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A novel AMPK-dependent FoxO3A-SIRT3 intramitochondrial complex sensing glucose levels

Abstract

Reduction of nutrient intake without malnutrition positively influences lifespan and healthspan from yeast to mice and exerts some beneficial effects also in humans. The AMPK-FoxO axis is one of the evolutionarily conserved nutrient-sensing pathways, and the FOXO3A locus is associated with human longevity. Interestingly, FoxO3A has been reported to be also a mitochondrial protein in mammalian cells and tissues. Here we report that glucose restriction triggers FoxO3A accumulation into mitochondria of fibroblasts and skeletal myotubes in an AMPK-dependent manner. A low-glucose regimen induces the formation of a protein complex containing FoxO3A, SIRT3, and mitochondrial RNA polymerase (mtRNAPol) at mitochondrial DNA-regulatory regions causing activation of the mitochondrial genome and a subsequent increase in mitochondrial respiration. Consistently, mitochondrial transcription increases in skeletal muscle of fasted mice, with a mitochondrial DNA-bound FoxO3A/SIRT3/mtRNAPol complex detectable also in vivo. Our results unveil a mitochondrial arm of the AMPK-FoxO3A axis acting as a recovery mechanism to sustain energy metabolism upon nutrient restriction.

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Abbreviations

GR:

Glucose restriction

FoxO:

Forkhead-box O

AMPK:

AMP-activated kinase

FoxO binding sites:

FHRE

mtDNA:

Mitochondrial DNA

MEFs:

Mouse embryonic fibroblasts

mtRNAPol:

Mitochondrial RNA polymerase

CR:

Calorie restriction

HG:

High glucose medium

LG:

Low glucose medium

CC:

Compound C

NAM:

Nicotinamide

DNP:

2,4-Dinitrophenol

FED:

Feeding

FAST:

Fasting

AcK:

Anti-acetyl lysine

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Acknowledgments

We thank Dr. Francesco Paolo Jori for his helpful discussion during the preparation of the manuscript and editorial assistance, Dr. Roberta Ledonne for preparing the illustrations and editing this manuscript, Drs. Michele Petruzzeli and Daniele Di Giandomenico for technical assistance, Dr. Antonio Moschetta for discussion, Dr. Karen Arden for generously providing Foxo3A−/− MEFs and Drs. Lucisano and Pellegrini (Unit of Biostatistics, Consorzio Mario Negri Sud) for statistical analysis. Image acquisition and image data analysis were performed at the Advanced Light and Electron Microscopy Facility of the Consorzio Mario Negri Sud. This work was partially supported by a ‘My First Grant 2007’ and an ‘Investigator Grant 2010’ (IG10177) (to C.S.) from the Italian Association for Cancer Research (AIRC).

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Correspondence to Cristiano Simone.

Electronic supplementary material

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Supplementary Fig. 1

FoxO3A accumulates into the mitochondria upon GR (LG 24h), as shown by immunogold labeling of murine C2C12-derived and murine NIH-3T3 fibroblasts (black dots represent gold particles recognizing FoxO3A immunocomplexes). (TIFF 15990 kb)

Supplementary Fig. 2.

Upon GR (LG 24h), but not glutamine deprivation or oxidative stress, FoxO3A localizes to mitochondria in primary IMR90 human fibroblasts, as shown by immunofluorescence analysis using the indicated antibodies and probes. (TIFF 15991 kb)

Supplementary Fig. 3

a-d GR induces the time-dependent upregulation of mitochondrial genes both at the RNA (a, c, d) and the protein level (b) in murine NIH-3T3 (a, b), C2C12-derived terminally differentiated myotubes (c) and primary IMR90 human fibroblasts (d). (a, c, d) black bars: ATPase 6 and 8 genes; white bars: COI, COII, and COIII genes; diagonally hatched bars: ND1, ND2, ND3, ND4, ND4L, ND5, and ND6 genes; checkered bars: cytochrome b gene. The dotted line corresponds to the expression levels detected in cells cultured in standard glucose conditions (HG). Data are presented as mean ± SEM. (TIFF 19782 kb)

Supplementary Fig. 4

a GR-induced time-dependent upregulation of mitochondrial genes occurs in the absence of mitochondrial biogenesis in C2C12-derived myotubes (LG 24h), primary IMR90 (LG 48h), and NIH-3T3 cells (LG 48h). Data are presented as mean ± SEM. b The efficacy of FoxO3A genetic ablation by a specific siRNA was confirmed by densitometric analysis of Western-blot signals (FoxO3A protein levels were reduced 62% to 79% in both HG and LG conditions compared to control siRNAs). (TIFF 14984 kb)

Supplementary Fig. 5

a The efficacy of AMPK genetic ablation by a specific siRNA was confirmed by Western-blot analysis. b, c AMPK activation in HG does not affect mitochondrial biogenesis in C2C12-derived myotubes (b) and NIH-3T3 cells (c), as revealed by quantification of the total amount of mitochondrial DNA 24h after the addition of AICAR, metformin or resveratrol. d Nicotinamide does not influence mitochondrial content in myotubes upon low-glucose conditions (LG 12h). Data are presented as mean ± SEM. (TIFF 15990 kb)

Supplementary Fig. 6.

ChIP experiments showing that sirtuin inhibition using nicotinamide (NAM) reduces FoxO3A binding to mtDNA in NIH-fibroblasts (FHRE#1, LG 16h). Unrel: unrelated antibody (anti-IgG). Data are presented as mean ± SEM. (TIFF 15986 kb)

Supplementary Fig. 7.

a, b Immunogold labeling of NIH-3T3 fibroblasts showing that (a) FoxO3A accumulates into the mitochondria in response to GR and AMPK exogenous activation (AICAR) and (b) AMPK ablation by a specific siRNA prevents FoxO3A accumulation even in the presence of low glucose conditions or addition of AICAR (black dots represent gold particles recognizing FoxO3A immunocomplexes). (TIFF 15954 kb)

Supplementary Fig. 7.

a, b Immunogold labeling of NIH-3T3 fibroblasts showing that (a) FoxO3A accumulates into the mitochondria in response to GR and AMPK exogenous activation (AICAR) and (b) AMPK ablation by a specific siRNA prevents FoxO3A accumulation even in the presence of low-glucose conditions or addition of AICAR (black dots represent gold particles recognizing FoxO3A immunocomplexes). (TIFF 15954 kb)

Supplementary Fig. 8.

Overnight fasting activates AMPK in tibialis and EDL skeletal muscles (18h FAST). (TIFF 19780 kb)

Supplementary material 10 (DOCX 30 kb)

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Peserico, A., Chiacchiera, F., Grossi, V. et al. A novel AMPK-dependent FoxO3A-SIRT3 intramitochondrial complex sensing glucose levels. Cell. Mol. Life Sci. 70, 2015–2029 (2013). https://doi.org/10.1007/s00018-012-1244-6

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Keywords

  • Glucose restriction
  • FoxO3A
  • AMPK
  • SIRT3
  • OXPHOS