The broad, long term objectives of this project are: a) to describe the regulation of long chain fatty acid synthesis in the rat in response to thyroid hormone and carbohydrate feeding; b) to determine the extent to which the changes observed can be accounted for by alterations in the levels of the mRNAs encoding the key lipogenic enzymes; c) to investigate the mechanisms by which these mRNA levels are altered. The synthesis of fatty acids is a crucial metabolic pathway, necessary for the storage of fuel and the provision of subcellular structural components, which is responsive to both dietary and hormonal inputs. Thyroid hormones and dietary carbohydrate are two important regulators, interacting with each other to achieve the level of fat synthesis required for a given environment. Lipogenesis will be measured, following alterations in thyroid hormone status and carbohydrate feeding, by determining the incorporation of 3H20 into fatty acids in vivo. A wide range of tissues will be analysed to see at which sites fatty acid synthesis responds to these stimuli. The kinetics of response will also be examined. Available evidence suggests that both thyroid hormones and diet regulate lipogenesis by increasing the synthesis of the enzymes involved. This will be checked by measuring the levels of the mRNAs encoding acetyl-coA carboxylase fatty acid synthase, malic enzyme and S14 (a protein of unknown function which has been strongly linked to the lipogenic process). The involvement of transcription will be assessed using nuclear run-off assays in vitro. The extent to which these mRNAs are coordinately regulated will be established, and also whether their stimulation by thyroid hormones requires ongoing protein synthesis. Experiments will also be performed with isolated hepatocytes, to confirm the directness of the stimulations and also to further investigate the mechanism. It is expected that a range of tissue specific responses to the two stimuli will be observed. The basis for this tissue specificity will be sought by isolating genomic clones encompassing the region of the acetyl-coA carboxylase and fatty acid synthase genes immediately upstream from the start site of transcription. The binding of nuclear proteins, extracted from responsive and non- responsive tissues, will be assessed using the techniques of gel mobility assays, DNase footprinting and exonuclease III digestion. Simple purification of these proteins will be attempted and their ability to stimulate RNA synthesis in an in vitro transcriptional system assessed.
| Freake, Hedley Christopher; Moon, Yangha Kim (2003) Hormonal and nutritional regulation of lipogenic enzyme mRNA levels in rat primary white and brown adipocytes. J Nutr Sci Vitaminol (Tokyo) 49:40-6 |
| Yeh, W J; Leahy, P; Freake, H C (1993) Regulation of brown adipose tissue lipogenesis by thyroid hormone and the sympathetic nervous system. Am J Physiol 265:E252-8 |
| Blennemann, B; Moon, Y K; Freake, H C (1992) Tissue-specific regulation of fatty acid synthesis by thyroid hormone. Endocrinology 130:637-43 |
