Final Report Summary - OBINNSC1 (The Physiological Control of Stem Cells: Obesity, Insulin, and Neural Stem Cell Dynamics.)
SUMMARY
Summary
Environmental factors that affect an organism’s milieu intérieur, such as diet, infection, or societal stress, can greatly impact stem cell activity, organ function, and health.
This Intra-European Fellowship has allowed a young researcher of proven ability to investigate this phenomenon in an internationally renowned laboratory.
New findings have been made, and avenues of research opened, in our understanding of how a high fat diet and obesity can impact stem cells in the brain.
Whilst it had been observed that the diet-induced obesity caused by long term exposure of mice to a high-fat diet correlated with a decrease in adult neurogenesis, we have found that this is preceded by a period of increased neurogenesis. This is an extremely interesting result; it suggests that a high fat diet can affect neural stem cells prior to the onset of obesity, and that the resulting increase in proliferation then leads to stem cell exhaustion. This has led us to expand our study to other strains of laboratory mice that do not develop diet-induced obesity, in order to understand whether this is a direct universal effect of a high-fat diet.
The study had initially planned to have a focus on the Insulin/IGF signalling pathway. However after failing to find the hallmarks of this pathway’s activity, we have taken a new research direction. In order to identify the molecular mechanisms responsible for the interaction of high-fat diet/obesity with adult neurogenesis, we are taking an unbiased approach based on RNA sequencing. We have used Laser Capture Microdissection to isolate stem cells, allowing us to isolate their RNA and compare gene expression between our experimental groups.
During the course of the fellowship the fellow has been able to learn and develop a number of skills important for career development and skill transfer. These include: attending international conferences; teaching undergraduates; mentoring PhD students; participating in the peer review process; presenting and sharing his work with his peers; gaining qualifications in animal husbandry and surgery; advancing his interest and skills in advanced light microscopy; and improving his grasp of the Swedish language.
The Karolinska Institute’s ‘visiting researcher’ framework has helped the fellow fully integrate into Swedish society.
The research performed gives us new insight into how dietary factors can affect the body at the level of the stem cell, and may provide new tools for researchers aiming to develop stem cell therapies or treat diseases caused in part by stem cell malfunction. The work performed thanks to this fellowship will form the core of a scientific publication that other researchers will be able to build on.
Context & Objectives
Stem cell populations are essential to the maintenance, repair, and function of the tissues in which they reside. In keeping with their importance, stem cells are subject to a myriad of regulatory signals and interactions that tightly control their proliferation and differentiation.
One mode of stem cell regulation is via systemic endocrine cues. In this way, environmental factors that affect an organism’s milieu intérieur, such as diet, infection, or societal stress, can greatly impact stem cell function.
Understanding how altered physiological states affect the dynamics of stem cell populations will provide a platform from which we may be able to therapeutically address aspects of illness and disease that are derived from altered stem cell function. Further, an increased understanding of how stem cell proliferation and differentiation are controlled may provide new tools for researchers trying to develop stem cell-based therapies.
Neural stem cells (NSCs) in the adult hippocampus serve as a powerful paradigm for the study of environment-stem cell interactions; the rate at which they divide and generate new neurons is acutely sensitive to environmental factors such as exercise and stress. The environmental variable we are interested in is diet: specifically, the effect of diet-induced obesity on adult hippocampal NSCs.
Obesity has become widespread in Europe, and the rest of the world. The World Health Organisation (WHO), based on the latest estimates from EU countries, reports that obesity affects between 10 and 30% of adults. Further, the WHO estimates that within the European Region nearly 10% of school age children are obese, which represents approximately 3.75 million children.
Obesity has been linked to poor performance in cognitive tests involving memory, the development of dementia in the old, and, though conflicting reports exist, depression. Strikingly, these conditions have also been linked to impaired hippocampal neurogenesis and function. In recent years a number of short reports have been published that suggest a high-fat diet, and resulting obesity, impairs hippocampal neurogenesis in rodents. However, the mechanism by which obesity can alter NSC behaviour and fate remains unknown. This project aims to marry advanced genetic manipulation and microscopy with the established C57BL/6J mouse model of diet-induced obesity to precisely define the effects of a high-fat diet on NSCs, and elucidate the molecular mechanisms responsible.
In order to elucidate the mechanisms by which a high-fat diet and obesity regulate neural stem cell behaviour our initial immediate objectives were:
• To use immunohistochemistry to define the changes in NSC behaviour, and signalling pathway activity, that occur during the development of obesity.
• To optimise tamoxifen dosage for, and perform genetic lineage tracing of hippocampal NSCs.
• To develop conditional knock-out models of the Insulin and Insulin-like Growth Factor Receptors for use in adult hippocampal NSCs.
Results & Foreground
We have analysed hippocampal NSCs at the population level with respect to proliferation and neuron production and how these parameters are altered in response to a high fat diet and obesity. Mice are shifted from a control to a high-fat diet at 6 weeks of age. At 22 weeks of age, following 16 weeks on a high-fat diet, obesity and hyperinsulinemia are fully developed. Proliferation and neuronal production were assayed, via immunofluorescence with antibodies against Ki67 and Doublecortin respectively, at various time points during these 16 weeks. After 16 weeks, as previously reported, we found a reduction in proliferation and neurogenesis of approximately 20%. Earlier time points provided a novel and very interesting result. We found that the reduction in neurogenesis in obese mice was preceded by an increased rate of neurogenesis. This result suggests that a high-fat diet acts to increase NSC proliferation, which then results in NSC “exhaustion” and the reduced levels of neurogenesis seen in obese mice after 16 weeks. We are now assaying total NSC numbers using an antibody against Nestin (a NSC marker) in combination with antibodies against Sox2 and GFAP, in order to determine the nature of any exhaustion.
That the change in NSC proliferation occurs prior to the development of obesity also suggests that dietary fat may have a more direct effect on stem cells than was previously thought i.e. it may not act via its effects on fat tissue. It may be that regardless of weight gain, simply eating a high-fat diet is enough to impair neurogenesis and brain function. In order to test this we have established our high-fat diet paradigm in two mouse strains that are not susceptible to diet-induced obesity (the F1 hybrid progeny of C57BL/6J and 129S2/SvPasCrl, and BALB/cByJ). Initial data suggests that a high fat diet may indeed have an effect on neurogenesis in these strains. However, the study is ongoing and more mice are currently being analysed; only after this analysis will it be possible to make a final, statistically sound, conclusion.
Whilst it has proven relatively straight forward to reduce tamoxifen dosage to a level where recombination is rare enough to allow a clonal analysis approach, transferring these protocols into an obesity model can be challenging. We have found that transgenes often have uncharacterised physiological phenotypes i.e. they do not respond to dietary fat in the same way as the C57BL/6J strain. It has subsequently been reported that such metabolic/physiological phenotypes are frequently present in transgenic animals but remain largely uncharacterised. Optimisation of lineage tracing protocols within the diet-induced obesity paradigm is ongoing. Whilst not allowing genetic labelling of a single stem cell and its progeny, we have promising data from a nestin-GFP transgenic mouse line in our high-fat diet paradigm. nestin-GFP seems to have the same response to a high-fat diet as the native C57BL/6J mice (i.e. early increased neurogenesis). nestin-GFP allows powerful population level analysis of NSCs (and their lineages to a limited extent) in the high-fat diet paradigm, and could also be extremely useful for the transcriptional analysis described below as it allows unambiguous identification of individual stem cells.
The prime candidate for a link between high-fat diet, obesity, and adult neurogenesis was Insulin/IGF signalling. However, analysis of pathway activation via phosphorylated pathway effectors such as AKT/PKB, S6K and 4EBP has suggested that Insulin/IGF signalling does not significantly change in response to high-fat diet in the proliferative zone of the hippocampus. Given the lack of evidence for the direct involvement of Insulin/IGF signalling, at the NSC level, in the regulation of proliferation and neurogenesis by dietary fat it was decided to postpone the genetic manipulation of these pathways and instead focus on an unbiased exploratory approach to identify the signalling mechanisms involved.
We have used laser capture microdissection (LCM) to isolate the subgranular zone –the small region of the hippocampus that houses the neural stem cells. High quality RNA can be extracted from these samples, enabling subsequent gene expression analysis. We are currently optimising an immunostaining protocol in combination with LCM to allow the identification and isolation of individual progenitor cells, allowing a more precise single cell analysis; and we have optimised the generation of high quality RNA samples from not just small groups of cells, but also single cells that were microdissected from the hippocampus (this step is critical to ensuring the generation of high quality data from the downstream RNA-seq transcriptional analysis). Comparison of the transcriptional snapshots generated between control mice and those on a high-fat diet will generate candidate pathways that might mediate the interaction between dietary fat and stem cell behaviour.
It is expected that the final results will identify the molecular mechanism by which a high fat diet influences NSC behaviour. This should happen in two steps: Firstly the identification of candidate pathways via transcriptional analysis of the hippocampal NSCs and subsequent immunohistochemistry; Secondly, the genetic and pharmacological interrogation of candidate pathways to identify the factors that are necessary and sufficient to mediate the effect of a high-fat diet.
The reason that we refer to future work and publication in this report is that the fellow will continue to work on this project in another lab. The fellow terminated his fellowship early to take up a post-doctoral position at KTH Royal Institute of Technology in a laboratory that specialises in the molecular and computational techniques required to perform and analyse next generation sequencing-based experiments. This will allow him to continue working on this research project in to the future whilst co-ordinating a collaboration with the lab which hosted his fellowship at the Karolinska Institute.
Impact, Implications & Dissemination
The knowledge gained about how high-fat diet regulates this NSC population may well be applicable to stem cell populations in other tissues, and provide a platform from which we may be able to therapeutically address aspects of illness and disease that are derived from altered stem cell function. Our initial findings already suggest that a high-fat diet may exert adverse effects on stem cell population dynamics and tissue function prior to, and perhaps independent from, weight gain; thus stressing the universal importance of a well-balanced diet.
The completion of this project will allow people to understand how the dietary choices they make can directly affect aspects of brain function derived from an adult stem cell population.
The data generated thus far will form the core of a scientific publication that the research community will be able to build on. At the Karolinska Institute significant publications are announced and explained on the home website as well as through the print, radio and television media. Further, a regular open public lecture series is held where researchers explain their published research and its implications.
ETHICAL & SOCIAL IMPLICATIONS
The Karolinska Institute enforces some of the highest animal welfare and ethical standards in Europe –going beyond minimum legal requirements. We worked within these high standards in regard to animal housing, maintenance, health monitoring, anaesthesia, and euthanasia.
We always sought to minimise any suffering and use as few animals as possible. No animals experienced significant suffering during this project (at most an animal received a single intraperitoneal injection).
The completion of this project will allow people to understand how the dietary choices they make can directly affect aspects of brain function derived from an adult stem cell population.
Any molecular mechanisms discovered may provide a platform from which we might therapeutically address aspects of illness and disease that are derived from altered stem cell function. Further, an increased understanding of how stem cell proliferation and differentiation are controlled may provide new tools for researchers trying to develop stem cell-based therapies.
The data generated thus far will form the core of a scientific publication that the research community will be able to build on. The work will be presented at international conferences. And, importantly, in order to disseminate the work to the general public we can utilise the Karolinska Institute framework that interacts with print, radio, and television media outlets; and also offers a public lecture platform.
Summary
Environmental factors that affect an organism’s milieu intérieur, such as diet, infection, or societal stress, can greatly impact stem cell activity, organ function, and health.
This Intra-European Fellowship has allowed a young researcher of proven ability to investigate this phenomenon in an internationally renowned laboratory.
New findings have been made, and avenues of research opened, in our understanding of how a high fat diet and obesity can impact stem cells in the brain.
Whilst it had been observed that the diet-induced obesity caused by long term exposure of mice to a high-fat diet correlated with a decrease in adult neurogenesis, we have found that this is preceded by a period of increased neurogenesis. This is an extremely interesting result; it suggests that a high fat diet can affect neural stem cells prior to the onset of obesity, and that the resulting increase in proliferation then leads to stem cell exhaustion. This has led us to expand our study to other strains of laboratory mice that do not develop diet-induced obesity, in order to understand whether this is a direct universal effect of a high-fat diet.
The study had initially planned to have a focus on the Insulin/IGF signalling pathway. However after failing to find the hallmarks of this pathway’s activity, we have taken a new research direction. In order to identify the molecular mechanisms responsible for the interaction of high-fat diet/obesity with adult neurogenesis, we are taking an unbiased approach based on RNA sequencing. We have used Laser Capture Microdissection to isolate stem cells, allowing us to isolate their RNA and compare gene expression between our experimental groups.
During the course of the fellowship the fellow has been able to learn and develop a number of skills important for career development and skill transfer. These include: attending international conferences; teaching undergraduates; mentoring PhD students; participating in the peer review process; presenting and sharing his work with his peers; gaining qualifications in animal husbandry and surgery; advancing his interest and skills in advanced light microscopy; and improving his grasp of the Swedish language.
The Karolinska Institute’s ‘visiting researcher’ framework has helped the fellow fully integrate into Swedish society.
The research performed gives us new insight into how dietary factors can affect the body at the level of the stem cell, and may provide new tools for researchers aiming to develop stem cell therapies or treat diseases caused in part by stem cell malfunction. The work performed thanks to this fellowship will form the core of a scientific publication that other researchers will be able to build on.
Context & Objectives
Stem cell populations are essential to the maintenance, repair, and function of the tissues in which they reside. In keeping with their importance, stem cells are subject to a myriad of regulatory signals and interactions that tightly control their proliferation and differentiation.
One mode of stem cell regulation is via systemic endocrine cues. In this way, environmental factors that affect an organism’s milieu intérieur, such as diet, infection, or societal stress, can greatly impact stem cell function.
Understanding how altered physiological states affect the dynamics of stem cell populations will provide a platform from which we may be able to therapeutically address aspects of illness and disease that are derived from altered stem cell function. Further, an increased understanding of how stem cell proliferation and differentiation are controlled may provide new tools for researchers trying to develop stem cell-based therapies.
Neural stem cells (NSCs) in the adult hippocampus serve as a powerful paradigm for the study of environment-stem cell interactions; the rate at which they divide and generate new neurons is acutely sensitive to environmental factors such as exercise and stress. The environmental variable we are interested in is diet: specifically, the effect of diet-induced obesity on adult hippocampal NSCs.
Obesity has become widespread in Europe, and the rest of the world. The World Health Organisation (WHO), based on the latest estimates from EU countries, reports that obesity affects between 10 and 30% of adults. Further, the WHO estimates that within the European Region nearly 10% of school age children are obese, which represents approximately 3.75 million children.
Obesity has been linked to poor performance in cognitive tests involving memory, the development of dementia in the old, and, though conflicting reports exist, depression. Strikingly, these conditions have also been linked to impaired hippocampal neurogenesis and function. In recent years a number of short reports have been published that suggest a high-fat diet, and resulting obesity, impairs hippocampal neurogenesis in rodents. However, the mechanism by which obesity can alter NSC behaviour and fate remains unknown. This project aims to marry advanced genetic manipulation and microscopy with the established C57BL/6J mouse model of diet-induced obesity to precisely define the effects of a high-fat diet on NSCs, and elucidate the molecular mechanisms responsible.
In order to elucidate the mechanisms by which a high-fat diet and obesity regulate neural stem cell behaviour our initial immediate objectives were:
• To use immunohistochemistry to define the changes in NSC behaviour, and signalling pathway activity, that occur during the development of obesity.
• To optimise tamoxifen dosage for, and perform genetic lineage tracing of hippocampal NSCs.
• To develop conditional knock-out models of the Insulin and Insulin-like Growth Factor Receptors for use in adult hippocampal NSCs.
Results & Foreground
We have analysed hippocampal NSCs at the population level with respect to proliferation and neuron production and how these parameters are altered in response to a high fat diet and obesity. Mice are shifted from a control to a high-fat diet at 6 weeks of age. At 22 weeks of age, following 16 weeks on a high-fat diet, obesity and hyperinsulinemia are fully developed. Proliferation and neuronal production were assayed, via immunofluorescence with antibodies against Ki67 and Doublecortin respectively, at various time points during these 16 weeks. After 16 weeks, as previously reported, we found a reduction in proliferation and neurogenesis of approximately 20%. Earlier time points provided a novel and very interesting result. We found that the reduction in neurogenesis in obese mice was preceded by an increased rate of neurogenesis. This result suggests that a high-fat diet acts to increase NSC proliferation, which then results in NSC “exhaustion” and the reduced levels of neurogenesis seen in obese mice after 16 weeks. We are now assaying total NSC numbers using an antibody against Nestin (a NSC marker) in combination with antibodies against Sox2 and GFAP, in order to determine the nature of any exhaustion.
That the change in NSC proliferation occurs prior to the development of obesity also suggests that dietary fat may have a more direct effect on stem cells than was previously thought i.e. it may not act via its effects on fat tissue. It may be that regardless of weight gain, simply eating a high-fat diet is enough to impair neurogenesis and brain function. In order to test this we have established our high-fat diet paradigm in two mouse strains that are not susceptible to diet-induced obesity (the F1 hybrid progeny of C57BL/6J and 129S2/SvPasCrl, and BALB/cByJ). Initial data suggests that a high fat diet may indeed have an effect on neurogenesis in these strains. However, the study is ongoing and more mice are currently being analysed; only after this analysis will it be possible to make a final, statistically sound, conclusion.
Whilst it has proven relatively straight forward to reduce tamoxifen dosage to a level where recombination is rare enough to allow a clonal analysis approach, transferring these protocols into an obesity model can be challenging. We have found that transgenes often have uncharacterised physiological phenotypes i.e. they do not respond to dietary fat in the same way as the C57BL/6J strain. It has subsequently been reported that such metabolic/physiological phenotypes are frequently present in transgenic animals but remain largely uncharacterised. Optimisation of lineage tracing protocols within the diet-induced obesity paradigm is ongoing. Whilst not allowing genetic labelling of a single stem cell and its progeny, we have promising data from a nestin-GFP transgenic mouse line in our high-fat diet paradigm. nestin-GFP seems to have the same response to a high-fat diet as the native C57BL/6J mice (i.e. early increased neurogenesis). nestin-GFP allows powerful population level analysis of NSCs (and their lineages to a limited extent) in the high-fat diet paradigm, and could also be extremely useful for the transcriptional analysis described below as it allows unambiguous identification of individual stem cells.
The prime candidate for a link between high-fat diet, obesity, and adult neurogenesis was Insulin/IGF signalling. However, analysis of pathway activation via phosphorylated pathway effectors such as AKT/PKB, S6K and 4EBP has suggested that Insulin/IGF signalling does not significantly change in response to high-fat diet in the proliferative zone of the hippocampus. Given the lack of evidence for the direct involvement of Insulin/IGF signalling, at the NSC level, in the regulation of proliferation and neurogenesis by dietary fat it was decided to postpone the genetic manipulation of these pathways and instead focus on an unbiased exploratory approach to identify the signalling mechanisms involved.
We have used laser capture microdissection (LCM) to isolate the subgranular zone –the small region of the hippocampus that houses the neural stem cells. High quality RNA can be extracted from these samples, enabling subsequent gene expression analysis. We are currently optimising an immunostaining protocol in combination with LCM to allow the identification and isolation of individual progenitor cells, allowing a more precise single cell analysis; and we have optimised the generation of high quality RNA samples from not just small groups of cells, but also single cells that were microdissected from the hippocampus (this step is critical to ensuring the generation of high quality data from the downstream RNA-seq transcriptional analysis). Comparison of the transcriptional snapshots generated between control mice and those on a high-fat diet will generate candidate pathways that might mediate the interaction between dietary fat and stem cell behaviour.
It is expected that the final results will identify the molecular mechanism by which a high fat diet influences NSC behaviour. This should happen in two steps: Firstly the identification of candidate pathways via transcriptional analysis of the hippocampal NSCs and subsequent immunohistochemistry; Secondly, the genetic and pharmacological interrogation of candidate pathways to identify the factors that are necessary and sufficient to mediate the effect of a high-fat diet.
The reason that we refer to future work and publication in this report is that the fellow will continue to work on this project in another lab. The fellow terminated his fellowship early to take up a post-doctoral position at KTH Royal Institute of Technology in a laboratory that specialises in the molecular and computational techniques required to perform and analyse next generation sequencing-based experiments. This will allow him to continue working on this research project in to the future whilst co-ordinating a collaboration with the lab which hosted his fellowship at the Karolinska Institute.
Impact, Implications & Dissemination
The knowledge gained about how high-fat diet regulates this NSC population may well be applicable to stem cell populations in other tissues, and provide a platform from which we may be able to therapeutically address aspects of illness and disease that are derived from altered stem cell function. Our initial findings already suggest that a high-fat diet may exert adverse effects on stem cell population dynamics and tissue function prior to, and perhaps independent from, weight gain; thus stressing the universal importance of a well-balanced diet.
The completion of this project will allow people to understand how the dietary choices they make can directly affect aspects of brain function derived from an adult stem cell population.
The data generated thus far will form the core of a scientific publication that the research community will be able to build on. At the Karolinska Institute significant publications are announced and explained on the home website as well as through the print, radio and television media. Further, a regular open public lecture series is held where researchers explain their published research and its implications.
ETHICAL & SOCIAL IMPLICATIONS
The Karolinska Institute enforces some of the highest animal welfare and ethical standards in Europe –going beyond minimum legal requirements. We worked within these high standards in regard to animal housing, maintenance, health monitoring, anaesthesia, and euthanasia.
We always sought to minimise any suffering and use as few animals as possible. No animals experienced significant suffering during this project (at most an animal received a single intraperitoneal injection).
The completion of this project will allow people to understand how the dietary choices they make can directly affect aspects of brain function derived from an adult stem cell population.
Any molecular mechanisms discovered may provide a platform from which we might therapeutically address aspects of illness and disease that are derived from altered stem cell function. Further, an increased understanding of how stem cell proliferation and differentiation are controlled may provide new tools for researchers trying to develop stem cell-based therapies.
The data generated thus far will form the core of a scientific publication that the research community will be able to build on. The work will be presented at international conferences. And, importantly, in order to disseminate the work to the general public we can utilise the Karolinska Institute framework that interacts with print, radio, and television media outlets; and also offers a public lecture platform.
