Doctoral Examination
IH - … Degree’s Examination by Fernando Castro
Chavez, Master in Sciences… Doctor in Sciences’ Title with
the work “Analysis of the Expression of Genes Implied in Lipid Metabolism in Perilipin Knock-Out Mouse Obesity Resistant”. Go
ahead, Fernando.
FDOCC – This work consists in the analysis of
lipid gene expression in the obesity resistant mouse due to the deliberate
inactivation of the perilipin gene. As introduction …
current methods basically consist in exercise and the adequate diet, but what
we pretend with this works is basically to discover what happens with mice
which never gets obese. Here, at the left side we have the obese mouse, which
is also sterile because it has a deletion in the leptin
receptor and, as you can see, don’t even needs to be
subjected by the tail because it can not move. At the right side we have the
non obese mouse which is also fertile, the perilipin
knock-out mouse, the theme of this work and when it is crossed with an obese
female, whose males are sterile, with a perilipin
knock-out male, from this product are fertile and lean males. This is why it is
said that when this knock-out animal interbreeds with genetically obese
animals, their offspring are “healed” from obesity, and are healthy, normal and
meager.
The antecedents are the next ones: We know that
obesity is a cardiovascular risk factor and for diabetes, 34 % of the adult
population, for example in the
Inactivation of this perilipin
gene that forms the protein that produces the storage box that surrounds
lipids, the surface protein of the lipid droplet, produced lean mice obesity
resistant.
Knock-out mice showed a constitutive lipolysis, due to perilipin
constantly phosphorilated, process that inactivates
it. Its metabolic processes were increased due to their oxygen consumption. The
mice were used in this work to clarify the mechanisms through it resists
obesity.
First we are going to see the human perilipin gene, localized in the 15th
chromosome, in this region (15q26.1). This is the size of the complete gene
(14,987 bases), including its introns; it is oriented
in the negative chain, its replication is through the
And to know what is perilipin
activating, recent studies done about the promotor
for this site, a new and original site, which is the site of union of PPAR, in
this case, PPAR gamma. It is a transcriptional factor that when it attaches to
this site, in DNA, perilipin expression is activated,
and now it is thought that when another PPAR, PPAR-delta attaches to this same
location in DNA, what happens is inactivation.
Now we will represent the protein, that being
different to the other members of the adipophilin
family, this is the one with the proper bolt that permits it to be like an
entrance guardian for the lipid degrading enzymes in the lipid droplet, due
that it is the only in this family that contains specific phosphorilation
sites. We know that when phosphorus is over peripilin,
what happens is that this perilipin opens up letting
to enter those lipid degrading proteins, in contrast with the other lacking
those phosphorilation sites. Which means that while
the others could be considered fill-up proteins, the only one that holds the
bolt, the one controlling the door, is perilipin.
Here, we have represented that after catecholamines stimuli, for example nor-epinephrine or
epinephrine, called before adrenaline, which attaches to the receptor and this activates the G protein, and once the G protein is
activated, it activates the cyclic AMP and inserts it in protein cynase A. When this protein cynase
A acquires phosphorus, what it does is to insert this phosphorus both to the
lipid degrading HSL, as well as to the entrance door to reach those lipids,
which is the function of perilipin. Which means that
phosphorus over the degrading enzyme it activates it to enable it to degrade
lipids, and the same phosphorus over the enzyme that protects the lipid droplet
it opens it allowing the entrance of the degrading enzyme.
Here we have the first results with microarrays, in whose obese animals were studied. Microarrays, where thousands of genes are ordered on a
slide, aligned. The double ob mouse the ob ob which
have the leptin gene deleted. What the researchers
were expecting was that the fatty acids synthesis genes to be increased in
these obese animals. But they were surprised when they studied the microarrays, then they discovered that precisely all those
genes that are increased in adipogenesis, the
beginning of the adipose tissue, instead of increasing, which is what happens
with normal adipogenesis. But in these obese animals,
all these genes for the fatty acid synthesis are reduced in its expression.
This was impressive for them and is impressive for us because the opposite
extreme, which is the perilipin knock-out mouse, has
the same characteristics, the fatty acids synthesis genes are downregulated, which means that the obese mouse is obese
even if eats as the normal one, everything gets stored and does not need to
produce its own fatty acids. Another characteristic of obese animals, that the plasminogen activator inhibitor 1 was highly expressed both
in obese mice and in diabetic mice.
How it was obtained this perilipin
knock-out mouse in
This is the figure that clarifies how heterocygotes are presenting both bands, while those that
we were studying were the homocygotes producing the
lighter band, the smaller.
Here we have the mice again, this is the one
under study, the one that never gets obese, no matter how many fat or how many
lipids it consumes. Here, at its side, is the normal mouse, then, the sterile
obese mouse, and at the end, the lean result of an obese female with a perilipin knock-out.
Another work group obtained a second perilipin knock-out, but this time they inserted the
cassette in a different location than the previous one. This new animal has a
deletion that takes three complete exons, two
complete introns, and almost all the introns that are further out.
We are going to see the similar characteristics
of these two mice and where they differ. The reasons why these are different, I
think is due to the different place in which the cassette was inserted. The
authors declared that it was due to a different strain of mice the one that
they used, but it may make more sense to think there being located some
regulators, or enhancers or delayers on the different deleted locations.
Basically the difference is the mouse that was obtained in
Here, one of the obesity resistant mouse
characteristics is that if its located at four degrees (Celsius), without food,
its temperature starts going down faster than the normal animal under the same
conditions, without food …Here the knock-out has more possibilities to die. But
if food is added at the same temperature, the knock-out keeps its internal
temperature.
In the last related work that I saw from Doctor
Chan’s Lab. … it is notable that if you add food, the perilipin
knock-out is able even to keep its body temperature more than the normal
animal, and it was significant. Which means that if it eats and eats there’s no
problem with cold or to die due to freezing at low temperatures…
What justifies this study is that this mouse
obesity resistant is like a model to learn metabolism, and when we learn its
metabolism, we can obtain possible treatments against obesity … if obese
persons have problems in different tissues of their organisms, we suppose that
also this antiobesity model is going to reflect
variations in its different tissues … we can explore treatments of the
metabolic pathways that are altered in this animal, specially those related to perilipin. Those altered genes at the beginning of the
metabolic pathways could be used as key sites to develop treatments against
obesity and also to counter collateral diseases like diabetes.
The hypothesis is that “the beta oxidation
genes for the metabolism of lipids are over expressed in this animal”. For that
reason, the general objective, in coordinated relation with the hypothesis is
“to determine the expression of genes involved in lipid metabolism in the perilipin knock-out
mice obesity resistant in the next tissues: white adipose, muscle, heart, liver
and kidney.”
The
particular objectives, derived from the general objective are: “how much it
increases the beta-oxidation gene expression?” which coincides with the
hypothesis, and to “determine the level of change of other genes that could be
related”, and to validate these results using a different method than microarrays, as will be seen in methodology.
Here, in
methodology, a normal animal is taken and the animal that never gets obese and
their tissues are extracted, and from each one of those tissues total RNA is
extracted, and from it the messenger RNA is taken, which is converted into
proteins. And from this messenger RNA we obtain the first complementary DNA
strand, from which the second is obtained, to do an in vitro transcription in which the biotin markers will be a inserted, that are going to be the colors that will be
evaluated at the end in the microarray. Then, these
complementary RNA’s are fragmented, and once these are fragmented they are
applied to the microarray, that big amount of aligned
genes on the slide. And from here, what is left is the analysis, the reading
and the results’ validation.
The
analysis in itself, as it can be seen in the computer is in this form, and
there each gene has twenty different probes, and apart of those twenty, another
twenty having their middle nucleotide modified, which means that each gene has
forty probes, twenty which are the perfect alignments and twenty that are the
imperfect alignments. And they are uniformly distributed, and the proper
software can recognize each of them and it puts them together to see where it
hybridized. And this is what can be seen, this is the curve of expression for
the twenty fragments from a particular gene with the perfect alignment and the
curve with the imperfect alignment is lower than the other. This, for example
was the knock-out animal and this is the normal animal. What the program does
is to calculate the area under the curve between the two alignments of the
normal animal and to divide it to the area under the curve from the knock-out
animal. Finally, done that tabulation, we see that the difference in the area
under the curve was increased, like in this case, and that tells us if it was
twice, or if it was 50 % and the program will put it like a 1.5 fold increase.
In the other side what we are looking here is reduction, and as some genes
could have some imperfect assemble, that imperfect curve could be eliminated,
and the program does it, and it is calculated only the difference of the bigger
curve, the perfect assemble of the knock-out animal compared to the wild type.
Then,
with that, what was obtained is, first, that white adipose tissue had more
reduction in the gene expression than increase. The rest of tissues had a
balanced increase and reduction. But in which we saw a big unbalance was in
adipose tissue, in which is the fats of this animal obesity resistant and that
is more the downregulation than the upregulation of its gene expression.
33 % of
those genes that were reduced in adipose tissue is
related to transcription and translation genes, here, there is a big amount of
genes. And the CEBP-alpha gene, that we studied by de la Dr. Christine
recommendation, appears within these genes that were reduced. So, we can state
now that the negative regulation of those genes for lipid metabolism are not
regulated by CEBP but rather by PPAR, derivates of PPAR-alpha repress the rest
of the lipid metabolic synthesis system. We saw that perilipin
is repressed by PPAR-delta and activated by PPAR-gama.
… While
more blue is the expression, that means the expression is lower and if more red
the expression, that means that the gene is higher in its expression. The
knock-out animal increased greatly the expression of its metabolic genes when
compared to the wild type, by the other side the knock-out animal dramatically
reduced its expression of fatty acids synthesis genes …the most impacting
result from this work is that the difference between the wild type and the antiobesity type, is very clear, and that its lipid
metabolism degrading was activated, and at the same time it reduced the
expression of those transcription and translation genes.
The
notable genes in the category of reduced expression are SCD1 and SCD2 which
fell within transcription genes. SCD2 in the wild type animal is expressed the
most in the white adipose tissue when compared with any other tissue which may
indicate the importance of this gene.
The
expression of these genes was evaluated using another methodology apart of microarrays, RT-PCR which discovered the same differential
relation. Seeing that those genes that were downregulated in microarrays were
also reduced here, and vice versa.
Then,
discussion is to put together first all those blue colors and the red ones to
make some sense of them. For them to make sense in metabolism, and when put in
order …it can be seen clearly that the gatekeepers for the entrance of acyl coenzyme A, which is a product of lipid degradation,
are increased in their expression, carnitine acylcarnitine translocase and carnitine O-palmitoyl transferase 2, these two were increased, which means that
the white adipose tissue organelles, that we know that are mainly mitochondrias, could be also peroxisomes
participating, but that study is left pending, it was not possible to see in
detail which percent those could be participating.
The acyl coenzyme A enters, and experiences beta - oxidation
with those enzymes that are increased, to obtain acetyl coenzyme A, that at its
own time enters the process in the Krebs cycle to end in the electronic
transport chin.
It was
contemplated the hypothesis that only beta oxidation genes were increased, but
it was seen that also the doorkeepers for acyl coenzyme
A, and for the Krebs cycle and the respiratory chain were also increased,
coordinately working from the start, while every internalizing of acetyl
coenzyme A that may be originated from carbohydrates, at least in white adipose
tissue, are jointly reduced, which means that, in adipose tissue, mitochondria
and some other organelle such as peroxisomes are
virtually concentrated in fat burning.
While as
in the case of the antecedents that we saw that obese mice had low expression
of fatty acid synthesis genes, curiously we saw something similar in the
obesity resistant animal. The diminishing on the gene expression involved in
the unsaturated fatty acid synthesis, such as the SCDs
that we saw in the graphics, having the blue colors, and also cholesterol
synthesis genes, are low in its expression in this animal.
Two of the genes that we present here have been used recently as target
sites to counter cholesterol. Then we had supposed that some of these downregulated routes could be studied to control obesity or
to reduce cholesterol, that, some researchers are already starting to use genes
within those pathways to reduce cholesterol.
… The enzymes related to desaturation, the
ones in-saturating fatty acids within our organism, reduced its expression, and
again they appear related to transcriptional and translational factors. SCD1
appears together with the translation initiation factor in all tissues, which
means that SCD1 is related somehow with translational processes.
When both desaturation enzymes are only
aligned in white adipose tissue, we can see that both align together (SCD1 and
SCD2) and are grouped with ATP citrate lyase, which
is the one that performs the synthesis of fatty acids in the cytoplasm, and is
as well downregulated at the same level than the
previous ones.
When we see the tissue comparison, it can be seen that heart and kidney
tend to follow a similar increase pathway, both in the Krebs cycle and in the
fatty acid degradation through beta oxidation similar to white adipose tissue.
It needs to be noticed that the chosen animals for this work were
animals between six and ten weeks of age, that have not been subjected to any
dietetic load no high fat diet or high in carbohydrates, thus, on a basal
level, these tissues, heart and kidney are collaborating in fat degrading. In
adult animals, in other experiments, it can be seen that muscle incorporates to
do the same… Which means that once the foods are digested, the stomach provides
a signal …it communicates with the brain, and for the short term energy is
stored in the liver and for the long term, storage is in the form of fats, its
energy is stored in adipose tissue. And when it is necessary, to do exercise
and activity, all tissues communicate, brain, liver and adipose tissue, to send
to the muscle the kind of energy, either at the short or at the long term that
be necessary. Then, this work that deals with an intense communication among
tissues, and all the changes that we saw … if one tissue got altered, also another…
Then this is something extra, it was not part of the hypothesis and
objectives, but is an important point that in the same adipose tissue it
appeared genes with genes from macrophages, from leucocytes, and also related
with globins… Talking to the Jury’s President, Doctor Ivan, he told me that
this first two (Cyba, Fmo5) could be involved in the cytochrome’s increase for the respiratory chain. Then we
can see here that there is some coordination in the expression of these genes.
And the last aspect of this job that it was not planned but that
appeared was the palindromic linker inside thousands
of transcripts present in genbank and in the Affymetrix kits. There is one palindromic
sequence that can bind gene fragments present in two different chromosomes, but
on studying with more detail it can be seen that the palindrome self-hibridizes and on doing this auto-hibridizing,
evades the attack of the restriction enzyme ready to digest it, then it stays
indigested and the enzyme is unable to capture it, and there are thousands, and
until now, in the last study I found more than ten thousands of this kind of
transcripts in el genbank, amd
from these, at least 10 % are inside Affymetrix.
Which means that if persons are not conscious that there is a false transcription
“positive”, which is an artifact, and is taken as real and as part of something
that happens in nature, and use it as targets for antibiotics or pharmaceutical
treatments, obviously this is going to provide a false result…
So, with
all that has been said, conclusions are that in this work we saw that the
metabolic pathways increased are the catabolic oxidative for fatty acids:
beta-oxidation, Krebs cycle and the electronic transport chain, and that it
were reduced the routs for the expression of genes for the lipids synthesis and
cholesterol. We saw that the transcripts for the proteins involved in
transcription processes and translation formed the 33% of the reduced genes in
white adipose tissue and we saw that it can also be due to transcriptional factors
or co-activators which are acting as mediators causing all these changes in the
expression …because those are freely swimming in cells of adipose tissue and
some others that could be excreted for the close communication between tissues
to explain those changes present in other tissues. The results can help us to
discover a treatment for the control of obesity, following the pathways of the
genes reduced in its expression and trying to study in more detail those genes,
if they are reduced, or even perilipin, in a
reversible way, it can be possible to control obesity in a temporary,
pharmacological way. And the discovery of that palindromic
linker that was not planned, however, it was found, and also mRNA for globins
and transcripts related to macrophages were found in the adipose tissue of
these altered animals.
The
limitations of this study are that only were used males and it was not analyzed
the mice brown adipose tissue.
Perspectives
as well are that anti-atherosclerotic studies could be done with these mice and
to study as well the gene expression of animals that recovered their fertility
because it will be very interesting, to learn which changes occurred in these
tissues. In the same time, to study genes which are specific of organisms like
a gene that is the only one, when it was done a comparison in the genbank, only matched in its expression the mouse, it lacks
of homologous in another organism (AA185432), this will be very interesting and
as well as the possible existence of genes, which are specific of species,
because it is already known that there are tissue specific genes, if a
treatment is followed for those genes tissue specific genes, in the Thesis ten
examples are included for white adipose tissue, and this could control
specifically one tissue…
I thank
my family, specially my wife, for her efforts and constant love and support.
Here the names of the collaborators
in this work, both from
The products of this work are attached
at the end of the Thesis, the article in Diabetes,
and the review article in Annals of Hepatology, the chapter in a specialized book on
obesity, related to the genes implied in obesity susceptibility and antiobesity genes.
Here are references of some authors
that wrote me, this is interesting because that work quotes a study done with
human beings and they try to apply the results of what we presented here as
something already happening in humans as well. This is another work at Baylor
were they used similar technique of validation, specifically RT-PCR. This one
quotes our article in the possible surge of new drugs …and that researcher put
our reference in his Ph. D. Thesis. He is studying changes in isolated cells of
adipocites. This is another work from
(The jury laughs)
These are the electronic emails that
I have received, I have reduced the quantity. The first one is specially
interesting, 20 % of the articles that I quote in the article of Diabetes including the one that writes
is, to be as its coauthor and it says that he thinks “interesting and
provocative the fact of having found these genes related to macrophages in
white adipose tissue”. This is also very important because is the one that
obtained the knock-out mouse for SCD1, the desaturase
of fatty acids, which is very similar to the mouse that we studied, except that
his mouse produces necrosis in the liver and is it not yet known why. This
other is interesting because they are studying the relation between adipose
tissue and macrophages. And this one discovered the protein that is stimulated
in fasting that in our case was increased in its expression. Here, this person
of the Pasteur Institute is also studying the comparison between macrophages
and adipocytes. As you see, this is very popular;
when a macrophage gets trapped in the arteries it starts to express some adipocyte characteristics and when adipocytes
are altered in mice that can not store fats, they start expressing genes that
are macrophage like. This one was interested, even told that he wanted to
collaborate, and even someone wrote me from a journal asking me to be the
referee for an article.
And, this is the last slide, this is
an acquaintance of Houston I tried to explain this and he told me: “hey, this
is very difficult to understand, very strange”, better if we fight obesity on
the streets, and his strategy has been basically, while the discovery of this
pharmaceutical reversible product to inhibit perilipin,
has been to do exercise and nutrition, that’s all.
(Audience laughs)
IH – I think that now we leave this
work to the consideration of the referees, questions?
CI – Congratulations, Fernando … a
work that also has a lot of future possibilities … I have a doubt related to
the sterile obese interbreeding with the perilipin
knock-out…
FDOCC – Both homocygotes
lacking of leptin (ob ob)
and the ones lacking leptin receptor (db db), when interbreed with the perilipin
knock-out their offspring are fertile. Evidently those parents need to be
female from those lines interbreeding with perilipin
knock-out males as males of these lines are regularly sterile… It is known that
perilipin is expressed in esteroidogenic
cells and it may be that this alteration in the case of perilipin
that is not expressed, it could compensate the other inactivation but this is
under study and is one of the most interesting perspectives, to see what is changing
in these animals… By the other side, the genes reduced in their expression,
related to transcription and translation were some 130 genes that still remain
open to research, as important prospects for the future.
SU – Congratulations …related to the
difference between perilipin knock-out models…
FDOCC - … they eat at normal
temperature, 30 % more than the normal animal to sep
their body temperature stable, by the other side, the animal of the other work
group eats the same as a wild type. This for me is one of the most dramatic
differences. Are both animals obesity resistant?, yes.
Are both animals fertile?, yes. Is their size normal?
Indeed. The oxygen consumption in both animal models is increased. The muscles
in both animals are increased they are more muscular without the need to do
exercise. Why? Because they need to burn energy… specially
the adult. The white adipose is more meager, with a more uniform
reduction in Tansey’s model but at the end, the white
adipose tissue has the same number of adipocytes than
the normal animal, only smaller in size because they don’t store the lipid
droplet. Plasmatic leptin presented differences, in
the animal of Javier is reduced, and what does leptin?
Is the one that gives signal to the brain that it is already full, that it is
not necessary to keep eating more. Then, if it is
reduced in Javier’s animal, that never gets obese, that jeans that it never
will receive the satiety signal, to stop eating … and here is where the main
differences start, where leptin is increased. Here leptin constantly is living signals to this animal telling
him “you are already full”, “you are filled”, you don’t need to sep eating,
which means that the animal that we are investigating lacks the limit and can
continue eating. Then, related to food consumption, in response to your
question that leptin is increased in the other model,
there the food consumption is normal. Which means that each time the signal is
increased the animal stops eating. In the animal that we studied, leptin is reduced, the animal lacks of a stopping point and
continues eating, having a food consumption a 30 % more than the normal animal.
In the case of glucose metabolism, in our model is normal, but in the other
model is damaged, maybe the constant leak of leptin,
that is also secreted by adipose tissue, had something to do, that had not yet
been studied, in order for this to happen, in the case of the glucose metabolic
damage in the other model. The animals of our model, in a recent article it was
reported that when having one year old they develop a light insulin resistance,
but they keep being normal because they have a normal amount of blood glucose.
Then, it can be said that the model that we used lacks of dangers of developing
pathological diabetes like in the other, the one since young already has the
glucose metabolism damaged. The other thing that is different is the plasma
glucose and insulin which is elevated in the other model, so in this, both
blood insulin and leptin are elevated, while in the
case that we worked on, glucose and insulin are normal both in the small animal
as in the one year old animal … (here the video was stopped).
(CG reads the Act of the Degree)
That act read by CG and concluded by
IH, declares in its last paragraphs:
“…the student presented his work for
his degree, on concluding this, the student was interrogated and his work
questioned, for him showing dominion on the topic and for him to defend his
position related to the different points of his work.
Once concluded, the jury members
proceeded, privately, to deliberate about the examination result and they
determined that the student was APPROVED.
Then, the Jury President proceeded
to take the corresponding protest to the student in the next terms:
Do you protest to practice the
profession with honesty, consecrate your exercise to the well being of the
collectivity, always keeping the good name of the
To this the student responded:
“YES I PROTEST”
The president of the Jury concluded:
“If you do so, your conscience and
the collectivity reward you and if not, they demand it to you”.
This session was concluded, being
Links to my work:
Diabetes: http://diabetes.diabetesjournals.org/cgi/content/abstract/52/11/2666
Annals of Hepatology: http://www.medigraphic.com/pdfs/hepato/ah-2004/ah044c.pdf
Obesity chapter in Spanish: http://www.oocities.org/plin9k/fdocc-genes-obesidad.pdf
Thesis in Spanish: http://www.oocities.org/plin9k/tesis-fdocc.zip
ISCID three articles:
http://www.iscid.org/papers/Chavez_Palindromati_101505.pdf
http://www.iscid.org/papers/Chavez_MolecularAnalysis_030804.pdf
http://www.iscid.org/boards/ubb-get_topic-f-6-t-000553.html
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