Uptake of cholesterol by small intestinal brush border membrane is protein-mediated

Absorption of cholesterol by small intestinal brush border membrane from either mixed micelles or small unilamellar vesicles is protein-mediated. It is a second-order reaction. The kinetic data are consistent with a mechanism involving collision-induced transfer of cholesterol. With micelles as the donor particle, there is net transfer of cholesterol while with small unilamellar vesicles as the donor, cholesterol is evenly distributed between the two lipid pools at equilibrium. The cholesterol absorption by brush border membrane from both mixed micelles and small unilamellar vesicles reveals saturation kinetics. Proteolytic treatment of brush border membrane with papain releases about 25% of the total membrane protein. As a result, the cholesterol uptake by brush border membrane changes from a second-order reaction to a first-order one. The reaction mechanism changes from collision-induced cholesterol uptake to a mechanism involving diffusion of monomeric cholesterol through the aqueous phase. The protein(s) released into the supernatant by papain treatment of brush border membrane exhibit(s) cholesterol exchange activity between two populations of small unilamellar vesicles. The supernate-protein(s) bind(s) the spin-labeled cholesterol analogue 3-doxyl-5 alpha-cholestane.     

Lipid raft organization and function in the small intestinal brush border

The enterocyte brush border of the small intestine is a highly specialized membrane designed to function both as a high capacity digestive/absorptive surface of dietary nutrients and a permeability barrier towards lumenal pathogens. It is characterized by an unusually high content of glycolipids (∼30% of the total microvillar membrane lipid), enabling the formation of liquid ordered microdomains, better known as lipid rafts. The glycolipid rafts are stabilized by galectin-4, a 36 kDa divalent lectin that cross-links galactosyl (and other carbohydrate) residues present on membrane lipids and several brush border proteins, including some of the major hydrolases. These supramolecular complexes are further stabilized by intelectin, a 35 kDa trimeric lectin that also functions as an intestinal lactoferrin receptor. As a result, brush border hydrolases, otherwise sensitive to pancreatic proteinases, are protected from untimely release into the gut lumen. Finally, anti-glycosyl antibodies, synthesized by plasma cells locally in the gut, are deposited on the brush border glycolipid rafts, protecting the epithelium from lumenal pathogens that exploit lipid rafts as portals for entry to the organism.     

Glucagon-like peptide 2 has limited efficacy to increase nutrient absorption in fetal and preterm pigs

Exogenous glucagon-like peptide 2 (GLP-2) prevents intestinal atrophy and increases nutrient absorption in term newborn pigs receiving total parenteral nutrition (TPN). We tested the hypothesis that the immature intestine of fetuses and preterm neonates has a diminished nutrient absorption response to exogenous GLP-2. This was accomplished using catheterized fetal pigs infused for 6 days (87-91% of gestation) with GLP-2 (25 nmol.kg(-1).day(-1) iv; n = 7) or saline (n = 7), and cesarean-delivered preterm pigs (92% of gestation) that received TPN with GLP-2 (25 nmol.kg(-1).day(-1) iv; n = 8) or saline (n = 7) for 6 days after birth. Responses to GLP-2 were assessed by measuring intestinal dimensions, absorption of nutrients (glucose, leucine, lysine, proline) by intact tissues and brush border membrane vesicles, and abundance of sodium-glucose cotransporter mRNA. Infusion of GLP-2 increased circulating GLP-2 levels in fetuses, but did not increase intestinal mass or absorption of nutrients by intact tissues and brush border membrane vesicles, except for lysine. Administration of exogenous GLP-2 to preterm TPN-fed pigs similarly did not increase rates of nutrient absorption, yet nutrient absorption capacities of the entire small intestine tended to increase (+10-20%, P < 0.10) compared with TPN alone due to increased intestinal mass (+30%, P < 0.05). GLP-2 infusion did not increase sodium-glucose cotransporter-1 mRNA abundance in fetuses or postnatal preterm pigs. Hence, the efficacy of exogenous GLP-2 to improve nutrient absorption by the intestine of fetal and preterm pigs is limited compared with term pigs and more mature animals and humans.     

Age-associated changes in intestinal fructose uptake are not explained by alterations in the abundance of GLUT5 or GLUT2

A reduction in nutrient absorption may contribute to malnourishment in the elderly. The objectives of this study were to determine the effects of aging on the absorption of fructose in rats, as well as the mechanisms of these adaptive changes. Male Fischer 344 rats aged 1, 9, and 24 months were fed standard Purina chow for 2 weeks (PMI #5001, PMI Nutritionals, Brentwood, MO). The uptake of (14)C-labeled D-fructose was determined in vitro using the intestinal sheet method. Intestinal samples were taken for RNA isolation and for brush border membrane (BBM) and basolateral membrane (BLM) preparation. Northern blotting, Western blotting, and immunohistochemistry were used to determine the effects of age and diet on GLUT5 and GLUT2. When expressed on the basis of intestinal or mucosal weights, aging was associated with a decline in jejunal and ileal fructose uptake, whereas jejunal fructose uptake was increased when expressed on the basis of serosal or mucosal surface area. The alterations in fructose uptake were not paralleled by changes in GLUT5 or GLUT2 abundance. These results indicate that 1) the effect of age on fructose uptake depends on the method used to express results, and 2) the age-associated changes in uptake are not explained by alterations in GLUT5 and GLUT2.     

Myosin-1a is critical for normal brush border structure and composition

To develop our understanding of myosin-1a function in vivo, we have created a mouse line null for the myosin-1a gene. Myosin-1a knockout mice demonstrate no overt phenotypes at the whole animal level but exhibit significant perturbations and signs of stress at the cellular level. Among these are defects in microvillar membrane morphology, distinct changes in brush-border organization, loss of numerous cytoskeletal and membrane components from the brush border, and redistribution of intermediate filament proteins into the brush border. We also observed significant ectopic recruitment of another short-tailed class I motor, myosin-1c, into the brush border of knockout enterocytes. This latter finding, a clear demonstration of functional redundancy among vertebrate myosins-I, may account for the lack of a whole animal phenotype. Nevertheless, these results indicate that myosin-1a is a critical multifunctional component of the enterocyte, required for maintaining the normal composition and highly ordered structure of the brush border.     

Ferrous iron uptake by rat duodenal brush border membrane vesicles: Effects of dietary iron level and competing minerals (Zn, Mn, and Ca)

We sought to confirm a recent report that Fe⁺² uptake into rat brush-border membrane vesicles is markedly increased by short-term consumption of iron-deficient diet, with no additional enhancement as the animal becomes functionally iron-deficient with continuing dietary Fe deprivation. In addition, we investigated whether previously observed in vivo absorption interactions of iron, zinc, and manganese occur in the brush border membrane vesicles uptake process, and whether short-term or long-term consumption of an iron-deficient diet affects the interaction at the uptake level. We did not observe any differences in Fe⁺² uptake between normal and iron-deficient brush border membrane vesicles, even when the iron status contrast was intensified by feeding a high iron versus iron-deficient diet for 3 weeks. Equimolar Zn⁺² and Mn⁺² decreased Fe⁺² uptake by 29 to 50% and 11 to 39%, respectively. Iron deficiency did not alter these effects. Equimolar Fe⁺² decreased Zn⁺² uptake by 13 to 22%. Calcium, included as a negative control, did not affect Fe⁺² uptake. Thus, some competition between Fe⁺² and similar divalent cations does occur at the level of the brush border membrane; the exact nature of this competition remains to be determined.     

DECREASED DENSITIES OF INTRAMEMBRANOUS PARTICLES AND CYTOCHEMICALLY DETECTABLE CHOLESTEROL IN MICROVILLI OF STARVED RAT ENTEROCYTES

The densities of intramembranous particles (IMPs) and of sterol complexes induced by treatment of filipin were studied by freeze-fracture replication of intact intestine and/or isolated brush border membranes (BBM) of well-fed and starved rats. The density of IMPs and filipin-sterol complexes (FSCs) decrease considerably during starvation. Biochemical estimations show a decrease in the levels of cholesterol and proteins with respect to phospholipids during starvation which is in agreement with morphological findings. It is suggested that these changes may play a role in regulating membrane fluidity which in turn affects absorption of nutrients through BBM.     

Proline transport across the intestinal microvillus membrane may be regulated by membrane physical properties.

There is now abundant evidence that integral membrane protein function may be modulated by the physical properties of membrane lipids. The intestinal brush border membrane represents a membrane system highly specialized for nutrient absorption and, thus, provides an opportunity to study the interaction between integral membrane transport proteins and their lipid environment. We have previously demonstrated that alterations in this environment may modulate the function of the sodium-dependent glucose transporter in terms of its affinity for glucose. In this communication we report that membrane lipid-protein interactions are distinctly different for the proline transport proteins. Maximal transport rates for L-proline by either the neutral brush border or imino transport systems are reduced 10-fold when the surrounding membrane environment is made more fluid over the physiological range that exists along the crypt-villus axis. Furthermore, in microvillus membrane vesicles prepared from enterocytes isolated from along the crypt-villus axis a similar gradient exists in the functional activity of these transport systems. This would imply that either the functional activity of these transporters are regulated by membrane physical properties or that the synthesis and insertion of these proteins is coordinated in concert with membrane physical properties as the enterocyte migrates up the crypt-villus axis.     

Neuroprotective role of taurine during aging

Aging of the brain is characterized by several neurochemical modifications involving structural proteins, neurotransmitters, neuropeptides and related receptors. Alterations of neurochemical indices of synaptic function are indicators of age-related impairment of central functions, such as locomotion, memory and sensory performances. Several studies demonstrate that ionotropic GABA receptors, glutamate decarboxylase (GAD), and somatostatinergic subpopulations of GABAergic neurons are markedly decreased in experimental animal brains during aging. Additionally, levels of several neuropeptides co-expressed with GAD decrease during aging. Thus, the age-related decline in cognitive functions could be attributable, at least in part, to decrements in GABA inhibitory neurotransmission. In this study, we showed that chronic supplementation of taurine to aged mice significantly ameliorated the age-dependent decline in spatial memory acquisition and retention. We also demonstrated that concomitant with the amelioration in cognitive function, taurine caused significant alterations in the GABAergic and somatostatinergic system. These changes included (1) increased levels of the neurotransmitters GABA and glutamate, (2) increased expression of both isoforms of GAD (65 and 67) and the neuropeptide somatostatin, (3) decreased hippocampal expression of the β3 subunits of the GABA_A receptor, (4) increased expression in the number of somatostatin-positive neurons, (5) increased amplitude and duration of population spikes recorded from CA1 in response to Schaefer collateral stimulation and (6) enhanced paired pulse facilitation in the hippocampus. These specific alterations of the inhibitory system caused by taurine treatment oppose those naturally occurring in the aging brain, suggesting a protective role of taurine in this process. An increased understanding of age-related neurochemical changes in the GABAergic system will be important in elucidating the underpinnings of the functional changes of aging. Taurine supplementation might help forestall the age-related decline in cognitive functions through interaction with the GABAergic system.     

Aging: its influence on the intestinal unstirred water layer thickness, surface area, and resistance in the unanesthetized rat

The intestinal absorption of some nutrients changes with aging. As the unstirred water layer (UWL) is an important rate limiting step in the absorption of nutrients in general and of lipid soluble nutrients in particular, we investigated possible changes in the UWL dimensions in the aging rat in vivo. We measured the thickness (d) of the UWL using rapid changes in the luminal sodium concentration to induce changes in the transmucosal potential differences. We assessed the surface area (Sw) and resistance (d/SwD) of the UWL at varying flow rates by using increasingly lipophilic medium chain saturated alcohols as probes. At high UWL resistance, d decreased from 318 to 268 microns between 1 and 29 months of age. As the animals aged, Sw changed from 114 to 106 cm2/100 cm and from 262 to 214 cm2/100 cm at low and high flow rates, respectively, using dodecanol as a probe. The resistance of the UWL (d/SwD) remained relatively stable at all ages studied. These experiments demonstrate that age-related changes in absorption are dependent on the aqueous diffusion coefficient and degree of lipid solubility of the specific nutrients. At low UWL resistance, absorption of compounds with higher diffusion coefficients and greater aqueous solubility is decreased with aging. In contrast, previous studies have demonstrated that the absorption of nutrients with low diffusion coefficients and high lipid solubility increases with aging especially when the resistance of the UWL is high.     

Isolation and characterization of the brush border fraction from newborn rat renal proximal tubule cells

A renal brush border fraction was isolated from newborn Sprague-Dawley rats, and its morphological and enzymatic characteristics were studied in comparison to that from the adult. Definite microvillar structures are seen by electron microscopy, and brush border preparations from the newborn are enriched in known marker enzymes. Though morphological development is more advanced and enzyme specific activities are greater in the adult, polyacrylamide gel electrophoresis of membrane proteins reveal no significant change in pattern with increasing age. These studies suggest that the brush border of the proximal tubule cell is present at birth as a significantly developed structure.     

Effect of the insecticidal Galanthus nivalis agglutinin on metabolism and the activities of brush border enzymes in the rat small intestine

With increasing use of lectin genes in crop plants to improve insect resistance, the dietary exposure of humans to lectins will rise and it is necessary to assess whether the presently most favored insecticidal lectin, Galanthus nivalis agglutinin, would be harmful for mammals. Effects of Galanthus nivalis agglutinin on gut and brush border enzymes were studied in rats over a 10-day dietary exposure and compared with those of a known antinutrient, phytohaemagglutinin. At a level that provides insecticidal protection for plants but did not reduce the growth of young rats, Galanthus nivalis agglutinin had negligible effects on the weight and length of the small intestine even though there was a slight, but significant hypertrophy of this tissue. However, the activities of brush border enzymes were affected; sucrase-isomaltase activity was nearly halved and those of alkaline phosphatase and aminopeptidase were significantly increased. Although most of the changes in gut metabolism caused by the incorporation of Galanthus nivalis agglutinin in the diet were less extensive than those found with toxic phytohaemagglutinin, some of them may be potentially deleterious. Thus, further and longer animal studies are needed to establish whether it is safe to use Galanthus nivalis agglutinin in transgenic plants destined for human consumption.     

Energetics of the Na+-dependent transport of D-glucose in renal brush border membrane vesicles.

The energetics of the Na+-dependent transport of D-glucose into osmotically active membrane vesicles, derived from the brush borders of the rabbit renal proximal tubule, was studied by determining how alterations in the electrochemical potential of the membrane induced by anions, ionophores, and a proton conductor affect the uptake of the sugar. The imposition of a large NaCl gradient (medium is greater than vesicle) resulted in the transient uptake of D-glucose into brush border membranes against its concentration gradient. In the presence of Na+ salts of isethionate or sulfate, both relatively impermeable anions, there was no accumulation of D-glucose above the equilibrium value. With Na+ salts of two highly permeable lipophilic anions, NO3- and SCN-, the transient overshoot was enhanced relative to that with Cl-. With Na+ salts whose mode of membrane translocation is electroneutral, i.e. acetate, bicarbonate, and phosphate, no overshoot was found. These findings suggest that only anions which penetrate the brush border membrane and generate an electrochemical potential, negative on the inside, permit the uphill Na+-dependent transport of D-glucose.     

Anti-glycosyl antibodies in lipid rafts of the enterocyte brush border: a possible host defense against pathogens

The pig small intestinal brush border is a glycoprotein- and glycolipid-rich membrane that functions as a digestive/absorptive surface for dietary nutrients as well as a permeability barrier for pathogens. The present work was performed to identify carbohydrate-binding (lectinlike) proteins associated with the brush border. Chromatography on lactose-agarose was used to isolate such proteins, and their localization was studied biochemically and by immunofluorescence microscopy and immunogold electron microscopy. IgG and IgM were the two major proteins isolated, indicating that naturally occurring anti-glycosyl antibodies are among the major lectinlike proteins in the gut. IgG and IgM as well as IgA were localized to the enterocyte brush border, and a brief lactose wash partially released all three immunoglobulins from the membrane, indicating that anti-glycosyl antibodies constitute a major part of the immunoglobulins at the lumenal surface of the gut. The antibodies were associated with lipid rafts at the brush border, and they frequently (52%) coclustered with the raft marker galectin 4. A lactose wash increased the susceptibility of the brush border toward lectin peanut agglutin and cholera toxin B, suggesting that anti-glycosyl antibodies compete with other carbohydrate-binding proteins at the lumenal surface of the gut. Thus anti-glycosyl antibodies constitute a major group of proteins associated with the enterocyte brush border membrane. We propose they function by protecting the lipid raft microdomains of the brush border against pathogens.     

Isolation and characterization of the brush border fraction from newborn rat renal proximal tubule cells.

A renal brush border fraction was isolated from newborn Sprague-Dawley rats, and its morphological and enzymatic characteristics were studied in comparison to that from the adult. Definite microvillar structures are seen by electron microscopy, and border preparations from the newborn are enriched in known marker enzymes. Though morphological development is more advanced and enzyme specific activities are greater in the adult, polyacrylamide gel electrophoresis of membrane proteins reveals no significant change in pattern with increasing age. These studies suggest that the brush border of the proximal tubule cell is present at birth as a significantly developed structure.     

Cytoskeletal protein and mRNA accumulation during brush border formation in adult chicken enterocytes

We have explored the development of the brush border in adult chicken enterocytes by analyzing the cytoskeletal protein and mRNA levels as enterocytes arise from crypt stem cells and differentiate as they move toward the villus. At the base of the crypt, a small population of cells contain a rudimentary terminal web and a few short microvilli with long rootlets. These microvilli appear to arise from bundles of actin filaments which nucleate on the plasma membrane. The microvilli apparently elongate via the addition of membrane supplied by vesicles that fuse with the microvillus and extend the membrane around the actin core. Actin, villin, myosin, tropomyosin and spectrin, but not myosin I (previously called 110 kD; see Mooseker and Coleman, J. Cell Biol. 108, 2395-2400, 1989) are already concentrated in the luminal cytoplasm of crypt cells, as seen by immunofluorescence. Using quantitative densitometry of cDNA-hybridized RNA blots from cells isolated from crypts, villus middle (mid), or villus tip (tip), we found a 2- to 3-fold increase in villin, calmodulin and tropomyosin steady-state mRNA levels; an increase parallel to morphological brush border development. Actin, spectrin and myosin mRNA levels did not change significantly. ELISA of total crypt, mid and tip cell lysates show that there are no significant changes in actin, myosin, spectrin, tropomyosin, myosin I, villin or alpha-actinin protein levels as the brush border develops. The G-/F-actin ratio also did not change with brush border assembly. We conclude that, although the brush border is not fully assembled in immature enterocytes, the major cytoskeletal proteins are present in their full concentration and already localized within the apical cytoplasm. Therefore brush border formation may involve reorganization of a pool of existing cytoskeletal proteins mediated by the expression or regulation of an unidentified key protein(s).     

Aminopeptidase N (CD13) is a molecular target of the cholesterol absorption inhibitor ezetimibe in the enterocyte brush border membrane

Intestinal cholesterol absorption is an important regulator of serum cholesterol levels. Ezetimibe is a specific inhibitor of intestinal cholesterol absorption recently introduced into medical practice; its mechanism of action, however, is still unknown. Ezetimibe neither influences the release of cholesterol from mixed micelles in the gut lumen nor the transfer of cholesterol to the enterocyte brush border membrane. With membrane-impermeable Ezetimibe analogues we could demonstrate that binding of cholesterol absorption inhibitors to the brush border membrane of small intestinal enterocytes from the gut lumen is sufficient for inhibition of cholesterol absorption. A 145-kDa integral membrane protein was identified as the molecular target for cholesterol absorption inhibitors in the enterocyte brush border membrane by photoaffinity labeling with photoreactive Ezetimibe analogues (Kramer, W., Glombik, H., Petry, S., Heuer, H., Schafer, H. L., Wendler, W., Corsiero, D., Girbig, F., and Weyland, C. (2000) FEBS Lett. 487, 293-297). The 145-kDa Ezetimibe-binding protein was purified by three different methods and sequencing revealed its identity with the membrane-bound ectoenzyme aminopeptidase N ((alanyl)aminopeptidase; EC 3.4.11.2; APN; leukemia antigen CD13). The enzymatic activity of APN was not influenced by Ezetimibe (analogues). The uptake of cholesterol delivered by mixed micelles by confluent CaCo-2 cells was partially inhibited by Ezetimibe and nonabsorbable Ezetimibe analogues. Preincubation of confluent CaCo-2 cells with Ezetimibe led to a strong decrease of fluorescent APN staining with a monoclonal antibody in the plasma membrane. Independent on its enzymatic activity, aminopeptidase N is involved in endocytotic processes like the uptake of viruses. Our findings suggest that binding of Ezetimibe to APN from the lumen of the small intestine blocks endocytosis of cholesterol-rich membrane microdomains, thereby limiting intestinal cholesterol absorption.     

Changes in the structural complexity of the aged brain

Structural changes of neurons in the brain during aging are complex and not well understood. Neurons have significant homeostatic control of essential brain functions, including synaptic excitability, gene expression, and metabolic regulation. Any deviations from the norm can have severe consequences as seen in aging and injury. In this review, we present some of the structural adaptations that neurons undergo throughout normal and pathological aging and discuss their effects on electrophysiological properties and cognition. During aging, it is evident that neurons undergo morphological changes such as a reduction in the complexity of dendrite arborization and dendritic length. Spine numbers are also decreased, and because spines are the major sites for excitatory synapses, changes in their numbers could reflect a change in synaptic densities. This idea has been supported by studies that demonstrate a decrease in the overall frequency of spontaneous glutamate receptor-mediated excitatory responses, as well as a decrease in the levels of alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid and N-methyl-d-aspartate receptor expression. Other properties such as gamma-aminobutyric acid A receptor-mediated inhibitory responses and action potential firing rates are both significantly increased with age. These findings suggest that age-related neuronal dysfunction, which must underlie observed decline in cognitive function, probably involves a host of other subtle changes within the cortex that could include alterations in receptors, loss of dendrites, and spines and myelin dystrophy, as well as the alterations in synaptic transmission. Together these multiple alterations in the brain may constitute the substrate for age-related loss of cognitive function.     

Prevention of alterations in the transport of nutrients in pyelonephritic rats by immunization with pili

The uptake of D-glucose, L-aspartate, L-lysine and L-proline was studied in renal brush border membrane vesicles prepared from control, infected and actively immunized-infected rats. The uptake of D-glucose, L-lysine and L-proline was decreased significantly (p less than 0.05) during the course of infection in the infected animals. However, the uptake of L-aspartate was increased significantly (p less than 0.05) in early stages and decreased significantly (p less than 0.05) in later stages of infection in the infected animals. When the animals were actively immunized with pili, still there were changes in the uptake of D-glucose and L-aspartate, but the changes appeared later and less pronounced. No change in the uptake of L-lysine and L-proline was observed in the immunized-infected animals. The findings demonstrated that active immunization with pili prevents alterations in the uptake of nutrients in pyelonephritic rats.     

Identification of binding proteins for cholesterol absorption inhibitors as components of the intestinal cholesterol transporter

To identify protein components of the intestinal cholesterol transporter, rabbit small intestinal brush border membrane vesicles were submitted to photoaffinity labeling using photoreactive derivatives of 2-azetidinone cholesterol absorption inhibitors. An integral membrane protein of M(r) 145.3+/-7.5 kDa was specifically labeled in brush border membrane vesicles from rabbit jejunum and ileum. Its labeling was concentration-dependently inhibited by the presence of cholesterol absorption inhibitors whereas bile acids, D-glucose, fatty acids or cephalexin had no effect. The inhibitory potency of 2-azetidinones to inhibit photolabeling of the 145 kDa protein correlated with their in vivo activity to inhibit intestinal cholesterol absorption. These results suggest that an integral membrane protein of M(r) 145 kDa is (a component of) the cholesterol absorption system in the brush border membrane of small intestinal enterocytes.