Pettersen 2015 Abstract MiPschool London 2015

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Metabolic adjustments after reversible induction of mitochondrial biogenesis via AMPK/NRF1 in HeLa cells.

Link:

Pettersen IKN, Nilsson LIH, Nikolaisen J, Micklem D, Lorens J, Tronstad KJ (2015)

Event: MiPschool London 2015

Regulation of mitochondrial biogenesis represents one of the major mechanisms the cell employs to adapt to changes in energy conditions [1,2]. By increasing the mitochondrial biomass, the cell strengthens the respiratory capacity and is able to produce more ATP. Recent studies have provided new insights into important roles of mitochondrial biogenesis in cellular adaptation to energy stress, mutations and aging. One of the major regulators of mitochondrial biogenesis is the transcription factor nuclear respiratory factor 1 (NRF1) [3]. We developed a reversible GFP reporter cell system in order to study the activity of NRF1 and the effects on mitochondrial properties. NRF1 is an essential regulator of genes involved in mitochondrial DNA transcription and components of the respiratory chain, among others. One of the major routes of NRF1 activation is via the cellular energy sensor AMP-activated protein kinase (AMPK)[4]. In this study, we addressed the role of NRF1 mediated mitochondrial biogenesis in cellular adaptation and stress tolerance.

The AMPK/NRF1 pathway was activated by 0.5 mM AICAR and GFP expression was monitored by flow cytometry and microscopy. Amount of mtDNA and mRNA of genes of interest was determined by quantitative PCR, and respiratory function was analysed with both Seahorse XF96 Analyzer and OOROBOROS Oxygraph-2k. Substrate preference for respiration was also investigated in the Seahorse XF96 Analyzer. Activation of AMPK by AICAR resulted in induction of NRF1 regulated transcription and increased levels of mtDNA, respiratory chain components and mitochondrial respiration. Gene expression analysis of an array of mitochondrial components demonstrated that multiple mitochondrial proteins were upregulated after AMPK/NRF1 activation. We found that NRF1 regulated GFP expression can be reversed by removal of AICAR (Fig 1). We also observed an increase in glucose/pyruvate driven respiration in AICAR treated cells but not in the presence of L-glutamine (Fig 2).

In conclusion, we have developed a GFP-based reporter system of NRF1 regulated gene transcription which enables non-invasive surveillance of mitochondrial biogenesis in living cells. We find correlation between NRF1 regulated GFP expression and regulation of mtDNA, mitochondrial respiration and mRNA expression levels of essential contributors to mitochondrial biogenesis in response to energetic stress.

Keywords: NRF1

O2k-Network Lab: NO Bergen Tronstad KJ


Labels: MiParea: Instruments;methods, mt-Biogenesis;mt-density, mtDNA;mt-genetics 




Regulation: Substrate 

Pathway:HRR: Oxygraph-2k 


Affiliations

1-Dept Biomed, Univ Bergen, Norway. - Ina.pettersen@uib.no

2-BergenBio AS, Norway

Figures

MiPschool2015 Pettersen Figure1.jpg

Figure 1. MiPschool2015 Pettersen Figure2.jpg Figure 2.


References

  1. Hock MB Kralli A (2009) Transcriptional control of mitochondrial biogenesis and function. Annu Rev Physiol 71:177-203.
  2. Scarpulla RC (2008)Transcriptional Paradigms in Mammalian Mitochondrial Biogenesis and Function. 88:611-38.
  3. Scarpulla RC (1997) Nuclear control of respiratory chain expression in mammalian cells. J Bioenerg Biomembr 29:109-19.
  4. Hardie DG (2011) AMP-activated protein kinase: a cellular energy sensor with a key role in metabolic disorders and in cancer. Biochem Soc Trans 39:1-13.