Regulation of inflammatory responses by the commensal microbiota.

It is well established that dysregulation of the interactions between the immune system and commensal bacteria is one factor that underpins the development and chronicity of a number of inflammatory diseases. Certain phyla of bacteria within the microbiota have been associated with 'health', but the mechanisms by which the presence of these bacteria supports a healthy environment are still being unravelled. Recent evidence indicates that one such mechanism involves the anti-inflammatory properties of fermentation products of fibre, short-chain fatty acids and their signalling through the G-protein coupled receptor GPR43. Recent findings also indicate that, even in health, bacterial communities harbour in the airways, indicating that direct exposure to bacterial products at this site may provide a further explanation for how commensal bacteria can regulate chronic airway inflammation.

Ocular antisense oligonucleotide delivery by cationic nanoemulsion for improved treatment of ocular neovascularization: an in-vivo study in rats and mice.

The efficacy of an antisense oligonucleotide (ODN17) cationic nanoemulsion directed at VEGF-R2 to reduce neovascularization was evaluated using rat corneal neovascularization and retinopathy of prematurity (ROP) mouse models. Application of saline solution or scrambled ODN17 solution on eyes of rats led to the highest extent of corneal neovascularization. The groups treated with blank nanoemulsion or scrambled ODN17 nanoemulsion showed moderate inhibition in corneal neovascularization with no significant difference with the saline and scrambled ODN17 control solution groups, while the groups treated with ODN17 solution or Avastin® (positive ODN17 control) clearly elicited marked significant inhibition in corneal neovascularization confirming the results reported in the literature. The highest significant corneal neovascularization inhibition efficiency was noted in the groups treated with ODN17 nanoemulsion (topical and subconjunctivally). However, in the ROP mouse model, the ODN17 in PBS induced a 34% inhibition of retinal neovascularization when compared to the aqueous-vehicle-injected eyes. A significantly higher inhibition of vitreal neovascularization (64%) was observed in the group of eyes treated with ODN17 nanoemulsion. No difference in extent of neovascularization was observed between blank nanoemulsion, scrambled ODN17 nanoemulsion, vehicle or non-treated eyes. The overall results indicate that cationic nanoemulsion can be considered a promising potential ocular delivery system and an effective therapeutic tool of high clinical significance in the prevention and forthcoming treatment of ocular neovascular diseases.

Total fatty acid composition in the characterization and identification of orchid mycorrhizal fungi Epulorhiza spp.

Rhizoctonia-like fungi are the main mycorrhizal fungi in orchid roots. Morphological characterization and analysis of conserved sequences of genomic DNA are frequently employed in the identification and study of fungi diversity. However, phytopathogenic Rhizoctonia-like fungi have been reliably and accurately characterized and identified through the examination of the fatty acid composition. To evaluate the efficacy of fatty acid composition in characterizing and identifying Rhizoctonia-like mycorrhizal fungi in orchids, three Epulorhiza spp. mycorrhizal fungi from Epidendrum secundum, two unidentified fungi isolated from Epidendrum denticulatum, and a phytopathogenic fungus, Ceratorhiza sp. AGC, were grouped based on the profile of their fatty acids, which was assessed by the Euclidian and Mahalanobis distances and the UPGMA method. Dendrograms distinguished the phytopathogenical isolate of Ceratorhiza sp. AGC from the mycorrhizal fungi studied. The symbionts of E. secundum were grouped into two clades, one containing Epulorhiza sp.1 isolates and the other the Epulorhiza sp.2 isolate. The similarity between the symbionts of E. denticulatum and Epulorhiza spp. fungi suggests that symbionts found in E. denticulatum may be identified as Epulorhiza. These results were corroborated by the analysis of the rDNA ITS region. The dendrogram constructed based on the Mahalanobis distance differentiated the clades most clearly. Fatty acid composition analysis proved to be a useful tool for characterizing and identifying Rhizoctonia-like mycorrhizal fungi.

Plac8 is an inducer of C/EBPβ required for brown fat differentiation, thermoregulation, and control of body weight.

Brown adipocytes oxidize fatty acids to produce heat in response to cold or to excessive energy intake; stimulation of brown fat development and function may thus counteract obesity. Brown adipogenesis requires activation of the transcription factor C/EBPβ and recruitment of the zinc finger protein Prdm16, but upstream inducers of these proteins are incompletely defined. Here, we show that genetic inactivation of Plac8, a gene encoding an evolutionarily conserved protein, induces cold intolerance, and late-onset obesity, as well as abnormal morphology and impaired function of brown adipocytes. Using brown preadipocyte lines we show that Plac8 is required for brown fat differentiation, that its overexpression induces C/EBPβ and Prdm16, and that upon induction of differentiation Plac8 associates with C/EBPβ and binds to the C/EBPβ promoter to induce its transcription. Thus, Plac8 is a critical upstream regulator of brown fat differentiation and function that acts, at least in part, by inducing C/EBPβ expression.

PPARbeta/delta regulates paneth cell differentiation via controlling the hedgehog signaling pathway.

BACKGROUND & AIMS: All 4 differentiated epithelial cell types found in the intestinal epithelium derive from the intestinal epithelial stem cells present in the crypt unit, in a process whose molecular clues are intensely scrutinized. Peroxisome proliferator-activated receptor beta (PPARbeta) is a nuclear hormone receptor activated by fatty acids and is highly expressed in the digestive tract. However, its function in intestinal epithelium homeostasis is understood poorly. METHODS: To assess the role of PPARbeta in the small intestinal epithelium, we combined various cellular and molecular approaches in wild-type and PPARbeta-mutant mice. RESULTS: We show that the expression of PPARbeta is particularly remarkable at the bottom of the crypt of the small intestine where Paneth cells reside. These cells, which have an important role in the innate immunity, are strikingly affected in PPARbeta-null mice. We then show that Indian hedgehog (Ihh) is a signal sent by mature Paneth cells to their precursors, negatively regulating their differentiation. Importantly, PPARbeta acts on Paneth cell homeostasis by down-regulating the expression of Ihh, an effect that can be mimicked by cyclopamine, a known inhibitor of the hedgehog signaling pathway. CONCLUSIONS: We unraveled the Ihh-dependent regulatory loop that controls mature Paneth cell homeostasis and its modulation by PPARbeta. PPARbeta currently is being assessed as a drug target for metabolic diseases; these results reveal some important clues with respect to the signals controlling epithelial cell fate in the small intestine.

High protein intake reduces intrahepatocellular lipid deposition in humans.

BACKGROUND: High sugar and fat intakes are known to increase intrahepatocellular lipids (IHCLs) and to cause insulin resistance. High protein intake may facilitate weight loss and improve glucose homeostasis in insulin-resistant patients, but its effects on IHCLs remain unknown. OBJECTIVE: The aim was to assess the effect of high protein intake on high-fat diet-induced IHCL accumulation and insulin sensitivity in healthy young men. DESIGN: Ten volunteers were studied in a crossover design after 4 d of either a hypercaloric high-fat (HF) diet; a hypercaloric high-fat, high-protein (HFHP) diet; or a control, isocaloric (control) diet. IHCLs were measured by (1)H-magnetic resonance spectroscopy, fasting metabolism was measured by indirect calorimetry, insulin sensitivity was measured by hyperinsulinemic-euglycemic clamp, and plasma concentrations were measured by enzyme-linked immunosorbent assay and gas chromatography-mass spectrometry; expression of key lipogenic genes was assessed in subcutaneous adipose tissue biopsy specimens. RESULTS: The HF diet increased IHCLs by 90 +/- 26% and plasma tissue-type plasminogen activator inhibitor-1 (tPAI-1) by 54 +/- 11% (P < 0.02 for both) and inhibited plasma free fatty acids by 26 +/- 11% and beta-hydroxybutyrate by 61 +/- 27% (P < 0.05 for both). The HFHP diet blunted the increase in IHCLs and normalized plasma beta-hydroxybutyrate and tPAI-1 concentrations. Insulin sensitivity was not altered, whereas the expression of sterol regulatory element-binding protein-1c and key lipogenic genes increased with the HF and HFHP diets (P < 0.02). Bile acid concentrations remained unchanged after the HF diet but increased by 50 +/- 24% after the HFHP diet (P = 0.14). CONCLUSIONS: Protein intake significantly blunts the effects of an HF diet on IHCLs and tPAI-1 through effects presumably exerted at the level of the liver. Protein-induced increases in bile acid concentrations may be involved. This trial was registered at www.clinicaltrials.gov as NCT00523562.

Protein turnover and thermogenesis in response to high-protein and high-carbohydrate feeding in men.

The rates of energy expenditure and wholebody protein turnover were determined during a 9-h period in a group of seven men while they received hourly isocaloric meals of high-protein (HP) or high-carbohydrate (HC) content. Their responses to feeding were compared with those to a short period of fasting (15-24 h). The 9-h thermic response to the repeated feeding of HP meals was found to be greater than that to the HC meals (9.6 +/- 0.6% vs 5.7 +/- 0.4% of the energy intake, respectively, means +/- SEM, p less than 0.01). The rate of whole-body nitrogen turnover over 9 h increased from 17.6 +/- 2.2 g on the fasting day to 27.4 +/- 1.4 g during HC feeding (NS) and there was a further increase to 58.2 +/- 5.3 g resulting from HP feeding (p less than 0.001). By using theoretical estimates (based upon ATP requirements) of the metabolic cost of protein synthesis, 36 +/- 9% of the thermic response to HC feeding and 68 +/- 3% of the response to HP feeding could be accounted for by the increases in protein synthesis compared with the fasting state.

Genome-wide association analysis identifies variants associated with nonalcoholic fatty liver disease that have distinct effects on metabolic traits.

Nonalcoholic fatty liver disease (NAFLD) clusters in families, but the only known common genetic variants influencing risk are near PNPLA3. We sought to identify additional genetic variants influencing NAFLD using genome-wide association (GWA) analysis of computed tomography (CT) measured hepatic steatosis, a non-invasive measure of NAFLD, in large population based samples. Using variance components methods, we show that CT hepatic steatosis is heritable (∼26%-27%) in family-based Amish, Family Heart, and Framingham Heart Studies (n = 880 to 3,070). By carrying out a fixed-effects meta-analysis of genome-wide association (GWA) results between CT hepatic steatosis and ∼2.4 million imputed or genotyped SNPs in 7,176 individuals from the Old Order Amish, Age, Gene/Environment Susceptibility-Reykjavik study (AGES), Family Heart, and Framingham Heart Studies, we identify variants associated at genome-wide significant levels (p<5×10(-8)) in or near PNPLA3, NCAN, and PPP1R3B. We genotype these and 42 other top CT hepatic steatosis-associated SNPs in 592 subjects with biopsy-proven NAFLD from the NASH Clinical Research Network (NASH CRN). In comparisons with 1,405 healthy controls from the Myocardial Genetics Consortium (MIGen), we observe significant associations with histologic NAFLD at variants in or near NCAN, GCKR, LYPLAL1, and PNPLA3, but not PPP1R3B. Variants at these five loci exhibit distinct patterns of association with serum lipids, as well as glycemic and anthropometric traits. We identify common genetic variants influencing CT-assessed steatosis and risk of NAFLD. Hepatic steatosis associated variants are not uniformly associated with NASH/fibrosis or result in abnormalities in serum lipids or glycemic and anthropometric traits, suggesting genetic heterogeneity in the pathways influencing these traits.

Retinoid X receptor and peroxisome proliferator-activated receptor activate an estrogen responsive gene independent of the estrogen receptor.

Estrogen receptors regulate transcription of genes essential for sexual development and reproductive function. Since the retinoid X receptor (RXR) is able to modulate estrogen responsive genes and both 9-cis RA and fatty acids influenced development of estrogen responsive tumors, we hypothesized that estrogen responsive genes might be modulated by RXR and the fatty acid receptor (peroxisome proliferator-activated receptor, PPAR). To test this hypothesis, transfection assays in CV-1 cells were performed with an estrogen response element (ERE) coupled to a luciferase reporter construct. Addition of expression vectors for RXR and PPAR resulted in an 11-fold increase in luciferase activity in the presence of 9-cis RA. Furthermore, mobility shift assays demonstrated binding of RXR and PPAR to the vitellogenin A2-ERE and an ERE in the oxytocin promoter. Methylation interference assays demonstrated that specific guanine residues required for RXR/PPAR binding to the ERE were similar to residues required for ER binding. Moreover, RXR domain-deleted constructs in transfection assays showed that activation required RXR since an RXR delta AF-2 mutant completely abrogated reporter activity. Oligoprecipitation binding studies with biotinylated ERE and (35)S-labeled in vitro translated RXR constructs confirmed binding of delta AF-2 RXR mutant to the ERE in the presence of baculovirus-expressed PPAR. Finally, in situ hybridization confirmed RXR and PPAR mRNA expression in estrogen responsive tissues. Collectively, these data suggest that RXR and PPAR are present in reproductive tissues, are capable of activating estrogen responsive genes and suggest that the mechanism of activation may involve direct binding of the receptors to estrogen response elements.

Effects of dexamethasone on the metabolic responses to mental stress in humans.

The haemodynamic effects of the sympathetic nervous system (SNS) activations elicited by hypoglycaemia, acute alcohol administration, or insulin can be prevented by a pretreatment with dexamethasone in humans. This suggests a possible role of central corticotropin releasing hormone (GRIT) release. Mental stress activates the SNS, and decreases systemic vascular resistances though a beta-adrenergic-mediated vasodilation thought to involve vascular nitric oxide release. It also increases insulin-mediated glucose disposal, an effect presumably related to vasodilation. In order to evaluate whether activation of SNS by mental stress is glucocorticoid-sensitive, we monitored the haemodynamic and metabolic effects of mental stress during hyperinsulinaemia in healthy humans with and without a 2-day treatment with 8 mg day(-1) dexamethasone. Mental stress decreased systemic vascular resistances by 21.9% and increased insulin-mediated glucose disposal by 2 8.4% without dexamethasone pretreatment. After 2 days of dexamethasone treatment, whole body insulin-mediated glucose disposal was decreased by 40.8%. The haemodynainic effects of mental stress were however, not affected. Mental stress acutely increased insulin-mediated glucose disposal by 28.0%. This indicates that mental stress elicits a stimulation of SNS through dexamethasone-insensitive pathway, distinct of those activated by insulin, alcohol, or hyperglycaemia.

Inhibition of Na(+)-K(+)-ATPase by digoxin and its relation with energy expenditure and nutrient oxidation rate.

This study investigates the effects of digoxin, an inhibitor of the Na+ pump (Na(+)-K(+)-ATPase), on resting metabolic rate (RMR), respiratory quotient (RQ), and nutrient oxidation rate. Twelve healthy male subjects followed a double-blind protocol design and received either 1 mg/day digoxin or a placebo 2 days before indirect calorimetry measurements. Digoxin induced a 0.22 +/- 0.07 kJ/min or 3.8 +/- 1.5% (mean +/- SE, P = 0.01) decrease in RMR and a 0.40 +/- 0.13 kJ/min (P = 0.01) decrease in fat oxidation rate, whereas carbohydrate and protein oxidation rates did not change significantly. A dose-response relationship between serum digoxin and RQ was observed. These results suggest that digoxin reduces not only RMR but also fat oxidation rate by mechanisms that remain to be elucidated. Because a linkage and an association between genes coding the Na(+)-K(+)-ATPase and the RQ have been previously observed, the present demonstration of an effect of Na(+)-K(+)-ATPase inhibition on fat oxidation rate strengthens the concept that the activity of this enzyme may play a role in body weight regulation.

Short-term overexpression of a constitutively active form of AMP-activated protein kinase in the liver leads to mild hypoglycemia and fatty liver.

AMP-activated protein kinase (AMPK) is a major therapeutic target for the treatment of diabetes. We investigated the effect of a short-term overexpression of AMPK specifically in the liver by adenovirus-mediated transfer of a gene encoding a constitutively active form of AMPKalpha2 (AMPKalpha2-CA). Hepatic AMPKalpha2-CA expression significantly decreased blood glucose levels and gluconeogenic gene expression. Hepatic expression of AMPKalpha2-CA in streptozotocin-induced and ob/ob diabetic mice abolished hyperglycemia and decreased gluconeogenic gene expression. In normal mouse liver, AMPKalpha2-CA considerably decreased the refeeding-induced transcriptional activation of genes encoding proteins involved in glycolysis and lipogenesis and their upstream regulators, SREBP-1 (sterol regulatory element-binding protein-1) and ChREBP (carbohydrate response element-binding protein). This resulted in decreases in hepatic glycogen synthesis and circulating lipid levels. Surprisingly, despite the inhibition of hepatic lipogenesis, expression of AMPKalpha2-CA led to fatty liver due to the accumulation of lipids released from adipose tissue. The relative scarcity of glucose due to AMPKalpha2-CA expression led to an increase in hepatic fatty acid oxidation and ketone bodies production as an alternative source of energy for peripheral tissues. Thus, short-term AMPK activation in the liver reduces blood glucose levels and results in a switch from glucose to fatty acid utilization to supply energy needs.

Changes of the mucosal N3 and N6 fatty acid status occur early in the colorectal adenoma-carcinoma sequence

Despite data favouring a role of dietary fat in colonic carcinogenesis, no study has focused on tissue n3 and n6 fatty acid (FA) status in human colon adenoma-carcinoma sequence. Thus, FA profile was measured in plasma phospholipids of patients with colorectal cancer (n = 22), sporadic adenoma (n = 27), and normal colon (n = 12) (control group). Additionally, mucosal FAs were assessed in both diseased and normal mucosa of cancer (n = 15) and adenoma (n = 21) patients, and from normal mucosa of controls (n = 8). There were no differences in FA profile of both plasma phospholipids and normal mucosa, between adenoma and control patients. There were considerable differences, however, in FAs between diseased and paired normal mucosa of adenoma patients, with increases of linoleic (p = 0.02), dihomogammalinolenic (p = 0.014), and eicosapentaenoic (p = 0.012) acids, and decreases of alpha linolenic (p = 0.001) and arachidonic (p = 0.02) acids in diseased mucosa. A stepwise reduction of eicosapentaenoic acid concentrations in diseased mucosa from benign adenoma to the most advanced colon cancer was seen (p = 0.009). Cancer patients showed lower alpha linolenate (p = 0.002) and higher dihomogammalinolenate (p = 0.003) in diseased than in paired normal mucosa. In conclusion changes in tissue n3 and n6 FA status might participate in the early phases of the human colorectal carcinogenesis.