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Lung regeneration after acute injury usually depends on stem cell migration and differentiation, and functional alveoli-like tissue and capillary structure formation. The homing of mesenchymal stem cells (MSCs) to injury sites promotes lung repair through damaged cell replacement and anti-inflammatory and anti-fibrotic effects. Here, we aimed to improve therapeutic effects of the endogenous MSCs by increasing their homing efficiency. We have identified a high-affinity leptin receptor (LEPR)-binding peptide using a phage display screening technique, as the LEPR is highly expressed in MSCs. The selected LEPR-binding peptides were modified with a collagen binding peptide for specifically tethering to a collagen scaffold. After implantation of the LEPR-binding peptide functionalized collagen scaffold in a rat model of acute lung injury, the endogenous LEPR+ MSCs were specifically recruited out of circulation to the scaffold, and their retention periods in the damaged area were significantly prolonged. The migrated MSCs in the functional scaffold promoted the differentiation of type Ⅱ alveolar epithelial cells to type Ⅰ alveolar epithelial cells and facilitated alveoli-like tissue and capillary formation, thus improved lung function recovery. These results suggest that tethering the LEPR binding peptides to the collagen scaffold significantly enhanced endogenous MSC recruitment and promoted functional regeneration of injured lung tissue.

The pleiotropic cytokine hormone leptin, by activating its receptor OB-R, plays a major role in many biological processes, including energy homeostasis, immune function, and cell survival and proliferation. Abnormal leptin action is associated with obesity, autoimmune diseases, and cancer. The pharmacological characterization of OB-R and the development of synthetic OB-R ligands are still in their infancy because currently available binding assays are not compatible with ligand saturation binding experiments and high-throughput screening (HTS) approaches. We have developed here a novel homogeneous time-resolved fluorescence-based binding assay that overcomes these limitations. In this assay, fluorescently labeled leptin or leptin antagonist binds to the SNAP-tagged OB-R covalently labeled with terbium cryptate (Tb). Successful binding is monitored by measuring the energy transfer between the Tb energy donor and the fluorescently labeled leptin energy acceptor. Ligand binding saturation experiments revealed high-affinity dissociation constants in the subnanomolar range with an excellent signal-to-noise ratio. The assay performed in a 384-well format shows high specificity and reproducibility, making it perfectly compatible with HTS applications to identify new OB-R agonists or antagonists. In addition, fluorescently labeled leptin and SNAP-tagged OB-R will be valuable tools for monitoring leptin and OB-R trafficking in cells and tissues.

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.

Rats selectively bred to develop diet-induced obesity (DIO) have an early onset reduction in the sensitivity of their ventromedial hypothalamic nucleus (VMN) neurons to leptin compared with diet-resistant (DR) rats. This reduced sensitivity includes decreased leptin receptor (Lepr-b) mRNA expression, leptin receptor binding, leptin-induced phosphorylation of STAT3 (pSTAT3), and impaired leptin excitation (LepE) of VMN neurons. When administered exogenously, the pancreatic peptide, amylin, acts synergistically to reduce food intake and body weight in obese, leptin-resistant DIO rats by increasing VMN leptin signaling, likely by stimulation of microglia IL-6, which acts on its receptor to increase leptin-induced pSTAT3. Here, we demonstrate that incubation of cultured VMN neurons of outbred rats with IL-6 increases their leptin sensitivity. Control, dissociated DIO VMN neurons express 66% less Lepr-b and 75% less Bardet Biedl Syndrome-6 (BBS6) mRNA and have reduced leptin-induced activation of LepE neurons compared with DR neurons. Incubation for 4 days with IL-6 increased DIO neuron Lepr-b expression by 77% and BBS6 by 290% and corrected their defective leptin activation of LepE neurons to DR levels. Since BBS6 enhances trafficking of Lepr-b to the cell membrane, the increases in Lepr-b and BBS6 expression appear to account for correction of the reduced leptin excitation of DIO LepE neurons to that of control DR rats. These data support prior findings suggesting that IL-6 mediates the leptin-sensitizing effects of amylin on VMN neurons and that the inherent leptin resistance of DIO rats can be effectively reversed at a cellular level by IL-6.

Because rearing rats in large litters (LLs) protects them from becoming obese, we postulated that LL rearing would protect rats selectively bred to develop diet-induced obesity (DIO) from becoming obese by overcoming their inborn central leptin resistance. Male and female DIO rats were raised in normal litters (NLs; 10 pups/dam) or LLs (16 pups/dam) and assessed for anatomical, biochemical, and functional aspects of leptin sensitivity at various ages when fed low-fat chow or a 31% fat high-energy (HE) diet. LL rearing reduced plasma leptin levels by postnatal day 2 (P2) and body weight gain by P8. At P16, LL DIO neonates had increased arcuate nucleus (ARC) binding of leptin to its extracellular receptors and at P28 an associated increase of their agouti-related peptide and alpha-MSH axonal projections to the paraventricular nucleus. Reduced body weight persisted and was associated with increased ARC leptin receptor binding and sensitivity to the anorectic effects of leptin, reduced adiposity, and enhanced insulin sensitivity in LL DIO rats fed chow until 10 wk of age. The enhanced ARC leptin receptor binding and reduced adiposity of LL DIO rats persisted after an additional 5 wk on the HE diet. Female LL DIO rats had similar reductions in weight gain on both chow and HE diet vs. normal litter DIO rats. We postulate that LL rearing enhances DIO leptin sensitivity by lowering plasma leptin levels and thereby increasing leptin receptor availability and that this both enhances the ARC-paraventricular nucleus pathway development and protects them from becoming obese.

In rats selectively bred to develop diet-induced obesity (DIO) 3 wk of postweaning exercise reduces weight and adipose regain for 10 wk after exercise cessation, despite intake of 31% fat high-energy (HE) diet. To test the hypothesis that this effect is due to increased central leptin sensitivity, 4-wk-old DIO rats were fed the HE diet and left sedentary (Sed), exercised for 3 wk, and then remained sedentary for 10 additional weeks (Ex/Sed) or continued exercise for a full 13 wk (Ex). After 3 wk, leptin (5 mg/kg ip) induced a 36% decrease in 24-h food intake in Ex rats, while Sed rats had no change in 24-h intake. Ex rats also had 23% more leptin-induced phospho-STAT3 (pSTAT3)-expressing neurons in the arcuate nucleus (ARC) and 95% and 68% higher 125I-labeled leptin receptor binding in the ventromedial and dorsomedial nuclei than did Sed rats, respectively. At 7 wk after onset, leptin decreased 24-h intake by 20% in Ex and 24% in Ex/Sed rats without altering Sed intake. After a total of 13 wk, compared with Sed rats, Ex and Ex/Sed rats had 58% and 38% less fat, respectively, but leptin failed to decrease food intake in any group. Nevertheless, Ex, but not Ex/Sed rats, still had 32% more ARC leptin-induced pSTAT3-expressing neurons than Sed rats. These data suggest that brief postweaning exercise in DIO rats that are inherently leptin resistant causes a sustained resistance to obesity on HE diet, which is, in part, due to increased central leptin sensitivity.

Obesity is a global health issue that has been identified as a risk factor for several types of cancer. High levels of body fat and circulating leptin are typical identifiers of obesity in humans and animals. Leptin is a 16kD protein hormone which is secreted by adipocytes, and maybe secreted from cancer cells, that functions to control satiation via leptin receptor binding. However, obese individuals often develop “leptin resistance”, which is a mechanism that leads to the accumulation of excess leptin. Increased binding of leptin to its receptor (OB-R) due to leptin resistance has been associated with disease progression and poor prognosis in human cancers. Our group has previously shown that leptin-mediated cancer cell proliferation is inhibited by the LPrA2 (leptin peptide receptor antagonist 2). We have prepared novel leptin antagonists and tested their ability to block leptin-induced survival and chemoresistance to Paclitaxel (TAX) and Gemcitabine (GEM) in cancer cells and derived tumorspheres (pancreatic: PANC-1, MiaPaca2 and triple negative breast cancer: MDA-MB231 and MDA-MB468). Western blot protein analyses showed the ability of the antagonists to specifically inhibit leptin-induced phosphorylation of STAT3, and expression of cyclin D, and Notch1 in cancer cells. Additionally, potential toxicity of antagonists was tested using MTT assay with concentrations up to 100X higher than the effective dose in non-malignant breast cells (MCF-10A). Data generated show no toxicity of the novel antagonists in vitro. Leptin-induced proliferation of breast cancer and pancreatic cancer cells (120-160%) was significantly inhibited by the novel antagonists. In addition, leptin-mediated progression of S-phase was also reduced by the antagonists. Leptin increased TAX and GEM chemoresistance in cells and tumorspheres that were efficiently inhibited by the antagonists. These data suggest that the new antagonists could be equally or more effective than LPrA2 for adjuvant treatment of cancer. Acknowledgements: This work has been supported by Pilot Project Award from MSM/Tuskegee University/UAB Cancer Center partnership grant 5U54CA118638; PC SPORE Grant from UAB to RRGP, and facilities and support services at Morehouse School of Medicine (1G12RR026250-03; NIH RR 03034 and 1C06 RR18386). Citation Format: Crystal C. Lipsey, Adriana Harbuzariu, Ruben R. Gonzalez-Perez. Targeting obesity-related cancer progression with novel leptin receptor antagonists [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3540. doi:10.1158/1538-7445.AM2017-3540

Specific [125I]leptin receptor binding sites have been identified in choroid plexus (CP), but have eluded regional localization within the brain parenchyma. To optimize specific [125I]leptin binding in brain loci, we ran experiments varying the pH of incubation buffers. We found that specific [125I]leptin binding in CP was strikingly pH dependent with the most acidic buffer, pH 5.5, resulting in a greater than 100% increase over the amount of specific binding measured at pH 7.5. While low pH permitted detection of specific binding in parenchymal loci, clear pH dependency was only observed in the CP. In the caudate putamen (CauP), a locus with low specific binding, values for specific binding did not differ significantly across the range of pH conditions tested. Using incubation buffers at pH 6.0 in subsequent binding experiments, we localized specific [125I]leptin binding in several brain loci including thalamus and hypothalamus. In CP and thalamus, where the range of OD permitted analysis of binding parameters, [125I]leptin binding was saturable with increasing concentrations of unlabelled leptin. In all loci, specific [125I]leptin binding was insensitive to competition by high concentrations of other unlabelled compounds. Our results varying pH conditions of the incubation buffer suggest leptin receptors may be divided into subclassifications based on pH sensitivity of the specific binding. Furthermore, our results suggest that although densities are low, high affinity leptin receptors are present in neural loci implicated in food intake and energy balance, and are more widespread in the forebrain than previously determined.

The leptin gene has received intensive attention and scientific investigation for its importance in energy homeostasis and reproductive regulation in mammals. Furthermore, study of the leptin gene is of crucial importance for public health, particularly for its role in obesity, as well as for other numerous physiological roles that it plays in mammals. In the present work, we report the identification of novel leptin genes in 4 species of Cetacea, and a comparison with 55 publicly available leptin sequences from mammalian genome assemblies and previous studies. Our study provides evidence for positive selection in the suborder Odontoceti (toothed whales) of the Cetacea and the family Phocidae (earless seals) of the Pinnipedia. We also detected positive selection in several leptin gene residues in these two lineages. To test whether leptin and its receptor evolved in a coordinated manner, we analyzed 24 leptin receptor gene (LPR) sequences from available mammalian genome assemblies and other published data. Unlike the case of leptin, our analyses did not find evidence of positive selection for LPR across the Cetacea and Pinnipedia lineages. In line with this, positively selected sites identified in the leptin genes of these two lineages were located outside of leptin receptor binding sites, which at least partially explains why co-evolution of leptin and its receptor was not observed in the present study. Our study provides interesting insights into current understanding of the evolution of mammalian leptin genes in response to selective pressures from life in an aquatic environment, and leads to a hypothesis that new tissue specificity or novel physiologic functions of leptin genes may have arisen in both odontocetes and phocids. Additional data from other species encompassing varying life histories and functional tests of the adaptive role of the amino acid changes identified in this study will help determine the factors that promote the adaptive evolution of the leptin genes in marine mammals.

Hashimoto’s thyroiditis, a common autoimmune disease, is associated with autoantibodies to thyroglobulin (Tg) and thyroid peroxidase (TPO). TPO, unlike abundant and easily purified Tg, is rarely investigated as an autoantigen in animals. We asked whether antibodies (Abs) develop to both TPO and Tg in thyroiditis in mice that is induced (C57BL/6 and DBA/1 strains) or arises spontaneously (NOD.H-2h4).ScreeningforTPOAbswasperformedbyflowcytometryusingmouseTPO-expressingeukaryotic cells. Sera were also tested for binding to purified mouse Tg and human TPO. The antibody data were compared with the extent of thyroiditis. Immunization with mouse TPO adenovirus broke self-tolerance to this protein in C57BL/6 mice, but thyroiditis was minimal and TgAbs wereabsent. InDBA/1micewithextensivegranulomatousthyroiditis inducedbyTgimmunization,TPOAbswerevirtuallyabsentdespite high levels of TgAbs. In contrast, antibodies to mouse TPO, with minimal cross-reactivity with human TPO, arose spontaneously in older (7–12 months) NOD.H-2h4 mice. Unexpectedly, TgAbs preceded TPOAbs, a time course paralleled in relatives of probands with juvenile Hashimoto’s thyroiditis. These findings demonstrate a novel aspect of murine and human thyroid autoimmunity, namely breaking B cell self-tolerance occurs first for Tg and subsequently for TPO. Inflammation and Obesity Pathogenesis: The Hypothalamus Heats Up Joshua P. Thaler and Michael W. Schwartz (Endocrinology, published June 23, 2010, 10.1210/en.2010-0336) ABSTRACT Obesity induced by high-fat (HF) feeding is associated with low-grade inflammation in peripheral tissues that predisposes to insulin resistance. Recent evidence suggests the occurrence of a similar process in the hypothalamus, which favors weight gain through impairment of leptin and insulin signaling. In addition to its implications for obesity pathogenesis, this hypothesis suggests that centrally targeted antiinflammatory therapies may prove effective in prevention and treatment of this disorder. This article highlights molecular and cellular mechanisms by which hypothalamic inflammation predisposes to diet-induced obesity.Obesity induced by high-fat (HF) feeding is associated with low-grade inflammation in peripheral tissues that predisposes to insulin resistance. Recent evidence suggests the occurrence of a similar process in the hypothalamus, which favors weight gain through impairment of leptin and insulin signaling. In addition to its implications for obesity pathogenesis, this hypothesis suggests that centrally targeted antiinflammatory therapies may prove effective in prevention and treatment of this disorder. This article highlights molecular and cellular mechanisms by which hypothalamic inflammation predisposes to diet-induced obesity. Large Litter Rearing Enhances Leptin Sensitivity and Protects Selectively Bred Diet-induced Obese Rats from Becoming Obese Christa M. Patterson, Sebastien G. Bouret, Sunny Park, Boman G. Irani, Ambrose A. Dunn-Meynell, and Barry E. Levin (Endocrinology, 10.1210/en.2010-0401) ABSTRACT Because rearing rats in large litters (LLs) protects them from becoming obese, we postulated that LL rearing would protect rats selectively bred to develop diet-induced obesity (DIO) from becoming obese by overcoming their inborn central leptin resistance. Male and female DIO rats were raised in normal litters (10 pups/dam) or LLs (16 pups/dam) and assessed for anatomical, biochemical, and functional aspects of leptin sensitivity at various ages when fed low-fat chow or a 31% fat high-energy (HE) diet. LL rearing reduced plasma leptin levels by postnatal d 2 (P) 2 and body weight gain by P8. At P16, LL DIO neonates had increased arcuate nucleus (ARC) binding of leptin to its extracellular receptors and at P28 an associated increase of their agouti-related peptide and -MSH axonal projections to the paraventricular nucleus. Reduced body weight persisted and was associated with increased ARC leptin receptor binding and sensitivity to the anorectic effects of leptin, reduced adiposity, and enhanced insulin T R A N S L A T I O N A L H I G H L I G H T S F R O M E N D O C R I N O L O G YBecause rearing rats in large litters (LLs) protects them from becoming obese, we postulated that LL rearing would protect rats selectively bred to develop diet-induced obesity (DIO) from becoming obese by overcoming their inborn central leptin resistance. Male and female DIO rats were raised in normal litters (10 pups/dam) or LLs (16 pups/dam) and assessed for anatomical, biochemical, and functional aspects of leptin sensitivity at various ages when fed low-fat chow or a 31% fat high-energy (HE) diet. LL rearing reduced plasma leptin levels by postnatal d 2 (P) 2 and body weight gain by P8. At P16, LL DIO neonates had increased arcuate nucleus (ARC) binding of leptin to its extracellular receptors and at P28 an associated increase of their agouti-related peptide and -MSH axonal projections to the paraventricular nucleus. Reduced body weight persisted and was associated with increased ARC leptin receptor binding and sensitivity to the anorectic effects of leptin, reduced adiposity, and enhanced insulin T R A N S L A T I O N A L H I G H L I G H T S F R O M E N D O C R I N O L O G Y

The arcuate nucleus (ARC) mediates the anorexic effects of leptin and expresses the long form (Ob-Rb) of the leptin receptor. To determine whether ARC leptin binding increases when plasma leptin levels are low during fasting, [125I]-leptin specific binding to rat brain slices was measured by quantitative autoradiography. [125I]-leptin specific binding was dense in the ARC and increased 2-fold after a 48-h fast (P

Endogene, funktionelle Teratogene wie auch chemische Noxen können in kritischen Entwicklungsperioden zur Zerstörung zentraler Reglerund damit zu lebenslang anhaltenden Fehlfunktionen endokriner Regelungsprozesse führen. In der vorliegenden Studie wurden die akutenÄnderungen in der Energiebilanz während der Säugephase an zwei unterschiedlichen Modellen untersucht, die durch Eingriffe in denersten Lebenstagen Adipositas im adulten Tier bewirken. Im ersten Adipositasmodell wurde eine postnatale Überernährung (PNO) 21 Tage alter Rattenwelpen durch die Aufzucht in einem Nest mitnur vier Tieren, gegenüber zwölf Tieren in normal großen Nestern, induziert, wobei neben Wistarratten entweder Zuckerratten, dieheterozygot für einen Defekt des Leptinrezeptors sind (+/fa), oder deren Wildtyp-Wurfgeschwister (+/+) untersucht wurden. Bei allenPNO-Tieren ergab sich gegenüber den im normalen Nest aufgezogenen Kontrolltieren sowohl eine größere fettfreie Trockenmasse undFettmasse, wobei die normalerweise phänotypisch kaum ausgeprägte genetische Prädisposition der +/fa-Tiere zu vermehrterFettakkumulation bei den PNO-Tieren erheblich verstärkt wurde. Neben Zunahmen der Körperfettmasse entwickelten die +/fa-PNO-Tiere,aber auch die PNO-Wistarratten, eine relative Hyperleptinämie. Diese war mit einer Leptinresistenz assoziiert, wie sich in einer nahezuvollständigen Insensitivität insbesondere der PNO-Wistarratten und der +/fa-PNO-Tiere gegenüber exogen appliziertem Leptin zeigte. EineBestimmung der hypothalamischen Leptinrezeptorbindung an Hypothalamus-Homogenaten verdeutlichte, dass diese weder bei denPNO-Tieren noch durch Leptinbehandlung im Vergleich zu den Kontrolltieren verändert wurde. Unabhängig davon wiesen zum einen +/fa-und zum anderen weibliche Tiere eine verminderte Leptinrezeptorbindung auf. Diese Befunde am Untersuchungsmodell I zeigen, dass eine erhöhte Energiezufuhr im Säuglingsalter nicht nur zu einem verstärktemWachstum, sondern auch zu einer exzessiven Fettdeposition führt. Zusätzlich konnte eine auffallende Interaktion zwischen einemdefinierten, normalerweise rezessiv eingestuften Merkmal und frühen postnatalen Unwelteinflüssen gezeigt werden, die modellhaft für dieProgrammierung der phänotypischen Ausprägung genetischer Störungen sein könnte. Unabhängig vom genetischen Hintergrund tritt dabeieine Leptinresistenz als Leitsymptom auf, die jedoch nicht auf einer Änderung der Rezeptorendichte beruht. Eine analoge Beziehungzwischen genetischem Hintergrund undÜberernährung wäre auch bei der Wistarratte zu vermuten, ist aber noch nicht identifiziert worden. Grundlage des zweiten Adipositasmodelles ist die chemische Läsionierung von Nervenzellkörpern durch Verabreichung vonMonosodiumglutamat (MSG), das in hypothalamischen Gebieten mit durchlässiger Blut-Hirnschranke wirksam wird. Die postnataleMSG-Behandlung führte bei den MSG-behandelten Tiere zu einer geringeren Fettdeposition gegenüber den NaCl-behandeltenKontrolltieren. Bei identischer Nahrungszufuhr künstlich über intraösophageale Katheter aufgezogener Tiere kam es zu einem nahezuvollständigen Verschwinden der Stoffwechselabsenkung im Minimum der Tiere und einer erhöhten Energiedissipation derMSG-behandelten Tiere. Auch die künstlich aufgezogenen Tiere zeigten eine starke Abnahme besonders des Körperfettgehaltes, was dieÄnderungen in der Energiebilanz der Tiere als Ursache bestätigt. Mit dem verminderten Körperfettgehalt ging auch eine Abnahme derPlasmaleptinspiegel einher. Um die Auswirkungen der MSG-Behandlung auf die für die Energiehomöostase wichtigen hypothalamischen Kerngebiete insbesonderedes Nucleus Arcuatus (ARC) zu untersuchen wurden kresylviolett-gefärbte coronale Gehirnschnitte mikroskopisch untersucht. Währendandere Kerngebiete nicht von der chemischen Läsionierung durch MSG betroffen waren, trat eine weitgehende Zerstörunghypothalamischer Neurone im ARC ein. Zudem wurde zur Funktionskontrolle am zehn Tage alten Rattenhirn die Reagibilität auf Leptinanhand des Nachweises eines 'immediate-early-gene' Produkts, des Fos Proteins, als Marker für neuronale Aktivierung nachgewiesenund die Verteilung des durch das Neuropeptid Y (NPY) als Transmitter gekennzeichneten, hirn-intrinsischen Neuronensystems, dessenBeitrag zur Stimulation der Nahrungsaufnahme bekannt ist, trotz der neuronalen Unreife der juvenilen Tiere nachgewiesen. Die Befundedes Adipositasmodells II zeigen, dass der MSG-induzierten Adipositas im Erwachsenenalter eine Phase erhöhten Energieumsatzesvorausgeht, die in ihrer Wirkung auf den circadianen Rhythmus der einer Leptinapplikation bei der juvenilen Ratte vergleichbar ist und vorallem zu einer Verringerung des Körperfettgehaltes führt. Bei der Deutung dieses Befundes sind die histochemischen Hinweise zubeachten, dass normalerweise bereits im Alter von zehn Tagen eine Fortleitung des Leptinsignals zum Nucleus paraventricularis alsintegrative Struktur für die Kontrolle der Energiebilanz existiert und dass insbesondere das an dieser Verbindung beteiligte NPY-Systementwickelt ist. Der beobachtete Zelluntergang im ARC wirkt sich akut wie eine durch Leptin hervorgerufene Enthemmung dernormalerweise wirksamen Maßnahmen der juvenilen Energieeinsparung im Tagesminimum aus. Zusammenfassend läßt sich feststellen, dass zwei völlig unterschiedliche Einflüsse, nämlich eine chemische Noxe, genauso wie einepostnatale Überernährung in einer kritischen juvenilen Entwicklungsphase, obwohl sie zunächst gegenläufige Veränderung derFettdeposition auslösen, schließlich zu Adipositas und deren assoziierten Begleitstörungen im adulten Tier führen können., Endogenous as well as chemical functional teratogens, if administered in critical periods of neuronal development, may destroy centralregulatory mechanisms and therefore lead to lifelong-lasting dysfunctions of endocrine regulatory loops. In this study acute changes inenergy balance during suckling period has been examined in two rat obesity models, which are known to induce an obese phenotype in theadult animal. In the first obesity model a postnatal overnutrition (PNO) has been induced by rearing pups in small litters of only 4 animals (small litter size)compared to pups reared under control conditions with 12 pups (normal litter size). Besides Wistar rats either Zucker rats, which areheterozygous for a leptin receptor defect 'fatty' (+/fa), or wildtyp Zucker rats have been used in this study. All PNO-animal showed anenhanced fat-free dry-mass (FFDM) as well as an increased body-fat mass compared to their control littermates, which had been reared inlitters of normal size. The enlarged fat deposition of heterozygous animals which is normally hardly detectable by phenotype is massivelyenhanced by the postnatal overnutrition. Besides the increase in body fat mass the +/fa-PNO as well as the PNO-Wistar rats developed arelative hyperleptinaemia. This was associated with a leptin resistance, which was expressed in a nearly total insensitivity exspecially of thePNO-Wistar- and PNO-+/fa-Zucker rats against peripheral leptin treatment. The investigation of hypothalamic leptin receptor binding inZucker rats revealed that neither PNO- nor leptin-treated animals had a different binding compared to the controll animals. Independent ofthese findings, a diminished leptin receptor binding of heterozygous and female animals could be detected. These results of the first obesity model show that a higher energy supply in postnatal life not only contributes to a higher body weight butexspecially supports an exaggerated fat deposition. Additionally, this study demonstrated an obvious interaction between an usuallyinconspicous genetic trait and early postnatal enviromental influences, which could be exemplary for the programming of phenotypicalexpression of genetic disorders. Independant of genetic background, a leptin resistance as a lead-symptom develops, which is not causedby an altered expression of the leptin receptor. In the second obesity model monosodiumglutamate (MSG) in high doses has been used to lesion neuronal cell bodies in specifichypothalamic areas without a blood-brain-barrier. Postnatal MSG-treatment causes a diminshed fat-deposition of the treated suckling-ageanimals compared to the control (NaCl-treated) animals. Artificial rearing which ensures asupply with identical amounts of milk viaintraosophageal catheters revealed a nearly complete suppression of the circadian torpor-like decreases of Tc and MR in the dailyminimum and therefore lead to a higher energy dissipation of the MSG-treated animals. The artificially reared animals showed also adiminished fat mass compared to the control animals in accordance with the observed changes in energy metabolism which thus werecausal for the reduction of body weight. To study the effect of MSG treatment on hypothalamic nuclei, which are known to be involved inregulation of energy homeostasis, cresyl-violet stained brain sections have been examined. A marked loss of cells in the arcuate nucleus(ARC) could be detected while other brain regions were not affected by the treatment. Additionally, to test whether those brain regions arealready functional at an age of ten days, the expression of the Fos protein, an immediate early-gene-product, which is known as a markerfor neuronal activity, could be demonstrated. Despite the neuronal immaturity of ten day old rat pups the NPY-ergic projections, which havebeen shown to be essential for signal transmission in the control of food intake, already exist. The results show that MSG-induced obesity, which develops in adulthood, is preceded by a period of expanded energy expenditure injuvenile life. With respect to their effects on energy metabolism leptin treatment and MSG lesioning of juvenile pups are comparable andlead mainly to a diminished fat deposition. For the interpretation of these findings one has to keep in mind the histological results, whichrevealed that even at this early age the transmission of the leptin signal to the nucleus paraventricularis is already possible, because theNPY-ergic connectivities are already existent. The neuronal cell death in the ARC leads to an leptin-like disinhibition of normal energysaving mechanisms in the minimum of the daily circadian rhythm in 10 day old pups. Summarizing the results, this study shows that two totally different influences - postnatal overnutrition or a chemical teratogene - ifadministered in periods that are critical for developing organisms, can induce changes in energy metabolism and nutritional state ofanimals which may lead to a higher obesity susceptibility in adulthood.

Because rearing rats in large litters (LLs) protects them from becoming obese, we postulated that LL rearing would protect rats selectively bred to develop diet-induced obesity (DIO) from becoming obese by overcoming their inborn central leptin resistance. Male and female DIO rats were raised in normal litters (10 pups/dam) or LLs (16 pups/dam) and assessed for anatomical, biochemical, and functional aspects of leptin sensitivity at various ages when fed low-fat chow or a 31% fat high-energy (HE) diet. LL rearing reduced plasma leptin levels by postnatal d 2 (P) 2 and body weight gain by P8. At P16, LL DIO neonates had increased arcuate nucleus (ARC) binding of leptin to its extracellular receptors and at P28 an associated increase of their agouti-related peptide and α-MSH axonal projections to the paraventricular nucleus. Reduced body weight persisted and was associated with increased ARC leptin receptor binding and sensitivity to the anorectic effects of leptin, reduced adiposity, and enhanced insulin sensitivity in LL DIO rats fed chow until 10 wk of age. The enhanced ARC leptin receptor binding and reduced adiposity of LL DIO rats persisted after an additional 5 wk on the HE diet. Female LL DIO rats had similar reductions in weight gain on both chow and HE diet vs. normal litter DIO rats. We postulate that LL rearing enhances DIO leptin sensitivity by lowering plasma leptin levels and thereby increasing leptin receptor availability and that this both enhances the ARC-paraventricular nucleus pathway development and protects them from becoming obese.

Endothelial progenitor cells (EPC) have been reported to contribute to neovascularization. We have previously shown that the adipocytokine leptin may enhance the adhesive properties of EPC by upregulating specific integrins. To investigate whether the angiogenic effects of leptin may be mediated by modulation of EPC function, mononuclear cells were isolated from healthy human volunteers and cultivated under endothelial cell conditions for 7 days. In the matrigel assay, pretreatment of EPC with recombinant leptin for 24 hours dose-dependently enhanced their incorporation into tubular structures provided by mature endothelial cells. For example, 138.3 ± 7.6% (P = 0.001) and 145.3 ± 5.5% (P = 0.0001) CM-DiI-labeled EPC were detected after stimulation with 10 and 100 ng/mL leptin, respectively (control-treated EPC defined as 100%). Furthermore, in the spheroid angiogenesis assay, stimulation of EPC with 10 ng/mL leptin increased the number of sprouts (P < 0.0001) and tube length (P < 0.0001) of coincubated mature endothelial cells, and the outgrowth of EPC (P < 0.0001). Addition of 100-fold excess of leptin-neutralizing or leptin-receptor-binding antibodies completely reversed these effects. Moreover, EPC adhesion onto endothelial cell tubules could be reduced by addition of RGD peptides (from 159 ± 13.7% to 101.8 ± 14.6%; P = 0.02), or of neutralizing antibodies against αvβ3 (from 165.3 ± 11.8% to 103.8 ± 13.3%; P = 0.006) or αvβ5 (to 93.5 ± 15.8%; P = 0.005). Further experiments using specific signal transduction inhibitors (10 μM of LY294002, PD98059, or SB203580), as well as Western blot analysis, revealed that leptin signaling in EPC involves phosphoinositide-3 kinase and p42/44, but not by p38 MAP kinase. The effects of leptin could also be confirmed under in vivo conditions. Stimulation of EPC with 100 ng/mL leptin potentiated the insprout of newly formed avian vessels into collagen onplants placed on the chorion allantoic membrane of chicken embryos (angiogenic index, 0.58 ± 0.24) compared to control-treated EPC (0.44 ± 0.27; P = 0.07) and endothelial basal medium alone (0.31 ± 0.26; P = 0.0007). Thus, our in vitro and in vivo results suggest that the angiogenic effects of leptin may partly depend on its specific interaction with endothelial progenitor cells.

Leptin is an adipocyte-derived hormone that acts as a major regulator for food intake and energy homeostasis. Leptin deficiency or resistance can result in profound obesity, diabetes, and infertility in humans. Since its discovery, our understanding of leptin's biological functions has expanded from anti-obesity to broad effects on reproduction, hematopoiesis, angiogenesis, blood pressure, bone mass, lymphoid organ homeostasis, and T lymphocyte systems. Leptin orchestrates complex biological effects through its receptors, expressed both centrally and peripherally. Leptin receptor belongs to the class I cytokine receptor superfamily. At least five isoforms of leptin receptor exist, primarily because of alternate splicing. The longest form is capable of full signal transduction. The short forms may serve as leptin binding proteins and play a role in leptin transporting across the blood-brain barrier. In this review, we present the crystal structure of leptin and the structural comparison with other four-helical cytokines, discuss the leptin-receptor binding models based on other cytokine-receptor complex structures, and summarize the most recent progress on leptin signal transduction pathways--especially its link to peripheral lipid metabolism through AMP-activated protein kinase and hepatic stearoyl-CoA desaturase-1 pathways. Furthermore, we propose the structure based design of leptin analogs with increased stability, improved potency, enhanced blood-brain barrier transport, and extended time action for future therapeutic application.

Leptin is an adipocyte-derived hormone that acts as a major regulator for food intake and energy homeostasis. Leptin deficiency or resistance can result in profound obesity, diabetes, and infertility in humans. Since its discovery, our understanding of leptin's biological functions has expanded from anti-obesity to broad effects on reproduction, hematopoiesis, angiogenesis, blood pressure, bone mass, lymphoid organ homeostasis, and T lymphocyte systems. Leptin orchestrates complex biological effects through its receptors, expressed both centrally and peripherally. Leptin receptor belongs to the class I cytokine receptor superfamily. At least five isoforms of leptin receptor exist, primarily because of alternate splicing. The longest form is capable of full signal transduction. The short forms may serve as leptin binding proteins and play a role in leptin transporting across the blood-brain barrier. In this review, we present the crystal structure of leptin and the structural comparison with other four-helical cytokines, discuss the leptin-receptor binding models based on other cytokine-receptor complex structures, and summarize the most recent progress on leptin signal transduction pathways--especially its link to peripheral lipid metabolism through AMP-activated protein kinase and hepatic stearoyl-CoA desaturase-1 pathways. Furthermore, we propose the structure based design of leptin analogs with increased stability, improved potency, enhanced blood-brain barrier transport, and extended time action for future therapeutic application.