Molecular Exercise Physiology Mitochondrial biogenesis Presentation 3 Henning Wackerhage

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Molecular Exercise PhysiologyMitochondrial
biogenesis Presentation 3Henning Wackerhage
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Learning outcomes

• At the end of this presentation you should be
  able to
• Explain the endosymbiosis hypothesis.
• Describe stimuli that activate mitochondrial
  biogenesis.
• Explain the effect of an overexpression of PGC-1
  in muscle.
• Explain how the expression of mitochondrial
  proteins encoded in nuclear and mitochondrial DNA
  is regulated.

Mitochondrial biogenesis is not an easy process
to understand. You will need to spend
considerable time on revision.
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Mitochondrial biogenesisPart 1Exercise and
mitochondrial biogenesis
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Mitochondrial biogenesis
Mitochondria are the power stations of our cells
and the sites of oxidative phosphorylation. The
enzymes for fat metabolism (b-oxidation), the
Krebs cycle, electron transport chain and finally
the F0F1 ATP synthase are located inside the
mitochondria. Hopefully, you know a bit about
oxidative phosphorylation from previous lectures.
Have a close look at the parts of a mitochondrion
in the figure below. All these parts need to be
synthesized during mitochondrial biogenesis.
Mitochondrial DNA Krebs cycle Electron transport
chain F0F1 ATP synthase
Pathways (e.g. b-oxidation) Transporters (e.g.
ATP/ADP, FAs)
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Task
How do mitochondria synthesize ATP? Name at least
two researchers that won a Nobel price on ATP
synthesis by mitochondria.
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Mitochondrial biogenesis in skeletal muscle

• Mitochondrial biogenesis can be stimulated in
  skeletal muscle
• Skeletal muscle mitochondria proliferate (divide
  and increase in numbers) in response to exercise
  (Holloszy et al 1967).
• Chronic electrical stimulation of a muscle also
  increasesd mitochondrial biogenesis (Williams et
  al. 1987).
• Thyroid hormones increase the metabolic rate and
  mitochondrial enzyme levels (Tat et al. 1963).
• Mitochondrial biogenesis also occurs during
  development.

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Chronic electrical stimulation activates
mitochondrial biogenesis
In this study, an electrode was connected to a
motor nerve innervating the hindlimbs of a rat.
The hindlimb muscles were stimulated for several
weeks. The stain is the nitro blue tetrazolium
stain which stains a mitochondrial enzyme. The
figure shows how chronic electrical stimulation
increases the mitochondrial content of this
muscle.
Control
Salmons, Jarvis, Higginson, Manolopoulos, Woods,
Wackerhage, unpublished data (2001)
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Endurance training increases the mitochondrial
content
Saltin et al. (1976)
Subjects endurance trained with one leg and
rested the other. After the training period, the
capillary density, mitochondrial content and peak
oxygen uptake achieved when cycling with that leg
were measured. The results showed that endurance
training increased the mitochondrial content of
the trained leg by ? 20 and also the peak oxygen
uptake that was achieved when working with the
trained leg only.
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Mitochondrial biogenesisPart 2Origin of
mitochondria
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Serial endosymbiosis hypothesis
Where do mitochondria come from? Lynn Margulis
published a book in 1981 entitled symbiosis in
cell evolution, where she stated the so-called
endosymbiosis hypothesis. According to this
hypothesis, cells with a pre-aerobic metabolism
invaded anaerobic, prokaryotic host cells and
formed a symbiosis. The most compelling evidence
for the endosymbiotic hypothesis is that
mitochondria have their own DNA. Less than 10
of the proteins of a mitochondrion are encoded in
the mitochondrial DNA. All other proteins are
encoded in the nuclear DNA.
Prof Lynn Margulis
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Serial endosymbiosis hypothesis
Ancestral host cell (anaerobic)
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Two DNAs encode mitochondrial proteins
Nuclear DNA encoding mitochondrial proteins
Mitochondrial DNA
Because there are two sources of DNA that encode
mitochondrial biogenesis, a regulatory system is
needed to regulate the expression of these
proteins, and their transport and assembly during
mitochondrial biogenesis.
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Mitochondrial biogenesis Part 4Mitochondrial
genetics
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Sequencing of mitochondrial DNA
All genes that encode mitochondrial proteins need
to be expressed during mitochondrial biogenesis.
Some of these genes are encoded in mitochondrial
DNA and others in the normal DNA inside the
nucleus. Researchers at the University of
Cambridge have published the DNA sequence of the
human mitochondrial DNA in 1981, which can be
considered as the start of the human genome
project Anderson S, Bankier AT, Barrell BG, de
Bruijn MH, Coulson AR, Drouin J, Eperon IC,
Nierlich DP, Roe BA, Sanger F, Schreier PH, Smith
AJ, Staden R, Young IG. Sequence and organization
of the human mitochondrial genome. Nature.
290457-465, 1981. This paper showed that a DNA
sequence that contained 16569 base pairs could be
sequenced with the DNA sequencing techniques
developed by Fred Sanger and others.
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Mitochondrial DNA
What is the structure of mitochondrial DNA?
Mitochondrial DNA contains 16568 base pairs and
it is transcribed as a single transcript that
encodes 13 proteins (see figure). The
mitochondrial DNA is economical there are very
few non-coding sequences.
Mitochondrial DNA encodes only 13 proteins which
is less than 10 of all the mitochondrial
proteins. All other proteins are encoded in the
nuclear DNA. Transcription factors that regulate
the transcription and replication (doubling) of
DNA bind to the displacement loop (D-loop, see
top of figure).
Anderson et al. (1981), Clayton (1991)
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Task
a) Identify the names of the genes that are
encoded in mitochondrial DNA. Name at least five
other proteins that are needed for the production
of mitochondria. b) How is mitochondrial DNA
inherited. Find out.
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Mitochondrial DNA
The respiratory chain in mitochondria consists of
four protein complexes plus F0F1 ATP synthase
which is synthesizing ATP. All complexes are
assembled from several proteins. The table shows
that some of these proteins are encoded in
nuclear DNA and others in mitochondrial DNA.
Complex Mitochondrial Nuclear encoded
proteins encoded proteins I 7 gt25 II 0
4 III 1 10 IV 3 10 F0F1 ATP
synthase 2 11
Poyton and McEwan (1996)
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Mitochondrial biogenesis Part 3Factors and
pathways involved
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Task
Assume you would have to design a regulatory
mechanism by which exercise activates
mitochonrial biogenesis. What would be a good
exercise signal in muscle? How would you activate
mitochondrial genes encoded in nuclear DNA? How
would you activate mitochondrial genes encoded in
mitochondrial DNA? What other steps and reactions
are needed?
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Regulation of mitochondrial biogenesis
Mitochondrial biogenesis requires co-ordination
of the synthesis of mitochondrial proteins and
other molecules and the assembly of these
molecules into a new mitochondrion. Researchers
have identified two classes of transcription
factors that are involved in this process a)
Transcription factors that regulate the
transcription and replication (DNA copying) of
mitochondrial DNA (mtDNA) b) Transcription
factors that regulate the transcription of
mitochondrial genes encoded in the nuclear DNA.
Transcription factor regulating mitochondrial
genes in nuclear DNA Transcription factor
regulating mitochondrial genes in nuclear DNA
Nucleus
Mitochondria
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Transcription factors acting on nuclear DNA
Several transcription factors have been
discovered by researchers. Leading in the field
are Richard Scarpulla, who has discovered the
nuclear respiratory factors (NRF-1, NFRF-2) and
Pere Puigserver who discovered the
transcriptional co-factor PPARg coactivator
(PGC-1). This co-factor is not a transcription
factor itself but binds to transcription factors.
These transcription and co-factors regulate the
expression of mitochondrial genes located in the
nucleus.
Richard C Scarpulla
Pere Puigserver
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Transcription factors acting on nuclear DNA
The nuclear respiratory factor (NRF-1) was
discovered by Evans and Scarpulla (1989, 1990).
Subsequent studies showed that NRF-1 had
transcription factor binding sites in genes that
encoded proteins of the respiratory chain, F0F1
ATP synthase, heme biosynthesis and protein
import into mitochondria. All these proteins are
located in the nuclear DNA. Many of these genes
also have binding sites for NRF-2. Puigserver et
al. (1998) then discovered the transcriptional
co-activator PGC-1. Both NRF-1, PGC-1 and other
transcription and co-factors bind together and
regulate the expression of mitochondrial genes
that are encoded in nuclear DNA.
PGC-1
Expression of mitochondrial genes
NRF
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Task
PGC-1 is a transcriptional co-activator. What is
the difference between a transcriptional
co-activator and a transcription factor? How do
they work?
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PGC-1 and mitochondrial biogenesis
AMP-activated protein kinase (AMPK Terada et al.
2003) and Calcium/calmodulin-activated kinase
(CamK IV Wu et al. 2003) have been shown to
stimulate PGC-1 expression. The action of p38
MAPK leads to an increased phosphorylation of
PGC-1 (Puigserver et al. 2003).
The story so far
AMPK
PGC-1
CamK
Endurance exercise
p38
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PGC-1 and mitochondrial biogenesis
An increased PGC-1 concentration then expression
of NRF-1, NRF-2 and mtTFA, i.e. transcription
factors that regulate mitochondrial genes encoded
in nuclear and mitochondrial DNA. In addition,
PGC-1 binds to NRF-1 (Wu et al. 1999). Thus,
PGC-1 appears to be the master regulator of
mitochondrial biogenesis.
The more detailed story
AMPK
PGC-1
Mitochondrial biogenesis
NRF
PGC-1
CamK
mtTFA
?
Endurance exercise
p38
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PGC-1 is the master regulator of mitochondrial
biogenesis
Lin et al. (2002) generated transgenic mice that
overexpressed PGC-1 in their muscles. In the
figures, WT refers to the wildtype (normal mice)
and TG to the transgenic mice. The transgenic
muscles have more cytochrome c, an enzyme found
in mitochondria. These data show that PGC-1 can
induce mitochondrial biogenesis in vivo.
WT TG
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PGC-1 is the master regulator of mitochondrial
biogenesis and also regulates other slow genes
Overexpression of PGC-1 did not only affect
mitochondrial biogenesis. The transgenic muscles
appear red due to the high myoglobin content (see
also the Western blot bottom left. The
mitochondrial content is increased as expected
(cytochrome C is a marker) and surprisingly also
motor proteins such as slow troponin (TnI) and
slow myosin are upregulated (Lin et al. 2002).
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AMPK induces PGC-1 and mitochondrial biogenesis
The following figure shows that swimming and
AICAR, an AMPK activator, increase PGC-1 (Terada
et al. 2002).
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AMPK was named here at Dundee
AMPK was named by Prof. Grahame Hardie, who know
works at the Wellcome Trust Biocentre at the
University of Dundee. AMPK regulates the
adaptation to exercise and is a treatment target
for type 2 diabetes mellitus and
obesity. http//www.dundee.ac.uk/biocentre/SLSBDIV
6dgh.htm
AMPK regulation
AMP
AMPK
ATP, PCr
Grahame Hardie
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CaMK IV induces PGC-1 and mitochondrial biogenesis
Wu et al. (2002) generated transgenic mice that
overexpressed calmodulin-dependent kinase IV
(CaMk IV) in their muscles. In the figures, WT
refers to the wildtype, the normal mouse and TG
to the transgenic mouse. The transgenic mice had
more mitochondria in their muscles (round objects
in the section). In addition they measure the
cytochrome B gene DNA which is a marker for the
mitochondrial DNA content.
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Transcription factors acting on mitochondrial DNA
OK, nuclear genes encoding mitochondrial proteins
are regulated by the co-factor PGC-1 and
transcription factors such as NRF-1. However, the
expression of these genes is not enough for
mitochondrial biogenesis. The question is (1)
Which transcription factors stimulate the
transcription of mitochondrial DNA and (2) the
replication of mitochondrial DNA? The latter is
important because the new mitochondria need their
own mitochondrial DNA. The following slide gives
an answer to this question.
?
(1)
mtDNA
(2)
Endurance exercise
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Transcription factors acting on mitochondrial DNA
NRF-1 (together with PGC-1) was also shown to
induce the mitochondrial transcription factor A
(mtTFA also known as Tfam, TCF6). mtTFA is
imported into mitochondria and binds to the
D-loop of the mitochondrial DNA. mtTFA binding to
mitochondrial DNA induces the replication and
transcription of mitochondrial DNA. Knockout mice
that do not synthesize mtTFA die in the uterus
because they do not synthesize mitochondria. The
sequence of events is shown below.
(3) mtTFA translation
mtTFA
mtTFA
(1) Endurance exercise
mtDNA
PGC-1
(2) mtTFA transcription
(4) mtTFA is imported to the mitochondrion and
binds to mitochondrial DNA which is then
replicated and transcribed.
NRF-1
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How mitochondrial biogenesis works

• Putting it all together (see schematical figure
  on the next slide)
• Exercise activates the AMPK, CamK and p38 signal
  transduction pathways, among other.
• A key consequence is the increased expression and
  phosphorylation of the transcriptional co-factor
  PGC-1. The exact mechanisms are largely unknown.
• PGC-1 with transcription factors such as MEF2 and
  PPAR is increasing the expression of
  mitochondrial transcription factors such as
  NRF-1.
• PGC-1 now together with transcription factors
  such as NRF-1 is causing an increased
  transcription of a) mitochondrial genes encoded
  in nuclear DNA and b) the mitochondrial
  transcription factor mtTFA.
• mtTFA is causing the transcription of the genes
  encoded in mitochondrial DNA and replication of
  mitochondrial DNA.
• All proteins encoded in nuclear and mitochondrial
  DNA together with all other parts are assembled
  as a mitochondrion.

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Task
Stop here. Try to draw a diagram from the
information given on the previous slide.
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Exercise-induced mitochondrial biogenesis
Exercise
CaMK
Cai?
?
p38
Mitochondrial biogenesis
Protein import and assembly
PGC-1
Expression of mtTFs and proteins
NRF
Replication and transcription
Nucleus
Mitochondrion
Skeletal muscle fibre
mtTFA and mtTFB
Mitochondrial protein encoded in nuclear
DNA Mitochondrial protein encoded in
mitochondrial DNA
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The End