SSA The impact of phytochemicals on adipose tissue function and obesity

Obesity is a major public health problem caused by a combination of genetic susceptibility, consumption of energy-dense, high fat/sugar foods and altered physical activity. Public health messages have had limited success in combating obesity. Reformulating foods using natural additives which improve metabolic health offers exciting potential to reduce obesity rates. Phytochemicals which are abundant in foods such as blueberries have been shown to counteract the effects of high fat diets in rodent model systems. These findings may explain the strong epidemiological evidence that a diet high in fruit and vegetables is protective against obesity and the metabolic syndrome. The molecular mechanisms by which phytochemical-rich diets improve metabolic health are unclear and need to be elucidated to allow their exploitation in novel food products. We have recently demonstrated that blueberry extract (BB) supplementation completely prevents obesity and its metabolic consequences in C57B/6 mice fed a high fat diet. Critically BB extracts increased energy expenditure and induced markers of white adipose tissue browning. The resulting 'brite' adipocytes increase lipid oxidation and thermogenesis thereby reducing adiposity and improving metabolic health. We therefore propose to delineate the metabolic impact of phytochemicals, especially those in blueberry (BB) extracts, on adipose tissue, using cell culture systems (cell lines and primary cells) and by exploiting the cutting edge 'thermomouse' in vivo model of adipocyte browning.

Firstly, we will assess the effect of phytochemicals on adipocyte differentiation. Immortalised and primary adipocyte cell models (3T3-L1, C3H10T1/2 and isolated stromal cells from inguinal adipose tissue) will be differentiated in the absence and presence of phytochemicals using established protocols. We will measure markers of differentiation (e.g. C/EBPb, C/EBPa, PPARg) and browning/lipid oxidation (e.g. UCP1, CPT1, PGC1a) using qPCR and Western blotting to determine whether phytochemicals can cause newly forming adipocytes to favour a brite, rather than white phenotype. Lipid accumulation will be determined by LipidTox staining. We will also perform similar experiments using fully differentiated cultured adipocytes to assess whether phytochemicals induce the browning of pre-existing white adipocytes. Where gene expression changes suggest increased lipid oxidation and energy dissipation, cells will also be assessed using Seahorse bioanalyser metabolic assays to confirm changes in mitochondrial function and lipid or glucose oxidation. Secondly, we will measure the impact of phytochemicals on adipose tissue inflammation in cell lines and primary cells (in isolation or in co-cultivation with macrophage cell lines). Inflammatory processes will be induced by treatment with LPS or hypoxia and be analysed by measuring expression of inflammatory markers (e.g. TNFa, IL1b, IL6). Mitochondrial function and membrane potential will be assessed using fluorescent reagents (e.g. JC-1, mitotracker red). Thirdly, we will use the 'thermomouse' in vivo model in which a luciferase reporter is expressed under the control of the UCP1 promoter (Jax Labs). Uniquely this mouse model allows determination of UCP1 activation, a key marker of adipocyte browning, in live mice by imaging of luciferase induction. These mice will be fed a high fat diet with or without BB extracts and imaged to determine changes in UCP1 activation. This will allow us to determine in real-time precisely which adipose depots exhibit browning in response to BB extracts and how rapidly this occurs. At the end of the experiment adipose depots will be collected for ex vivo analysis of oxidative gene expression and metabolic function.