The lab has previously shown that adipsin, a mouse adipocyte-derived serine protease, is secreted into the bloodstream and is markedly deficient in several models of genetic and acquired obesity, suggesting a possible functional role for this protein in the systemic or local regulations of energy balance. The overall goal of the proposed work is two-fold: to understand the role of adipsin in adipose physiology and obesity and to use the adipsin gene as a model to understand how obesity genes regulate the function of other gene promoters. Both of these goals are novel and will increase our understanding of the biochemical and genetic basis of obesity. Adipsin's direct role in the physiology of adipose cells will be approached in vitro by treating fat cells with adipsin and measuring changes in lipogenesis and lipolysis. Since adipsin activates the alternative pathway of complement via its intrinsic Factor D catalytic activity, the ability of various components of the alternative complement pathway to regulate adipose cell physiology will be studied, including complement-derived ligands such as C3a, C5a and Ba. In addition to an in vitro approach, adipsin will be administered acutely and chronically to obese and lean animals to ascertain the role of this protein and the alternative complement pathway in systemic energy balance in vivo. Multiple measures of systemic energy balance and hormonal regulation will be measured. Preliminary pharmacokinetic studies in lean and obese mice support the feasibility of altering adipsin levels in vivo by administration of the pure protein. New data in transgenic mice shows that a rodent obesity gene (db) suppresses adipsin expression through its action (direct or indirect) on the adipsin promoter. Complementary cellular and biochemical approaches will be used to study this pathway of signal transduction and identify the cis- and trans-acting factors involved in the """"""""obesity-response element"""""""". Cultured adipocytes will be treated with serum from lean and obese mice and with various hormones to model the in vivo effects of obesity on the endogenous adipsin gene. By transfection of chimeric constructions and a series of deletions and mutations from the adipsin gene promoter into these cells, sequences involved in a putative """"""""obesity-response element"""""""" will be mapped. A biochemical approach will involve mapping this response element in the adipsin promoter via DNA footprinting (and gelshift) analysis of the adipsin promoter DNA using nuclear extracts from lean and obese animals. The identity of putative """"""""obesity-response elements"""""""" will be critically tested in vivo using point mutagenesis in the adipsin promoter and the construction of transgenic, obese mice. The trans-acting factor(s) binding to this response element and ultimately responsible for aberrant adipsin expression in obesity will be cloned and characterized by biochemical and new molecular genetic techniques.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
5R01DK042539-02
Application #
3243652
Study Section
Physiological Chemistry Study Section (PC)
Project Start
1990-09-01
Project End
1995-08-31
Budget Start
1991-09-01
Budget End
1992-08-31
Support Year
2
Fiscal Year
1991
Total Cost
Indirect Cost
Name
Dana-Farber Cancer Institute
Department
Type
DUNS #
149617367
City
Boston
State
MA
Country
United States
Zip Code
02215
Peraldi, P; Xu, M; Spiegelman, B M (1997) Thiazolidinediones block tumor necrosis factor-alpha-induced inhibition of insulin signaling. J Clin Invest 100:1863-9
Hotamisligil, G S; Arner, P; Atkinson, R L et al. (1997) Differential regulation of the p80 tumor necrosis factor receptor in human obesity and insulin resistance. Diabetes 46:451-5
Hu, E; Liang, P; Spiegelman, B M (1996) AdipoQ is a novel adipose-specific gene dysregulated in obesity. J Biol Chem 271:10697-703
Hotamisligil, G S; Peraldi, P; Budavari, A et al. (1996) IRS-1-mediated inhibition of insulin receptor tyrosine kinase activity in TNF-alpha- and obesity-induced insulin resistance. Science 271:665-8
Hotamisligil, G S; Arner, P; Caro, J F et al. (1995) Increased adipose tissue expression of tumor necrosis factor-alpha in human obesity and insulin resistance. J Clin Invest 95:2409-15
Hotamisligil, G S; Murray, D L; Choy, L N et al. (1994) Tumor necrosis factor alpha inhibits signaling from the insulin receptor. Proc Natl Acad Sci U S A 91:4854-8
Hotamisligil, G S; Budavari, A; Murray, D et al. (1994) Reduced tyrosine kinase activity of the insulin receptor in obesity-diabetes. Central role of tumor necrosis factor-alpha. J Clin Invest 94:1543-9
Platt, K A; Claffey, K P; Wilkison, W O et al. (1994) Independent regulation of adipose tissue-specificity and obesity response of the adipsin promoter in transgenic mice. J Biol Chem 269:28558-62
Hotamisligil, G S; Shargill, N S; Spiegelman, B M (1993) Adipose expression of tumor necrosis factor-alpha: direct role in obesity-linked insulin resistance. Science 259:87-91
White, R T; Damm, D; Hancock, N et al. (1992) Human adipsin is identical to complement factor D and is expressed at high levels in adipose tissue. J Biol Chem 267:9210-3