Defining the molecular roles of peripheral CB1 and CB2 cannabinoid receptors in age-induced changes in energy and metabolic homeostasis.

Lead Research Organisation: University of Dundee
Department Name: School of Life Sciences

Abstract

There is immense interest at present in targeting the action of a class of compounds, termed cannabinoids, in the treatment of obesity and metabolic-related disorders. Cannabinoids are present in cannabis, but our bodies naturally create cannabinoid-like chemicals, known as endocannabinoids that lock-on to protein molecules found on the surface of cells called cannabinoid receptors (i.e. CB1 and CB2). During obesity, diabetes and, as we have recently discovered, during ageing the CB1 is notably over-activated by endocannabinoids produced by the body resulting in impaired insulin action and dysregulation of energy balance in metabolically important tissues such as muscle, fat and liver. CB1 over-activation has been linked strongly with development of insulin resistance and increased adiposity. In contrast, emerging data indicates that CB2 may confer a protective physiological effect. In line with this idea, we find that CB2 inhibitors (antagonists) augment the insulin-desensitising effects of the endocannabinoid, anandamide (AEA) in muscle cells, whereas CB2 activators (agonists) ameliorate the loss in insulin signalling. Moreover, it is noteworthy that whilst CB1 expression is enhanced in ageing muscle that of CB2 is significantly decreased, consistent with the reduced insulin sensitivity that prevails in ageing skeletal muscle.

Intriguingly, CB1 antagonists (e.g. rimonabant) promote glucose tolerance, stimulate energy expenditure and reduce body weight in obese animals by mechanisms independent of their appetite-reducing effect. Consistent with such findings, we find rimonabant ameliorates age-related tissue insulin resistance and fat mass gain in older animals. Precisely how CB1 antagonism elicits these beneficial effects is unclear, but our recent work indicates that CB1 blockade induces activation of AMPK - a molecule that not only senses cellular energy but promotes the breakdown and burning of fat in mitochondria (the cell's energy factory). The molecular events linking CB1 blockade to AMPK activation and the effect of the latter on enzymes involved in fat breakdown/burning and mitochondrial dynamics remain poorly understood. The proposed studies will utilise cultured muscle and fat cells to understand how CB1 inhibition or CB2 activation impacts upon molecules implicated in insulin action, energy balance and mitochondrial function and integrity. Our molecular analyses will involve biochemical and state-of-the-art imaging techniques for visualising mitochondrial staining in muscle and fat cells. These cell-based studies will be complemented with analysis of tissues from young and old mice genetically deficient in CB2 or experiments in young and aged mice administered a pharmacologically active dose of a CB1 antagonist or CB2 agonist for two weeks. During this period we will monitor food intake, glucose tolerance, energy expenditure, physical activity and fat mass before sampling blood/tissue for experimental analyses designed to dissect the mechanisms by which CB1 blockade or CB2 activation improves the metabolic status of aged animals. We also aim to test the effects of exercise in aged animals given that physical activity is known to help sustain tissue sensitivity to anabolic hormones such as insulin and preserve tissue functionality during aging. These studies will help unveil whether exercise curtails age-related changes in tissue CB1 and CB2 expression and, if so, whether these correlate favourably with measures of whole body energetics (i.e. body fat, glucose tolerance and energy expenditure).

The proposed research will specifically expand our fundamental understanding of how modulating peripheral CB receptor activity influences energy balance and insulin action. The findings that will emerge will advance our knowledge of these key issues and prove invaluable in designing therapies that selectively target the peripheral ECS for treatment of age and obesity-related metabolic disorders.

Technical Summary

There is growing appreciation that sustained over-activation of the peripheral type I cannabinoid receptor (CB1) contributes significantly to the development of insulin resistance and promotes disturbances in both energy and metabolic homeostasis in tissues such as muscle, liver and adipose tissue. Reduced insulin sensitivity and increased metabolic dysfunction are prominent features of ageing tissue and we have discovered that expression of CB1 is significantly enhanced in aging muscle, liver and fat. Administration of a selective CB1 antagonist (rimonabant) to aged animals not only induces beneficial effects upon tissue insulin signalling and energy balance, but reduces fat mass by mechanisms that are, as yet, poorly understood. Intriguingly, unlike CB1, expression of CB2 is significantly reduced in aging tissue consistent with emerging data suggesting it may confer a protective insulin-sensitising function. Remarkably, we reveal that both CB1 antagonism and CB2 agonism invoke AMPK activation and that this is likely to support greater fat oxidation via enhanced PGC1alpha-induced mitochondrial biogenesis/respiratory capacity, thereby promoting favourable gains in insulin sensitivity and metabolic function within peripheral tissues. The fact that CB1 inhibition suppresses expression of proadipogenic genes (FAS, SREBP-1, PPAR-gamma2) whilst inducing that of the lipolytic enzyme, ATGL in aged animals strongly supports this proposition. This proposal will investigate how CB1 blockade/CB2 stimulation (using peripherally acting receptor antagonists/agonists), activate AMPK and how this impacts mechanistically, at the molecular level, on mitochondrial function/energy homeostasis and insulin sensitivity in muscle, fat and liver during aging. We will also explore whether exercise mitigates age-related changes in tissue CB1 and CB2 receptor expression and, if so, whether these correlate with enhanced retention of insulin sensitivity and metabolic function.

Planned Impact

Who will benefit from this research?
Academics: Our understanding of how the peripheral endocannabinoid (EC) system affects key anabolic responses in tissues such skeletal muscle, liver and adipose tissue is still very much in its infancy. Consequently, our findings will have benefits for other academic researchers, especially those working on ageing research, regulation of fuel/energy homeostasis and insulin action.
Private Sector: Our findings will appeal to pharmaceutical companies with an interest in the EC system, especially with respect to therapies that help maintain tissue response/function during ageing.
Government: The findings may help inform policy on healthy ageing and benefits of exercise in relation to maintenance of tissue mass/function at both national (e.g. DH) and international (e.g. EU Healthy Ageing Initiative, WHO) levels.
Public and Charitable Sectors: Individuals working for public health/sports-related disciplines (e.g. physiotherapists, exercise instructors) and scientific advisors to Medical Charities will benefit from the findings in terms of helping to devise appropriate strategies that alleviate age-related decline in tissue health, as well as advising their clients of recent advances.
General Public: Target beneficiaries include the elderly, especially those presenting with significant insulin resistance or sarcopenia.

How will they benefit from this research?
Our pilot studies have identified significant age-related changes in EC receptor activity in tissues such as skeletal muscle, fat and liver that we believe contribute to the pathogenesis of tissue insulin resistance. This reduced anabolic response to insulin will impact on tissue mass/function thus contributing to increased frailty, reduced life quality and increased healthcare costs. Our work indicates that drugs countering age-induced changes in EC receptor function will not only ameliorate loss in peripheral tissue insulin sensitivity but promote loss of adiposity. Our research will also address whether age-related changes in EC receptor expression and the associated metabolic sequalae can be mitigated by implementing a physical activity program. Such work will be appeal to other academics with an interest in ageing research and those in the pharmaceutical sector with research programmes targeting the EC system. Ultimately, the work will be of particular benefit to the elderly in terms of counteracting age-related changes in tissue mass/function, thereby improving health/quality of life and reducing overall healthcare costs. The discoveries, materials and expertise will be made available to other academics and interested commercial beneficiaries through publications, meetings and Material Transfer Agreements, which may benefit the UK economic competitiveness in biopharmaceutical products. Appointed staff will benefit 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 by way of the 2011 BBSRC Excellence with Impact Award to the College of Life Sciences (CLS). Impact was also a key measure in REF2014, and CLS was recently rated best in biological sciences of any UK University. 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) as well as professional bodies that they are members of (e.g. Diabetes UK and Royal Society of Edinburgh) who interact directly with the public. The impact of our research is publicised on our respective College websites or, where appropriate, through press releases from our Publicity Offices or engagement with our Technology Transfer Offices in matters concerning Intellectual Property Rights and commercial development.

Publications

10 25 50

publication icon
Lipina C (2017) Lipid modulation of skeletal muscle mass and function. in Journal of cachexia, sarcopenia and muscle

publication icon
Lipina C (2016) Is REDD1 a Metabolic Éminence Grise? in Trends in endocrinology and metabolism: TEM

publication icon
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

 
Description There is considerable interest in the clinical potential of a class of compounds, termed cannabinoids, for the treatment of obesity and diabetes. Cannabinoids were originally identified in the cannabis sativa plant but the mammalian body also produces cannabinoid-like molecules, termed endocannabinoids, which exert their effects by "locking onto" specific binding molecules, known as cannabinoid (CB1 and CB2) receptors, present in the membrane of cells. Two well characterised endocannabinoids are anandamide (AEA) and 2-arachidonoylglycerol (2-AG), which serve as activators for the CB1 and CB2 cannabinoid receptors, respectively. The focus of this project has been to understand how the action of AEA and 2-AG at these receptors impacts upon cellular responses that may be of relevance in the development or prevention of metabolic dysfunction as seen in tissues, for example, in diabetes and during the course of aging.

A key observation over the past year has been our finding that AEA and 2-AG can promote anti-inflammatory effects in muscle cells. Chronic low grade inflammation is a feature of obesity and a risk factor implicated in the pathogenesis of insulin resistance. Previous studies from the Hundal lab have shown that sustained exposure of muscle cells to saturated fatty acids, such as palmitate, as commonly seen during obesity induces signalling events promoting a heightened pro-inflammatory state (characterised by increased expression of genes encoding proinflammatory cytokines, e.g. IL-6, CINC-1 and COX-2). Strikingly, we have discovered that coincubation of muscle cells with palmitate and AEA or 2-AG helps to antagonise the proinflammatory drive initiated by palmitate as evidenced by a reduction in proinflammatory gene expression. It is important to stress that in the absence of palmitate, neither AEA nor 2-AG has any capacity to modify proinflammatory gene expression when incubated with myotubes for up to 24h.

It is plausible that the anti-inflammatory effects we observe with AEA and 2-AG stem from their stimulating action at CB1 and CB2. However, we find that palmitate-incubated myotubes treated with AEA or 2-AG in the absence of presence of inhibitors/blockers that act at CB1 or CB2 does not result in any detectable repression in the anti-inflammatory action of either AEA or 2-AG. These latter observations thus imply that it is highly unlikely the observed anti-inflammatory effects of AEA and 2-AG are mediated via CB1 or CB2. Consistent with this proposition, incubating palmitate-treated myotubes with synthetic pharmacological activators such as WIN 55-212 (a mixed CB1/CB2 activator) also fail to replicate the anti-inflammatory action of AEA or 2-AG. Consequently, these findings support the view that AEA and 2-AG may exert their effects by, as yet, undefined interactions with other membrane receptors. In this regard, it is noteworthy that there is some evidence that these endocannabinoids may interact with GPR55; a membrane receptor tentatively tagged as CB3 because of its ability to bind and be activated by plant cannabinoids. Exploring whether inhibitors selectively block GPR55 activity negate the anti-inflammatory action of AEA and 2-AG will help resolve this issue. If they do, the findings raise the intriguing possibility that some of the previously ascribed actions of AEA and 2-AG may in fact arise via CB1 and CB2 independent routes.

Another aspect of this project is to understand the potential mechanism(s) by which CB2 may confer protection against insulin resistance induced by a sustained activation of CB1 in muscle and liver cells. Our studies reveal that CB2 in particular confers a protective metabolic effect since drugs that inhibit CB2 augment the decline in insulin sensitivity triggered by CB1 receptor activation using AEA in muscle. In contrast, CB2 activators ameliorate AEA-induced reduction in insulin signalling by a mechanism that may involve activation of molecule known as AMPK, widely regarded as the cells master energy sensor. The insulin-sensitising potential of CB2 is also seen in liver cells in which expression of an enzyme called glucose 6 phosphatase (whose activity contributes significantly to the increased liver to blood sugar output during insulin resistant disorders such as diabetes) is reduced by CB2 activators. These observations provide yet further support for our assertion that CB1 and CB2 differentially modulate insulin sensitivity and energy-regulating enzymes (i.e. AMPK) and that targeting CB1 for inhibition or CB2 for activation may help mitigate disturbances in insulin action and metabolic dysfunction associated with diabetes and aging.
Exploitation Route We still have a year of funding left on this project and although we have met some of our stated objectives our research is still on-going and so still too early to indicate how we will take the work forward when completed.
Sectors Education,Healthcare,Pharmaceuticals and Medical Biotechnology

URL https://www.harihundal.com/endocannabinoid-system
 
Description Delineating the roles of GPR55 in cellular metabolism and energy homeostasis
Amount £411,233 (GBP)
Funding ID BB/S00033X/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 01/2019 
End 12/2021
 
Description Lipid-induced insulin resistance and metabolic dysfunction: the role of caveolins and cavins
Amount £93,500 (GBP)
Funding ID 15/0005350 
Organisation Diabetes UK 
Sector Charity/Non Profit
Country United Kingdom
Start 10/2016 
End 09/2019
 
Description Nutrient-induced mitochondrial dysfunction and its reciprocal control by inflammatory signalling: implications for skeletal muscle insulin action.
Amount £203,494 (GBP)
Organisation Diabetes UK 
Sector Charity/Non Profit
Country United Kingdom
Start 06/2017 
End 05/2020
 
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 thatGPR55 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 Blairgowrie Primary School, Angus 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Schools
Results and Impact In 2015, my Research Division hosted a public engagement activity called "Incredible Immunology" at Dundee Science Centre. This event involved all members of the division engaging with the attending public and discussing, in lay terms, the basis of their research programme and its relevance to public health. The event was extremely well received and based on the feedback that was received the Division decided to host another Incredible Immunology event but to hold it in a regional community school centre so as to widen access to audiences out with the city of Dundee. In May 2017 the Division held a 2 day public engagement activity in the town of Blairgowrie in Angus that targeted primary school children on day 1 and the local town community on day 2. My lab, and others from my Division, utilised intelligent game play and props that members of Division had created to inform the children and local public of our research methods and findings. The two day activity got local press coverage and the feedback suggested that the attending public thought the event both "informative and enjoyable" and one that they would like see repeated in the future.
Year(s) Of Engagement Activity 2017
 
Description Dundee Science Centre - Incredible Immunology 
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 Members of the Division of Cell Signalling & Immunology (CSI) held a two day event at Dundee Science centre - the first day engaged students from the Tayside region and the second day was a family fun day. Both days were aimed at conveying the relevance of the research being done by members of CSI to the public ranging from infection, immunity and nutritional disorders. Numerous stands with interactive activities were set up to inform how are research matters to the public at large. The event in total had more than 200 visitors and those coming from the schools reported that they found the event to be fun, informative and a great learning experience
Year(s) Of Engagement Activity 2015
URL http://discovery.dundee.ac.uk/portal/en/researchunits/cell-signalling-and-immunology(cf577b0d-98ed-4...
 
Description Edinburgh International Science Festival 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Public/other audiences
Results and Impact I attended and presented in a session called 'Ask a Researcher' at the opening night party of the Edinburgh International Science Festival in March 2018, which was staged at the city's Art Centre. During the course of the evening over 600 members of the public were welcomed to the opening event and were able to participate in numerous hands on activities that had been arranged by the organisers and various vendors/participants. I met a significant number of the visiting public spanning the full age spectrum from young children to individuals in their nineties. Appropriately, I was discussing the importance of nutrition, exercise and healthy living during the human life span and fielded numerous questions during the night in relation to healthy and unhealthy living. I was able to utilise tools that my institution had provided (for measurement of muscle grip strength, skin-fold callipers, blood pressure monitors etc.) that allowed me to engage with the visiting public in a meaningful way that also allowed me to discuss my research and its significance to public health issues such as obesity and diabetes. I believe I had a positive impact on individuals that I met based on a shift in their understanding and request for further information.
Year(s) Of Engagement Activity 2018
URL https://edinburghfestival.list.co.uk/festival/edinburgh-international-science-festival/
 
Description Life Sciences - Open Doors Day 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Public/other audiences
Results and Impact This event was held on 7/11/2015 and enables members of the public to look more closely at the outstanding technologies within our research labs that are crucial to our success. The event allows the public to appreciate how biologists, physicists, chemists, clinicians and computing specialists work together to address the most challenging scientific questions and to develop research tools to advance scientific discovery.
We invite the public to tour Dundee's world-class technology research laboratories and discover, through hands on activities, how research science really works.
Year(s) Of Engagement Activity 2015
URL http://www.lifesci.dundee.ac.uk/impact/public-engagement