Intragastric Administration of Casein Leads to Nigrostriatal Disease Progressed Accompanied with Persistent Nigrostriatal-Intestinal Inflammation Activited and Intestinal Microbiota-Metabolic Disorders Induced in MPTP Mouse Model of Parkinson's Disease.

Gut microbial dysbiosis and alteration of gut microbiota composition in Parkinson's disease (PD) have been increasingly reported, no recognized therapies are available to halt or slow progression of PD and more evidence is still needed to illustrate its causative impact on gut microbiota and PD and mechanisms for targeted mitigation. Epidemiological evidence supported an association between milk intake and a higher incidence of Parkinson's disease (PD), questions have been raised about prospective associations between dietary factors and the incidence of PD. Here, we investigated the significance of casein in the development of PD. The mice were given casein (6.75 g/kg i.g.) for 21 days after MPTP (25 mg/kg i.p. × 5 days) treatment, the motor function, dopaminergic neurons, inflammation, gut microbiota and fecal metabolites were observed. The experimental results revealed that the mice with casein gavage after MPTP treatment showed a persisted dyskinesia, the content of dopamine in striatum and the expression of TH in midbrain and ileum were decreased, the expression of Iba-1, CD4, IL-22 in midbrain and ileum increased continuously with persisted intestinal histopathology and intestinal barrier injury. Decreased intestinal bile secretion in addition with abnormal digestion and metabolism of carbohydrate, lipids and proteins were found, whereas these pathological status for the MPTP mice without casein intake had recovered after 24 days, no significant differences were observed with regard to only treated with casein. Our study demonstrates that intestinal pathologic injury, intestinal dysbacteriosis and metabolism changes promoted by casein in MPTP mice ultimately exacerbated the lesions to dopaminergic neurons.

The dominant protein phosphatase PP1c isoform in smooth muscle cells, PP1cß, is essential for smooth muscle contraction.

Contractile force development of smooth muscle is controlled by balanced kinase and phosphatase activities toward the myosin regulatory light chain (RLC). Numerous biochemical and pharmacological studies have investigated the specificity and regulatory activity of smooth muscle myosin light-chain phosphatase (MLCP) bound to myosin filaments and comprised of the regulatory myosin phosphatase target subunit 1 (MYPT1) and catalytic protein phosphatase 1cß (PP1cß) subunits. Recent physiological and biochemical evidence obtained with smooth muscle tissues from a conditional MYPT1 knockout suggests that a soluble, MYPT1-unbound form of PP1cß may additionally contribute to myosin RLC dephosphorylation and relaxation of smooth muscle. Using a combination of isoelectric focusing and isoform-specific immunoblotting, we found here that more than 90% of the total PP1c in mouse smooth muscles is the ß isoform. Moreover, conditional knockout of PP1cα or PP1cγ in adult smooth muscles did not result in an apparent phenotype in mice up to 6 months of age and did not affect smooth muscle contractions ex vivo In contrast, smooth muscle-specific conditional PP1cß knockout decreased contractile force development in bladder, ileal, and aortic tissues and reduced mouse survival. Bladder smooth muscle tissue from WT mice was selectively permeabilized to remove soluble PP1cß to measure contributions of total (α-toxin treatment) and myosin-bound (Triton X-100 treatment) phosphatase activities toward phosphorylated RLC in myofilaments. Triton X-100 reduced PP1cß content by 60% and the rate of RLC dephosphorylation by 2-fold. These results are consistent with the selective dephosphorylation of RLC by both MYPT1-bound and -unbound PP1cß forms in smooth muscle.

Treatment of Intestinal Fibrosis in Experimental Inflammatory Bowel Disease by the Pleiotropic Actions of a Local Rho Kinase Inhibitor.

BACKGROUND: Intestinal fibrosis resulting in (sub)obstruction is a common complication of Crohn's disease (CD). Rho kinases (ROCKs) play multiple roles in TGFß-induced myofibroblast activation that could be therapeutic targets. Because systemic ROCK inhibition causes cardiovascular side effects, we evaluated the effects of a locally acting ROCK inhibitor (AMA0825) on intestinal fibrosis. METHODS: Fibrosis was assessed in mouse models using dextran sulfate sodium (DSS) and adoptive T-cell transfer. The in vitro and ex vivo effects of AMA0825 were studied in different cell types and in CD biopsy cultures. RESULTS: ROCK is expressed in fibroblastic, epithelial, endothelial, and muscle cells of the human intestinal tract and is activated in inflamed and fibrotic tissue. Prophylactic treatment with AMA0825 inhibited myofibroblast accumulation, expression of pro-fibrotic factors, and accumulation of fibrotic tissue without affecting clinical disease activity and histologic inflammation in 2 models of fibrosis. ROCK inhibition reversed established fibrosis in a chronic DSS model and impeded ex vivo pro-fibrotic protein secretion from stenotic CD biopsies. AMA0825 reduced TGFß1-induced activation of myocardin-related transcription factor (MRTF) and p38 mitogen-activated protein kinase (MAPK), down-regulating matrix metalloproteinases, collagen, and IL6 secretion from fibroblasts. In these cells, ROCK inhibition potentiated autophagy, which was required for the observed reduction in collagen and IL6 production. AMA0825 did not affect pro-inflammatory cytokine secretion from other ROCK-positive cell types, corroborating the selective in vivo effect on fibrosis. CONCLUSIONS: Local ROCK inhibition prevents and reverses intestinal fibrosis by diminishing MRTF and p38 MAPK activation and increasing autophagy in fibroblasts. Overall, our results show that local ROCK inhibition is promising for counteracting fibrosis as an add-on therapy for CD.

Marked Enteropathy in an Accelerated Macaque Model of AIDS.

Enteropathy in HIV infection is not eliminated with combination antiretroviral therapy and is possibly linked to microbial translocation. We used a rapidly progressing SIV/pigtailed macaque model of HIV to examine enteropathy and microbial translocation. Histologic evidence of intestinal disease was observed in only half of infected macaques during late-stage infection (LSI). Combination antiretroviral therapy initiated during acute infection prevented intestinal disease. In the ileum and colon, enteropathy was associated with increased caspase-3 staining, decreased CD3+ T cells, and increased SIV-infected cells. CD3+ T cells were preserved in LSI animals without intestinal disease, and levels of CD3 staining in all LSI animals strongly correlated with the number of infected cells in the intestine and plasma viral load. Unexpectedly, there was little evidence of microbial translocation as measured by soluble CD14, soluble CD163, lipopolysaccharide binding protein, and microbial 16s ribosomal DNA. Loss of epithelial integrity indicated by loss of the tight junction protein claudin-3 was not observed during acute infection despite significantly fewer T cells. Claudin-3 was reduced in LSI animals with severe intestinal disease but did not correlate with increased microbial translocation. LSI animals that did not develop intestinal disease had increased T-cell intracytoplasmic antigen 1-positive cytotoxic T lymphocytes, suggesting a robust adaptive cytotoxic T-lymphocyte response may, in part, confer resilience to SIV-induced intestinal damage.

FEATURES OF HISTOCHEMICAL CHANGES IN THE ACTIVITY OF SUCCINATE DEHYDROGENASE OF ARTIFICIAL BLADDER IN DYNAMICS (EXPERIMENTAL STUDY).

The gold standard for the treatment of invasive bladder cancer recognized throughout the world is radical cystectomy with orthotopic ileocystoplasty using the ileal intestinal tract. The study of the effect of urine on the adaptation of the mucosa of the artificial bladder continues for the last twenty years. According to the researchers, the results are quite contradictory, as some scientists note the hypersecretion of sulphomucins, sialomucins, progressive atrophy of microvilli, adenomatous hyperplasia and dysplasia. The aim of investigation to study the features of the histochemically revealed activity of succinate dehydrogenase in the wall of the artificial bladder and ileum in experimental animals. The material of the present study were the results obtained from the study of 18 female mini-pigs aged 4-5 months and weighing 8-10 kg. The modeling of the artificial bladder was performed in experimental animals, by cystectomy and subsequent ileo-cystoplasty. Experimental animals with a bladder model in groups of 6 animals were withdrawn from the experiment 3, 6 and 12 months after operational modeling. As for the wall of the official bladder, the changes in the activity of the studied enzymes were significant and showed not only possible changes in the activity of the Krebs cycle, but also about periodic displacements of the accents of substrate maintenance. These changes, in our view, are related to the transformation processes in the structural elements of the ileum wall, from which an unproblem has been formed to fulfill new functional duties. Signs of a violation of energy metabolism indicate the processes of hypoxia in the tissue of the artificial bladder and require further study and observation.

Activity of SHIP, Which Prevents Expression of Interleukin 1ß, Is Reduced in Patients With Crohn's Disease.

BACKGROUND & AIMS: Crohn's disease (CD) is associated with a dysregulated immune response to commensal micro-organisms in the intestine. Mice deficient in inositol polyphosphate 5'-phosphatase D (INPP5D, also known as SHIP) develop intestinal inflammation resembling that of patients with CD. SHIP is a negative regulator of PI3Kp110α activity. We investigated mechanisms of intestinal inflammation in Inpp5d(-/-) mice (SHIP-null mice), and SHIP levels and activity in intestinal tissues of subjects with CD. METHODS: We collected intestines from SHIP-null mice, as well as Inpp5d(+/+) mice (controls), and measured levels of cytokines of the interleukin 1 (IL1) family (IL1α, IL1ß, IL1ra, and IL6) by enzyme-linked immunosorbent assay. Macrophages were isolated from lamina propria cells of mice, IL1ß production was measured, and mechanisms of increased IL1ß production were investigated. Macrophages were incubated with pan-phosphatidylinositol 3-kinase inhibitors or PI3Kp110α-specific inhibitors. Some mice were given an antagonist of the IL1 receptor; macrophages were depleted from ilea of mice using clodronate-containing liposomes. We obtained ileal biopsies from sites of inflammation and peripheral blood mononuclear cells (PBMCs) from treatment-naïve subjects with CD or without CD (controls), and measured SHIP levels and activity. PBMCs were incubated with lipopolysaccharide and adenosine triphosphate, and levels of IL1ß production were measured. RESULTS: Inflamed intestinal tissues and intestinal macrophages from SHIP-null mice produced higher levels of IL1B and IL18 than intestinal tissues from control mice. We found PI3Kp110α to be required for macrophage transcription of Il1b. Macrophage depletion or injection of an IL1 receptor antagonist reduced ileal inflammation in SHIP-null mice. Inflamed ileal tissues and PBMCs from patients with CD had lower levels of SHIP protein than controls (P < .0001 and P < .0002, respectively). There was an inverse correlation between levels of SHIP activity in PBMCs and induction of IL1ß production by lipopolysaccharide and adenosine triphosphate (R(2) = .88). CONCLUSIONS: Macrophages from SHIP-deficient mice have increased PI3Kp110α-mediated transcription of Il1b, which contributes to spontaneous ileal inflammation. SHIP levels and activity are lower in intestinal tissues and peripheral blood samples from patients with CD than controls. There is an inverse correlation between SHIP activity and induction of IL1ß production by lipopolysaccharide and adenosine triphosphate in PBMCs. Strategies to reduce IL1B might be developed to treat patients with CD found to have low SHIP activity.

The nitric oxide synthase 2 pathway is targeted by both pro- and anti-inflammatory treatments in the immature human intestine.

BACKGROUND AND AIM: NO synthase 2 (NOS2) was recently identified as one the most overexpressed genes in intestinal samples of premature infants with necrotizing enterocolitis (NEC). NOS2 is widely implicated in the processes of epithelial cell injury/apoptosis and host immune defense but its specific role in inflammation of the immature human intestinal mucosa remains unclear. Interestingly, factors that prevent NEC such as epidermal growth factor (EGF) attenuate the inflammatory response in the mid-gestation human small intestine using serum-free organ culture while drugs that are associated with NEC occurrence such as the non-steroidal anti-inflammatory drug, indomethacin (INDO), exert multiple detrimental effects on the immature human intestine. In this study we investigate the potential role of NOS2 in modulating the gut inflammatory response under protective and stressful conditions by determining the expression profile of NOS2 and its downstream pathways in the immature intestine. METHODS: Gene expression profiles of cultured mid-gestation human intestinal explants were investigated in the absence or presence of a physiological concentration of EGF (50 ng/ml) or 1 µM INDO for 48 h using Illumina whole genome microarrays, Ingenuity Pathway Analysis software and quantitative PCR to investigate the expression of NOS2 and NOS2-pathway related genes. RESULTS: In the immature intestine, NOS2 expression was found to be increased by EGF and repressed by INDO. Bioinformatic analysis identified differentially regulated pathways where NOS2 is known to play an important role including citrulline/arginine metabolism, epithelial cell junctions and oxidative stress. At the individual gene level, we identified many differentially expressed genes of the citrulline/arginine metabolism pathway such as ARG1, ARG2, GLS, OAT and OTC in response to EGF and INDO. Gene expression of tight junction components such as CLDN1, CLDN2, CLDN7 and OCN and of antioxidant markers such as DUOX2, GPX2, SOD2 were also found to be differentially modulated by EGF and INDO. CONCLUSION: These results suggest that the protective effect of EGF and the deleterious influence of INDO on the immature intestine could be mediated via regulation of NOS2. Pathways downstream of NOS2 involved with these effects include metabolism linked to NO production, epithelial barrier permeability and antioxidant expression. These results suggest that NOS2 is a likely regulator of the inflammatory response in the immature human gut and may provide a mechanistic basis for the protective effect of EGF and the deleterious effects of INDO.

Intestinal alkaline phosphatase deficiency leads to dysbiosis and bacterial translocation in the newborn intestine.

BACKGROUND: Intestinal alkaline phosphatase (IAP) has been shown to help maintain intestinal homeostasis. Decreased expression of IAP has been linked with pediatric intestinal diseases associated with bacterial overgrowth and subsequent inflammation. We hypothesize that the absence of IAP leads to dysbiosis, with increased inflammation and permeability of the newborn intestine. METHODS: Sprague-Dawley heterozygote IAP cross-matches were bred. Pups were dam fed ad lib and euthanized at weaning. The microbiotas of terminal ileum (TI) and colon was determined by quantitative real-time polymerase chain reaction (qRT-PCR) of subphylum-specific bacterial 16S ribosomal RNA. RT-PCR was performed on TI for inflammatory cytokines. Intestinal permeability was quantified by fluorescein isothiocyanate-dextran permeability and bacterial translocation by qRT-PCR for bacterial 16S ribosomal RNA in mesenteric lymph nodes. Statistical analysis was done by chi-square analysis. RESULTS: All three genotypes had similar concentrations of bacteria in the TI and colon. However, IAP knockout (IAP-KO) had significantly decreased diversity of bacterial species in their colonic stool compared with heterozygous and wild-type (WT). IAP-KO pups had a nonstatistically significant 3.9-fold increased inducible nitric oxide synthase messenger RNA expression compared with WT (IAP-KO, 3.92 ± 1.36; WT, 1.0 ± 0.27; P = 0.03). IAP-KO also had significantly increased bacterial translocation to mesenteric lymph nodes occurred in IAP-KO (IAP-KO, 7625 RFU/g ± 3469; WT, 4957 RFU/g ± 1552; P = 0.04). Furthermore, IAP-KO had increased permeability (IAP-KO, 0.297 mg/mL ± 0.2; WT, 0.189 mg/mL ± 0.15 P = 0.07), but was not statistically significant. CONCLUSIONS: Deficiency of IAP in the newborn intestine is associated with dysbiosis and increased inflammation, permeability, and bacterial translocation.

Low-protein diets affect ileal amino acid digestibility and gene expression of digestive enzymes in growing and finishing pigs.

The objective of this study was to evaluate effects of dietary crude protein (CP) intake on ileal amino acid digestibilities and expression of genes for digestive enzymes in growing and finishing pigs. In Experiment 1, 18 growing pigs (average initial BW = 36.5 kg) were assigned randomly into one of three treatments (n = 6/treatment group) representing normal (18 % CP), low (15 % CP), and very low (12 % CP) protein intake. In Experiment 2, 18 finishing pigs (average initial BW = 62.3 kg) were allotted randomly into one of three treatments (n = 6/treatment group), representing normal (16 % CP), low (13 % CP) and very low (10 % CP) protein intake. In both experiments, diets with low and very low CP were supplemented with crystalline amino acids to achieve equal content of standardized ileal digestible Lys, Met, Thr, and Trp, and were provided to pigs ad libitum. Daily feed intake, BW, and feed/gain ratios were determined. At the end of each experiment, all pigs were slaughtered to collect pancreas, small-intestine samples, and terminal ileal chymes. Samples were used for determining expression of genes for digestive enzymes and ileal amino acid digestibilities. Growing pigs fed the 12 % CP and 15 % CP diets had lower final body weight (P < 0.01) and ADG (P < 0.0001) when compared with pigs fed the 18 % dietary CP diet. Growing pigs fed with the 12 % CP diet showed higher digestibilities for CP (P < 0.05), DM (P < 0.05), Lys (P < 0.0001), Met (P < 0.01), Cys (P < 0.01), Thr (P < 0.01), Trp (P < 0.05), Val (P < 0.05), Phe (P < 0.05), Ala (P < 0.05), Cys (P < 0.01), and Gly (P < 0.05) than those fed the 18 % CP diet. Finishing pigs fed the 16 % CP diet had a higher (P < 0.01) final body weight than those fed the 10 % CP diet. mRNA levels for digestive enzymes (trypsinogen, chymotrypsin B, and dipeptidases-II and III) differed among the three groups of pigs (P < 0.05), and no difference was noted in the genes expression between control group and lower CP group. These results indicated that a reduction of dietary CP by a six-percentage value limited the growth performance of growing-finishing pigs and that a low-protein diet supplemented with deficient amino acids could reduce the excretion of nitrogen into the environment without affecting weight gain.

Expression of small intestinal nutrient transporters in embryonic and posthatch turkeys.

Nutrients are absorbed in the small intestine through a variety of transporter proteins, which have not been as well characterized in turkeys as in chickens. The objective of this study was to profile the mRNA expression of amino acid and monosaccharide transporters in the small intestine of male and female turkeys. Jejunum was collected during embryonic development (embryonic d 21 and 24, and d of hatch (DOH)) and duodenum, jejunum, and ileum were collected in a separate experiment during posthatch development (DOH, d 7, 14, 21, and 28). Real-time PCR was used to determine expression of aminopeptidase N (APN), one peptide (PepT1), 6 amino acid (ASCT1, b(o,+)AT, CAT1, EAAT3, LAT1, y(+)LAT2) and 3 monosaccharide (GLUT2, GLUT5, SGLT1) transporters. Data were analyzed by ANOVA using JMP Pro 11.0. APN, b(o,+)AT, PepT1, y(+)LAT2, GLUT5, and SGLT1 showed increased expression from embryonic d 21 and 24 to DOH. During posthatch, all genes except GLUT2 and SGLT1 were expressed greater in females than males. GLUT2 was expressed the same in males as females and SGLT1 was expressed greater in males than females. All basolateral membrane transporters were expressed greater during early development then decreased with age, while the brush border membrane transporters EAAT3, GLUT5, and SGLT1 showed increased expression later in development. Because turkeys showed high-level expression of the anionic amino acid transporter EAAT3, a direct comparison of tissue-specific expression of EAAT3 between chicken and turkey was conducted. The anionic amino acid transporter EAAT3 showed 6-fold greater expression in the ileum of turkeys at d 14 compared to chickens. This new knowledge can be used not only to better formulate turkey diets to accommodate increased glutamate transport, but also to optimize nutrition for both sexes.

N-glycosylation is essential for ileal ASBT function and protection against proteases.

The bile acid transporter ASBT is a glycoprotein responsible for active absorption of bile acids. Inhibiting ASBT function and bile acid absorption is an attractive approach to lower plasma cholesterol and improve glucose imbalance in diabetic patients. Deglycosylation of ASBT was shown to decrease its function. However, the exact roles of N-glycosylation of ASBT, and how it affects its function, is not known. Current studies investigated the roles of N-glycosylation in ASBT protein stability and protection against proteases utilizing HEK-293 cells stably transfected with ASBT-V5 fusion protein. ASBT-V5 protein was detected as two bands with molecular mass of ~41 and ~35 kDa. Inhibition of glycosylation by tunicamycin significantly decreased ASBT activity and shifted ASBT bands to ~30 kDa, representing a deglycosylated protein. Treatment of total cellular lysates with PNGase F or Endo H glycosidases showed that the upper 41-kDa band represents a fully mature N-acetylglucosamine-rich glycoprotein and the lower 35-kDa band represents a mannose-rich core glycoprotein. Studies with the glycosylation deficient ASBT mutant (N10Q) showed that the N-glycosylation is not essential for ASBT targeting to plasma membrane. However, mature glycosylation significantly increased the half-life and protected ASBT protein from digestion with trypsin. Incubating the cells with high glucose (25 mM) for 48 h increased mature glycosylated ASBT along with an increase in its function. These results unravel novel roles for N-glycosylation of ASBT and suggest that high levels of glucose alter the composition of the glycan and may contribute to the increase in ASBT function in diabetes mellitus.

Heterogeneity of subordination of the IL-18/IFN-γ axis to caspase-1 among patients with Crohn's disease.

In Crohn's disease (CD), hierarchical architecture of the inflammatory network, including subordination of IL-18, an IFN-γ-inducing cytokine, to the inflammasome, have remained undeciphered. Heterogeneity among patients of such a subordination cannot be evaluated by animal models, monofactorial in their etiology and homogenous in disease progression. To address these issues, we set up an ex vivo model of inflamed mucosa explant cultures from patients with active long-standing CD. Th1 cytokine production, especially IFN-γ and IL-18, was assessed in relation with inflammation intensity. Subordination of the Th1 response to caspase-1, effector of the inflammasome, was determined in explant cultures subjected to pharmacological inhibition of caspase-1 by YVAD. We showed a correlation between secreted IFN-γ/IL-18 levels, and caspase-1 activation, with inflammation intensity of intestinal CD mucosa explants. Inhibition of caspase-1 activation using the specific inhibitor YVAD identified a homogenous non responder group featuring a caspase-1-independent IL-18/IFN-γ response, and a heterogenous responder group, in which both IL-18 and IFN-γ responses were caspase-1-dependent, with a 40-70% range of inhibition by YVAD. These findings bring out the concept of heterogeneity of subordination of the Th1 response to inflammasome activation among CD patients. This ex vivo model should have therapeutic relevance in allowing to determine eligibility of CD patients for new targeted therapies.

Intestine-specific deletion of SIRT1 in mice impairs DCoH2-HNF-1α-FXR signaling and alters systemic bile acid homeostasis.

BACKGROUND & AIMS: Sirtuin 1 (SIRT1), the most conserved mammalian oxidized nicotinamide adenine dinucleotide-dependent protein deacetylase, is an important metabolic sensor in many tissues. However, little is known about its role in the small intestine, which absorbs and senses nutrients. We investigated the functions of intestinal SIRT1 in systemic bile acid and cholesterol metabolism in mice. METHODS: SIRT1 was specifically deleted from the intestines of mice using the flox-Villin-Cre system (SIRT1 iKO mice). Intestinal and hepatic tissues were collected, and bile acid absorption was analyzed using the everted gut sac experiment. Systemic bile acid metabolism was studied in SIRT1 iKO and flox control mice placed on standard diets, diets containing 0.5% cholic acid or 1.25% cholesterol, or lithogenic diets. RESULTS: SIRT1 iKO mice had reduced intestinal farnesoid X receptor (FXR) signaling via hepatocyte nuclear factor 1α (HNF-1α) compared with controls, which reduced expression of the bile acid transporter genes Asbt and Mcf2l (encodes Ost) and absorption of ileal bile acids. SIRT1 regulated HNF-1α/FXR signaling partially through dimerization cofactor of HNF-1a (Dcoh2) Dcoh2, which increases dimerization of HNF-1α. SIRT1 was found to deacetylate Dcoh2, promoting its interaction with HNF-1α and inducing DNA binding by HNF-1α. Intestine-specific deletion of SIRT1 increased hepatic bile acid biosynthesis, reduced hepatic accumulation of bile acids, and protected animals from liver damage from a diet high in levels of bile acids. CONCLUSIONS: Intestinal SIRT1, a key nutrient sensor, is required for ileal bile acid absorption and systemic bile acid homeostasis in mice. We delineated the mechanism of metabolic regulation of HNF-1α/FXR signaling. Reagents designed to inhibit intestinal SIRT1 might be developed to treat bile acid-related diseases such as cholestasis.

Vitamin D receptor activation down-regulates the small heterodimer partner and increases CYP7A1 to lower cholesterol.

BACKGROUND & AIMS: Little is known about the effects of the vitamin D receptor (VDR) on hepatic activity of human cholesterol 7α-hydroxylase (CYP7A1) and cholesterol metabolism. We studied these processes in mice in vivo and mouse and human hepatocytes. METHODS: Farnesoid X receptor (Fxr)(-/-), small heterodimer partner (Shp)(-/-), and C57BL/6 (wild-type control) mice were fed normal or Western diets for 3 weeks and were then given intraperitoneal injections of vehicle (corn oil) or 1α,25-dihydroxyvitamin D3 (1,25[OH]2D3; 4 doses, 2.5 µg/kg, every other day). Plasma and tissue samples were collected and levels of Vdr, Shp, Cyp7a1, Cyp24a1, and rodent fibroblast growth factor (Fgf) 15 expression, as well as levels of cholesterol, were measured. We studied the regulation of Shp by Vdr using reporter and mobility shift assays in transfected human embryonic kidney 293 cells, quantitative polymerase chain reaction with mouse tissues and mouse and human hepatocytes, and chromatin immunoprecipitation assays with mouse liver. RESULTS: We first confirmed the presence of Vdr mRNA and protein expression in livers of mice. In mice fed normal diets and given injections of 1,25(OH)2D3, liver and plasma concentrations of 1,25(OH)2D3 increased and decreased in unison. Changes in hepatic Cyp7a1 messenger RNA (mRNA) correlated with those of Cyp24a1 (a Vdr target gene) and inversely with Shp mRNA, but not ileal Fgf15 mRNA. Similarly, incubation with 1,25(OH)2D3 increased levels of Cyp24a1/CYP24A1 and Cyp7a1/CYP7A1 mRNA in mouse and human hepatocytes, and reduced levels of Shp mRNA in mouse hepatocytes. In Fxr(-/-) and wild-type mice with hypercholesterolemia, injection of 1,25(OH)2D3 consistently reduced levels of plasma and liver cholesterol and Shp mRNA, and increased hepatic Cyp7a1 mRNA and protein; these changes were not observed in Shp(-/-) mice given 1,25(OH)2D3 and fed Western diets. Truncation of the human small heterodimer partner (SHP) promoter and deletion analyses revealed VDR-dependent inhibition of SHP, and mobility shift assays showed direct binding of VDR to enhancer regions of SHP. In addition, chromatin immunoprecipitation analysis of livers from mice showed that injection of 1,25(OH)2D3 increased recruitment of Vdr and rodent retinoid X receptor to the Shp promoter. CONCLUSIONS: Activation of the VDR represses hepatic SHP to increase levels of mouse and human CYP7A1 and reduce cholesterol.

Role of adenosine 5'-monophosphate-activated protein kinase in α-linolenic acid-induced intestinal lipid metabolism.

n-3 Long-chain PUFA up-regulate intestinal lipid metabolism. However, whether these metabolic effects of PUFA on intestine are mediated by AMP-activated protein kinase (AMPK) remains to be elucidated. To determine the effects of α-linolenic acid (ALA) on intestinal fatty acid (FA) metabolism and whether these effects were affected by AMPK deletion, mice deficient in the catalytic subunit of AMPKα1 or AMPKα2 and wild-type (WT) mice were fed either a high-fat diet (HF) or HF supplemented with ALA (HF-A). The results showed that ALA supplementation decreased serum TAG content in WT mice. ALA also increased mRNA expression of genes (carnitine palmitoyltransferase 1a, acyl-CoA oxidase 1, medium-chain acyl-CoA dehydrogenase, cytochrome P450 4A10 and pyruvate dehydrogenase kinase isoenzyme 4a) involved in intestinal lipid oxidation and mRNA expression of TAG synthesis-related genes (monoacylglycerol O-acyltransferase 2, diacylglycerol O-acyltransferases 1 and 2) in WT mice. Consistent with these, expression levels of phosphorylated AMPKα1 and AMPKα2 were also increased in WT mice after ALA addition. However, in the absence of either AMPKα1 or AMPKα2, ALA supplementation failed to increase intestinal lipid oxidation. In addition, no significant effects of either diet (HF and HF-A) or genotype (WT, AMPKα1(-/-) and AMPKα2(-/-)) on FA uptake in the intestine and faecal TAG output were observed. Our results suggest that AMPK is indispensable for the effects of ALA on intestinal lipid oxidation.

SHIP-1 deficiency in the myeloid compartment is insufficient to induce myeloid expansion or chronic inflammation.

SHIP-1 has an important role in controlling immune cell function through its ability to downmodulate PI3K signaling pathways that regulate cell survival and responses to stimulation. Mice deficient in SHIP-1 display several chronic inflammatory phenotypes including antibody-mediated autoimmune disease, Crohn's disease-like ileitis and a lung disease reminiscent of chronic obstructive pulmonary disease. The ileum and lungs of SHIP-1-deficient mice are infiltrated at an early age with abundant myeloid cells and the mice have a limited lifespan primarily thought to be due to the consolidation of lungs with spontaneously activated macrophages. To determine whether the myeloid compartment is the key initiator of inflammatory disease in SHIP-1-deficient mice, we examined two independent strains of mice harboring myeloid-restricted deletion of SHIP-1. Contrary to expectations, conditional deletion of SHIP-1 in myeloid cells did not result in consolidating pneumonia or segmental ileitis typical of germline SHIP-1 deficiency. In addition, other myeloid cell abnormalities characteristic of germline loss of SHIP-1, including flagrant splenomegaly and enhanced myelopoiesis, were absent in mice lacking SHIP-1 in myeloid cells. This study indicates that the spontaneous inflammatory disease characteristic of germline SHIP-1 deficiency is not initiated solely by LysM-positive myeloid cells but requires the simultaneous loss of SHIP-1 in other hematolymphoid lineages.

Atorvastatin induces bile acid-synthetic enzyme Cyp7a1 by suppressing FXR signaling in both liver and intestine in mice.

Statins are effective cholesterol-lowering drugs to treat CVDs. Bile acids (BAs), the end products of cholesterol metabolism in the liver, are important nutrient and energy regulators. The present study aims to investigate how statins affect BA homeostasis in the enterohepatic circulation. Male C57BL/6 mice were treated with atorvastatin (100 mg/kg/day po) for 1 week, followed by BA profiling by ultra-performance LC-MS/MS. Atorvastatin decreased BA pool size, mainly due to less BA in the intestine. Surprisingly, atorvastatin did not alter total BAs in the serum or liver. Atorvastatin increased the ratio of 12α-OH/non12α-OH BAs. Atorvastatin increased the mRNAs of the BA-synthetic enzymes cholesterol 7α-hydroxylase (Cyp7a1) (over 10-fold) and cytochrome P450 27a1, the BA uptake transporters Na⁺/taurocholate cotransporting polypeptide and organic anion transporting polypeptide 1b2, and the efflux transporter multidrug resistance-associated protein 2 in the liver. Noticeably, atorvastatin suppressed the expression of BA nuclear receptor farnesoid X receptor (FXR) target genes, namely small heterodimer partner (liver) and fibroblast growth factor 15 (ileum). Furthermore, atorvastatin increased the mRNAs of the organic cation uptake transporter 1 and cholesterol efflux transporters Abcg5 and Abcg8 in the liver. The increased expression of BA-synthetic enzymes and BA transporters appear to be a compensatory response to maintain BA homeostasis after atorvastatin treatment. The Cyp7a1 induction by atorvastatin appears to be due to suppressed FXR signaling in both the liver and intestine.

A novel approach to maintain gut mucosal integrity using an oral enzyme supplement.

OBJECTIVE: To determine the role of intestinal alkaline phosphatase (IAP) in enteral starvation-induced gut barrier dysfunction and to study its therapeutic effect as a supplement to prevent gut-derived sepsis. BACKGROUND: Critically ill patients are at increased risk for systemic sepsis and, in some cases, multiorgan failure leading to death. Years ago, the gut was identified as a major source for this systemic sepsis syndrome. Previously, we have shown that IAP detoxifies bacterial toxins, prevents endotoxemia, and preserves intestinal microbiotal homeostasis. METHODS: WT and IAP-KO mice were used to examine gut barrier function and tight junction protein levels during 48-hour starvation and fed states. Human ileal fluid samples were collected from 20 patients postileostomy and IAP levels were compared between fasted and fed states. To study the effect of IAP supplementation on starvation-induced gut barrier dysfunction, WT mice were fasted for 48 hours +/- IAP supplementation in the drinking water. RESULTS: The loss of IAP expression is associated with decreased expression of intestinal junctional proteins and impaired barrier function. For the first time, we demonstrate that IAP expression is also decreased in humans who are deprived of enteral feeding. Finally, our data demonstrate that IAP supplementation reverses the gut barrier dysfunction and tight junction protein losses due to a lack of enteral feeding. CONCLUSIONS: IAP is a major regulator of gut mucosal permeability and is able to ameliorate starvation-induced gut barrier dysfunction. Enteral IAP supplementation may represent a novel approach to maintain bowel integrity in critically ill patients.

Distinct patterns of aging effects on the expression and activity of carboxylesterases in rat liver and intestine.

The age-associated alteration in expression levels of carboxylesterases (CESs) can affect both intestinal and hepatic first-pass metabolism after oral administration of xenobiotic esters such as prodrugs. In this study, the age-related expression of CES isozymes and hydrolase activities were simultaneously investigated in liver, jejunum, and ileum from 8-, 46-, and 90-week-old rats. Rat liver expresses three major CES1 isozymes, Hydrolase A, Hydrolase B, and Hydrolase C, as well as one minor CES1 (Egasyn) and three minor CES2 isozymes (RL4, AY034877, and D50580). The mRNA and protein levels of major hepatic CES1 isozymes were decreased in an age-dependent manner, while those of minor CESs were maintained in all age groups. The hepatic hydrolase activity for temocapril was decreased in an age-dependent manner, accompanied by downregulation of Hydrolase B/C mRNA, while age-independent hydrolysis of propranolol derivatives was observed in rat liver, due to the contribution of Egasyn. Rat small intestine expresses one major CES2 (RL4) and four minor CESs (Hydrolase B, Hydrolase C, Egasyn, and AY034877). Interestingly, the expression of RL4 was age-dependently increased in both jejunum and ileum, while minor isozymes showed a constant expression across a wide age range. The up-regulation of RL4 expression with aging led to an increase of intestinal hydrolase activities for temocapril and propranolol derivatives. Consequently, age-dependent changes in the expression of CES isozymes affect the hydrolysis of xenobiotics in both rat liver and small intestine.