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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.

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.

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

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.