1
AU - Sinha MK
AU - Opentanova I
AU - Ohannesian JP
AU - Kolaczynski JW
AU - Heiman ML
AU - Hale J
AU - Becker GW
AU - Bowsher RR
AU - Stephens TW
AU - Caro JFTI - Evidence of free and bound leptin in human circulation.Studies in lean and obese subjects and during short-term fasting.AB - Little is known about leptin's interaction with other circulating proteins which could be important for its biological effects.Sephadex G-100 gel filtration elution profiles of 125I-leptin-serum complex demonstrated 125I-leptin eluting in significant proportion associated with macromolecules. The 125I-leptin binding to circulating macromolecules was specific, reversible, and displaceable with unlabeled leptin (ED50: 0.73 +/- 0.09 nM, mean +/- SEM, n = 3). Several putative leptin binding proteins were detected by leptin-affinity chromatography of which either 80- or 100-kD proteins could be the soluble leptin receptor as approximately 10%of the bound 125I-leptin was immunoprecipitable with leptin receptor antibodies. Significantly higher (P < 0.001) proportions of total leptin circulate in the bound form in lean (46.5 +/- 6.6%) compared with obese (21.4 +/- 3.4%) subjects.In lean subjects with 21% or less body fat, 60-98% of the total leptin was in the bound form. Short-term fasting significantly decreased basal leptin levels in three lean (P < 0.0005) and three obese (P < 0.005) subjects while refeeding restored it to basal levels. The effects of fasting on free leptin levels were more pronounced in lean subjects (basal vs. 24-h fasting: 19.6 +/- 1.9 vs. 1.3 +/- 0.4 ng/ml) compared with those in obese subjects (28.3 +/- 9.8 vs. 14.7 +/- 5.3). No significant (P > 0.05) decrease was observed in bound leptin in either group. These studies suggest that in obese individuals the majority of leptin circulates in free form, presumably bioactive protein, and thus obese subjects are resistant to free leptin. In lean subjects with relatively low adipose tissue, the majority of circulating leptin is in the bound form and thus may not be available to brain receptors for its inhibitory effects on food intake both under normal and food deprivation states.MH - Carrier Proteins|*AN/*MEMH - Proteins|*AN/*ME/PHSO - J Clin Invest 1996 Sep; 98(6):1277-82DP - 1996 SepTA - J Clin InvestPG - 1277-82IP - 6VI - 98UI - 964205932
AU - Baumann H
AU - Morella KK
AU - White DW
AU - Dembski M
AU - Bailon PS
AU - Kim H
AU - Lai CF
AU - Tartaglia LATI - The full-length leptin receptor has signaling capabilities of interleukin 6-type cytokine receptors.AB - The leptin receptor (OB-R) is a single membrane-spanning protein that mediates the weight regulatory effects of leptin (OB protein). The mutant allele (db) of the OB-R gene encodes a protein with a truncated cytoplasmic domain that is predicted to be functionally inactive. Several mRNA splice variants encoding OB-Rs with different length cytoplasmic domains have been detected in various tissues.Here we demonstrate that the full-length OB-R (predominantly expressed in the hypothalamus), but not a major naturally occurring truncated form or a mutant from found in db/db mice, can mediate activation of signal transducer and activator of transcription (STAT) proteins and stimulate transcription through interleukin 6 responsive gene elements. Reconstitution experiments suggest that, although OB-R mediates intracellular signals with a specificity similar to interleukin 6-type cytokine receptors, signaling appears to be independent of the gp130 signal transducing component of the interleukin 6-type cytokine receptors.MH - Antigens, CD|*PHMH - Carrier Proteins|*PHMH - Receptors, Interleukin|*PHSO - Proc Natl Acad Sci U S A 1996 Aug; 93(16):8374-8DP - 1996 AugTA - Proc Natl Acad Sci U S APG - 8374-8IP - 16VI - 93UI - 963232293
AU - Campfield LA
AU - Smith FJ
AU - Burn PTI - The OB protein (leptin) pathway--a link between adipose tissue mass and central neural networks.AB - OB protein (also known as leptin), a previously unknown protein signal, is secreted from adipose tissue, circulates in the blood, probably bound to a family of binding proteins,and acts on central neural networks that regulate ingestive behavior and energy balance. OB protein provides a communication link from fat tissue and the brain. Rapidly accumulating evidence suggests that OB protein appears to play a major role in the control of body fat stores through coordinated regulation of feeding behavior, metabolism, autonomic nervous system and body energy balance in rodents, primates and humans. The field has rapidly moved from cloning of the ob gene to demonstration of complex regulation of ob gene expression in adipose tissue in rats and humans, and then the demonstration of potent biological activity of OB protein in ob/ob, diet-induced, and lean mice as well as obese and lean rats but not in db/db obese mice. A significant milestone was our demonstration that central administration of OB protein lead to reductions in food intake, body weight and alterations in metabolism consistent with activation of the autonomic nervous system. These findings were followed by the identification of a central binding site for labelled OB protein in the choroid plexus in ob/ob, db/db and lean mice as well as lean and obese Zucker rats. The expression cloning of a central receptor, OB-R, from the mouse choroid plexus soon followed. The OB-R receptor was found to be expressed in the choroid plexus, the hypothalamus as well as several peripheral tissues. OB-R exists in multiple forms; the two major forms are a short form (with a truncated intracellular domain) and long form (with the complete intracellular domain). The long form is thought to be the form that signals and mediates the biological effects of OB protein. Initial in situ hybridization studies have demonstrated the mRNA for the long form OB-R receptor to be localized to the hypothalamus as well as peripheral sites. Recently, it was demonstrated that the db gene encodes the OB-R receptor. Evidence has been provided for a specific transport system for OB protein to cross the blood-brain-barrier and enter the brain of mice, rats and humans. The rate of transport can be decreased by high plasma concentrations of OB protein. Thus, reduced entry of OB protein to the brain may be one of the mechanisms of reduced sensitivity of the OB protein pathway in obese individuals. OB protein appears to also play a role in the important neuroendocrine adaptive responses to fasting and in the control of reproduction.Therapeutic approaches to the treatment of obesity based on OB protein ranging from OB protein by injection to OB-R receptor agonists and to upregulation of OB signalling pathways are under intense investigation.MH - Adipose Tissue|*MH - Neural Pathways|*MH - Obesity|*/ET/THMH - Proteins|GE/*PHSO - Horm Metab Res 1996 Dec; 28(12):619-32DP - 1996 DecTA - Horm Metab ResPG - 619-32IP - 12VI - 28UI - 971658384
AU - Mercer JG
AU - Hoggard N
AU - Williams LM
AU - Lawrence CB
AU - Hannah LT
AU - Morgan PJ
AU - Trayhurn PTI - Coexpression of leptin receptor and preproneuropeptide Y mRNA in arcuate nucleus of mouse hypothalamus.AB - Leptin, the protein product of the adipose tissue-specific ob (obese) gene (1), reduces the body weight, adiposity and food intake of obese ob/ob mice on peripheral or central injection (2, 3, 4). [125I]leptin binding has been detected in mouse choroid plexus (5), from which a leptin receptor gene was expression cloned (5). The gene has at least 6 splice variants (6, 7). Leptin receptor mRNA was localized in the hypothalamus by in situ hybridization being particularly abundantly expressed in the arcuate nucleus (8). There is evidence linking the physiological effects of injected leptin with hypothalamic neuropeptide Y (9, 10) (NPY), which has potent central effects on food intake and energy balance (11), and is also expressed in the arcuate nucleus.Here we report dual in situ hybridization studies for leptin receptor and NPY gene expression in the mouse arcuate nucleus,where the majority of cells examined expressed both genes.This provides the first direct evidence that leptin acts on cells that express NPY mRNA.MH - Arcuate Nucleus|*ME/ULMH - Carrier Proteins|*BI/GEMH - Neuropeptide Y|*BI/GEMH - Protein Precursors|*BI/GEMH - RNA, Messenger|GE/*MESO - J Neuroendocrinol 1996 Oct; 8(10):733-5DP - 1996 OctTA - J NeuroendocrinolPG - 733-5IP - 10VI - 8UI - 970673885
AU - Vaisse C
AU - Halaas JL
AU - Horvath CM
AU - Darnell JE Jr
AU - Stoffel M
AU - Friedman JMTI - Leptin activation of Stat3 in the hypothalamus of wild-type and ob/ob mice but not db/db mice.AB - Leptin, a hormone secreted by adipocytes, regulates the size of the adipose tissue mass through effects on satiety and energy metabolism. Leptin's precise sites of action are not known. The leptin receptor (Ob-R) is found in many tissues in several alternatively spliced forms raising the possibility that leptin exerts effects on many tissues including the hypothalamus. Ob-R is a member of the gp130 family of cytokine receptors which are known to stimulate gene transcription via activation of cytosolic STAT proteins.In order to identify the sites of leptin action in vivo,we assayed for activation of STAT proteins in mice treated with leptin. The STAT proteins bind to phosphotyrosine residues in the cytoplasmic domain of the ligand-activated receptor where they are phosphorylated. The activated STAT proteins dimerize and translocate to the nucleus where they bind DNA and activate transcription. The activation of STAT proteins in response to leptin was assayed in a variety of mouse tissues known to express Ob-R. Leptin injection activated Stat3 but no other STAT protein in the hypothalamus of ob/ob and wild-type mice but not db/db mice, mutants that lack an isoform of the leptin receptor.Leptin did not induce STAT activation in any of the other tissues tested. Activation of Stat3 by leptin was dose dependent and first observed after 15 minutes and maximal at 30 minutes. Our data indicate the hypothalamus is a direct target of leptin action and that this activation is critically dependent on the gp-130-like leptin receptor isoform missing in C57BLKS/J db/db mice. This is the first in vivo demonstration of leptin signal transduction.MH - DNA-Binding Proteins|*MEMH - Hypothalamus|*MEMH - Proteins|*PHMH - Trans-Activators|*MESO - Nat Genet 1996 Sep; 14(1):95-7DP - 1996 SepTA - Nat GenetPG - 95-7IP - 1VI - 14UI - 963769786
AU - Imagawa K
AU - Numata Y
AU - Katsuura G
AU - Sakaguchi I
AU - Morita A
AU - Kikuoka S
AU - Matumoto Y
AU - Tsuji T
AU - Tamaki M
AU - Sasakura K
AU - Teraoka H
AU - Hosoda
AU - K
AU - Ogawa Y
AU - Nakao KTI - Structure-function studies of human leptin.AB - To elucidate the structural requirement of human leptin for its functions, the wild-type, mutant-type, C-terminal deletion, and N-terminal deletion were expressed in Escherichia coli and purified in soluble forms. These leptin analogs were intracerebroventrically injected into C57BL/6J ob/ob mice, and their in vivo biological activities were evaluated.The mutant-type leptin lacking a C-terminal disulfide bond reduced food intake at doses of more than 15 pmol/mouse,which was as effective as the wild-type leptin. C-terminal deletion without the loop structure, also significantly,but to a lesser extent, reduced food intake at doses of more than 90 pmol/mouse. However, N-terminal deletions showed no effect on food intake. We also evaluated the effects of the leptin analogs on radiolabeled leptin binding to its receptor in the choroid plexus using autoradiography.An excess of unlabeled mutant-type leptin as well as wild-type leptin led to complete inhibition of binding. C-terminal deletions led to weak inhibitory activity, whereas N-terminal deletions caused no inhibitory activity. These results clearly demonstrate that the N-terminal region of leptin is essential for both its biological and receptor binding activities. The amino acid sequence of the C-terminal loop structure is also important for enhancing these actions,whereas the C-terminal disulfide bond is not needed.MH - Carrier Proteins|*MEMH - Obesity|*MEMH - Proteins|GE/*PDAD - Research and Development Diagnostic Science DivisionAD - Osaka 566-0022AD - Japan.SO - J Biol Chem 1998 Dec 25; 273(52):35245-9DP - 1998 Dec 25TA - J Biol ChemPG - 35245-9IP - 52VI - 273UI - 990743097
AU - da Silva BA
AU - Bjçrbaek C
AU - Uotani S
AU - Flier JSTI - Functional properties of leptin receptor isoforms containing the gln-->pro extracellular domain mutation of the fatty rat [see comments]AB - Mutations of the leptin receptor have been found to cause obesity in rodents. The fa mutation that is responsible for obesity in Zucker rats is a missense mutation (269 gln-->pro) in the extracellular domain of the leptin receptor.We have characterized the effects of this mutation on the two major isoforms of the leptin receptor, Ob-Rb and Ob-Ra, by studying cell-surface expression, leptin binding affinity, signaling capacity, and receptor-mediated internalization and degradation of leptin in transfected mammalian cell lines. Both Ob-Rb(269 gln-->pro) and Ob-Ra(269 gln-->pro)have decreased cell-surface expression and decreased leptin binding affinity. Ob-Rb(269 gln-->pro) was shown to have defective signaling to the JAK-STAT pathway and markedly diminished ability to activate transcription of the egr-1 promoter. Constitutive ligand-independent activation of Ob-Rb(269 gln-->pro) was observed for activation of egr-1-luc but only under conditions when JAK2 was coexpressed with Ob-Rb(269 gln-->pro), Finally, Ob-Ra(269 gln-->pro)has an increased ability to internalize leptin but is less efficient at degrading leptin, as compared with Ob-Ra. In conclusion, both Ob-Ra(269 gln-->pro) and Ob-Rb(269 gln-->pro) have multiple functional defects.MH - Carrier Proteins|*GE/ME/*PHMH - Mutation|*PHMH - Obesity|*GE/*PPMH - Rats, Zucker|*GE/*PHSO - Endocrinology 1998 Sep; 139(9):3681-90DP - 1998 SepTA - EndocrinologyPG - 3681-90IP - 9VI - 139UI - 983894118
AU - Golden PL
AU - Maccagnan TJ
AU - Pardridge WMTI - Human blood-brain barrier leptin receptor. Binding and endocytosis in isolated human brain microvessels.AB - The peripheral production of leptin by adipose tissue and its putative effect as a signal of satiety in the central nervous system suggest that leptin gains access to the regions of the brain regulating energy balance by crossing the brain capillary endothelium, which constitutes the blood-brain barrier in vivo. The present experiments characterize the binding and internalization of mouse recombinant leptin in isolated human brain capillaries, an in vitro model of the human blood-brain barrier. Incubation of 125I-leptin with isolated human brain capillaries resulted in temperature-dependent binding: at 37 degrees C, approximately 65% of radiolabeled leptin was bound per milligram of capillary protein. Two-thirds of the bound radioactivity was resistant to removal by acid wash, demonstrating endocytosis of 125I-leptin into capillary cells. At 4 degrees C, binding to isolated capillaries was reduced to approximately 23%/mg of protein, the majority of which was acid wash resistant.Binding of 125I-leptin to brain capillary endothelial plasma membranes was saturable, described by a two-site binding model with a high-affinity dissociation constant of 5.1+/-2.8 nM and maximal binding capacity of 0.34+/-0.16 pmol/mg of membrane protein. Addition of porcine insulin or insulin-like growth factor at a final concentration of 100 nM had a negligible effect on leptin binding. These results provide evidence for a leptin receptor that mediates saturable, specific, temperature-dependent binding and endocytosis of leptin at the human blood-brain barrier.MH - Blood-Brain Barrier|*PHMH - Carrier Proteins|*PHMH - Endocytosis|*SO - J Clin Invest 1997 Jan; 99(1):14-8DP - 1997 JanTA - J Clin InvestPG - 14-8IP - 1VI - 99UI - 971487659
AU - Kieffer TJ
AU - Heller RS
AU - Habener JFTI - Leptin receptors expressed on pancreatic beta-cells.AB - Leptin (Ob protein) is a recently isolated hormone produced by adipocytes and is a powerful regulator of satiety centers in the brain. A defect in either leptin production or transmission of the leptin signal in animal models, i.e. ob/ob and db/db mice, respectively, results in a syndrome of obesity and diabetes which closely resembles that which occurs in humans. Leptin release is regulated in part by nutritional status and its expression in adipose tissue is up-regulated by insulin. Since hyperinsulinemia is a primary defect in ob/ob and db/db mice which manifests early in the disease,we postulated that leptin may also regulate insulin release as part of a "adipoinsular' feedback loop. We demonstrate the expression of leptin receptor mRNA in primary rat pancreatic islets and in the insulinoma cell line beta TC-3. Furthermore,we find binding of 125I-leptin to beta TC-3 cells which is significantly displaced by leptin. These findings suggest the possibility that the binding of leptin to its receptor in beta-cells may modulate insulin expression in a negative feedback loop, and thereby may have an anti-obesity effect.MH - Carrier Proteins|*BI/MEMH - Islets of Langerhans|*MEMH - Proteins|*MESO - Biochem Biophys Res Commun 1996 Jul; 224(2):522-7DP - 1996 JulTA - Biochem Biophys Res CommunPG - 522-7IP - 2VI - 224UI - 9629552010
AU - Ghilardi N
AU - Ziegler S
AU - Wiestner A
AU - Stoffel R
AU - Heim MH
AU - Skoda RCTI - Defective STAT signaling by the leptin receptor in diabetic mice.AB - Leptin and its receptor, obese receptor (OB-R), comprise an important signaling system for the regulation of body weight. Splice variants of OB-R mRNA encode proteins that differ in the length of their cytoplasmic domains. We cloned a long isoform of the wild-type leptin receptor that is preferentially expressed in the hypothalamus and show that it can activate signal transducers and activators of transcription (STAT)-3, STAT-5, and STAT-6. A point mutation within the OB-R gene of diabetic (db) mice generates a new splice donor site that dramatically reduces expression of this long isoform in homozygous db/db mice. In contrast, an OB-R protein with a shorter cytoplasmic domain is present in both db/db and wild-type mice. We show that this short isoform is unable to activate the STAT pathway. These data provide further evidence that the mutation in OB-R causes the db/db phenotype and identify three STAT proteins as potential mediators of the anti-obesity effects of leptin.MH - Carrier Proteins|GE/*PHMH - Diabetes Mellitus, Experimental|*MEMH - DNA-Binding Proteins|*MEMH - Signal Transduction|*GEMH - Trans-Activators|*MESO - Proc Natl Acad Sci U S A 1996 Jun; 93(13):6231-5DP - 1996 JunTA - Proc Natl Acad Sci U S APG - 6231-5IP - 13VI - 93UI - 9627052011
AU - Antczak M
AU - Van Blerkom JTI - Oocyte influences on early development: the regulatory proteins leptin and STAT3 are polarized in mouse and human oocytes and differentially distributed within the cells of the preimplantation stage embryo.AB - Unique protein domains, concentration gradients, and asymmetric protein distributions or polarities are principle forces establishing the identity and fate of individual cells during early development in lower vertebrates and invertebrates.Here, we present evidence that these same forces exist during mammalian development in the form of two representative regulatory proteins, leptin and STAT3. Leptin, the 16 kDa cytokine product of the obese gene (ob) is involved in the activation of STAT3, a member of the signal transducer and activation of transcription family of proteins. We examined the temporal and spatial aspects of leptin and STAT3 immunofluorescence in mouse and human oocytes and preimplantation stage embryos. The findings demonstrate that both leptin and STAT3 are polarized in the oocyte and, as a consequence of their location and the position of the cleavage planes with respect to these protein domains:(i) differences in allocation of these proteins between blastomeres occur at the first cell division such that by the 8-cell stage; (ii) unique cellular domains consisting of leptin/STAT3 rich and leptin/STAT3 poor populations of cells are generated. By the morula stage, a cell-borne concentration gradient of these proteins extending along the surface of the embryo is observed. A potential role of these proteins in early development is indicated at the morula stage where the 'inner' cells consist of blastomeres that contain little, if any, leptin/STAT3 while 'outer'cells contain both leptin/STAT3 rich and poor cells. This pattern persists through the hatched blastocyst stage with little, if any, leptin/STAT3 detected in the inner cell mass and populations of leptin/STAT3 rich and poor cells forming the trophoblast. We have examined oocytes from mutant C57BL/6J ob/ob mice which are both obese and infertile (although fertility can be restored by the exogenous provision of leptin) and have found STAT3 and the mutant (truncated)leptin protein to be present and polarized, suggesting the possibility that the truncated leptin protein may still contain operational domains which are functional during oocyte development and early embryogenesis. Furthermore,analysis of leptin and STAT3 in intact ovarian follicles suggests that these proteins may be maternally derived and in particular, that a subpopulation of follicle cells may be partly responsible for the establishment of their polarized distribution in the oocyte. The results are discussed with respect to the proposition that leptin and STAT3 have critical roles in early mammalian development, and may be involved in the determination of the animal pole of the oocyte and in the establishment of the inner cell mass and trophoblast in the preimplantation stage embryo.MH - Cell Polarity|*PHMH - DNA-Binding Proteins|IP/*ME/PHMH - Fetal Development|*PHMH - Oocytes|ME/*PHMH - Preimplantation Phase|*PHMH - Proteins|IP/*ME/PHMH - Trans-Activators|IP/*ME/PHSO - Mol Hum Reprod 1997 Dec; 3(12):1067-86DP - 1997 DecTA - Mol Hum ReprodPG - 1067-86IP - 12VI - 3UI - 9812449912
AU - Campbell FM
AU - Gordon MJ
AU - Hoggard N
AU - Dutta Roy AKTI - Interaction of free fatty acids with human leptin.AB - Relatively high concentrations of leptin are present in plasma and it is thought to play a major role in lipid homeostasis. Leptin is reported to lower tissue triglyceride content by increasing intracellular oxidation of free fatty acids (FFA). However very little is known regarding the interaction between leptin and plasma FFA. We studied the interaction of FFA with leptin using a direct radiolabelled fatty acid binding assay, a fluorescence assay, electrophoretic mobility and autoradiobinding. All these data indicate that binding of FFA with leptin is reversible and shows a positive co-operativity. The binding of FFA to leptin produces a change in the pI value of the leptin and also increased the electrophoretic mobility of the protein in native polyacrylamide gels. The change in leptin's electrophoretic mobility depends on the chain length and the number of double bonds of the fatty acid, as stearic acid, 18:0, had no effect whereas oleic acid, 18:1n-9, linoleic acid,18:2n-6, arachidonic acid, 20:4n-6, and docosahexaneoic acid, 22:6n-3, affected leptin's mobility to different degrees. The physiological implication of leptin-FFA interaction is not known, however the interaction may depend on the plasma FFA composition and concentration which are known to vary in different pathological/physiological conditions.MH - Fatty Acids|*MEMH - Proteins|*MESO - Biochem Biophys Res Commun 1998 Jun; 247(3):654-8DP - 1998 JunTA - Biochem Biophys Res CommunPG - 654-8IP - 3VI - 247UI - 9832118213
AU - Siegrist Kaiser CA
AU - Pauli V
AU - Juge Aubry CE
AU - Boss O
AU - Pernin A
AU - Chin WW
AU - Cusin I
AU - Rohner Jeanrenaud F
AU - Burger AG
AU - Zapf J
AU - Meier CATI - Direct effects of leptin on brown and white adipose tissue.AB - Leptin is thought to exert its actions on energy homeostasis through the long form of the leptin receptor (OB-Rb), which is present in the hypothalamus and in certain peripheral organs, including adipose tissue. In this study, we examined whether leptin has direct effects on the function of brown and white adipose tissue (BAT and WAT, respectively) at the metabolic and molecular levels. The chronic peripheral intravenous administration of leptin in vivo for 4 d resulted in a 1.6-fold increase in the in vivo glucose utilization index of BAT, whereas no significant change was found after intracerebroventricular administration compared with pair-fed control rats, compatible with a direct effect of leptin on BAT. The effect of leptin on WAT fat pads from lean Zucker Fa/ fa rats was assessed ex vivo, where a 9- and 16-fold increase in the rate of lipolysis was observed after 2 h of exposure to 0.1 and 10 nM leptin, respectively.In contrast, no increase in lipolysis was observed in the fat pads from obese fa/fa rats, which harbor an inactivating mutation in the OB-Rb. At the level of gene expression,leptin treatment for 24 h increased malic enzyme and lipoprotein lipase RNA 1.8+/-0.17 and 1.9+/-0.14-fold, respectively,while aP2 mRNA levels were unaltered in primary cultures of brown adipocytes from lean Fa/fa rats. Importantly, however, no significant effect of leptin was observed on these genes in brown adipocytes from obese fa/fa animals.The presence of OB-Rb receptors in adipose tissue was substantiated by the detection of its transcripts by RT-PCR, and leptin treatment in vivo and in vitro activated the specific STATs implicated in the signaling pathway of the OB-Rb. Taken together, our data strongly suggest that leptin has direct effects on BAT and WAT, resulting in the activation of the Jak/STAT pathway and the increased expression of certain target genes, which may partially account for the observed increase in glucose utilization and lipolysis in leptin-treated adipose tissue.MH - Adipose Tissue|*DE/MEMH - Brown Fat|*DE/MEMH - Proteins|*PDSO - J Clin Invest 1997 Dec; 100(11):2858-64DP - 1997 DecTA - J Clin InvestPG - 2858-64IP - 11VI - 100UI - 9805259014
AU - Gavrilova O
AU - Barr V
AU - Marcus Samuels B
AU - Reitman MTI - Hyperleptinemia of pregnancy associated with the appearance of a circulating form of the leptin receptor.AB - Leptin is a hormone produced in adipose cells that regulates energy expenditure, food intake, and adiposity. In mice,we observed that circulating leptin levels increase 20-40-fold during pregnancy. Pregnant ob/ob females had no detectable serum leptin, demonstrating that the heterozygous conceptus was not the source of the leptin. However, leptin RNA and protein levels in maternal adipose tissue were not elevated. The circulating leptin was in a high molecular weight complex, suggesting that the rise in leptin was due to expression of a binding protein. Indeed, quantitative assays of serum leptin binding capacity revealed a 40-fold increase, coincident with the rise in serum leptin. Leptin binding activity reached a capacity of 207 +/- 15 nmol/liter of serum at day 18 of gestation, and half-maximal binding was observed with approximately 3 nM leptin. The binding protein was purified and partially sequenced, revealing sequence identity to the extracellular domain of the leptin receptor. We found that the placenta produces large amounts of the OB-Re isoform of leptin receptor mRNA, which encodes a soluble binding protein. Thus, the extreme hyperleptinemia of late pregnancy is attributable to binding of the leptin by a secreted form of the leptin receptor made by the placenta.MH - Carrier Proteins|CH/*MEMH - Mice, Obese|*BLMH - Proteins|*MESO - J Biol Chem 1997 Nov; 272(48):30546-51DP - 1997 NovTA - J Biol ChemPG - 30546-51IP - 48VI - 272UI - 9804376415
AU - Yamashita T
AU - Murakami T
AU - Otani S
AU - Kuwajima M
AU - Shima KTI - Leptin receptor signal transduction: OBRa and OBRb of fa type.AB - We report herein the characterization of activities of signal transduction for three types of leptin receptors (OBRs) from rats, the OBRa, OBRb, and OBRb with fa mutation (OBRb-fa), by measurement of the levels of tyrosine phosphorylation of STAT3 (signal transducers and activators of transcription 3) and MAPK (mitogen-activated protein kinase), which are induced by leptin stimulation of CHO cells stably expressing the OBR (CHO-OBRb, CHO-OBRa, or CHO-OBRb-fa cells). As the result of leptin stimulation, enhanced levels of tyrosine phosphorylation of STAT3 and MAPK were detected in CHO-OBRb cells. In CHO-OBRb-fa cells, enhancement levels for both were lower than those in CHO-OBRb cells. In CHO-OBRa cells, only the phosphorylation of MAPK was detected. These data suggest that these reduced signaling activities cause obesity in fa/fa rats and that OBRa, which has been generally thought to be inactive at signaling, actually transmits signals through the MAPK pathway.MH - Carrier Proteins|CL/GE/*MEMH - Proteins|*MEMH - Receptors, Cytokine|CL/GE/*MESO - Biochem Biophys Res Commun 1998 May; 246(3):752-9DP - 1998 MayTA - Biochem Biophys Res CommunPG - 752-9IP - 3VI - 246UI - 9828960316
AU - Diamond FB Jr
AU - Eichler DC
AU - Duckett G
AU - Jorgensen EV
AU - Shulman D
AU - Root AWTI - Demonstration of a leptin binding factor in human serum.AB - Serum leptin levels are elevated in subjects with exogenous obesity, indicating that obesity is associated with leptin resistance. Since in man no abnormalities have yet been found in either the genes for leptin or its receptor, the mechanism of leptin resistance in obesity remains unknown.To determine if resistance might be related to leptin binding by a serum component, we assessed the carrier status of leptin in serum. The presence of a specific leptin binding factor in human serum has been established by (1) demonstrating [125I]-leptin binding to a serum component that is saturable and specifically displaceable only by unlabeled leptin and not by human growth hormone, pork insulin, insulin-like growth factors I and II, luteinizing or follicle stimulating hormones, transforming growth factor-beta 1, interleukin-6, or leukemia inhibiting factor; (2) fractionating the leptin bound serum complex and the serum leptin binding component on a molecular sieving column revealing a mass of approximately 450 kDa; and (3) identifying an inverse correlation between the concentration of serum leptin and the quantity of the leptin binding component. It is suggested that binding of leptin by this serum component may influence the physiologic response to leptin.MH - Carrier Proteins|*BL/CH/IPMH - Proteins|*MESO - Biochem Biophys Res Commun 1997 Apr; 233(3):818-22DP - 1997 AprTA - Biochem Biophys Res CommunPG - 818-22IP - 3VI - 233UI - 9731249917
AU - Birkenmeier G
AU - Kmpfer I
AU - Kratzsch J
AU - Schellenberger WTI - Human leptin forms complexes with alpha 2-macroglobulin which are recognized by the alpha 2-macroglobulin receptor/low density lipoprotein receptor-related protein.AB - OBJECTIVE: To identify binding proteins of leptin in human plasma. METHODS: Binding was evaluated by electrophoresis,size exclusion chromatography (SEC), Western blotting, and radioisotope labeling. Quantification of leptin and the different forms of alpha2-macroglobulin (alpha2-M) was performed by ELISA. RESULTS: Leptin interacts with the proteinase inhibitor, alpha2-M. 125I-labeled leptin specifically binds to the transformed inhibitor, which arises by reaction with proteinases or with reactive primary amines. No leptin binding was observed to the native alpha2-M, which abundantly occurs in plasma. The complex formation between leptin and alpha2-M was found to proceed within minutes and was stable, as it resisted separation by SEC and electrophoresis. The Kd of the complex was 2.14 +/-0.78 micromol/l. Complex formation with transformed alpha2-M did not interfere with the immunological determination of leptin in plasma. The leptin-alpha2-M complex was found to be recognized by the alpha2-M receptor/low density lipoprotein receptor-related protein. By computer analysis, a simple model is presented showing that the degree of transformation of alpha2-M may significantly influence the leptin concentration in blood. CONCLUSIONS: The proteinase inhibitor, alpha2-M, may act as a leptin-binding protein in human plasma.Binding of leptin to transformed alpha2-M and its rapid clearance by the alpha2-M receptor may significantly influence the bioavailability of leptin in human plasma.MH - alpha-Macroglobulins|*MEMH - Proteins|*MEMH - Receptors, Immunologic|*BLMH - Receptors, LDL|*BLSO - Eur J Endocrinol 1998 Aug; 139(2):224-30DP - 1998 AugTA - Eur J EndocrinolPG - 224-30IP - 2VI - 139UI - 9838947418
AU - Carpenter LR
AU - Farruggella TJ
AU - Symes A
AU - Karow ML
AU - Yancopoulos GD
AU - Stahl NTI - Enhancing leptin response by preventing SH2-containing phosphatase 2 interaction with Ob receptor.AB - Leptin is an adipocyte-derived cytokine that regulates food intake and body weight via interaction with its Ob receptor (ObR). Serum leptin levels are chronically elevated in obese humans, suggesting that obesity may be associated with leptin resistance and the inability to generate an adequate ObR response. Evidence suggests that transcriptional activation of target genes by STAT3 (signal transducer and activator of transcription) in the hypothalamus is a critical pathway that mediates leptin's action. Herein we report that activation of ObR induces the tyrosine phosphorylation of the tyrosine phosphatase SH2-containing phosphatase 2 (SHP-2) and demonstrate that Tyr986 within the ObR cytoplasmic domain is essential to mediate phosphorylation of SHP-2 and binding of SHP-2 to ObR. Surprisingly, mutation of Tyr986 to Phe, which abrogates SHP-2 phosphorylation and binding to the receptor, dramatically increases gene induction mediated by STAT3. Our findings indicate that SHP-2 is a negative regulator of STAT3-mediated gene induction after activation of ObR and raise the possibility that blocking the interaction of SHP-2 with ObR could overcome leptin resistance by boosting leptin's weight-reducing effects in obese individuals.MH - Carrier Proteins|GE/*MEMH - DNA-Binding Proteins|*MEMH - Protein-Tyrosine-Phosphatase|*MEMH - Proteins|*MEMH - Signal Transduction|*MH - Trans-Activators|*MESO - Proc Natl Acad Sci U S A 1998 May; 95(11):6061-6DP - 1998 MayTA - Proc Natl Acad Sci U S APG - 6061-6IP - 11VI - 95UI - 9826330819
AU - Quinton ND
AU - Smith RF
AU - Clayton PE
AU - Gill MS
AU - Shalet S
AU - Justice SK
AU - Simon SA
AU - Walters S
AU - Postel Vinay MC
AU - Blakemore AI
AU - Ross RJTI - Leptin binding activity changes with age: the link between leptin and puberty.AB - The timing of the physical transition from child to adult is determined by a biological clock that switches off the pituitary gonadal axis during infancy until puberty. Body composition (and in particular, fat mass), through leptin,are critical signals to this clock. However, no direct relationship between leptin and puberty has been demonstrated.Leptin is bound in the circulation by a high-affinity binding protein, which has been identified as a soluble leptin receptor. We found circulating levels of leptin binding activity (LBA) to be low at birth, to be high in the prepubertal years, to fall through puberty, and then to remain stable during adult life. LBA correlated with pubertal status in both boys and girls. We postulate that the fall in LBA,associated with increasing age and puberty, reflects a reduction in expression of truncated leptin receptors, and leptin is then available to the full-length receptor,which transmits the biological signal for leptin. The high levels of LBA occur during the years when the pituitary gonadal axis is quiescent. Thus, the change in LBA could explain how leptin regulates puberty.MH - Aging|*PHMH - Proteins|*ME/PHMH - Puberty|*PHSO - J Clin Endocrinol Metab 1999 Jul; 84(7):2336-41DP - 1999 JulTA - J Clin Endocrinol MetabPG - 2336-41IP - 7VI - 84UI - 9933168520
AU - Liu C
AU - Liu XJ
AU - Barry G
AU - Ling N
AU - Maki RA
AU - De Souza EBTI - Expression and characterization of a putative high affinity human soluble leptin receptor.AB - Leptin, a circulating 16-kDa protein secreted by adipocytes,decreases body weight by reducing food intake and enhancing energy utilization. Leptin receptors that share homology to the glycoprotein gp130 have been recently cloned. In addition, differentially spliced leptin receptor messenger RNAs have been identified. Functional mutations in either the leptin or leptin receptor gene cause obesity. In the present study, expression of the full length human leptin receptor complementary DNA encoding the long cytoplasmic domain of leptin receptor in COS7 cells resulted in high affinity membrane binding of 125I-leptin (Ki approximately 200 pM); no detectable binding was present in the medium.In addition, we expressed the extracellular domain of human leptin receptor in COS7 cells and identified a soluble leptin receptor in the conditioned medium that binds human and mouse leptin with high affinity comparable with the full length membrane receptor. Transfected COS7 cells expressing the soluble leptin receptor also demonstrated modest specific 125I-leptin binding in whole cells, presumably due to association of the soluble leptin receptor to cell membrane proteins.Data from cross-linking studies identified two specific bands in the 125I-leptin/soluble leptin receptor complex with molecular masses of approximately 130-150 kDa and 300 kDa. The 130-150 kDa molecular mass was confirmed in Western blot analysis and Coomassie staining of the purified soluble receptor and probably represents the glycosylated form of the receptor. The 300-kDa band most likely represents a homodimer of the soluble leptin receptor complex because HPLC gel filtration analysis of the 125I-leptin/soluble leptin receptor complex identified a single peak corresponding to a molecular mass of approximately 340 kDa. The soluble leptin receptor antagonized 125I-leptin binding to the membrane receptor, suggesting its potential utility as a functional tool for determining the role of endogenous leptin.MH - Carrier Proteins|*BI/*GE/MESO - Endocrinology 1997 Aug; 138(8):3548-54DP - 1997 AugTA - EndocrinologyPG - 3548-54IP - 8VI - 138UI - 9737549321
AU - Nakashima K
AU - Narazaki M
AU - Taga TTI - Leptin receptor (OB-R) oligomerizes with itself but not with its closely related cytokine signal transducer gp130.AB - Leptin (OB) exerts weight-reducing effects in mice. The structure of the receptor for this factor, OB-R, is considerably similar to those of gp130, the common signal transducing receptor component for the interleukin-6 (IL-6) family of cytokines, and leukemia inhibitory factor receptor (LIFR). Since the IL-6 family of cytokines signal through gp130 homodimer or gp130/LIFR heterodimer, we have examined in this study the possible involvement of gp130 and LIFR in leptin signaling through OB-R. Leptin stimulation induces tyrosine phosphorylation of neither gp130 nor LIFR, while LIF stimulation does both. As examined by using two differently epitope-tagged OB-R molecules, the spontaneous homo-oligomerization of OB-R has been elucidated. Ba/F3 cells, which do not express gp130, are non-responsive to leptin and exhibit increased DNA synthesis in response to leptin after transfection of OB-R cDNA alone. OB-R appears to transduce the signal via its homo-oligomerization without interaction with gp130 or LIFR.MH - Antigens, CD|CH/*MEMH - Carrier Proteins|*CH/GE/*MEMH - Membrane Glycoproteins|CH/*MEMH - Receptors, Cytokine|*MESO - FEBS Lett 1997 Feb; 403(1):79-82DP - 1997 FebTA - FEBS LettPG - 79-82IP - 1VI - 403UI - 9719020922
AU - Barr VA
AU - Lane K
AU - Taylor SITI - Subcellular localization and internalization of the four human leptin receptor isoforms.AB - There are four known isoforms of the human leptin receptor (HLR) with different C-terminal cytoplasmic domains (designated by the number of unique C-terminal amino acids). In cells expressing HLR-5, -15, or -274, 15-25% of the leptin binding sites were located at the plasma membrane. In contrast,in cells expressing HLR-67, only 5% of the total binding sites were at the plasma membrane. Immunofluorescent microscopy showed that all four isoforms partially co-localized with calnexin and beta-COP, markers of the endoplasmic reticulum and the Golgi, respectively. All isoforms were also detected in an unidentified punctate compartment. All isoforms were internalized via clathrin-mediated endocytosis, but at different rates. After 20 min at 37 degrees C, 45% of a bound cohort of labeled ligand had been internalized by HLR-15, 30% by HLR-67, 25% by HLR-274, and 15% by HLR-5.Degradation of internalized leptin occurred in lysosomes.Overnight exposure to leptin down-regulated all isoforms,but to a variable extent. HLR-274 displayed the greatest down-regulation and also appeared to reach lysosomes more quickly than the other isoforms. The faster degradation of HLR-274 may help to terminate leptin signaling.MH - Carrier Proteins|GE/*MESO - J Biol Chem 1999 Jul; 274(30):21416-24DP - 1999 JulTA - J Biol ChemPG - 21416-24IP - 30VI - 274UI - 9934008623
AU - Combatsiaris TP
AU - Charron MJTI - Downregulation of uncoupling protein 2 mRNA in white adipose tissue and uncoupling protein 3 mRNA in skeletal muscle during the early stages of leptin treatment.AB - The mechanisms underlying the increase in energy expenditure during leptin treatment are not clear. We recently showed that a 5-h intravenous or intracerebroventricular infusion of leptin elevated basal glucose uptake in skeletal muscle (SM) and brown adipose tissue and increased whole-body glucose turnover in C57Bl/6J mice (Kamohara S, Burcelin R, Halaas JL, Friedman JM, Charron MJ: Acute stimulation of glucose metabolism in mice by leptin treatment. Nature 389:374-377, 1997). We extended the previous study by measuring steady-state levels of uncoupling protein (UCP)-2 mRNA and UCP-3 mRNA in white adipose tissue (WAT) and SM. Leptin by intravenous or intracerebroventricular infusion for 5 h was associated with a decrease in UCP-2 mRNA in WAT (47-52%) and UCP-3 mRNA in SM (33-37%). Because overexpression of UCP-2 or UCP-3 can depolarize the inner mitochondrial membrane, suppression of UCP-2 mRNA and UCP-3 mRNA may in fact lower respiratory demands in WAT and SM. This is consistent with the parallel suppression of cytochrome oxidase subunit IV (COX-IV) mRNA in WAT (35-39%) after leptin infusion. COX-IV mRNA in SM did not respond to acute leptin treatment. Mitochondrial inorganic phosphate carrier (P1C) mRNA was also suppressed in WAT (33-35%) by either method of leptin infusion, but only intravenous infusion of leptin reduced P1C mRNA in SM (40%). Denervation suppressed mRNA levels for UCP-2 (49%), UCP-3 (36%), and COX-IV (59%) and eliminated the acute response to leptin in SM. The comparable response to leptin under intravenous or intracerebroventricular infusion and the loss of responsiveness after denervation strongly suggest that the acute effects of leptin involve central signaling pathways.MH - Adipose Tissue|*MEMH - Carrier Proteins|*GEMH - Muscle, Skeletal|*MEMH - Proteins|*GE/*PDMH - RNA, Messenger|*MEAD - Department of BiochemistryAD - Albert Einstein College of MedicineAD - BronxAD - New York 10461AD - USA.SO - Diabetes 1999 Jan; 48(1):128-33DP - 1999 JanTA - DiabetesPG - 128-33IP - 1VI - 48UI - 9910725124
AU - Wang MY
AU - Koyama K
AU - Shimabukuro M
AU - Mangelsdorf D
AU - Newgard CB
AU - Unger RHTI - Overexpression of leptin receptors in pancreatic islets of Zucker diabetic fatty rats restores GLUT-2, glucokinase,and glucose-stimulated insulin secretion.AB - The high-Km glucose transporter, GLUT-2, and the high-Km hexokinase of beta cells, glucokinase (GK), are required for glucose-stimulated insulin secretion (GSIS). GLUT-2 expression in beta cells of Zucker diabetic fatty (ZDF)rats is profoundly reduced at the onset of beta-cell dysfunction of diabetes. Because ZDF rats are homozygous for a mutation in their leptin receptor (OB-R) gene and are therefore leptin-insensitive, we expressed the wild-type OB-R gene in diabetic islets by infusing a recombinant adenovirus (AdCMV-OB-Rb) to determine whether this reversed the abnormalities.Leptin induced a rise in phosphorylated STAT3, indicating that the transferred wild-type OB-R was functional. GLUT-2 protein rose 17-fold in AdCMV-OB-Rb-treated ZDF islets without leptin, and leptin caused no further rise. GK protein rose 7-fold without and 12-fold with leptin. Preproinsulin mRNA increased 64% without leptin and rose no further with leptin, but leptin was required to restore GSIS. Clofibrate and 9-cis-retinoic acid, the partner ligands for binding to peroxisome proliferator-activator receptor alpha (PPARalpha)and retinoid X receptor, up-regulated GLUT-2 expression in islets of normal rats, but not in ZDF rats, in which PPARalpha is very low. Because the fat content of islets of diabetic ZDF rats remains high unless they are treated with leptin, it appears that restoration of GSIS requires normalization of intracellular nutrient homeostasis, whereas up-regulation of GLUT-2 and GK is leptin-independent, requiring only high expression of OB-Rb.MH - Carrier Proteins|*GEMH - Diabetes Mellitus, Non-Insulin-Dependent|*GE/*PPMH - Islets of Langerhans|DE/*ME/PHMH - Obesity|*GE/*PPSO - Proc Natl Acad Sci U S A 1998 Sep; 95(20):11921-6DP - 1998 SepTA - Proc Natl Acad Sci U S APG - 11921-6IP - 20VI - 95UI - 9842625425
AU - Zhang Y
AU - Olsen DR
AU - Nguyen KB
AU - Olson PS
AU - Rhodes ET
AU - Mascarenhas DTI - Expression of eukaryotic proteins in soluble form in Escherichia coli.AB - At the optimum temperature for its growth (37 degrees C), Escherichia coli tends to accumulate heterologous proteins in insoluble form. Fusion protein technology has been used to increase the solubility of overexpressed proteins in this organism, but with variable degrees of success. Fusion to a mutant form of DsbA (DsbAmut) confers higher levels of solubility to heterologous proteins in a reproducible way, even when E. coli is grown at 37 degrees C. We have shown this to be true with a diverse sample of eukaryotic proteins: IGF-I, IGFBP-3, 3C proteinase, TGF beta-2, sTGF beta-RII, BDNF, GDNF, mEGFBP, leptin, and GFP. In addition,we have investigated the effects of charge average and proline content on the solubility of DsbAmut fusions. Coexpression of a protein prolyl isomerase [cyclophilin (L-)] and modification of selected asparagine residues to aspartic acid appear to have beneficial effects on the accumulation of soluble heterologous proteins.MH - Escherichia coli|GE/*MEMH - Gene Expression Regulation, Bacterial|*GEMH - Recombinant Fusion Proteins|*BI/CH/GESO - Protein Expr Purif 1998 Mar; 12(2):159-65DP - 1998 MarTA - Protein Expr PurifPG - 159-65IP - 2VI - 12UI - 9819187626
AU - Shimizu H
AU - Ohtani K
AU - Tsuchiya T
AU - Takahashi H
AU - Uehara Y
AU - Sato N
AU - Mori MTI - Leptin stimulates insulin secretion and synthesis in HIT-T 15 cells.AB - Leptin, an ob gene product, corrects hyperinsulinemia in ob/ob mice. The leptin receptor may exist in pancreatic islets. The present studies were undertaken to determine the direct effect of 1-100 ng/ml recombinant leptin on insulin secretion and synthesis in HIT-T 15 cells by using static culture system. The addition of recombinant leptin significantly increased insulin secretion for 20 min at the highest concentration (100 ng/ml). The addition of recombinant leptin dose-dependently increased insulin secretion for 24 h in the 7 mM glucose-containing F-12 K medium. The incubation with recombinant leptin for 24 h increased preproinsulin mRNA expression, assessed with reverse transcription-polymerase chain reaction (RT-PCR) method. It was furthermore demonstrated that HIT-T 15 cells possessed the specific binding site for [125I]-labeled leptin. The present study demonstrated the existence of the leptin-specific binding sites that mediate its stimulatory effect on insulin secretion and synthesis in HIT-T 15 cells.MH - Insulin|BI/*SEMH - Islets of Langerhans|ME/*SEMH - Proteins|ME/*PDSO - Peptides 1997; 18(8):1263-6DP - 1997TA - PeptidesPG - 1263-6IP - 8VI - 18UI - 9805785327
AU - Albertsson Wikland K
AU - Boguszewski M
AU - Karlberg JTI - Children born small-for-gestational age: postnatal growth and hormonal status.AB - It is generally recognized that children born small-for-gestational age (SGA) have a 5-7 times higher risk of short stature than children born at normal size. It has been suggested that the programming of the endocrine axes occurs during critical phases of fetal development and is affected by intrauterine growth retardation. This study was undertaken to characterize the postnatal growth pattern and the final height of children born SGA, as part of a population- based study (n = 3,650), from birth to final height, and to evaluate the hormonal status in another group of prepubertal children born SGA (n = 134) without postnatal catch-up growth. The majority (88%) of 'healthy' full-term singleton SGA infants achieved catch-up growth during the first 2 years of life,and most of the increase in height occurred by 2 months of age. The SGA children who remained short at 2 years of age had a higher risk of short stature later in life.The risk of having a short final height (<-2 SDS) was five times higher for children with a low birth weight and seven times higher for those with a low birth length in comparison with children with a normal birth size. Moreover, about 20% of all children of short stature were born SGA. As a group, children born SGA will have a final height, expressed in SDS, as they had during the prepubertal years. This is in contrast to children, who became short postnatally.During puberty, these short children will have a mean height gain of 0.6 SDS for girls and 0.7 SDS for boys. The mean estimated secretion rate for growth hormone (GH) was lower in the short children born SGA compared with the reference groups born at an appropriate size for gestational age,of either short (p < 0.05) or normal stature (p < 0.001). Moreover, in the youngest children born SGA (2-6 years of age) a different pattern of GH secretion was found, with a high basal GH level, low peak amplitude, and high peak frequency. The majority of the children born SGA had levels of GH-binding protein within the range previously reported for normal children. However, the levels of insulin-like growth factor I (IGF-I), IGF-binding protein-3 (IGFBP-3) and leptin were significantly reduced compared with the reference values (p < 0.001, p < 0.01 and p < 0.001,respectively). In conclusion, the low spontaneous GH secretion rate and a disturbed GH secretion pattern, together with low serum levels of IGF-I, IGFBP-3 and leptin, might contribute to the reduced postnatal growth in some of the subgroup of children born SGA who remained short during childhood.MH - Infant, Small for Gestational Age|BL/*GDSO - Horm Res 1998; 49 Suppl 2():7-13DP - 1998TA - Horm ResPG - 7-13VI - 49 Suppl 2UI - 9840250028
AU - Eichler DC
AU - Root AW
AU - Duckett G
AU - Moore KL
AU - Diamond FBTI - A spun-column assay for determination of leptin binding in serum.AB - Serum collected from 27 patients was assayed simultaneously using a spun-column assay (SPC) and a traditional exclusion gel-filtration assay (GFC) to determine specific leptin binding. The levels of serum leptin binding determined by either assay correlated inversely with serum leptin levels (SPC, r = 0.63, P < 0.001; GFC, r = 0.79, P < 0.0001). Although specific leptin binding as determined by the traditional exclusion gel-filtration assay was generally higher than that obtained by the spun-column assay (mean = 18.3% vs 14.0%, P < 0. 02, respectively); the values obtained between the two assay methods were highly correlative (r = 0.89, P < 0.0001). By varying either the amount of 125I-leptin or the amount of competitor, analysis was carried out using the spun-column assay to determine the intrinsic properties of serum leptin binding. Results yielded a Kd = 0.3 nM, where each variable amount of leptin or competitor was carried out in duplicate. The complete analysis was carried out in the time that it typically takes for a single sample determination by the traditional exclusion gel-filtration assay. We conclude that the "spun-column" assay is a useful method for rapid and accurate quantification of leptin binding in serum. Copyright 1999 Academic Press.MH - Blood Chemical Analysis|*MTMH - Chromatography, Gel|*MTMH - Proteins|*MESO - Anal Biochem 1999 Feb; 267(1):100-3DP - 1999 FebTA - Anal BiochemPG - 100-3IP - 1VI - 267UI - 9911928129
AU - Chung CD
AU - Liao J
AU - Liu B
AU - Rao X
AU - Jay P
AU - Berta P
AU - Shuai KTI - Specific inhibition of Stat3 signal transduction by PIAS3.AB - The signal transducer and activator of transcription-3 (Stat3) protein is activated by the interleukin 6 (IL-6)family of cytokines, epidermal growth factor, and leptin.A protein named PIAS3 (protein inhibitor of activated STAT)that binds to Stat3 was isolated and characterized. The association of PIAS3 with Stat3 in vivo was only observed in cells stimulated with ligands that cause the activation of Stat3. PIAS3 blocked the DNA-binding activity of Stat3 and inhibited Stat3-mediated gene activation. Although Stat1 is also phosphorylated in response to IL-6, PIAS3 did not interact with Stat1 or affect its DNA-binding or transcriptional activity. The results indicate that PIAS3 is a specific inhibitor of Stat3.MH - Carrier Proteins|CH/GE/*ME/PDMH - DNA-Binding Proteins|GE/*MEMH - Signal Transduction|*MH - Trans-Activators|*MESO - Science 1997 Dec; 278(5344):1803-5DP - 1997 DecTA - SciencePG - 1803-5IP - 5344VI - 278UI - 9804961530
AU - Seufert J
AU - Kieffer TJ
AU - Leech CA
AU - Holz GG
AU - Moritz W
AU - Ricordi C
AU - Habener JFTI - Leptin suppression of insulin secretion and gene expression in human pancreatic islets: implications for the development of adipogenic diabetes mellitus.AB - Previously we demonstrated the expression of the long form of the leptin receptor in rodent pancreatic beta-cells and an inhibition of insulin secretion by leptin via activation of ATP-sensitive potassium channels. Here we examine pancreatic islets isolated from pancreata of human donors for their responses to leptin. The presence of leptin receptors on islet beta-cells was demonstrated by double fluorescence confocal microscopy after binding of a fluorescent derivative of human leptin (Cy3-leptin). Leptin (6.25 nM) suppressed insulin secretion of normal islets by 20% at 5.6 mM glucose.Intracellular calcium responses to 16.7 mM glucose were rapidly reduced by leptin. Proinsulin messenger ribonucleic acid expression in islets was inhibited by leptin at 11.1 mM, but not at 5.6 mM glucose. Leptin also reduced proinsulin messenger ribonucleic acid levels that were increased in islets by treatment with 10 nM glucagon-like peptide-1 in the presence of either 5.6 or 11.1 mM glucose. These findings demonstrate direct suppressive effects of leptin on insulin-producing beta-cells in human islets at the levels of both stimulus-secretion coupling and gene expression.The findings also further indicate the existence of an adipoinsular axis in humans in which insulin stimulates leptin production in adipocytes and leptin inhibits the production of insulin in beta-cells. We suggest that dysregulation of the adipoinsular axis in obese individuals due to defective leptin reception by beta-cells may result in chronic hyperinsulinemia and may contribute to the pathogenesis of adipogenic diabetes.MH - Gene Expression|*DEMH - Insulin|*SEMH - Islets of Langerhans|CH/DE/*MEMH - Obesity in Diabetes|*MEMH - Proinsulin|*GEMH - Proteins|*PDAD - Laboratory of Molecular EndocrinologyAD - Massachusetts General HospitalAD - Howard Hughes Medical InstituteAD - Harvard Medical SchoolAD - Boston 02114AD - USA.SO - J Clin Endocrinol Metab 1999 Feb; 84(2):670-6DP - 1999 FebTA - J Clin Endocrinol MetabPG - 670-6IP - 2VI - 84UI - 9914503431
AU - Bjçrbaek C
AU - Uotani S
AU - da Silva B
AU - Flier JSTI - Divergent signaling capacities of the long and short isoforms of the leptin receptor.AB - Leptin receptors include a long form (OBRl) with 302 cytoplasmic residues that is presumed to mediate most or all of leptins signaling, and several short forms, including one (OBRs)that has 34 cytoplasmic residues, is widely expressed, and is presumed not to signal but to mediate transport or clearance of leptin. We studied the abilities of these two receptor isoforms to mediate signaling in transfected cells. In response to leptin, OBRl, but not OBRs, underwent tyrosine phosphorylation that was enhanced by co-expression with JAK2. In cells expressing receptors and JAK2, both OBRs and OBRl mediated leptin-dependent tyrosine phosphorylation of JAK2, and this was abolished with OBRs when the Box 1 motif was mutated. In cells expressing receptors, JAK2 and IRS-1, leptin induced tyrosine phosphorylation of IRS-1 through OBRs and OBRl. In COS cells expressing hemagglutinin-ERK1 and receptors, leptin increased ERK1 kinase activity through OBRl, with the magnitude increased by co-expression of JAK1 or JAK2, and to a lesser degree through OBRs, despite greater receptor expression. In stable Chinese hamster ovary cell lines expressing OBRs or OBRl, leptin stimulated endogenous ERK2 phosphorylation. Whereas leptin stimulated tyrosine phosphorylation of hemagglutinin-STAT3 and induction of a c-fos luciferase reporter plasmid through OBRl, OBRs was without effect in these assays. In conclusion, OBRl is capable of signaling to IRS-1 and mitogen-activated protein kinase via JAK, in addition to activating STAT pathways. Although substantially weaker than OBRl, OBRs is capable of mediating signal transduction via JAK, but these activities are of as yet unknown significance for leptin biology in vivo.MH - Carrier Proteins|CH/GE/*MEMH - Signal Transduction|*SO - J Biol Chem 1997 Dec; 272(51):32686-95DP - 1997 DecTA - J Biol ChemPG - 32686-95IP - 51VI - 272UI - 9807045332
AU - Chance WT
AU - Sheriff S
AU - Moore J
AU - Peng F
AU - Balasubramaniam ATI - Reciprocal changes in hypothalamic receptor binding and circulating leptin in anorectic tumor-bearing rats.AB - Although reduced biological activity of the obese gene product, leptin, has been associated with obesity, little information is available concerning leptin alterations during anorexia. Therefore, we measured circulating leptin concentrations and hypothalamic leptin binding in anorectic tumor-bearing and pair-fed control rats. Plasma concentrations of leptin decreased in tumor-bearing rats early in the course of tumor growth, and fell to nearly non-detectable levels during severe anorexia. The pair-fed control rats that ate the same amount of food as did the anorectic tumor-bearing rats exhibited a 50% decrease in plasma leptin concentration. Concentrations of free fatty acids were elevated in both tumor-bearing and pair-fed groups, while circulating levels of triglycerides were increased only in anorectic tumor-bearing rats. Leptin receptor density was doubled in the hypothalamus of tumor bearing rats, while binding affinity was decreased by 50%. These results suggest that peripheral leptin production is down-regulated,perhaps due to increased lipolysis in tumor-bearing rats.It appears that hypothalamic leptin systems up-regulate receptor numbers in response to decreased blood leptin level, however, the decrease in binding affinity may compensate for these alterations. Therefore, the influence of leptin on hypothalamic neuropeptide Y feeding systems may be minimal in anorectic tumor-bearing rats. Copyright 1998 Published by Elsevier Science B.V.MH - Anorexia|BL/*MEMH - Carrier Proteins|*MEMH - Hypothalamus|*ME/PHMH - Proteins|*MEMH - Sarcoma, Experimental|BL/*MESO - Brain Res 1998 Aug; 803(1-2):27-33DP - 1998 AugTA - Brain ResPG - 27-33IP - 1-2VI - 803UI - 9839837733
AU - Matsuoka T
AU - Tahara M
AU - Yokoi T
AU - Masumoto N
AU - Takeda T
AU - Yamaguchi M
AU - Tasaka K
AU - Kurachi H
AU - Murata YTI - Tyrosine phosphorylation of STAT3 by leptin through leptin receptor in mouse metaphase 2 stage oocyte.AB - Leptin is the product of the obese gene (ob), and is secreted in plasma from mature adipocytes. It has been recently reported that leptin is synthesized in granulosa and cumulus cells within the follicle of the ovary, and is present in mature human oocytes, suggesting possible roles of leptin in several aspects of pre- and post-ovulatory follicular development. On the other hand, STAT (Signal Transducer and Activator of Transcription) transcription factors are involved in leptin-associated signal transduction. In this report, we studied the expression of leptin receptor and STAT3 activation by leptin in metaphase 2 stage (M2) oocytes.Reverse transcriptase-polymerase chain reaction (RT-PCR)and immunoblotting showed that mRNA and protein of leptin receptor were expressed in M2 stage oocyte. Leptin at 15 ng/ml, the concentration observed in follicular fluid, caused tyrosine phosphorylation of STAT3 in mouse M2 stage oocytes. These results suggest possible roles of leptin in several aspects during oocyte maturation by activating the STAT signal transduction pathway. Copyright 1999 Academic Press.MH - Carrier Proteins|GE/*MEMH - DNA-Binding Proteins|*MEMH - Oocytes|*DE/GD/MEMH - Phosphotyrosine|*MEMH - Proteins|GE/PD/*PHMH - Trans-Activators|*MESO - Biochem Biophys Res Commun 1999 Mar; 256(3):480-4DP - 1999 MarTA - Biochem Biophys Res CommunPG - 480-4IP - 3VI - 256UI - 9918231534
AU - Ghilardi N
AU - Skoda RCTI - The leptin receptor activates janus kinase 2 and signals for proliferation in a factor-dependent cell line.AB - The antiobesity effects of leptin are mediated by the obese receptor (OB-R), a member of the cytokine receptor superfamily.Several isoforms of OB-R that differ in the length of the cytoplasmic domain have been described. An isoform with a long cytoplasmic domain of 302 amino acids, termed OB-Rb, contains the conserved box 1 and box 2 motifs and is likely to be responsible for leptin-induced signaling. A point mutation in the OB-R gene of diabetes (db) mice generates a new splice donor that interferes with the correct splicing of the OB-Rb mRNA and is predicted to cause absence of the OB-Rb protein in db/db mice. Here we examined the signaling potential of the long isoform, OB-Rb, and of a short isoform, OB-Ra, in BaF3 cells, a factor-dependent hematopoietic cell line. The long isoform was able to generate a proliferative signal and upon leptin binding, activated janus kinase 2 (Jak2). Consistently, antibodies directed against the extracellular domain of OB-R coprecipitated Jak2. The short isoform, OB-Ra, was inactive in both proliferation and Jak activation. These results provide further support for the long isoform, OB-Rb, being the principal mediator of the effects of leptin and help to explain why db/db mice are resistant to leptin, despite the presence of the short OB-R isoforms.MH - Carrier Proteins|GE/*MEMH - Protein-Tyrosine Kinase|*MEMH - Receptors, Cytokine|GE/*MEMH - Signal Transduction|*SO - Mol Endocrinol 1997 Apr; 11(4):393-9DP - 1997 AprTA - Mol EndocrinolPG - 393-9IP - 4VI - 11UI - 9724655335
AU - Li C
AU - Friedman JMTI - Leptin receptor activation of SH2 domain containing protein tyrosine phosphatase 2 modulates Ob receptor signal transduction.AB - Leptin exerts its weight-reducing effects by binding to its receptor and activating signal transduction in hypothalamic neurons and other cell types. To identify the components of the leptin signal transduction pathway, an approach was developed in which bacterially expressed phosphorylated fragments of Ob receptor b (Ob-Rb) were used as affinity agents. Leptin binding to the Ob-Rb form of the leptin receptor leads to tyrosyl phosphorylation of the cytoplasmic domain of its receptor. Two of the three cytoplasmic tyrosines of Ob-Rb, at positions 985 and 1138, are phosphorylated after leptin treatment. Affinity chromatography using a tyrosine-phosphorylated fragment spanning Tyr 985 of Ob-Rb was used to identify proteins that bind to this site.The SH2 domain containing protein tyrosine phosphatase 2 (SHP-2) was isolated from bovine and mouse hypothalamus by using this method. After cotransfection of Ob-Rb, Janus kinase 2 (JAK2), and SHP-2 into 293T cells, leptin results in direct binding of SHP-2 to the phosphorylated Tyr 985.The bound SHP-2 is itself tyrosine phosphorylated after leptin treatment. SHP-2 is not phosphorylated after leptin treatment when a Y-->F 985 receptor mutant is cotransfected.In the absence of SHP-2 phosphorylation, the level of JAK2 phosphorylation was increased. Tyrosyl phosphorylation of the leptin receptor and signal transducer and activater of transcription 3 (STAT3) are not affected by phosphorylation of SHP-2. These data suggest that activation of SHP-2 by the leptin receptor results in a decreased phosphorylation of JAK2 and may act to attenuate leptin signal transduction.The method used in this report can in principle be used to isolate additional components of the leptin, or other,signal transduction pathway.MH - Carrier Proteins|*MEMH - Obesity|*PPMH - Protein-Tyrosine-Phosphatase|*MEMH - Signal Transduction|*SO - Proc Natl Acad Sci U S A 1999 Aug; 96(17):9677-82DP - 1999 AugTA - Proc Natl Acad Sci U S APG - 9677-82IP - 17VI - 96UI - 9938057836
AU - Haft CR
AU - de la Luz Sierra M
AU - Barr VA
AU - Haft DH
AU - Taylor SITI - Identification of a family of sorting nexin molecules and characterization of their association with receptors.AB - Sorting nexin 1 (SNX1) is a protein that binds to the epidermal growth factor (EGF) receptor and is proposed to play a role in directing EGF receptors to lysosomes for degradation (R. C. Kurten, D. L. Cadena, and G. N. Gill, Science 272:1008-1010, 1996). We have obtained full-length cDNAs and deduced the amino acid sequences of three novel homologous proteins, which were denoted human sorting nexins (SNX2,SNX3, and SNX4). In addition, we identified a presumed splice variant isoform of SNX1 (SNX1A). These molecules contain a conserved domain of approximately 100 amino acids,which was termed the phox homology (PX) domain. Human SNX1 (522 amino acids), SNX1A (457 amino acids), SNX2 (519 amino acids), SNX3 (162 amino acids), and SNX4 (450 amino acids)are part of a larger family of hydrophilic molecules including proteins identified in Caenorhabditis elegans and Saccharomyces cerevisiae. Despite their hydrophilic nature, the sorting nexins are found partially associated with cellular membranes.They are widely expressed, although the tissue distribution of each sorting nexin mRNA varies. When expressed in COS7 cells, epitope-tagged sorting nexins SNX1, SNX1A, SNX2,and SNX4 coimmunoprecipitated with receptor tyrosine kinases for EGF, platelet-derived growth factor, and insulin. These sorting nexins also associated with the long isoform of the leptin receptor but not with the short and medium isoforms.Interestingly, endogenous COS7 transferrin receptors associated exclusively with SNX1 and SNX1A, while SNX3 was not found to associate with any of the receptors studied. Our demonstration of a large conserved family of sorting nexins that interact with a variety of receptor types suggests that these proteins may be involved in several stages of intracellular trafficking in mammalian cells.MH - Carrier Proteins|*CH/PHMH - Receptors, Epidermal Growth Factor-Urogastrone|*MESO - Mol Cell Biol 1998 Dec; 18(12):7278-87DP - 1998 DecTA - Mol Cell BiolPG - 7278-87IP - 12VI - 18UI - 9903823237
AU - Crouse JA
AU - Elliott GE
AU - Burgess TL
AU - Chiu L
AU - Bennett L
AU - Moore J
AU - Nicolson M
AU - Pacifici RETI - Altered cell surface expression and signaling of leptin receptors containing the fatty mutation.AB - Leptin and the leptin receptor are key players in the regulation of body weight. In an attempt to dissect the molecular mechanism of the Zucker fatty rat leptin receptor mutation (Gln269 --> Pro) we analyzed the effects of this mutation on leptin receptor signaling and expression in three different expression systems: 1) 32D cells expressing leptin/erythropoietin receptor chimeras, 2) COS-7 cells expressing a leptin receptor short form, and 3) 293 cells expressing soluble receptor forms. To determine if the Gln269 --> Pro mutation is critical for the observed phenotype, we made a similar Gln --> Pro mutation at a vicinal residue two amino acids upstream of the fatty mutation to see if it would have similar effects.Incorporation of either of the Gln --> Pro mutations into wild type receptor forms did not interfere with leptin binding, but it resulted in a signaling-incompetent receptor.In addition, the majority of the mutant receptor protein was localized intracellularly. Our results suggest that the obese phenotype resulting from the Gln269 --> Pro mutation in the leptin receptor of the Zucker fatty rat may be due not only to a reduced cell surface expression of this form of the leptin receptor, but also to a post-leptin binding malfunction of the receptor that interferes with subsequent signal transduction.MH - Carrier Proteins|*GE/MEMH - Signal Transduction|*/GESO - J Biol Chem 1998 Jul; 273(29):18365-73DP - 1998 JulTA - J Biol ChemPG - 18365-73IP - 29VI - 273UI - 9832504738
AU - Serradeil Le Gal C
AU - Raufaste D
AU - Brossard G
AU - Pouzet B
AU - Marty E
AU - Maffrand JP
AU - Le Fur GTI - Characterization and localization of leptin receptors in the rat kidney.AB - Characterization and localization of leptin binding sites were investigated in rat kidneys using [125I]leptin as a ligand. [125I]Leptin specific binding was found in high amounts in rat renomedullary membranes. This binding was specific, saturable, time-dependent (K(obs) = 0.055 +/-0.008 min(-1)) and the dissociation of receptor-bound ligand was slowly reversible (K(-1) = 0.048 +/- 0.013 min(-1)). From saturation experiments, a single class of high-affinity binding sites for leptin was identified with an apparent K(d) of 0.57 +/- 0.14 nM and a B(max) of 45 +/- 10 fmol/mg protein. [125I]Leptin binding was inhibited in a dose-dependent manner by cold leptin and was highly selective since not displaceable by a number of other hormones or peptides. Autoradiographic experiments performed on adult rat kidney sections showed the intense presence of [125I]leptin receptors only in specific areas of the renal inner medulla and also consistent labeling associated with vascular structures in the corticomedullary region. The study of the postnatal developmental expression of leptin receptors in the kidney showed very low expression during the early postnatal period (8-21 days). Full expression of leptin sites was achieved at about 30 days and remained stable throughout adulthood (60 days and upwards). Moreover, in vivo administration of leptin (0.5 mg/kg i.p.) induced a significant and rapid diuretic effect in normally hydrated conscious rats. Thus, these data constitute the first characterization and mapping of [125I]leptin specific binding sites in the rat kidney and raise the possibility of a renal control by leptin.MH - Aging|*MEMH - Carrier Proteins|*MEMH - Diuresis|*DEMH - Kidney|GD/*MEMH - Proteins|*ME/PDSO - FEBS Lett 1997 Mar; 404(2-3):185-91DP - 1997 MarTA - FEBS LettPG - 185-91IP - 2-3VI - 404UI - 9722794439
AU - Haniu M
AU - Arakawa T
AU - Bures EJ
AU - Young Y
AU - Hui JO
AU - Rohde MF
AU - Welcher AA
AU - Horan TTI - Human leptin receptor. Determination of disulfide structure and N-glycosylation sites of the extracellular domain.AB - The leptin receptor (OB-R) is a member of the class I cytokine receptor family and mediates the weight regulatory effects of its ligand through interaction with cytoplasmic kinases.The extracellular domain of this receptor is comprised of two immunoglobulin-like and cytokine-receptor homology domains each and type III fibronectin domains. The extracellular domain of human leptin receptor was expressed in and purified from Chinese hamster ovary cells and was found to contain extensive N-glycosylation (approximately 36% of the total protein). The purified protein had a molecular weight of approximately 145,000 and exhibited ligand binding ability as evidenced by formation of ligand-receptor complex, followed by chemical cross-linking. The determined disulfide motif of the soluble leptin receptor contained several distinct cystine knots as well as 10 free cysteines. The N-glycosylation analysis revealed that Asn624 of the WSXWS motif (residues 622-626) within the C-terminal cytokine receptor homology domain was glycosylated, indicating that this region is solvent-exposed. On the other hand, the N-terminal WSXWS motif was not glycosylated.MH - Carrier Proteins|CH/IP/*MEMH - Disulfides|*CHSO - J Biol Chem 1998 Oct; 273(44):28691-9DP - 1998 OctTA - J Biol ChemPG - 28691-9IP - 44VI - 273UI - 9900321140
AU - Devos R
AU - Guisez Y
AU - Van der Heyden J
AU - White DW
AU - Kalai M
AU - Fountoulakis M
AU - Plaetinck GTI - Ligand-independent dimerization of the extracellular domain of the leptin receptor and determination of the stoichiometry of leptin binding.AB - The leptin receptor is a class I transmembrane protein with either a short or a long cytoplasmic domain. Using chemical cross-linking we have analyzed the binding of leptin to its receptor. Cross-linking of radiolabeled leptin to different isoforms of the leptin receptor expressed on COS-1 cells reveals leptin receptor monomer, homodimer,and oligomer complexes. Cotransfection of the long and short form of the leptin receptor did not provide any evidence for the formation of heterodimer complexes. Soluble forms consisting of either the entire extracellular domain or the two cytokine receptor homologous domains of the leptin receptor were purified to homogeneity from recombinant baculovirus-infected insect cells by leptin affinity chromatography.Gel filtration chromatography showed that these proteins exist in a dimeric form. Analysis of the complex formed between soluble leptin receptor and leptin by native polyacrylamide gel electrophoresis, and data obtained from the amino acid composition of the complex provide direct evidence that the extracellular domain of the leptin receptor binds leptin in a 1:1 ratio.MH - Carrier Proteins|*CH/*MEMH - Proteins|*MESO - J Biol Chem 1997 Jul; 272(29):18304-10DP - 1997 JulTA - J Biol ChemPG - 18304-10IP - 29VI - 272UI - 9736476041
AU - Laron Z
AU - Silbergeld A
AU - Lilos P
AU - Blum FWTI - Serum leptin in obese patients with Laron syndrome before and during IGF-I treatment.AB - Fifteen patients with primary GH resistance (Laron syndrome,LS) were studied before and during 6 months of daily replacement treatment with IGF-I. The main findings were that patients with LS and normal or high serum GH binding protein (GHBP)were less obese than those with a negative GHBP, and that serum leptin levels varied with body mass as in other types of obesity.MH - Insulin-Like Growth Factor I|AN/DF/*TUMH - Mutation|*MH - Obesity|*BL/DT/*GEMH - Proteins|*ANMH - Receptors, Somatotropin|*GEAD - Endocrine & Diabetes Research UnitAD - Schneider Children's Medical Center of IsraelAD - Petah TikvaAD - Israel.SO - J Pediatr Endocrinol Metab 1998 Sep-Oct; 11(5):653-6DP - 1998 Sep-OctTA - J Pediatr Endocrinol MetabPG - 653-6IP - 5VI - 11UI - 9904665742
AU - Commins SP
AU - Marsh DJ
AU - Thomas SA
AU - Watson PM
AU - Padgett MA
AU - Palmiter R
AU - Gettys TWTI - Norepinephrine is required for leptin effects on gene expression in brown and white adipose tissue.AB - Exogenous leptin enhances energy utilization in ob/ob mice by binding its hypothalamic receptor and selectively increasing peripheral fat oxidation. Leptin also increases uncoupling protein 1 (UCP1) expression in brown adipose tissue (BAT), but the neurotransmitter that mediates this effect has not been established. The present experiments sought to determine whether leptin regulates UCP1 expression in BAT and its own expression in white adipose tissue (WAT) through the long or short forms of leptin receptor and modulation of norepinephrine release. Mice lacking dopamine beta-hydroxylase (Dbh-/-), the enzyme responsible for synthesizing norepinephrine and epinephrine from dopamine, were treated with leptin (20 microg/g body weight/day) for 3 days before they were euthanized. UCP1 messenger RNA (mRNA) and protein expression were 5-fold higher in BAT from control (Dbh+/-) compared with Dbh-/- mice. Leptin produced a 4-fold increase in UCP1 mRNA levels in Dbh+/- mice but had no effect on UCP1 expression in Dbh-/-. The beta3-adrenergic agonist, CL-316,243 increased UCP1 expression and established that BAT from both groups of mice was capable of responding to beta-adrenergic stimulation. Similarly, exogenous leptin reduced leptin mRNA in WAT from Dbh+/- but not Dbh-/- mice.In separate experiments, leptin produced comparable reductions in food intake in both Dbh+/- and Dbh-/- mice, illustrating that norepinephrine is not required for leptin's effect on food intake. Lastly, db/db mice lacking the long form of the leptin receptor failed to increase UCP1 mRNA in response to exogenous leptin but increased UCP1 mRNA in response to CL-316,243. These studies establish that norepinephrine is required for leptin to regulate its own expression in WAT and UCP1 expression in BAT and indicate that these effects are likely mediated through the centrally expressed long form of the leptin receptor.MH - Adipose Tissue|*PHMH - Brown Fat|*PHMH - Carrier Proteins|*MEMH - Gene Expression|*PHMH - Membrane Proteins|*MEMH - Norepinephrine|*PHMH - Proteins|GE/PD/*PHSO - Endocrinology 1999 Oct; 140(10):4772-8DP - 1999 OctTA - EndocrinologyPG - 4772-8IP - 10VI - 140UI - 9942788343
AU - Florkowski CM
AU - Barnard R
AU - Livesey JH
AU - Veveris T
AU - Espiner EA
AU - Donald RATI - Growth hormone binding protein correlates strongly with leptin and percentage body fat in GH-deficient adults, is increased by GH replacement but does not predict IGF-I response.AB - GH-binding protein (GHBP) corresponds to the extracellular domain of the GH receptor (GHR) and has been shown to be closely related to body fat. This study aimed to examine the inter-relationship between GHBP, leptin and body fat,and to test the hypothesis that GHBP is modified by GH replacement in GH-deficient adults and predicts IGF-I response.Twenty adults, mean age 47 years (range 20-69) with proven GH deficiency were randomly allocated to either GH (up to 0.25 U/kg/week in daily doses) or placebo for 3 months before cross-over to the opposite treatment. Plasma GHBP and leptin were measured at baseline and 2, 4, 8 and 12 weeks after each treatment. Whole body composition was measured at baseline by dual-energy X-ray absorptiometry (DEXA). There was a strong correlation between baseline leptin and GHBP (r = 0.88, P < 0.0001) and between baseline GHBP and percentage body fat, (r = 0.83, P < 0.0001). Mean GHBP levels were higher on GH compared with placebo, 1.53 +/- 0.28 vs 1.41 +/- 0.25nM, P = 0.049. There was no correlation between baseline IGF-I and GHBP (r = -0.049,P = 0.84), and GHBP did not predict IGF-I response to GH replacement. The close inter-relationship between GHBP,leptin and body fat suggests a possible role for GHBP in the regulation of body composition. GHBP is increased by GH replacement in GH-deficient adults, but does not predict biochemical response to GH replacement.MH - Adipose Tissue|*AHMH - Body Composition|*MH - Carrier Proteins|*BLMH - Hormone Replacement Therapy|*MH - Hypopituitarism|ET/PA/*PP/THMH - Insulin-Like Growth Factor I|*ME/SEMH - Proteins|*ME/SEMH - Somatropin|*DF/*TUSO - Growth Horm IGF Res 1999 Feb; 9(1):35-40DP - 1999 FebTA - Growth Horm IGF ResPG - 35-40IP - 1VI - 9UI - 9922391344
AU - Igel M
AU - Becker W
AU - Herberg L
AU - Joost HGTI - Hyperleptinemia, leptin resistance, and polymorphic leptin receptor in the New Zealand obese mouse.AB - New Zealand Obese (NZO) mice exhibit a polygenic syndrome of hyperphagia, obesity, hyperinsulinemia, and hyperglycemia similar to that observed in young diabetes mutant mice on the C57BLKS/J background (C57BLKS/J-Lepr(db)/Lepr(db)). Here we show that in NZO this syndrome is accompanied by a marked elevation of the leptin protein in adipose tissue and serum. The promoter region and the complementary DNA of the ob gene of NZO mice, including its 5'-untranslated region, are identical with the wild-type sequence (C57BL,BALB/c), except that the transcription start is located 5 bp upstream of the reported site. In contrast to C57BLKS/J+/+ and C57BL/6J-Lep(ob)/Lep(ob) mice, NZO mice failed to respond to recombinant leptin (7.2 microg/g) with a reduction of food intake. Leptin receptor messenger RNA as detected by PCR appears as abundant in hypothalamic tissue of NZO mice as in tissue from lean mice. Ten nucleotide polymorphisms are found in the complementary DNA of the leptin receptor, resulting in two conservative substitutions (V541I and V651I) in the extracellular part of the receptor and one nonconservative substitution (T1044I) in the intracellular domain between the presumed Jak and STAT binding boxes.However, these mutations are also present in the related lean New Zealand Black strain (body fat at 9 weeks: New Zealand Black, 6.2 +/- 1.3%; NZO, 17.0 +/- 1.7%). Thus,the polymorphic leptin receptor seems to play only a minor,if any, role in the obesity and hyperleptinemia of the NZO mouse. It is suggested that the main defect in NZO is located distal from the leptin receptor or at the level of leptin transport into the central nervous system.MH - Carrier Proteins|AN/GE/*MEMH - Obesity|*ME/PPMH - Polymorphism (Genetics)|*MH - Proteins|GE/*ME/*PDSO - Endocrinology 1997 Oct; 138(10):4234-9DP - 1997 OctTA - EndocrinologyPG - 4234-9IP - 10VI - 138UI - 9746270845
AU - Llopis MA
AU - Granada ML
AU - Cuatrecasas G
AU - Formiguera X
AU - Sßnchez Planell L
AU - Sanmart A
AU - Alastru A
AU - Rull M
AU - Corominas A
AU - Foz MTI - Growth hormone-binding protein directly depends on serum leptin levels in adults with different nutritional status.AB - The aim of this work was to assess the relationship between GH-binding protein (GHBP) and leptin. Both peptides are nutritionally regulated, but the recent implication of a role for leptin in the GH axis requires further study.To avoid the sexual dimorphism in leptin values, we performed leptin standardization according to gender (SD score-leptin). The relationship between SD score-leptin and GHBP was studied in 128 adults with different nutritional status [8 groups according to body mass index (BMI)], ranging from severely underweight anorexia nervosa to highly morbid obesity. Both GHBP and SD score-leptin significantly increased according to BMI within the range from 18-27 kg/m2, whereas no significant differences were found among underweight groups (BMI, < 18 kg/m2) or among obesity grades (BMI, > 27 kg/m2). We found a strong correlation between GHBP and SD score-leptin (r = 0.8; P < 0.0001). Multiple regression analysis revealed SD score-leptin to be a significant determinant of GHBP, accounting for 64% of the variation, whereas BMI did not contribute further to explaining changes in GHBP.This suggests a physiological pathway involving both GHBP (the soluble fraction of GH receptor) and leptin. Thus,we might speculate that leptin could be the signal that induces the related nutritional changes observed in GHBP/GH receptor expression.MH - Carrier Proteins|*MEMH - Nutritional Status|*MH - Proteins|*MESO - J Clin Endocrinol Metab 1998 Jun; 83(6):2006-11DP - 1998 JunTA - J Clin Endocrinol MetabPG - 2006-11IP - 6VI - 83UI - 9828937246
AU - Bjarnason R
AU - Boguszewski M
AU - Dahlgren J
AU - Gelander L
AU - Kristr÷m B
AU - Rosberg S
AU - Carlsson B
AU - Albertsson Wikland K
AU - Carlsson LMTI - Leptin levels are strongly correlated with those of GH-binding protein in prepubertal children.AB - OBJECTIVE: Nutritional status is an important determinant of growth, and previous studies have indicated that this is due, at least in part, to an increased target-tissue sensitivity to GH. An attractive candidate for mediating this effect is leptin, a hormone secreted by the adipose tissue. The aim of this study was to investigate if there was a connection between GH-binding protein (GHBP) and leptin. DESIGN AND METHODS: We investigated the relationship between serum levels of leptin and those of GHBP in 229 prepubertal children. These included 107 healthy children with normal GH secretion, 55 GH-deficient (GHD) children and 55 children born small for gestational age (SGA) sampled on one occasion for GHBP and leptin, and 12 healthy children followed longitudinally at monthly interval for 1 year.RESULTS: In the healthy children and in those born SGA,the serum concentration of GHBP was positively correlated with that of leptin (r = 0.65, P < 0.001; r = 0.74, P <0.001 respectively). There was no correlation between GHBP and leptin in the group of children with GHD (r = 0.27,not significant). This means that leptin alone explained 42% of the variation of GHBP in the healthy group and 55%in the SGA group. The correlation remained after adjustment for body mass index and age in the healthy children (r = 0.57, P < 0.0001, r2 = 0.33) and for children born SGA (r = 0.74, P < 0.0001, r2 = 0.55). There was a positive correlation between the intra-individual monthly changes in GHBP and changes in leptin respectively, in the 12 healthy children followed longitudinally, the mean of the correlation coefficients was 0.38 (median = 0.29; range 0.03 to 0.86;P < 0.05). CONCLUSIONS: There was a highly significant correlation between serum levels of leptin and those of GHBP, except in children with GHD. The possibility that leptin could mediate the effects of body fat mass on GH sensitivity, therefore, merits further investigation.MH - Carrier Proteins|*BLMH - Proteins|*MESO - Eur J Endocrinol 1997 Jul; 137(1):68-73DP - 1997 JulTA - Eur J EndocrinolPG - 68-73IP - 1VI - 137UI - 9738622047
AU - Schffler A
AU - Palitzsch KD
AU - Watzlawek E
AU - Drobnik W
AU - Schwer H
AU - Sch÷lmerich J
AU - Schmitz GTI - Frequency and significance of the A-->G (-3826) polymorphism in the promoter of the gene for uncoupling protein-1 with regard to metabolic parameters and adipocyte transcription factor binding in a large population-based Caucasian cohort.AB - BACKGROUND: The recently described A-->G (-3826) point mutation within the distal region of the UCP-1 promoter is possibly involved in the development of obesity, diabetes and related metabolic disorders. It was the aim of this study to examine the allelic frequency and the prevalence of the three UCP-1 genotypes in a broad caucasian cohort and to investigate the significance of this polymorphism for obesity and diabetes. METHODS: 1020 subjects were randomly chosen from 6450 participants in the Diabetomobile Study.The UCP-1 genotype was determined by genomic PCR and Bcl-I-RFLP analysis in 1020 subjects and tested for association with a variety of metabolic parameters. In addition, the influence of this mutation on adipocyte nuclear factor binding was investigated by electrophoretic mobility shift assays (EMSA). RESULTS: The genotype frequencies in 1020 subjects were: AA genotype, 57.0%; AG genotype, 35.4%; GG genotype, 7.6%; with allelic frequencies of 0.75 for allele A and 0.25 for allele G. No significant differences between the genotypes and age, gender, BMI, leptin, glucose,fasting insulin, C-peptide, HbA1c, diabetes manifestation,total cholesterol, and HDL cholesterol were found. Analysis of the Trp64Arg polymorphism of the beta3-adrenergic receptor in a subgroup of 343 subjects revealed no additive effect to the UCP-1 polymorphism. An yet unknown adipocyte-specific factor of nuclear extracts from 3T3-L1 adipocytes during differentiation is able to bind specifically to the distal UCP-1 promoter region and this binding ability can not be abolished by the mutation. CONCLUSIONS: We determined the genotype and allelic frequency of the UCP-1 promoter polymorphism in the largest known population-based study.The results from genotyping demonstrate clearly that this polymorphism does not play a major role in the pathogenesis obesity and diabetes. A yet unknown adipocyte derived and differentiation-dependent regulated transcription factor is able to bind to the distal UCP-1 promoter surrounding -3826 bp. This binding is not affected by presence of the mutation.MH - Carrier Proteins|*GEMH - Caucasoid Race|*GEMH - Diabetes Mellitus, Non-Insulin-Dependent|*GEMH - Membrane Proteins|*GEMH - Obesity|*GEMH - Polymorphism (Genetics)|*SO - Eur J Clin Invest 1999 Sep; 29(9):770-9DP - 1999 SepTA - Eur J Clin InvestPG - 770-9IP - 9VI - 29UI - 9939836748
AU - Cao GY
AU - Considine RV
AU - Lynn RBTI - Leptin receptors in the adrenal medulla of the rat.AB - Leptin is the protein product of the recently cloned obesity gene. Leptin receptor mRNA is found in a number of central and peripheral locations. The hypothalamus is a presumed site of action. However, little is known about the specific locations of the receptor in peripheral organs. Epinephrine has potent anorectic effects and can cause weight loss by a variety of mechanisms. Excretion of epinephrine is reduced in the ob/ob mouse, which lacks leptin, suggesting an effect by leptin on the adrenal medulla. In the current study, the presence of the leptin receptor was identified on epinephrine-secreting cells in the adrenal medulla. Immunohistochemical studies found dense leptin receptor-like immunoreactivity in the adrenal medulla with no labeling in the adrenal cortex. Double immunofluorescent labeling confirmed that the leptin receptor was present on cells that were phenylethanolamine N-methyltransferase-like immunoreactive and therefore were epinephrine-secreting cells. Leptin receptor mRNA in the adrenal medulla was detected by reverse transcriptase-polymerase chain reaction, with the majority of the mRNA coding for the short isoform (Ob-Ra) of the receptor. Finally, autoradiography was performed using 125I-labeled leptin; specific binding was found in the adrenal medulla, with no specific binding in the adrenal cortex. These results suggest that leptin may have a direct effect on epinephrine-secreting cells in the adrenal medulla.Epinephrine may play a role in mediating the effects of leptin to reduce body weight.MH - Adrenal Medulla|*MEMH - Carrier Proteins|GE/*MESO - Am J Physiol 1997 Aug; 273(2 Pt 1):E448-52DP - 1997 AugTA - Am J PhysiolPG - E448-52IP - 2 Pt 1VI - 273UI - 9742345849
AU - Ceddia RB
AU - William WN Jr
AU - Lima FB
AU - Carpinelli AR
AU - Curi RTI - Pivotal role of leptin in insulin effects.AB - The OB protein, also known as leptin, is secreted by adipose tissue, circulates in the blood, probably bound to a family of binding proteins, and acts on central neural networks regulating ingestive behavior and energy balance. The two forms of leptin receptors (long and short forms) have been identified in various peripheral tissues, a fact that makes them possible target sites for a direct action of leptin.It has been shown that the OB protein interferes with insulin secretion from pancreatic islets, reduces insulin-stimulated glucose transport in adipocytes, and increases glucose transport, glycogen synthesis and fatty acid oxidation in skeletal muscle. Under normoglycemic and normoinsulinemic conditions, leptin seems to shift the flux of metabolites from adipose tissue to skeletal muscle. This may function as a peripheral mechanism that helps control body weight and prevents obesity. Data that substantiate this hypothesis are presented in this review.MH - Adipose Tissue|*MEMH - Insulin|*SEMH - Islets of Langerhans|*MEMH - Muscle, Skeletal|*MEMH - Proteins|*PHSO - Braz J Med Biol Res 1998 Jun; 31(6):715-22DP - 1998 JunTA - Braz J Med Biol ResPG - 715-22IP - 6VI - 31UI - 9836397150
AU - Saito K
AU - Tobe T
AU - Minoshima S
AU - Asakawa S
AU - Sumiya J
AU - Yoda M
AU - Nakano Y
AU - Shimizu N
AU - Tomita MTI - Organization of the gene for gelatin-binding protein (GBP28).AB - GBP28 is a novel human plasma gelatin-binding protein that is encoded by apM1 mRNA, expressed specifically in adipose tissue. Three overlapping clones (two lambda clones and one BAC clone) containing the human plasma gelatin-binding protein (GBP28) gene were isolated and characterized. The GBP28 gene spans 16kb and is composed of three exons from 18bp to 4277bp in size with consensus splice sites. The sizes of the two introns were 0.8 and 12kb, respectively.The gene's regulatory sequences contain putative promoter elements, but no typical TATA box.The third exon of this gene contains a long 3'-untranslated sequence containing three Alu repeats. The exon-intron organization of this gene was very similar to that of obese gene, encoding leptin.We also report the chromosome mapping of this gene by fluorescence in situ hybridization (FISH) using a genomic DNA fragment as a probe. The GBP28 gene was located on human chromosome 3q27. The nucleotide sequence data reported in this paper will appear in the DDBJ/EMBL/GenBank nucleotide sequence databases with the accession numbers ABO12163, ABO12164 or ABO12165.MH - Carrier Proteins|*GESO - Gene 1999 Mar; 229(1-2):67-73DP - 1999 MarTA - GenePG - 67-73IP - 1-2VI - 229UI - 9919698451
AU - Kratzsch J
AU - Dehmel B
AU - Pulzer F
AU - Keller E
AU - Englaro P
AU - Blum WF
AU - Wabitsch MTI - Increased serum GHBP levels in obese pubertal children and adolescents: relationship to body composition, leptin and indicators of metabolic disturbances.AB - OBJECTIVE: The serum concentration of the high-affinity growth hormone-binding protein (GHBP) is increased in obesity but the mechanisms are poorly understood. This study assessed the physiological mechanisms involved in the regulation of GHBP in adiposity. SUBJECTS AND MEASUREMENTS: We tested a number of obesity specific parameters for their association with GHBP. In this study, 199 normal or overweight children and adolescents (101 boys, 98 girls, aged (mean +/- s.d.): 13.7 +/- 2.3 y) underwent an anthropometric evaluation (circumference measurements and bioimpedance analysis) combined with blood withdrawal for the measurement of insulin-like growth factor-I (IGF-I), insulin, leptin and GHBP (by specific RIA), uric acid, triglycerides and cholesterol.RESULTS: By linear regression analysis GHBP correlated significantly (P < 0.001) with percent body fat mass (r = 0.71), waist (r = 0.73) and hip (r = 0.69) circumference,weight (r = 0.61) waist hip ratio (WHR) (r = 0.54), as well as with the serum concentrations of leptin (r = 0.64), uric acid (r = 0.54), insulin (r = 0.45), LDL-cholesterol (r = 0.43), cholesterol (r =0.33), LDL/HDL ratio (r = 0.47), triglycerides (r = 0.30) and with height standard deviations scores (SDS) (r = 0.23). Age, gender and pubertal stage had no impact on GHBP. In a multiple regression analysis containing age and gender, as well as the anthropometric variables, percent fat mass and waist circumference, as independent variables, associations between GHBP and leptin (P < 0.001), cholesterol (P < 0.01), LDL-cholesterol (P = 0.01), LDL/HDL ratio (P = 0.02), triglycerides (P = 0.01) remained significant. In a final model using the stepwise analysis involving age, gender and all the independent predictors of GHBP, waist circumference (P < 0.001), accounted for 49.5% of the 60.0% total variability in GHBP, while the implication of leptin (P < 0.001), age (P < 0.01) and cholesterol (P < 0.05) increased the predicted variability for 7.5%, 1.9%, and 1.0%, respectively. Serum GHBP was significantly reduced in a subgroup of 104 overweight or obese patients during a diet-induced weight loss programme,the coefficient of correlation between GHBP and leptin after (r = 0.45, P < 0.001) and before weight reduction (r = 0.41, P < 0.001) were comparable. CONCLUSION: Waist circumference, an indicator of abdominal body fat mass,is a major determinant of GHBP levels during childhood,while leptin may be one candidate for a signal linking adipocytes to the growth hormone receptor related GHBP release. Additionally, elevated serum levels of GHBP may reflect metabolic disturbances of adiposity.MH - Aging|*BL/ME/PHMH - Body Composition|*MH - Carrier Proteins|*BLMH - Obesity|*BL/ME/PPMH - Proteins|*MEMH - Puberty|*BL/ME/PHSO - Int J Obes Relat Metab Disord 1997 Dec; 21(12):1130-6DP - 1997 DecTA - Int J Obes Relat Metab DisordPG - 1130-6IP - 12VI - 21UI - 9808776052
AU - Williams LM
AU - Adam CL
AU - Mercer JG
AU - Moar KM
AU - Slater D
AU - Hunter L
AU - Findlay PA
AU - Hoggard NTI - Leptin receptor and neuropeptide Y gene expression in the sheep brain.AB - Leptin, a protein secretory product of adipocytes, is important in appetite control, energy balance and reproduction. In rodents, the physiological effects of leptin are centrally mediated, in part via the neuropeptide Y (NPY) system in the hypothalamus. The role of leptin in ruminants, where appropriate nutrition and reproductive status are of major economic concern, is largely unknown. To elucidate the function of leptin in sheep we have investigated putative sites of action for leptin in the brain and pituitary gland using both in-situ hybridization to detect expression of the signalling form of the leptin receptor (OB-Rb) and in-vitro autoradiography using (125I)leptin to detect sites of specific leptin binding. OB-Rb gene expression occurred in the hippocampus, cerebral cortex, preoptic area, stria terminalis and choroid plexus, and within the hypothalamus in the paraventricular (PVN), ventromedial (VHM) and arcuate (ARC) nuclei. OB-Rb gene expression in the ovine pituitary gland was not detected by in-situ hybridization. Sites of OB-Rb and NPY gene expression were compared using both in-situ hybridization on adjacent sections containing the arcuate and ventromedial nuclei, and dual in-situ hybridization on sections containing these areas. In serial sections,OB-Rb expression was found to correspond closely with that of NPY over the arcuate nuclei. Using dual in-situ hybridization,NPY expressing neurones in the arcuate nuclei were also positive for OB-Rb gene expression. Therefore, it appears that leptin may partly act via OB-Rb located on NPY neurones in the sheep hypothalamus as in the rodent.MH - Brain|*MEMH - Carrier Proteins|*GEMH - Gene Expression|*MH - Neuropeptide Y|*GESO - J Neuroendocrinol 1999 Mar; 11(3):165-9DP - 1999 MarTA - J NeuroendocrinolPG - 165-9IP - 3VI - 11UI - 9921599453
AU - Rosenblum CI
AU - Tota M
AU - Cully D
AU - Smith T
AU - Collum R
AU - Qureshi S
AU - Hess JF
AU - Phillips MS
AU - Hey PJ
AU - Vongs A
AU - Fong TM
AU - Xu L
AU - Chen HY
AU - Smith RG
AU - Schindler C
AU - Van der Ploeg LHTI - Functional STAT 1 and 3 signaling by the leptin receptor (OB-R); reduced expression of the rat fatty leptin receptor in transfected cells.AB - The leptin receptor (OB-R) bears homology to members of the class I cytokine receptor family. We demonstrate that leptin binding to OB-R stimulates formation of STAT-1 and STAT-3 complexes, thereby defining transcriptional motifs for genes that are under leptin control. Transfected fa OB-R bound leptin with equal affinity to that of wild type OB-R. fa OB-R abundance was about 7 fold reduced compared to control cells. Surprisingly, the low level of fa OB-R is fully capable of activating the STAT signal transduction pathway. We discuss plausible explanations for the obese phenotype in Zucker fatty rats.MH - Carrier Proteins|*BI/*PHMH - DNA-Binding Proteins|*MEMH - Signal Transduction|*MH - Trans-Activators|*MESO - Endocrinology 1996 Nov; 137(11):5178-81DP - 1996 NovTA - EndocrinologyPG - 5178-81IP - 11VI - 137UI - 9705066854
AU - Hamann A
AU - Matthaei STI - Regulation of energy balance by leptin.AB - The high prevalence of obesity and its well documented association with the cardiovascular risk factors diabetes mellitus, dyslipidemia and hypertension represents a major problem for the general health status of industrialized societies. Although numerous studies have shown that genetic factors have a major influence on the regulation of energy homeostasis and the susceptibility to obesity, the genes and predisposing mutations involved are insufficiently understood. Among several known rodent models of obesity due to single gene mutations, mice homozygous for the obese (ob) gene exhibit massive early-onset obesity, hyperphagia,non-insulin-dependent diabetes mellitus, defective thermoregulation and infertility. Recently the ob gene was identified by positional cloning and shown to be mutated in ob/ob mice.Leptin, the product of the ob gene, is a 167-amino acid secreted protein that is synthesized exclusively in adipose tissue. With the exception of ob/ob mice, circulating plasma leptin is elevated in obesity. Administration of recombinant leptin to ob/ob mice reduces fat mass, food intake, hyperglycemia and hyperinsulinemia. The various effects of the hormone are mediated by leptin receptors expressed at high levels in the hypothalamus, but also in several other non-neuronal tissues. A mutation in the leptin receptor gene is responsible for the obese phenotype of db/db mice. Plasma leptin in humans is positively correlated with body fat mass, suggesting that leptin resistance rather than leptin deficiency is a common feature of human obesity. This review briefly summarizes the current status of the rapidly growing evidence that leptin plays an important role in the regulation of body weight and fat deposition.MH - Adipocytes|*MEMH - Carrier Proteins|GE/*MEMH - Energy Metabolism|*PHMH - Obesity|*ET/GE/MEMH - Proteins|AD/BI/*MESO - Exp Clin Endocrinol Diabetes 1996; 104(4):293-300DP - 1996TA - Exp Clin Endocrinol DiabetesPG - 293-300IP - 4VI - 104UI - 9704147455
AU - Cohen B
AU - Novick D
AU - Rubinstein MTI - Modulation of insulin activities by leptin [see comments]AB - Leptin mediates its effects on food intake through the hypothalamic form of its receptor OB-R. Variants of OB-R are found in other tissues, but their function is unknown.Here, an OB-R variant was found in human hepatic cells.Exposure of these cells to leptin, at concentrations comparable with those present in obese individuals, caused attenuation of several insulin-induced activities, including tyrosine phosphorylation of the insulin receptor substrate-1 (IRS-1), association of the adapter molecule growth factor receptor-bound protein 2 with IRS-1, and down-regulation of gluconeogenesis.In contrast, leptin increased the activity of IRS-1-associated phosphatidylinositol 3-kinase. These in vitro studies raise the possibility that leptin modulates insulin activities in obese individuals.MH - Insulin|*PDMH - Proteins|ME/*PDSO - Science 1996 Nov; 274(5290):1185-8DP - 1996 NovTA - SciencePG - 1185-8IP - 5290VI - 274UI - 9705360456
AU - Schwartz MW
AU - Seeley RJ
AU - Campfield LA
AU - Burn P
AU - Baskin DGTI - Identification of targets of leptin action in rat hypothalamus.AB - The hypothesis that leptin (OB protein) acts in the hypothalamus to reduce food intake and body weight is based primarily on evidence from leptin-deficient, ob/ob mice. To investigate whether leptin exerts similar effects in normal animals,we administered leptin intracerebroventricularly (icv) to Long-Evans rats. Leptin administration (3.5 microg icv)at the onset of nocturnal feeding reduced food intake by 50% at 1 h and by 42% at 4 h, as compared with vehicle-treated controls (both P < 0.05). To investigate the basis for this effect, we used in situ hybridization (ISH) to determine whether leptin alters expression of hypothalamic neuropeptides involved in energy homeostasis. Two injections of leptin (3.5 microg icv) during a 40 h fast significantly decreased levels of mRNA for neuropeptide Y (NPY, which stimulates food intake) in the arcuate nucleus (-24%) and increased levels of mRNA for corticotrophin releasing hormone (CRH, an inhibitor of food intake) in the paraventricular nucleus (by 38%) (both P < 0.05 vs. vehicle-treated controls). To investigate the anatomic basis for these effects, we measured leptin receptor gene expression in rat brain by ISH using a probe complementary to mRNA for all leptin receptor splice variants. Leptin receptor mRNA was densely concentrated in the arcuate nucleus, with lower levels present in the ventromedial and dorsomedial hypothalamic nuclei and other brain areas involved in energy balance.These findings suggest that leptin action in rat hypothalamus involves altered expression of key neuropeptide genes, and implicate leptin in the hypothalamic response to fasting.MH - Corticotropin-Releasing Hormone|GE/*MEMH - Eating|*DEMH - Gene Expression Regulation|*DEMH - Hypothalamus|AH/*DE/MEMH - Neuropeptide Y|GE/*MEMH - Proteins|*PDSO - J Clin Invest 1996 Sep; 98(5):1101-6DP - 1996 SepTA - J Clin InvestPG - 1101-6IP - 5VI - 98UI - 9637965857
AU - Considine RV
AU - Caro JFTI - Leptin: genes, concepts and clinical perspective.AB - Obesity is a complex disease which results from the interaction of multiple genes and the environment. The recently discovered genes for leptin (ob gene) and the leptin receptor appear to play a major regulatory role in body energy balance and adipose tissue deposition. Furthermore, defects in the ob gene and leptin receptor gene have been demonstrated to be the cause of obesity in several rodent models. These observations raise the possibility that human obesity may also be due to defects in the leptin signal system. This review will summarize the current findings on the ob gene,leptin and the leptin receptor in both animals and humans.These observations will be discussed in the context of potential defects in the system and the possibility that these defects result in obesity in humans.MH - Carrier Proteins|*MEMH - Obesity|*/BL/GE/MEMH - Proteins|*/AN/GE/MEMH - Receptors, Cytokine|*MESO - Horm Res 1996; 46(6):249-56DP - 1996TA - Horm ResPG - 249-56IP - 6VI - 46UI - 9713739758
AU - Bennett BD
AU - Solar GP
AU - Yuan JQ
AU - Mathias J
AU - Thomas GR
AU - Matthews WTI - A role for leptin and its cognate receptor in hematopoiesis.AB - BACKGROUND. Hematopoiesis entails the production of multiple blood cell lineages throughout the lifespan of the organism.This is accomplished by the regulated expansion and differentiation of hematopoietic precursors that originate from self-renewing hematopoietic stem cells. Studies of lineage commitment and proliferation have shown that the cytokine family of growth factors plays an important role in hematopoietic differentiation. However, in hematopoiesis, as in most self-renewing biological systems, the molecules that regulate the stem cells directly remain largely unknown. In this study, we have undertaken a search for novel cytokines that may influence the fate of hematopoietic stem cells.RESULTS. We have cloned three splice variants of a novel cytokine receptor from human hematopoietic stem cells expressing the CD34 antigen, one of which is identical to the leptin receptor. Expression analysis revealed that the leptin receptor is expressed in both human and murine hematopoietic stem cell populations, and that leptin is expressed by hematopoietic stroma. We show that leptin provides a proliferative signal in hematopoietic cells. Importantly, we demonstrate that leptin provides a proliferative signal in BAF-3 cells and increases the proliferation of hematopoietic stem cell populations. The proliferative effects of leptin seem to be at the level of a multilineage progenitor, as shown by increased myelopoiesis, erythropoiesis and lymphopoiesis.Analysis of db/db mice, in which the leptin receptor is truncated, revealed that the steady-state levels of peripheral blood B cells and CD4-expressing T cells were dramatically reduced, demonstrating that the leptin pathway plays an essential role in lymphopoiesis. Colony assays performed using marrow from db/db and wild-type mice indicated that db/db marrow has a deficit in lymphopoietic progenitors;furthermore, db/db mice are unable to fully recover the lymphopoietic population following irradiation insult, and although the levels of peripheral blood erythrocytes are normal in db/db mice, spleen erythrocyte production is severely compromized. CONCLUSIONS. We have discovered that leptin and its cognate receptor constitute a novel hematopoietic pathway that is required for normal lymphopoiesis.This pathway seems to act at the level of the hematopoietic stem/progenitor cell, and may well also impact upon erythropoiesis,particularly in anemic states that may require output from the spleen. These findings offer a new perspective on the role of the fat cell in hematopoiesis.MH - Carrier Proteins|GE/*PHMH - Hematopoiesis|*PHMH - Proteins|*PHSO - Curr Biol 1996 Sep; 6(9):1170-80DP - 1996 SepTA - Curr BiolPG - 1170-80IP - 9VI - 6UI - 9639896859
AU - Gainsford T
AU - Willson TA
AU - Metcalf D
AU - Handman E
AU - McFarlane C
AU - Ng A
AU - Nicola NA
AU - Alexander WS
AU - Hilton DJTI - Leptin can induce proliferation, differentiation, and functional activation of hemopoietic cells.AB - Many cytokines exert their biological effect through members of the hemopoietin receptor family. Using degenerate oligonucleotides to the common WSXWS motif, we have cloned from human hemopoietic cell cDNA libraries various forms of the receptor that was recently shown to bind the obesity hormone, leptin.mRNAs encoding long and short forms of the human leptin receptor were found to be coexpressed in a range of human and murine hemopoietic organs, and a subset of cells from these tissues bound leptin at the cell surface. Ectopic expression in murine Ba/F3 and M1 cell lines revealed that the long, but not the short, form of the leptin receptor can signal proliferation and differentiation, respectively.In cultures of murine or human marrow cells, human leptin exhibited no capacity to stimulate cell survival or proliferation,but it enhanced cytokine production and phagocytosis of Leishmania parasites by murine peritoneal macrophages. Our data provide evidence that, in addition to its role in fat regulation, leptin may also be able to regulate aspects of hemopoiesis and macrophage function.MH - Carrier Proteins|GE/*MEMH - Hematopoietic Stem Cells|*CY/DE/MEMH - Proteins|*PDSO - Proc Natl Acad Sci U S A 1996 Dec; 93(25):14564-8DP - 1996 DecTA - Proc Natl Acad Sci U S APG - 14564-8IP - 25VI - 93UI - 9712142660
AU - Caro JF
AU - Kolaczynski JW
AU - Nyce MR
AU - Ohannesian JP
AU - Opentanova I
AU - Goldman WH
AU - Lynn RB
AU - Zhang PL
AU - Sinha MK
AU - Considine RVTI - Decreased cerebrospinal-fluid/serum leptin ratio in obesity:a possible mechanism for leptin resistance [see comments]AB - BACKGROUND: A receptor for leptin has been cloned from the choroid plexus, the site of cerebrospinal-fluid (CSF)production and the location of the blood/cerebrospinal-fluid barrier. Thus, this receptor might serve as a transporter for leptin. We have studied leptin concentrations in serum and (CSF). METHODS AND FINDINGS: We demonstrated by radioimmunoassay and western blot the presence of leptin in human CSF. We then measured leptin in CSF and serum in 31 individuals with a wide range of bodyweight. Mean serum leptin was 318% higher in 8 obese (40.2 [SE 8.6] ng/mL) than in 23 lean individuals (9.6 [1.5] ng/mL, p < 0.0005). However,the CSF leptin concentration in obese individuals (0.337 [0.04] ng/mL) was only 30% higher than in lean people (0.259 [0.26] ng/mL, p < 0.1). Consequently, the leptin CSF/serum ratio in lean individuals (0.047 [0.010]) was 4.3-fold higher than that in obese individuals (0.011 [0.002], p < 0.05). The relation between CSF leptin and serum leptin was best described by a logarithmic function (r = 0 x 52, p < 0.01). INTERPRETATION: Our data suggest that leptin enters the brain by a saturable transport system.The capacity of leptin transport is lower in obese individuals,and may provide a mechanism for leptin resistance.MH - Obesity|*MEMH - Proteins|*ME/PHSO - Lancet 1996 Jul; 348(9021):159-61DP - 1996 JulTA - LancetPG - 159-61IP - 9021VI - 348UI - 9629529761
AU - Malik KF
AU - Young WS 3rdTI - Localization of binding sites in the central nervous system for leptin (OB protein) in normal, obese (ob/ob), and diabetic (db/db) C57BL/6J mice.AB - Leptin (OB protein) fused to the FLAG epitope and a kinase recognition site was expressed in bacteria, immunopurified,and phosphorylated using [gamma-(33)P] ATP. The resulting probe was used to characterize the distribution of leptin binding sites within brain sections of normal, ob/ob, and db/db C57BL/6J male mice. Leptin binding sites were found in leptomeninges and choroid plexus. Leptin binding within the choroid plexus is slightly elevated in ob/ob mice when compared to normal males (p<0.05). Binding of leptin by the choroid plexus of db/db male mice is lower than in normal males (p<0.05), but normally distributed. Based on the association and dissociation rates of leptin binding on tissue sections, we estimate the K(D) of the choroid plexus site at 0.25X10(-9) M. From our results, we hypothesize that the binding of leptin to its site may cause the release or transport of uncharacterized factor(s) into the cerebral spinal fluid (CSF) to affect neuronal populations controlling feeding and metabolism.MH - Central Nervous System|CH/*MEMH - Diabetes Mellitus|GE/*MEMH - Mice, Obese|*MEMH - Obesity|GE/*MEMH - Proteins|*MESO - Endocrinology 1996 Apr; 137(4):1497-500DP - 1996 AprTA - EndocrinologyPG - 1497-500IP - 4VI - 137UI - 9617859062
AU - Houseknecht KL
AU - Mantzoros CS
AU - Kuliawat R
AU - Hadro E
AU - Flier JS
AU - Kahn BBTI - Evidence for leptin binding to proteins in serum of rodents and humans: modulation with obesity.AB - Many hormones circulate bound to serum proteins that modulate ligand bioactivity and bioavailability. To understand the biology of leptin action, we investigated the presence of leptin binding proteins in serum. 125I-labeled leptin binds competitively to at least three serum macromolecules with molecular masses of approximately 85, approximately 176, and approximately 240 kDa in rodents and approximately 176 and approximately 240 kDa in humans. The ability to bind appears to involve sulfhydryl/disulfide interactions because it is inhibited under reducing conditions. When serum is added to recombinant 125I-leptin, there is a shift in sedimentation of 125I-leptin as analyzed by sucrose gradient centrifugation from approximately S1.9 to approximately S4.3. This shift is markedly attenuated in serum from obese mice (ob/ob, db/db, brown-fat ablated, gold-thioglucose treated, high-fat fed) compared with that from nonobese controls. The size distribution of endogenous serum leptin as determined by radioimmunoassay (RIA) after sucrose gradient centrifugation is also consistent with saturation of binding in hyperleptinemic obesity. In humans, free leptin increases with BMI. Thus, in lean rodents and humans a large proportion of leptin circulates bound to several serum proteins. Free leptin is increased in serum of obese subjects, which may alter leptin bioactivity, transport, and/or clearance.MH - Blood Proteins|IP/*MEMH - Obesity|*BLMH - Proteins|IP/*MESO - Diabetes 1996 Nov; 45(11):1638-43DP - 1996 NovTA - DiabetesPG - 1638-43IP - 11VI - 45UI - 9702018863
AU - Igel M
AU - Becker W
AU - Herberg L
AU - Joost HGTI - Evidence that reduced leptin levels, but not an aberrant sequence of leptin or its receptor, contribute to the obesity syndrome in NON mice.AB - NON mice exhibit a polygenic syndrome of mild obesity which is less pronounced than that of the ob and db strains. Here, we have shown that the syndrome is accompanied by a rise in leptin mRNA levels in adipose tissue, corresponding with the increase in adipose tissue mass. Surprisingly,levels of the leptin protein in adipose tissue and serum were comparable to those of lean control animals (BL57/Ksj-+/+), and markedly lower than those in db/db-mice. The coding regions of the cDNA sequences of both leptin and the leptin receptor from NON mice were identical with those of the wild-type sequences. We suggested that low levels of leptin in adipose tissue and serum contribute to the obesity of NON mice.MH - Carrier Proteins|*GE/*MEMH - Obesity|*GE/MEMH - Proteins|*GE/*MESO - Horm Metab Res 1996 Dec; 28(12):669-73DP - 1996 DecTA - Horm Metab ResPG - 669-73IP - 12VI - 28UI - 9716584664
AU - Leclercq Meyer V
AU - Considine RV
AU - Sener A
AU - Malaisse WJTI - Do leptin receptors play a functional role in the endocrine pancreas?AB - It was recently speculated that leptin may exert a direct inhibitory effect upon insulin release from the pancreatic B-cell. This proposal meets, however, with two objections.First, although the message for leptin receptors is indeed detected in rat pancreatic islets, the short form of this receptor, for which no signalling function is known, represents the major species present in islet cells. Second, in the isolated perfused rat pancreas, leptin (1.0 nM) fails to affect the release of either insulin or glucagon.MH - Carrier Proteins|GE/*MEMH - Insulin|*SEMH - Pancreas|*MEMH - Proteins|*MESO - Biochem Biophys Res Commun 1996 Dec; 229(3):794-8DP - 1996 DecTA - Biochem Biophys Res CommunPG - 794-8IP - 3VI - 229UI - 9711565965
AU - Kallen CB
AU - Lazar MATI - Antidiabetic thiazolidinediones inhibit leptin (ob) gene expression in 3T3-L1 adipocytes.AB - Lack of leptin (ob) protein causes obesity in mice. The leptin gene product is important for normal regulation of appetite and metabolic rate and is produced exclusively by adipocytes. Leptin mRNA was induced during the adipose conversion of 3T3-L1 cells, which are useful for studying adipocyte differentiation and function under controlled conditions. We studied leptin regulation by antidiabetic thiazolidinedione compounds, which are ligands for the adipocyte-specific nuclear receptor peroxisome proliferator-activated receptor gamma (PPARgamma) that regulates the transcription of other adipocyte-specific genes. Remarkably,leptin gene expression was dramatically repressed within a few hours after thiazolidinedione treatment. The ED50 for inhibition of leptin expression by the thiazolidinedione BRL49653 was between 5 and 50 nM, similar to its Kd for binding to PPARgamma. The relatively weak, nonthiazolidinedione PPAR activator WY 14,643 also inhibited leptin expression,but was approximately 1000 times less potent than BRL49653.These results indicate that antidiabetic thiazolidinediones down-regulate leptin gene expression with potencies that correlate with their abilities to bind and activate PPARgamma.MH - Adipocytes|*DE/MEMH - Gene Expression Regulation|*DEMH - Hypoglycemic Agents|ME/*PDMH - Obesity|*GEMH - Proteins|*GEMH - Pyrimidines|ME/*PDMH - Thiazoles|ME/*PDSO - Proc Natl Acad Sci U S A 1996 Jun; 93(12):5793-6DP - 1996 JunTA - Proc Natl Acad Sci U S APG - 5793-6IP - 12VI - 93UI - 9623404266
AU - Iida M
AU - Murakami T
AU - Ishida K
AU - Mizuno A
AU - Kuwajima M
AU - Shima KTI - Substitution at codon 269 (glutamine --> proline) of the leptin receptor (OB-R) cDNA is the only mutation found in the Zucker fatty (fa/fa) rat.AB - We recently cloned one of spliced variant forms of rat leptin receptor (OB-R), which contains a short intracellular domain, and found obese-phenotype-linked nucleotide alteration in the extracellular domain of the cDNA from the Zucker (fa/fa) rat, which results in a glutamine269 to proline269 amino acid substitution. Reported herein are the cloning and sequencing of another spliced variant forms of rat OB-R cDNA with a long intracellular domain. Both forms of OB-R cDNA share the same extracellular domain. In the Zucker (fa/fa) rat, no changes in either the gene structure nor in the nucleotide sequence of the long intracellular domain were observed. However, the expression level of OB-R mRNA in the brain of Zucker (fa/fa) rat was higher than for lean littermates. These facts suggest that the substitution at codon 269 of the OB-R cDNA represents the crucial mutation which results in the obese phenotype of Zucker (fa/fa) rat.MH - Carrier Proteins|CH/*GEMH - Glutamine|*MH - Obesity|*GEMH - Point Mutation|*MH - Proline|*MH - Rats, Zucker|*GESO - Biochem Biophys Res Commun 1996 Jul; 224(2):597-604DP - 1996 JulTA - Biochem Biophys Res CommunPG - 597-604IP - 2VI - 224UI - 9629553167
AU - Mercer JG
AU - Hoggard N
AU - Williams LM
AU - Lawrence CB
AU - Hannah LT
AU - Trayhurn PTI - Localization of leptin receptor mRNA and the long form splice variant (Ob-Rb) in mouse hypothalamus and adjacent brain regions by in situ hybridization.AB - Expression of the leptin receptor gene has been examined in mouse hypothalamus and other brain regions by in situ hybridization. With a probe recognizing all the known splice variants, receptor mRNA was evident in several brain regions (cortex, hippocampus, thalamus), with strong expression in the hypothalamus (arcuate, ventromedial, paraventricular and ventral premammillary nuclei), choroid plexus and leptomeninges.A probe specific to the long splice variant of the leptin receptor (Ob-Rb), containing the putative intracellular signaling domain, again revealed strong expression in the hypothalamus; there was, however, minimal hybridization to choroid plexus and leptomeninges. These results indicate that the hypothalamus is a key site of leptin action, although other brain regions are also targeted.MH - Alternative Splicing|*MH - Brain|*MEMH - Carrier Proteins|BI/*GEMH - Hypothalamus|*MESO - FEBS Lett 1996 Jun; 387(2-3):113-6DP - 1996 JunTA - FEBS LettPG - 113-6IP - 2-3VI - 387UI - 9624450568
AU - Lynn RB
AU - Cao GY
AU - Considine RV
AU - Hyde TM
AU - Caro JFTI - Autoradiographic localization of leptin binding in the choroid plexus of ob/ob and db/db mice.AB - The obese gene product, leptin, is synthesized in adipose tissue and is a circulating factor regulating body weight.To identify the location of leptin receptors in the brain we have performed an autoradiographic study of the binding of [(125)I]leptin to frozen sections of mouse brain. Dense specific binding of [(125)I]leptin was found only in the choroid plexus which is located in the dorsal part of the third ventricle and lateral ventricles. Specific binding of [(125)I]leptin was found the ob/ob and db/db mice. These findings further our understanding of the sites and mechanism of action of leptin on brain centers regulating body weight.MH - Brain|*MEMH - Carrier Proteins|*MEMH - Obesity|*GE/*MEMH - Obesity in Diabetes|GE/*MEMH - Proteins|*MESO - Biochem Biophys Res Commun 1996 Feb; 219(3):884-9DP - 1996 FebTA - Biochem Biophys Res CommunPG - 884-9IP - 3VI - 219UI - 9621674569
AU - Tartaglia LA
AU - Dembski M
AU - Weng X
AU - Deng N
AU - Culpepper J
AU - Devos R
AU - Richards GJ
AU - Campfield LA
AU - Clark FT
AU - Deeds J
AU - et alTI - Identification and expression cloning of a leptin receptor,OB-R.AB - The ob gene product, leptin, is an important circulating signal for the regulation of body weight. To identify high affinity leptin-binding sites, we generated a series of leptin-alkaline phosphatase (AP) fusion proteins as well as [125I]leptin. After a binding survey of cell lines and tissues, we identified leptin-binding sites in the mouse choroid plexus. A cDNA expression library was prepared from mouse choroid plexus and screened with a leptin-AP fusion protein to identify a leptin receptor (OB-R). OB-R is a single membrane-spanning receptor most related to the gp130 signal-transducing component of the IL-6 receptor,the G-CSF receptor, and the LIF receptor. OB-R mRNA is expressed not only in choroid plexus, but also in several other tissues, including hypothalamus. Genetic mapping of the gene encoding OB-R shows that it is within the 5.1 cM interval of mouse chromosome 4 that contains the db locus.MH - Obesity|*GE/MEMH - Proteins|*GE/IP/MESO - Cell 1995 Dec; 83(7):1263-71DP - 1995 DecTA - CellPG - 1263-71IP - 7VI - 83UI - 9612812970
AU - Considine RV
AU - Considine EL
AU - Williams CJ
AU - Hyde TM
AU - Caro JFTI - The hypothalamic leptin receptor in humans: identification of incidental sequence polymorphisms and absence of the db/db mouse and fa/fa rat mutations.AB - Leptin-receptor gene expression in hypothalamic tissue from lean and obese humans was examined. The full-length leptin receptor, that is believed to transmit the leptin signal, is expressed in human hypothalamus. There was no difference in the amount of leptin-receptor mRNA In seven lean (BMI 23.3 +/- 0.9 kg/m2) and eight obese (BMI 36.9 +/- 1.5) subjects as determined by reverse transcription-polymerase chain reaction. A sequence polymorphism (A-->G) was detected at position 668 of the leptin receptor cDNA. This second base substitution changed a glutamine to an arginine at position 223 of the leptin receptor protein.Of 15 subjects analyzed, 11 were heterozygous for this base change and 3 were