Molecular Exercise Physiology
Mitochondrial biogenesis
Presentation 3
Henning Wackerhage
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.
Mitochondrial biogenesis
Part 1
Exercise and mitochondrial biogenesis
Mitochondrial biogenesis
Mitochondria are the power stations of our cells and the sites of
oxidative phosphorylation. The enzymes for fat metabolism (boxidation), 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
Pathways (e.g. b-oxidation)
F0F1 ATP synthase
Transporters (e.g. ATP/ADP, FAs)
Task
How do mitochondria synthesize ATP? Name at least two
researchers that won a Nobel price on ATP synthesis by
mitochondria.
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.
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
Increased mitochondrial density
after chronic electrical stimulation
Salmons, Jarvis, Higginson, Manolopoulos,
Woods, Wackerhage, unpublished data (2001)
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.
Mitochondrial biogenesis
Part 2
Origin of mitochondria
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
Serial endosymbiosis hypothesis
Ancestral
host cell
(anaerobic)
Ancestral prokaryote
(pre-aerobic
metabolism)
Eukaryote with
aerobic metabolism
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.
Mitochondrial biogenesis
Part 4
Mitochondrial genetics
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. 290:457-465, 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.
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)
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.
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
I
II
III
IV
Mitochondrial
encoded proteins
7
0
1
3
F0F1 ATP synthase
2
Nuclear
encoded proteins
>25
4
10
10
11
Poyton and McEwan (1996)
Mitochondrial biogenesis
Part 3
Factors and pathways involved
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?
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
replication (DNA copying) of mitochondrial DNA (mtDNA);
b) Transcription factors that regulate the
mitochondrial genes encoded in the nuclear DNA.
transcription
and
of
Transcription
factor
regulating
mitochondrial genes in nuclear DNA
Nucleus
Mitochondria
Transcription
factor
regulating
mitochondrial genes in nuclear DNA
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
Transcription factors acting on nuclear DNA
The nuclear respiratory factor (NRF-1) was discovered by Evans
and Scarpulla (1989, 1990). Subsequent studies showed that NRF1 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 coactivator 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
NRF
Expression of
mitochondrial genes
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?
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
CamK
Endurance
exercise
p38
PGC-1
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, PGC1 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
CamK
Endurance
exercise
PGC-1
PGC-1
?
p38
NRF
mtTFA
Mitochondrial
biogenesis
PGC-1 is the master regulator of mitochondrial
biogenesis
WT
TG
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.
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).
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).
PGC-1 is also induced by CamK IV (see next slide) and
phosphorylated by p38 MAPK. All three, AMPK, CamK and p38
have been shown to be activated by exercise, induce or activate
PGC-1 and induce mitochondrial biogenesis.
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/SLSBDIV6dgh.htm
AMPK regulation
AMP
AMPK
ATP, PCr
Grahame Hardie
CaMK IV induces PGC-1 and mitochondrial biogenesis
Wu et al. (2002) generated
transgenic
mice
that
overexpressed
calmodulindependent 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.
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)
(2)
Endurance
exercise
mtDNA
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
(1) Endurance exercise
mtTFA
mtDNA
PGC-1
NRF-1
(2) mtTFA
transcription
(4) mtTFA is imported to the
mitochondrion and binds to
mitochondrial DNA which is
then replicated and transcribed.
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.
Task
Stop here. Try to draw a diagram from the information given on
the previous slide.
Exercise-induced mitochondrial biogenesis
AMPK
AMP, PCr
CaMK
PGC-1
expression
?
PGC-1
PPAR MEF2
PGC-1
NRF
NRFexpression
[Ca]i
Exercise
p38
Protein import
and assembly
Mitochondrial
biogenesis
Expression of mtTFs
and proteins
Nucleus
Replication and
transcription
Mitochondrion
Skeletal muscle fibre
Mitochondrial protein encoded in nuclear DNA
mtTFA and mtTFB
Mitochondrial protein encoded in mitochondrial DNA
The End