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.

Fig. 1

FoxO3A accumulates into the mitochondria upon GR. a, b, c Upon GR (LG), FoxO3A accumulates into the mitochondria as shown by immunofluorescence analysis (a) in murine C2C12-derived (LG 24 h) and primary satellite cell-derived myotubes (LG 36 h), and by immunogold labeling (b) in murine C2C12-derived myotubes and NIH-3T3-fibroblasts (LG 24 h) (black dots represent gold particles recognizing FoxO3A immunocomplexes). These data (n = 3) were quantified by scoring the percentage of FoxO3A-positive cells and counting the number of gold particles per single mitochondria. Data are presented as mean ± SEM and significance was calculated with Student’s t test (c). d Cellular fractionation of murine NIH-3T3 and human primary IMR90 fibroblasts shows mitochondrial enrichment of FoxO3A under low glucose conditions (LG 16 and 24 h, respectively). The values indicated are the results of the densitometric analysis of FoxO3A normalized against the mitochondrial fractionation control VDAC (arbitrary units, HG = 1). Cellular fractionation controls: nuclei: PCNA; cytoplasm: GAPDH; mitochondria: VDAC. Asterisks non-specific band

Fig. 2

GR-dependent FoxO3A mitochondrial import leads to increased mitochondrial respiration. a, b Under GR conditions (LG 12 h), FoxO3A binds to two different target sites (FHRE#1: 14,963–15,110 bp; FHRE#2: 15,400–15,469 bp) on mitochondrial DNA in C2C12 myotubes (n = 5) (a) and NIH-3T3 fibroblasts (n = 5) (b). Signal specificity is confirmed by the analysis of a distal region not containing FHRE sites (NON FHRE, 8,653–8,768 bp). Unrel unrelated antibody (anti-IgG). Data are presented as mean ± SEM and significance was calculated with Student’s t test. c–g GR (LG) induces the upregulation of mitochondrial genes both at the RNA (c, e, n = 5) and the protein level (d, f, n = 3) in C2C12-derived (c LG 12 h, d LG 24 h) and primary satellite cell-derived myotubes (e LG 36 h, f LG 48 h). Upregulation of mitochondrial genes upon GR (LG 24 h) is blunted by the absence of FoxO3A in primary MEFs (n = 5) (g). h Upon genetic ablation of FoxO3A using a specific siRNA, GR (LG 36 h) fails to induce an increase in DNP-uncoupled mitochondrial endogenous respiration in murine NIH-3T3 fibroblasts (left panel n = 9, right panel n = 6). Data are presented as mean ± SEM and significance was calculated with Student’s t test. (c, e, g) black bars ATPase 6 and 8 genes, white bars COI, COII and COIII genes, grey bars ND1, ND2, ND3, ND4, ND4L, ND5, and ND6 genes, light grey bar 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, *p < 0.05

Fig. 3

AMPK mediates GR-dependent FoxO3A mitochondrial enrichment. a GR (LG) induces phospho-activation of AMPK in NIH-3T3 fibroblasts (LG 24 h) and C2C12-derived myotubes (LG 12 h). b GR-dependent mitochondrial accumulation of FoxO3A significantly decreases upon pharmacological inhibition of AMPK using compound C(CC) in NIH-3T3 fibroblasts (LG 16 h). c Exogenous activation of AMPK using AICAR, metformin or resveratrol induces FoxO3A accumulation into the mitochondria in NIH-3T3 fibroblasts. d FoxO3A accumulates into the mitochondria in response to GR and AMPK exogenous activation (AICAR), while AMPK ablation by a specific siRNA prevents FoxO3A accumulation even in low glucose (LG 24 h) or in the presence of an AMPK-activator (AICAR) in high glucose (HG 24 h), as shown by immunogold labeling of NIH-3T3 fibroblasts (black dots represent gold particles recognizing FoxO3A immunocomplexes). e These data (n = 3) were quantified by scoring the percentage of FoxO3A-positive cells and counting the number of gold particles per single mitochondria. Data are presented as mean ± SEM and significance was calculated with Student’s t test

Fig. 4

AMPK mediates the GR-dependent FoxO3A mitochondrial transcriptional program. a Inhibition of AMPK using compound C(CC) abrogates GR-dependent FoxO3A binding to mitochondrial DNA at both its target sites (FHRE#1: 14,963–15,110 bp; FHRE#2: 15,400–15,469 bp) in myotubes (n = 3). Unrel unrelated antibody (anti-IgG). Data are presented as mean ± SEM and significance was calculated with Student’s t test. b, c Exogenous activation of AMPK using AICAR or resveratrol promotes the upregulation of mitochondrial genes in C2C12-derived myotubes (b n = 3) and NIH-3T3 fibroblasts (c n = 3) in standard culture conditions (HG 24 h). d The AMPK activator AICAR (HG 24 h) fails to induce mitochondrial gene expression in primary MEFs derived from FoxO3A^−/− mice (n = 3). e siRNA-mediated AMPK knock-down interferes with FoxO3A-dependent upregulation of mitochondrial genes induced by low glucose (LG 24 h) and addition of AICAR (HG 24 h) in NIH-3T3 fibroblasts (n = 3). The dotted line corresponds to the expression levels detected in cells transfected with a control siRNA and cultured in high-glucose conditions. b–e black bars ATPase 6 and 8 genes, white bars COI, COII and COIII genes, grey bars ND1, ND2, ND3, ND4, ND4L, ND5, and ND6 genes, light grey bar 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, *p < 0.05

Fig. 5

A FoxO3A/SIRT3/mtRNAPol protein complex is assembled on mtDNA upon GR. a, b FoxO3A, mtRNAPol, and SIRT3 are co-recruited on mitochondrial DNA upon GR as shown by chromatin immunoprecipitation (ChIP) experiments in C2C12 myotubes (a n = 3, LG 12 h) and NIH-3T3 fibroblasts (b n = 3, LG 16 h). Unrel unrelated antibody (anti IgG). Data are presented as mean ± SEM * p < 0.05. c mtRNAPol immunocomplexes are enriched in both FoxO3A and SIRT3 in glucose-restricted NIH-3T3 fibroblasts upon GR (LG 16 h) as shown by co-immunoprecipitation analysis

Fig. 6

SIRT3 activity was required for GR-dependent expression of the mitochondrial genome. a Upregulation of mitochondrial genes upon GR (LG, 24 h) is blunted by the absence of SIRT3 in primary MEFs (n = 5). b–d Sirtuin inhibition using nicotinamide (NAM) reduces the upregulation of mitochondrial genes (b n = 5) and FoxO3A binding to mtDNA (i.e., FHRE#1) (d n = 3) in myotubes (LG 12 h), but not its accumulation into the mitochondria in NIH-3T3 fibroblasts (c) upon low-glucose conditions (LG 24 h). Data in d are presented as mean ± SEM and significance was calculated with Student’s t test. e Co-IP performed in glucose-restricted (LG 24 h) primary IMR90 human fibroblasts. mtRNAPol immunocomplexes were blotted with an anti-acetyl lysine antibody (AcK), then stripped and re-blotted with anti-mtRNAPol, anti-FoxO3A, and anti-SIRT3 antibodies. a, b black bars ATPase 6 and 8 genes, white bars COI, COII, and COIII genes, grey bars ND1, ND2, ND3, ND4, ND4L, ND5, and ND6 genes, light grey bar 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, *p < 0.05

Fig. 7

FoxO3A binds to mtDNA in muscle of fasted mice. a The transcription of mitochondrial genes increases in fasted mice (FAST 18 h) in both glycolytic (tibialis/EDL, n = 5), oxidative (gastrocnemius/soleus, n = 5) and mixed type (quadriceps, n = 10) skeletal muscles. black bars ATPase 6 and 8 genes, white bars COI, COII, and COIII genes, grey bars ND1, ND2, ND3, ND4, ND4L, ND5, and ND6 genes, light grey bar cytochrome b gene. The dotted line corresponds to the expression levels detected in fed mice. b FoxO3A and mtRNAPol are both enriched at regulatory sites of mtDNA in tibialis and EDL skeletal muscles of fasted mice, as shown by ChIP experiments (n = 3). Data are presented as mean ± SEM, *p < 0.05. c In vivo co-immunoprecipitation analyses revealed that mtRNAPol immunocomplexes are enriched in both FoxO3A and SIRT3 in tibialis and EDL skeletal muscles during fasting (FAST 18 h)

Fig. 8

The AMPK-FoxO3A-SIRT3 pathway induces mitochondrial transcription and respiration upon GR. A representative scheme depicted to summarize the findings of this work. Activation of AMPK by glucose restriction (GR) or AMPK agonists lead to FoxO3A mitochondrial enrichment. Into the mitochondria, SIRT3 mediates FoxO3A binding to mtDNA and transcription of mitochondrial-encoded core or catalytic subunits of the OXPHOS machinery, resulting in increased mitochondrial respiration