Leptin and the obesity receptor (OB-R) in the small intestine and colon: a colocalization study

Abstract

Leptin is a hormone that plays an important role in overall body energy homeostasis, and the obesity receptor, OB-R, is widely distributed in the organism. In the intestine, a multitude of leptin actions have been reported, but it is currently unclear to what extent the hormone affects the intestinal epithelial cells by an endocrine or exocrine signaling pathway. To elucidate this, the localization of endogenous porcine leptin and OB-R in enterocytes and colonocytes was studied. By immunofluorescence microscopy, both leptin and OB-R were mainly observed in the basolateral membrane of enterocytes and colonocytes but also in the apical microvillar membrane of the cells. By electron microscopy, coclustering of hormone and receptor in the plasma membrane and localization in endosomes was frequently detected at the basolateral surface of the epithelial cells, indicative of leptin signaling activity. In contrast, coclustering occurred less frequently at the apical cell surface, and subapical endosomal localization was hardly detectable. We conclude that leptin action in intestinal epithelial cells takes place at the basolateral plasma membrane, indicating that the hormone uses an endocrine pathway both in the jejunum and colon. In contrast, the data obtained did not provide evidence for an exocrine, lumenal action of the hormone in the intestine.

Figures

Figure 1

Localization by immunofluorescence microscopy of OB-R in jejunum and colon. Cryosections of jejunum (A–C) or colon (D) were labeled with an antibody to OB-R (A,D) or Na+/K+-ATPase (B). In the jejunum, OB-R labeling was most intense along the basolateral surface (arrowheads in A) of the enterocytes, as shown by its colocalization with the basolateral marker Na+/K+-ATPase (C), but patchy labeling at the apical brush border was also observed (arrows in A). In colonocytes, OB-R was mainly seen along the basolateral surface (arrowheads in D) and only very weakly at the apical membrane (arrows in D). C, colonocytes; E, enterocytes; LP, lamina propria. Bar = 10 μm.

Figure 2

Localization by immunofluorescence microscopy of endogeneous leptin in jejunum and colon. Cryosections of jejunum (A,B) or colon (C) were labeled with an antibody to leptin. In enterocytes of both villus (A) and crypt (B) regions, the labeling was punctate and most intense along the basolateral surface (arrowheads). In colonocytes (C), the labeling was most distinct at the basal surface (arrowheads), and some amorphous labeling was present at the apical surface in the crypt lumen (L). In both tissues, labeling over the lamina propria was also observed. E, enterocytes; LP, lamina propria. Bar = 10 μm.

Figure 3

Double immunogold labeling for OB-R and leptin in jejunal enterocytes. Electron micrographs of the apical brush border (A–C) show a scattered labeling for both proteins along the microvilli. Colocalization of OB-R (large particles) and leptin (small particles) was only occasionally observed (arrows), but labeling at the bottom of the microvilli was not detected. By comparison, labeling along the basolateral plasma membrane was more intense (D). At a higher magnification of a section of the same view (E), most of the small and large gold particles were seen to be colocalized along the membrane (arrows). In addition, both OB-R and leptin were seen in endosomes in the vicinity of the basolateral plasma membrane (F), where they also often colocalized (arrows). Bar = 0.2 μm.

Figure 4

Double immunogold labeling for OB-R and leptin in colonocytes. Electron micrographs of the apical surface (A–C) showed no (A) or little (B,C) labeling for both OB-R (large particles) and leptin (small particles) along the microvilli, and colocalization was only very rarely detected (arrow). Labeling at the flat, non-microvillar part of the apical plasma membrane was not observed. At the basolateral surface (D,E) and in nearby endosomes (F), labeling for both proteins was much more abundant, and colocalization was frequently observed (arrows). Bar = 0.1 μm.

Figure 5

Localization of leptin in subcellular fractions of jejunum and colon by immunoblotting. Samples of soluble (Sol), Mg2+-precipitated (Mg), and microvillar (Mic) fractions of jejunum and soluble (Sol) and total membrane (Mem) fractions of colon were subjected to SDS/PAGE in a 15% gel together with a sample of pure leptin (Lep, 1 μg). After transfer onto an Immobilon membrane, leptin was detected by immunoblotting. Molecular mass value of leptin is indicated by the arrow.

Figure 6

Localization of OB-R and aminopeptidase N (ApN) in subcellular fractions. Samples of soluble (Sol), Mg2+-precipitated (Mg), and microvillar (Mic) fractions of jejunum and soluble (Sol) and total membrane (Mem) fractions of colon were subjected to SDS/PAGE in 10% gel. OB-R and ApN were detected by immunoblotting. Molecular mass values are indicated by arrows.

Figure 7

Detergent-resistant membrane (DRM) analysis of OB-R and intestinal alkaline phosphatase (IAP). DRMs were prepared from the Mg2+-precipitated membrane fraction by extraction with Triton X-100 followed by sucrose gradient ultracentrifugation as described in Materials and Methods. The gradient fractions were subjected to SDS/PAGE in a 10% gel, and OB-R and IAP were detected by immunoblotting. Molecular mass values and the position in the gradient of soluble proteins and DRMs are indicated by arrows.