Hydrophobic Bile Salts Induce Pro-Fibrogenic Proliferation of Hepatic Stellate Cells through PI3K p110 Alpha Signaling.

Bile salts accumulating during cholestatic liver disease are believed to promote liver fibrosis. We have recently shown that chenodeoxycholate (CDC) induces expansion of hepatic stellate cells (HSCs) in vivo, thereby promoting liver fibrosis. Mechanisms underlying bile salt-induced fibrogenesis remain elusive. We aimed to characterize the effects of different bile salts on HSC biology and investigated underlying signaling pathways. Murine HSCs (mHSCs) were stimulated with hydrophilic and hydrophobic bile salts. Proliferation, cell mass, collagen deposition, and activation of signaling pathways were determined. Activation of the human HSC cell line LX 2 was assessed by quantification of α-smooth muscle actin (αSMA) expression. Phosphatidyl-inositol-3-kinase (PI3K)-dependent signaling was inhibited both pharmacologically and by siRNA. CDC, the most abundant bile salt accumulating in human cholestasis, but no other bile salt tested, induced Protein kinase B (PKB) phosphorylation and promoted HSC proliferation and subsequent collagen deposition. Pharmacological inhibition of the upstream target PI3K-inhibited activation of PKB and pro-fibrogenic proliferation of HSCs. The PI3K p110α-specific inhibitor Alpelisib and siRNA-mediated knockdown of p110α ameliorated pro-fibrogenic activation of mHSC and LX 2 cells, respectively. In summary, pro-fibrogenic signaling in mHSCs is selectively induced by CDC. PI3K p110α may be a potential therapeutic target for the inhibition of bile salt-induced fibrogenesis in cholestasis.

Multidisciplinary lifestyle intervention is associated with improvements in liver damage and in surrogate scores of NAFLD and liver fibrosis in morbidly obese patients.

PURPOSE: Non-alcoholic fatty liver disease (NAFLD) is the hepatic manifestation of the metabolic syndrome. Particularly morbidly obese patients are at risk of developing progressive liver disease. Nutritional and lifestyle intervention is recommended as the standard of care in NAFLD. However, there is a striking lack of evidence to support the efficacy of lifestyle intervention to treat NAFLD in morbidly obese patients. Here, we aimed to assess the impact of lifestyle intervention on NAFLD in the morbidly obese in a real-world setting. METHODS: 136 obese patients were included in an industry-independent, multiprofessional lifestyle intervention program with a lead-in phase of 12 weeks of formula diet and a total of 48 weeks intensive counselling. Body weight and markers of the metabolic syndrome were analyzed. Presence of NAFLD was screened for by use of non-invasive markers of fatty liver, non-alcoholic steatohepatitis and liver fibrosis. RESULTS: Weight loss goals (i.e. > 5% or > 10% of initial body weight, respectively, depending on baseline BMI) were achieved in 89.7% of subjects in the intention-to-treat analysis and 93.9% in the per-protocol analysis. This was associated with a pronounced improvement in serum ALT values. The percentage of subjects who fulfilled non-invasive criteria for fatty liver dropped from 95.2 to 54.8%. Risk of NASH improved and the number of patients at risk of liver fibrosis declined by 54.1%. CONCLUSION: Lifestyle intervention was associated with a marked improvement of serum ALT and an improvement of surrogate scores indicative of NAFLD and, importantly, advanced fibrosis, in a real-world cohort of morbidly obese patients.

Development of Protocols for Regeneration and Transformation of Apomitic and Sexual Forms of Dallisgrass (Paspalum dilatatum Poir.).

Paspalum dilatatum (common name dallisgrass), a productive C4 grass native to South America, is an important pasture grass found throughout the temperate warm regions of the world. It is characterized by its tolerance to frost and water stress and a higher forage quality than other C4 forage grasses. P. dilatatum includes tetraploid (2n = 40), sexual, and pentaploid (2n = 50) apomictic forms, but is predominantly cultivated in an apomictic monoculture, which implies a high risk that biotic and abiotic stresses could seriously affect the grass productivity. The obtention of reproducible and efficient protocols of regeneration and transformation are valuable tools to obtain genetic modified grasses with improved agronomics traits. In this review, we present the current regeneration and transformation methods of both apomictic and sexual cultivars of P. dilatatum, discuss their strengths and limitations, and focus on the perspectives of genetic modification for producing new generation of forages. The advances in this area of research lead us to consider Paspalum dilatatum as a model species for the molecular improvement of C4 perennial forage species.

IL-18 but Not IL-1 Signaling Is Pivotal for the Initiation of Liver Injury in Murine Non-Alcoholic Fatty Liver Disease.

Non-alcoholic fatty liver disease (NAFLD) is rising in prevalence, and a better pathophysiologic understanding of the transition to its inflammatory phenotype (NASH) is key to the development of effective therapies. To evaluate the contribution of the NLRP3 inflammasome and its downstream effectors IL-1 and IL-18 in this process, we applied the true-to-life "American lifestyle-induced obesity syndrome" (ALiOS) diet mouse model. Development of obesity, fatty liver and liver damage was investigated in mice fed for 24 weeks according to the ALiOS protocol. Lipidomic changes in mouse livers were compared to human NAFLD samples. Receptor knockout mice for IL-1 and IL-18 were used to dissect the impact of downstream signals of inflammasome activity on the development of NAFLD. The ALiOS diet induced obesity and liver steatosis. The lipidomic changes closely mimicked changes in human NAFLD. A pro-inflammatory gene expression pattern in liver tissue and increased serum liver transaminases indicated early liver damage in the absence of histological evidence of NASH. Mechanistically, Il-18r-/-- but not Il-1r-/- mice were protected from early liver damage, possibly due to silencing of the pro-inflammatory gene expression pattern. Our study identified NLRP3 activation and IL-18R-dependent signaling as potential modulators of early liver damage in NAFLD, preceding development of histologic NASH.

Prevalence of a First-Degree Relative With Colorectal Cancer and Uptake of Screening Among Persons 40 to 54 Years Old.

BACKGROUND & AIMS: People with a first-degree relative with colorectal cancer (CRC) are recommended to start CRC screening at age 40. However, there is limited information on how many people in different age groups have a known family history of CRC and how many of them have had a colonoscopy. METHODS: We set up a multicenter, cross-sectional, population-based study in Germany to determine what proportions of persons in age groups from 40 to 54 years old have a known family history of CRC. We invited 160,000 persons to participate in an online survey from 2015 through 2016. We investigated what proportions of persons in each age group reported a family history of CRC and what proportions of persons underwent a colonoscopy examination using descriptive statistics and multiple logistic regression models. RESULTS: Of 28,711 responders to the online questionnaire (8428 were age 40-44 years, 9879 were age 45-49 years, and 10,404 were age 50-54 years), 2705 stated that they had a first-degree relative with CRC (9.4%). The prevalence of a first-degree relative with CRC increased with age: 7.5%, 9.6%, and 10.9% for people 40 to 44 years old, 45 to 49 years old, and 50 to 54 years old, respectively. The prevalence of a first-degree relative who received a diagnosis of CRC at age 70 years or older increased steadily with each age group. Although a greater proportion of people with a family history of CRC had undergone a colonoscopy examination (54.5%) than people without a family history of CRC (25.7%; P < .0001), large proportions of people within this risk group were not in compliance with the guidelines (54.8%, 47.6%, and 38.6% for ages 40-44 y, 45-49 y, and 50-54 y, respectively). CONCLUSIONS: One in 10 persons in Germany age 40 to 54 years old has a first-degree relative with CRC. Guidelines recommend initiation of screening at ages 40 to 45 years for people with a family history, yet at this age many people do not have a family history of CRC yet, and almost half of persons 40 to 54 years old with a family history of CRC have not yet received a screening colonoscopy.

Lifestyle intervention for morbid obesity: effects on liver steatosis, inflammation, and fibrosis.

The prevalence of obesity-related nonalcoholic fatty liver disease (NAFLD) is rising. NAFLD may result in nonalcoholic steatohepatitis (NASH), progressing to liver cirrhosis. Weight loss is recommended to treat obesity-related NASH. Lifestyle intervention may improve NASH; however, pertinent trials have so far focused on overweight patients, whereas patients with obesity are at highest risk of developing NAFLD. Furthermore, reports of effects on liver fibrosis are scarce. We evaluated the effect of lifestyle intervention on NAFLD in a real-life cohort of morbidly obese patients. In our observational study, 152 patients underwent lifestyle intervention, with a follow-up of 52 weeks. Noninvasive measures of obesity, metabolic syndrome, liver steatosis, liver damage, and liver fibrosis were analyzed. Treatment response in terms of weight loss was achieved in 85.1% of patients. Dysglycemia and dyslipidemia improved. The proportion of patients with fatty liver dropped from 98.1 to 54.3% ( P < 0.001). Weight loss >10% was associated with better treatment response ( P = 0.0009). Prevalence of abnormal serum transaminases fell from 81.0 to 50.5% ( P < 0.001). The proportion fibrotic patients, as determined by the NAFLD fibrosis score, dropped from 11.8 to 0% ( P < 0.05). Low serum levels of adiponectin correlated with degree of liver damage, i.e., serum liver transaminases ( r = -0,32, P < 0.05). Serum levels of adiponectin improved with intervention. In conclusion, lifestyle intervention effectively targeted obesity and the metabolic syndrome. Liver steatosis, damage and fibrosis were ameliorated in this real-life cohort of morbidly obese patients, mediated in part by changes in the adipokine profile. Patients with weight loss of >10% seemed to benefit most. NEW & NOTEWORTHY We demonstrate new evidence that lifestyle intervention is effective in treating NAFLD in the important group of patients with (morbid) obesity. Although current guidelines on the therapy of NASH recommend weight loss of 5-7%, weight reduction >10% may be favorable in morbid obesity. Serum levels of adipokines correlate with liver damage, which is indicative of their pathogenetic importance in human NASH. Our study adds to the limited body of evidence that NAFLD-associated liver fibrosis may resolve with lifestyle intervention.

γ-Catenin acts as a tumor suppressor through context-dependent mechanisms in colorectal cancer.

PURPOSE: γ-Catenin is a protein closely related to ß-catenin. While the overexpression of ß-catenin has been linked with impaired prognosis and survival in various malignancies, both oncogenic and tumor suppressor functions have been described for γ-catenin. Thus, its role in cancer remains controversial. In this study, we examined the impact of γ-catenin expression on the malignant potential of colorectal cancer cells. METHODS: γ-Catenin was knocked down by short interfering RNA in the γ-catenin-proficient DLD-1 cell line and stably overexpressed in the γ-catenin-deficient cell line RKO. The effects of these molecular manipulations on the malignant potential of the cell lines were tested in vitro and in vivo in a xenograft tumor model. RESULTS: γ-Catenin contributed to Wnt signaling independent of the cellular context. Unlike its sister molecule ß-catenin, γ-catenin inhibited cellular invasion and anoikis in cells endogenously expressing γ-catenin. In line with this tumor suppressor function, its de novo expression in RKO cells inhibited proliferation via cell cycle arrest. In a xenograft tumor model, overexpression of γ-catenin starkly reduced tumor growth in vivo. CONCLUSIONS: This is the first report demonstrating a tumor-suppressive effect of γ-catenin in colorectal cancer both in vitro and in vivo. Detailed in vitro analysis revealed that effects of γ-catenin differ in γ-catenin proficient and deficient cells, indicating that its function in colorectal cancer is dependent on the cellular context. This finding adds to our understanding of γ-catenin and may have implications for future studies of catenin/Wnt targeted cancer therapies.

Role of interleukin-1 and its antagonism of hepatic stellate cell proliferation and liver fibrosis in the Abcb4(-/-) mouse model.

AIM: To study the interleukin-1 (IL-1) pathway as a therapeutic target for liver fibrosis in vitro and in vivo using the ATP-binding cassette transporter b4(-/-) (Abcb4(-/-)) mouse model. METHODS: Female and male Abcb4(-/-) mice from 6 to 13 mo of age were analysed for the degree of cholestasis (liver serum tests), extent of liver fibrosis (hydroxyproline content and Sirius red staining) and tissue-specific activation of signalling pathways such as the IL-1 pathway [quantitative polymerase chain reaction (qPCR)]. For in vivo experiments, murine hepatic stellate cells (HSCs) were isolated via pronase-collagenase perfusion followed by density gradient centrifugation using female mice. Murine HSCs were stimulated with up to 1 ng/mL IL-1ß with or without 2.5 µg/mL Anakinra, an IL-1 receptor antagonist, respectively. The proliferation of murine HSCs was assessed via the BrdU assay. The toxicity of Anakinra was evaluated via the fluorescein diacetate hydrolysis (FDH) assay. In vivo 8-wk-old Abcb4(-/-) mice with an already fully established hepatic phenotype were treated with Anakinra (1 mg/kg body-weight daily intraperitoneally) or vehicle and liver injury and liver fibrosis were evaluated via serum tests, qPCR, hydroxyproline content and Sirius red staining. RESULTS: Liver fibrosis was less pronounced in males than in female Abcb4(-/-) animals as defined by a lower hydroxyproline content (274 ± 64 µg/g vs 436 ± 80 µg/g liver, respectively; n = 13-15; P < 0.001; Mann-Whitney U-test) and lower mRNA expression of the profibrogenic tissue inhibitor of metalloproteinase-1 (TIMP) (1 ± 0.41 vs 0.66 ± 0.33 fold, respectively; n = 13-15; P < 0.05; Mann-Whitney U-test). Reduced liver fibrosis was associated with significantly lower levels of F4/80 mRNA expression (1 ± 0.28 vs 0.71 ± 0.41 fold, respectively; n = 12-15; P < 0.05; Mann-Whitney U-test) and significantly lower IL-1ß mRNA expression levels (1 ± 0.38 vs 0.44 ± 0.26 fold, respectively; n = 13-15; P < 0.001; Mann-Whitney U-test). No gender differences in the serum liver parameters [bilirubin; alanine aminotransferase (ALT); aspartate aminotransferase and alkaline phosphatase (AP)] were found. In vitro, the administration of IL-1ß resulted in a significant increase in HSC proliferation [0.94 ± 0.72 arbitrary units (A.U.) in untreated controls, 1.12 ± 0.80 A.U. at an IL-1ß concentration of 0.1 ng/mL and 1.18 ± 0.73 A.U. at an IL-1ß concentration of 1 ng/mL in samples from n = 6 donor animals; P < 0.001; analyses of variance (ANOVA)]. Proliferation was reduced significantly by the addition of 2.5 µg/mL Anakinra (0.81 ± 0.60 A.U. in untreated controls, 0.92 ± 0.68 A.U. at an IL-1ß concentration of 0.1 ng/mL, and 0.91 ± 0.69 A.U. at an IL-1ß concentration of 1 ng/mL; in samples from n = 6 donor animals; P < 0.001; ANOVA) suggesting an anti-proliferative effect of this clinically approved IL-1 receptor antagonist. The FDH assay showed this dose to be non-toxic in HSCs. In vivo, Anakinra had no effect on the hepatic hydroxyproline content, liver serum tests (ALT and AP) and pro-fibrotic (collagen 1α1, collagen 1α2, transforming growth factor-ß, and TIMP-1) and anti-fibrotic [matrix metalloproteinase 2 (MMP2), MMP9 and MMP13] gene expression after 4 wk of treatment. Furthermore, the hepatic IL-1ß and F4/80 mRNA expression levels were unaffected by Anakinra treatment. CONCLUSION: IL-1ß expression is associated with the degree of liver fibrosis in Abcb4(-/-) mice and promotes HSC proliferation. IL-1 antagonism shows antifibrotic effects in vitro but not in Abcb4(-/-) mice.

1,25-(OH)2-vitamin D3 prevents activation of hepatic stellate cells in vitro and ameliorates inflammatory liver damage but not fibrosis in the Abcb4(-/-) model.

BACKGROUND/PURPOSE OF THE STUDY: Vitamin D3-deficiency is common in patients with chronic liver-disease and may promote disease progression. Vitamin D3-administration has thus been proposed as a therapeutic approach. Vitamin D3 has immunomodulatory effects and may modulate autoimmune liver-disease such as primary sclerosing cholangitis. Although various mechanisms of action have been proposed, experimental evidence is limited. Here we test the hypothesis that active 1,25-(OH)2-vitamin D3 inhibits activation of hepatic stellate cells (HSC) in vitro and modulates liver-injury in vivo. METHODS: Proliferation and activation of primary murine HSC were assessed by BrdU- and PicoGreen(®)-assays, immunoblotting, immunofluorescence-microscopy, quantitative-PCR, and zymography following calcitriol-treatment. Wild-type and ATP-binding cassette transporter b4(-/-) (Abcb4(-/-))-mice received calcitriol for 4 weeks. Liver-damage, inflammation, and fibrosis were assessed by serum liver-tests, Sirius-red staining, quantitative-PCR, immunoblotting, immunohistochemistry and hydroxyproline quantification. RESULTS: In vitro, calcitriol inhibited activation and proliferation of murine HSC as shown by reduced α-smooth muscle actin and platelet-derived growth factor-receptor-ß-protein-levels, BrdU and PicoGreen®-assays. Furthermore, mRNA-levels and activity of matrix metalloproteinase 13 were profoundly increased. In vivo, calcitriol ameliorated inflammatory liver-injury reflected by reduced levels of alanine aminotransferase in Abcb4(-/-)-mice. In accordance, their livers had lower mRNA-levels of F4/80, tumor necrosis factor-receptor 1 and a lower count of portal CD11b positive cells. In contrast, no effect on overall fibrosis was observed. CONCLUSION: Calcitriol inhibits activation and proliferation of HSCs in vitro. In Abcb4(-/-)-mice, administration of calcitriol ameliorates inflammatory liver-damage but has no effect on biliary fibrosis after 4 weeks of treatment.

Pilot randomized trial of selective internal radiation therapy vs. chemoembolization in unresectable hepatocellular carcinoma.

BACKGROUND & AIMS: To compare selective internal radiation therapy (SIRT) with transarterial chemoembolization (TACE), the standard-of-care for intermediate-stage unresectable, hepatocellular carcinoma (HCC), as first-line treatment. METHODS: SIRTACE was an open-label multicenter randomized-controlled pilot study, which prospectively compared primarily safety and health-related quality of life (HRQoL) changes following TACE and SIRT. Patients with unresectable HCC, Child-Pugh ≤B7, ECOG performance status ≤2 and ≤5 liver lesions (≤20 cm total maximum diameter) without extrahepatic spread were randomized to receive either TACE (at 6-weekly intervals until tumour enhancement was not observed on MRI or disease progression) or single-session SIRT (yttrium-90 resin microspheres). RESULTS: Twenty-eight patients with BCLC stage A (32.1%), B (46.4%) or C (21.4%) received either a mean of 3.4 (median 2) TACE interventions (N = 15) or single SIRT (N = 13). Both treatments were well tolerated. Despite SIRT patients having significantly worse physical functioning at baseline, at week-12, neither treatment had a significantly different impact on HRQoL as measured by Functional Assessment of Cancer Therapy-Hepatobiliary total or its subscales. Both TACE and SIRT were effective for the local control of liver tumours. Best overall response-rate (RECIST 1.0) of target lesions were 13.3% and 30.8%, disease control rates were 73.3% and 76.9% for TACE and SIRT, respectively. Two patients in each group were down-staged for liver transplantation (N = 3) or radiofrequency ablation (N = 1). CONCLUSIONS: Single-session SIRT appeared to be as safe and had a similar impact on HRQoL as multiple sessions of TACE, suggesting that SIRT might be an alternative option for patients eligible for TACE.

Walnut-enriched diet reduces fasting non-HDL-cholesterol and apolipoprotein B in healthy Caucasian subjects: a randomized controlled cross-over clinical trial.

BACKGROUND: Walnut consumption is associated with reduced risk of coronary heart disease (CHD). OBJECTIVE: We assessed the effect of walnuts on lipid and glucose metabolism, adipokines, inflammation and endothelial function in healthy Caucasian men and postmenopausal women ≥50years old. DESIGN: Forty subjects (mean±SEM: age 60±1years, BMI 24.9±0.6kg/m(2); 30 females) were included in a controlled, cross-over study and randomized to receive first a walnut-enriched (43g/d) and then a Western-type (control) diet or vice-versa, with each lasting 8weeks and separated by a 2-week wash-out. At the beginning and end of each diet phase, measurements of fasting values, a mixed meal test and an assessment of postprandial endothelial function (determination of microcirculation by peripheral artery tonometry) were conducted. Area under the curve (AUC), incremental AUC (iAUC) and treatment×time interaction (shape of the curve) were evaluated for postprandial triglycerides, VLDL-triglycerides, chylomicron-triglycerides, glucose and insulin. RESULTS: Compared with the control diet, the walnut diet significantly reduced non-HDL-cholesterol (walnut vs. control: -10±3 vs. -3±2mg/dL; p=0.025) and apolipoprotein-B (-5.0±1.3 vs. -0.2±1.1mg/dL; p=0.009) after adjusting for age, gender, BMI and diet sequence. Total cholesterol showed a trend toward reduction (p=0.073). Fasting VLDL-cholesterol, LDL-cholesterol, HDL-cholesterol, triglycerides and glucose, insulin, HOMA-IR, and HbA1c did not change significantly. Similarly, fasting adipokines, C-reactive protein, biomarkers of endothelial dysfunction, postprandial lipid and glucose metabolism and endothelial function were unaffected. CONCLUSION: Daily consumption of 43g of walnuts for 8weeks significantly reduced non-HDL-cholesterol and apolipoprotein-B, which may explain in part the epidemiological observation that regular walnut consumption decreases CHD risk.

Anti-melanin-concentrating hormone treatment attenuates chronic experimental colitis and fibrosis.

Fibrosis represents a major complication of several chronic diseases, including inflammatory bowel disease (IBD). Treatment of IBD remains a clinical challenge despite several recent therapeutic advances. Melanin-concentrating hormone (MCH) is a hypothalamic neuropeptide shown to regulate appetite and energy balance. However, accumulating evidence suggests that MCH has additional biological effects, including modulation of inflammation. In the present study, we examined the efficacy of an MCH-blocking antibody in treating established, dextran sodium sulfate-induced experimental colitis. Histological and molecular analysis of mouse tissues revealed that mice receiving anti-MCH had accelerated mucosal restitution and lower colonic expression of several proinflammatory cytokines, as well as fibrogenic genes, including COL1A1. In parallel, they spared collagen deposits seen in the untreated mice, suggesting attenuated fibrosis. These findings raised the possibility of perhaps direct effects of MCH on myofibroblasts. Indeed, in biopsies from patients with IBD, we demonstrate expression of the MCH receptor MCHR1 in α-smooth muscle actin(+) subepithelial cells. CCD-18Co cells, a primary human colonic myofibroblast cell line, were also positive for MCHR1. In these cells, MCH acted as a profibrotic modulator by potentiating the effects of IGF-1 and TGF-ß on proliferation and collagen production. Thus, by virtue of combined anti-inflammatory and anti-fibrotic effects, blocking MCH might represent a compelling approach for treating IBD.

Salutary effects of adiponectin on colon cancer: in vivo and in vitro studies in mice.

BACKGROUND: Obesity and a high-fat diet are associated with the risk and progression of colon cancer. Low adiponectin levels may play an important role in the development of colon and other obesity-related malignancies. No previous studies have directly investigated the mechanistic effects of adiponectin on colon cancer in the settings of obesity, a high-fat diet and/or adiponectin deficiency. OBJECTIVE: To investigate the effects of adiponectin on the growth of colorectal cancer in adiponectin-deficient or wild-type-C57BL/6 mice fed a low-fat or high-fat diet. RESULTS: Mice fed a high-fat-diet gained more weight and had larger tumours than mice fed a low-fat-diet. Adiponectin administration suppressed implanted tumour growth, causing larger central necrotic areas. Adiponectin treatment also suppressed angiogenesis assessed by CD31 staining and VEGFb and VEGFd mRNA expression in tumours obtained from mice fed a high-fat-diet and from adiponectin-deficient mice. Adiponectin treatment decreased serum insulin levels in mice on a high-fat-diet and increased serum-interleukin (IL)-12 levels in adiponectin-deficient mice. In vitro, it was found that adiponectin directly controls malignant potential (cell proliferation, adhesion, invasion and colony formation) and regulates metabolic (AMPK/S6), inflammatory (STAT3/VEGF) and cell cycle (p21/p27/p53/cyclins) signalling pathways in both mouse MCA38 and human HT29, HCT116 and LoVo colon cancer cell lines in a LKB1-dependent way. CONCLUSION: These new mechanistic and pathophysiology studies provide evidence for an important role of adiponectin in colon cancer. The data indicate that adiponectin or analogues might be useful agents in the management or chemoprevention of colon cancer.

Increased susceptibility of melanin-concentrating hormone-deficient mice to infection with Salmonella enterica serovar Typhimurium.

Melanin-concentrating hormone (MCH) was initially identified in mammals as a hypothalamic neuropeptide regulating appetite and energy balance. However, the wide distribution of MCH receptors in peripheral tissues suggests additional functions for MCH which remain largely unknown. We have previously reported that mice lacking MCH develop attenuated intestinal inflammation when exposed to Clostridium difficile toxin A. To further characterize the role of MCH in host defense mechanisms against intestinal pathogens, Salmonella enterocolitis (using Salmonella enterica serovar Typhimurium) was induced in MCH-deficient mice and their wild-type littermates. In the absence of MCH, infected mice had increased mortality associated with higher bacterial loads in blood, liver, and spleen. Moreover, the knockout mice developed more-severe intestinal inflammation, based on epithelial damage, immune cell infiltrates, and local and systemic cytokine levels. Paradoxically, these enhanced inflammatory responses in the MCH knockout mice were associated with disproportionally lower levels of macrophages infiltrating the intestine. Hence, we investigated potential direct effects of MCH on monocyte/macrophage functions critical for defense against intestinal pathogens. Using RAW 264.7 mouse monocytic cells, which express endogenous MCH receptor, we found that treatment with MCH enhanced the phagocytic capacity of these cells. Taken together, these findings reveal a previously unappreciated role for MCH in host-bacterial interactions.

Reduced intestinal tumorigenesis in APCmin mice lacking melanin-concentrating hormone.

BACKGROUND: Melanin-concentrating hormone (MCH) is an evolutionary conserved hypothalamic neuropeptide that in mammals primarily regulates appetite and energy balance. We have recently identified a novel role for MCH in intestinal inflammation by demonstrating attenuated experimental colitis in MCH deficient mice or wild type mice treated with an anti-MCH antibody. Therefore, targeting MCH has been proposed for the treatment of inflammatory bowel disease. Given the link between chronic intestinal inflammation and colorectal cancer, in the present study we sought to investigate whether blocking MCH might have effects on intestinal tumorigenesis that are independent of inflammation. METHODOLOGY: Tumor development was evaluated in MCH-deficient mice crossed to the APCmin mice which develop spontaneously intestinal adenomas. A different cohort of MCH-/- and MCH+/+ mice in the APCmin background was treated with dextran sodium sulphate (DSS) to induce inflammation-dependent colorectal tumors. In Caco2 human colorectal adenocarcinoma cells, the role of MCH on cell survival, proliferation and apoptosis was investigated. RESULTS: APCmin mice lacking MCH developed fewer, smaller and less dysplastic tumors in the intestine and colon which at the molecular level are characterized by attenuated activation of the wnt/beta-catenin signaling pathway and increased apoptotic indices. Form a mechanistic point of view, MCH increased the survival of colonic adenocarcinoma Caco2 cells via inhibiting apoptosis, consistent with the mouse studies. CONCLUSION: In addition to modulating inflammation, MCH was found to promote intestinal tumorigenesis at least in part by inhibiting epithelial cell apoptosis. Thereby, blocking MCH as a therapeutic approach is expected to decrease the risk for colorectal cancer.

Dietary walnuts inhibit colorectal cancer growth in mice by suppressing angiogenesis.

OBJECTIVE: Animal studies have demonstrated that dietary supplementation with flaxseed oil inhibits colorectal cancer growth. Recent data indicate that walnuts have strong antiproliferative properties against colon cancer cells in vitro but no previous study has assessed the effects of walnuts in vivo or performed a joint evaluation of flaxseed oil and walnuts. The aim of the present study was to examine the effect of dietary walnuts on colorectal cancer in vivo and to comparatively evaluate their efficacy in relation to flaxseed oil. METHODS: HT-29 human colon cancer cells were injected in 6-wk-old female nude mice. After a 1-wk acclimation period, mice (n = 48) were randomized to diets containing ∼19% of total energy from walnuts, flaxseed oil, or corn oil (control) and were subsequently studied for 25 d. RESULTS: Tumor growth rate was significantly slower in walnut-fed and flaxseed-fed mice compared with corn oil-fed animals (P < 0.05) by 27% and 43%, respectively. Accordingly, final tumor weight was reduced by 33% and 44%, respectively (P < 0.05 versus control); the differences between walnut and flaxseed diets did not reach significance. We found no differences among groups in metabolic and hormonal profile, serum antioxidant capacity, or inflammation (P > 0.05). However, walnuts and flaxseed oil significantly reduced serum expression levels of angiogenesis factors, including vascular endothelial growth factor (by 30% and 80%, respectively), and approximately doubled total necrotic areas despite smaller tumor sizes (P < 0.05 versus control). Dietary walnuts significantly decreased angiogenesis (CD34 staining; P = 0.017 versus control), whereas this effect did not reach significance in the flaxseed oil group (P = 0.454 versus control). CONCLUSION: We conclude that walnuts in the diet inhibit colorectal cancer growth by suppressing angiogenesis. Further studies are needed to confirm our findings in humans and explore underlying mechanisms.

γ-Catenin is an independent prognostic marker in early stage colorectal cancer.

PURPOSE: Expression and role of γ-catenin in colorectal carcinogenesis is not well understood. We aimed at characterizing γ-catenin's expression pattern during colorectal carcinogenesis. METHODS: The expression pattern of γ-catenin was characterized in adenomas, primary colorectal carcinomas, and their corresponding metastases. Since this descriptive immunohistochemical analysis revealed upregulation of γ-catenin in the invasive front of both primary tumors and metastases, a tissue microarray (TMA) was performed, allowing for correlation of subcellular expression patterns with disease recurrence and cancer-specific survival. Comparison of γ-catenin expression with that of ß-catenin was performed. RESULTS: In normal colonic epithelium and adenomas, γ-catenin was weakly expressed at the membrane. In central areas of primary colorectal carcinomas, membranous and cytoplasmatic expression was present, with cytoplasmatic and nuclear upregulation of γ-catenin in the invasive fronts. Expression patterns found in metastases resembled those of their respective primary tumors. Subsequent TMA analysis showed that upregulation of cytoplasmatic γ-catenin in the invasive fronts of curatively resected early T2 and T3 colorectal carcinomas was associated with shortened disease-free survival and an increased risk of death (p=0.003; hazard ratio = 2.98; 95% confidence interval, 1.44-6.18). CONCLUSIONS: The correlation of upregulated cellular γ-catenin levels with higher recurrences and impaired survival suggests a tumor promoting role of γ-catenin in colorectal cancer. γ-Catenin may therefore serve as a marker for identifying patients who are at increased risk of disease recurrence who may benefit from closer follow-up and adjuvant therapy.

Insulin glargine and NPH insulin increase to a similar degree epithelial cell proliferation and aberrant crypt foci formation in colons of diabetic mice.

The insulin analog glargine has a higher binding affinity than regular insulin for the insulin-like growth factor 1 receptor in vitro, raising questions about increased mitogenicity in vivo. Observational studies in humans have recently reported a potential differential association between insulin glargine and malignancies, but available evidence remains inconclusive. We directly compared glargine vs. neutral protamine Hagedorn (NPH) insulin's effects on cell proliferation in colonic mucosa and on formation of aberrant crypt foci in diabetic mice, i.e., early stages of colorectal cancer development. Mice (BKS.Cg-+Lepr(db)/+Lepr(db)/OlaHsd) were treated with insulin glargine (G), NPH insulin (NPH), or saline (NaCl). We assessed epithelial proliferation after long-term insulin treatment (18 weeks) by 5-bromo-2'-deoxyuridine and Ki67 staining and analyzed the formation of aberrant crypt foci (ACF) in mice treated with insulin glargine or NPH insulin or 10 weeks after initiation with 1,2-dimethylhydrazine. Insulin glargine treatment did not result in significantly different epithelial colonic proliferation compared to NPH insulin (G, 137 ± 22; NPH, 136 ± 15; NaCl, 100 ± 20 (relative proliferation index)), but both insulin-treated groups of mice had a higher proliferation index compared to the NaCl control group (p<0.001). Similarly, we observed no difference in ACF formation between glargine- and NPH-insulin-treated mice (G, 132 ± 12; NPH, 138 ± 9; NaCl, 100 ± 7 (relative number of ACF)), but ACF formation was significantly higher in insulin-treated mice than in NaCl-treated control mice (p=0.001). Chronic insulin treatment results in higher colonic epithelial proliferation and ACF formation, but the use of insulin glargine vs. NPH insulin is not associated with increased risk.

The effect of telmisartan on glucose and lipid metabolism in nondiabetic, insulin-resistant subjects.

Intervention studies have shown that angiotensin receptor blocker therapy may reduce the incidence of type 2 diabetes mellitus. It is unknown whether short-term angiotensin receptor blocker therapy can improve glucose and lipid metabolism in insulin-resistant subjects. We evaluated the effect of telmisartan (40 mg/d, 12 weeks) in 20 subjects with insulin resistance (body mass index, 31.8 +/- 3.31 kg/m(2); triglycerides, 179 +/- 98 mg/dL; glucose, 104 +/- 9 mg/dL; homeostasis model assessment index, 3.78 +/- 1.52) in a randomized, placebo-controlled, double-blind, cross-over study. At the end of each treatment phase, oral and intravenous glucose tolerance tests including C-peptide and insulin measurements were performed, and fasting and postprandial lipids were determined. Compared to placebo, telmisartan resulted in a reduction in homeostasis model assessment index (-11%, P = .06) and glucose area under the curve during intravenous glucose tolerance (-11%, P = .04). We observed an increase (+32%, P = .05) in the insulinogenic index indicating an improved beta-cell function. Fasting and postprandial lipid parameters did not change. We observed an increase in adiponectin (6%, P = .09), whereas IL-6, high-sensitivity C-reactive protein, fibrinogen, and free fatty acid concentrations did not change. This indicates that the improvement in glucose metabolism is rather mediated by direct effects, such as activation of PPARgamma. Our data indicate that in insulin-resistant persons 12 weeks of telmisartan result in a significant improvement in glucose metabolism with a predominant improvement in beta-cell function.

Genetic variants of adiponectin receptor 2 are associated with increased adiponectin levels and decreased triglyceride/VLDL levels in patients with metabolic syndrome.

BACKGROUND: Adiponectin acts as an antidiabetic, antiinflammatory and antiatherogenic adipokine. These effects are assumed to be mediated by the recently discovered adiponectin receptors AdipoR1 and AdipoR2. AIM: The purpose of this study was to determine whether variations in the AdipoR1 and AdipoR2 genes may contribute to insulin resistance, dyslipidemia and inflammation. METHODS: We sequenced all seven coding exons of both genes in 20 unrelated German subjects with metabolic syndrome and tested genetic variants for association with glucose, lipid and inflammatory parameters. RESULTS: We identified three AdipoR2 variants (+795G/A, +870C/A and +963C/T) in perfect linkage disequilibrium (r2 = 1) with a minor allele frequency of 0.125. This haplotype was associated with higher plasma adiponectin levels and decreased fasting triglyceride, VLDL-triglyceride and VLDL-cholesterol levels. No association, however, was observed between the AdipoR2 SNP cluster and glucose metabolism. CONCLUSION: To our knowledge, this is the first study to identify an association between genetic variants of the adiponectin receptor genes and plasma adiponectin levels. Furthermore, our data suggest that AdipoR2 may play an important role in triglyceride/VLDL metabolism.