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Investigating the role of CART in energy balance using novel genetic techniques

Reference	BB/C515398/1
Principal Investigator / Supervisor	Professor Kevin Murphy
Co-Investigators / Co-Supervisors
Institution	Imperial College London
Department	Div of Investigative Science
Funding type	Research
Value (£)	245,321
Status	Completed
Type	Research Grant
Start date	06/06/2005
End date	05/06/2008
Duration	36 months

Abstract

The hypothalamus is the central nervous system region crucial for the regulation of energy balance. Cocaine- and amphetamine-regulated transcript (CART) codes for a neuropeptide secreted by a key hypothalamic neurone in the regulation of energy homeostasis. Two key neuronal populations in the arcuate nucleus (ARC) of the hypothalamus regulate appetite. The first co-expresses Neuropeptide Y and agouti related peptide and is orexigenic. The second co-expresses CART and pro-opiomelanocortin (POMC) which is processed to the anorectic neuropeptide, alpha-melanocyte stimulating hormone. Initially CART was thought to act primarily as an anorectic neuropeptide in the hypothalamus, based mainly on intracerebroventricular (ICV) injection experiments. However, ICV administration of CART produces profound akinesia suggesting that the decrease in food intake is due to motor disturbances. Significantly, CART dramatically increases food intake when injected into discrete hypothalamic nuclei. Chronically increasing CART signalling in the ARC greatly increases food intake. I hypothesise that ARC CART acts as an auto-inhibitor of POMC neurons and plays an important physiological role in the regulation of energy intake and expenditure. I will use three different genetic models of disrupted CART expression and signalling to establish the physiological importance of CART in energy balance. A) Targeted disruption of CART synthesis in the POMC neurone in transgenic mice. Using BAC will allow the generation of transgenic mice with gene expression targeted only to the POMC neuron. In order to specifically target disruption of CART synthesis to POMC neurones in the ARC, transgenic mice will be created expressing CART antisense mRNA driven by the POMC promoter. This will inhibit CART synthesis specifically in neurons that express POMC. Founder transgenic mice will be bred to F1 mice and characterisation studies performed on these litters. B) Disruption of CART synthesis in discrete hypothalamic nuclei of the adult rat. AAV-antisense CART, AAV-RNAi CART or control AAV-GFP will be stereotactically injected into discrete hypothalamic nuclei (ARC, PVN or VMN) of adult male Wistar rats. Correct location of injection will be confirmed in tissue slices by in situ hybridisation and immunofluoresence techniques prior to characterisation of the effects of disrupted CART synthesis. C) Conditional knockout of CART in transgenic mice. Transgenic mice will be created to allow cre-recombinase mediated deletion of the CART gene in the adult. Recombinant AAV expressing cre-recombinase cDNA (AAV-CRE) will be generated. In order to delete the CART gene, AAV-CRE will be introduced by ICV injection, or by direct intranuclear injection into the ARC, PVN or VMN of floxed mice. In each model, disruption of CART synthesis will be confirmed in vitro and phenotypic changes measured. Localisation of CART and alpha-MSH expression and synthesis, hypothalamic CART release, and the effect on food intake and energy expenditure will be established.

Summary

unavailable

Committee	Closed Committee - Animal Sciences (AS)
Research Topics	Diet and Health, Neuroscience and Behaviour
Research Priority	X – Research Priority information not available
Research Initiative	X - not in an Initiative
Funding Scheme	X – not Funded via a specific Funding Scheme

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