Intestinal epithelial cells modulate antigen-presenting cell responses to bacterial DNA.

Intestinal epithelial cells and antigen-presenting cells orchestrate mucosal innate immunity. This study investigated the role of bacterial DNA in modulating epithelial and bone marrow-derived antigen-presenting cells (BM-APCs) and subsequent T-lymphocyte responses. Murine MODE-K epithelial cells and BM-APCs were treated with DNA from either Bifidobacterium breve or Salmonella enterica serovar Dublin directly and under coculture conditions with CD4(+) T cells. Apical stimulation of MODE-K cells with S. Dublin DNA enhanced secretion of cytokines from underlying BM-APCs and induced interleukin-17 (IL-17) and gamma interferon (IFN-γ) secretion from CD4(+) T cells. Bacterial DNA isolated from either strain induced maturation and increased cytokine secretion from BM-APCs. Conditioned medium from S. Dublin-treated MODE-K cells elicited an increase in cytokine secretion similar to that seen for S. Dublin DNA. Treatment of conditioned medium from MODE-K cells with RNase and protease prevented the S. Dublin-induced increased cytokine secretion. Oral feeding of mice with B. breve DNA resulted in enhanced levels of colonic IL-10 and transforming growth factor ß (TGFß) compared with what was seen for mice treated with S. Dublin DNA. In contrast, feeding mice with S. Dublin DNA increased levels of colonic IL-17 and IL-12p70. T cells from S. Dublin DNA-treated mice secreted high levels of IL-12 and IFN-γ compared to controls and B. breve DNA-treated mice. These results demonstrate that intestinal epithelial cells are able to modulate subsequent antigen-presenting and T-cell responses to bacterial DNA with pathogenic but not commensal bacterial DNA inducing effector CD4(+) T lymphocytes.

VSL#3 probiotic upregulates intestinal mucosal alkaline sphingomyelinase and reduces inflammation.

BACKGROUND: Alkaline sphingomyelinase, an enzyme found exclusively in bile and the intestinal brush border, hydrolyzes sphingomyelin into ceramide, sphingosine and sphingosine-1-phosphate, thereby inducing epithelial apoptosis. Reduced levels of alkaline sphingomyelinase have been found in premalignant and malignant intestinal epithelia and in ulcerative colitis tissue. Probiotic bacteria can be a source of sphingomyelinase. OBJECTIVE: To determine the effect of VSL#3 probiotic therapy on mucosal levels of alkaline sphingomyelinase, both in a mouse model of colitis and in patients with ulcerative colitis. METHODS: Interleukin-10 gene-deficient (IL10KO) and wild type control mice were treated with VSL#3 (10(9) colony-forming units per day) for three weeks, after which alkaline sphingomyelinase activity was measured in ileal and colonic tissue. As well, 15 patients with ulcerative colitis were treated with VSL#3 (900 billion bacteria two times per day for five weeks). Alkaline sphingomyelinase activity was measured through biopsies and comparison of ulcerative colitis disease activity index scores obtained before and after treatment. RESULTS: Lowered alkaline sphingomyelinase levels were seen in the colon (P=0.02) and ileum (P=0.04) of IL10KO mice, as compared with controls. Treatment of these mice with VSL#3 resulted in upregulation of mucosal alkaline sphingomyelinase activity in both the colon (P=0.04) and the ileum (P=0.01). VSL#3 treatment of human patients who had ulcerative colitis decreased mean (+/- SEM) ulcerative colitis disease activity index scores from 5.3+/-1.8946 to 0.70+/-0.34 (P=0.02) and increased mucosal alkaline sphingomyelinase activity. CONCLUSION: Mucosal alkaline sphingomyelinase activity is reduced in the intestine of IL10KO mice with colitis and in humans with ulcerative colitis. VSL#3 probiotic therapy upregulates mucosal alkaline sphingomyelinase activity.

Acidosis and lipopolysaccharide from Escherichia coli B:055 cause hyperpermeability of rumen and colon tissues.

The objective of the present investigation was to evaluate the effects of acidic pH of the perfusate and presence of lipopolysaccharide (LPS) on permeability of rumen and colon mucosal tissues to mannitol and LPS using the Ussing chamber system. Rumen and colon tissues (n = 8), obtained from slaughtered feedlot steers, were tested for changes in permeability to (3)H-mannitol under pH of 4.5, 5.5, and 6.5 for rumen and at 5.5, 6.5, and 7.4 for colon, with or without LPS from Escherichia coli B:055 at 500 microg/mL. The (3)H-Mannitol was added at 10 microL (525.4 GBq/mmol) on the mucosal side of the Ussing chamber to detect changes in permeability, and 4 samples were taken at 20, 25, 30, and 35 min from the serosal side. Permeability of rumen and colon mucosa to (3)H-mannitol increased 6- and 5-fold, respectively, at acidic pH values of 4.5 and 5.5 and in the presence of 500 micro/mL of LPS. In contrast, LPS did not affect rumen and colon permeability at pH that ranged from 5.5 and 7.4. Translocation of LPS across the rumen and colon mucosa of cattle was not pH dependent. The LPS translocated through these tissues if present at the mucosal side. In conclusion, the permeability of rumen and colon tissues to (3)H-mannitol increased in presence of LPS and under acidic pH, whereas LPS permeated through mucosal tissues independently of the pH of the perfusate. Further research is warranted to understand the mechanism(s) by which acidic pH of the rumen digesta and presence of LPS make rumen and colon tissues "leaky".

Fecal Microbial Transplantation in Inflammatory Bowel Disease: A Movement Too Big to Be Ignored.

The development of new therapies for inflammatory bowel disease is plagued by high costs, potential side effects, and variable levels of effectiveness. Fecal microbial transplant in inflammatory bowel diseases can offer an alternative to traditionally developed pharmacologic therapies and has demonstrated the ability to induce disease remission in randomized control trials. However, questions remain about the ultimate role of this therapy in disease management, including long term safety, and the optimal composition of transplanted stool.

PGM1 deficiency: Substrate use during exercise and effect of treatment with galactose.

Mutations in PGM1 (phosphoglucomutase 1) cause Glycogen Storage Disease type XIV, which is also a congenital disorder of protein N-glycosylation. It presents throughout life as myopathy with additional systemic symptoms. We report the effect of oral galactose treatment during five months in a patient with biochemically and genetically confirmed PGM1 deficiency. The 12-minute-walking distance increased by 225 m (65%) and transferrin glycosylation was restored to near-normal levels. The exercise assessments showed a severe exercise intolerance due to a block in skeletal muscle glycogenolytic capacity and that galactose treatment tended to normalize skeletal muscle substrate use from fat to carbohydrates during exercise.

Oral vanadate reduces Na(+)-dependent glucose transport in rat small intestine.

The effects of oral vanadate supplementation on intestinal morphometry and glucose transport were examined in STZ-induced diabetic and age-matched control male Sprague-Dawley rats. Animals received 0.1 mg/ml vanadium pentoxide in their drinking water over 14 days. Vanadate reduced intestinal glucose maximal transport capacity in both diabetic and control animals. In jejunum tissue, this decrease in glucose absorption was a direct consequence of downregulation of the glucose carrier and was not related to changes in mucosal morphometry. In the ileum tissue of control animals, the vanadate-induced decrease in glucose maximal transport capacity occurred in conjunction with an increase in carrier affinity and mucosal morphometric measurements. In the ileum tissue of diabetic animals, the vanadate-induced decrease in glucose maximal transport capacity occurred with a decrease in mucosal morphometric measurements. Na(+)-K(+)-adenosine triphosphatase activity was affected by vanadate only in diabetic animals. These results demonstrate that oral vanadate supplementation results in downregulation of the small intestinal sodium-dependent glucose carrier in both diabetic and nondiabetic rats. Furthermore, the vanadate effect may be occurring at the cellular level.

Orally administered immunosuppressants modify intestinal uptake of nutrients in rabbits.

The effect on intestinal nutrient transport of the immunosuppressive drugs cyclosporin A (CsA), cyclosporin G (CsG), and rapamycin (RAP) was determined in New Zealand white rabbits. Rabbits received oral doses of CsA (20 mg/kg/day), CsG (10 mg/kg/day), or RAP (1 mg/kg/day) for 10 days. Animals receiving RAP had decreased food intake and weight gain compared with controls. This correlated with a decrease in both total ileal weight and corresponding mucosal weight. CsA and CsG administration had no effect on food intake, total weight gain, or intestinal weight. Villus surface area was significantly decreased in all groups as compared with controls. Jejunal uptake of D-glucose as well as 1 medium and 4 long chain fatty acids was not affected by drug administration, while both mucosal-to-serosal and net 3-0-methylglucose fluxes were increased (P < 0.05) in the jejunum by all 3 drugs. In the ileum, the rates of uptake of D-glucose as well as stearic and linoleic acids were increased in animals treated with RAP compared with controls. There was an increase in the ileal values of the maximal transport rate (Vmax) and apparent Michaelis constant (Km*) in RAP, and a fall in the Vmax and Km* in CsG. CsG administration resulted in a decreased cholesterol uptake in both jejunum and ileum, and a decreased D-glucose uptake in the ileum compared with controls. These differences in glucose uptake among groups could not be attributed to variations in body, intestinal, or mucosal weights. It is unlikely that the changes observed in CsA- and CsG-treated animals would have nutritional importance, as these animals gained weight normally. In addition, in these animals the changes mainly occurred in the ileum, not in the jejunum, where most glucose is absorbed, and the associated alterations in the values of the Vmax and Km* would lead to reciprocal changes in the rates of uptake of varying luminal concentrations of glucose. In contrast, these changes are likely to be of more importance in RAP-treated animals, since they failed to gain weight normally. The significance of these findings needs to be established in chronically treated animals.

Interleukin-10 gene-deficient mice develop a primary intestinal permeability defect in response to enteric microflora.

The normal intestinal epithelium provides a barrier relatively impermeable to luminal constituents. However, patients with inflammatory bowel disease experience enhanced intestinal permeability that correlates with the degree of injury. IL-10 gene-deficient mice were studied to determine whether increased intestinal permeability occurs as a primary defect before the onset of mucosal inflammation or is secondary to mucosal injury. At 2 weeks of age, IL-10 gene-deficient mice show an increase in ileal and colonic permeability in the absence of any histological injury. This primary permeability defect is associated with increased mucosal secretion of interferon-gamma and tumor necrosis factor-alpha, and does not involve an increase in nitric oxide synthase activity. Colonic permeability remains elevated as inflammation progresses, while ileal permeability normalizes by 6 weeks of age. IL-10 gene-deficient mice raised under germ-free conditions have no inflammation, and demonstrate normal permeability and cytokine levels. This data suggests that the intestinal permeability defect in IL-10 gene-deficient mice occurs due to a dysregulated immune response to normal enteric microflora and, furthermore, this permeability defect exists prior to the development of mucosal inflammation.

Effects of physical activity, body weight and composition, and muscular strength on bone density in young women.

OBJECTIVE: The purpose of this study was to examine the relationship between body weight and composition, muscular strength, physical activity, and bone mineral density (BMD) in eumenorrheic college-aged women. METHODS: BMD and bone mineral content (BMC) of the total body, and BMD of the lumbar spine (L2-L4) and femoral neck (via dual energy x-ray absorptiometry), as well as body composition and muscular strength, were measured in 60 college-aged women. The women were divided into three groups: 1) low body weight athletes involved in weight-bearing, collegiate sports (N = 20), 2) matched low body weight and sedentary (N = 20), and 3) average body weight and sedentary (N = 20). All groups were matched for height, age, and age at menarche. RESULTS: The athletes had significantly greater (P < 0.05) (mean +/- SD) total body BMD (1.164 +/- 0.06 g x cm[-2]), L2-L4 BMD (1.240 +/- 0.13 g x cm[-2]), femoral neck BMD (1.144 +/- 0.13 g x cm[-2]) and total body BMC (2.44 +/- 0.30 kg) than the low body weight, sedentary (LWS) group, but were only greater than the average body weight sedentary group (AWS) for femoral neck BMD. Significant correlations were found between lean body mass (LBM) and all BMD variables (P < 0.001). A significant correlation (P < 0.01) was found between fat mass and all BMD variables in the sedentary subjects alone (N = 40), but with inclusion of the athletes (N = 60), none of the correlations between fat mass and BMD were significant. Arm and leg strength isometric torque values corrected for muscle + bone cross-sectional area (M + B CSA) were not significantly different between the athletes and LWS group, but the athletes were greater (P < 0.05) than the AWS group for both arm and leg strength/M + B CSA. No significant, site-specific correlations were found between strength/M + B and BMD. CONCLUSIONS: In summary, the athletes had significantly greater BMD, BMC, and LBM than the LWS group and, except for a greater femoral neck BMD, similar BMD, BMC, and LBM as the AWS group. These results suggest that LBM and weight-bearing exercise both enhance BMD in eumenorrheic young adult women.

The use of probiotics in gastrointestinal disease.

Probiotics are living microorganisms that can affect the host in a beneficial manner. Prebiotics are nondigestible food ingredients that stimulate the growth and activity of probiotic bacteria already established in the colon. Efficacy of probiotic compounds has been shown in a wide range of gastrointestinal diseases. Lactobacillus GG alone, or the combination of Bifidobacterium bifidum and Streptococcus thermophilus, is effective in the treatment of Clostridium difficile, as well as in preventing the frequency and severity of infectious acute diarrhea in children. Prevention of antibiotic-induced diarrhea with the concomitant administration of either Lactobacillus GG or Saccharomyces boulardii has been demonstrated. The most successful studies involve the use of Lactobacillus GG at a dose of 1 x 1010 viable organisms per day and the yeast boulardii at a dose of 1 g/day. A probiotic preparation (VSL#3 - 6 g/day) that uses a combination of three species of Bifidobacterium, four strains of Lactobacillus and one strain of Streptocccus has shown promise in maintaining remission in ulcerative colitis and pouchitis, as well as in preventing the postoperative recurrence of Crohn's disease. The mechanism of action of probiotics may include receptor competition, effects on mucin secretion or probiotic immunomodulation of gut-associated lymphoid tissue. Oral administration of probiotic compounds has been demonstrated to be well tolerated and safe. However, while probiotics have the potential to improve human health and to prevent and treat some diseases, major improvements are needed in labelling and quality assurance procedures for probiotic compounds. In addition, well planned and controlled clinical studies are necessary to delineate fully the potential for probiotic compounds.

Patients with medium-chain acyl-coenzyme a dehydrogenase deficiency have impaired oxidation of fat during exercise but no effect of L-carnitine supplementation.

BACKGROUND: It is not clear to what extent skeletal muscle is affected in patients with medium-chain acyl-coenzyme A dehydrogenase deficiency (MCADD). l-Carnitine is commonly used as a supplement in patients with MCADD, although its beneficial effect has not been verified. DESIGN: We investigated (1) fuel utilization during prolonged low-intensity exercise in patients with MCADD and (2) the influence of 4 weeks of oral l-carnitine supplementation on fuel utilization during exercise. METHODS: Four asymptomatic patients with MCADD and 11 untrained, healthy, age- and sex-matched control subjects were included. The subjects performed a 1-hour cycling test at a constant workload corresponding to 55% of Vo2max, while fat and carbohydrate metabolism was assessed, using the stable isotope technique and indirect calorimetry. The patients ingested 100 mg/kg/d of l-carnitine for 4 weeks, after which the cycling tests were repeated. RESULTS: At rest, palmitate oxidation and total fatty acid oxidation (FAO) rates were similar in patients and healthy control subjects. During constant workload cycling, palmitate oxidation and FAO rates increased in both groups, but increased 2 times as much in healthy control subjects as in patients (P = .007). Palmitate oxidation and FAO rates were unchanged by the l-carnitine supplementation. CONCLUSION: Our results indicate that patients with MCADD have an impaired ability to increase FAO during exercise but less so than that observed in patients with a number of other disorders of fat oxidation, which explains the milder skeletal muscle phenotype in MCADD. The use of carnitine supplementation in MCADD cannot be supported by the present findings.

VSL#3 probiotics provide protection against acute intestinal ischaemia/reperfusion injury.

Acute intestinal ischaemia/reperfusion injury (AII/R) is an adaptive physiologic response during critical illness, involving mesenteric vasoconstriction and hypoperfusion. Prevention of AII/R in high risk patient populations would have a significant impact on morbidity and mortality. The purpose of this study was to investigate the protective effects of VSL#3 probiotic treatment in a murine model of AII/R. Adult 129/SvEv mice were subjected to an experimental AII/R model using superior mesenteric artery occlusion. Animals were pre-treated with either three days or two weeks of VSL#3 probiotics. Local tissue injury markers were assessed by levels of myeloperoxidase and activation of nuclear factor kappa B (NFкB). Systemic and local cytokines, including interleukin (IL)-1ß, IL- 10, TNFα, and interferon gamma were measured by ELISA and multiplex fluorescent detection. VSL#3 probiotics reduced local tissue inflammation and injury due to AII/R. A two-week course of VSL#3 was more effective than a shorter three-day course. The reduction in local inflammation from the two-week course of VSL#3 is correlated to a significant reduction in levels of active IL-1ß, and tissue levels of myeloperoxidase. Levels of active NFкB were significantly elevated in the vehicle-fed AII/R mice, corroborating with tissue inflammation, which were attenuated by VSL#3 administrations. VSL#3 did not cause any systemic inflammation or lung injury. VSL#3 probiotics are effective in reducing local tissue injury from AII/R by down-regulating pro-inflammatory mediators and immune cell recruitment. This study highlights a potential role for VSL#3 in management of patients at high risk for AII/R.

Barley-derived ß-glucans increases gut permeability, ex vivo epithelial cell binding to E. coli, and naive T-cell proportions in weanling pigs.

Weaning in young animals is associated with an increased incidence of gastrointestinal infections. ß-glucans exert numerous physiological effects, including altering immune function. The objective of this study was to determine the effects of feeding barley (Hordeum vulgare L.)-derived ß-glucans on immune and intestinal function in weanling pigs (Sus scrofa). Thirty-one individually-housed Dutch Landrace pigs (21 d; initial BW, 6,298 ± 755 g) were weaned and fed a wheat-based diet (control) or a low (Lo-BG), medium (Med-BG), or high ß-glucan-containing barley-based diet (Hi-BG) for 2 wk with 7 or 8 pigs/treatment. Intestinal segments were analyzed for permeability using Ussing chambers and K88 Escherichia coli adhesion to enterocytes was assessed ex vivo. Immune cells from mesenteric lymph nodes, peripheral blood, and Peyer's patches were analyzed for lymphocyte subsets by indirect immunofluorescence and the ability to respond ex vivo to mitogens by (3)H-thymidine incorporation. Hematology and neutrophil function were determined by flow cytometry. Neutrophil burst, size, and granularity, lymphocyte proliferation, and B-cell distribution in peripheral blood lymphocytes, Peyer's patches, and mesenteric lymph nodes were not affected by ß-glucans content of the diet. The ß-glucans content of the diet altered blood concentrations of erythrocytes and leukocytes, CD4, CD45RA, and CD8 blood cells (P < 0.05). In addition, feeding ß-glucan resulted in increased (P < 0.05) percentage CD45RA positive cells in peripheral blood lymphocytes, Peyer's patches, and mesenteric lymph nodes. Mannitol permeability and tissue conductance were increased (P < 0.05) in Hi-BG fed pigs compared with control pigs. Percentage maximum K88-E.coli binding was increased in proportion to the ß-glucan content of the diet (P < 0.05). Although ß-glucan feeding during the weaning period increased blood lymphocytes and the proportion of naïve T-cells, it also increased E. coli-enterocyte binding and intestinal permeability. ß-glucan may alter immune and intestinal function of weaning pigs.

BAR domains, amphipathic helices and membrane-anchored proteins use the same mechanism to sense membrane curvature.

The internal membranes of eukaryotic cells are all twists and bends characterized by high curvature. During recent years it has become clear that specific proteins sustain these curvatures while others simply recognize membrane shape and use it as "molecular information" to organize cellular processes in space and time. Here we discuss this new important recognition process termed membrane curvature sensing (MCS). First, we review a new fluorescence-based experimental method that allows characterization of MCS using measurements on single vesicles and compare it to sensing assays that use bulk/ensemble liposome samples of different mean diameter. Next, we describe two different MCS protein motifs (amphipathic helices and BAR domains) and suggest that in both cases curvature sensitive membrane binding results from asymmetric insertion of hydrophobic amino acids in the lipid membrane. This mechanism can be extended to include the insertion of alkyl chain in the lipid membrane and consequently palmitoylated and myristoylated proteins are predicted to display similar curvature sensitive binding. Surprisingly, in all the aforementioned cases, MCS is predominantly mediated by a higher density of binding sites on curved membranes instead of higher affinity as assumed so far. Finally, we integrate these new insights into the debate about which motifs are involved in sensing versus induction of membrane curvature and what role MCS proteins may play in biology.

Inflammatory bowel disease: lessons from the IL-10 gene-deficient mouse.

The pathogenesis of Crohn's disease likely involves multifactorial interactions between genetic factors and environmental triggers. The most recent studies suggest that luminal bacteria are a significant factor in the onset and chronicity of inflammation. In interleukin-10 (IL-10) gene-deficient mice a Crohn's-like colitis develops when the mice are raised under conventional animal care facilities but fails to develop when they are raised under germ-free conditions. These mice demonstrate significant alterations in the species and the levels of bacteria colonizing the colon, suggesting that genetic factors in the host may be critical in controlling bacterial colonization. In addition, early treatment of IL-10 gene-deficient mice with antibiotics can prevent the development of colitis in later life, suggesting that early events during the neonatal period can influence later disease progression. Recent work has focused on using probiotic bacterial mixtures to alter the microbial balance in the colon in attempts to reduce inflammation. The use of the VSL-3 probiotic mixture in the IL-10 gene-deficient mouse resulted in a complete normalization of physiological transport function and barrier integrity, in conjunction with a reduction in mucosal secretion of TNF-alpha and IFN-gamma. Further, it would appear that a soluble factor is released from a bacterium found in the VSL-3 mixture that can act directly on the epithelium to enhance barrier integrity. Results from animal models of inflammatory bowel disease suggest that genetically susceptible hosts can mount a pathogenic cellular immune response to specific nonpathogenic bacterial species, as a consequence of defective immunologic tolerance and lack of appropriate mucosal defences. Probiotic bacteria appear to be a promising new alternative for the treatment of clinical conditions that are associated with alterations in gut barrier function, including Crohn' s disease.

Vanadate treatment rapidly improves glucose transport and activates 6-phosphofructo-1-kinase in diabetic rat intestine.

The effect of oral vanadate on intestinal sodium-dependent glucose transport and 6-phosphofructo-1-kinase (EC 2.7.1.11) activity was examined in male Sprague-Dawley rats following a 30-day period of non-treated streptozotocin-induced diabetes. Non-treated diabetic rats were hyperglycaemic and demonstrated increased intestinal sodium-dependent glucose transport and Na,K-ATPase activity compared with controls. These increases were associated with a significant decrease in the total activity and activity ratios (activity at 0.5 mmol/l fructose 6-phosphate at pH 7.0/activity at pH 8.0) of intestinal 6-phosphofructo-1-kinase and decreased levels of fructose 2,6-bisphosphate. Supplementation of drinking water with vanadate (0.5 mg/ml) resulted in a rapid decline in blood glucose levels to a slightly hyperglycaemic level. Jejunal glucose transport and Na,K-ATPase activity were normalized after 48 h of vanadate treatment. In contrast, ileal glucose transport was significantly reduced 12 h following beginning vanadate treatment even though Na,K-ATPase activity did not normalize until 36 h later. Km was significantly decreased in both jejunum and ileum by vanadate treatment indicating an increased affinity of the sodium-dependent intestinal glucose transporter for glucose. 6-phosphofructo-1-kinase total activity and susceptibility to ATP inhibition was completely restored after 12 h of vanadate treatment. This increase was associated with a rise in fructose 2,6-bisphosphate levels. Fasting rats for 12 h had no effect on glucose transport or 6-phosphofructo-1-kinase activity, indicating the anorectic effect of vanadate was not responsible for changes in either parameter. In contrast, cycloheximide prevented both the rise in 6-phosphofructo-1-kinase activity and the rise in fructose 2,6-bisphosphate levels, and the subsequent reduction in glucose transport, indicating a requirement for protein synthesis. The removal of vanadate resulted in an immediate return to pre-treatment blood glucose levels. In contrast, intestinal glucose transport and 6-phosphofructo-1-kinase activity remained at treatment levels up until 72 h, indicating that oral vanadate treatment can have prolonged beneficial effects on intestinal function. In conclusion, the treatment of streptozotocin-induced diabetic rats with oral vanadate results in an activation of 6-phosphofructo-1-kinase coupled with a normalization of intestinal sodium-dependent glucose transport. Vanadate may thus have a beneficial effect on intestinal function and may prove useful as oral adjunctive diabetic therapy.

Basolateral membrane lipid dynamics alter Na-K ATPase activity in rabbit small intestine.

The role of basolateral membrane fluidity in regulating Na-K ATPase activity along the crypt-villus axis in rabbit distal small intestine was assessed. Basolateral membranes were prepared from isolated villus and crypt enterocytes at 24- to 28-fold enhancement. Villus basolateral membranes were significantly (p < 0.001) more fluid than crypt basolateral membranes as measured by 1,6-diphenyl-1,3,5-hexatriene. No difference was seen between the two groups as measured by either 2-(9-anthroyloxy)-stearic fatty acid or 16-(9-anthroyloxy)-palmitic acid. Fluidity alterations were accompanied by an increased phospholipid content in villus membranes, which resulted in a decreased cholesterol:phospholipid ratio and an increased lipid:protein molar ratio. Na-K ATPase activity was significantly (p < 0.01) greater in villus basolateral membranes than in crypt membranes, and demonstrated a greater sensitivity to ouabain inhibition. Ouabain inhibition curves calculated from villus data fit well (p < 0.001) with a two binding site model, with a high affinity (Ki 16 nM) and a low affinity (Ki 4.2 microM) ouabain binding site. In crypt basolateral membranes, only a low affinity site was apparent (Ki 3.0 microM). Fluidizing crypt basolateral membranes in vitro with benzyl alcohol to levels seen in villus basolateral membranes resulted in the appearance of a high affinity ouabain binding site (Ki 110 nM) and an increased sensitivity of Na-K ATPase to ouabain inhibition. The fluidization of villus basolateral membranes eliminated the binding associated with the high affinity site. Treatment with methanol, as a control, did not alter Na-K ATPase activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Vanadate reduces sodium-dependent glucose transport and increases glycolytic activity in LLC-PK1 epithelia.

The effect of vanadate pentoxide on apical sodium-dependent glucose transport in LLC-PK1 epithelia was examined. Epithelia grown in the presence or absence of 1 microM vanadate formed confluent monolayers and exhibited no differences in DNA, protein, or ultrastructure. Vanadate-supplemented epithelia demonstrated a lower steady-state alpha-methyl-D-glucopyranoside (AMG) concentrating capacity and a twofold reduction in apical AMG uptake Jmax. This decreased AMG transport occurred as a consequence of a reduction in the number of transport carriers and was not associated with a change in the sodium electrochemical gradient. The vanadate-induced reduction in apical glucose carrier functional activity and expression was accompanied by a stimulation of intracellular glycolytic flux activity, as evidenced by increased glucose consumption, lactate production, PFK-1 activity, and intracellular ATP. There was no difference in intracellular cAMP levels between vanadate-supplemented and non-supplemented epithelia. These results demonstrate an association between stimulation of glycolytic pathway activity and an adaptive response in the form of a reduction in the function and expression of the sodium-dependent apical glucose transporter in LLC-PK1 epithelia.

Insulin downregulates diabetic-enhanced intestinal glucose transport rapidly in ileum and slowly in jejunum.

The effect of insulin on intestinal sodium-dependent glucose transport and brush border membrane surface area was examined in male Sprague-Dawley rats following a 30-day period of nontreated streptozocin-induced diabetes. Nontreated diabetic rats were hyperglycemic and demonstrated increased jejunal and ileal Na-dependent glucose transport Jmax (maximal transport capacity) and Na-K ATPase activity compared with controls. Daily administration of insulin resulted in a steady decline in blood glucose levels over a period of 6 days. Jejunal Jmax was normalized after 2 days of insulin therapy, while ileal Jmax was normalized 12 h following a single insulin injection. The normalization of Na-K ATPase activity lagged behind Na-dependent glucose transport regulation by 24 h in both jejunum and ileum. Brush border surface area was increased in the ileum of diabetic rats as a result of an increase in microvillus height. Insulin treatment resulted in a decrease in ileal microvillus height to control values by 12 h, which correlated directly with the decrease in Na-dependent glucose transport. Insulin had no effect on jejunal brush border surface area. In conclusion, these findings indicate that the jejunum and ileum respond differentially to experimentally induced diabetes, and these regions also utilize different adaptive mechanisms to regulate Na-dependent glucose transport.