A fundamental system of cellular energy homeostasis regulated by PGC-1alpha

Abstract

Maintenance of ATP levels is a critical feature of all cells. Mitochondria are responsible for most ATP synthesis in eukaryotes. We show here that mammalian cells respond to a partial chemical uncoupling of mitochondrial oxidative phosphorylation with a decrease in ATP levels, which recovers over several hours to control levels. This recovery occurs through an increased expression of the transcriptional coactivator peroxisome proliferator-activated receptor-coactivator 1alpha (PGC-1alpha) and mitochondrial genes. Cells and animals lacking PGC-1alpha lose this compensatory mechanism and cannot defend their ATP levels or increase mitochondrial gene expression in response to reduced oxidative phosphorylation. The induction of PGC-1alpha and its mitochondrial target genes is triggered by a burst of intracellular calcium, which causes an increase in cAMP-response-element-binding protein and transducer of regulated cAMP-response-element-binding proteins actions on the PGC-1alpha promoter. These data illustrate a fundamental transcriptional cycle that provides homeostatic control of cellular ATP. In light of this compensatory system that limits the toxicity of mild uncoupling, the use of chemical uncoupling of mitochondria as a means of treating obesity should be re-evaluated.

Fig. 1.

Chemical uncoupling induces multiple metabolic changes. (A) Cells were treated with 0, 25, 50, 100, or 200 μM FCCP for 16 or 24 h, and viability was measured by using the LDH assay (P = 0.01 compared with untreated cells at 24 h). (B) ATP levels were measured after fibroblasts were treated with 0 or 50 μM FCCP for 0, 2, 8, 16, or 24 h (P = 0.01 compared with the untreated cells at 0.5 h). (C) Lactate was measured after fibroblasts were treated with 0 or 50 μM FCCP for 2, 8, 16, or 24 h (P = 0.01 compared with the untreated cells). (D) Mitochondrial volume density in cells treated with 0 and 25 μM FCCP for 72 h (P = 0.01).

Fig. 2.

Chemical uncoupling induces OXPHOS genes and the PGC-1 coactivators. (A) Fibroblasts were treated with 0 or 50 μM FCCP for 16 h. RNA was measured by using real-time PCR (all points are significant except for tubulin, P < 0.01). (B) The experiment in A was repeated, but cells were treated for 72 h with 0 or 25 μM FCCP (all points are significant except for tubulin and hexokinase, P < 0.01). (C) WT and PGC-1α^−/− preadipocytes (KO) were treated with 0 or 50 μM FCCP for 16 h, and mRNAs were measured. The PGC-1α measurement represents exon 2, which is still present in the KO cells (P < 0.05 compared with the WT FCCP-treated cells). (D) WT and PGC-1α KO preadipocytes were treated with 0, 25, 50, 100, and 200 μM FCCP for 16 or 24 h. Viability was measured by using the LDH cytotoxicity assay (P < 0.01 compared with the WT FCCP-treated cells). (E) ATP levels in the WT and PGC-1α KO cells were measured after treatment with 50 μM FCCP (P = 0.02 compared with the WT FCCP-untreated cells at 16 h). (F) Lactate levels in the media of WT and KO cells treated with 200 μM FCCP for the were measured (P = 0.005 compared with the WT FCCP-treated cells at 16 h).

Fig. 3.

The induction of PGC-1α, PGC-1β, and OXPHOS genes requires increased [Ca²⁺]_i levels. (A) Fibroblasts were loaded with Fura-2, and [Ca²⁺]_i levels were measured (34, 54) as 50 μM FCCP was added. (B) [Ca²⁺]_i levels were measured as in A first with the addition 10 μM BAPTA and the subsequent addition of FCCP. (C) Fibroblasts were pretreated for 1 h with 0 or 10 μM BAPTA followed by 16 h of treatment with 0 or 50 μM FCCP, and mRNAs were measured (P < 0.01 compared with the FCCP-treated cells). (D) Fibroblasts were treated with 0 or 5 μM A23187 for 16 h (all points are significant except for tubulin, P < 0.01), and mRNAs were measured.

Fig. 4.

AMPK and CREB are involved in the mechanism inducing PGC-1α. (A) Fibroblasts were pretreated for 1 h with 0 or 25 μM compound C (CC) followed by 16 h of treatment with 0 or 50 μM FCCP. mRNAs were measured (P < 0.02 compared with FCCP-treated cells). (B) Fibroblasts were treated as in Fig. 3C. Western blots were used to measure protein levels. (C) PGC-1α promoter constructs with a luciferase reporter containing mutations in the MEF or CRE site were transfected into fibroblasts (PGC-1α WT is the unmutated PGC-1α 2-kb promoter), then treated with FCCP. Luciferase units were normalized to β-galactosidase and to the cells treated with 0 μM FCCP (P = 0.01). (D) ACREB was cotransfected with the PGC-1α 2-kb promoter into fibroblasts. The cells were treated with FCCP, and luciferase units were measured and normalized as in C (P = 0.01).

Fig. 5.

TORC plays a critical role in the uncoupling-mediated induction of PGC-1α and mitochondrial genes. (A) Fibroblasts were treated as described in Fig. 3C (P < 0.01 compared with the FCCP-treated samples), and mRNAs for TORC1, TORC2, and TORC3 were measured. (B) Fibroblasts were transfected with the PGC-1α 2-kb promoter, cotransfected with T1–44eGFP, and then treated with FCCP. Luciferase units were normalized to the cells treated with 0 μM FCCP (P = 0.005). (C) Preadipocytes constitutively expressing T1–44eGFP or the eGFP control were treated with either 0 or 50 μM FCCP for 16 h, and mRNAs were measured (P < 0.01 compared with FCCP-treated control cells). (D) ChIP was used to measure phospho-CREB protein and TORC protein bound to the PGC-1α promoter upon treatment with FCCP. Fibroblasts were treated as described in Fig. 3C. Cells were cross-linked, and protein–DNA complexes were harvested by using phospho-CREB or PAN-TORC antibodies. The amount of PGC-1α promoter was then quantified by using PCR and real-time PCR with PGC-1α-specific primers (P = 0.02).

Fig. 6.

The uncoupling-mediated induction of mRNA for PGC-1α, PGC-1β, and mitochondrial genes occurs in vivo. (A and B) WT and muscle-specific PGC-1α^−/− female mice were injected with either saline or 50 mg/kg DNP. Skeletal muscle (A) and liver (B) were harvested after 5 h (P < 0.01 compared with the WT DNP-treated mice, n = 8).