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

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.

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.

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.