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 United States, they are overweight.


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 Okazaki method, and it has four possible alternative splicing products, which means that is the same gene that produces different protein variants in humans... The homologies are only in their exons, in these percents in other animals. It is interesting that the human perilipin homology with mice (84.24 %) is even higher than with dog (81.84 %) or rat (83.01 %), this talks about non lineal homologies … and of course, if we include introns in such homologies, this difference will increase…


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 Baylor College, in Houston? It was through cassette insertion that interrupted part of the exon and part of the next intron. Exons are the sites that are translated into proteins, while introns, which alternate them, are not translated. In this case, when the cassette was introduced, the gene was completely altered, and as Javier was able to detect it, through Southern Blot when he used the restriction enzyme, XbaI, he realized that in normal mice there was a specific pattern with this length: 8.5 kb, while in the animals with the insertion, to be certain that they were altered, they had a smaller pattern (7 kb).


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 Baylor College does not develop extreme diabetes, but a light insulin resistance but it balances in such a way that it has normal blood glucose levels. I think that it is because the exon deletion is smaller and of an intron part. And the other however, develops diabetes, I think that it is due to a wider destruction, three exons and introns


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 Texas University and Baylor College.


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 Spain in which it is reviewed the de microarrays for the study of nutrigenomics and nutrigenetics. This, I don’t know what it says, it is in Norway


(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 Guadalajara University?


To this the student responded:




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 13:00 thirteen hours, signing for evidence those that participated on it.” (Appear 9 signatures, myself, my six juries, from the outstanding coordinator of the program and the academic secretary).


Links to my work:




Annals of Hepatology:


Obesity chapter in Spanish:


Thesis in Spanish:


ISCID three articles:

Tasters of the Word (YouTube), videos recientes: "Astronomía y Nacimiento de Jesucristo: Once de Septiembre Año Tres A.C.", "Estudio sobre Sanidades" (en 20 episodios), "Jesus Christ, Son or God?" and "We've the Power to Heal":

Tasters of the Word (the blog, with: "Astronomy and the Birth of Jesus Christ"):


And a commercial before we go:

Window Cleaning of Ronnie Petree, where my wife works (smile): Good Looking Glass of Houston (serving also at: Katy, Surgarland, Conroe, Kingwood, Woodlands, Galveston).