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The Study of Hornets Has Led to the Development of the Sports Drink, VAAM
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| Discovery of VAAM | |||
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After about four years of research in the US, Dr. Abe launched the study of hornet poison at RIKEN from 1978. "In the US, I conducted research on three different topics of research at three different laboratories, including research into vitamin A and the retina. These laboratories provided us a rich, free, and extremely functional environment. After returning home, I thought that if I chose the same topic of research as was being conducted in the US, I definitely would not be able to compete with them. Thus I have tried to focus my research on hornet poison, which is unique in Japan because hornets (genus Vespa) do not live in the US and Europe." Dr. Abe, however, had to meet extraordinary hardships for the sake of his selection. "I was often stung by hornets because, not being an expert, I began collecting them without adequate preparation. At one time, I was stung on the face by a hornet. In 30 minutes, my blood pressure went down rapidly, and I became paralyzed, although I remained conscious. My pupils dilated. My sight was fogged by halation when I tried to look at bright areas. It was indeed a beautiful world (joke). It was then that I formed a serious desire to conduct research into hornet poison. I learned firsthand that hornet poison is nothing less than astonishing." In the meantime, Dr. Abe succeeded in discovering a hornet neurotoxin, mandaratoxin, in 1981, thus making good progress in his research. However, he needed more hornets for his research activities. "I began to think that I was stung by hornets because I did not know them, and that I should observe the ecology of hornets in order to collect them safely and efficiently." One day, Dr. Abe found that many hornets resting on a sappy sawtooth oak were dead after a few days because they were unable to return to their nest. "There was any number of prey insects around there. I wondered why the strongest hornets had died although they are located at the top of the ecological tree of insects. Adult hornets only feed on liquid or liquid food because they have narrow esophagi located in their pinched-in waists. They hunt their prey not for food, but they bring back their prey in a meatball form to feed their larvae. The larvae, in turn, produce a nutrient liquid to feed the adult hornets, thus exchanging food by means of trophallaxis (upper picture on the cover page: adult giant hornets flocking to tree sap and their larvae that produce a nutrient liquid). Until then, many bionomists had already observed trophallaxis among social insects. However, nothing was clarified about the functions and importance of the nutrient liquid. "The nutrient liquid is essential for the survival of adult hornets. When they lose their hives, they starve to death because they cannot get the nutrient liquid. I thought that it is the exchange between the nutrient liquid and the meatball that forms the bond on which a society consisting of about 1,000 hornets is based. Thus I tried to investigate the components of the nutrient liquid." The analysis of the nutrient liquid produced by hornet larvae revealed that it is rich in proline, glycine, and alanine, and is a mixture of 17 out of the 20 essential amino acids that are the raw materials of proteins. "The liquid is significantly different in amino acid composition from casein (major component of milk) that is considered to be an ideal protein for mammals like us. I tried to further investigate the nutrient liquids produced by four kinds of hornet larvae living in the Kanto area, and found that they are similar in amino acid composition. Consequently, I deduced that this shows the amino acid composition necessary for worker-hornets to have their common capabilities, such as enhanced stamina. Thus I termed this amino acid mixture "VAAM (Vespa Amino Acid Mixture): amino acid mixture produced by hornet larvae" for further investigation." |
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| VAAM causes fat to burn | |||
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A hornet, only 5 cm in length, can fly about 100 km a day. It is capable of expanding and contracting its flight muscle 2,000 times a minute. "The flight muscles of hornets are the best in performance of all living beings on earth. They need to efficiently produce much energy to boost their stamina and support their flight muscles. Accordingly, I thought that the energy production has something to do with VAAM. Moreover, I found the fact that fat burns efficiently when the energy metabolism of amino acids in the flight muscles is activated. To store fat as a life-supporting energy reserve, life has established an energy metabolism system where fat does not easily burn, through a long evolution process, thus enabling living things to make great progress. In order for the fat to change into energy, active acetic acid (acetyl-CoA) decomposed from pyruvic acid needs to be given to the reaction channel called the "citric acid cycle (or TCA cycle)" (Figure 1). However, note that the active acetic acid is not given to the circuit unless it combines with oxaloacetic acid. In other words, the fat cannot change into energy unless the TCA cycle is activated to produce the oxaloacetic acid in advance, because the fat cannot produce the oxaloacetic acid. The TCA cycle generally starts up when both the active acetic acid and the oxaloacetic acid, decomposed from the pyruvic acid, are combined to produce the citric acid. In fact, when a person exercises, sugar is firstly decomposed to initiate the TCA cycle. It takes about 10 to 15 minutes until the fat burns efficiently. "I formed a hypothesis I called the 'amino acid engine hypothesis,' because I thought that the TCA cycle can be initiated only by proline, glycine, alanine, and glutamic acid (proved to be glutamine afterwards) (Figure 1). The hypothesis was based on much evidence in enzyme metabolism including the fact that the energy metabolism of amino acids is active in flight muscles." In other words, the proline, glycine, and alanine make individual circuits which, together with the glutamine, play the role of initiating the TCA cycle. Thus, the fat burns efficiently to produce much energy from the beginning of physical exercise. If the hypothesis is correct, VAAM must have an effect on mice and human beings too because the TCA cycle is commonly observed among not only insects but also vertebrates. Dr. Abe tried to give VAAM to some mice and let them swim in a pool. The analysis showed that the mice were able to continue swimming one or more hours longer than when he gave other nutrient liquids to them. He also investigated their blood after 30 minutes of swimming and found that the mice with VAAM produced lower levels of lactic acid with almost the same level of glucose, and that they showed extremely high levels of fat (free fatty acids)(Figure 2) . These data showed that little glucose was consumed in the TCA cycle whereas fat was efficiently consumed. When glucose burns, lactic acid is produced, which is a substance that creates fatigue in the body and that leads to difficulty in physical movement. In contrast, VAAM has an anti-fatigue effect because it maintains the lactic acid at a low level. Furthermore, the data on other metabolic substances involved in the TCA cycle supported the amino acid engine hypothesis.
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| The key toward practical use of VAAM | |||
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An article titled "Like inexhaustible children" can be found in RIKEN News, No. 110 (May, 1991). This is a small article of only 400 characters, in which the effect of VAAM was introduced by Dr. Abe, Sponsored Senior Scientist. "This article brought many representatives from the food business and amino acid business to our laboratory. However, they initially refused to believe the effect of VAAM. This seems reasonable because I was the first to use VAAM to clarify the concept that amino acid mixtures deliver new functions, although an amino acid had been known to have a specific physiological activity independently. Some businesses asked us to take VAAM to their laboratories to confirm its effect by animal experiments. They conducted animal experiments using different kinds of animals and concluded that VAAM without doubt does work." Thus, an approach toward practical use was launched. At first, a company tried to commercially produce VAAM at a cost of \100 per can, but in vain because of the cost and taste restrictions. Next, Meiji Dairies Corporation implemented new price and marketing strategies to work toward the practical use of VAAM. Researchers at Meiji Dairies Corporation put their efforts into solving the challenge of taste, and finally succeeded in launching VAAM in 1995 as a new type of sports drink, VAAM with a grapefruit taste. Several years later, they improved the taste, which resulted in an explosion of popularity. Today it is estimated that more than one million users drink VAAM on a daily basis. Furthermore, thanks to the success of VAAM, many companies tried to place various amino acid drinks on sale. "Japan accounts for more than 80% of the world total in amino acid production. Japan, based on the traditional fermentation technology typified by fermented soybeans, soybean paste, and soybean sauce, has lead the world in amino acid production. The present amino acid boom has been created by the appearance of a new commercial product named VAAM, which indicated new possibilities for amino acids." VAAM is earning the biggest royalty profits among the patents licensed to RIKEN. Dr. Abe mentions the key toward the practical use of VAAM like this: "Close communication with the company was very important in developing its practical use because I was only the person who knew the details of VAAM. It was the strong feeling of camaraderie among us that lead to the success of VAAM." |
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| Future possibilities of amino acid mixtures | |||
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Dr. Abe found that VAAM, besides activating the TCA cycle, increases the blood concentration of adrenaline (epinephrine), or noradrenaline (norepinephrine), which are hormones for the central nerves and peripheral nerves and have diverse influences on the nervous system, and that it prompts the disintegration of body fat. In other words, VAAM has effects on hormones and the nervous system such as regulation of the autonomic nervous system, a depressing effect on central fatigue, etc. Furthermore, many other functions of VAAM including hepatic function protection against alcohol are being clarified. There are a number of possible amino acid combinations other than the one for VAAM. Thus new types of amino acid mixtures with new functions will be developed in the future. Dr. Abe says, "The internal organs of the body must be mutually controlling each others' functions through the amino acid composition." "It has been known that when cancer cells develop in the body, a special amino acid composition is observed in the blood. I think that in the future, we will try to clarify the fact that amino acids are the medium for communication in the body, and advance the research on life adjustment functions under an amino acid environment." The research on the amino acid mixtures opened by VAAM is surely taking another promising turn. 
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A new type of sports drink, VAAM (Meiji Dairies Corporation) is famous for the fact that many top athletes including the women's marathon winner at the Sydney Olympics, Naoko Takahashi consumed it to boost their stamina. This is an artificially reproduced mixture of 17 amino acids, the mixing ratio of which is equivalent to the nutrient liquid produced by hornet larvae. The liquid was analyzed and clarified by Dr. Takashi Abe, Special Chief Scientist at RIKEN. VAAM is considered to enable body fat to burn efficiently during exercise. "Amino acids have been known to show specific physiological activities. However, I was the first to show the concept that amino acid mixtures deliver new functions," says Dr. Abe. His research is revealing the fact that besides body fat combustion, VAAM has various functions such as protecting liver function from alcohol, and controlling the autonomic nervous system.
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The Origin and Properties of Germ Cells Towards Generating Totipotency
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| The origin of mammalian germ cells | |||
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Individual organisms can survive without germ cells. However, only germ cells are capable of inheriting genetic information from their ancestors and passing it to their offspring. "Why do germ cells have totipotency, the ability to produce offspring? And what is the mechanism of germ cell formation? Germ cells harbor hints to the riddle of bringing out the full potential of the genome. And it is this that I want to reveal," explains Saitou about the theme of his work. Germ cells are produced in different ways between mammals and, for example, frogs and Drosophila. In the ova of frogs and the like, a structure for germ cell production known as 'germ plasm' is already present. Upon fertilization, an ovum begins repeated cell division and differentiates into various kinds of somatic cells, but only those cells that contain germ plasm differentiate into germ cells. Germ plasm suppresses the expression of the genes essential for differentiation into somatic cells, and the cells containing them differentiate into germ cells while retaining the latent totipotency. On the other hand, no germ plasm are present in the ova of mammals such as mice and humans. In mice, an ovum becomes a blastocyst 3.5 days after fertilization (Figure 1). Outside cells (trophectoderm) become the placenta and other tissues that support fetal growth, whereas inside cells (inner cell mass) become the fetal body. Every cell of the inner cell mass is capable of multiple differentiation into all kinds of cells. The cells of the inner cell mass begin differentiating into somatic cells, some of which, however, depart from their course to somatic cells and differentiate into germ cells. Then, how are the different fates of somatic cells and germ cells determined? "When I began studying mammalian germ cells at the end of the 1990s, there was little knowledge about when, where, and by which genes germ cells are produced. I wanted to start my research into mammalian germ cells by understanding their origin." In those days, it was known that when a mouse embryo is stained by a certain method at about seven days after fertilization, the region for the formation of primordial germ cells, from which germ cells are produced, stains characteristically. Saitou and others attempted to excise the region and determine which genes are functioning. Each fragment comprised about 250 cells, including both primordial germ cells and somatic cells, all of which are mutually undistinguishable by appearance, even when examined under a microscope. Saitou and others divided the fragment into discrete cells, and examined the genes expressed in individual cells using an advanced technique known as 'single-cell gene expression analysis.' They succeeded in sorting the primordial germ cells and somatic cells based on the gene expression pattern (i.e., which genes are expressed and which genes are not). Hence, they advocated a molecular mechanism of germ cell formation. The key point resides in the specific suppression of a group of genes expressed in somatic cells, known as 'the Hox gene cluster.' Being determinants of the nature of somatic cells, the Hox genes are completely suppressed in primordial germ cells. Furthermore, Saitou and others discovered Blimp1, a gene expressed specifically in primordial germ cells. "When a fertilized egg with the Blimp1 gene destroyed artificially was allowed to develop, no germ cells were produced. Blimp1 is considered to play a key role in determining the fate of germ cells." Then, when does Blimp1 begin to be expressed? Yasuhide Ohinata, a researcher at Saitou's laboratory, examined embryos for Blimp1 expression by artificial luminescence, and found a particular site to be lightening 6.25 days after fertilization (Figure 1) (lower panel on front cover). In 2005, its image was featured on the front cover of the July 14 issue of the British scientific journal Nature. "Traditionally, it had generally been considered that new-generation germ cells emerge about seven days after fertilization; however, we found for the first time that germ cell formation begins in epiblast cells much earlier than expected on day 6.25." It is postulated that Blimp1 is expressed upon receipt of a signal from adjoining extraembryonic ectoderm (a region becoming tissues that support fetal growth, including placenta). "As such, Blimp1 may suppress the expression of the Hox genes. We also found, however, that other unique genes essential for differentiation into germ cells exist, in addition to Blimp1. The mechanism of mammalian germ cell formation appears to be highly complex." Saitou and others succeeded in developing the 'single-cell microarray,' a technique for comprehensive analysis of gene expression in single cells, and are exploring the mechanism of germ cell formation in more detail using the new tool. 
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| Epigenetic markings controlling gene expression | |||
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Both somatic cells and germ cells have a genome, which carries all genetic information for the organism. Then, why do only germ cells acquire totipotency, by which they are capable of differentiating into all kinds of cells and producing offspring? What are different between somatic cells and germ cells? It is considered that the major difference resides in how the 'marks' for controlling gene expression are attached onto the genome. There are roughly two modes of marking onto the genome: 'DNA methylation,' in which a methyl group attaches to the DNA base cytosine, and 'histone modification,' in which histone, a protein around which DNA winds, is methylated or acetylated. In DNA methylation, regions serving as switches for gene expression (promoters etc.) are methylated so that switching becomes unlikely. In histone modification, the likelihood of switching and the extent of gene expression suppression vary depending on which portion of the histone is methylated or acetylated and other factors. Some marks allow immediate gene expression upon a particular stimulation. For example, there is a phenomenon characteristic of mammals, known as 'genomic imprinting,' in which the expression of particular genes is controlled by the mode of mark attachment. We have two sets of genomes, coming from mother and father, respectively. The two had been considered to have absolutely the same potential. However, if a fertilized egg is artificially produced from a combination of paternal and paternal genomes, the placenta portion becomes gigantic relative to the embryo proper. Conversely, if a fertilized egg is produced from maternal and maternal genomes, almost no placenta is formed. In both cases, embryogenesis ceases prematurely. Hence, the maternal and paternal genomes work differently. This is because genomic imprinting takes place in the maternal and paternal germ cells, in which marks for specific control of gene expression attach to different sites on the genome to produce ova and spermatozoon, respectively. Normally, gene expression occurs with the expression of genes of both maternal and paternal origin. However, genes undergoing genomic imprinting are expressed only from the genome of either maternal or paternal origin. "It is postulated that the paternal genome works to enlarge the placenta so as to maximize nutrition from the mother's body, whereas the maternal genome works to prevent the placenta from becoming too large so as to protect the mother's body. Normal development cannot proceed without a good balance between the two genomes. Some congenital developmental disorders are known to occur due to genome imprinting abnormalities. Hence, genome imprinting is deemed to be a biological system essential for mammals to nurture their fetuses using the placenta in the uterus." |
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| Totipotency acquired by epigenetic reprogramming | |||
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Saitou and others are working to clarify how the marks on the genome change in germ cells (Figure 2). Ova and spermatozoon, which are cells specialized for fertilization, have the respective due marks attached to them. After fertilization, the marks are detached; the relative amount of DNA methylation for the entire genome minimizes at the blastocyst stage on day 3.5. However, the genome imprinting mark alone persists in the blastocyst. Subsequently, the number of marks increases with the progress of differentiation into somatic cells. In the cells that have become somatic cells, the genome imprinting mark persists throughout the lifespan of the organism. "In somatic cells, a large number of marks attach to unwanted genes to strongly suppress their expression during the course of differentiation, so as to, for example, prevent the muscle cell genes from functioning in nerve cells. This is why somatic cells are incapable of differentiating into various kinds of cells." However, Yoshiyuki Seki, a trainee at the Laboratory for Mammalian Germ Cell Biology, discovered that in germ cells, the suppressive marks begin diminishing again on day 8.0, near completion of germ cell formation (Figure 2, H3K9me2/methylated DNA). "Exclusively in germ cells, the suppressive marks on the genome, which are normally stable, are cleared, and the genome is initialized to a 'new' state. Subsequently, a mark characteristic to the initial embryo and inner cell mass (Figure 2, H3K27me3) attaches." Germ cells are considered to acquire totipotency through these processes of initialization and reprogramming of the marks on the genome. Several days later the genome imprinting mark disappears. In recent years, somatic cell cloning technology has been the topic of the day. It comprises incorporating the somatic cell genome into a fertilized egg. However, the success rate for the birth of a cloned animal is as low as 1%, and cloned animals, if born alive, have a variety of abnormalities. This is attributable to the fact that the somatic cell genome has a large number of highly suppressive marks that are unlikely to be detached, and the marks remain attached during the developmental process. "However, mating a male and female of an abnormal cloned animal species will produce normal offspring. This is because the marks are cleared and the genomes of the cloned animal are initialized only in germ cells." If the molecular mechanism behind the reprogramming of marks on the genome in germ cells is elucidated, it may be possible in the future to freely manipulate the once-determined fate of cells. It may also be feasible to intentionally change the fate of somatic cells to find new medical applications. "It should be noted, however, that much remains unknown about the molecular mechanism of stable marking and other aspects. We believe such problems can be solved through further research into the basic mechanisms behind the early development of germ cells. As my ultimate goal, I want to establish how to create an ovum logically from a somatic cell, because the ovum is the only kind of cell that conveys all the mystery of embryogenesis." The ongoing study of germ cells in Saitou's laboratory explores the mystery of embryogenesis, and potentially will have a major impact on medicine in the future.
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Multicellular organisms have two types of cells: germ cells and somatic cells. A germ cell refers to an ovum, a spermatozoon, or a cell that serves as the source of them. All cells that constitute our body, except germ cells, are somatic cells. Somatic cells seldom change their fate of differentiating into a particular kind of cell, performing an intrinsic function, and eventually dying. The only kind of cell that has totipotency, that is the ability to differentiate into any cell and produce offspring, is the fertilized egg which is the fusion of an ovum and a spermatozoon. How do germ cells acquire this totipotency in the course of their development? Mitinori Saitou, Team Leader of the Laboratory for Mammalian Germ Cell Biology at the Center for Developmental Biology, and others are investigating the origin of mammalian germ cells to elucidate the mechanisms behind the ability to acquire totipotency.