In order to quantify the role of incretins in first- and second-phase insulin secretion (ISR) in type 2 diabetes mellitus (T2DM), a double-blind, randomized study with 12 T2DM subjects and 12 healthy subjects (HS) was conducted using the hyperglycemic clamp technique together with duodenal nutrition perfusion and intravenous infusion of the glucagon-like peptide 1 (GLP-1) receptor antagonist exendin(9-39). Intravenous glucose alone resulted in a significantly greater first- and second-phase ISR in HS compared with T2DM subjects. Duodenal nutrition perfusion augmented both first- and second-phase ISR but first-phase ISR more in T2DM subjects (approximately eight- vs. twofold). Glucose-related stimulation of ISR contributed only 20% to overall ISR. Infusion with exendin(9-39) significantly reduced first- and second-phase ISR in both HS and T2DM subjects. Thus, both GLP-1 and non–GLP-1 incretins contribute to the incretin effect. In conclusion, both phases of ISR are impaired in T2DM. In particular, the responsiveness to glucose in first-phase ISR is blunted. GLP-1 and glucose-dependent insulinotropic polypeptide (GIP) secretions are unaltered. The absolute incretin effect is reduced in T2DM; its relative importance, however, appears to be increased, highlighting its role as an important amplifier of first-phase ISR in T2DM.
Diabetes is generally diagnosed too late. Therefore, biomarkers indicating early stages of β-cell dysfunction and mass reduction would facilitate timely counteraction. Transgenic pigs expressing a dominant-negative glucose-dependent insulinotropic polypeptide receptor (GIPRdn) reveal progressive deterioration of glucose control and reduction of β-cell mass, providing a unique opportunity to study metabolic changes during the prediabetic period. Plasma samples from intravenous glucose tolerance tests of 2.5- and 5-month-old GIPRdn transgenic and control animals were analyzed for 163 metabolites by targeted mass spectrometry. Analysis of variance revealed that 26 of 163 parameters were influenced by the interaction Genotype × Age (P ≤ 0.0001) and thus are potential markers for progression within the prediabetic state. Among them, the concentrations of seven amino acids (Phe, Orn, Val, xLeu, His, Arg, and Tyr) were increased in 2.5-month-old but decreased in 5-month-old GIPRdn transgenic pigs versus controls. Furthermore, specific sphingomyelins, diacylglycerols, and ether phospholipids were decreased in plasma of 5-month-old GIPRdn transgenic pigs. Alterations in plasma metabolite concentrations were associated with liver transcriptome changes in relevant pathways. The concentrations of a number of plasma amino acids and lipids correlated significantly with β-cell mass of 5-month-old pigs. These metabolites represent candidate biomarkers of early phases of β-cell dysfunction and mass reduction.
The G protein-coupled receptor 55 (GPR55) is a novel cannabinoid (CB) receptor, whose role in the gastrointestinal (GI) tract remains unknown. Here we studied the significance of GPR55 in the regulation of GI motility.
GPR55 mRNA and protein expression were measured by RT-PCR and immunohistochemistry. The effects of the GPR55 agonist O-1602 and a selective antagonist cannabidiol (CBD) were studied in vitro and in vivo and compared to a non-selective cannabinoid receptor agonist WIN55,212-2. CB1/2−/− and GPR55−/− mice were employed to identify the receptors involved.
GPR55 was localized on myenteric neurons in mouse and human colon. O-1602 concentration-dependently reduced evoked contractions in muscle strips from the colon (∼60%) and weakly (∼25%) from the ileum. These effects were reversed by CBD, but not by CB1 or CB2 receptor antagonists. I.p. and i.c.v. injections of O-1602 slowed whole gut transit and colonic bead expulsion; these effects were absent in GPR55−/− mice. WIN55,212-2 slowed whole gut transit effects, which were counteracted in the presence of a CB1 antagonist AM251. WIN55,212-2, but not O-1602 delayed gastric emptying and small intestinal transit. Locomotion, as a marker for central sedation, was reduced following WIN55,212-2, but not O-1602 treatment.
GPR55 is strongly expressed on myenteric neurons of the colon and it is selectively involved in the regulation of colonic motility. Since activation of GPR55 receptors is not associated with central sedation, the GPR55 receptor may serve as a future target for the treatment of colonic motility disorders.
•G protein-coupled receptor 55 (GPR55) is a binding site for cannabinoids.•No conclusive information was available on function of GPR55 in the GI tract.•We found that targeting GPR55 at peripheral or central sites slows GI motility.•Slowing effect of GPR55 activation on GI motility is primarily observed in colon.•Targeting GPR55 may be a future tool for treatment of colonic motility disorders.
Cannabidiol; Cannabinoid; Colon; Gastrointestinal motility; GPR55; CBD, cannabidiol; CB1, cannabinoid-1; CB2, cannabinoid-2; cDNA, complementary DNA; COX-2, cyclooxygenase-2; GE, gastric emptying; GPR55, G protein-coupled receptor 55; EFS, electrical field stimulation; ECS, endocannabinoid system; FAAH, fatty acid amide hydrolase; GI, gastrointestinal; i.c.v., intracerebroventricular administration; i.p., intraperitoneal; KRS, Krebs–Ringer solution; LMMP, longitudinal muscle-myenteric plexus layer; MAGL, monoacylglycerol lipase; PPARα, peroxisome proliferator-activated receptor-alpha; RT-PCR, reverse transcription polymerase chain reaction; TRPV1, transient receptor potential vanilloid 1
Obesity is associated with abnormal fasting and postprandial lipids, which may link obesity with atherosclerosis. We explored fasting and postprandial lipids in morbidly obese patients treated with sleeve gastrectomy and in control subjects.
After fasting for 12 h 15 morbidly obese patients (BMI 51.4±6.5 kg/m2, 43.7±12.6 years) received a standardized oral fat load before and 3 months after bariatric surgery (sleeve gastrectomy). Controls (n=9, BMI 23.1±1.4 kg/m2) were studied once. Plasma was obtained fasting and then postprandially every 2 h for 8 h. Triglycerides (TG), chylomicron-TG (CM-TG), VLDL/chylomicron-remnant (VLDL/CR)-TG, cholesterol, LDL-cholesterol, VLDL/CR-cholesterol and HDL-cholesterol were isolated by ultracentrifugation at each time point. Postprandial values were expressed as area under the curve (AUC) and incremental area under the curve (iAUC). In addition, fasting glucose and insulin values and HOMA-IR-Index was measured (n=14).
Compared to controls morbidly obese patients had elevated TG and slightly altered postprandial lipids. Following surgery (weight loss 23.4 kg±6.2 kg; p<0.001) fasting TG (−19.1%; p=0.04), VLDL/CR-TG (−20.0%; p=0.05) decreased significantly, while fasting cholesterol, VLDL-, HDL- and LDL-cholesterol did not change. AUC and iAUC decreased significantly for VLDL/CR-TG (−20.4%, p=0.04 and −38.5%, p=0.04, respectively). Neither fasting nor postprandial changes correlated with the change in weight. In patients with preoperatively elevated TG (>150 mg/dl) a similar pattern was observed. Fasting insulin and HOMA were reduced significantly (−51.9%; p=0.004 and −47.9%; p=0.011).
Three months after sleeve gastrectomy fasting and postprandial lipoprotein metabolism and glucose metabolism is improved in morbidly obese patients. The potential mechanisms may relate to decreased caloric intake but also to hormonal changes.
Postprandial lipids; Chylomicron; Chylomicron remnant; Dyslipidemia; VLDL-triglycerides
Agonistic antibodies targeting TRAIL-receptors 1 and 2 (TRAIL-R1 and TRAIL-R2) are being developed as a novel therapeutic approach in cancer therapy including pancreatic cancer. However, the cellular distribution of these receptors in primary pancreatic cancer samples has not been sufficiently investigated and no study has yet addressed the issue of their prognostic significance in this tumor entity.
Aims and Methods
Applying tissue microarray (TMA) analysis, we performed an immunohistochemical assessment of TRAIL-receptors in surgical samples from 84 consecutive patients affected by pancreatic adenocarcinoma and in 26 additional selected specimens from patients with no lymph nodes metastasis at the time of surgery. The prognostic significance of membrane staining and staining intensity for TRAIL-receptors was evaluated.
The fraction of pancreatic cancer samples with positive membrane staining for TRAIL-R1 and TRAIL-R2 was lower than that of cells from surrounding non-tumor tissues (TRAIL-R1: p<0.001, TRAIL-R2: p = 0.006). In addition, subgroup analyses showed that loss of membrane staining for TRAIL-R2 was associated with poorer prognosis in patients without nodal metastases (multivariate Cox regression analysis, Hazard Ratio: 0.44 [95% confidence interval: 0.22−0.87]; p = 0.019). In contrast, analysis of decoy receptors TRAIL-R3 and -R4 in tumor samples showed an exclusively cytoplasmatic staining pattern and no prognostic relevance.
This is a first report on the prognostic significance of TRAIL-receptors expression in pancreatic cancer showing that TRAIL-R2 might represent a prognostic marker for patients with early stage disease. In addition, our data suggest that loss of membrane-bound TRAIL-receptors could represent a molecular mechanism for therapeutic failure upon administration of TRAIL-receptors-targeting antibodies in pancreatic cancer. This hypothesis should be evaluated in future clinical trials.
Background & Aims
Genome-wide association studies identified the autophagy gene IRGM to be strongly associated with Crohn's disease (CD) but its impact in ulcerative colitis (UC), its phenotypic effects and potential epistatic interactions with other IBD susceptibility genes are less clear which we therefore analyzed in this study.
Genomic DNA from 2060 individuals including 817 CD patients, 283 UC patients, and 961 healthy, unrelated controls (all of Caucasian origin) was analyzed for six IRGM single nucleotide polymorphisms (SNPs) (rs13371189, rs10065172 = p.Leu105Leu, rs4958847, rs1000113, rs11747270, rs931058). In all patients, a detailed genotype-phenotype analysis and testing for epistasis with the three major CD susceptibility genes NOD2, IL23R and ATG16L1 were performed.
Our analysis revealed an association of the IRGM SNPs rs13371189 (p = 0.02, OR 1.31 [95% CI 1.05–1.65]), rs10065172 = p.Leu105Leu (p = 0.016, OR 1.33 [95% CI 1.06–1.66]) and rs1000113 (p = 0.047, OR 1.27 [95% CI 1.01–1.61]) with CD susceptibility. There was linkage disequilibrium between these three IRGM SNPs. In UC, several IRGM haplotypes were weakly associated with UC susceptibility (p<0.05). Genotype-phenotype analysis revealed no significant associations with a specific IBD phenotype or ileal CD involvement. There was evidence for weak gene-gene-interaction between several SNPs of the autophagy genes IRGM and ATG16L1 (p<0.05), which, however, did not remain significant after Bonferroni correction.
Our results confirm IRGM as susceptibility gene for CD in the German population, supporting a role for the autophagy genes IRGM and ATG16L1 in the pathogenesis of CD.
The fact that the receptors for the TNF-related apoptosis inducing ligand (TRAIL) are almost invariably expressed in colorectal cancer (CRC) represents the rationale for the employment of TRAIL-receptors targeting compounds for the therapy of patients affected by this tumor. Yet, first reports on the use of these bioactive agents provided disappointing results. We therefore hypothesized that loss of membrane-bound TRAIL-R might be a feature of some CRC and that the evaluation of membrane staining rather than that of the overall expression of TRAIL-R might predict the response to TRAIL-R targeting compounds in this tumor.
Aim and Methods
Thus, we evaluated the immunofluorescence pattern of TRAIL-receptors and E-cadherin to assess the fraction of membrane-bound TRAIL-receptors in 231 selected patients with early-stage CRC undergoing surgical treatment only. Moreover, we investigated whether membrane staining for TRAIL-receptors as well as the presence of KRAS mutations or of microsatellite instability (MSI) had an effect on survival and thus a prognostic effect.
As expected, almost all CRC samples stained positive for TRAIL-R1 and 2. Instead, membrane staining for these receptors was positive in only 71% and 16% of samples respectively. No correlation between KRAS mutation status or MSI-phenotype and prognosis could be detected. TRAIL-R1 staining intensity correlated with survival in univariate analysis, but only membranous staining of TRAIL-R1 and TRAIL-R2 on cell membranes was an independent predictor of survival (cox multivariate analysis: TRAIL-R1: p = 0.019, RR 2.06[1.12–3.77]; TRAIL-R2: p = 0.033, RR 3.63[1.11–11.84]).
In contrast to the current assumptions, loss of membrane staining for TRAIL-receptors is a common feature of early stage CRC which supersedes the prognostic significance of their staining intensity. Failure to achieve therapeutic effects in recent clinical trials using TRAIL-receptors targeting compounds might be due to insufficient selection of patients bearing tumors with membrane-bound TRAIL-receptors.
We reported the induction of tumor-selective iodide uptake and therapeutic efficacy of 131I in a hepatocellular carcinoma (HCC) xenograft mouse model, using novel polyplexes based on linear polyethylenimine (LPEI), shielded by polyethylene glycol (PEG), and coupled with the epidermal growth factor receptor-specific peptide GE11 (LPEI-PEG-GE11). The aim of the current study in the same HCC model was to evaluate the potential of biodegradable nanoparticle vectors based on pseudodendritic oligoamines (G2-HD-OEI) for systemic sodium iodide symporter (NIS) gene delivery and to compare efficiency and tumor specificity with LPEI-PEG-GE11. Transfection of HCC cells with NIS cDNA, using G2-HD-OEI, resulted in a 44-fold increase in iodide uptake in vitro as compared with a 22-fold increase using LPEI-PEG-GE11. After intravenous application of G2-HD-OEI/NIS HCC tumors accumulated 6–11% ID/g 123I (percentage of the injected dose per gram tumor tissue) with an effective half-life of 10 hr (tumor-absorbed dose, 281 mGy/MBq) as measured by 123I scintigraphic gamma camera or single-photon emission computed tomography computed tomography (SPECT CT) imaging, as compared with 6.5–9% ID/g with an effective half-life of only 6 hr (tumor-absorbed dose, 47 mGy/MBq) for LPEI-PEG-GE11. After only two cycles of G2-HD-OEI/NIS/131I application, a significant delay in tumor growth was observed with markedly improved survival. A similar degree of therapeutic efficacy had been observed after four cycles of LPEI-PEG-GE11/131I. These results clearly demonstrate that biodegradable nanoparticles based on OEI-grafted oligoamines show increased efficiency for systemic NIS gene transfer in an HCC model with similar tumor selectivity as compared with LPEI-PEG-GE11, and therefore represent a promising strategy for NIS-mediated radioiodine therapy of HCC.
Klutz and colleagues evaluate the therapeutic efficacy of biodegradable nanoparticle vectors based on pseudodendritic oligoamines (G2-HD-OEI) for systemic sodium iodide symporter gene delivery in a xenograft mouse model of hepatocellular carcinoma. This approach led to a significant delay in tumor growth and marked improvement in survival.
We reported the therapeutic efficacy of 131I in hepatocellular carcinoma (HCC) cells stably expressing the sodium iodide symporter (NIS) under the control of the tumor-specific α-fetoprotein (AFP) promoter. In the current study we investigated the efficacy of adenovirus-mediated in vivo NIS gene transfer followed by 131I and 188Re administration for the treatment of HCC xenografts. We used a replication-deficient adenovirus carrying the human NIS gene linked to the mouse AFP promoter (Ad5-AFP-NIS) for in vitro and in vivo NIS gene transfer. Functional NIS expression was confirmed by in vivo γ-camera imaging, followed by analysis of NIS protein and mRNA expression. Human HCC (HepG2) cells infected with Ad5-AFP-NIS concentrated 50% of the applied activity of 125I, which was sufficiently high for a therapeutic effect in an in vitro clonogenic assay. Four days after intratumoral injection of Ad5-AFP-NIS (3×109 plaque-forming units) HepG2 xenografts accumulated 14.5% injected dose (ID)/g 123I with an effective half-life of 13 hr (tumor-absorbed dose, 318 mGy/MBq 131I). In comparison, 9.2% ID/g 188Re was accumulated in tumors with an effective half-life of 12.8 hr (tumor-absorbed dose, 545 mGy/MBq). After adenovirus-mediated NIS gene transfer in HepG2 xenografts administration of a therapeutic dose of 131I or 188Re (55.5 MBq) resulted in a significant delay in tumor growth and improved survival without a significant difference between 188Re and 131I. In conclusion, a therapeutic effect of 131I and 188Re was demonstrated in HepG2 xenografts after tumor-specific adenovirus-mediated in vivo NIS gene transfer.
Targeted gene transfer of sodium iodide symporter (NIS) into tumors followed by radionuclide administration is a promising therapeutic approach. In this report, Klutz and colleagues investigate the in vivo efficacy of transcriptionally targeted adenovirus vector-mediated NIS gene transfer followed by 131I and 188Re administration for the treatment of hepatocellular carcinoma (HCC) xenografts. The authors report that significant delays in tumor growth and improved survival were seen in the HCC-xenograft mouse model that received this treatment course.
HCC is diagnosed in approximately half a million people per year, worldwide. Staging is a more complex issue than in most other cancer entities and, mainly due to unique geographic characteristics of the disease, no universally accepted staging system exists to date. Focusing on survival rates we analyzed demographic, etiological, clinical, laboratory and tumor characteristics of HCC-patients in our institution and applied the common staging systems. Furthermore we aimed at identifying the most suitable of the current staging systems for predicting survival.
Overall, 405 patients with HCC were identified from an electronic medical record database. The following seven staging systems were applied and ranked according to their ability to predict survival by using the Akaike information criterion (AIC) and the concordance-index (c-index): BCLC, CLIP, GETCH, JIS, Okuda, TNM and Child-Pugh. Separately, every single variable of each staging system was tested for prognostic meaning in uni- and multivariate analysis. Alcoholic cirrhosis (44.4%) was the leading etiological factor followed by viral hepatitis C (18.8%). Median survival was 18.1 months (95%-CI: 15.2–22.2). Ascites, bilirubin, alkaline phosphatase, AFP, number of tumor nodes and the BCLC tumor extension remained independent prognostic factors in multivariate analysis. Overall, all of the tested staging systems showed a reasonable discriminatory ability. CLIP (closely followed by JIS) was the top-ranked score in terms of prognostic capability with the best values of the AIC and c-index (AIC 2286, c-index 0.71), surpassing other established staging systems like BCLC (AIC 2343, c-index 0.66). The unidimensional scores TNM (AIC 2342, c-index 0.64) and Child-Pugh (AIC 2369, c-index 0.63) performed in an inferior fashion.
Compared with six other staging systems, the CLIP-score was identified as the most suitable staging system for predicting prognosis in a large German cohort of predominantly non-surgical HCC-patients.
IL12B encodes the p40 subunit of IL-12, which is also part of IL-23. Recent genome-wide association studies identified IL12B and IL23R as susceptibility genes for inflammatory bowel disease (IBD). However, the phenotypic effects and potential gene-gene interactions of IL12B variants are largely unknown.
We analyzed IL12B gene variants regarding association with Crohn's disease (CD) and ulcerative colitis (UC). Genomic DNA from 2196 individuals including 913 CD patients, 318 UC patients and 965 healthy, unrelated controls was analyzed for four SNPs in the IL12B gene region (rs3212227, rs17860508, rs10045431, rs6887695). Our analysis revealed an association of the IL12B SNP rs6887695 with susceptibility to IBD (p = 0.035; OR 1.15 [95% CI 1.01–1.31] including a trend for rs6887695 for association with CD (OR 1.41; [0.99–1.31], p = 0.066) and UC (OR 1.18 [0.97–1.43], p = 0.092). CD patients, who were homozygous C/C carriers of this SNP, had significantly more often non-stricturing, non-penetrating disease than carriers of the G allele (p = 6.8×10−5; OR = 2.84, 95% CI 1.66–4.84), while C/C homozygous UC patients had less often extensive colitis than G allele carriers (p = 0.029; OR = 0.36, 95% CI 0.14–0.92). In silico analysis predicted stronger binding of the minor C allele of rs6887695 to the transcription factor RORα which is involved in Th17 differentiation. Differences regarding the binding to the major and minor allele sequence of rs6887695 were also predicted for the transcription factors HSF1, HSF2, MZF1 and Oct-1. Epistasis analysis revealed weak epistasis of the IL12B SNP rs6887695 with several SNPs (rs11889341, rs7574865, rs7568275, rs8179673, rs10181656, rs7582694) in the STAT4 gene which encodes the major IL-12 downstream transcription factor STAT4 (p<0.05) but there was no epistasis between IL23R and IL12B variants.
The IL12B SNP rs6887695 modulates the susceptibility and the phenotype of IBD, although the effect on IBD susceptibilty is less pronounced than that of IL23R gene variants.
Genome-wide association studies identified PTPN2 (protein tyrosine phosphatase, non-receptor type 2) as susceptibility gene for inflammatory bowel diseases (IBD). However, the exact role of PTPN2 in Crohn's disease (CD) and ulcerative colitis (UC) and its phenotypic effect are unclear. We therefore performed a detailed genotype-phenotype and epistasis analysis of PTPN2 gene variants.
Genomic DNA from 2131 individuals of Caucasian origin (905 patients with CD, 318 patients with UC, and 908 healthy, unrelated controls) was analyzed for two SNPs in the PTPN2 region (rs2542151, rs7234029) for which associations with IBD were found in previous studies in other cohorts. Our analysis revealed a significant association of PTPN2 SNP rs2542151 with both susceptibility to CD (p = 1.95×10−5; OR 1.49 [1.34–1.79]) and UC (p = 3.87×10−2, OR 1.31 [1.02–1.68]). Moreover, PTPN2 SNP rs7234029 demonstrated a significant association with susceptibility to CD (p = 1.30×10−3; OR 1.35 [1.13–1.62]) and a trend towards association with UC (p = 7.53×10−2; OR 1.26 [0.98–1.62]). Genotype-phenotype analysis revealed an association of PTPN2 SNP rs7234029 with a stricturing disease phenotype (B2) in CD patients (p = 6.62×10−3). Epistasis analysis showed weak epistasis between the ATG16L1 SNP rs2241879 and PTPN2 SNP rs2542151 (p = 0.024) in CD and between ATG16L1 SNP rs4663396 and PTPN2 SNP rs7234029 (p = 4.68×10−3) in UC. There was no evidence of epistasis between PTPN2 and NOD2 and PTPN2 and IL23R. In silico analysis revealed that the SNP rs7234029 modulates potentially the binding sites of several transcription factors involved in inflammation including GATA-3, NF-κB, C/EBP, and E4BP4.
Our data confirm the association of PTPN2 variants with susceptibility to both CD and UC, suggesting a common disease pathomechanism for these diseases. Given recent evidence that PTPN2 regulates autophagosome formation in intestinal epithelial cells, the potential link between PTPN2 and ATG16L1 should be further investigated.
The aim of this study was to assess the clinical sensitivities of the tumor markers chromogranin A (CgA), urinary 5-hydroxyindoleacetic acid (5-HIAA) and alkaline phosphatase (AP) in neuroendocrine tumors (NETs) of the GastroEnteroPancreatic-(GEP-) system depending on tumor primary location and metastatic spread. In a retrospective single-center series, sensitivities were evaluated in serum samples from 110 patients with midgut (n = 62) and pancreatic (n = 48) NETs. CgA levels were analyzed by a commercially-available immunoradiometric assay (CIS-bio) during routine follow-up in the years 2000–2009. CgA showed a higher sensitivity for midgut (68%) than pancreatic (54%) NETs. A higher CgA sensitivity and significantly higher median CgA values were found in patients with liver metastases than in those without, and in patients with hepatic and additionally extra-hepatic metastases than in those with hepatic and nodal metastases alone, respectively. We found an overall sensitivity for elevated 5HIAA excretion of 69% for midgut NETs and a significant correlation between median CgA and 5-HIAA values. The sensitivity of AP and the correlations of AP/CgA-data-pairs were low in both midgut and pancreatic NETs, although highest for metastatic pancreatic NETs. The sensitivity of CgA measurement depends on the NET primary location and spread of disease. 5-HIAA and CgA showed comparable sensitivity in midgut NETs, while AP does not seem to be useful as a tumor marker in GEP-NETs.
neuroendocrine tumors; gastroenteropancreatic system; sensitivity; chromogranin A; CIS-bio assay; alkaline phosphatase; urinary 5-hydroxyindoleacetic acid; liver metastases
Chemerin is a recently discovered hepatoadipokine that regulates adipocyte differentiation as well as chemotaxis and activation of dendritic cells and macrophages. Chemerin was reported to modulate insulin sensitivity in adipocytes and skeletal muscle cells in vitro and to exacerbate glucose intolerance in several mouse models in vivo. In humans, chemerin was shown to be associated with multiple components of the metabolic syndrome including BMI, triglycerides, HDL cholesterol, and hypertension. This study aimed to examine the effect of chemerin on weight, glucose and lipid metabolism, as well as atherosclerosis in vivo.
RESEARCH DESIGN AND METHODS
We used recombinant adeno-associated virus to express human chemerin in LDL receptor knockout mice on high-fat diet.
Expression of chemerin did not significantly alter weight, lipid levels, and extent of atherosclerosis. Chemerin, however, significantly increased glucose levels during the intraperitoneal glucose tolerance test without affecting endogenous insulin levels and the insulin tolerance test. Chemerin reduced insulin-stimulated Akt1 phosphorylation and activation of 5′AMP-activated protein kinase (AMPK) in the skeletal muscle, but had no effect on Akt phosphorylation and insulin-stimulated AMPK activation in the liver and gonadal adipose tissue.
Chemerin induces insulin resistance in the skeletal muscle in vivo. Chemerin is involved in the cross talk between liver, adipose tissue, and skeletal muscle.
CXCL16 mediates adhesion and phagocytosis of both Gram-negative and Gram-positive bacteria and is a strong chemoattractant for CXCR6+ T cells. In this study, we determined the so far unknown expression and signal transduction of the novel CXCL16-CXCR6 chemokine-ligand receptor system in intestinal inflammation in vivo and in vitro.
CXCL16 mRNA was measured by quantitative PCR in human colonic biopsies of patients with Crohn’s disease (CD) as well as in the TNFΔARE mouse model of ileitis and in murine cytomegalovirus (MCMV)-induced colitis. CXCL16 serum levels were analyzed by ELISA. CXCL16-induced signal transduction was analyzed in IEC with phospho-specific antibodies for MAP kinases and Akt.
We found an inverse expression pattern of CXCL16 and CXCR6 with highest CXCL16 mRNA levels in the proximal murine small intestine and highest CXCR6 mRNA expression in the distal colon. CXCL16 and CXCR6 mRNA were expressed in colorectal cancer (CRC)-derived IEC lines. CRC-expressed CXCR6 was functional as demonstrated by CXCL16-induced MAP kinase and Akt activation. Intestinal CXCL16 expression was elevated in the TNFΔARE mouse model of ileitis and in MCMV-induced colitis (p<0.05) and in the sera and colons of patients with CD (p<0.05), where its expression correlated highly with CXCR6 and IL-8 levels (r=0.85 and 0.89, respectively).
CRC-derived IEC express the functional CXCL16 receptor CXCR6. CXCL16 mRNA and protein expression is up-regulated in intestinal inflammation in vitro and in CD patients, suggesting an important role for this chemokine in intestinal inflammation.
Crohn’s disease; ulcerative colitis; inflammatory bowel disease; chemokine; CXCL16; CXCR6; mouse model; intestinal inflammation; intestinal epithelial cells; cytokine; signaling
The aim of this study was to examine postnatal islet and beta-cell expansion in healthy female control mice and its disturbances in diabetic GIPRdn transgenic mice, which exhibit an early reduction of beta-cell mass. Pancreata of female control and GIPRdn transgenic mice, aged 10, 45, 90 and 180 days were examined, using state-of-the-art quantitative-stereological methods. Total islet and beta-cell volumes, as well as their absolute numbers increased significantly until 90 days in control mice, and remained stable thereafter. The mean islet volumes of controls also increased slightly but significantly between 10 and 45 days of age, and then remained stable until 180 days. The total volume of isolated beta-cells, an indicator of islet neogenesis, and the number of proliferating (BrdU-positive) islet cells were highest in 10-day-old controls and declined significantly between 10 and 45 days. In GIPRdn transgenic mice, the numbers of islets and beta-cells were significantly reduced from 10 days of age onwards vs. controls, and no postnatal expansion of total islet and beta-cell volumes occurred due to a reduction in islet neogenesis whereas early islet-cell proliferation and apoptosis were unchanged as compared to control mice. Insulin secretion in response to pharmacological doses of GIP was preserved in GIPRdn transgenic mice, and serum insulin to pancreatic insulin content in response to GLP-1 and arginine was significantly higher in GIPRdn transgenic mice vs. controls. We could show that the increase in islet number is mainly responsible for expansion of islet and beta-cell mass in healthy control mice. GIPRdn transgenic mice show a disturbed expansion of the endocrine pancreas, due to perturbed islet neogenesis.
The members of the Suppressor of Cytokine Signaling (SOCS) protein family mainly modulate the Janus kinase (JAK)/signal transducer and activator of transcription (STAT) pathway. SOCS-1 and SOCS-3 have already been shown to influence growth and apoptosis of pancreatic β-cells. We hypothesized that SOCS-2, which is expressed in pancreatic islets, also contributes to β-cell physiology. We tested this hypothesis in vivo in SOCS-2−/− knockout mice and in vitro in Ins-1E rat insulinoma cells. We found that SOCS-2−/− mice have normal islet insulin secretion and unchanged glucose and insulin tolerance compared to wildtype controls. SOCS-2−/− are bigger than wildtype mice but body weight-corrected β-cell mass and islet morphology were normal. Growth hormone-induced proliferation of Ins-1E cells was not affected by either siRNA-mediated SOCS-2 knockdown or stable SOCS-2 overexpression. Interleukin-1β mediated cell death in vitro was unchanged after SOCS-2 knockdown. Similarly, autoimmune destruction of β-cells in vivo after multiple low-dose injections of streptozotocin (STZ) was not altered in SOCS-2−/− mice.
In summary, SOCS-2−/− knockout mice have a normal function of insulin-producing pancreatic β-cells, a fully adapted β-cell mass and a normal morphology of the endocrine islets. Based on in vitro evidence, the increased β-cell mass in the mutants is likely due to indirect adaptive mechanisms and not the result of altered growth hormone signaling within the β-cells. Immune mediated β-cell destruction is also not affected by SOCS-2 ablation in vitro and in vivo.
SOCS1; SOCS2; SOCS3; C57Bl/6; α-cell; δ-cell; glucagon; somatostatin; pancreatic polypeptide
Colorectal cancer (CRC) is one of the leading causes of cancer related morbidity and death. Despite the fact that the mean age at diagnosis of CRC is lower in men, screening by colonoscopy or fecal occult blood test (FOBT) is initiated at same age in both genders. The prevalence of the common CRC precursor lesion, advanced adenoma, is well documented only in the screening population. The purpose of this study was to assess the risk of advanced adenoma at ages below screening age.
Methods and Findings
We analyzed data from a census of 625,918 outpatient colonoscopies performed in adults in Bavaria between 2006 and 2008. A logistic regression model to determine gender- and age-specific risk of advanced neoplasia was developed. Advanced neoplasia was found in 16,740 women (4.6%) and 22,684 men (8.6%). Male sex was associated with an overall increased risk of advanced neoplasia (odds ratio 1.95; 95% confidence interval, CI, 1.91 to 2.00). At any age and in any indication group, more colonoscopies were needed in women than in men to detect advanced adenoma or cancer. At age 75 14.8 (95% CI, 14.4–15.2) screening, 18.2 (95% CI, 17.7–18.7) diagnostic, and 7.9 (95% CI, 7.6–8.2) colonoscopies to follow up on a positive FOBT (FOBT colonoscopies) were needed to find advanced adenoma in women. At age 50 39.0 (95% CI, 38.0–40.0) diagnostic, and 16.3 (95% CI, 15.7–16.9) FOBT colonoscopies were needed. Comparable numbers were reached 20 and 10 years earlier in men than in women, respectively.
At any age and independent of the indication for colonoscopy, men are at higher risk of having advanced neoplasia diagnosed upon colonoscopy than women. This suggests that starting screening earlier in life in men than in women might result in a relevant increase in the detection of asymptomatic preneoplastic and neoplastic colonic lesions.
The insulinotropic action of the incretin glucose-dependent insulinotropic polypeptide (GIP) is impaired in type 2 diabetes, while the effect of glucagon-like peptide-1 (GLP-1) is preserved. To evaluate the role of impaired GIP function in glucose homeostasis and development of the endocrine pancreas in a large animal model, we generated transgenic pigs expressing a dominant-negative GIP receptor (GIPRdn) in pancreatic islets.
RESEARCH DESIGN AND METHODS
GIPRdn transgenic pigs were generated using lentiviral transgenesis. Metabolic tests and quantitative stereological analyses of the different endocrine islet cell populations were performed, and β-cell proliferation and apoptosis were quantified to characterize this novel animal model.
Eleven-week-old GIPRdn transgenic pigs exhibited significantly reduced oral glucose tolerance due to delayed insulin secretion, whereas intravenous glucose tolerance and pancreatic β-cell mass were not different from controls. The insulinotropic effect of GIP was significantly reduced, whereas insulin secretion in response to the GLP-1 receptor agonist exendin-4 was enhanced in GIPRdn transgenic versus control pigs. With increasing age, glucose control deteriorated in GIPRdn transgenic pigs, as shown by reduced oral and intravenous glucose tolerance due to impaired insulin secretion. Importantly, β-cell proliferation was reduced by 60% in 11-week-old GIPRdn transgenic pigs, leading to a reduction of β-cell mass by 35% and 58% in 5-month-old and 1- to 1.4-year-old transgenic pigs compared with age-matched controls, respectively.
The first large animal model with impaired incretin function demonstrates an essential role of GIP for insulin secretion, proliferation of β-cells, and physiological expansion of β-cell mass.
The carcinoembryonic antigen-related cell adhesion molecule 6 (CEACAM6) acts as a receptor for adherent-invasive E. coli (AIEC) and its ileal expression is increased in patients with Crohn's disease (CD). Given its contribution to the pathogenesis of CD, we aimed to investigate the role of genetic variants in the CEACAM6 region in patients with inflammatory bowel diseases (IBD).
In this study, a total of 2,683 genomic DNA samples (including DNA from 858 CD patients, 475 patients with ulcerative colitis (UC), and 1,350 healthy, unrelated controls) was analyzed for eight CEACAM6 SNPs (rs10415946, rs1805223 = p.Pro42Pro, rs4803507, rs4803508, rs11548735 = p.Gly239Val, rs7246116 = pHis260His, rs2701, rs10416839). In addition, a detailed haplotype analysis and genotype-phenotype analysis were performed. Overall, our genotype analysis did not reveal any significant association of the investigated CEACAM6 SNPs and haplotypes with CD or UC susceptibility, although certain CEACAM6 SNPs modulated CEACAM6 expression in intestinal epithelial cell lines. Despite its function as receptor of AIEC in ileal CD, we found no association of the CEACAM6 SNPs with ileal or ileocolonic CD. Moreover, there was no evidence of epistasis between the analyzed CEACAM6 variants and the main CD-associated NOD2, IL23R and ATG16L1 variants.
This study represents the first detailed analysis of CEACAM6 variants in IBD patients. Despite its important role in bacterial attachment in ileal CD, we could not demonstrate a role for CEACAM6 variants in IBD susceptibility or regarding an ileal CD phenotype. Further functional studies are required to analyze if these gene variants modulate ileal bacterial attachment.
The aims were to analyze two novel NOD2 variants (rs2066843 and rs2076756) in a large cohort of patients with inflammatory bowel disease and to elucidate phenotypic consequences.
Genomic DNA from 2700 Caucasians including 812 patients with Crohn's disease (CD), 442 patients with ulcerative colitis (UC), and 1446 healthy controls was analyzed for the NOD2 SNPs rs2066843 and rs2076756 and the three main CD-associated NOD2 variants p.Arg702Trp (rs2066844), p.Gly908Arg (rs2066847), and p.Leu1007fsX1008 (rs2066847). Haplotype and genotype-phenotype analyses were performed. The SNPs rs2066843 (p = 3.01×10−5, OR 1.48, [95% CI 1.23-1.78]) and rs2076756 (p = 4.01×10−6; OR 1.54, [95% CI 1.28-1.86]) were significantly associated with CD but not with UC susceptibility. Haplotype analysis revealed a number of significant associations with CD susceptibility with omnibus p values <10−10. The SNPs rs2066843 and rs2076756 were in linkage disequilibrium with each other and with the three main CD-associated NOD2 mutations (D'>0.9). However, in CD, SNPs rs2066843 and rs2076756 were more frequently observed than the other three common NOD2 mutations (minor allele frequencies for rs2066843 and rs2076756: 0.390 and 0.380, respectively). In CD patients homozygous for these novel NOD2 variants, genotype-phenotype analysis revealed higher rates of a penetrating phenotype (rs2076756: p = 0.015) and fistulas (rs2076756: p = 0.015) and significant associations with CD-related surgery (rs2076756: p = 0.003; rs2066843: p = 0.015). However, in multivariate analysis only disease localization (p<2×10−16) and behaviour (p = 0.02) were significantly associated with the need for surgery.
The NOD2 variants rs2066843 and rs2076756 are novel and common CD susceptibility gene variants.
Specific differences in signaling and antiviral properties between the different Lambda-interferons, a novel group of interferons composed of IL-28A, IL-28B and IL-29, are currently unknown. This is the first study comparatively investigating the transcriptome and the antiviral properties of the Lambda-interferons IL-28A and IL-29.
Expression studies were performed by microarray analysis, quantitative PCR (qPCR), reporter gene assays and immunoluminometric assays. Signaling was analyzed by Western blot. HCV replication was measured in Huh-7 cells expressing subgenomic HCV replicon. All hepatic cell lines investigated as well as primary hepatocytes expressed both IFN-λ receptor subunits IL-10R2 and IFN-λR1. Both, IL-28A and IL-29 activated STAT1 signaling. As revealed by microarray analysis, similar genes were induced by both cytokines in Huh-7 cells (IL-28A: 117 genes; IL-29: 111 genes), many of them playing a role in antiviral immunity. However, only IL-28A was able to significantly down-regulate gene expression (n = 272 down-regulated genes). Both cytokines significantly decreased HCV replication in Huh-7 cells. In comparison to liver biopsies of patients with non-viral liver disease, liver biopsies of patients with HCV showed significantly increased mRNA expression of IL-28A and IL-29. Moreover, IL-28A serum protein levels were elevated in HCV patients. In a murine model of viral hepatitis, IL-28 expression was significantly increased.
IL-28A and IL-29 are up-regulated in HCV patients and are similarly effective in inducing antiviral genes and inhibiting HCV replication. In contrast to IL-29, IL-28A is a potent gene repressor. Both IFN-λs may have therapeutic potential in the treatment of chronic HCV.
Interleukin 31 (IL31), primarily expressed in activated lymphocytes, signals through a heterodimeric receptor complex consisting of the IL31 receptor alpha (IL31Rα) and the oncostatin M receptor (OSMR). The aim of this study was to analyse IL31 receptor expression, signal transduction, and specific biological functions of this cytokine system in intestinal inflammation.
Expression studies were performed by RT‐PCR, quantitative PCR, western blotting, and immunohistochemistry. Signal transduction was analysed by western blotting. Cell proliferation was measured by MTS assays, cell migration by restitution assays.
Colorectal cancer derived intestinal epithelial cell (IEC) lines express both IL31 receptor subunits, while their expression in unstimulated primary murine IEC was low. LPS and the proinflammatory cytokines TNF‐α, IL1β, IFN‐γ, and sodium butyrate stimulation increased IL31, IL31Rα, and OSMR mRNA expression, while IL31 itself enhanced IL8 expression in IEC. IL31 mediates ERK‐1/2, Akt, STAT1, and STAT3 activation in IEC resulting in enhanced IEC migration. However, at low cell density, IL31 had significant antiproliferative capacities (p<0.005). IL31 mRNA expression was not increased in the TNFΔARE mouse model of ileitis but in inflamed colonic lesions compared to non‐inflamed tissue in patients with Crohn's disease (CD; average 2.4‐fold increase) and in patients with ulcerative colitis (UC; average 2.6‐fold increase) and correlated with the IL‐8 expression in these lesions (r = 0.564 for CD; r = 0.650 for UC; total number of biopsies analysed: n = 88).
IEC express the functional IL31 receptor complex. IL31 modulates cell proliferation and migration suggesting a role in the regulation of intestinal barrier function particularly in intestinal inflammation.
interleukin; Crohn's disease; ulcerative colitis; inflammatory bowel disease; intestinal epithelial cell
Inactivation of the Fanconi anemia (FA) pathway through defects in one of 13 FA genes occurs at low frequency in various solid cancer entities among the general population. As FA pathway inactivation confers a distinct hypersensitivity towards DNA interstrand-crosslinking (ICL)-agents, FA defects represent rational targets for individualized therapeutic strategies. Except for pancreatic cancer, however, the prevalence of FA defects in gastrointestinal (GI) tumors has not yet been systematically explored.
A panel of GI cancer cell lines was screened for FA pathway inactivation applying FANCD2 monoubiquitination and FANCD2/RAD51 nuclear focus formation and a newly identified FA pathway-deficient cell line was functionally characterized. The hepatocellular carcinoma (HCC) line HuH-7 was defective in FANCD2 monoubiquitination and FANCD2 nuclear focus formation but proficient in RAD51 focus formation. Gene complementation studies revealed that this proximal FA pathway inactivation was attributable to defective FANCC function in HuH-7 cells. Accordingly, a homozygous inactivating FANCC nonsense mutation (c.553C > T, p.R185X) was identified in HuH-7, resulting in partial transcriptional skipping of exon 6 and leading to the classic cellular FA hypersensitivity phenotype; HuH-7 cells exhibited a strongly reduced proliferation rate and a pronounced G2 cell cycle arrest at distinctly lower concentrations of ICL-agents than a panel of non-isogenic, FA pathway-proficient HCC cell lines. Upon retroviral transduction of HuH-7 cells with FANCC cDNA, FA pathway functions were restored and ICL-hypersensitivity abrogated. Analyses of 18 surgical HCC specimens yielded no further examples for genetic or epigenetic inactivation of FANCC, FANCF, or FANCG in HCC, suggesting a low prevalence of proximal FA pathway inactivation in this tumor type.
As the majority of HCC are chemoresistant, assessment of FA pathway function in HCC could identify small subpopulations of patients expected to predictably benefit from individualized treatment protocols using ICL-agents.
Recent studies demonstrated an association of STAT4 variants with systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA), indicating that multiple autoimmune diseases share common susceptibility genes. We therefore investigated the influence of STAT4 variants on the susceptibility and phenotype of inflammatory bowel diseases (IBD) in a large patient and control cohort.
Genomic DNA from 2704 individuals of Caucasian origin including 857 patients with Crohn's disease (CD), 464 patients with ulcerative colitis (UC), and 1383 healthy, unrelated controls was analyzed for seven SNPs in the STAT4 gene (rs11889341, rs7574865, rs7568275, rs8179673, rs10181656, rs7582694, rs10174238). In addition, a detailed genotype-phenotype analysis was performed. Our analysis revealed an association of the STAT4 SNP rs7574865 with overall decreased susceptibility to CD (p = 0.047, OR 0.86 [95% CI 0.74–0.99]). However, compared to CD patients carrying the wild type genotype, the STAT4 SNP rs7574865 was significantly associated with early CD onset (p = 0.021) and colonic CD (p = 0.008; OR = 4.60, 95% CI 1.63–12.96). For two other STAT4 variants, there was a trend towards protection against CD susceptibility (rs7568275, p = 0.058, OR 0.86 [95% CI 0.74–1.00]; rs10174238, p = 0.057, OR 0.86 [95% CI 0.75–1.00]). In contrast, we did not observe any association with UC susceptibility. Evidence for weak gene-gene interaction of STAT4 with the IL23R SNP rs11209026 was lost after Bonferroni correction.
Our results identified the STAT4 SNP rs7574865 as a disease-modifying gene variant in colonic CD. However, in contrast to SLE and RA, the effect of rs7574865 on CD susceptibility is only weak.