Induction of leptin receptor expression in the liver by leptin and food deprivation

Leptin resistance is a common feature of obesity and the metabolic syndrome. However, the regulated expression of the leptin receptor (Ob-R) has not been studied in detail. Expression profiling of liver mRNA in leptin-treated wild-type mice revealed a marked increase in leptin receptor mRNA levels, which had not previously been described. This was confirmed by isoform-specific real-time PCR, which showed a >25-fold increase in the mRNAs encoding the short forms (Ob-Ra, Ob-Rc) and a >10-fold increase in the mRNA encoding the long (Ob-Rb) form of the leptin receptor in liver. In parallel, we also observed induction of plasma-soluble leptin receptor (SLR) protein by leptin administration, pair feeding, and short term food restriction. However, induction of SLR by leptin is abolished in mice with selective deletion of Ob-R from liver using Cre-LoxP technology. These data suggest that the liver is a major source of Ob-R mRNA expression under conditions of negative energy balance. Membrane-bound Ob-R is then shed into the circulation as SLR. Our study thus reveals an unexpected role of the liver in modulating total circulating leptin levels and possibly its biological activity.     

瘦素、胰岛素及IL-6对平滑肌细胞瘦素受体mRNA表达的影响

目的研究瘦素、胰岛素及IL-6对平滑肌细胞瘦素受体(Ob-R)mRNA表达的影响。方法采用六孔板培养皿培养鼠平滑肌细胞,5个孔作为一个浓度组,通过半定量的RT-PCR方法,分别观察了不同浓度的大鼠瘦素、胰岛素及IL-6对平滑肌细胞瘦素受体mRNA表达水平的影响。结果通过半定量RT-PCR,我们检测了短型瘦素受体(Ob-Ra)和长型瘦素受体(Ob-Rb)的mRNA表达水平,瘦素受体(Ob-R)mRNA表达水平随着瘦素及IL-6浓度增加而上升,随着胰岛素的浓度增加而下降。结论瘦素及IL-6可在体外上调平滑肌细胞瘦素受体的表达,胰岛素可下调平滑肌细胞瘦素受体的表达。这三个因子参与了瘦素受体的表达调控,对我们了解瘦素抵抗的发生机制有重要意义。     

Molecular cloning and expression of bovine (Bos taurus) leptin receptor isoform mRNAs

We characterized Bos taurus leptin receptor (Ob-R) isoform mRNAs as well as their expression in different tissues, including some adipose depots (perirenal, subcutaneous and intermuscular adipose tissues). Based on the GenBank database sequences of the bovine partial Ob-R, primers were designed to amplify cDNAs of bovine Ob-R isoforms. The full-length cDNAs of bovine the Ob-R isoforms were cloned by combination with 3'-and 5'-RACE. Three bovine Ob-R isoform cDNAs were cloned and the sequence analyses revealed that these cDNAs were bovine Ob-R isoforms, i.e., the long form (Ob-Rb), the middle form (Ob-Ra) and the short form (Ob-Rc). The open reading frames of Ob-Ra, Ob-Rb and Ob-Rc gene were 2688, 3498 and 2673 bp, respectively. The deduced amino acid sequences suggested that the isoforms were single transmembrane proteins, and differed in the C-terminal amino acid sequences. The amino acid sequence of these bovine Ob-R isoforms showed 73-75% identity compared with the corresponding mouse isoforms. The tissue-specific expression of the bovine Ob-R isoforms were measured by semi-quantitative RT-PCR. Expression of Ob-Rb was highest in liver, heart, spleen and kidney, with lower expression in lung and testis, and slight expression in muscle. Ob-Ra was highly expressed in liver and spleen, whereas moderate expression was observed in heart, testis, and muscle, and its expression was the lowest in lung and kidney. Ob-Rc mRNA was expressed in the liver, heart, testis, kidney and muscle, but not in the lung and spleen. In adipose tissues, higher expression of Ob-Ra and Ob-Rb mRNA was observed in intermuscular adipose tissue than in subcutaneous or perirenal adipose tissues. Ob-Ra mRNA level was positively correlated with Ob-Rb mRNA level in the adipose tissues (r=0.81, P<0.05). The results demonstrated that each Ob-R isoform mRNA was differentially expressed in various tissues of cattle, which may be involved in the difference of peripheral actions for leptin.     

High-dose leptin activates human leukocytes via receptor expression on monocytes

Leptin is capable of modulating the immune response. Proinflammatory cytokines induce leptin production, and we now demonstrate that leptin can directly activate the inflammatory response. RNA expression for the leptin receptor (Ob-R) was detectable in human PBMCs. Ob-R expression was examined at the protein level by whole blood flow cytometry using an anti-human Ob-R mAb 9F8. The percentage of cells expressing leptin receptor was 25 +/- 5% for monocytes, 12 +/- 4% for neutrophils, and 5 +/- 1% for lymphocytes (only B lymphocytes). Incubation of resting PBMCs with leptin induced rapid expression of TNF-alpha and IL-6 mRNA and a dose-dependent production of TNF-alpha and IL-6 by monocytes. Incubation of resting PBMCs with high-dose leptin (250 ng/ml, 3-5 days) induced proliferation of resting cultured PBMCs and their secretion of TNF-alpha (5-fold), IL-6 (19-fold), and IFN-gamma (2.5-fold), but had no effect on IL-4 secretion. The effect of leptin was distinct from, and additive to, that seen after exposure to endotoxin or activation by the mixed lymphocyte reaction. In conclusion, Ob-R is expressed on human circulating leukocytes, predominantly on monocytes. At high doses, leptin induces proinflammatory cytokine production by resting human PBMCs and augments the release of these cytokines from activated PBMCs in a pattern compatible with the induction of Th1 cytokines. These results demonstrate that leptin has a direct effect on the generation of an inflammatory response. This is of relevance when considering leptin therapy and may partly explain the relationship among leptin, proinflammatory cytokines, insulin resistance, and obesity.     

Short-term and long-term leptin exposure differentially affect human natural killer cell immune functions

Epidemiological studies have indicated that obesity is associated with a higher risk for certain cancers caused by elevated levels of adipocyte-derived hormones. Leptin, one such hormone produced by adipocytes, is a major regulator of metabolism and has also been shown to modulate immunity. However, its role in regulating human natural killer (NK) cell functions is largely unknown. Here, we show that the leptin receptor (Ob-R) is expressed on 5% of NK cells isolated from blood donors, as measured with flow cytometry, and expression of the signal-transducing long form of the leptin receptor Ob-Rb was confirmed with quantitative PCR. The Ob-R+ subpopulation displayed a lower expression of CD16, a cell surface receptor mediating antibody-dependent activation. Short-term stimulation with leptin increased IFNγ secretion, CD69 activation marker expression, and cytotoxic lysis of tumor cells; this was mediated by an improved conjugate forming between NK cells and tumor cells as well as higher expression of tumor necrosis factor-related apoptosis-inducing ligand. On the contrary, long-term incubation with leptin significantly impaired these NK cell immune functions and decreased cell proliferation. In addition, phosphorylation of Jak-2 after leptin stimulation was reduced in peripheral mononuclear blood cells from obese humans compared with normal-weight controls. NK cells represent an immune cell population that is crucial for an effective antitumor response. Here, we show that long-term exposure to leptin, similarly to the situation in obese individuals with elevated serum leptin levels, significantly impairs integral parts of NK cell immune functions, possibly linking leptin to increased cancer susceptibility in obesity.     

High circulating leptin receptors with normal leptin sensitivity in liver-specific insulin receptor knock-out (LIRKO) mice

Liver-specific insulin receptor knock-out (LIRKO) mice display hyperinsulinemia, abnormal glucose metabolism, and progressive liver dysfunction. In addition, circulating leptin levels appear to be increased more than 10-fold. However, food intake, body weight, and adipose mass are not significantly altered in LIRKO mice compared with wild-type littermates. Using a ligand immunofunctional assay, we found that the apparent increase in circulating leptin in LIRKO mice is because of an 80-fold increased serum level of soluble leptin receptor. Gene expression analysis by microarray and real time PCR reveals the liver as the source of soluble leptin receptor in LIRKO mice, with an increase in expression of the short (Ob-Ra), long (Ob-Rb), and soluble (Ob-Re) forms of the leptin receptor. Direct control of leptin receptor expression by insulin could also be demonstrated in isolated hepatocytes from normal mice. Despite the markedly increased levels of leptin receptor in their circulation, LIRKO mice exhibit normal or even enhanced leptin sensitivity, as assessed by their physiological and molecular responses to exogenous leptin administration and their lower base-line hypothalamic levels of SOCS3 mRNA. Thus, insulin signaling in the liver plays an important role in control of leptin receptor expression and shedding. In the LIRKO mouse, this is lost, leading to markedly increased leptin receptors into the circulation. These high levels of circulating leptin receptor bind leptin and likely alter its clearance, but do not inhibit leptin action and may actually potentiate leptin action. In this manner, insulin signaling in liver plays an important role in leptin homeostasis and fine modulation of leptin action.     

Melatonin enhances leptin expression by rat adipocytes in the presence of insulin

Leptin and melatonin play an important role in the regulation of body mass and energy balance. Both hormones show a circadian rhythm, with increasing values at night. In addition, melatonin receptors were recently described in adipocytes, where leptin is synthesized. Here, we investigated the influence of melatonin and its interaction with insulin and dexamethasone on leptin expression. Isolated rat adipocytes were incubated with melatonin (1 nM) alone or in combination with insulin (5 nM) and/or dexamethasone (7 nM) for 6 h. Melatonin or insulin alone did not affect leptin expression, but together they increased it by 120%. Dexamethasone increased leptin mRNA content (105%), and this effect was not enhanced by melatonin. Simultaneous treatment with the three hormones provoked a further increase in leptin release (250%) and leptin mRNA (100%). Melatonin prevented the forskolin-induced inhibition (95%) of leptin expression. In addition, melatonin's ability to stimulate leptin release (in the presence of insulin) was completely blocked by pertussis toxin and luzindole. To gain further insight into the molecular basis of melatonin and insulin synergism, the insulin-signaling pathway was investigated. Melatonin increased the insulin-induced insulin receptor-beta tyrosine phosphorylation, which led to an increased serine phosphorylation of the downstream convergent protein Akt. We concluded that melatonin interacts with insulin and upregulates insulin-stimulated leptin expression. These effects are caused by melatonin binding to the pertussis toxin-sensitive G(i) protein-coupled membrane receptor (MT1 subtype) and the cross talk with insulin, since insulin receptor and its convergent target Akt are coactivated by melatonin.     

Rat Insulinoma-Derived Pancreatic β-cells Express a Functional Leptin Receptor That Mediates a Proliferative Response

In addition to its interaction at hypothalamic sites to affect feeding and energy expenditure, leptin has been shown to exhibit a proliferative response in erythropoietic cells. The functional leptin receptor is also present in pancreatic islets and we now demonstrate that a commonly used clonal insulin secreting β-cell line, RINm5F, expresses high levels of the Ob-Rb mRNA. Leptin causes an increase in tyrosine phosphorylation of a number of intracellular proteins and a dose related (10nM–200nM) increase in expression of the immediate-early gene, c-fos. This precedes a leptin induced proliferative response in serum-deprived RINm5F cells, which suggests that leptin might be involved in the complex regulation of proliferation of the pancreatic β-cell.     

Novel regulatory mechanisms for generation of the soluble leptin receptor: implications for leptin action

Background

The adipokine leptin realizes signal transduction via four different membrane-anchored leptin receptor (Ob-R) isoforms in humans. However, the amount of functionally active Ob-R is affected by constitutive shedding of the extracellular domain via a so far unknown mechanism. The product of the cleavage process the so-called soluble leptin receptor (sOb-R) is the main binding protein for leptin in human blood and modulates its bioavailability. sOb-R levels are differentially regulated in metabolic disorders like type 1 diabetes mellitus or obesity and can, therefore, enhance or reduce leptin sensitivity.

Methodology/Principal Findings

To describe mechanisms of Ob-R cleavage and to investigate the functional significance of differential sOb-R levels we established a model of HEK293 cells transiently transfected with different human Ob-R isoforms. Using siRNA knockdown experiments we identified ADAM10 (A Disintegrin And Metalloproteinase 10) as a major protease for constitutive and activated Ob-R cleavage. Additionally, the induction of lipotoxicity and apoptosis led to enhanced shedding shown by increased levels of the soluble leptin receptor (sOb-R) in cell supernatants. Conversely, high leptin concentrations and ER stress reduced sOb-R levels. Decreased amounts of sOb-R due to ER stress were accompanied by impaired leptin signaling and reduced leptin binding.

Conclusions

Lipotoxicity and apoptosis increased Ob-R cleavage via ADAM10-dependent mechanisms. In contrast high leptin levels and ER stress led to reduced sOb-R levels. While increased sOb-R concentrations seem to directly block leptin action, reduced amounts of sOb-R may reflect decreased membrane expression of Ob-R. These findings could explain changes of leptin sensitivity which are associated with variations of serum sOb-R levels in metabolic diseases.     

Vasoactive intestinal peptide: autocrine growth factor in neuroblastoma.

Neuroblastoma is the most common solid tumor of children less than 5 years of age; yet the biology of this tumor is poorly understood. Neuroblastoma tumors are derived from neural crest precursors; they synthesize both adrenergic and peptidergic neurotransmitters. This study determined VIP receptor expression in primary neuroblastoma tumors prior to chemotherapy. The VIP receptor was expressed in 12 of 15 neuroblastoma tumors as determined by direct binding studies (KD = 1.3-12.4 nM) and VIP-mediated stimulation of adenylate cyclase. The VIP stimulation index for adenylate cyclase in the primary tumor was inversely correlated with the VIP content of the tumor, suggesting that VIP regulates its own receptor expression. Similar observations were made in vitro by comparison of two human neuroblastoma cell lines, IMR32 and SKNSH. Both cell lines were demonstrated to express specific, high affinity VIP receptors (KD = 4 nM and 2.5 nM for IMR32 and SKNSH, respectively). IMR32 cells contained very low levels of VIP (0.6 pg VIP/10(6) cells). Exogenous VIP stimulated adenylate cyclase 22-fold over basal activity and VIP inhibited proliferation of IMR32 cells by 49% in 6-day cultures. On the other hand, SKNSH cells synthesized high levels of VIP (6.3 pg/10(6) cells), metabolized VIP rapidly and demonstrated a low level of VIP-mediated stimulation of adenylate cyclase; their proliferation rate was minimally inhibited by exogenous VIP. These observations help validate the hypothesis that VIP serves as an autocrine growth factor in neuroblastoma.