Leptin Receptor Isoforms Expressed in Human Adipose Tissue D. Kielar, J.S.C. Clark, A. Ciechanowicz, G. Kurzawski, T. Sulikowski, and M. Naruszewicz Leptin and its structural gone, Ob, are exclusively expressed in adipose tissue. Leptin is secreted into the blood and is responsible for fat mass regulation via leptin receptors in the hypothalamus. This has been considered the major role of leptin, but leptin receptor isoforms are expressed not only in the brain but also in most other tissues in humans and rodents: heart, placenta, lung, liver, muscle, kidney, pancreas, spleen, thymus, prostate, testes, ovary, small intestine, and colon. This implicates leptin regulation in other systems apart from fat mass regulation, and leptin action has been demonstrated in human fetal development and reproductive development, liver metabolism, hematopoiesis, and insulin secretion. Four splice variants of the leptin receptor have been identified in humans: the long isoform huOb-R and the shorter isoforms B219.1 to B219.3. It is known that the long isoform has full intracellular signaling capacity, and is responsible for anorectic action in the hypothalamus. The roles of the other isoforms are yet to be elucidated. Here, we report the identification by reverse transcriptase-polymerase chain reaction (RT-PCR) of three leptin receptor isoforms coexpressed in human visceral adipose tissue: the long isoform huOb-R and the short isoforms huB219.1 and huB219.3. The possible roles of these isoforms are discussed. Copyright © 1998 by W.B. Saunders Company A DIPOSE TISSUE has recently been discovered to have an endocrine character, and specifically expresses the hor- mone leptin. A well-established role of leptin is in fat mass regulation via hypothalamic control of food intake and thermo- genesis, v3 However, leptin can affect fat mass regulation directly, without involvement of the brain. Recently published data indicate that leptin can directly affect adipocyte metabo- lism: leptin inhibits adipocyte acetyl coenzyme A carboxylase gone expression and activity, 4 suppresses in vitro lipid synthe- sis, 4,5 and counteracts induction of lipid synthesis by glucocorti- colds and insulin. 4 Identification of the possible leptin receptor isoforms involved in adipose tissue is the main purpose of the present study. A second direct effect of leptin has been demonstrated on human hepatoma cells (hepG2) expressing the Ob-R receptor. 6 Exposure of these cells to leptin attenuated several insulin- induced activities, including insulin receptor signaling, and downregulated gluconeogenesis. The investigators suggested a possible role of hyperleptinemia in obesity-associated insulin resistance. There is a third direct effect of leptin on pancreatic [3 cells, as 125I-leptin has been found to bind to [3 insulinoma cells, 7 and Ob-Rb (long isoform of leptin receptor) is expressed in pancreatic islets of ob/ob and wild-type mice. s Moreover, it has been shown that leptin reduces basal and glucose-stimulated insulin secretion in a perfused pancreas preparation from ob/ob mice but not from Zuckerfa/fa rats or db/db mice, 8 which have mutated Ob-Rb receptors. Presumably, the latter two direct effects both result in hyperinsulinemia, hyperglycemia, and diabetes in vivo in the case of leptin resistance. In addition to fat mass regulation, leptin has several impor- tant roles that have already been clarified, and probably others awaiting discovery. Leptin receptors have a wide distribution in humans and rodents, and therefore many peripheral actions, including regulation of hematopoiesis, 9 stimulation of cytokine From the Departments of Clinical Biochemistry, Genetics, and Surgery, Pomeranian Medical Academy, Szczecin, Poland. Submitted September 17, 1997; accepted December 19, 1997. Address reprint requests to M. Naruszewicz, PhD, Department of Clinical Biochemistry, 70-111 Szczecin, Al. Powstar[c6w Wlkp 72. Copyright © 1998 by W.B. Saunders Company 0026-0495/98/4707-0015503.00/0 production and macrophage phagocytosis, 1° and control of the development of reproductive systems.11-13 Taken together, these findings point to the importance of identifying the presence of the different leptin receptor isoforms in different tissues. Elucidation of their function and intracellu- lar signaling, plus mutations or disruptions in these, may enable the prevention of the effects of leptin resistance and its consequences. MATERIALS AND METHODS Preparation of RNA Biopsies of visceral adipose tissue were obtained from female patients in the fasted state undergoing laparoscopic cholecystectomy, with cholelithiasis but no other serious illness, after approval of the protocol by the Pomeranian Medical Academy Ethical Committee. All participants provided written consent after being informed of the nature, purpose, and possible risks of the study. Columns from a Total RNA Isolation kit from A and A Biotechnology (Gdansk, Poland) were used to isolate RNA from this tissue. About 0.5 g frozen tissue was first pulverized in Fenozol solution (1 mL, from the kit) and homogenized using a glass cylinder homogenizer for 3 minutes, and then the manufacturer's instructions for the kit were followed (modified from Chomczynski and Sacchi14). Solutions of RNA were quantified spectrophotometrically at 260 nm, and the integrity of the RNA was verified using the absorption ratio at 260/280 nm and agarose gels. Solutions of RNA were suspended in water and stored at - 80°C. Leukocytes were isolated from fresh blood samples (10 mL each) from two overweight female patients, using an osmotic gradient method with Gradisol G (Polfa, Kutno, Poland) and the manufacturer's protocol. RNA was isolated exactly following the method of Chomczyn- ski and Sacchi. 14 Primers The original terminology for the leptin receptor isoforms 9 is used here, but it should be noted that huOb-R is homologous to muOb-Rb and huB219.3 is homologous to muOb-Ra. (The human isoforms are sometimes referred to as huOb-Rb and huOb-Ra, respectively1°). A set of primers (Table 1) for the Ob receptor isoforms were designed using the software PrimerSelect (DNASTAR, Madison, USA). Four reverse primers, B1R, B2R, B3R, and Ob-RR, were designed--one for each of the receptor isoforms already identified 9 plus another reverse primer, BOR, designed to produce a product with any B219/Ob-R transcript. All reverse primers were theoretically compatible with the forward primer, B3E 844 Metabolism, Vol 47, No 7 (July), 1998: pp 844-847