Lise-Lotte Niels-Christiansen

Cholera toxin entry into pig enterocytes occurs via a lipid raft- and clathrin-dependent mechanism

The small intestinal brush border is composed of lipid raft microdomains, but little is known about their role in the mechanism whereby cholera toxin gains entry into the enterocyte. The present work characterized the binding of cholera toxin B subunit (CTB) to the brush border and its internalization. CTB binding and endocytosis were performed in organ-cultured pig mucosal explants and studied by fluorescence microscopy, immunogold electron microscopy, and biochemical fractionation. By fluorescence microscopy CTB, bound to the microvillar membrane at 4 degrees C, was rapidly internalized after the temperature was raised to 37 degrees C. By immunogold electron microscopy CTB was seen within 5 min at 37 degrees C to induce the formation of numerous clathrin-coated pits and vesicles between adjacent microvilli and to appear in an endosomal subapical compartment. A marked shortening of the microvilli accompanied the toxin internalization whereas no formation of caveolae was observed. CTB was strongly associated with the buoyant, detergent-insoluble fraction of microvillar membranes. Neither CTBs raft association nor uptake via clathrin-coated pits was affected by methyl-beta-cyclodextrin, indicating that membrane cholesterol is not required for toxin binding and entry. The ganglioside GM(1) is known as the receptor for CTB, but surprisingly the toxin also bound to sucrase-isomaltase and coclustered with this glycosidase in apical membrane pits. CTB binds to lipid rafts of the brush border and is internalized by a cholesterol-independent but clathrin-dependent endocytosis. In addition to GM(1), sucrase-isomaltase may act as a receptor for CTB.

Endocytic trafficking from the small intestinal brush border probed with FM dye

The small intestinal brush border functions as the bodys main portal for uptake of dietary nutrients and simultaneously acts as the largest permeability barrier against pathogens. To enable this, the digestive enzymes of the brush border are organized in lipid raft microdomains stabilized by cross-linking galectins and intelectin, but little is known about the dynamic properties of this highly specialized membrane. Here, we probed the endocytic membrane trafficking from the brush border of organ-cultured pig intestinal mucosal explants by use of a fixable, lipophilic FM dye. The fluorescent dye readily incorporated into the brush border, and by 15 min faint but distinct punctae were detectable approximately 1 microm beneath the brush border, indicative of a constitutive endocytosis. The punctae represented a subpopulation of early endosomes confined to the actomyosin-rich terminal web region, and their number and intensity increased by 1 h, but trafficking further into the enterocyte was not observed except in immature epithelial cells of the crypts. A powerful ligand for receptor-mediated endocytosis, cholera toxin B subunit, increased apical endocytosis and caused membrane trafficking to proceed to compartments localized deeper into the cytoplasm of the enterocytes. Two major raft-associated brush border enzymes, alkaline phosphatase and aminopeptidase N, were excluded from endocytosis. We propose that the terminal web cytoskeleton, by inhibiting traffic from apical early endosomes further into the cell, contributes to the overall permeability barrier of the gut.

Crucial role of alkaline sphingomyelinase in sphingomyelin digestion: a study on enzyme knockout mice

Alkaline sphingomyelinase (alk-SMase) hydrolyses sphingomyelin (SM) to ceramide in the gut. To evaluate the physiological importance of the enzyme, we generated alk-SMase knockout (KO) mice by the Cre-recombinase-Locus of X-over P1(Cre-LoxP) system and studied SM digestion. Both wild-type (WT) and KO mice were fed 3H-palmitic acid labeled SM together with milk SM by gavage. The lipids in intestinal content, intestinal tissues, serum, and liver were analyzed by TLC. In KO mice, nondigested 3H-SM in the intestinal content increased by 6-fold and the formation of 3H-ceramide decreased markedly, resulting in 98% reduction of 3H-ceramide/3H-SM ratio 1 h after gavage. The absorbed 3H-palmitic acid portion was decreased by 95%. After 3 h, a small increase in 3H-ceramide was identified in distal intestine in KO mice. In feces, 3H-SM was increased by 243% and ceramide decreased by 74% in the KO mice. The KO mice also showed significantly decreased radioactivity in liver and serum. Furthermore, alkaline phosphatase activity in the mucosa was reduced by 50% and histological comparison of two female littermates preliminarily suggested mucosal hypertrophy in KO mice. This study provides definite proof for crucial roles of alk-SMase in SM digestion and points to possible roles in regulating mucosal growth and alkaline phosphatase function.

Leptin and the Obesity Receptor (OB-R) in the Small Intestine and Colon: A Colocalization Study

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.

Localization and biosynthesis of aminopeptidase N in pig fetal small intestine

BACKGROUND & AIMS Little is known about the expression of brush border enzymes in fetal enterocytes. The aim of this study was to describe the localization and biosynthesis of porcine fetal aminopeptidase N. METHODS This study was performed using histochemistry and immunoelectron microscopy and [35S]methionine labeling of cultured mucosal explants. RESULTS Enzyme activity was present in the brush border membrane and extended into the apical cytoplasm. The protein was colocalized with cationized ferritin at the surface of endocytic structures including coated pits, vesicles, tubules, and large vacuoles in the apical cytoplasm. The transient high mannose-glycosylated form of fetal aminopeptidase N was processed to the mature complex-glycosylated form at a markedly slower rate than the enzyme in adult intestine. Likewise, dimerization occurred slowly compared with the adult form of aminopeptidase N, and it took place mainly after the Golgi-associated complex glycosylation. The enzyme had a biphasic appearance in the Mg(2+)-precipitated and microvillar fractions, indicating that the bulk of newly made aminopeptidase N is transported to the brush border membrane before appearing in the apical endocytic structures. CONCLUSIONS In comparison with the adult enzyme, fetal aminopeptidase N has a more widespread subcellular distribution with substantial amounts present in apical endocytic compartments characteristic of the fetal enterocyte.

Permeabilization of enterocytes induced by absorption of dietary fat

Abstract Absorption of dietary fat in the small intestine involves epithelial exposure to potentially harmful molecules such as bile salts and free fatty acids. We used organ culture of porcine jejunal explants incubated with a pre-digested mixture of fat (plant oil), bile and pancreatin to mimick the physiological process of dietary fat absorption, and short exposures to the fat mixture caused fat droplet accumulation within villus enterocytes. Lucifer yellow (LY), a fluorescent membrane-impermeable polar tracer was included to monitor epithelial integrity. Both in controls and during fat absorption LY penetrated the epithelium and accumulated in the basal lamina and the lamina propria. LY was also seen in the paracellular space, whereas villus enterocytes were generally only weakly labeled except for small amounts taken up by apical endocytosis. In the crypts, however, fat absorption induced cell permeabilization with LY accumulating in the cytosol and nucleus. Morphologically, both apical and basolateral membranes appeared intact, indicating that the leakiness was caused by minor lesions in the membrane. Albeit to a lesser extent, bile alone was capable of permeabilizing crypt cells, implying that the surfactant properties of bile salts are involved in the process. In addition to LY, crypt enterocytes also became permeable for albumin, ovalbumin and insulin. In conclusion, during fat absorption the permeability of the gut epithelium is increased mainly in the crypts. A possible explanation is that cell membranes of immature crypt cells, lacking detergent-resistant lipid raft microdomains, are less resistant to the deleterious effects of bile salts and free fatty acids.

Dietary free fatty acids form alkaline phosphatase-enriched microdomains in the intestinal brush border membrane.

Abstract Free fatty acids released during intralumenal digestion of dietary fat must pass through the enterocyte brush border membrane before triacylglycerol reassembly and subsequent chylomicron delivery to the lymph system. In the present work fluorescent BODIPY fatty acid analogs were used to study this membrane passage in organ cultured intestinal mucosal explants. We found that in addition to a rapid uptake into the cytoplasm, a fraction of the fatty acid analogs were inserted directly into the brush border membrane. Furthermore, a brief exposure of microvillar membrane vesicles to a fat mixture mimicking a physiological solution of dietary mixed micelles, rearranged the lipid raft microdomain organization of the membranes. Thus, the fat mixture generated a low-density subpopulation of microvillar detergent resistant membranes (DRMs) highly enriched in alkaline phosphatase (AP). Since this GPI-linked enzyme is the membrane protein in the brush border with the highest affinity for lipid rafts, this implies that free fatty acids selectively insert stably into these membrane microdomains. We have previously shown that absorption of dietary lipids transiently induce a selective endocytosis of AP from the brush border, and from work by others it is known that fat absorption is accompanied by a rise in serum AP and secretion of surfactant-like particles from enterocytes. We propose that these physiological processes may be triggered by the sequestering of dietary free fatty acids in lipid raft microdomains of the brush border.