Differential effects of high dose omega-3 fatty acids on metabolism and inflammation in patients with obesity: eicosapentaenoic and docosahexaenoic acid supplementation.

Background: Obesity is complicated by low-grade chronic inflammation characterised by increases in inflammatory proteins and cells in peripheral blood. It has been known that omega-3 fatty acids (FA) like eicosapentaenoic (EPA) and docosahexaenoic (DHA) could modulate the inflammatory process and improve metabolic markers. Objective: This study aimed to determine the effect of high-dose omega-3 FA on metabolic and inflammatory markers among patients with obesity and healthy volunteers. Methods: This prospective study included 12 women with obesity (body mass index [BMI] ≥ 35.0 kg/m2) and 12 healthy women (BMI < 24.0 kg/m2) who were supplemented with a dose of 4.8 g/day (3.2 g EPA plus 1.6 g DHA) for 3 months followed by no treatment for 1 month. Plasma metabolic and inflammatory markers and levels of mRNA transcripts of CD4+ T lymphocyte subsets were determined monthly. Results: None of the participants exhibited changes in weight or body composition after study completion. EPA and DHA supplementation improved metabolic (insulin, Homeostatic Model Assessment of Insulin Resistance [HOMA-IR], triglyceride [TG]/ high-density lipoprotein [HDL] ratio, TG, and arachidonic acid [AA]/EPA ratio) and tumor necrosis factor-alpha (TNF-α). Moreover, the levels of mRNA transcripts of T CD4+ lymphocyte subsets (TBX21, IFNG, GATA-3, interleukin [IL]-4, FOXP3, IL-10 IL-6, and TNF-α), were down-regulated during the intervention phase. After 1 month without supplementation, only insulin, HOMA-IR and the mRNA transcripts remained low, whereas all other markers returned to their levels before supplementation. Conclusion: Supplementation with high-dose omega-3 FAs could modulate metabolism and inflammation in patients with obesity without weight loss or changes in body composition. However, these modulatory effects were ephemeral and with clear differential effects: short-duration on metabolism and long-lasting on inflammation.

Cholecalciferol supplementation increases FGF23 in peritoneal dialysis patients with hypovitaminosis D: a randomized clinical trial.

BACKGROUND: Vitamin D deficiency is common in peritoneal dialysis (PD) patients, so its supplementation has been advocated as potentially beneficial. METHODS: Double-blind, placebo-controlled, randomized clinical trial. Subjects on PD treated with high calcium peritoneal dialysate (Ca 3.5 mEq/l) and serum levels of 25-hydroxi vitamin D (25D) < 20 ng/ml were randomized to receive cholecalciferol (4800 IU/daily) or placebo for 16 weeks. The outcome measures were the effects on the osteogenic biomarkers osteoprotegerin (primary endpoint), intact fibroblast growth factor-23 (iFGF23), osteocalcin, osteopontin, iPTH, 1,25-dyhydroxivitamin D (1,25D), and interleukin-6. RESULTS: Fifty-eight subjects were randomly assigned. Baseline characteristics were similar in both groups. Cholecalciferol supplemented subjects had a significant increase in serum 25D (from 11.4 ± 5.0 to 28.3 ± 10.3 ng/ml), 1,25D and iFGF23 compared with placebo group. iFGF23 levels increased an average of 10,875 pg/ml per month (95% CI 11,778-88,414) in the cholecalciferol group and was unchanged in the placebo group (2829 pg/ml, 95% CI - 2181 to 14,972). Extremely high iFGF23 levels (> 30,000 pg/ml) were observed in 74% of subjects receiving cholecalciferol although iFGF23 returned to baseline values after 32 weeks of withdrawal. The observed changes in iFGF23 correlated with 1,25D levels and were not modified by other variables. No difference was observed between groups in osteoprotegerin or other osteogenic biomarkers levels. CONCLUSIONS: Cholecalciferol supplementation increases serum 25D levels in subjects on PD exposed to high calcium dialysate, yet it induces an exponential increase of iFGF23 in most patients, which disappear after withdrawal of supplementation and may be a major concern for this maneuver.

Biomarkers Associated with Vascular Calcification in Peritoneal Dialysis.

UNLABELLED: ♦ BACKGROUND: Vascular calcification is strongly associated with cardiovascular disease and mortality. However, some factors related to vascular calcification in patients with end-stage renal disease receiving peritoneal dialysis (PD) remain unknown. This study aimed to evaluate the associations of osteoprotegerin (OPG), osteopontin (OPN), osteocalcin (OCN), fibroblast growth factor 23 (FGF-23), magnesium, and phosphate clearance with vascular calcification in PD subjects, assessed by plain radiographs. ♦ METHODS: Simple vascular calcification scores (SVCS) obtained from plain X-rays of the pelvis and hands, and the Kauppila Index (KI) from lateral lumbar X-rays were assessed in 76 adults receiving PD for ≥ 6 months (43 women, median age 39 years, median time on PD 1.4 years). Levels of OPG, OPN, OCN, and FGF-23 were determined by luminometry. ♦ RESULTS: Serum OPG levels were higher in subjects with vascular calcification (n = 22 with SVCS > 3; n = 19 with KI > 7) compared with those with less calcification (p < 0.001). Spearman's correlation coefficients between OPG and SVCS and KI were r = 0.49 and r = 0.51, respectively (both p < 0.001). Subjects with vascular calcification had significantly lower renal phosphate clearance. Multiple regression analysis showed that vascular calcification assessed by SVCS was associated with age (r = 0.2, p = 0.042), diabetes mellitus (r = 2.4, p < 0.001), body mass index (BMI) (r = 0.09, p = 0.037), and OPG (r = 0.22, p = 0.001). Vascular calcification assessed by KI was associated with age (r = 0.16, p < 0.001), time on PD (r = 0.54, p = 0.001) and OPG (r = 0.08, p = 0.04). Osteocalcin, OPN, FGF-23, and magnesium were not associated with vascular calcification. ♦ CONCLUSIONS: Higher levels of OPG were consistently associated with vascular calcification in subjects on PD.

Novel association of the R230C variant of the ABCA1 gene with high triglyceride levels and low high-density lipoprotein cholesterol levels in Mexican school-age children with high prevalence of obesity.

BACKGROUND AND AIMS: Metabolic syndrome (MetS) is a disorder that includes a cluster of several risk factors for the development of type 2 diabetes and cardiovascular disease. The R230C variant of the ABCA1 gene has been associated with low HDL-cholesterol in several studies, but its association with MetS in children remains to be determined. The aim of this study was to analyze the association of the R230C variant with MetS and other metabolic traits in school-aged Mexican children. METHODS: The study was performed in seven urban primary schools in the State of Mexico. Four hundred thirty-two Mexican school-age children 6-13 years old were recruited. MetS was identified using the International Diabetes Federation definition. The R230C variant of the ABCA1 gene was genotyped to seek associations with MetS and other metabolic traits. RESULTS: The prevalence of MetS was 29% in children aged 10-13 years. The R230C variant was not associated with MetS (OR = 1.65; p = 0.139). Furthermore, in the whole population, the R230C variant was associated with low HDL-cholesterol levels (ß coefficient = -3.28, p <0.001). Interestingly, in the total population we found a novel association of this variant with high triglyceride levels (ß coefficient = 14.34; p = 0.027). CONCLUSIONS: We found a new association of the R230C variant of the ABCA1 gene with high triglyceride levels. Our findings also replicate the association of this variant with low HDL-cholesterol levels in Mexican school-age children.

Injury induces localized airway increases in pro-inflammatory cytokines in humans and mice.

BACKGROUND: Secretory immunoglobulin A (sIgA) increases in the airways of humans and mice after injury to protect against infection. The pro-inflammatory cytokines tumor necrosis factor (TNF)-α, interleukin (IL)-1ß, and IL-6 are linked molecularly to sIgA production and secretion and are required for sIgA increases in the airway after injury in a mouse model. We investigated the injury effect on airway and serum concentrations to determine the source of the cytokines involved in the airway IgA response. METHODS: In the first experiment, TNF-α, IL-1ß, and IL-6 concentrations in bronchoalveolar lavage (BAL) fluid and serum obtained from 11 ventilated trauma patients within 30 h of admission were compared with those in eight elective surgical patients. In the second experiment, male ICR mice received no injury (n = 7) or injury with sham celiotomy and neck incisions (n = 8) with sacrifice of all animals at 8 h for BAL fluid and serum cytokine measurements by enzyme-linked immunosorbent assay. RESULTS: Injured patients had significantly higher BAL fluid and serum TNF-α, IL-1ß, and IL-6 concentrations, with greater increases in the BAL fluid than in the serum. Injured mice had significantly increased BAL fluid concentrations of TNF-α, IL-1ß, and IL-6 without significant changes in serum TNF-α or IL-1ß. Serum IL-6 increased significantly. CONCLUSIONS: Injury significantly increases human and mouse airway TNF-α, IL-1ß, and IL-6. Increases are greater in the airway than in serum, implying a local rather than a systemic stress response to injury.

Parenteral feeding depletes pulmonary lymphocyte populations.

BACKGROUND: The effect of parenteral nutrition (PN) on lymphocyte mass in the lung is unknown, but reduced mucosal lymphocytes are hypothesized to play a role in the reduced immunoglobulin A-mediated immunity in both gut and lung. The ability to transfer and track cells between mice may allow study of diet-induced mucosal immune function. The objectives of this study are to characterize lung T-cell populations following parenteral feeding and to study distribution patterns of transferred donor lung T cells in recipient mice. METHODS: In experiment 1, cannulated male Balb/c mice are randomized to receive chow or PN for 5 days. Lung lymphocytes are obtained via collagenase digestion, and flow cytometric analysis is used to identify total T (CD3+) and B (CD45/B220+) cells. In experiment 2, isolated lung T cells from chow-fed male Balb/c mice are pooled and labeled in vitro with a fluorescent dye (carboxyfluorescein diacetate succinimidyl ester [CFSE]), and 1.1 x 10(8) CFSE+ cells (3.1 x 10(6) T cells) are transferred to chow-fed Balb/c recipients. Cells recovered from recipient lungs and intestinal lamina propria (LP) are analyzed by flow cytometry to determine CFSE/CD3+ T cells at 1, 2, and 7 days. In experiment 3, cells are transferred to PN-fed recipients. RESULTS: In experiment 1, PN significantly decreases lung T- and B-cell populations compared with chow feeding. In experiment 2, CFSE+ T-cell retention is highest on day 1 in lung and LP, and decreases on day 2. Cells are gone by day 7; 98.1% of retained donor lung T cells migrate to recipient lungs and 1.9% to the intestine on day 1. Similar results are seen in experiment 3 after transfer of cells to PN-fed recipients. CONCLUSIONS: PN reduces pulmonary lymphocyte populations consistent with impaired respiratory immunity. Transferred lung T cells preferentially localize to recipient lungs rather than intestine with maximal accumulation at 24 hours. Limited cross-talk of transferred lung T cells to the intestine indicates that mucosal lymphocyte traffic might be programmed to localize to specific effector sites.

Parenteral nutrition maintains pulmonary IgA antibody transport capacity, but not active transport, following injury.

BACKGROUND: Parenteral nutrition (PN) increases post-trauma pneumonia versus enteral feeding. PN impairs murine immunoglobulin A (IgA) airway defenses and abrogates a normal IgA increase following injury. This work investigates the effect of type/route of nutrition on lung IgA and its transport protein, polymeric immunoglobulin receptor (pIgR), after injury. METHODS: Catheterized mice were randomized to Chow or PN for 5 days and sacrificed without injury (Chow: n = 12; PN n = 11), or 8 hours after laparotomy + neck incisions (Chow-injury: n = 11, PN-injury: n = 13). Bronchoalveolar lavage (BAL) and lung IgA levels were analyzed by enzyme-linked immunosorbent assay (ELISA) and lung pIgR by Western blot. RESULTS: BAL IgA levels increased in Chow-injury versus PN-injury (P <.01) with no differences in pIgR. PN-injury tissue IgA levels decreased versus Chow (P <.01), Chow-injury (P <.01), and PN (P <.05). CONCLUSIONS: PN impairs the airway IgA response to injury but not due to impaired IgA transport capacity/pIgR level.

Parenteral nutrition induces organ specific alterations in polymeric immunoglobulin receptor levels.

BACKGROUND: Secretory immunoglobulin A (IgA) prevents pathogen adherence at mucosal surfaces to prevent infection. Polymeric immunoglobulin receptor (pIgR), located on the basolateral surface of mucosal cells, binds dimeric IgA produced by B cells with the cooperation of T cells in the lamina propria. This IgA-pIgR complex is transported apically, where it is exocytosed as secretory IgA to the mucosal surface. Our prior work shows that parenteral nutrition (PN) impairs both airway and small intestine mucosal immunity by reducing T and B cells and IgA levels. This work examines intestinal and respiratory tissue-specific pIgR responses to PN. METHODS: Cannulated male Institute of Cancer Research mice were randomized to Chow (n = 10) or PN (n = 10). After 5 days, animals were sacrificed and lavages obtained from the small intestine, lung (BAL = bronchoalveolar lavage), and nasal airways (NAL). Small intestine, lung, and nasal passage tissues were also collected. Lavage and tissue homogenate IgA levels were quantified by enzyme-linked immunosorbent assay and pIgR by Western blot. RESULTS: PN group SIL and NAL IgA levels dropped significantly compared with Chow. PN significantly reduced pIgR levels in the SI while no pIgR change was noted in nasal passages and lung pIgR actually increased with PN. Tissue homogenate IgA levels did not change with PN in the SI while levels in the nasal passage and lung decreased. CONCLUSIONS: PN impairs airway mucosal immunity by reduction in IgA available for transport rather than via a reduction in pIgR levels. In the small intestine, diminished pIgR is implicated in the deterioration of antibody-mediated mucosal immunity.

Parenteral nutrition inhibits tumor necrosis factor-alpha-mediated IgA response to injury.

BACKGROUND: Parenteral nutrition (PN) increases the incidence of pneumonia in severely injured patients compared with enteral feeding (ENT). Injury induces an innate airway IgA response in severely injured patients; similar responses occur in mice. Tumor necrosis factor-alpha (TNF-alpha) and interleukin-1 beta (IL-1beta) stimulate the production of polymeric immunoglobulin receptor (pIgR), the protein required to transport immunoglobulin A (IgA) to mucosal surfaces. We have shown that PN alters levels of lung and nasal passage IgA and several IgA-stimulating cytokines. We hypothesized that TNF-alpha and IL-1beta blockade, as well as PN, would blunt the airway IgA response to injury. METHODS: Male Institute of Cancer Research (ICR) mice were randomized to uninjured controls (n = 10) or to intra-peritoneal phosphate-buffered saline (PBS) (n = 9), antagonistic TNF-alpha antibody (100 mcg, n = 7), or antagonistic IL-1beta antibody (50 mcg, n = 8) 30 min prior to surgical stress with laparotomy and neck incisions. Mice were sacrificed at 8 h for nasal and bronchoalveolar lavage (NAL, BAL) to measure IgA by enzyme-linked immunosorbent assay. In a separate experiment, 12 mice underwent intravenous cannulation followed by chow (n = 5) or PN (n = 7) feeding for 5 days prior to the same stress and IgA measurement. RESULTS: Injury significantly increased NAL and BAL IgA (225 +/- 104 ng) compared with baseline (145 +/- 38 ng; p = 0.01). Blockade of TNF-alpha eliminated the innate airway IgA response to injury (130 +/- 47 ng; p = 0.01), whereas IL-1beta blockade blunted and PN eliminated it completely. CONCLUSIONS: Tumor necrosis factor-alpha is involved in the respiratory IgA immune response to injury. Both TNF-alpha blockade and PN impair this innate response, and blockade of IL-1beta impairs it to a degree. We hypothesize that these cytokines blunt this response via their known effects on the polymeric immunoglobulin receptor (pIgR), whereas the PN-induced deficit likely is multifactorial.

Injury stimulates an innate respiratory immunoglobulin a immune response in humans.

BACKGROUND: Secretory immunoglobulin A (SIgA) is the specific immune antibacterial defense. Since pneumonia frequently complicates the course of trauma patients, we studied early airway immune responses after injury. METHODS: Twelve severely injured, intubated (expected for >/=5 d) patients had tracheal and bilateral lung lavage (BAL) within 30 hours of injury (n = 12). Epithelial lining fluid (ELF) volume and SIgA were measured by urea dilution and enzyme-linked immunosorbent assay (ELISA), respectively. Control BAL specimens were obtained from eight healthy elective surgical patients. Anatomically based comparisons were made between groups with Welch's unpaired t test. To verify human data, 30 male mice received no injury (time 0, n = 7) or injury with abdominal and neck incisions and were killed for airway IgA at 4 (n = 7), 8 (n = 8), and 24 (n = 8) hours. Analysis of variance (ANOVA) and Fisher's protected least significant difference testing was used to analyze animal data. RESULTS: Initial trauma patient SIgA concentration (SIgA/mL ELF) increased compared with control in the lungs bilaterally (p < 0.05 both right and left). ELF volume was significantly higher in the right lung (p = 0.02) and just missed statistical significance (p = 0.07) on the left. Mouse IgA increased 8 hours after stress (p < 0.05 versus 0, 4, and 24 hours) and returned to normal by 24 hours. CONCLUSION: A previously unrecognized innate human airway mucosal immune response with increased airway SIgA and ELF occurs after severe injury and is reproducible experimentally. This accessible, quantifiable human response allows study of clinical strategies to reduce infections via mucosal immune therapies.

Decreased enteral stimulation alters mucosal immune chemokines.

BACKGROUND: Migration of lymphocytes into and through the mucosal immune system depends upon adhesion molecules to attract circulating cells and chemokines to stimulate diapedesis into tissues. Decreased enteral stimulation significantly reduces mucosal addressin cellular adhesion molecule-1 (MAdCAM-1) levels, an adhesion molecule critical for homing of T and B cells to Peyer's patches (PP), which reduces PP and intestinal T and B cells. We studied the effect of type and route of nutrition on tissue specific chemokines in PP (CXCL-12, -13 and CCL-19, -20 and -21), small intestine (SI; CCL-20, -25 and -28) and lung (CXCL-12, CCL-28). METHODS: Intravenously cannulated male Institute of Cancer Research (ICR) mice were randomized to chow or parenteral nutrition (PN) for 5 days. PP, SI, and lung chemokine mRNA levels were measured using real-time qRT-polymerase chain reaction, and analyzed semiquantitatively by the DeltaDeltaCt method. Protein levels were quantified using enzyme-linked immunosorbent assay (ELISA) techniques, and groups compared using Student's t-test. RESULTS: PP CXCL13 protein significantly decreased, whereas CCL21 protein increased significantly in the parenterally fed group. Parenteral feeding significantly decreased SI CCL20 and CCL 25 protein levels. CCL28 decreased significantly in the SI and lung of intravenously fed animals. mRNA levels changed in the opposite direction (compared with protein) for all chemokines except CCL28. CONCLUSIONS: Decreased enteral stimulation significantly alters key mucosal immune chemokine protein levels at multiple sites. In general, PN (and concomitant lack of enteral stimulation) results in decreased levels of chemokines that control lymphocyte migration within the mucosal immune system.

Enteral feeding of a chemically defined diet preserves pulmonary immunity but not intestinal immunity: the role of lymphotoxin beta receptor.

BACKGROUND: Compared with chow or a complex enteral diet (CED), IV administration of a parenteral nutrition solution (IV-PN) impairs intestinal and respiratory mucosal immunity, resulting in cellular and immunoglobulin A (IgA) defects in the intestine and impaired respiratory antiviral and antibacterial defenses. PN given intragastrically (IG-PN) impairs intestinal immunity similar to IV-PN but preserves antiviral defences and partially preserves antibacterial defenses. Lymphotoxin beta receptor (LTbetaR) is a molecule essential for development and organization of lymphoid tissue. It controls many molecules important in mucosal immune integrity. This study examines effects of route (IV or enteral) and type (PN, CED, or chow) on murine intestine and lung LTbetaR expression. METHODS: Forty-three mice randomly received IV-PN (n = 12), IG-PN (n = 11), IV saline + chow (chow; n = 11), or a CED (n = 9). After 5 days of feeding, intestinal and lung samples were obtained and processed for levels of LTbetaR by Western blot. RESULTS: IV-PN significantly reduced intestinal and lung LTbetaR compared with CED and chow. IG-PN reduced LTbetaR levels only in the intestine but preserved lung levels. CONCLUSIONS: Route and type of nutrition differentially influence molecular events in the intestinal and respiratory mucosal immune systems. Enteral feeding with any diet (complex or chemically defined) maintains lung LTbetaR expression, whereas intestinal LTbetaR levels are maintained only with CEDs (chow and CED). We hypothesize that LTbetaR is responsible for the observed preservation of respiratory tract immunity with administration of a noncomplex, chemically defined enteral diet, whereas intestinal immunity is compromised with this diet.

Effects of lymphotoxin beta receptor blockade on intestinal mucosal immunity.

BACKGROUND: Mucosal addressin cellular adhesion molecule-1 (MAdCAM-1) directs lymphocyte migration into gut-associated lymphoid tissue (GALT) through Peyer's patches (PPs). Parenteral nutrition (PN) impairs mucosal immunity by reducing PPs MAdCAM-1 expression, T and B cells in GALT, and intestinal and respiratory immunoglobulin (Ig) A levels. We previously showed that PN reduces lymphotoxin beta receptor blockade (LTbetaR) in PPs and intestine, and that stimulation with LTbetaR agonist antibodies reverses these defects. To confirm that LTbetaR regulates transcription of MAdCAM-1 message and more fully understand the effects of LTbetaR on MAdCAM-1 function within the mucosal immune system, we studied the effect of LTbetaR blockade with a chimeric LTbetaR Ig-fusion protein on MAdCAM-1 mRNA levels, PP lymphocyte mass and IgA levels in the intestinal and respiratory tracts. METHODS: Mice were cannulated and killed 3 days after receiving chow + control Ig, chow + LTbetaR-Ig fusion protein (100 microg IV), or PN + control Ig. The PPs of half of the animals were processed for lymphocyte count, and the other half were processed for complementary DNA and subsequent polymerase chain reaction (PCR). mRNA levels of MAdCAM-1 were determined by real-time PCR; intestinal and respiratory IgA levels were measured by ELISA. RESULTS: PN significantly reduced PP lymphocyte mass, MAdCAM-1 mRNA, and intestinal IgA. As anticipated, LTbetaR blockade significantly decreased PP cells and MAdCAM-1 mRNA, but not intestinal IgA because chow feeding was maintained. Both LTbetaR blockade and PN decreased nasal IgA, but not significantly. CONCLUSIONS: LTbetaR blockade in chow animals significantly reduces transcription of MAdCAM-1 gene and PPs lymphocyte mass. These data implicate inadequate LTbetaR signaling as a major mechanism for decreased GALT cells with lack of enteral stimulation, and further establish the role of LTbetaR in the mucosal immune system.

Intestinal polymeric immunoglobulin receptor is affected by type and route of nutrition.

BACKGROUND: Secretory immunoglobulin A (SIgA) prevents adherence of pathogens at mucosal surfaces to prevent invasive infection. Polymeric immunoglobulin receptor (pIgR) is located on the basolateral surface of epithelial cells and binds dimeric immunoglobulin A (IgA) produced by plasma cells in the lamina propria. This IgA-pIgR complex is transported apically, where IgA is exocytosed as SIgA to the mucosal surface. Our prior work shows that mice fed intragastric (IG, an elemental diet model) and IV parenteral nutrition (PN) solution have reduced intestinal T and B cells, SIgA, and interleukin-4 (IL-4) compared with mice fed chow or a complex enteral diet (CED). Prior work also demonstrates a reduction in IgA transport to mucosal surfaces in IV PN-fed mice. Because IL-4 up-regulates pIgR production, this work studies the effects of these diets on intestinal pIgR. METHODS: Male Institute of Cancer Research (ICR) mice were randomized to chow (n = 11) with IV catheter, CED (n = 10) or IG PN (n = 11) via gastrostomy and IV PN (n = 12) for 5 days. CED and PN were isocaloric and isonitrogenous. Small intestine was harvested for pIgR and IL-4 assays after mucosal washing for IgA. IgA and IL-4 levels were analyzed by enzyme-linked immunosorbent assay and pIgR by Western blot. RESULTS: Small intestinal pIgR expression, IgA levels, and IL-4 levels decreased significantly in IV PN and IG PN groups. CONCLUSIONS: Lack of enteral stimulation affects multiple mechanisms responsible for decreased intestinal SIgA levels, including reduced T and B cells in the lamina propria, reduced Th-2 IgA-stimulating cytokines, and impaired expression of the IgA transport protein, pIgR.

Parenteral nutrition and fasting reduces mucosal addressin cellular adhesion molecule-1 (MAdCAM-1) mRNA in Peyer's patches of mice.

BACKGROUND: Mucosal addressin cellular adhesion molecule-1 (MAdCAM-1) in Peyer's patches (PP) is the gateway molecule for cellular migration into the mucosal immune system. Lack of enteral feeding during parenteral nutrition (PN) rapidly decreases PP MAdCAM-1, leading to drops in mucosal T and B cells and intestinal and respiratory IgA. We determined the molecular events associated with MAdCAM-1 mRNA and protein during PN (short and long term) and fasting (1 and 2 days). METHODS: Experiment 1: Cannulated mice received PN for 8 hours (short-term PN, n = 6) or chow + saline (chow, n = 6). Experiment 2: Cannulated mice received PN (long-term PN, n = 4) or chow (n = 3) for 5 days. Experiment 3: Noncannulated chow mice were fasted for 1 and 2 days (n = 2/time). Total cellular RNA from the PP was quantified for MAdCAM-1 mRNA by real-time polymerase chain reaction (PCR). MAdCAM-1 protein was measured by Western blot. RESULTS: PN rapidly down-regulated MAdCAM-1 gene expression. After 8 hours of PN with lack of enteral feeding, MAdCAM-1 mRNA levels dropped 20% (0.8-fold vs chow, p > .05); 5 days of PN reduced MAd-CAM-1 levels 64% (0.34-fold vs chow, p < .05). PN reduced MAdCAM-1 protein levels by 30% (chow: 329 +/- 14 vs PN: 230 +/- 35, p < .05) after 5 days. Fasting of uncannulated mice decreased MAdCAM-1 mRNA levels by 16% (0.84-fold, p < .05) at day 1 and 30% (0.7-fold, p < .05) by day 2 compared with chow. CONCLUSIONS: Both PN with lack of enteral feeding and fasting down-regulate MAdCAM-1 mRNA and protein levels in PP. The MAdCAM-1 changes are due to lack of enteral stimulation rather than toxic effects of PN.

Redox reactions of dehydroepiandrosterone and its metabolites in differentiating 3T3-L1 adipocytes: A liquid chromatographic-mass spectrometric study.

Dehydroepiandrosterone is known to depress fatty acid formation in differentiating 3T3-L1 adipocytes. The metabolism of dehydroepiandrosterone and four of its natural metabolites in differentiating adipocytes was studied by liquid chromatography-mass spectrometry. Adipocytes rapidly converted dehydroepiandrosterone to androst-5-ene-3beta,17beta-diol. 7alpha-Hydroxy-DHEA was interconverted with 7-oxo-DHEA and 7beta-hydroxy-DHEA and the corresponding 17beta reduced products. Dehydroepiandrosterone and its derivatives were detected only in the culture medium suggesting that dehydroepiandrosterone is metabolized via enzymes located in close proximity to, or that are integral parts of the cell membrane. Alternatively, there may be efficient mechanisms at play for extrusion of the steroids to the aqueous media rather than being retained in the lipid-rich cell. An interesting aspect of the study was finding androstenediol as the major metabolite of dehydroepiandrosterone. Androst-5-ene-3beta,17beta-diol has been implicated in prostate cancer. The contribution of adipose cells to the circulating supply of androst-5-ene-3beta,17beta-diol may therefore be considered in managing prostate cancer.

Parenteral nutrition impairs gut-associated lymphoid tissue and mucosal immunity by reducing lymphotoxin Beta receptor expression.

OBJECTIVE: To determine the effects of parenteral nutrition (PN) on LTbetaR in gut-associated lymphoid tissue (GALT), particularly the intestine and Peyer's patches (PP). SUMMARY BACKGROUND DATA: Lack of enteral stimulation with PN impairs mucosal immunity and reduces IgA levels through depression of GALT cytokines (IL-4 and IL-10) and GALT specific adhesion molecules. We have shown that each is critical to intact mucosal immunity through effects on lymphocyte homing, IgA production, and resistance to antibacterial and antiviral immunity. IgA is the principal specific immunologic mucosal defense. LTbetaR stimulation controls production of IL-4, the adhesion molecule MAdCAM-1, and other key components of GALT, all of which are important in increasing IgA levels and maintaining mucosal defenses. METHODS: Experiment 1: LTbetaR expression in intestine and PP was analyzed by Western blot after 5 days of chow, a complex enteral diet (CED), or PN. Diets were isocaloric and isonitrogenous except for chow. Experiment 2: After completing pilot experiments to determine the appropriate dose of the LTbetaR agonistic antibody, mice received chow, PN + 5 mug of anti-LTbetaR mAb (2 times/d, i.v.) or PN + isotype control antibody. PP lymphocytes and intestinal IgA levels were measured after 2 days. RESULTS: Lack of enteral stimulation with PN significantly decreased LTbetaR expression in intestine and PP compared with chow and CED. LTbetaR stimulation with an agonistic anti-LTbetaR mAb significantly increased PP lymphocyte counts and intestinal IgA in PN fed-mice. CONCLUSIONS: LTbetaR expression is critical for GALT control mechanisms and intact mucosal immunity. PN reduces LTbetaR expression, PP lymphocytes, and intestinal IgA production. Exogenous LTbetaR stimulation reverses PN-induced depression of gut mucosal immunity.

Effects of sterculic acid on stearoyl-CoA desaturase in differentiating 3T3-L1 adipocytes.

The effects of sterculic acid on cell size, adiposity, and fatty acid composition of differentiating 3T3-L1 adipocytes are correlated with stearoyl-CoA desaturase (SCD) expression (mRNA and protein levels) and enzyme activity. Fluorescence-activated cell scanning (FACS) analysis showed that adipocytes differentiated with methylisobutylxanthine, dexamethasone, and insulin (MDI) plus 100 microM sterculic acid comprised a population of predominantly large cells with reduced adiposity compared to MDI-treated cells. Although both groups had similar amounts of total fat, their fatty acid profiles were strikingly different: MDI-treated cells had high levels of the unsaturated palmitoleic (Delta(9)-16:1) and oleic (Delta(9)-18:1) acids, whereas the cells cultured with MDI plus sterculic acid accumulated palmitic (16:0) and stearic (18:0) acids together with a marked reduction in Delta(9)-16:1. Although the cells treated with MDI plus sterculic acid had similar levels of scd1 and scd2 mRNAs and antibody-detectable SCD protein as the MDI-treated cells, the SCD enzyme activity was inhibited more than 90%. The accumulation of 16:0 and 18:0, together with normal levels of fatty acid synthase (FAS) and aP2 mRNAs, shows that de novo synthesis and elongation of fatty acids, as well as cell differentiation, were not affected by sterculic acid. Because of the increase in cell size in the sterculic acid-treated cells, the insulin-stimulated 2-deoxyglucose (2-DOG) uptake was determined. Compared to MDI-treated cells, the 2-DOG uptake in the cells treated with sterculic acid was not affected. These results indicate that sterculic acid directly inhibits SCD activity, possibly by a turnover-dependent reaction, without affecting the processes required for adipocyte differentiation, scd gene expression or SCD protein translation.

Molecular differences caused by differentiation of 3T3-L1 preadipocytes in the presence of either dehydroepiandrosterone (DHEA) or 7-oxo-DHEA.

The effects of dehydroepiandrosterone (DHEA) and 7-oxo-DHEA on the cell size, adiposity, and fatty acid composition of differentiating 3T3-L1 preadipocyte cells are correlated with stearoyl-CoA desaturase (SCD) expression (mRNA and protein levels) and enzyme activity. Fluorescence-activated cell sorting shows that preadipocyte cells treated with methylisobutylxanthine, dexamethasone, and insulin (MDI) plus DHEA comprise a population distribution of predominantly large cells with reduced adiposity. In contrast, cells treated with MDI plus 7-oxo-DHEA comprise a population distribution of almost equal proportions of small and large cells that have an adiposity equivalent to cells differentiated with MDI alone. The cells treated with MDI plus DHEA have significantly reduced levels of total fatty acid, mainly due to a dramatic reduction in the level of palmitoleic (Delta(9)-16:1) acid. The cells treated with MDI plus 7-oxo-DHEA have a significantly increased level of total fat, primarily due to increased levels of Delta(9)-16:1 and palmitic (16:0) acids. At the molecular level, the DHEA-treated cells contain lowered amounts of SCD1 mRNA and antibody-detectable desaturase protein, while 7-oxo-DHEA-treated cells contained elevated levels of SCD1 mRNA and protein. Inhibition of differentiation in DHEA-treated cells was also suggested by a reduction in the mRNA level of the adipogenic gene aP2. At the level of microsomal enzymatic activity, SCD activity was decreased in DHEA-treated cells while the SCD activity was increased in 7-oxo-DHEA-treated cells. The changes in mRNA levels and enzyme activity were concentration-dependent and appeared as early as day 3 of the differentiation protocol. The results show that DHEA and 7-oxo-DHEA have distinct modes of action with respect to the complex transcriptional cascade required for differentiation. Furthermore, differences in the insulin-stimulated uptake of 2-deoxyglucose and in the activity of carnitine palmitoyl transferase observed from either DHEA- or 7-oxo-DHEA-treated cells support the ability of DHEA to produce a thermogenic effect in differentiating preadipocytes, while 7-oxo-DHEA promotes differentiation without other changes typical of thermogenesis.