Protein tyrosine phosphatase 1B negatively regulates leptin signaling in a hypothalamic cell line Wiweka Kaszubska a, *, H. Douglas Falls a , Verlyn G. Schaefer a , Deanna Haasch a , Leigh Frost b , Paul Hessler b , Paul E. Kroeger b , David W. White c , Michael R. Jirousek a,1 , James M. Trevillyan a a Metabolic Disease Research, Global Pharmaceutical Products Division, Department 47R, Bldg. AP10, Abbott Laboratories, 100 Abbott Park Road, Abott Park, IL 60064-6099, USA b Genomics and Molecular Biology, Global Pharmaceutical Products Division, Abbott Laboratories, Abbott Park, IL 60064, USA c Metabolic Disease Biology, Millennium Pharmaceuticals, Inc., Cambridge, MA 02139, USA Received 17 April 2002; accepted 29 May 2002 Abstract Protein tyrosine phosphatase 1B (PTP1B) has recently been implicated in the regulation of body weight. A surprising phenotype of PTP1B-deficient mice is their resistance to diet-induced obesity. Since leptin is one of the primary hormones involved in the regulation of body weight and energy homeostasis, we investigated whether PTP1B affects leptin receptor (lepR) signaling directly. A mouse hypothalamic cell line, GT1-7, was established as a suitable cell model for the study of leptin signaling. Stimulation of GT1-7 cells by leptin caused tyrosine phosphorylation of endogenous STAT3 and activation of a STAT-dependent luciferase reporter gene. Over-expression of PTP1B in GT1-7 cells resulted in a dose-dependent decrease in endogenous JAK2 and STAT3 tyrosine phosphorylation compared with cells transfected with lepR alone. Consistent with inhibition of JAK  /STAT signaling, PTP1B over-expression caused a dose-dependent decrease in leptin-induced, STAT-dependent luciferase reporter gene activation in GT1-7 cells. Furthermore, over-expression of PTP1B led to a decrease in mRNA accumulation of suppressor-of-cytokine-signalling- 3 (SOCS3) and c -fos , genes that are acutely induced by leptin. Using gene microarray analysis, we confirmed that PTP1B reduces the level of gene expression of SOCS3 and showed that the expression level of other leptin-regulated genes was affected. Genes up- regulated by leptin were decreased in cells over-expressing PTP1B. Conversely, the expression of genes down-regulated by leptin was enhanced by PTP1B over-expression in GT1-7 cells. Our findings indicate that PTP1B is a negative regulator of leptin signaling and suggest that PTP1B inhibitors might be efficacious in the treatment of obesity by increasing leptin sensitivity. # 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Protein  /tyrosine phosphatase 1B; Leptin; JAK2; STAT3; SOCS3; GT1-7 cell line 1. Introduction Leptin is a 16 kDa hormone secreted from adipocytes in proportion to body fat mass (Houseknecht et al., 1998). It acts on its receptor in the hypothalamus to regulate food intake, energy expenditure and neuroen- docrine function. Leptin imparts its effects on energy homeostasis by regulating a complex network of neuro- nal circuitry leading to the up-regulation of anorexigenic peptides and the down-regulation of orexigenic ones, resulting in the reduction of food intake (Schwartz et al., 2000). Lack of functional leptin produces an obese and diabetic phenotype in rodents that can be normalized by the addition of exogenous leptin (Halaas et al., 1995; Pelleymounter et al., 1995; Rentsch et al., 1995). The condition of human obesity, however, does not seem to be generally characterized by leptin-deficiency since obese individuals have elevated plasma leptin levels and are resistant to pharmacological treatment with * Corresponding author. Tel.:  /1-847-938-0497; fax:  /1-847-938- 1674 E-mail address: wiweka.kaszubska@abbott.com (W. Kaszubska). 1 Present address: Pfizer Global Research and Development, La Jolla Laboratories, 10770 Science Center Drive, San Diego, CA 92121- 1187, USA. Molecular and Cellular Endocrinology 195 (2002) 109  /118 www.elsevier.com/locate/mce 0303-7207/02/$ - see front matter # 2002 Elsevier Science Ireland Ltd. All rights reserved. PII:S0303-7207(02)00178-8