The title of this lecture is on a blue background in white text: "Nutrition in Metabolic Diseases: too-much or too-little?". To the right of this is an inset webcam video showing Professor Gyan Tripathi, Head of Human Sciences Research Centre, University of Derby. He is wearing a suit and tie and headphones.+
I will be discussing my current research as well as my past experiences and research because of which I am at the moment at my current position. So today's research topic which I have chosen to discuss is 'Nutrition in Metabolic Diseases' when is it too much and or too little?
A white slide entitled "Current Research" appears with a list of items in blue. It reads: "Translational Medicine: Obesity & Type 2 Diabetes. Nutrient-gene interaction: Role of micronutrients in metabolic health. Maternal nutrition and foetal programming." Below that reads "Disease Mechanisms: Mechanisms leading to insulin resistance. Mechanisms of cell organelle dysfunction: Endoplasmic reticulum and Mitochondria".
So my current research is mainly in the area of translational medicine, with a key focus on obesity and obesity-mediated metabolic disorders. I am particularly interested in understanding the causes and novel mechanisms of metabolic diseases so that we can find new drug targets against these disorders. In that respect, I am studying mechanisms which lead to insulin resistance in adipose tissue and also the cell organelle dysfunctions which are one of the primary causes for inducing these insulin resistance in adipocytes.
A new slide shows a cartoon figure of an overweight man. Next to him are an illustrated list of "Causes" labelled: Eating too much, Genetics, Certain medications, Stress, Poor sleep, and Hormonal imbalance. To the right is another illustrated list, this time of "Consequences" which includes Heart disease, Diabetes, Kidney disease, Fatty liver, Cancer and High blood pressure.
When we talk of obesity and metabolism many of you might have heard about Metabolic Syndrome. Metabolic Syndrome is the name basically given to a group of health problems which lead to higher risk of diseases of the heart and blood vessels, such as heart attacks, strokes and any other cardiovascular disease. It's a very common disorder and one in four adults in the UK are thought to have some kind of Metabolic Syndrome. The risk factors which cause Metabolic Syndrome primarily, the biggest one is obesity or being overweight. Another one is insulin resistance or high blood pressure. And one of the main causes are having poor sleep, stress, among many factors.
A slide comes up with the title in red, "Where did it all go wrong?" A diagram showing six figures, each depicting the stages of development from ape on the left marked "5 million years ago" to "250,000 years ago" is illustrated by a picture of a healthy male Homo sapiens. The final picture in the set shows a bulging overweight man wearing only shorts and carrying a super-size soft-drink cup with a straw. He is labelled "Today".
So I would like to discuss what happened to us and where did we go wrong? It has taken five million years for us to become homo sapiens, and around 200,000 years to become homo sapiens from neanderthals, but if you look back our diet has been consistent for mostly even before 100 years ago, but in the past 100 years, all of us can visualize the changes our nutrition, our diet and eating habits have taken. Not only this diet, it's also our lifestyle. If you think, if I remember in my childhood my dad used to cycle to work, a few, around five, six miles every day. And five-six kilometres going to school was common by walk. Now I have my kids: it's two kilometres away, the school, and they will wait at the door to be dropped off. So there are various changes which we have taken over but we haven't adapted the diet accordingly or the nutrition which we get.
Under the images of the evolution of Homo Sapiens, a table fades in showing that in 1960 1% of men were obese and 2% of women were. In 2010 25% of men were obese compared with 27% women. Finally, it predicts that by 2050 60% of men and 50% of women could be obese.
If you think in 1960 in the western world only one percent of males were obese and two percent female. Then it increased to 25 percent men and 27 percent females, in just a few decades. And by 2050 we are predicted that more than half our population will be obese. So you can understand the amount of a challenge we are facing and the amount of diseases, there is increase in cancer, there is increase in metabolic diseases, cardiovascular diseases, you can name it. Since we are talking about evolution, my research also has evolved with time.
The next slide is entitled "MSc Biotechnology: Indian Institute of Technology Bombay, Mumbai India". There is an old photograph showing some University Students in India, some are holding red books. Further photographs appear alongside it. One is titled "Prof N. S. Punekar, IIT Bombay" and shows a man with a moustache. Another is taken through a microscope and shows mould growing. Next to it, more mould is growing in a petri-dish. Finally, there is a chemical diagram labelled "AMP deaminase in Aspergillus niger".
I started as a microbiologist, using molecular biology techniques in fungi. So my first project was under Professor Punekar at IIT Bombay where we were studying Aspergillus Niger. Aspergillus Niger is the common fungus which you will see first thing when you notice a stale bread as a black spot or a dark green growth. You can also find them on onions, the black shapes which you see when it sporulates, so it does look very pretty under an electron microscope, but it smells disgusting. And one of the reasons we were studying actually is to look at its industrial potential.
It is used for the production of citric acid as gluconic acid industrially. And the pathway which I was investigating was the role of AMP deaminase in that respect. But to take you back, AMP deaminase is one of the targets for metformin. Metformin is the first-line drug which is given to type 2 diabetics. Obviously, I didn't know it at that time, but it is given so that it can stop this reaction and so that there is a huge accumulation of adenosine monophosphate which can then be converted into ATP.
From there I received a fellowship from the Council of Scientific and Industrial Research after an entrance exam which funded me for the next five years. And then it gave me the opportunity to choose the institute and supervisor wherever I wanted to work.
Another slide shows some more photographs, this time of a large white building and separately some bamboo growing and flowering. The title at the top in red reads: "PhD: National Chemical Laboratory Pune, India (1993-1999)". Another photo joins these, showing a group of Indian students, some are in western clothes and some are dressed more traditionally or in a mixture of the two. The final photo shows Gyan working in a lab with some petri-dishes and bottles. At the bottom, it reads: "Genetically Engineer bacteria for overproduction of biodegradable polymer".
So at that time, I was for some reason interested in bamboo and bamboo plants. Why? Because I saw a paper that they have a team in The National Chemical Laboratory Pune have managed to flower them in a petri dish or test tube. Why it is interesting is bamboo actually flowers only once in a lifetime and that is between 60 to 120 years, so it looked like a massive achievement and that's why the specific varieties of bamboo propagation is a challenge and is still to date.
So armed with my fellowship I visited The National Chemical Laboratory in Pune. Unfortunately, I could not meet that Dr. Muskenhas who led the project, but then I started looking around working with other teams, and one of the teams which were again in plant tissue culture, the rest of the teams were working on plants, Dr S K Rowell said: "oh I have got an interesting project and I have got some money to investigate biodegradable biopolymers." I said, "[W]hat's so interesting about that?" He said "This polymer actually has very similar functions to polyethylene, and it can replace polyethylene, and we can overproduce it and then the pathway matches with the fatty acid or lipid pathway within plants. So think if we can start producing this polymer in plants." So I went back looked at a few publications, and I thought actually it does make sense and it looks like a very interesting topic and it may change the world, the way we look at it. But anyway, 20 years on we are still at the same stage. So what happens?
This is all the research fellows at that time when I was doing PhD. So I said which organism to look at so he said "l-collision centrifuge that accumulates around 50% of the dry cell weight." I said, "[B]ut that's patented so we can't really work on it." So there is a National Collection of Industrial Micro-organisms within NCL, so I went and visited the microbiology department and came back with six or seven varieties. Here I am culturing those cells on a petri dish and looking at which ones to select. After southern blotting and lot of numerous trials I found one organism, Streptomyces aureofaciens, which is an actinomycete.
The next slide is labelled "Creating Recombinant E. coli for PHA Production (1993-1999)". There is a small picture of S. aureofaciens being cultured in a petri-dish. This is followed by some blue arrows indicating that it will be one of a series of pictures, which appear over the next few seconds. A complicated pathway diagram appears on the screen with many arrows and chemical names. This is the "TCA Cycle". A series of black dots in a grid pattern within a circle labelled "Genomic Library" appears. A fuzzy indistinct grey image appears, showing two dark spots on a hazy background. This is labelled "pGTSa067 and pGTSa240". Lastly an image showing E. coli cells glowing orange on a dark background comes to join the rest. This is labelled "Recombinant E. coli producing PHA".
And my first challenge was isolating DNA from it because it forms a very weird layer around it, similar to plants, and we did not have any protocols available. So I merged the bacterial isolation protocol with the plant isolation protocol and published it. Actually it is still one of very highly-cited papers which was just submitted as a small paper. So I isolated DNA from that and then started investigating the pathway for the polyhydroxyalkonades. And there were three genes which were involved in the synthesis of it. One is 3-ketothiolase, Acetoacetyl CoA reductase and PHA synthase. So the first two are usually found in most of the organisms, including us, because they are part of the TCA and Krebs cycle is the PHA synthase which is the novel enzyme here.
So after I created a genomic library from its DNA into e-coli and then initial screening gave me more than, I think, 300 clones, or something like that. And then step by step I had to isolate the clones for each gene. At the end, I ended up with just two. And I named it after myself pGTSa067 and pGTSa240 because they contained all the three genes, and to my surprise when I cultured them and looked under fluorescence microscope after staining they just glowed beautifully. These recombinant e coli was accumulating so much polyhydroxy alkylates I could not believe it at the first instance.
And I got lucky there. Lucky because these three genes were located in tandem one after another in a 4.5 kilobase pair of DNA and that was the biggest fluke of my life, actually.
A new slide titled: "Effect of Nitrogen Source in PHB production in Recombinant E. coli" features two graphs in black and white showing the "Effect of nutrients on PHB production" data being discussed.
So another thing was if you changed the carbon source or the nitrogen source within this polymer, it tends to not only change its growth rate, it also changes the property of the polymer which is produced. So you can produce polyhydroxy butyrate, polyhydroxybutyrate validate, polyhydroxyalkanoate, and each polymer gives its own property. Some are brittle, some are elastic, some have more piezoelectricity, so they can be used as bone implants. So its use is enormous if exploited. This is how it looks under electron microscope.
A black and white image showing lots of different sized bobbles appears next to the graphs. It is labelled: "Scanning electron micrograph of PHB obtained from recombinant Escherichia coli harbouring pGTSa067".
The next challenge was to sequence the 4.5 kilobase pair, and believe it or not this is the old-fashioned sequencing.
This slide is called "Old Fashioned Slab Based DNA Sequencer". There is a photograph of that machine in a lab surrounded by bottles and tubes and cables and droppers.
It was almost a one-metre long acrylamide gel electrophoresis, and every time you do it even a single bubble can destroy your whole thing, and so for everything you have to sequence it using S35 which is radioactive. So you have to pour the gel, do the radioactive reaction, remove the top plate and then dry it and then carry it around after drying and put extra film in a darkroom.
Next to the photograph of the Sequencer a strip of clear film is held in somebody's hand. It is marked with distinctive black bars of varying thicknesses which show a DNA sequence. On the same slide, a third image shows a closeup section of the DNA diagram.
So if you get it, so maximum one read will give you around 200 bases. And everything, with a ruler, you have to sit and you have to really feed somebody, treat them well so that they sit next to you to write all the sequences. So that took me like three, four months of my nights and days. So if you think in today's time it will probably take a couple of days by a commercial company. If you compared say with the Human Genome Project. With the Human Genome Project, total expenditure worldwide was 2.7 billion dollars and same level of sequencing is, suppose I want to get it done now, a draft sequence will cost me just under 1000 dollars, and I'll get it done in a few days. So that's the challenge. So technology does improve quite a lot.
A new slide is titled "Research Fellow: University of Aberdeen". It shows a small and very colourful photograph of about 20 students smiling all gathered around Professor Alistair Brown at the University of Aberdeen. The photo is titled "The Lads and Lasses of 'The Broon's' Lab".
And from there then I started looking for a post-doctoral fellowship, and one of the advantages of working in a good institute was that we always have a stream of good speakers. And professor Al Brown had visited the National Chemical Laboratory once and presented a seminar. After his seminar, I had a chat and he said "Okay". So I thought that he would have forgotten, but when the time came I sent an email and said "I am just starting thinking to start writing my thesis, would you have any positions available?" He said "Oh it's good timing, I have just acquired a BBSRC grant and in a couple of weeks I've been interviewing people, and so would you be ready to give an interview in a couple of weeks? What's your phone number?" So I gave him my phone number and I was interviewed on the phone in my PhD supervisor's house, so he was next door listening to me.
At the end of the interview, Al said: "Look, so far whoever I have offered the position they have never refused." I said, "That's great but what does that mean?" He said, "If I offer you, can you, would you accept it?" I said "Great! I haven't applied anywhere else, so that's fine!" I knew about his work as well, so I was quite pleased. And he said, "The latest you can delay is till March '99." And that was November 1998. I said, "God, I have to write my thesis, defence and everything has to be done in that time!"
So anyway it all worked out fine and by 23rd March '99, I was in his lab. And this is his lab, he had like eight, nine postdocs, and each of the postdocs were given a few PhD students to supervise, to not only work on their project but work on other projects as well. He used to work on pathogen candida albicans and the project we were investigating was looking at GCN4, how it coordinates morphogenesis transcription as in candida albicans.
Next to the colourful photograph, a series of images and diagrams of cells appear, entitled "GCN4 coordinates morphogenetic and metabolic responses in Candida Albicans".
Morphogenesis is very important because for candida albicans to become pathogenic it has to move from yeast form to hyphal form. That's why this was being investigated as one of the drug targets. If you think GCN4 actually regulates nitrogen metabolism within candida or yeast, including humans, and isoform of it is called EIF2 alpha. So if you imagine a normal gene looks like this:
A blank white slide appears titled "GCN4 Translation Control in C. albicans" in red text. Below it are the words "Normal Gene Structure". Beneath it is a green box labelled "promoter" which is joined to a larger blue box labelled "Genes".
There is a promoter, various other elemental binding sites on the gene and then it's transcribed from here to here as the open reading frame. So what's so different about GCN4? How does it sense there's the nitrogen deficiency?
A more complicated diagram arrives. This one is called "GCN4 Gene Structure". Another green box labelled "promoter" is joined to another large blue box labelled "Gcn4". This time, between these two, are a series of small blue boxes numbered from one to four.
The way it senses it, it has got not one open reading frame but four open reading frames at top of with the promoter, so when the RNA is synthesized it has got all the four. So when the translation starts the protein synthesis what happens is the first one is recognized and then terminated. So the methionine which is the first amino acid to be added is unavailable here. So that means there is a lot of nitrogen because it will start here, stop here, start here, stop here. But by the time it will reach here, there won't be any methionine available, so it will not initiate. But when there is a deficiency it will start here, stop here. But methylene won't be available for one of these, but it does become available at this GCN4. That's how it senses it. Some of this research is now textbook materials, actually.
Below the two diagrams, the words "Amino acid starvation induces filamentous growth in Candida albicans" appear with some small images of cells under magnification.
So what we did was to investigate whether GCN4 is activated or not. We knocked out GCN4 from candida albicans, and you can see as you - this is the heterozygous one and this is the homozygous one. It has got the full two GCN4 genes. It has got just one. And then there is reduction. There is still filamentous formation when you start with amino acids, but then if you knock out the whole GCN4, even if you starve it or whatever you do, it does not transform into hyphal form, so it becomes totally non-pathogenic.
A new slide labelled "Gcn4 co-ordinates morphogenetic and metabolic responses" arrives. It shows a chemical pathway diagram on the left and some tables showing DNA data on the right.
So this was the research which I carried out at Aberdeen University as my first project. And this is the pathway. Its main role is to synthesize and regulate amino acid biosynthesis, and these are some of the amino acids which are not synthesized when it's knocked out, yet, but its additional role is also to regulate morphogenesis where the yeast gets converted to hyphal form.
After this project, I was getting interested into getting more mainstream into mammalian research and I spoke to Al and he said "I have got another project coming which I helped write actually and was successful" he said, "You can continue on this one." I said "But my heart is set on moving into mammalian research." And as a postdoc, when you want to move into mammalian research from one system to another it is quite challenging because people expect you to come experienced.
The next slide is entitled "Research Scientist: Babraham Institute, University of Cambridge". An aerial photograph shows the historic Institute building in sunshine standing in acres of idyllic green lawns and trees. Next to the aerial photograph is a picture of Dr. Jenny Pell labelled "30/03/1955 to 20/11/2014, Fellow of Sidney Sussex College, University of Cambridge". It looks as though the photograph of Dr. Jenny Pell was taken on a fun night out. She is grinning at the camera and holding up a glass of wine.
Anyway I applied to Cambridge and I wasn't expecting to get even an interview here. And so Jenny, to her full... I don't know what she saw in my CV because at that time all my papers were also in writing, they were not published. So she gave me the opportunity. And she was investigating myogenesis, specifically the role of insulin growth factor, insulin-like growth factor IGF-1 and the binding proteins, and their role in myogenesis.
A third photograph arrives, this time showing strands or fibres glowing green under a microscope.
So the muscle is quite interesting, the skeletal muscles, they all, the skeletal muscles have satellite cells embedded in them. They are more like stem cells so when the muscle gets injured some of these pathways get triggered to heal the injury and that's where the myogenesis process happens. And these binding proteins and IGF plays an important role. So that was the premise of the project.
A new slide appears showing lots of small different coloured shapes. It is titled "IGF-independent effects of insulin-like growth factor binding protein-5 (Igfbp5)".
So these are the binding proteins. There are around seven. Depending on the tissue type, a different one is synthesized in each individual tissue.
The coloured shapes are joined by a much larger, more intricate pathway diagram showing "IGF-independent effects of insulin-like growth factor binding protein-5 (Igfbp5)".
IGF-1 has got a huge role in cellular proliferation, cell division and also metabolism, so it needs to be regulated very tightly and that's why these IGF binding proteins are there, to regulate its level within the circulation.
Now three images of dissected baby mice appear, labelled respectively "Control", "wtBP5" and "mutBP5 (line 4)".
But what happens, we were also interested in finding in these binding proteins, do they just regulate IGF or do they do some other functions which is independent of IGF? Because the number of proteins we have got is quite limited, so we investigated.
So what we did was we made transgenic mice, where we had two types of transgenic mice. One which synthesized the y-type form of binding protein 5 which is specifically expressed in muscle and other metabolic tissues, then another transgenic mice we created which has, we have mutated the binding site of the IGL so it is free circulating VP5 which doesn't bind to IGF, so it is not regulating IGF, so IGF is freely circulating as well. So what we expected that yes, these mice will be smaller with the y-type form of IGF-B5, but when we mutate the IGF-1 and IGF is really free, those mice may get larger than even the control. But to our surprise actually they, even the mutated IGA BP5 also had was the same size and was growth-retarded same as the y-type muted five.
Next to the dissected mice, a line graph arrives comparing their weights in grammes and ages in weeks.
And that gave us the reason to believe that BP5 has IGF-independent function as well. So we further investigated its role in myogenesis, how biotopes are formed and how it can play a role in cancer and other stuff.
In the remaining space, two small images in blue and green appear, labelled respectively; "MHC/DAPI" and "Myogenin/DAPI".
It was a highly successful period in my career in terms of research outputs. We published around six papers in just a little over three years’ time. From there then I moved to the actual Cambridge University campus next to the Addenbrookes Hospital on a Leukaemia Research Foundation fellowship for five years, and I started doing work on transcription factors which induce erythropoiesis or cell blood formation. But what happened, we were still working on mice and the way we were investigating was the transcription factor, how this is regulated from five kilobases down or upstream or downstream, and that did not give me enough satisfaction. I said, "It's never going to launch or take off as a drug or anything." So I saw this thing, then I went to Warwick University and met Professor Sudesh Kumar.
A new slide entitled "RCUK Academic Fellow: Warwick Medical School, University of Warwick" comes up. There is a photograph of a large modern building with ample carparking in front, followed by two staff photographs of middle-aged men.
I said I showed my interest and they wanted RCUK Academic Fellowship they have acquired and they wanted to advertise it. So I spoke to him and to my surprise when he invited me I thought it was my interview, but it was just to show the facility and walk me around, and they haven't even shortlisted it. So anyway I got shortlisted and from there on I was made assistant Prof. as well as RCUK Academic Fellow, and I was given the opportunity to form my own research team. And that's where all this started, and I found a very good relationship with Dr. Philip Metron, and now he's a professor at Nottingham Trent University and Head of Biosciences.
Next to arrive is a Christmas party photograph of ten people sitting around a restaurant table, all wearing Christmas hats and smiling. Two more photos arrive; one shows Professor Gyan Tripathi with some of his students all in matching blue shirts and smiling, the other shows Professor Gyan Tripathi at about 13,000 feet with his parachute just opening behind and above him in an empty bright blue sky. He looks very excited.
This is our team. We used to work closely together on several projects. So in addition to doing research, we also used to have fun sometimes: this is myself, I'm doing skydiving for charity, for Diabetes UK, jumping from 13,000 feet was quite - yeah, you can imagine - but I would do it again if I had the opportunity. So there we, everybody in the team, was working on adipose tissue and I had no experience in adipose tissue, I had come from skeletal muscle background and neuron models.
A new slide pops up entitled "Types of Adipose Tissues in Mammals" featuring a series of internal human diagrams with adipose tissue highlighted in orange. There are also diagrams of the different types of cells being described.
Here we were using human adipose tissue, getting adipose tissue from plastic surgery units or you can call it liposuction units from Birmingham and places like that, so at first, I was given full opportunity to continue doing what I was doing at Cambridge, at Babraham.
Then I looked at the resources and I came across a huge resource which was, they had a huge collection of not only just adipose tissue but also adipose-derived stem cells or stromal vascular fractions. So adipose tissue is quite interesting in terms... for a long time it was seen as a very sedentary organ which just accumulates lipid, and if you see there are various depots, it's not just located at one side like a liver or any other organ you can find that posterior tissue at various places and that's how it gets its name, where it is located. You have subcutaneous which is just beneath the skin and abdominal region and subcutaneous in gluteal femoral region, you also have a visceral adipose tissue which is embedded between the organs, are also known as mental adipose tissue. You have epicardial adipose tissue which is around the heart, then mesenteric adipose tissue which is in between the gut, so it is in close proximity with a lot of important organs. A lot of organs where it can influence their functions.
Currently, there are three types of adipose tissue which we have identified. One is the major depots are White adipose tissue which has a huge lipid droplet and accumulates a lot of lipid, but very few mitochondria and other cell organelles, but it is very active. Then there is a brown adipose tissue. It is brown because it has got a large amount of mitochondria. And then there is an intermediate one also called beige because of its colour or bright which has got intermediate number of mitochondria. So the mitochondria makes brown adipose tissue regulate body temperature and especially in cold. So it's mainly found in mammals who go into hibernation, say for example polar bears and bears and other, even rats have, rats and mice have lots of brown adipose tissue. Until recently people thought we don't have any brown adipose tissue left.
Babies are born with the brown adipose tissue, that's why they don't shiver because they can generate heat using that brown adipose tissue, but over time we lose it. Until recently - then somebody was doing scanning of thyroids PET scan and found these things lit up because of cold temperature and then they started thinking "What is it?" And on further investigation, they found actually these are brown adipose tissue which even adults have.
A PET scan of a human chest with arrows pointing out the cells in the shoulders and throat, highlighted in shades of orange and yellow. Beneath this, an MRI scan arrives, shaded in greens.
And you won't be surprised that the brownest adipose tissue in humans in the UK is found in people who work on construction sites, because they spend a lot of their time working in the cold outside. So we did a project, a small project with the Warwick Hospital. We were looking for cheap methods or a quick method of identifying brown adipose tissue by MRI scan instead of doing a PET scan. So using AI as well as the scanning technology we have formed an algorithm which you apply with the image then you can actually identify brown adipose tissue by MRI itself.
So one school of thought is that if we have more brown adipose tissue and if we expressed or increased the number of cells within our body, our metabolic rate will increase and we will lose weight naturally. If any of you are thinking the natural way of increasing your brown adipose tissue is spending at least two hours below 19 degrees outside or having cold showers regularly, it's not been proven, but that's one of the thoughts, that's when it lights up because we put a cold jacket on these people before the scanning.
A new slide entitled "Role of Adipose Tissue: I am fat but very active. Don't underestimate me" shows a spider diagram centred around a drawing of some adipose tissue, which Professor Gyan Tripathi will discuss.
So as you think, as you may know, that by this time that adipose tissue was thought to be an organ or a tissue which just there accumulates lipid and does nothing, but suddenly the whole perception changed.
Adipose tissue is believed to secrete, known to secrete, more than 200 cytokines. They're also called adipocytes. Adipose tissue itself when in cellular forms can be called liposite or adipocytes. Because of these cytokines, or adipokines, they are able to influence most of the metabolic organs in the body including the brain where they secrete leptin which regulates our satiety or hunger. So if you have less leptin you are a lot more likely to feel hungry more. Actually, there was a boy which had leptin mutation so he used to eat a lot. He was very active but very obese even though he was very active and the Sir Steve O'Reilly's group found that his leptin gene was mutated. And when they gave him leptin actually he became very thin, and so there are a lot of things it can induce because of its adipokine roles.
A slide entitled "Isolation of Adipose-Derived Stem Cells (ASC's) and Multipotency" comes up, showing the process under discussion. It begins with a photograph of the bucket of fat removed during liposuction which is then processed and can be used to build many tissues of the body.
So this is what we are interested in. I am interested in stem cells, mainly adipose-derived ones. So this is a type of fat which we used to get, say for example, after liposuction, a bucket full of them and then we'll spin them. You can isolate normal adipocytes at that time itself and study on them or you can further fraction them down into stromal vascular fraction or adult stem cells and freeze them and use it later. They are quite pluripotent, they can be converted into fat cells obviously, a bone, muscle, cartilage, skin. There are a lot of companies now trying to produce a skin graft using this because almost everybody has extra adipose tissue.
This slide is called "Nutrient Excess: Cell Organelle Dysfunction" and shows an enlarged diagram of an adipose cell. Next to the adipose cell are two lists. The first reads: "Endoplasmic Reticulum - Synthesis, Folding, Modification and Transport of Proteins, Calcium storage". The second list reads: "Mitochondria - Powerhouse of cell, Heat production, Calcium ions storage, Cell Metabolism, Cell growth and Death".
So a normal adipose tissue looks like this. It has a big lipid droplet and then it has got endoplasmic reticulum around it and then a few mitochondria. The role of endoplasmic reticulum which is my interest is basically it does protein synthesis. When the RNA is transcribed, ribosomes bind to it, and when the protein is translated it comes to the endoplasmic reticulum and there, that's where it gets folded, modified. And most of the secretory proteins do pass through that, especially for modifications. It also stores calcium. And then mitochondria. The mitochondria, as you know, is the powerhouse of the cell which produces energy. It is also involved in heat production, cellular metabolism, cell growth and cell death. So one of the biggest changes we see as soon as there is a nutrient access is the changes within endoplasmic reticulum and mitochondria.
So my interest is mainly in both these cell organelles and how they - if they don't work properly or there is a dysfunction in these two cell organelles due to excess nutrition or low nutrition - how it impacts metabolism.
The slide is entitled "ER Stress in Human Adipose Tissue". A very complex pathway diagram arrives on the left showing the functions being discussed.
So there is, this is the ER stress pathway. You see It gets switched on in times of abundance or excess nutrition. There is a demand of protein synthesis that also increases, if the demand of protein synthesis increases, there is demand of protein folding and protein modification increases. In normal circumstances, ER is able to manage all that process, but under extreme cases mainly, some of the factors understood are excess nutrition, viral infection and hypoxia, which may induce this process of ER stress.
So what it immediately does is it switches on a response called unfolded protein response and switches one of the pathways, say for example will activate and shut down any protein synthesis within the cell. Another pathway will switch on within the ER which will actually switch on, only the essential proteins which are required, those transcription factors for the survival of the cell, and the third pathway actually will switch on when everything has gone wrong and a cell has no chance of survival it will switch on the apoptotic pathway and kill the cell so that the bad thing does not affect. One of the things which have we have identified is that when the ER stress is massive in obesity it also induces high inflammation which could be the primary cause of insulin resistance.
The diagram is now joined on the right by a bar chart comparing the "Relative fold expression" in groups of lean and obese people. The results for the obese group are marked in red and are much higher than the lean group’s blue ones in most cases. Text beneath the bar chart reads, “Obese non-diabetic females had significantly higher ER stress levels in AbSc AT than lean”.
So this is one of the slides where we measured the markers of ER stress in lean people and obese people, and as you can see they have increased ER stress. Obesity does cause increased ER stress.
A new slide entitled "Weight loss alleviates ER stress in human adipose tissue" arrives showing diagrams of several internal organs. Beneath this are the words "ER stress is induced by High Glucose, Saturated Fatty Acids and Endotoxin in adipocytes".
To understand whether if these subjects lost weight will the ER stress be reduced and will their insulin sensitivity will be increased, for that then we collaborated with a group in Czech Republic who were doing regular bariatric surgery. And they had collected samples before bariatric surgery and also six months after bariatric surgery with weight loss.
Next to the previous diagrams, another bar graph appears, this entitled "ATF6 and IRE1a UPR effector gene expression".
And what we found was that indeed after bariatric surgery ER stress does go down and these people do become insulin-sensitive.
A new slide arrives entitled "Mitochondrial Dynamics", showing a series of flow charts illustrating "Healthy Mitochondrial Dynamics".
Similarly, we did a mitochondrial study where we investigated whether the mitochondrial dynamics is affected or mitochondrial structure is affected.
The next slide is headed "Weight loss improves mitochondrial dynamics" and shows a series of 12 small bar charts that Professor Gyan Tripathi will refer to.
And we found that with - please ignore the number of figures on this slide - but this tells us that the mitochondrial dynamics where the mitochondria constantly divides or fuses to maintain mitochondrial health, actually does improve after weight loss.
Mitochondria if you know is mostly or almost 100% from mother. It does not come from father so it does not have any sexual cycle to improve its health, so it has to constantly fuse and divide. In that way, it can maintain the mitochondrial health. The bad ones, when they divide they get destroyed by the autophagy, while the good ones will live and then fuse and become bigger mitochondria and metabolic. That's why metabolic health and mitochondrial dynamics is key to understanding this.
This slide is called "ER stress mediates mitochondrial dysfunction in obesity". Beneath it is a collection of small graphs and images concerning ER stress effects.
And then that's why we studied the mitochondrial morphology. And mitochondrial morphology if you see here what we did, we wanted to investigate whether ER stress also leads to mitochondrial dysfunction, so we induced artificially ER stress by using one of the molecules called tunicamycin which inhibits glycosylation or the protein modification. So as you can see the mitochondrial fragmentation increase, the swollen mitochondria increases and also the mitochondrial number increases. Sometimes an increase in mitochondrial number is good, but not always, especially if the mitochondrial number has increased due to mitochondrial fragmentation which is totally undesirable.
Under the diagrams, a small list of bullet-points appears labelled "The induction of ER stress in adipocytes". Point 1 reads: "Compromised mitochondrial function". Point 2 reads: "Reduced ATP production". Point 3 reads: "Reduced basal respiration rate".
By this time I was thinking about other avenues of research in terms of looking at drug targets and also disease prevention
A new slide pops up entitled "The Human Genome Project". Text at the top reads: GWAS (Genome-Wide Association Studies) studies have shown that the genetic component of common diseases usually explains only a small proportion of the phenotypic variance". It also shows an image of a foetus with two possibilities indicated for its future by arrows: one is the healthy homo sapiens man we saw in an earlier slide. Possibility two is exemplified by the very obese modern man in shorts carrying his huge soft-drink cup which we saw in the same slide. Further text reads: "The case of the missing heritability" and quotes from nature 2008: "When scientists opened up the human genome, they expected to find the genetic components of common traits and diseases. But they were nowhere to be seen..."
And that happened when I came across one of the lectures delivered by Dr. Ranjin Yajnik at work and that day he opened my eyes that there is other stuff you can investigate.
This slide is titled "Maternal vitamin B12 & folate and insulin resistance (6 years)". The first image is of Ranjan Yajnik, he has glasses and a white beard. A 3-dimensional bar graph appears next to Ranjan labelled "Yajnik, C S et al. ‘Vitamin B12 and folate concentrations during pregnancy and insulin resistance in the offspring: the Pune Maternal Nutrition Study".
And he was looking at vitamin B12, so what he showed, he's been working for 30, 40 years with the tribal population in India near Pune, and what he found, he looked at the mothers at the age of pregnancy, during pregnancy and followed up their children as well. And what he found that B12 deficiency, the mothers who had lower level of the guidance which says B12 deficiency is there, the mother's B12 deficiency, their children actually developed insulin resistance as early as - are showing signs of - as early as six years of age. So after his talk, I started discussing with another colleague, Dr. Server from NHS and who was also interested in vitamin B12 and it's a deficiencies. So what Ranjan told me, that yes I have got all this hypothesis and beautiful data to show that insulin resistance happens, but I have no proof or no mechanism to show why it is happening.
A new slide displays a large table with many categories and the detailed statistics of the women studied. Its title is "Vitamin B12 deficiency is associated with adverse lipid profile".
So I said, "Okay, we'll investigate this in the UK population." So we chose two populations in women of childbearing age; one was UK population with 315, and then we chose another population in Saudi Arabia with King Saudi University. And I was surprised to see that actually, these women who had B12 deficiency had higher cholesterol, higher BMI, higher triglycerides and higher homocysteine, all those things indicating towards metabolic syndrome.
A slide entitled "Vitamin B12 deficiency: adverse lipid profile" shows 4 separate scatter diagrams depicting the results in the European and Indian populations discussed. Text next to these reads: "Low B12 associated with high triglycerides and high cholesterol".
Then we further investigated, and in Europeans and Indian populations living in Europe, and what we found, that was in Caucasian as well as south Asian origin populations, again we found that if you had a low B12 or B12 deficiency you're more likely to have high triglycerides and high cholesterol in your circulation.
The next slide is very similar and is entitled "Association of maternal B12 and metabolic risk factors of neonates". Text reads: "In neonates maternal low B12 associated with high triglycerides, high HOMA-IR, low HDL, high homocysteine and high leptin". This time 5 separate scatter diagrams are shown.
Then we looked at the mothers who had given birth and looked at their B12 profile and then looked at the factors, mitral metabolic syndrome factors, within the babies. And we found that in the mothers who had B12 deficiency, their babies or the neonates had higher triglycerides, higher HOMA-IR which is an indicator of insulin resistance. They had low HDL which is good cholesterol, high homocysteine and high leptin as well.
Now a slide reads simply: "B12 deficiency may lead to: hyperhomocysteinemia, insulin resistance and adverse lipid profile".
So I was quite interested. So far what I had seen was the B12 deficiency is quite broad as well as it's quite significant, as it causes homocysteine increase which has always been linked with increased cardiovascular risk.
A new slide depicting a complex flow chart arrives titled: "Vitamin B12: 1-carbon metabolism".
So I went back and looked at the pathway. B12 actually plays an important role in DNA and RNA synthesis through its one carbon metabolism, and one key reaction where it acts as a cofactor is the methionine synthase conversion of homocysteine in methionine. So naturally, if there was low B12 you will have increased homocysteine and less methionine. What methionine also does convert into s-adenosine methionine, which is the donor of methyl group to DNA protein and RNA for their modifications or methylation process. It also has a role as cofactor in the conversion of succinyl COA which is used in the Krebs cycle for ATP generation and because of its deficiency MMA is an inhibitor of beta oxidation so fatty oscillopsia which leads to the lipid accumulation within the body.
A new slide labelled "Low B12 increases total cholesterol and homocysteine" shows the respective bar charts for cholesterol and homocysteine levels, as well as a photograph of some cells under magnification.
So to study that I designed several experiments on a primary adipocyte model and we started with 12 conditions, but settled down with just three because it was just going out of hand, so we had a condition of control which has optimum B12 level in the cell culture media, than low, and a condition where there was no B12 at all. And what we found was that when there is a B12 deficiency or low B12 then again there was increased cholesterol and homocysteine within the system itself.
Another slide arrives bearing the title in red: "Microarray analysis on human adipocytes treated with B12". Beneath this is a colourful diagram or chart showing "differently expressed genes". Next to this, two charts of detailed tabulated data appear corresponding to the diagram.
Then we did a microarray study from these samples and what we found, there were significant changes in low B12 and no B12 conditions and the genes are totally either highly expressed or have a lower expression compared to control. And to my surprise, the pathways which came up with significantly modified was cholesterol biosynthesis and unfolded protein response which is ER stress pathway, and these are the sum of the genes which were included in cholesterol biosynthesis.
The next slide is entitled "Low B12 increases gene expression of cholesterol biosynthesis" and it shows 5 separate bar graphs in shades of blue.
So I looked at the expression of these biosynthesis genes and we found that indeed these genes were highly expressed in low B12 or B12-deficiency conditions.
A new slide called "Regulation of Cholesterol" bears a chemical diagram labelled to show "Golgi", "High Sterol", "Low Sterol" and "ER".
The cholesterol, the way it is regulated is that once the cell senses there is a low cholesterol within the cell which is an essential molecule for survival of the cell this molecule translocates to the nucleus and targets these genes which regulate either cholesterol or synthesize cholesterol.
A new slide shows 6 blue bar charts headed "Low B12 increases transcriptional regulator of cholesterol - SREBs”.
So we looked at these regulatory genes as well and they were highly expressed whenever there was B12 deficiency, so it was quite clear that B12 deficiency leads to upregulation of cholesterol biosynthesis. So what was the actual mechanism?
A new slide entitled "Effect of B12 on Genome-Wide Methylation Profile" appears showing some of the detailed charts and diagrams that are being referred to. A table is labelled "Low B12 causes hypomethylation".
So as we explored earlier, during the pathway that whenever there is a B12 deficiency there is a reduced methylation potential.
A slide entitled "ChIP-Seq PPARy and C/EBPa sites" appears.
And what we found is that after a whole genome-wide methylation study we found that when there is vital deficiency there is a genome-wide methylation low, low methylation within all the genes and cholesterol regulatory genes such as STREBF1 and LDLR actually have CPG islands which have low methylation that makes them open for higher transcription. You can ignore the rest of the picture basically.
A new slide entitled "Gene expression in human adipose tissue (ScAT)" showing four separate bar chats in black and white labelled A, B, C and D respectively comes up on the screen. They show the difference between control and LB with relative mRNA expression for SREBF1, SREBF2, LDLR and HMGCR. All show LB with from two to four times the expression of Control.
So we wanted to investigate whether this is actually happening in mothers which have undergone pregnancy, so what we did was actually we collected adipose tissue from mothers who were undergoing caesarean section and we formed two groups: mothers who were into B12 deficient category and mothers who had a higher B12 or B12 surface and category and in that adipose tissue then we measured the cholesterol biosynthesis as well as regulatory genes, and we found indeed when you have B12 deficiency these genes were expressed and that's why these mothers had had cholesterol and also those babies during pregnancy were exposed to higher cholesterol as well, so they are at a more highly likely risk of developing metabolic syndrome when they grow up. That's why maternal nutrition is so important.
A colourful diagram is shown depicting the "Regulation of glucose transport".
Another thing which I have taken is the insulin resistance. Insulin resistance, the way it works is when you eat something insulin is produced by beta cells and most of the cells such as adipose cells and skeletal muscles they have insulin receptors so insulin binds, and then through various signalling it activates glut4 vesicles to the cell surface once the glut4 vesicles comes to the cell surface then they bind to the glucose and internalize it for energy production, so it's a simple pathway. What happens in insulin resistance is you may have a lot of insulin in circulation but something in this circulation goes wrong in this pathway that can lead to glut4 not moving to the cell surface, and if that happens then glucose stays in circulation and causes all kinds of secondary effects like cardiovascular disease and arterial defects and all those things.
The next slide is called "B12 deficiency causes insulin resistance" and first shows a colourful bar graph charting the "Glucose uptake assay" in three separate groups.
So we did investigate in B12 deficiency conditions actually the cells were compromised with the B12 intake. So here suppose, for in this graph you can see this is the control when you add insulin, the cells are taking more glucose but they're already compromised here so, and even if you add insulin, the cells are unable to take internalized glucose and they hence can't metabolize it.
The bar graph is joined by another in black and white showing the "Expression of phospho-AKTser473" with Control getting a pAkt:Akt ratio of close to 1 while LB and NoB are closer to 0.6 and 0.5 respectively.
And then we pinpointed that one of the key kinases are the enzymes which activates glut4 for translocation to the cell membrane was downregulated.
A new slide, this is titled "B12 deficiency induces PTEN and Trb3", shows two more bar graphs in black and white with genetic data.
Why that enzyme was downregulated was there were two proteins - one is phosphatase which stops the activation of AKT and another one trip-3 which directly binds to AKT so once they are activated they stop that AKT to mobilize the glutathione to the cell surface.
A slide titled "PTEN inhibition and Trb3 knockdown improves insulin sensitivity" shows a triple bar graph labelled: "Insulin-stimulated glucose uptake" for Control, LB and NoB.
So if you think from the drugs point of view these two become a good target from for drug discovery as you will see in this slide that when I inhibit these two proteins by SIRNA technology or drug technology the insulin uptake almost reaches to the normal control levels.
A new slide headed "GLUT4 translocation with PTEN inhibition" shows an image of blue shapes on a black background. There are smaller green shapes behind them.
This is a slide which shows in real-time when this is the cell which is insulin resistant because of P10 and you can see these green dots are glued for vesicle within the cell the blue one is nucleus.
Now the first image is joined by another similar one. This is labelled "BpV" and some of the blue shapes are much larger.
When I inhibit p10 with a drug or SIRNA and you can see these glut4 moving to the cell surface so that shows that it has started functioning and now it has become insulin sensitive.
A new slide, this time without a title, shows a flow chart which starts with a red box labelled "Vitamin B12". At the bottom of the diagram, another red box is labelled "Diabetes and CVD risk" with many arrows and the different possibilities in between, centred on abnormal gene expression and adipose tissue dysfunction.
So far what we have seen is vitamin B12 deficiency can cause abnormal gene expression. It can also cause hyper-homocysteine anaemia, it can cause insulin resistance, it can cause adipose tissue dysfunction and hence metabolic syndrome, not only in the person who is going through but also in future generations.
This slide is entitled "Message". On the left is a line drawing of a pregnant woman labelled "Suboptimal diet". Flowing down from this are a series of arrows indicating the consequences of a suboptimal diet as discussed in the video, ending finally in "Metabolic disease". In the centre of the slide is a flow chart from Vitamin B12 deficiency; Hypomethylation of key genes; Effects on tissue function; Metabolic disease. There is also a picture of a human foetus with three question-marks beneath it to the right of the slide.
And that's why the message is to put mothers on a proper diet so that their future generation is at less risk of metabolic. This is just one of the factors, there are multiple factors which could be studied this way to find treatments.
A slide titled "Wnt10b: role in adipogenesis (case study)" appears. It shows two bar charts, some small images of orange-coloured cells under a microscope and the statistics of the 19-year-old woman being discussed: Female, 19 years old, BMI 62, weight 143.2kg, fat mass 59.8% of body weight.
Just a couple of slides more: this is one of the interesting patients which we encountered in the clinic. This is a female, 19 years, who is metabolically active and healthy and has a mutation of Wnt10b. This is a gene which actually inhibits adipogenesis. Even though she is metabolically active her BMI is 62, it is higher than many bariatric surgery patients. And her weight was 143.2 at the age of 19. It was still metabolically active, so what we did, we took an adipose tissue sample from her and cultured it. So compared to allene, her adipose tissue accumulate large amount of lipids. Actually, adipose tissue, if it kept doing its role properly of accumulating lipids and lysing it when required there is no issue. The issue is when it does not function properly because of several other reasons, or other factors.
Now we are presented with a slide titled "Wnt10b: role in adipogenesis" which Gyan will discuss.
So this is the pathway for adipogenesis which is regulated by Wnt10b so in her because Wnt10b is off all these transcription factors which promote adipogenesis are switched on. That's the main reason for obesity and that also is the reason why we should not treat every patient the same. Because normally if we won't have investigated it further we won't know why she is obese and probably she would have had to go through multiple liposuctions or bariatric surgery and all that unnecessary steps.
A colourful slide is headed "Facility for OMICS research in metabolism (FORM)". An illustrated list reads: "Genetic & epigenetic factors, Ageing, Diseases, Drugs, Nutrition, Lifestyle, Environment" all pointing toward “Phenotype”, with clipart images illustrating each of these. To the right is the word “Form” with a flow chart beneath leading downward from DNA Genomics> RNA Transcriptomics> Proteins Proteomics> Biochemical Metabolomics> Biological Phenotype.
So coming to personalized medicine, we have been very lucky to acquire this funding to create a facility for omix research and metabolism at the University of Derby through local enterprise partnership funding as well as funding from Derby University which have made equal contributions, so total funding is around 1.7 million. Now we are in a great position to investigate not just genetic but also environmental factors and any other factors which may be leading to metabolic diseases in humans.
An illustrated list reads: "DNA - Genomics - 40,000 genes. RNA - Transcriptomics - 150,000 transcripts. Protein - Proteomics - 1000,000 proteins. Metabolites - 3000 compounds. An arrow pointing down indicates that we know less about the items earlier in the list and more about those later. A second arrow pointing down is labelled "Environmental influence".
Now we are in a position to investigate the whole genome, also the transcriptome, all the RNA which is overexpressed under disease condition. We can investigate all the protein molecules and also all the metabolites produced either in pathogenic conditions or in any other pathology, or something good we can also look at is the impact of various nutrients with this facility.
This slide is entitled "Why personalised medicine? We are all different". A photograph shows Winston Churchill along with details about him which Gyan will describe.
So why personalise medicine? Because we are all different. If you think Winston Churchill lived to the ripe old age of 90 and he was, most of his life, he was overweight, he drank, he smoked, and he never exercised and he wasn't known to have a healthy diet - yet he was reasonably okay and lived a healthy life.
The photograph of Winston Churchill is replaced by one of Jim Fixx, titled “Author: The Complete Book of Running.”
And there, on the other hand, we have Jim Fixx who wrote a book on 'The Complete Book of Running', and you would be surprised to know he died of a heart attack while running at an early age of 52. That's why it's very important because of some conditions where he was genetically predisposed for cardiac disease or something equivalent. So that's why it is important to treat every patient on the causes of the disease rather than treating them universally like we do.
This slide is titled "Acknowledgements" and shows several corporate logos and a long list of names under the headings of Diabetes Team, Systems Biology, Heart of England Hospital, QMUL, Southampton, India, CSRL, Czech Republic and King Saud University.
I have worked with a lot of people and it is impossible to acknowledge everyone, but some of them in the past 10 years who have made significant contributions are listed here and some of them might be listening to this talk so thank you, everybody, for your contribution in my research and for continued collaboration. If I acknowledged everyone it would be longer than the Avengers film tribute which goes on for more than six minutes.
Now the words "Thank you" appear in large red type on a white background.
Finally thank you, and I would like to show my personal gratitude to my parents.
Professor Gyan Tripathi's face fills the screen as he speaks. Behind him is a poster of a periodic table of fruits and nuts and above that, a bucolic picture of cottages by a river.
My father who was a man in the air force and my mother who never had the opportunity to go to school herself but made sure all her children were educated to the highest level possible. Secondly, I would like to thank my eldest brother who kept me on track with his constant advice during my studies during maths as well as he supported my any shortfall in funding while I was doing my graduate studies. And then finally I would like to thank my great uncle, my grandfather's younger brother, who had, the way I think, the way I do things, has a lot to do with what he taught me during my early summer vacations which was spent like three, four hours every day with him, so thank you very much.
The screen turns dark blue and the University of Derby logo with its three peaks appears in white in the centre.
Audio described version of Nutrition in Metabolic Diseases - Professor Gyan Tripathi inaugural lecture video