Short Isoforms Of Leptin Receptor Research Articles

The effects of Vitis vinifera L. phenolic compounds on a blood-brain barrier culture model: Expression of leptin receptors and protection against cytokine-induced damage.

The medicinal properties of grapes (Vitis vinifera L.) are well known since ancient times. Ethnobotanical grape preparations, like the Ayurvedic Darakchasava are used as cardiotonic and for the treatment of cardiovascular diseases. Dried grape products are also applied in Iranian traditional medicine for memory problems, which are linked to the pathology of brain microvessels, a special part of the cardiovascular system. The anti-inflammatory and protective effects of these traditional preparations on the cardiovascular system are related to their bioactive phenolic compounds. The blood-brain barrier (BBB), formed by brain capillaries, is not only involved in inflammatory and other diseases of the central nervous system, but also in many systemic diseases with an inflammatory component. Dietary obesity is a systemic chronic inflammatory condition in which the peripheral and central vascular system is affected. Among the cerebrovascular changes in obesity defective leptin transport across the BBB related to central leptin resistance is observed. Our aim was to study the protective effects of grape phenolic compounds epicatechin (EC), gallic acid (GA) and resveratrol (RSV) and grape-seed proanthocyanidin-rich extract (GSPE) on a cytokine-induced vascular endothelial inflammation model. Using a culture model of the BBB we investigated cytokine-induced endothelial damage and changes in the expression of leptin receptors and leptin transfer. For the BBB model, primary cultures of rat brain endothelial cells, glial cells and pericytes were used in co-culture. Cells were treated by tumor necrosis factor-α (TNF-α) and interleukin-1 β (IL-1β) (10 ng/ml each) to induce damage. Cell toxicity was evaluated by the measurement of impedance. The expression of leptin receptors was assessed by RT-qPCR and western blot. The production of reactive oxygen species (ROS) and nitric oxide (NO) were detected by fluorescent probes. GSPE (10 μg/ml), EC (10 μM), GA (1 μM) or RSV (10 μM) did not change the viability of brain endothelial cells. The gene expression of the short leptin receptor isoform, Ob-Ra, was up-regulated by GSPE, EC and RSV, while the mRNA levels of Lrp2 and clusterin, clu/ApoJ were not affected. The tested compounds did not change the expression of the long leptin receptor isoform, Ob-Rb. RSV protected against the cytokine-induced increase in albumin permeability of the BBB model. GSPE and EC exerted an antioxidant effect and GSPE increased NO both alone and in the presence of cytokines. The cytokine-induced nuclear translocation of transcription factor NF-κB was blocked by GSPE, GA and RSV. Cytokines increased the mRNA expression of Lrp2 which was inhibited by EC. RSV increased Ob-Ra and Clu in the presence of cytokines. Cytokines elevated leptin transfer across the BBB model, which was not modified by GSPE or RSV. Our results obtained on cell culture models confirm that natural grape compounds protect vascular endothelial cells against inflammatory damage in accordance with the ethnopharmacological use of grape preparations in cardiovascular diseases. Furthermore, grape compounds and GSPE, by exerting a beneficial effect on the BBB, may also be considered in the treatment of obesity after validation in clinical trials.

Leptin Receptor as a Potential Target to Inhibit Human Testicular Seminoma Growth

Although in past decades the adipokine leptin and its own receptor have been considered as significant cancer biomarkers, their potential involvement in human testicular seminoma growth and progression remains unexplored. Here, we showed that the expression of leptin and its receptor was significantly higher in human testicular seminoma compared with normal adult testis. Human seminoma cell line TCam-2 also expressed leptin along with the long and short isoforms of leptin receptor, and in response to leptin treatment showed enhanced activation of its downstream effectors. In line with these results, leptin stimulation significantly increased the proliferation and migration of TCam-2 cells. Treatment of TCam-2 cells with the peptide Leu-Asp-Phe-Ile (LDFI), a full leptin-receptor antagonist, completely reversed the leptin-mediated effects on cell growth and motility as well as reduced the expression of several leptin-induced target genes. More importantly, the invivo xenograft experiments showed that LDFI treatment markedly decreased seminoma tumor growth. Interestingly, LDFI-treated tumors showed reduced levels of the proliferation marker Ki-67 as well as decreased expression of leptin-regulated genes. Taken together, these data identify, for the first time, leptin as a key factor able to affect testicular seminoma behavior, highlighting leptin receptor as a potential target for novel potential treatments in this type of cancer.

Novel Role of Lck in Leptin-Induced Inflammation and Implications for Renal Aging.

Aging is associated with increased fat mass and elevated serum leptin levels (hyperleptinemia), causing proinflammation in the kidneys where it plays a primary role in the removal of endogenous leptin from the circulation. Lymphocyte-specific kinase (Lck) is a positive regulator of inflammatory signaling and a potential treatment target for age-related diseases, but its role in leptin signaling is unknown. Here, we investigated how Lck influences hyperleptinemia-induced inflammation in kidney tissues from 6- and 21-month-old rats. Results indicate that Lck expression and activation increased significantly in aged rat kidneys, especially at renal tubules. Furthermore, we identified interactions between Lck and short leptin-receptor isoforms, suggesting that Lck is a protein tyrosine kinase regulating leptin signaling. We further investigated whether increased Lck expression in renal tubular epithelial cells and macrophage infiltration are associated with leptin-induced inflammation. We then demonstrated that leptin activates Lck and proinflammatory transcription factors (STAT3 and NF-κB), while Lck knockdown modulates the expression of both transcription factors. Collectively, these data implicate that Lck leads to development of leptin-induced renal inflammation during aging. Inhibition of this protein tyrosine kinase may therefore be an appropriate therapeutic option for protection against age-related hyperleptinemia.

Hunting for the functions of short leptin receptor isoforms

Hunting for the functions of short leptin receptor isoforms

Leptin-mediated reactive oxygen species production does not significantly affect primary mouse hepatocyte functions in vitro

Direct and indirect effects of leptin on hepatic stellate cells (HSCs) have been documented in the literature, whereas little is known about leptin's actions on hepatocytes. Leptin mediates its profibrogenic and proinflammatory effects on HSCs in part through the production of intracellular reactive oxygen species (ROS). In this study, we focus our analysis on leptin-induced ROS production in hepatocytes. The expression of leptin receptor isoforms on primary mouse liver cells was examined by real-time quantitative-PCR and western blotting. Cultures were exposed to leptin in combination with inhibitors for reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, MAP kinase/ERK kinase 1 (MEK1) or janus kinase 2 (JAK2). ROS levels were quantified by measuring fluorescence. The effects of leptin on hepatocyte functions and programmed cell death were evaluated by fluorescent or luminescent assays. Leptin induced ROS production in primary hepatocytes by 150-450%, compared with a 20-30% increase in HSCs and liver sinusoidal endothelial cells (LSECs). This ROS production could be inhibited by NADPH oxidase, MEK1 and JAK2 inhibitors. Western blotting indicated that mouse HSCs and LSECs mainly express short leptin receptor isoforms, whereas hepatocytes appeared to express both short and long isoform(s). Leptin-induced ROS production in db/db hepatocytes did not differ from wild-type mice. Finally, leptin had no negative influence on primary hepatocyte functions. Leptin induced higher ROS levels in primary hepatocytes than in LSECs and HSCs, depending on NADPH oxidase, MEK1 and JAK2 signalling but not on the long leptin receptor isoform. Furthermore, leptin exposure did not influence primary hepatocyte functionality negatively.

Effects of leptin on lipopolysaccharide-induced myometrial apoptosis in an in vitro human model of chorioamnionitis

This study was aimed at assessing the role of leptin on human myometrium, by studying its receptor expression in pregnant myometrium and the interaction of leptin with inflammation-induced apoptosis. Myometrial samples were obtained from women with uncomplicated pregnancies who underwent cesarean delivery at term before labor onset. The effect of leptin on apoptosis was assessed by the incubation of myometrial strips with leptin (10(-10) to 10(-8) mol/L; 48 hours) before lipopolysaccharide treatment (10 μg/mL; 48 hours). Long and short leptin receptor isoforms were expressed in myometrial cells of pregnant women. Leptin prevented lipopolysaccharide-induced apoptosis, in a concentration-dependent manner, by down-regulating cleaved caspase-3 and BCL2-associated X protein and up-regulating BCL2 expression. This effect was mediated specifically through leptin receptor stimulation, followed by ERK1/2 signaling pathway activation. These results suggest a new potential pathway that is involved in delivery disorders of obese women and propose a role for the leptin-induced inhibition of myometrial apoptosis in the development of such disorders.

Effects of leptin and dexamethasone on long and short leptin receptor mRNA

Leptin exerts its effects by using both long (OB-Rb) and short (OB-Ra) receptors. Although leptin resistance or insensitivity is reported to be a cause of human obesity, there have not been enough studies to clarify regulation of leptin receptors. The authors studied leptin receptor regulation by leptin and dexamethasone in the human hepatocellular carcinoma cell (HepG2). Using a quantitative RT-PCR technique, the authors demonstrate expression of OB-Ra and OB-Rb mRNA after the incubation for 24 h with leptin or dexamethasone at various concentrations (10(-9)-10(-6) M). Leptin (10(-7)-10(-6) M) significantly inhibited expression of OB-Rb mRNA, with maximum inhibition (43% of control) at 10(-6) M. Expression of OB-R219.1 and OB-R219.3, two short isoforms of leptin receptor, were also reduced in cells treated with leptin most remarkably at 10(-7) M for OB-R219.1 (44% of the control) and at 10(-6) M for Ob-R219.3 (49% of the control). In contrast, dexamethasone (10(-8)-10(-6) M) significantly increased OB-Rb mRNA levels, with a maximum increase (204% of the control) at 10(-7) M, and OB-R219.1 and OB-R219.3 mRNA expression was also markedly increased at 10(-9)-10(-6) M. The peak values were 254% of the control for OB-R219.1 and 246% of the control for OB-R219.3 at 10(-7) M. In HepG2 cells, leptin inhibits and dexamethasone increases OB-Ra and OB-Rb mRNA expression. It is suggested that glucocorticoids as well as leptin itself contribute to regulatory effects of leptin through changes in gene expression of leptin receptors.

Activation of the Leptin Receptor by a Ligand-induced Conformational Change of Constitutive Receptor Dimers

Binding of leptin to the leptin receptor is crucial for body weight and bone mass regulation in mammals. Leptin receptors were shown to exist as dimers, but the role of dimerization in receptor activation remains unknown. Using a quantitative Bioluminescence Resonance Energy Transfer approach, we show here in living cells that approximately 60% of the leptin receptor exists as constitutive dimers at physiological expression levels in the absence of leptin. No further increase in leptin receptor dimerization was detected in the presence of leptin. Importantly, in cells expressing the short leptin receptor isoform, leptin promoted a robust enhancement of energy transfer signals that reflect specific conformational changes of pre-existing leptin receptor dimers and that may be used as read-out in screening assays for leptin receptor ligands. Both leptin receptor dimerization and the leptin-induced energy transfer were Janus kinase 2-independent. Taken together, our data support a receptor activation model based on ligand-induced conformational changes rather than ligand-induced dimerization.

Functional expression of the short isoform of the murine leptin receptor Ob-Rc (muB1.219) in Xenopus laevis oocytes.

Leptin, a hormone mainly secreted by the adipose tissue, acts on the hypothalamus to regulate food intake and thermogenesis. Six leptin receptor isoforms have been identified and localized in different tissues. While it is clear that leptin action in the brain occurs by binding to the long receptor isoform, several studies have shown that the short isoforms could be involved in the transcellular transport of the hormone from the blood to the brain. Based on these works, we decided to investigate whether the murine short leptin receptor isoform Ob-Rc (muB1.219) could transport leptin when expressed in Xenopus laevis oocytes. MuB1.219 cRNA was injected into the oocytes and functional studies were performed by incubating the oocytes in the presence of 2.5 nM [125I]-leptin, under different conditions. Results showed that leptin binding to the injected oocytes was four to eight-fold higher than the binding to the non-injected oocytes. This was blocked by 250 nM of non-radiolabeled leptin, suggesting that the binding was specific. Leptin internalization was observed from 30 min incubation onwards. Coexpression of the human Na+/glucose cotransporter and the leptin receptor showed that leptin increased sugar uptake into the oocytes. These results demonstrate that the short leptin receptor Ob-Rc is able to mediate binding and internalization of the hormone when expressed in oocytes and that it may perform intracellular signaling.

Leptin regulates interleukin-1β expression in the brain via the STAT3-independent mechanisms

Leptin is known to be an important circulating signal for regulation of food intake and body weight. These effects were suggested to be mediated through the hypothalamic center via the Ob-Rb receptor (long isoform of leptin receptor). Although short isoforms of leptin receptor exist in many regions of the brain, there has been little in vivo functional study of these areas such as for leptin’s target site. We report here that peripherally applied leptin increased interleukin (IL)-1β transcripts in many regions of the brain such as the hypothalamus, the hippocampus, the cortex, the cerebellum and the brainstem. Although leptin did not induce STAT3 activation or suppressor of cytokine signaling3 (SOCS3) expression in the hypothalamus of the db/db mice, which lack a functional Ob-Rb receptor, leptin increased the IL-1β levels to similar extents as normal mice. Therefore, a novel function of leptin is suggested as the induction of IL-1β expression in many regions of the brain via STAT3-independent mechanisms.

Acute stimulation of glucose uptake by leptin in l6 muscle cells.

The adipocyte hormone, leptin, acts via the central nervous system to modulate glucose metabolism by skeletal muscle, but the direct effects of leptin on glucose metabolism by skeletal muscle are unclear. In this study, we have examined effects of leptin on glucose uptake by cultured L6 muscle cells assessed with the non-metabolised glucose analogue 2-deoxy-D-glucose. Reverse-transcriptase polymerase chain reaction (RT-PCR) analysis of RNA showed that L6 muscle cells express a short isoform of the leptin receptor (ObRa), but not the long isoform (ObRb). In the absence of added insulin, incubation of L6 muscle cells with murine leptin (10( -11)-10( -8) M) for 10 min and 1 h increased glucose uptake by 15 % - 23 %. This effect of leptin was lost by 4 h. Leptin (10( -10) - 10( -9) M) initially (after 10 min) suppressed insulin-stimulated glucose uptake by 14 - 16 %, but had no effect in the longer term. Leptin-stimulated glucose uptake was inhibited by the phosphatidylinositol 3-kinase (PI3K) inhibitor wortmannin, but not by the janus kinase-2 (JAK-2) inhibitor tyrphostin AG490. The results suggest that leptin can act directly on L6 muscle cellsvia a short leptin receptor isoform to acutely stimulate basal (but not insulin-stimulated) glucose uptake via a PI3K-dependent pathway.

Transcellular transport of leptin by the short leptin receptor isoform ObRa in Madin-Darby Canine Kidney cells.

Leptin is an adipocyte-derived hormone that acts in specific regions of the brain to regulate body weight and neuroendocrine function. The mechanism by which leptin enters the brain is unknown. We previously reported that rat brain microvessels, which constitute the blood-brain barrier, contain large amounts of messenger RNA encoding a short form of the leptin receptor (ObRa), suggesting that this site may be important for receptor-mediated transport of leptin into the brain. The purpose of this study was to determine whether ObRa is capable of transcellular transport of intact leptin. A transwell system in which Madin-Darby Canine Kidney (MDCK) cells stably expressing ObRa are grown in a monolayer was used to determine receptor distribution on apical or basolateral cell surfaces and the capacity for directional transport of 125I-leptin. Binding of 125I-leptin was greater on the apical vs. the basolateral cell surface and transport of 125I-leptin occurred only in the apical to basolateral direction. 11% of transported radioactivity appearing in the basolateral chamber represented intact leptin as assessed by TCA precipitation analysis and by SDS-PAGE. Parental MDCK cells did not express leptin receptors and did not bind or transport 125I-leptin. Epidermal growth factor (EGF) binding and transport via endogenous EGF receptors in MDCK cells also was assessed. In contrast to leptin, specific binding of 125I-EGF occurred primarily on the basolateral cell surface and transport of 125I-EGF occurred predominantly in the basolateral to apical direction. These data show that ObRa is preferentially targeted to the apical cell membrane in MDCK cells and that leptin transport occurs, albeit at a low rate, in a unidirectional manner in the apical to basolateral direction. These findings may be relevant to the putative role of ObRa in receptor-mediated transport of leptin from the circulation into the brain.

Transcellular Transport of Leptin by the Short Leptin Receptor Isoform ObRa in Madin-Darby Canine Kidney Cells

Transcellular Transport of Leptin by the Short Leptin Receptor Isoform ObRa in Madin-Darby Canine Kidney Cells

Expression of leptin receptor isoforms in rat brain microvessels.

Leptin acts on specific brain regions to affect body weight regulation. As leptin is made by white adipose tissue, it is thought that leptin must cross the blood-brain barrier or the blood-cerebrospinal fluid barrier to reach key sites of action within the brain. High expression of a short form leptin receptor has been reported in the choroid plexus. However, whether one or more of the known leptin receptor isoforms is expressed in brain capillaries is unknown. To identify and quantitate leptin receptor isoforms in rat brain microvessels, we applied quantitative RT-PCR to RNA from purified rat brain microvessels in parallel with in situ hybridization. The results show that the amount of short form leptin receptor messenger RNA (mRNA) in brain microvessels is extremely high, exceeding that in choroid plexus. In contrast, low levels of this mRNA were detected in the cerebellum, hypothalamus, and meninges. The long form leptin receptor mRNA is only present at low levels in the microvessels, but surprisingly, its level in cerebellum is 5 times higher than that in the hypothalamus. In situ hybridization experiments confirmed strong expression of short leptin receptors in microvessels, choroid plexus, and leptomeninges. The distribution and type of leptin receptor mRNA isoforms in brain microvessels are consistent with the possibility that receptor-mediated transport of leptin across the blood-brain barrier is mediated by the short leptin receptor isoform.

Leptin activates PI-3 kinase in C2C12 myotubes via janus kinase-2 (JAK-2) and insulin receptor substrate-2 (IRS-2) dependent pathways.

We have recently shown that leptin mimicks insulin effects on glucose transport and glycogen synthesis through a phosphatidylinositol-3 (PI) kinase dependent pathway in C2C12 myotubes. The aim of the present study was to identify the signalling path from the leptin receptor to the PI-3 kinase. We stimulated C2C12 myotubes with insulin (100 nmol/l, 5 min) or leptin (0.62 nmol/l, 10 min) and determined PI-3 kinase activity in immunoprecipitates with specific non-crossreacting antibodies against insulin-receptor substrate (IRS 1/IRS 2) and against janus kinase (JAK 1 and JAK 2). While insulin-stimulated PI-3 kinase activity is detected in IRS-1 and IRS-2 immunoprecipitates, leptin-stimulated PI-3 kinase activity is found only in IRS-2 immunoprecipitates, suggesting that the leptin signal to PI-3 kinase occurs via IRS-2 and not IRS-1. Leptin-, but not insulin-stimulated PI-3 kinase activity is also detected in immunoprecipitates with antibodies against JAK-2, but not JAK-1. The data suggest that JAK-2 and IRS-2 couple the leptin signalling pathway to the insulin signalling chain. Since we have also detected leptin-stimulated tyrosine phosphorylation of JAK-2 and IRS-2 in C2C12 myotubes it can be assumed that leptin activates JAK-2 which induces tyrosine phosphorylation of IRS-2 leading to activation of PI-3 kinase. As we could not detect the long leptin receptor isoform in C2C12 myotubes we conclude that this signalling pathway is activated by a short leptin receptor isoform.