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Molecular adaptations to hypoxia in adipose tissue during fat deposition: implications for the endocrine and signalling role of white fat

Reference	BB/C006364/1
Principal Investigator / Supervisor	Professor Paul Trayhurn
Co-Investigators / Co-Supervisors	Dr Ian Wood
Institution	University of Liverpool
Department	Clinical Sciences
Funding type	Research
Value (£)	247,885
Status	Completed
Type	Research Grant
Start date	01/07/2005
End date	30/06/2008
Duration	36 months

Abstract

White adipose tissue, primarily through its endocrine and signalling role, is an important component of the regulation of energy balance. The tissue secretes a wide range of protein factors (adipokines), some of which are direct signals in energy balance regulation, while others are involved in processes such as angiogenesis, haemostatis, insulin sensitivity, and inflammation. Major changes in the production of a number of these adipokines occur with the expansion of adipose tissue mass in the obese state; this has important implications for normal functions such as insulin sensitivity and vascular health. The overall aim of this proposal is to examine the hypothesis that hypoxia may occur during the major accretion of adipose tissue in obesity, and that this leads to the activation of a series of hypoxia-inducible genes in adipocytes through the HIF-1 (hypoxia inducible factor-1) transcription factor. The project focuses on those genes associated with anaerobic metabolism and the inflammatory response, the increased synthesis of inflammation-related proteins evident when adipose tissue mass expands being potentially linked to hypoxia. A combination of studies on adipose tissue from lean and obese rodents and from human subjects, together with cell culture will be used. The cell culture studies will employ mouse 3T3-L1 cells and humans SGBS and hMADS adipocytes; the latter is a new immortalised human stem cell-derived line. The studies will examine gene expression and protein levels in tissues and in adipocytes in culture, and will include adipokine secretion. Hypoxia will be induced in cultured cells chemically and by reducing the oxygen tension. The expression of specific candidate genes will be screened by RT-PCR, while qPCR will be used for the quantitative measurement of mRNA. Commercial microarrays will be employed for assessing global responses to hypoxia, utilising arrays containing hypoxia-sensitive and inflammation-related genes. Proteins will be measuredby western blotting and ELISA. RNAi will be used to determine the effects of HIF-1a knockdown on the expression of hypoxia-sensitive genes to identify those regulated through the HIF-1 transcription. The specific objectives are to: (i) establish whether the HIF-1a gene is expressed, and HIF-1 protein present, in adipose tissues of rodents and when there are quantitative differences between depots; (ii) determine whether the molecular markers of hypoxia (HIF-1a mRNA, HIF-1) are elevated in fat depots of obese animals (ob ob mice, fa fa rats, dietary obese models). The expression of hypoxia-sensitive genes and the encoded proteins will also be measured: physiological fattening, as in late pregnancy, will be examined; (iii) employ 3T3-L1 cells to examine whether a number of genes are hypoxia-sensitive in adipocytes ¿ particularly those linked to glycolysis, inflammation and signalling. Gene expression studies will be paralleled by protein analysis, intracellular and the medium, for non-secreted proteins and adipokines; (iv) examine whether the HIF-1a gene is expressed, and HIX-1 present, in human adipose tissues (subcutaneous, omental) and whether the levels are increased in obese subjects: expressions of hypoxia-sensitive genes and their encoded proteins will be determined in parallel; (v) employ microarrays to examine the more global response to hypoxia in 3T3-L1 adipocytes, to identify genes beyond the candidates investigated in Objective 3; (vi) extend the results from 3T3-L1 adipocytes to human adipocytes, focusing on the candidate genes investigated with the 3T3-L1 cells and any relevant new genes identified from the microarray studies; (vii) employ siRNA with 3T3-L1 adipocytes to block HIF-1a mRNA, to examine which hypoxia-sensitive genes are regulated through HIF-1, focusing on key hypoxia-sensitive genes in adipocytes (identified in Objectives 3 and 5) and the level and release of the encoded proteins.

Summary

unavailable

Committee	Closed Committee - Agri-food (AF)
Research Topics	X – not assigned to a current Research Topic
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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