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Colonic leptin: source of a novel proinflammatory cytokine involved in IBD¹

Department of Medicine, Division of Digestive Diseases,
^* Department of Pathology, Emory University School of Medicine, Atlanta, GA, 30322, USA

² Correspondence: Emory University, Department of Medicine, Division of Digestive Diseases, 615 Michael St., Atlanta, GA 30322, USA. E-mail: dmerlin{at}emory.edu

SPECIFIC AIMS

Leptin, the obesity gene cloned in 1994, is produced mainly by adipocytes and is the key hormone involved in central regulation of body weight. Leptin acts through specific receptors in the brain to control food intake. Studies have shown that nonadipose tissues such as skeletal muscle, pituitary gland, and stomach can also produce leptin; it therefore it reaches the small intestine in an active form and can locally initiate biological processes involved in regulating functions, such as intestinal absorption. The aim of the present study is to determine whether colonic epithelial cells produce leptin and might contribute to pathological states such as inflammatory bowel disease (IBD).

PRINCIPAL FINDINGS

1. Inflamed colonic epithelial cells express leptin
In the present report we show for the first time that inflamed colonic epithelial cells express leptin concentrated at a subapical part of colonic cells (Fig. 1 B1–B3); leptin was not detected in normal colonic epithelial cells (Fig. 1A1-A3 ). These results suggest that the colon may represent a source of leptin in inflamed tissues. To verify this, we examined colonic lavages from normal patients and patients with ulcerative colitis. We demonstrate that colonic lavage fluid leptin concentrated from patients with mild to severe IBD is >15-fold that detected in normal patients. These results suggest that inflamed colonic epithelial cells secrete leptin into the intestinal lumen and may act on the colonic epithelial cells that normally express the functional leptin receptor at their apical membranes. Using Caco2-BBE cell monolayers to model colonic epithelia cells, we demonstrate that leptin was not detected in these cells. Interferon , which is known to be present at high levels in IBD tissues, induces expression and secretion of leptin in Caco2-BBE monolayers. These results agree with our in vivo results suggesting that leptin expression and secretion can be induced in inflamed colonic cells.

View larger version (106K): [in this window] [in a new window]   Figure 1. Leptin is expressed in inflamed human colon but not in normal human colon. Frozen sections were stained with anti-leptin antibody. Sections were counterstained for nuclei with hematoxylin. A1–A3) In normal human colon (normal colon mucosa, female male 59 years old), no staining of leptin is observed. B1–B3) In a patient with chronic ulcerative colitis, up-regulation of leptin expression in colonic epithelia is noted (colonic mucosa with chronic ulcerative colitis, female 74 years old). C1–C3) When human colon with ulcerative colitis was immunostained with anti-leptin antibody in the presence of 10 µmol/L peptide, no staining was observed. Similar results were observed in two additional control and ulcerative colitis patients respectively. x40, x20, x10 depict magnification.

2. Luminal leptin activate NF-B
We next investigated whether luminal leptin could act as a proinflammatory cytokine for colonic epithelial cells. Many proinflammatory cytokines activate nuclear transcription factors NF-B, and activation of NF-B has been implicated in the pathogenesis of IBD. To investigate whether luminal leptin is able to activate NF-B, we used Caco2-BBE monolayers that express leptin receptor at their apical plasma membranes. Electrophoretic mobility shift assays using [³²P] NF-B consensus sequence showed that nuclear extracts from Caco2-BBE monolayers stimulated with 2 nM luminal leptin evoked an enhanced association of nuclear proteins with NF-B consensus sequences. Treatment of nuclear extracts with an excess of cold NF-B consensus sequences resulted in a suppression of the luminal leptin effect on the upper band. Progressive increased levels of p65 in the nucleus were observed by confocal analysis in Caco2-BBE monolayers treated with 2 nM luminal leptin. NF-B activation by most inducers requires degradation of the isoform IB-. Therefore, we looked for luminal leptin-induced degradation of IB- in the cytoplasmic extracts of Caco2-BBE monolayers treated with luminal leptin. Western blot analysis, using an antibody against IB-, indicated that luminal leptin reduced the cytoplasmic levels of IB-. To verify that NF-B activation was induced by leptin and not by a bacterial contaminant, we boiled our leptin solution for 20 min. This treatment resulted in a complete loss of its bioactivity, demonstrating that NF-B activation was not induced by a bacterial contaminant. These results suggest that luminal leptin induces a proinflammatory response in colonic epithelial cells that is NF-B dependent.

3. Luminal leptin induces colonic inflammation in vivo
The inflammatory effect of luminal leptin was investigated in mice. Recombinant leptin (2 mg/mL) or vehicle was administrated intrarectally and mice were killed 48 h later. Leptin-induced wall damage with epithelial exfoliation and colonic cells are blunted compared with control mice.

CONCLUSIONS AND SIGNIFICANCE

Leptin receptor (Ob-R) is a member of the cytokine family of receptors. It consists of a single membrane-spanning protein that, through alternative mRNA splicing, exists in several isoforms. These isoforms differ only in the length of their intracellular domains and can be divided into those with a full-length intracellular domain, often referred to as the long isoform of the leptin receptor, and the short isoforms.

Leptin receptor isoforms have been reported in a wide variety of human and rodent tissues: heart, placenta, lung, liver, muscle, kidney, pancreas, spleen, thymus, prostate, testes, ovary, small intestine, and colon. It has been demonstrated that leptin receptors, including the long isoform, are expressed in human brush border enterocytes and colonic epithelial cells. The presence of leptin receptors (expressed in the brush border) along the small intestine suggest that luminal leptin might initiate biological processes involved in controlling functions such as absorption. We recently demonstrated that luminal leptin affect butyrate transport in colonic epithelial cells. Butyrate is the major intestinal fuel even when competing substrates such as glucose and glutamine are available. Beside its function as the dominant energy source for the colonocytes, butyrate affects cellular proliferation, differentiation, and apoptosis. It has been shown that under intestinal inflammation induced by toxin A, expression of the leptin receptor Ob-Rb is increased on epithelial and subepithelial cells.

It is conceivable that luminal gastric leptin reaches the small intestine, but it is unlikely that luminal leptin (from stomach) reaches the colon. Our preliminary results confirm that luminal leptin is barely detectable in colonic lavages from normal patients. In contrast, we demonstrate that colonic lavage fluid leptin concentration in patients with mild to severe IBD is >15 fold that of normal patients. Plasma leptin could easily cross a leaky epithelium and be present in the intestinal lumen. However, we have demonstrated that inflamed colonic epithelial cells express leptin that is concentrated at the subapical part of the colonic epithelial cells. In contrast, leptin was not detected in normal colonic epithelial cells. Together, these results suggest that inflamed colonic epithelial cells secrete leptin into the intestinal lumen which may act on the colonic epithelial cells that normally express the functional leptin receptor at their apical membranes (Fig. 2 ). Using in vitro approaches, we have confirmed that proinflammatory cytokine interferon is able to up-regulate the leptin expression and secretion by epithelial cells. The mechanism by which interferon induces leptin expression and secretion is unknown, and further study is needed to delineate its mode of action. Interferon present in high levels in IBD tissues may up-regulate leptin expression and secretion by colonic epithelial cells.

View larger version (48K): [in this window] [in a new window]   Figure 2. Schematic diagram showing luminal leptin secretion induced by proinflammatory stimuli in colonic epithelial cells. Colonic luminal leptin activates NF-B, which induces inflammatory responses.

We thus investigated whether luminal leptin could act as a proinflammatory cytokine on colonic epithelial cells. It is known that many proinflammatory cytokines activate nuclear transcription factors NF-B and AP-1, and the activation of NF-B has been implicated in the pathogenesis of inflammatory bowel disease (IBD). It has been demonstrated that leptin activated both AP-1 and NF-B in human umbilical vein endothelial cells. In the present study, we demonstrate that luminal leptin evoked enhanced association of nuclear proteins with NF-B consensus sequence. We suggest that luminal leptin acts as a proinflammatory cytokine on colonic epithelial cells and disseminates the inflammatory stimuli to the noninflamed areas in IBD tissue (Fig. 2 ).

Our in vivo experiments demonstrate that the presence of luminal leptin in the mice colon induced epithelial wall damage with epithelial exfoliation and colonic cell blunting compared with control mice. These data represent a characteristic histological finding in acute intestinal inflammation thought to contribute to the epithelial dysfunction characterizing such disorders.

In conclusion, we demonstrate that inflamed colonic cells are an unequivocal source of luminal leptin. Luminal leptin activates nuclear transcription factors NF-B, thus acting as a novel proinflammatory cytokine in colon (Fig. 2 ). These results suggest a new pathophysiological role for intraluminal leptin during states of intestinal inflammation such as IBD. These findings have potential mechanistic and therapeutic implications in the pathogenesis and treatment of inflammatory bowel disease.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.03-0422fje; doi: 10.1096/fj.03-0422fje

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