099 cells were permeabilized and cytosolic ADP concentrations were clamped. OCR were measured in the presence of 5 mM pyruvate, 3 mM malate, 0.1 mM palmitoyl-CoA, 0.5 mM carnitine and 5 mM ADP. Cells were treated with either 100 nM, 1 µM or 10 µM UK5099. Data shows maximal state 3 OCR normalized to maximal complex 4 activity from n=3 individual experiments. Note that when ATP demand is clamped, UK5099 had no effect on energy expenditure.

(D) Schematic representation of lipid cycling. HK, hexokinase; PFK, phosphofructokinase; ATGL, adipose triglyceride lipase; ACS, Acyl-CoA synthetase; TAG, triacylglyceride; DAG, diacylglyceride; MAG, monoacylglyceride; LPA, lysophosphatidic acid; FFA, free fatty acid; G3P, glycerol-3-phosphate

(E) Effect of MPC inhibition on glycerol 3-phosphate (G3P) and dihydroxyacetone phosphate (DHAP) generation. G3P/DHAP is used as a measure of acylglycerol synthesis. Brown adipocytes were treated with either vehicle, 100 nM UK5099 or 1 µM norepinephrine (NE) for 24 h from n=4 individual experiments. * p < 0.05 by ANOVA, relative to vehicle.

(F,G) Contribution of lipolysis to UK5099 induced energy demand. Primary brown adipocytes were pre-treated with either vehicle (DMSO), 100 nM UK5099, 40 µM Atglistatin or a combination of Atglistatin with UK5099 for 2 h. OCR were measured in respirometry media supplemented with 5 mM glucose and 3 mM glutamine in the presence of vehicle, UK5099, or Atglistatin. (F) Quantification of non-stimulated OCR from n=4 individual experiments. * p < 0.05, ** p < 0.01, *** p < 0.001 by ANOVA. (G) Quantification of ATP-linked respiration from n=4 individual experiments. Data were normalized to vehicle for each individual experiment. * p < 0.05, ** p < 0.01, *** p < 0.001 by ANOVA.

(H,I) Contribution of TAG synthesis to UK5099 induced energy demand. Primary brown adipocytes were pre-treated with either vehicle (DMSO), 100 nM UK5099, 1 µM DGAT1 and 1 µM DGAT2 or a combination of DGAT1/2 inhibitors with UK5099 for 2 h. OCR were measured in respirometry media supplemented with 5 mM glucose and 3 mM glutamine in the presence of vehicle, UK5099, DGAT1/2 inhibitors, or UK5099 + DGAT1/2 inhibitors. (H) Quantification of non-stimulated OCR from n=6 individual experiments. Data were normalized to vehicle for each individual experiment. ns p>0.05, * p < 0.05 by ANOVA. (I) Quantification of ATP-linked OCR from n=6 individual experiments. Data were normalized to vehicle for each individual experiment. ns p>0.05, ** p < 0.01 by ANOVA.

(J,K) Contribution of ACS to UK5099 induced energy demand. Primary brown adipocytes were pre-treated with either vehicle (DMSO), 100 nM UK5099, 5 µM Triacsin C or a combination of 5 µM Triacsin C with UK5099 for 2 h. OCR were measured in respirometry media supplemented with 5 mM glucose and 3 mM glutamine in the presence of vehicle, UK5099, Triacsin C, or UK5099 + Triacsin C.

(J) Quantification of non-stimulated OCR from n=4 individual experiments. Data were normalized to vehicle for each individual experiment. * p < 0.05 by ANOVA.

(K) Quantification of ATP-linked OCR from n=4 individual experiments. Data were normalized to vehicle for each individual experiment. * p < 0.05 by ANOVA. Note that MPC-inhibition induced energy demand requires lipolysis and ACS dependent fatty acid activation, but not DGAT-dependent TAG synthesis, suggesting the activation of a sub-cycle in lipid cycling pathway.

Data information: All data are presented as mean ± SEM.