Delineating the roles of GPR55 in cellular metabolism and energy homeostasis
Lead Research Organisation:
University of Dundee
Department Name: School of Life Sciences
Abstract
The G-protein coupled receptor (GPCR) superfamily play crucial roles in cell communication. As such, they mediate the effects of circulating hormones and other biologically active molecules across the blood-facing membranes of cells to regulate diverse cell/tissue processes including, for example, sensory perception, metabolism and satiety. Given their involvement in neurological disorders, inflammatory and metabolic diseases, diabetes and cardiac dysfunction they represent the largest and most successful class of "druggable" targets in the human body. However, despite the immense current interest in GPCR biology, the function of numerous members of this family remain poorly understood, but which may well represent important therapeutic targets for treatment of major public health issues, such as obesity, diabetes and cardiovascular disease.
This project aims to explore links between a lipid sensing GPCR, called GPR55, and processes influencing adiposity, inflammation, cardiac function and response to insulin within key metabolic tissues, such as white fat, liver, skeletal muscle and heart. These tissues are major targets for insulin in the body and represent the principal sites where sugar (glucose) and fat are stored and metabolised in response to the hormone. GPR55 is stimulated by a circulating lipid called LPI, which we find improves the response of these metabolic tissues to insulin and also helps lower inflammatory drive in cells derived from them. Crucially, this LPI-mediated response is lost if cells are co-treated with a GPR55 inhibitor. Strikingly, we have discovered that animals deficient in this lipid sensor exhibit reduced tissue responsiveness to insulin, impaired metabolic capacity and a decline in cardiac performance. Metabolic capacity is crucially dependent upon mitochondria; structures within cells representing the cell's "energy generator". Significantly, animals lacking GPR55 show changes in mitochondrial biology consistent with a reduced ability to "burn" fat. In line with this, we find animals lacking GPR55 develop obesity and that inhibiting the receptor in cultured adipocytes (fat cells) induces proteins that help make more fat, which would augment the process of obesity. Precisely how GPR55 links to the molecular regulation of the above processes is currently unclear.
The studies described in this application will utilise cells in culture from rodent and human origin as well as mouse tissues for laboratory-based analysis to help dissect out the role GPR55 plays not only with respect to insulin action and inflammation, but in control of tissue adiposity (fatness) and cardiac function. The project will also explore whether GPR55 activation helps mitigate the increase in fat gain, the loss in tissue sensitivity to insulin and cardiac dysfunction in mice fed a high fat calorie diet. Tissues taken from animals at the end of such studies will be processed for biochemical analysis and state-of-the-art whole cell/tissue protein profiling - an approach that will identify which proteins become up- or down-regulated in tissues of mice lacking GPR55 or in cells in which GPR55 has been activated/inhibited with selective drugs. This methodology will generate a wealth of information, potentially unveiling novel proteins that connect with GPR55 to regulate how insulin works or fat is stored or "burnt". Importantly, the large scale protein profiling may flag-up proteins that have not previously been linked to GPR55, but which may be central to the work of researchers in other fields thus providing an invaluable data resource to the scientific community.
Collectively, our pilot studies indicate GPR55 may function as a novel metabolic regulator within tissues and suggest that understanding how it regulates insulin action, lipid metabolism and cardiac function may offer new pharmacological opportunities for treatment of metabolic disorders associated with conditions such as obesity and type II diabetes.
This project aims to explore links between a lipid sensing GPCR, called GPR55, and processes influencing adiposity, inflammation, cardiac function and response to insulin within key metabolic tissues, such as white fat, liver, skeletal muscle and heart. These tissues are major targets for insulin in the body and represent the principal sites where sugar (glucose) and fat are stored and metabolised in response to the hormone. GPR55 is stimulated by a circulating lipid called LPI, which we find improves the response of these metabolic tissues to insulin and also helps lower inflammatory drive in cells derived from them. Crucially, this LPI-mediated response is lost if cells are co-treated with a GPR55 inhibitor. Strikingly, we have discovered that animals deficient in this lipid sensor exhibit reduced tissue responsiveness to insulin, impaired metabolic capacity and a decline in cardiac performance. Metabolic capacity is crucially dependent upon mitochondria; structures within cells representing the cell's "energy generator". Significantly, animals lacking GPR55 show changes in mitochondrial biology consistent with a reduced ability to "burn" fat. In line with this, we find animals lacking GPR55 develop obesity and that inhibiting the receptor in cultured adipocytes (fat cells) induces proteins that help make more fat, which would augment the process of obesity. Precisely how GPR55 links to the molecular regulation of the above processes is currently unclear.
The studies described in this application will utilise cells in culture from rodent and human origin as well as mouse tissues for laboratory-based analysis to help dissect out the role GPR55 plays not only with respect to insulin action and inflammation, but in control of tissue adiposity (fatness) and cardiac function. The project will also explore whether GPR55 activation helps mitigate the increase in fat gain, the loss in tissue sensitivity to insulin and cardiac dysfunction in mice fed a high fat calorie diet. Tissues taken from animals at the end of such studies will be processed for biochemical analysis and state-of-the-art whole cell/tissue protein profiling - an approach that will identify which proteins become up- or down-regulated in tissues of mice lacking GPR55 or in cells in which GPR55 has been activated/inhibited with selective drugs. This methodology will generate a wealth of information, potentially unveiling novel proteins that connect with GPR55 to regulate how insulin works or fat is stored or "burnt". Importantly, the large scale protein profiling may flag-up proteins that have not previously been linked to GPR55, but which may be central to the work of researchers in other fields thus providing an invaluable data resource to the scientific community.
Collectively, our pilot studies indicate GPR55 may function as a novel metabolic regulator within tissues and suggest that understanding how it regulates insulin action, lipid metabolism and cardiac function may offer new pharmacological opportunities for treatment of metabolic disorders associated with conditions such as obesity and type II diabetes.
Technical Summary
Proper control of metabolic signalling in skeletal muscle, liver and adipose tissue, which are major sites for fuel utilisation/storage, is crucial for maintaining glucose and lipid homeostasis. Consequently, disorders such as insulin resistance and type 2 diabetes may arise due to metabolic impairments in these tissues, by mechanisms that remain poorly defined. Recently we discovered that G-protein coupled receptor GPR55 functions to modulate several metabolic processes, and that mice deficient for this receptor exhibit impaired insulin sensitivity and heightened inflammation. Allied to this, GPR55-null mice also display reduced abundance of proteins regulating mitochondrial lipid oxidation within key metabolic tissues including the heart, coinciding with the development of cardiac dysfunction. Strikingly, we find GPR55 activation enhances insulin sensitivity and upregulates activity/expression of proteins involved in promoting mitochondrial biogenesis and respiration in muscle, hepatocytes and adipocytes. Intriguingly, impaired muscle insulin action in GPR55-null mice concurs with reduced protein abundance of IRS-1, a critical insulin signalling intermediate. In contrast, liver and adipose tissue deficient for GPR55 show no change in IRS-1 content but, instead, exhibit elevated expression of PTEN, a key repressor of insulin action. In addition, GPR55-deficient mice show increased adiposity and lipogenic drive, a phenotype mimicked in cultured fat cells treated with a GPR55 antagonist. We hypothesise that GPR55 stimulation would help alleviate diet-induced obesity, insulin resistance, impaired fuel utilisation/storage and cardiac dysfunction by altering expression and/or function of key insulin signalling components, as well as suppressing lipogenic drive and/or improving mitochondrial function. This project will delineate, mechanistically and functionally, how GPR55 regulates these metabolic processes in skeletal muscle, liver, adipose tissue, and the heart.
Planned Impact
Who will benefit from this research?
Academics: Our understanding of how GPR55 affects key anabolic responses in tissues such as skeletal muscle, adipose tissue, liver and heart is very much in its infancy. Consequently, our findings benefit other academic researchers, especially those working in areas related to metabolic and cardiovascular dysfunction (e.g. obesity and diabetes).
Private Sector: Our findings will appeal to pharmaceutical companies with an interest in the endocannabinoid system (ECS), especially with respect to therapies that help maintain/improve tissue response/function, for example, during obesity and Type II diabetes.
Government: The findings may help inform national (e.g. DoH) and international (e.g. Healthy Living matters, WHO) policy on healthy living in relation to maintenance of tissue function.
Public and Charitable Sectors: Individuals working for public health disciplines (e.g. nutritionists/dieticians etc) and scientific advisors to Medical Charities may benefit from the findings by helping to devise appropriate advice to counter diet-induced decline in tissue health, as well as advising their clients of recent advances.
General Public: Target beneficiaries include the public who may lead poor dietary/sedentary life styles and in whom insulin resistance and obesity-related metabolic dysfunction may be an issue.
How will they benefit from this research?
Our studies reveal that metabolically active tissues (e.g. muscle, fat, liver and heart) lacking GPR55 display reduced insulin sensitivity, heightened tissue inflammation and altered fuel metabolism that promote adiposity and cardiovascular dysfunction. The proposed research will break new ground by unravelling, at the molecular level, how GPR55 links to pathways responsive to insulin or those mediating inflammation and influencing energy metabolism. Such information may inform the design of novel strategies targeting GPR55 to help ameliorate the decline in insulin sensitivity or metabolic function seen in major public health conditions, such as obesity and diabetes, with the ultimate benefit of improved life quality and reduced healthcare costs. We believe our work will be appeal to other academics with an interest in insulin action, tissue inflammation, fuel/energy metabolism and cardiovascular physiology, as well as those involved in pharmaceutical drug discovery programmes focussing on the ECS. Discoveries, materials and expertise made available to other academics and interested commercial beneficiaries via publications, meetings and Material Transfer Agreements will benefit the UK economic competitiveness in biopharmaceutical products. Appointed staff will profit from institutional initiatives promoting career development and training in public engagement.
What will be done to ensure that they benefit from this research?
Both lead and non-lead institutions are fully committed to maximizing their research impact. This commitment was recognised by the BBSRC "Excellence with Impact" and the first UK Gold Engage Watermark Award by the National Co-ordinating Centre for Public Engagement to the School of Life Sciences (SLS). Impact was also a key measure in REF2014 in which SLS was rated top in biological sciences of any UK University and RGU's UoA3 impact rating was 100% 3*/4*. The applicants have established networks for communicating their research and its benefits via public engagement/outreach activities (e.g. via hosting public visits, Café Science, Royal Society Summer Science Exhibition) and their professional bodies (e.g. Diabetes UK, BHF, British Pharmacological Society and Royal Society of Biology) who interact directly with the public. The impact of our research is publicised on our respective School websites or, where appropriate, through press releases from our Publicity Offices or engagement with our Technology Transfer Offices in matters of Intellectual Property Rights and commercial development.
Academics: Our understanding of how GPR55 affects key anabolic responses in tissues such as skeletal muscle, adipose tissue, liver and heart is very much in its infancy. Consequently, our findings benefit other academic researchers, especially those working in areas related to metabolic and cardiovascular dysfunction (e.g. obesity and diabetes).
Private Sector: Our findings will appeal to pharmaceutical companies with an interest in the endocannabinoid system (ECS), especially with respect to therapies that help maintain/improve tissue response/function, for example, during obesity and Type II diabetes.
Government: The findings may help inform national (e.g. DoH) and international (e.g. Healthy Living matters, WHO) policy on healthy living in relation to maintenance of tissue function.
Public and Charitable Sectors: Individuals working for public health disciplines (e.g. nutritionists/dieticians etc) and scientific advisors to Medical Charities may benefit from the findings by helping to devise appropriate advice to counter diet-induced decline in tissue health, as well as advising their clients of recent advances.
General Public: Target beneficiaries include the public who may lead poor dietary/sedentary life styles and in whom insulin resistance and obesity-related metabolic dysfunction may be an issue.
How will they benefit from this research?
Our studies reveal that metabolically active tissues (e.g. muscle, fat, liver and heart) lacking GPR55 display reduced insulin sensitivity, heightened tissue inflammation and altered fuel metabolism that promote adiposity and cardiovascular dysfunction. The proposed research will break new ground by unravelling, at the molecular level, how GPR55 links to pathways responsive to insulin or those mediating inflammation and influencing energy metabolism. Such information may inform the design of novel strategies targeting GPR55 to help ameliorate the decline in insulin sensitivity or metabolic function seen in major public health conditions, such as obesity and diabetes, with the ultimate benefit of improved life quality and reduced healthcare costs. We believe our work will be appeal to other academics with an interest in insulin action, tissue inflammation, fuel/energy metabolism and cardiovascular physiology, as well as those involved in pharmaceutical drug discovery programmes focussing on the ECS. Discoveries, materials and expertise made available to other academics and interested commercial beneficiaries via publications, meetings and Material Transfer Agreements will benefit the UK economic competitiveness in biopharmaceutical products. Appointed staff will profit from institutional initiatives promoting career development and training in public engagement.
What will be done to ensure that they benefit from this research?
Both lead and non-lead institutions are fully committed to maximizing their research impact. This commitment was recognised by the BBSRC "Excellence with Impact" and the first UK Gold Engage Watermark Award by the National Co-ordinating Centre for Public Engagement to the School of Life Sciences (SLS). Impact was also a key measure in REF2014 in which SLS was rated top in biological sciences of any UK University and RGU's UoA3 impact rating was 100% 3*/4*. The applicants have established networks for communicating their research and its benefits via public engagement/outreach activities (e.g. via hosting public visits, Café Science, Royal Society Summer Science Exhibition) and their professional bodies (e.g. Diabetes UK, BHF, British Pharmacological Society and Royal Society of Biology) who interact directly with the public. The impact of our research is publicised on our respective School websites or, where appropriate, through press releases from our Publicity Offices or engagement with our Technology Transfer Offices in matters of Intellectual Property Rights and commercial development.
Publications
Shah DS
(2020)
Caveolin-3 deficiency associated with the dystrophy P104L mutation impairs skeletal muscle mitochondrial form and function.
in Journal of cachexia, sarcopenia and muscle
Stretton C
(2019)
CDK7 is a component of the integrated stress response regulating SNAT2 (SLC38A2)/System A adaptation in response to cellular amino acid deprivation.
in Biochimica et biophysica acta. Molecular cell research
Lipina C
(2019)
GPR55 deficiency is associated with increased adiposity and impaired insulin signaling in peripheral metabolic tissues.
in FASEB journal : official publication of the Federation of American Societies for Experimental Biology
Nisr RB
(2019)
Proinflammatory NFkB signalling promotes mitochondrial dysfunction in skeletal muscle in response to cellular fuel overloading.
in Cellular and molecular life sciences : CMLS
| Description | The overarching aim of this project is to explore the links between a lipid sensing receptor molecule, called GPR55, with processes influencing adiposity, inflammation, cardiac function and insulin responsiveness in key metabolic tissues including the liver, skeletal muscle, fat tissue. These tissues are major targets for insulin action in the body and represent principal sites where sugar (glucose) and fat are stored and metabolised in response to the hormone. GPR55 is stimulated by a circulating lipid called LPI (lysophosphatidylinositol), which we find improves the responsiveness of these metabolic tissues to insulin. Crucially, this LPI-driven response is lost if cells are co-treated with drugs that inhibit GPR55 function. Strikingly, we have discovered that animals deficient in this lipid sensor exhibit reduced tissue responsiveness to insulin, increased body fat content, impaired metabolic capacity and a decline in cardiac performance. Metabolic capacity is crucially dependent upon mitochondria; structures within cells representing the cell's "energy generator". Significantly, animals lacking GPR55 show changes in mitochondrial biology consistent with a reduced ability to "burn" fat. In line with this, we find animals lacking GPR55 develop obesity and that inhibiting the receptor in cultured adipocytes (fat cells) induces proteins that help make more fat, which would augment the process of obesity. Moreover, our analysis has further revealed that levels of these fat-making molecules such as fatty acid synthase are also increased in liver and heart tissue of mice deficient for GPR55, suggesting that this lipid sensor may play a protective role in preventing the development of fatty liver disease (a condition observed in mice lacking GPR55) as well as blocking the build-up of excess fat in heart tissue which can lead to a decline in heart function. We have recently discovered that distinct forms of the lipid LPI can impact differently on the ability of cultured human adipocytes to undergo a process resulting in the formation of mature fat cells. Strikingly, we have discovered that two specific LPI molecules known as LPI 17:1 and LPI 18:1 can potently suppress the maturation of human fat cells and impair their ability to produce and store more fat. We have shown this by measuring levels of fat inside these cells as well as staining them visually for lipids, and in both cases we observe a dramatic reduction in fat accumulation in response to LPI 17:1 and LPI 18:1. Consistent with this, we have shown that this coincides with the ability of these LPI molecules to decrease the abundance of enzymes directly involved in fat production including fatty acid synthase and acetyl CoA carboxylase. Moreover, we have found that incubating cultured human adipocyte cells with a GPR55 inhibitor molecule can mitigate the ability of LPI 17:1 and LPI 18:1 to impair fat cell maturation and fat accumulation thereby supporting a key role for GPR55 in mediating the fat suppressing actions of LPI 17:1 and LPI 18:1. Importantly, we have identified that the activity of a signalling molecule known as ROCK (Rho-associated protein kinase) plays a fundamental role in mediating the fat suppressing actions of LPI 17:1 and LPI 18:1, and that blocking ROCK activity leads to increased fat accumulation in cultured human adipocytes. Crucially these findings raise a potential role for these LPI molecules, acting through GPR55 and ROCK, in the control of human obesity. An additional protein we have also identified which is responsive to LPI 17:1 is STAT3, whose abundance in fat cells is reduced following exposure to the GPR55 activating lipid. Notably, STAT3 is a molecule known to stimulate fat producing capacity in adipocytes, and therefore its repression by LPI 17:1 would facilitate the anti-obesity action of the GPR55 activating lipid. It is also noteworthy that our work has shown that whilst LPI 17:1 acts to suppress fat production in human adipocytes, it does not appear to alter fat breakdown (by a process called lipolysis). To further improve our understanding of these novel findings, we have performed a large-scale screen of proteins (proteomic screen) in cultured human fat cells treated with LPI 17:1 with more than 2000 different protein molecules were detected using this experimental method. This analysis helped us identify a number of proteins molecules whose abundance is altered in response to LPI 17:1 provision in human derived adipocytes. One such example included carnitine palmitoyl transferase 1A (CPT1A) whose abundance was found to be increased in the presence of LPI 17:1. Notably, CPT1A is an enzyme which functions to transport fats to mitochondria where they are metabolised as a fuel source. LPI 17:1 treatment in adipocytes was also found to increase the abundance of the protein Peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1?) which functions to increase the production of mitochondria (energy generating machinery) inside cells. Therefore, by increasing the levels of CPT1A and PGC-1? in adipocytes, we would anticipate that LPI 17:1 may act to increase the fat burning capacity in human adipocytes. Data from our large-scale protein screen has also revealed that LPI 17:1 treatment in adipocytes acts to downregulate protein abundance of an enzyme known as neprilysin which targets other protein molecules leading to their degradation and inactivation. Notably neprilysin has been shown to target and inactivate hormones involved in the modulation of sugar and fat metabolism, as well as those implicated in controlling heart and cardiovascular function. Consequently, neprilysin is thought to play an important role in regulating whole body metabolism as well as blood pressure control. To this end, previous published studies have demonstrated that inhibition of neprilysin can improve insulin action in animal models of insulin resistance. In accordance with these observations made in cultured human adipocytes, we have recently developed a method for detecting and measuring levels of distinct LPI lipid species in human plasma (using a technique called Liquid Chromatography- Mass Spectrometry; LC-MS). Using this state-of-the-art LC-MS based method, we have been able to detect the presence of at least four different LPI molecules in human plasma, namely LPI 16:0, LPI 18:0, LPI 18:1 and LPI 20:4. Moreover, we have been able to demonstrate that the circulating levels of these LPI species are increased in plasma of obese versus lean individuals, thereby suggesting elevated LPI production in response to increased adiposity. As one of the key objectives of our planned study, we have now completed a comprehensive analysis of proteins expressed in fat, skeletal muscle and heart tissues of mice lacking GPR55. Utilising tissue harvested from these animals, samples were processed and used to perform a large scale proteomic screen to help us identify molecules whose abundance is altered in response to GPR55 activation and/or following a loss of GPR55 function. After performing this state-of-the-art analysis, we can reveal a number of previously unknown proteins whose levels are changed in fat tissue of GPR55 deficient mice which fulfil a key modulatory role in adipocyte function and energy metabolism. For example, one particular protein of interest whose levels were found to be markedly decreased in fat tissue of mice lacking GPR55 is Ehd2 (Eps15-homology domain containing protein 2), a molecule whose levels have been reported to be reduced in fat tissue of obese patients, and whose loss is associated with enhanced uptake of fatty acids (the building blocks of fat) and increased lipid droplet size in adipocytes. This raises the intriguing possibility that GPR55 may also act to control the formation and/or size of lipid droplets inside human adipocytes and by doing so would modulate fat storage capacity in these cells. This observation is also consistent with our observations made in cultured human adipocytes treated with LPI 17:1 which show a markedly reduced abundance of lipid droplets as determined by lipid staining. Our proteomic screen of mouse adipose tissue also revealed that the abundance of galectin-3, a key carbohydrate binding protein, is significantly increased in fat tissue of GPR55 deficient mice. Notably, levels of galectin-3 have been previously reported to be elevated in obese individuals concomitant with impaired glucose homeostasis. In addition, galectin-3 has also been shown to promote differentiation (or maturation) of precursor fat cells into mature adipocytes, thereby suggesting that its raised levels may contribute to increased adiposity observed in mice lacking GPR55. Another molecule we have identified which is implicated in the control of adipocyte maturation and fat production is Cathepsin S, whose abundance is similarly increased in fat tissue of mice lacking GPR55. Cathepsin S is an enzyme which acts to cleave certain extracellular proteins and whose circulating levels have been shown to be increased in obese versus lean individuals. Therefore these findings provide a novel insight into how GPR55 may act to promote its anti-obesogenic actions through its ability to modulate the activity and/or abundance of proteins such as Galectin-3 and Cathepsin S in human fat cells. Another key protein of interest we have identified whose abundance is markedly elevated in fat tissue of mice lacking GPR55 is natriuretic peptide receptor 3 (Npr3). Interestingly, previous published work has reported that mice which lack Npr3 specifically in fat tissue are resistant to diet-induced obesity and display improved insulin sensitivity. Importantly, we have recently discovered that incubating cultured human adipocytes with ML-184, a synthetic GPR55 activating molecule, leads to a marked reduction in Npr3 protein abundance. Moreover, this ML-184 mediated reduction in Npr3 is prevented by co-treatment with the GPR55 inhibiting molecule ML-191. These findings in mice and in cultured human adipocytes have allowed to us to identify a novel function for GPR55 in the regulation of Npr3, a protein which is also known to play a critical role in mediating the cardioregulatory actions of circulating natriuretic peptide hormones. In addition to work performed in mouse adipose tissue and human derived fat cells, we have also explored the metabolic and functional responses that altering GPR55 activity may convey in skeletal muscle and heart tissue. Notably, our analysis has revealed that the protein abundance of GPR55 is significantly reduced in skeletal (gastrocnemious) muscle tissue following high fat feeding. To this end, we have performed a large-scale screen of proteins expressed in skeletal muscle and heart tissue of normal mice fed either a normal or high fat diet, and compared this with their abundance in the corresponding tissues of mice which lack GPR55. Among a number of proteins altered in heart tissue of GPR55 deficient mice that we identified included decreased abundance of molecules that are involved in controlling the energy producing function of mitochondria such as cytochrome c oxidase 6C, cytochrome c oxidase 7C, and ATP8 (mitochondrially encoded ATP synthase membrane subunit 8). Similar comparative analysis in skeletal muscle of GPR55 deficient mice revealed further reductions in the levels of proteins implicated in mitochondrial respiratory activity and energy production including isocitrate dehydrogenase, ATPAF2 (ATP synthase mitochondrial F1 complex assembly factor 2), ATP synthase and NADH-ubiquinone oxidoreductase. A substantial reduction is also observed in the GPR55 deficient muscle levels of MICU3 (mitochondrial calcium uptake family member 3), a molecule which is involved in facilitating the uptake of calcium into mitochondria, helping to maintain their optimal energy producing activity. Notably, it has been previously reported that downregulation of MICU3 in muscle cells is associated with decreased myogenesis (formation of muscular fibres). Moreover, decreased MICU3 abundance has also been observed in skeletal muscle of aged mice. In addition, we have also discovered that gastrocnemius muscle tissue obtained from mice treated with the GPR55 activating molecule ML-184 exhibits a marked upregulation in the abundance of Ndufb7 (NADH:ubiquinone oxidoreductase subunit B7), an accessory subunit of the mitochondrial membrane respiratory chain enzyme NADH dehydrogenase (ubiquinone) implicated in cellular energy production. Notably, muscle tissue Ndufb7 abundance was not significantly altered by ML-184 treatment in GPR55 deficient mice. Given these notable changes in proteins implicated in the proper functioning of mitochondria in response to GPR55 activation or loss, we also explored the possibility that GPR55 may itself reside within these energy producing structures. Strikingly, we were able to detect the presence of GPR55 protein in isolated mitochondrial fractions derived from human skeletal muscle cells. This intriguing finding suggests that this receptor molecule may exert at least some of its metabolic actions at sites present at the mitochondria themselves. Strikingly, our initial observations also indicate that incubating muscle cells with LPI may increase the abundance of mitochondria present as evidenced by an increase in the levels of the mitochondrial marker protein TOMM20 following LPI treatment. These exciting findings provide a novel insight into how LPI interacting with GPR55 within mitochondria may act to control cellular energetics and metabolism, and provide an important platform for future studies examining links between mitochondrial function and GPR55. In addition to altering the levels of proteins implicated in energy production, we further report that inhibiting GPR55 using a pharmacological drug in human-derived cultured muscle cells leads to a significant reduction in cellular respiration. Respiration is the process by which organisms combine oxygen with nutrients and fuel substrates such as glucose to generate the cellular energy molecule ATP (adenosine triphosphate), a key molecule which is required to sustain proper cell functions, including the synthesis of new proteins. Allied to this, we have been able to demonstrate that inhibiting GPR55 also suppresses the synthesis of new proteins in muscle cells, which would be anticipated to negatively impact upon muscle growth and development. Allied to the repressive actions of GPR55 inhibition upon mitochondrial respiration, we have also obtained evidence that inhibition of GPR55 function is associated with increased mitophagy - a process which involves removing damaged or defective mitochondria from cells. Intriguingly, we have been able to demonstrate for the first time that levels of the protein known as FUNDC1, a key mitophagy signalling molecule, become markedly increased in skeletal muscle tissue of mice lacking GPR55 as well as in muscle tissue of normal mice fed a high fat diet. To support this finding, we have also been able to show that inhibiting GPR55 by pharmacological blockade similarly increases protein abundance of FUNDC1, and that of another mitophagy marker called PINK1 (PTEN induced kinase 1), in cultured human skeletal muscle cells. Intriguingly, GPR55 inhibition was associated with enhanced activation of the protein kinase enzyme ULK1 which itself has been reported to activate FUNDC1. Therefore, our work indicates that inhibition or loss of GPR55 leads to the activation of the ULK1-FUNDC1 mitophagic pathway, potentially as a response to damaged or defective mitochondria. Our proteomic analysis in muscle tissue has also uncovered a substantial reduction in the expression of glycogenin-1 in GPR55 deficient mice, a key protein involved in the synthesis of a large carbohydrate storage molecule known as glycogen derived from sugar. We are currently exploring how changes in the abundance of these proteins impacts upon energy metabolism in these tissues and how they are regulated by altered GPR55 activity. It is noteworthy that we have also demonstrated in skeletal muscle of mice lacking GPR55 that a protein called IRS-1 (insulin receptor substrate 1), which plays a key role in promoting insulin action, undergoes a modification called ubiquitylation which targets it for degradation within cells. Indeed, this finding may help explain our previous observation that IRS-1 protein abundance is reduced in skeletal muscle of mice lacking GPR55, concomitant with their reduced insulin responsiveness. Moreover, recently we have found that the expression of a protein called MEMO1 (mediator of ErbB2-driven cell motility) is significantly reduced in skeletal muscle from mice lacking GPR55. Intriguingly, MEMO1 is known to bind directly with IRS1 to promote insulin signalling and therefore our finding raises the possibility that GPR55 acts to alter the interaction of IRS-1 with MEMO1 to regulate insulin sensitivity in muscle cells. Notably, MEMO1 levels are also reduced in skeletal muscle tissue following high fat feeding in normal mice, thereby indicating a potential role for GPR55 mediated regulation of MEMO-1 in the development of obesity induced insulin resistance. Data retrieved from our large-scale protein screens in skeletal (gastrocnemius) muscle of mice has also uncovered a potential role for GPR55 in the control of skeletal muscle development and function. For example, we have discovered that skeletal muscle tissue in mice lacking GPR55 exhibit significantly reduced levels of the proteins CREG1 (Cellular Repressor of E1A Stimulated Genes 1), creatine kinase, Mlip (Muscle enriched Lamin Interacting Protein) and MyHC15 (Myosin Heavy Chain 15). CREG1 is a protein known to improve the capacity of skeletal muscle to respond to exercise endurance. Specifically, CREG1 has been shown to be localised at mitochondria where loss-of-function mutations in CREG1 have been reported to alter the activity and morphology of these key energy producing organelles in cultured skeletal muscle cells. Creatine kinase is an enzyme which plays a critical role in controlling energy delivery in skeletal muscle, whereby its absence in mice causes impaired voluntary running capacity as well as adverse functional, metabolic and structural muscle alterations. Muscle Enriched Lamin Interacting Protein (Mlip) functions as a transcriptional co-factor wherein previous work has demonstrated that inhibiting Mlip in cultured muscle cells blocks the function of myogenic regulatory factors such as MyoD (myoblast determination protein 1) and significantly inhibits myogenic differentiation. MyHC15 (Myosin Heavy Chain 15) is a member of a family of proteins which function to convert chemical energy inside cells into a mechanical force thereby playing a critical role in muscle function. In addition to these altered proteins, we have also discovered that administering mice with the GPR55 activator molecule ML-184 causes a marked increase in the abundance of tetranectin, a molecule which has been shown to be induced during skeletal muscle differentiation (mature muscle fibre formation) and which may also have a potential role in myogenesis and muscle regeneration. In addition, we have found that ML-184 treatment in mice increases the abundance of KLHL20 (Kelch like family member 20), a member of the Kelch family of proteins which have been implicated in skeletal muscle development. Notably, the ability of ML-184 to increase levels of tetranectin and KLHL20 proteins in muscle tissue is lost in mice lacking GPR55. Our proteomic analysis has also revealed that feeding mice lacking GPR55 a high fat diet leads to a marked reduction in the protein abundance of IGFBP5 (Insulin like growth factor binding protein 5) in skeletal muscle. However, this diet-induced loss of IGFBP5 is not observed in muscle tissue of control mice expressing GPR55. Importantly, IGFBP5 has been reported to facilitate muscle differentiation and maturation and its loss, alongside that of CREG1, creatine kinase, KLHL20 and Mlip, would be anticipated to convey an adverse effect upon muscle development, function and/or regenerative capacity. This is particularly important given the critical role that skeletal muscle tissue plays as the major site of glucose uptake in the body, as well as its other important physiological functions. Consequently, we are currently assessing whether GPR55 ligands act to alter the differentiation of cultured murine and human derived muscle cells (using myogenic marker molecules), alongside monitoring any changes in the abundance of the fore-mentioned muscle expressing proteins identified in our large-scale protein screens. Other notable proteins which are significantly altered in muscle tissue of mice lacking GPR55 include DIAPH1, KIF5B, and MAP2K1. Our data shows that MAP2K1 (also known as MEK1) is increased in skeletal muscle of GPR55 deficient mice, as well as in response to high fat feeding in normal mice. Importantly, MAP2K1 has been implicated in the development of insulin resistance in skeletal muscle cells derived from type II diabetic patients. In addition, levels of DIAPH1 protein were also found to be markedly increased in GPR55 deficient skeletal muscle tissue, similar to elevated levels of DIAPH1 observed in muscle tissue of normal mice fed a high fat diet. DIAPH1 is a key component of the signalling pathway activated by advanced glycation end products (AGEs) which along with their molecular target known as the receptor for AGE (RAGE) show increased levels in association with insulin resistance in human obesity. In addition, our proteomic analysis also revealed that the abundance of a protein known as KIF5B is significantly reduced in muscle tissue of mice lacking GPR55. Interestingly, KIF5B is implicated in controlling the movement of glucose transporter molecules in response to insulin, as well as key cellular organelles such as nuclei and mitochondria during muscle differentiation (maturation). Moreover, loss of KIF5B has been associated with skeletal muscle dysfunction in mice. In addition, our data also shows that KIF5B protein abundance is significantly reduced in skeletal muscle of normal mice following consumption of a high fat diet. Collectively, these findings implicate a potential role for GPR55 dependent signalling in regulating MAP2K1, DIAPH1 and KIF5B levels to modulate insulin sensitivity and skeletal muscle function. It is possible that some of the effects elicited by GPR55 stimulation or loss upon metabolic function may be linked to inflammatory status. To this end, our data indicates that GPR55 and its ligands may act to modulate tissue inflammation, whereby heightened inflammation is known to adversely impact upon energy homeostasis and metabolism. For example, our findings reveal that the abundance of the protein I?B? (NF-Kappa-B Inhibitor Alpha), a molecule which functions to suppress inflammation, is markedly reduced in liver tissue of GPR55 deficient mice, thereby indicating enhanced inflammation in the fatty livers of these animals. In accordance with this, we have discovered that levels of MCP-1 (monocyte chemoattractant protein 1), an inflammatory marker protein implicated in immune cell activation, is concomitantly increased in liver tissue of GPR55 KO mice. It is noteworthy that increased inflammation is a classic signature associated with the development of fatty liver disease. Conversely, we have been able to demonstrate that treatment of human fat cells with the GPR55 activating lipid LPI 17:1 increases levels of CRIP2 (Cysteine Rich Protein 2) - a molecule known to suppress inflammation. Strikingly, we have also recently discovered that GPR55 stimulation by ML-184 administration in mice leads to a marked downregulation in the fat tissue levels of C-reactive protein (CRP), a key marker of inflammation whose circulating levels have been reported to be increased in obese individuals. In addition, administering ML-184 in mice was similarly found to reduce levels of COMMD8 (COMM domain containing 8), a protein implicated in facilitating the degradation of I?B? and activation of pro-inflammatory signalling. Notably, we find that this ability of ML-184 to decrease CRP or COMMD8 protein levels in epidydimal fat is lost in mice lacking GPR55. To further support a role for GPR55 in the control of inflammation, our protein screen of gastrocnemius muscle tissue samples reveals that feeding GPR55 deficient mice a high fat diet causes a substantial increase in the abundance of Ubxn6 (Ubiquitin regulatory X domain-containing protein 6)(UBXN6) protein. Interestingly, UBXN6 is a protein which has been reported to positively modulate innate immune responses, contributing to increased inflammation. Moreover, our proteomic analysis has also revealed that treating mice with the GPR55 activating drug ML-184 causes the marked upregulation in the abundance of ENTPD2 (ecto-nucleoside triphosphate diphosphohydrolase 2) protein in skeletal muscle tissue. Notably, ENTPD2 protein abundance is not altered by ML-184 treatment in muscle tissue of GPR55 deficient mice. Intriguingly, ENTPD2 functions to hydrolyse extracellular ATP (adenosine triphosphate) which acts as a pro-inflammatory signalling molecule. Moreover, stimulation of GPR55 may further act to convey an anti-inflammatory action by hydrolysing extracellular ATP to produce adenosine, which in turn has anti-inflammatory properties. Together, our findings support a novel role for GPR55 as a suppressor of inflammation in key metabolic tissues and have provided new insight into how this receptor may convey its anti-inflammatory actions. In addition to its anti-inflammatory actions, we have discovered that activation of GPR55 can also suppress the production of damaging oxygen containing molecules known as reactive oxygen species (ROS) which can damage cells and impair their function if present at excess levels. More specifically, we have been able to demonstrate that GPR55 activating molecules can mitigate chemically induced production of ROS in cultured skeletal muscle cells. In addition, superoxide dismutase 2 (SOD2; an antioxidant enzyme which mitigates free radical formation and alleviates mitochondrial damage) was found to be increased in fat cells treated with the GPR55 activating lipid LPI 17:1. Consistent with this, our large scale protein screen has revealed that the abundance of two proteins which function to clear damaging ROS molecules (known as catalase and glutathione peroxidase 3), are decreased in heart tissue of mice lacking GPR55. Therefore, we are continuing to explore whether altering GPR55 activity can impact upon cellular and mitochondrial health by controlling levels of harmful free radicals. In a separate study, we have recently uncovered evidence suggesting that exercise, which is known to improve obesity and insulin resistance, may alter levels of lipids that activate GPR55. Specifically, protein analysis performed in skeletal muscle tissue isolated from aged mice that were subjected to voluntary running exercise revealed an increase in protein levels of an enzyme called phospholipase A2 (cPLA2), which functions to stimulate production of the GPR55 activating ligand LPI inside cells. This exciting observation therefore suggest that physical exercise may act to influence GPR55 function in the body, either through changing the abundance of GPR55 protein or altering levels of GPR55 activating lipids. The overarching aim of this project is to explore the links between a lipid sensing receptor molecule, called GPR55, with processes influencing adiposity, inflammation, cardiac function and insulin responsiveness in key metabolic tissues including the liver, skeletal muscle, fat tissue. These tissues are major targets for insulin action in the body and represent principal sites where sugar (glucose) and fat are stored and metabolised in response to the hormone. GPR55 is stimulated by a circulating lipid called LPI (lysophosphatidylinositol), which we find improves the responsiveness of these metabolic tissues to insulin. Crucially, this LPI-driven response is lost if cells are co-treated with drugs that inhibit GPR55 function. Strikingly, we have discovered that animals deficient in this lipid sensor exhibit reduced tissue responsiveness to insulin, increased body fat content, impaired metabolic capacity and a decline in cardiac performance. Metabolic capacity is crucially dependent upon mitochondria; structures within cells representing the cell's "energy generator". Significantly, animals lacking GPR55 show changes in mitochondrial biology consistent with a reduced ability to "burn" fat. In line with this, we find animals lacking GPR55 develop obesity and that inhibiting the receptor in cultured adipocytes (fat cells) induces proteins that help make more fat, which would augment the process of obesity. Moreover, our analysis has further revealed that levels of these fat-making molecules such as fatty acid synthase are also increased in liver and heart tissue of mice deficient for GPR55, suggesting that this lipid sensor may play a protective role in preventing the development of fatty liver disease (a condition observed in mice lacking GPR55) as well as blocking the build-up of excess fat in heart tissue which can lead to a decline in heart function. We have recently discovered that distinct forms of the lipid LPI can impact differently on the ability of cultured human adipocytes to undergo a process resulting in the formation of mature fat cells. Strikingly, we have discovered that two specific LPI molecules known as LPI 17:1 and LPI 18:1 can potently suppress the maturation of human fat cells and impair their ability to produce and store more fat. We have shown this by measuring levels of fat inside these cells as well as staining them visually for lipids, and in both cases we observe a dramatic reduction in fat accumulation in response to LPI 17:1 and LPI 18:1. Consistent with this, we have shown that this coincides with the ability of these LPI molecules to decrease the abundance of enzymes directly involved in fat production including fatty acid synthase and acetyl CoA carboxylase. Moreover, we have found that incubating cultured human adipocyte cells with a GPR55 inhibitor molecule can mitigate the ability of LPI 17:1 and LPI 18:1 to impair fat cell maturation and fat accumulation thereby supporting a key role for GPR55 in mediating the fat suppressing actions of LPI 17:1 and LPI 18:1. Importantly, we have identified that the activity of a signalling molecule known as ROCK (Rho-associated protein kinase) plays a fundamental role in mediating the fat suppressing actions of LPI 17:1 and LPI 18:1, and that blocking ROCK activity leads to increased fat accumulation in cultured human adipocytes. Crucially these findings raise a potential role for these LPI molecules, acting through GPR55 and ROCK, in the control of human obesity. An additional protein we have also identified which is responsive to LPI 17:1 is STAT3, whose abundance in fat cells is reduced following exposure to the GPR55 activating lipid. Notably, STAT3 is a molecule known to stimulate fat producing capacity in adipocytes, and therefore its repression by LPI 17:1 would facilitate the anti-obesity action of the GPR55 activating lipid. It is also noteworthy that our work has shown that whilst LPI 17:1 acts to suppress fat production in human adipocytes, it does not appear to alter fat breakdown (by a process called lipolysis). To further improve our understanding of these novel findings, we have performed a large-scale screen of proteins (proteomic screen) in cultured human fat cells treated with LPI 17:1 with more than 2000 different protein molecules were detected using this experimental method. This analysis helped us identify a number of proteins molecules whose abundance is altered in response to LPI 17:1 provision in human derived adipocytes. One such example included carnitine palmitoyl transferase 1A (CPT1A) whose abundance was found to be increased in the presence of LPI 17:1. Notably, CPT1A is an enzyme which functions to transport fats to mitochondria where they are metabolised as a fuel source. LPI 17:1 treatment in adipocytes was also found to increase the abundance of the protein Peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1?) which functions to increase the production of mitochondria (energy generating machinery) inside cells. Therefore, by increasing the levels of CPT1A and PGC-1? in adipocytes, we would anticipate that LPI 17:1 may act to increase the fat burning capacity in human adipocytes. Data from our large-scale protein screen has also revealed that LPI 17:1 treatment in adipocytes acts to downregulate protein abundance of an enzyme known as neprilysin which targets other protein molecules leading to their degradation and inactivation. Notably neprilysin has been shown to target and inactivate hormones involved in the modulation of sugar and fat metabolism, as well as those implicated in controlling heart and cardiovascular function. Consequently, neprilysin is thought to play an important role in regulating whole body metabolism as well as blood pressure control. To this end, previous published studies have demonstrated that inhibition of neprilysin can improve insulin action in animal models of insulin resistance. In accordance with these observations made in cultured human adipocytes, we have recently developed a method for detecting and measuring levels of distinct LPI lipid species in human plasma (using a technique called Liquid Chromatography- Mass Spectrometry; LC-MS). Using this state-of-the-art LC-MS based method, we have been able to detect the presence of at least four different LPI molecules in human plasma, namely LPI 16:0, LPI 18:0, LPI 18:1 and LPI 20:4. Moreover, we have been able to demonstrate that the circulating levels of these LPI species are increased in plasma of obese versus lean individuals, thereby suggesting elevated LPI production in response to increased adiposity. As one of the key objectives of our planned study, we have now completed a comprehensive analysis of proteins expressed in fat, skeletal muscle and heart tissues of mice lacking GPR55. Utilising tissue harvested from these animals, samples were processed and used to perform a large scale proteomic screen to help us identify molecules whose abundance is altered in response to GPR55 activation and/or following a loss of GPR55 function. After performing this state-of-the-art analysis, we can reveal a number of previously unknown proteins whose levels are changed in fat tissue of GPR55 deficient mice which fulfil a key modulatory role in adipocyte function and energy metabolism. For example, one particular protein of interest whose levels were found to be markedly decreased in fat tissue of mice lacking GPR55 is Ehd2 (Eps15-homology domain containing protein 2), a molecule whose levels have been reported to be reduced in fat tissue of obese patients, and whose loss is associated with enhanced uptake of fatty acids (the building blocks of fat) and increased lipid droplet size in adipocytes. This raises the intriguing possibility that GPR55 may also act to control the formation and/or size of lipid droplets inside human adipocytes and by doing so would modulate fat storage capacity in these cells. This observation is also consistent with our observations made in cultured human adipocytes treated with LPI 17:1 which show a markedly reduced abundance of lipid droplets as determined by lipid staining. Our proteomic screen of mouse adipose tissue also revealed that the abundance of galectin-3, a key carbohydrate binding protein, is significantly increased in fat tissue of GPR55 deficient mice. Notably, levels of galectin-3 have been previously reported to be elevated in obese individuals concomitant with impaired glucose homeostasis. In addition, galectin-3 has also been shown to promote differentiation (or maturation) of precursor fat cells into mature adipocytes, thereby suggesting that its raised levels may contribute to increased adiposity observed in mice lacking GPR55. Another molecule we have identified which is implicated in the control of adipocyte maturation and fat production is Cathepsin S, whose abundance is similarly increased in fat tissue of mice lacking GPR55. Cathepsin S is an enzyme which acts to cleave certain extracellular proteins and whose circulating levels have been shown to be increased in obese versus lean individuals. Therefore these findings provide a novel insight into how GPR55 may act to promote its anti-obesogenic actions through its ability to modulate the activity and/or abundance of proteins such as Galectin-3 and Cathepsin S in human fat cells. Another key protein of interest we have identified whose abundance is markedly elevated in fat tissue of mice lacking GPR55 is natriuretic peptide receptor 3 (Npr3). Interestingly, previous published work has reported that mice which lack Npr3 specifically in fat tissue are resistant to diet-induced obesity and display improved insulin sensitivity. Importantly, we have recently discovered that incubating cultured human adipocytes with ML-184, a synthetic GPR55 activating molecule, leads to a marked reduction in Npr3 protein abundance. Moreover, this ML-184 mediated reduction in Npr3 is prevented by co-treatment with the GPR55 inhibiting molecule ML-191. These findings in mice and in cultured human adipocytes have allowed to us to identify a novel function for GPR55 in the regulation of Npr3, a protein which is also known to play a critical role in mediating the cardioregulatory actions of circulating natriuretic peptide hormones. In addition to work performed in mouse adipose tissue and human derived fat cells, we have also explored the metabolic and functional responses that altering GPR55 activity may convey in skeletal muscle and heart tissue. Notably, our analysis has revealed that the protein abundance of GPR55 is significantly reduced in skeletal (gastrocnemious) muscle tissue following high fat feeding. To this end, we have performed a large-scale screen of proteins expressed in skeletal muscle and heart tissue of normal mice fed either a normal or high fat diet, and compared this with their abundance in the corresponding tissues of mice which lack GPR55. Among a number of proteins altered in heart tissue of GPR55 deficient mice that we identified included decreased abundance of molecules that are involved in controlling the energy producing function of mitochondria such as cytochrome c oxidase 6C, cytochrome c oxidase 7C, and ATP8 (mitochondrially encoded ATP synthase membrane subunit 8). Similar comparative analysis in skeletal muscle of GPR55 deficient mice revealed further reductions in the levels of proteins implicated in mitochondrial respiratory activity and energy production including isocitrate dehydrogenase, ATPAF2 (ATP synthase mitochondrial F1 complex assembly factor 2), ATP synthase and NADH-ubiquinone oxidoreductase. A substantial reduction is also observed in the GPR55 deficient muscle levels of MICU3 (mitochondrial calcium uptake family member 3), a molecule which is involved in facilitating the uptake of calcium into mitochondria, helping to maintain their optimal energy producing activity. Notably, it has been previously reported that downregulation of MICU3 in muscle cells is associated with decreased myogenesis (formation of muscular fibres). Moreover, decreased MICU3 abundance has also been observed in skeletal muscle of aged mice. In addition, we have also discovered that gastrocnemius muscle tissue obtained from mice treated with the GPR55 activating molecule ML-184 exhibits a marked upregulation in the abundance of Ndufb7 (NADH:ubiquinone oxidoreductase subunit B7), an accessory subunit of the mitochondrial membrane respiratory chain enzyme NADH dehydrogenase (ubiquinone) implicated in cellular energy production. Notably, muscle tissue Ndufb7 abundance was not significantly altered by ML-184 treatment in GPR55 deficient mice. Given these notable changes in proteins implicated in the proper functioning of mitochondria in response to GPR55 activation or loss, we also explored the possibility that GPR55 may itself reside within these energy producing structures. Strikingly, we were able to detect the presence of GPR55 protein in isolated mitochondrial fractions derived from human skeletal muscle cells. This intriguing finding suggests that this receptor molecule may exert at least some of its metabolic actions at sites present at the mitochondria themselves. Strikingly, our initial observations also indicate that incubating muscle cells with LPI may increase the abundance of mitochondria present as evidenced by an increase in the levels of the mitochondrial marker protein TOMM20 following LPI treatment. These exciting findings provide a novel insight into how LPI interacting with GPR55 within mitochondria may act to control cellular energetics and metabolism, and provide an important platform for future studies examining links between mitochondrial function and GPR55. In addition to altering the levels of proteins implicated in energy production, we further report that inhibiting GPR55 using a pharmacological drug in human-derived cultured muscle cells leads to a significant reduction in cellular respiration. Respiration is the process by which organisms combine oxygen with nutrients and fuel substrates such as glucose to generate the cellular energy molecule ATP (adenosine triphosphate), a key molecule which is required to sustain proper cell functions, including the synthesis of new proteins. Allied to this, we have been able to demonstrate that inhibiting GPR55 also suppresses the synthesis of new proteins in muscle cells, which would be anticipated to negatively impact upon muscle growth and development. Allied to the repressive actions of GPR55 inhibition upon mitochondrial respiration, we have also obtained evidence that inhibition of GPR55 function is associated with increased mitophagy - a process which involves removing damaged or defective mitochondria from cells. Intriguingly, we have been able to demonstrate for the first time that levels of the protein known as FUNDC1, a key mitophagy signalling molecule, become markedly increased in skeletal muscle tissue of mice lacking GPR55 as well as in muscle tissue of normal mice fed a high fat diet. To support this finding, we have also been able to show that inhibiting GPR55 by pharmacological blockade similarly increases protein abundance of FUNDC1, and that of another mitophagy marker called PINK1 (PTEN induced kinase 1), in cultured human skeletal muscle cells. Intriguingly, GPR55 inhibition was associated with enhanced activation of the protein kinase enzyme ULK1 which itself has been reported to activate FUNDC1. Therefore, our work indicates that inhibition or loss of GPR55 leads to the activation of the ULK1-FUNDC1 mitophagic pathway, potentially as a response to damaged or defective mitochondria. Our proteomic analysis in muscle tissue has also uncovered a substantial reduction in the expression of glycogenin-1 in GPR55 deficient mice, a key protein involved in the synthesis of a large carbohydrate storage molecule known as glycogen derived from sugar. We are currently exploring how changes in the abundance of these proteins impacts upon energy metabolism in these tissues and how they are regulated by altered GPR55 activity. It is noteworthy that we have also demonstrated in skeletal muscle of mice lacking GPR55 that a protein called IRS-1 (insulin receptor substrate 1), which plays a key role in promoting insulin action, undergoes a modification called ubiquitylation which targets it for degradation within cells. Indeed, this finding may help explain our previous observation that IRS-1 protein abundance is reduced in skeletal muscle of mice lacking GPR55, concomitant with their reduced insulin responsiveness. Moreover, recently we have found that the expression of a protein called MEMO1 (mediator of ErbB2-driven cell motility) is significantly reduced in skeletal muscle from mice lacking GPR55. Intriguingly, MEMO1 is known to bind directly with IRS1 to promote insulin signalling and therefore our finding raises the possibility that GPR55 acts to alter the interaction of IRS-1 with MEMO1 to regulate insulin sensitivity in muscle cells. Notably, MEMO1 levels are also reduced in skeletal muscle tissue following high fat feeding in normal mice, thereby indicating a potential role for GPR55 mediated regulation of MEMO-1 in the development of obesity induced insulin resistance. Data retrieved from our large-scale protein screens in skeletal (gastrocnemius) muscle of mice has also uncovered a potential role for GPR55 in the control of skeletal muscle development and function. For example, we have discovered that skeletal muscle tissue in mice lacking GPR55 exhibit significantly reduced levels of the proteins CREG1 (Cellular Repressor of E1A Stimulated Genes 1), creatine kinase, Mlip (Muscle enriched Lamin Interacting Protein) and MyHC15 (Myosin Heavy Chain 15). CREG1 is a protein known to improve the capacity of skeletal muscle to respond to exercise endurance. Specifically, CREG1 has been shown to be localised at mitochondria where loss-of-function mutations in CREG1 have been reported to alter the activity and morphology of these key energy producing organelles in cultured skeletal muscle cells. Creatine kinase is an enzyme which plays a critical role in controlling energy delivery in skeletal muscle, whereby its absence in mice causes impaired voluntary running capacity as well as adverse functional, metabolic and structural muscle alterations. Muscle Enriched Lamin Interacting Protein (Mlip) functions as a transcriptional co-factor wherein previous work has demonstrated that inhibiting Mlip in cultured muscle cells blocks the function of myogenic regulatory factors such as MyoD (myoblast determination protein 1) and significantly inhibits myogenic differentiation. MyHC15 (Myosin Heavy Chain 15) is a member of a family of proteins which function to convert chemical energy inside cells into a mechanical force thereby playing a critical role in muscle function. In addition to these altered proteins, we have also discovered that administering mice with the GPR55 activator molecule ML-184 causes a marked increase in the abundance of tetranectin, a molecule which has been shown to be induced during skeletal muscle differentiation (mature muscle fibre formation) and which may also have a potential role in myogenesis and muscle regeneration. In addition, we have found that ML-184 treatment in mice increases the abundance of KLHL20 (Kelch like family member 20), a member of the Kelch family of proteins which have been implicated in skeletal muscle development. Notably, the ability of ML-184 to increase levels of tetranectin and KLHL20 proteins in muscle tissue is lost in mice lacking GPR55. Our proteomic analysis has also revealed that feeding mice lacking GPR55 a high fat diet leads to a marked reduction in the protein abundance of IGFBP5 (Insulin like growth factor binding protein 5) in skeletal muscle. However, this diet-induced loss of IGFBP5 is not observed in muscle tissue of control mice expressing GPR55. Importantly, IGFBP5 has been reported to facilitate muscle differentiation and maturation and its loss, alongside that of CREG1, creatine kinase, KLHL20 and Mlip, would be anticipated to convey an adverse effect upon muscle development, function and/or regenerative capacity. This is particularly important given the critical role that skeletal muscle tissue plays as the major site of glucose uptake in the body, as well as its other important physiological functions. Consequently, we are currently assessing whether GPR55 ligands act to alter the differentiation of cultured murine and human derived muscle cells (using myogenic marker molecules), alongside monitoring any changes in the abundance of the fore-mentioned muscle expressing proteins identified in our large-scale protein screens. Other notable proteins which are significantly altered in muscle tissue of mice lacking GPR55 include DIAPH1, KIF5B, and MAP2K1. Our data shows that MAP2K1 (also known as MEK1) is increased in skeletal muscle of GPR55 deficient mice, as well as in response to high fat feeding in normal mice. Importantly, MAP2K1 has been implicated in the development of insulin resistance in skeletal muscle cells derived from type II diabetic patients. In addition, levels of DIAPH1 protein were also found to be markedly increased in GPR55 deficient skeletal muscle tissue, similar to elevated levels of DIAPH1 observed in muscle tissue of normal mice fed a high fat diet. DIAPH1 is a key component of the signalling pathway activated by advanced glycation end products (AGEs) which along with their molecular target known as the receptor for AGE (RAGE) show increased levels in association with insulin resistance in human obesity. In addition, our proteomic analysis also revealed that the abundance of a protein known as KIF5B is significantly reduced in muscle tissue of mice lacking GPR55. Interestingly, KIF5B is implicated in controlling the movement of glucose transporter molecules in response to insulin, as well as key cellular organelles such as nuclei and mitochondria during muscle differentiation (maturation). Moreover, loss of KIF5B has been associated with skeletal muscle dysfunction in mice. In addition, our data also shows that KIF5B protein abundance is significantly reduced in skeletal muscle of normal mice following consumption of a high fat diet. Collectively, these findings implicate a potential role for GPR55 dependent signalling in regulating MAP2K1, DIAPH1 and KIF5B levels to modulate insulin sensitivity and skeletal muscle function. It is possible that some of the effects elicited by GPR55 stimulation or loss upon metabolic function may be linked to inflammatory status. To this end, our data indicates that GPR55 and its ligands may act to modulate tissue inflammation, whereby heightened inflammation is known to adversely impact upon energy homeostasis and metabolism. For example, our findings reveal that the abundance of the protein I?B? (NF-Kappa-B Inhibitor Alpha), a molecule which functions to suppress inflammation, is markedly reduced in liver tissue of GPR55 deficient mice, thereby indicating enhanced inflammation in the fatty livers of these animals. In accordance with this, we have discovered that levels of MCP-1 (monocyte chemoattractant protein 1), an inflammatory marker protein implicated in immune cell activation, is concomitantly increased in liver tissue of GPR55 KO mice. It is noteworthy that increased inflammation is a classic signature associated with the development of fatty liver disease. Conversely, we have been able to demonstrate that treatment of human fat cells with the GPR55 activating lipid LPI 17:1 increases levels of CRIP2 (Cysteine Rich Protein 2) - a molecule known to suppress inflammation. Strikingly, we have also recently discovered that GPR55 stimulation by ML-184 administration in mice leads to a marked downregulation in the fat tissue levels of C-reactive protein (CRP), a key marker of inflammation whose circulating levels have been reported to be increased in obese individuals. In addition, administering ML-184 in mice was similarly found to reduce levels of COMMD8 (COMM domain containing 8), a protein implicated in facilitating the degradation of I?B? and activation of pro-inflammatory signalling. Notably, we find that this ability of ML-184 to decrease CRP or COMMD8 protein levels in epidydimal fat is lost in mice lacking GPR55. To further support a role for GPR55 in the control of inflammation, our protein screen of gastrocnemius muscle tissue samples reveals that feeding GPR55 deficient mice a high fat diet causes a substantial increase in the abundance of Ubxn6 (Ubiquitin regulatory X domain-containing protein 6)(UBXN6) protein. Interestingly, UBXN6 is a protein which has been reported to positively modulate innate immune responses, contributing to increased inflammation. Moreover, our proteomic analysis has also revealed that treating mice with the GPR55 activating drug ML-184 causes the marked upregulation in the abundance of ENTPD2 (ecto-nucleoside triphosphate diphosphohydrolase 2) protein in skeletal muscle tissue. Notably, ENTPD2 protein abundance is not altered by ML-184 treatment in muscle tissue of GPR55 deficient mice. Intriguingly, ENTPD2 functions to hydrolyse extracellular ATP (adenosine triphosphate) which acts as a pro-inflammatory signalling molecule. Moreover, stimulation of GPR55 may further act to convey an anti-inflammatory action by hydrolysing extracellular ATP to produce adenosine, which in turn has anti-inflammatory properties. Together, our findings support a novel role for GPR55 as a suppressor of inflammation in key metabolic tissues and have provided new insight into how this receptor may convey its anti-inflammatory actions. In addition to its anti-inflammatory actions, we have discovered that activation of GPR55 can also suppress the production of damaging oxygen containing molecules known as reactive oxygen species (ROS) which can damage cells and impair their function if present at excess levels. More specifically, we have been able to demonstrate that GPR55 activating molecules can mitigate chemically induced production of ROS in cultured skeletal muscle cells. In addition, superoxide dismutase 2 (SOD2; an antioxidant enzyme which mitigates free radical formation and alleviates mitochondrial damage) was found to be increased in fat cells treated with the GPR55 activating lipid LPI 17:1. Consistent with this, our large scale protein screen has revealed that the abundance of two proteins which function to clear damaging ROS molecules (known as catalase and glutathione peroxidase 3), are decreased in heart tissue of mice lacking GPR55. Therefore, we are continuing to explore whether altering GPR55 activity can impact upon cellular and mitochondrial health by controlling levels of harmful free radicals. In a separate study, we have recently uncovered evidence suggesting that exercise, which is known to improve obesity and insulin resistance, may alter levels of lipids that activate GPR55. Specifically, protein analysis performed in skeletal muscle tissue isolated from aged mice that were subjected to voluntary running exercise revealed an increase in protein levels of an enzyme called phospholipase A2 (cPLA2), which functions to stimulate production of the GPR55 activating ligand LPI inside cells. This exciting observation therefore suggest that physical exercise may act to influence GPR55 function in the body, either through changing the abundance of GPR55 protein or altering levels of GPR55 activating lipids. |
| Exploitation Route | We believe that our findings to date indicate that GPR55 may function as a novel metabolic regulator within tissues and suggest that understanding how it regulates insulin action, lipid metabolism and cardiac function may offer new pharmacological opportunities for treatment of metabolic disorders associated with conditions such as obesity and type II diabetes |
| Sectors | Education,Healthcare,Pharmaceuticals and Medical Biotechnology |
| Description | The project which finished in August 2022 revealed novel insights into the different ways that GPR55 and its ligands act to control energy metabolism. As part of this project we conducted large scale protein screening in mouse derived tissues as well as performing state of the art lipid analysis in human plasma samples which has generated extensive novel data sets, particularly given the fact that, to our knowledge, we utilized a drug that had not been previously tested in vivo for this purpose. We intend to include this data in manuscripts being prepared for publication which would provide a valuable information resource for both the academic and biopharmaceutical sectors involved in the study and/or development of treatments of metabolic and obesity related diseases. To this end, we have previously engaged/communicated with researchers from a leading biopharmaceutical company involved in exploring how GPR55 may impact upon metabolic health status. Moreover, our work has also attracted the attention of researchers based within our own multi-disciplinary facility (through internal communications) who have expressed a keen interest in collaborating with us in order to explore how some of our findings may be applied within the field of immunology and immune-related disease. |
| Sector | Education,Pharmaceuticals and Medical Biotechnology |
| Description | C5aR, a novel immunomodulator of metabolic signalling and energy metabolism in skeletal muscle |
| Amount | £274,686 (GBP) |
| Funding ID | 18/0005875 |
| Organisation | Diabetes UK |
| Sector | Charity/Non Profit |
| Country | United Kingdom |
| Start | 02/2019 |
| End | 01/2022 |
| Description | GPR55 its role as a modulator of insulin action and metabolism |
| Organisation | Robert Gordon University |
| Country | United Kingdom |
| Sector | Academic/University |
| PI Contribution | We have partnered with investigators at RGU to provide our knowledge and expertise on insulin signalling and metabolism |
| Collaborator Contribution | Our collaborators are in possession of a GPR55 mouse knock-out model. Tissues from these mice have been made available to us for analysis of protein expression and tissue signalling experiments |
| Impact | We have very recently published an article on the effects that GPR55 deficiency has upon adiposity and insulin signalling in peripheral metabolic tissues. This work has cemented a three way academic collaboration with researchers at the University of Dundee, University of Aberdeen and Robert Gordons University |
| Start Year | 2015 |
| Description | Use of a novel human adipocyte cell line for analysis of the role played by CB2 in metabolic signalling |
| Organisation | University of Ulm |
| Country | Germany |
| Sector | Academic/University |
| PI Contribution | We have entered into a collaboration with Professor Dr Martin Wabitsch at Ulm University who has established a human pre-adipocyte cell line from an infant with Simpson-Golabi-Behmel Syndrome (SGBS). The cell strain exhibits a high capacity for adipose differentiation, resulting in mature fat cells which are biochemically and functionally similar to human adipocytes. It is the only available human preadipocyte cell line and consequently represents an invaluable tool for studies assessing human adipocyte biology, lipid metabolism and energy metabolism. Professor Wabitsch has provided the SGBS human adipocyte cell line to us for use in our BBSRC funded project to interrogate the role that the endocannabinoid system may play in adipocyte physiology. We have now established use of SGBS adipocytes in the Hundal lab and have found that these express the CB2 cannabinoid receptor. Intriguingly, activation of this receptor initiates signalling events within cells that result in the activation of a key energy sensing kinase called AMPK, which is known to influence fuel oxidation in mitochondria and also function as an anti-inflammatory signalling molecule. The Hundal lab hosted a summer 2017 intern student from Singapore Polytechnic who was able to show that this AMPK activation by CB2 receptor activation was critically dependent on inducing nitric oxide (NO) production. inhibiting nitric oxide synthase within SGBS cells attenuated the CB2-dependent activation of AMPK. Precisely how CB2 induces NO production and how this mechanistically linked to AMPK is currently unknown, but the subject of on-going studies that will hopefully delineate the nature of these molecular links. |
| Collaborator Contribution | The utility of SGBS cells as a research tool for studies assessing adipocyte signalling and metabolism requires an extensive characterisation of their responses to hormonal and nutritional cues as well as a thorough understanding of how such responses compare to those of freshly isolated human adipocytes. The Wabitsch lab has performed detailed characterisation of the cells they have provided for use in our studies and we have access to data (some unpublished) that is useful in helping us to design and formulate the experiments we are carrying out in these cells. |
| Impact | No outputs to report as yet as studies are still ongoing. |
| Start Year | 2017 |
| Description | High School Engagement |
| Form Of Engagement Activity | Participation in an activity, workshop or similar |
| Part Of Official Scheme? | No |
| Geographic Reach | Local |
| Primary Audience | Schools |
| Results and Impact | The Hundal lab participated as part of a Divisional Public Engagement event entitled "Incredible Immunology" . This was a school-based engagement activity put on by the Division of Cell Signalling and Immunology based in the School of Life Sciences at the University of Dundee that raised pupil awareness and knowledge around immunology, metabolism, vaccines and well-being. Due to the coronavirus restrictions in place in 2021 the "Incredible Immunology" event shifted focus from in-person engagement to remote. Researchers and students prepared 'resource boxes' full of books, games and other interactive activities for pupils to use in class, and others filmed and prepared an ELISA practical that teachers could use to explore practical, hands-on science that was relevant to the research programmes being undertaken by investigators within the School of Life Sciences. |
| Year(s) Of Engagement Activity | 2019,2021 |
| Description | Incredible Immunology |
| Form Of Engagement Activity | Participation in an activity, workshop or similar |
| Part Of Official Scheme? | No |
| Geographic Reach | Local |
| Primary Audience | Schools |
| Results and Impact | Incredible Immunology is an event focused on helping school pupils and teachers to learn about the human immune system and the research taking place at the University of Dundee School of Life Sciences. During the event researchers and students from the Division of Cell Signalling and Immunology facilitated interactive activities at 'stands' at Arbroath High for over 120 S1 pupils and science teachers. These stands touched on topics like vaccination, herd immunity, and immune responses. My research was represented in this program of work by demonstrating the role that components of the endocannabinoid system (CB1, CB2 and GPR55) play in regulating whole body energy metabolism and how this might impact on the function of tissues such as skeletal muscle and cells of the immune system. Feedback from pupils from Arbroath High and public members showed an increased interest in science as a topic and increased familiarity with concepts and vocabulary associated with immunology. The School of Life Sciences has since been invited back to repeat the activity, and the school has also visited us on campus to take part in laboratory activities linked to the curriculum. |
| Year(s) Of Engagement Activity | 2019 |
| Description | Pint of Science |
| Form Of Engagement Activity | Participation in an activity, workshop or similar |
| Part Of Official Scheme? | No |
| Geographic Reach | Regional |
| Primary Audience | Public/other audiences |
| Results and Impact | Pint of Science has become an annual worldwide science festival that is run by a non-profit organisation that invites science researchers to share what they do and why they do it with the public over three days in May. The event takes place in casual venues such as pubs and cafes where researchers give a talk and the audience have the opportunity to ask them any questions in relation to their research and to chat with them in an informal setting. Professor Hundal was invited to participate in a "Pint of Science" event that was held in Dundee in May 2019 where he gave a 30 minute presentation entitled "The metabolic price of gluttony". The event was held in a local pub and tickets to his talk were sold out on the night. |
| Year(s) Of Engagement Activity | 2019 |