Ralph Melcher

Novel findings on the metabolic effects of the low glycaemic carbohydrate isomaltulose (Palatinose

The slow digestible disaccharide isomaltulose (iso; Palatinose™) is available as novel functional carbohydrate ingredient for manufacturing of low glycaemic foods and beverages. Although basically characterised, various information on physiological effects of iso are still lacking. Thus, the objective of the present study was to expand scientific knowledge of physiological characteristics of iso by a set of three human intervention trials. Using an ileostomy model, iso was found to be essentially absorbed, irrespective of the nature of food (beverage and solid food). Apparent digestibility of 50 g iso from two different meals was 95·5 and 98·8 %; apparent absorption was 93·6 and 96·1 %, respectively. In healthy volunteers, a single dose intake of iso resulted in lower postprandial blood glucose and insulin responses than did sucrose (suc), while showing prolonged blood glucose delivery over 3 h test. In a 4-week trial with hyperlipidaemic individuals, regular consumption of 50 g/d iso within a Western-type diet was well tolerated and did not affect blood lipids. Fasting blood glucose and insulin resistance were lower after the 4-week iso intervention compared with baseline. This would be consistent with possible beneficial metabolic effects as a consequence of the lower and prolonged glycaemic response and lower insulinaemic burden. However, there was no significant difference at 4 weeks after iso compared with suc. In conclusion, the study shows that iso is completely available from the small intestine, irrespective of food matrix, leading to a prolonged delivery of blood glucose. Regular iso consumption is well tolerated also in subjects with increased risk for vascular diseases.

Butyrate Inhibits Leukocyte Adhesion to Endothelial Cells via Modulation of VCAM-1

BackgroundLeukocyte recruitment to areas of inflammation depends on Integrin-VCAM/ICAM interaction. Blocking the vascular cell adhesion molecule (VCAM-1) and the intracellular adhesion molecule (ICAM-1) may have therapeutic benefit for the inflammatory component of bowel disease. Notably, the induction of ICAM and VCAM is mediated by a nuclear factor kappaB (NF-&kgr;B)-dependent mechanism. We investigated whether the anti-inflammatory properties of butyrate are mediated via the modulation of VCAM and ICAM on human endothelial cells. MethodsVCAM-1 and ICAM-1 expression on human endothelial cells upon tumor necrosis factor-&agr; (TNF-&agr;) stimulation was assessd by FACS analysis. A monocyte adhesion assay was performed to evaluate the relevance of a modulated CAM-expression. Electrophoretic mobility shift assays were applied to investigate NF-&kgr;B activation. ResultsThe observed butyrate-associated inhibition of monocyte adhesion to endothelial cells is associated with an inhibition of NF-&kgr;B activation in human endothelial cells. In this context, the observed suppression of the TNF-&agr; induced VCAM-1 expression is likely to play an essential role. ConclusionsButyrate inhibits VCAM-1 mediated leukocyte adhesion to human endothelial cells. This inhibition may contribute to the anti-inflammatory effects of butyrate in patients with distal ulcerative colitis.

Spectral karyotyping of the human colon cancer cell lines SW480 and SW620

The cell lines SW480 and SW620, derived from different stages of colon carcinoma in the same patient, have been used for a number of biochemical, immunological, and genetic studies on colon cancer. A comparative analysis of their karyotypes may identify chromosomal aberrations that might represent markers for metastatic spread. In the present study spectral karyotyping (SKY) was applied to these two colon cancer cell lines. Compared to previously reported G-banded karyotypes, 9 (SW480) and 7 (SW620) markers were identical, 3 (SW480) and 3 (SW620) markers could be redefined, 5 (SW480) and 8 (SW620) markers were newly identified, and 4 (SW480) and 5 (SW620) of the previous described markers could not be confirmed. The redefined aberrations include very complex rearrangements, such as a der(16) t(3;16;1;16;8;16; 1;16;10) and a der(18)t(18;15;17)(q12; p11p13;??) in SW620 and a der(19)t(19;8;19;5) in SW480, that have not been identified by conventional banding techniques. The resulting chromosome gains (5q11→5q15, 7pter→q22, 11, 13q14→qter, 20pter→p12, X) and losses (8pter→p2, 18q12→qter, Y) found in both SW480 and SW620 were in good agreement with those frequently described in colorectal tumors as primary changes in the stem cell. Abnormalities found exclusively in SW620 cells only (gains of 5pter→5q11, 12q12→q23, 15p13→p11, and 16q21→q24 and losses of 2pter→2p24, 4q28→qter, and 6q25→qter) can be viewed as changes that occurred in a putative metastatic founder cell.

The Ussing type chamber model to study the intestinal transport and modulation of specific tight-junction genes using a colonic cell line.

Polyphenols in apples, such as various hydroxycinnamic acids and flavonoids, have positive health effects that strongly depend on their bioavailability. In order to show that the Ussing-type chamber is a useful model to study metabolism, transport, and tightness of cell monolayers in one experimental setup, monolayers of the T84 colon carcinoma cell line mounted in Ussing-type chambers were incubated in the presence of physiological concentrations of various hydroxycinnamic acids (including ferulic, isoferulic, cinnamic, and hydrocinnamic acids) and flavonoids for 4 h. Concentrations of each tested polyphenol in the apical chamber, basolateral chamber, and those associated with the cells were then determined using HPLC with DAD (HPLC-DAD). The transport studies showed that the amounts of the tested polyphenols that passed from the apical to the basolateral side of the T84 monolayers depended on their polarity. Metabolites, such as glucuronides and sulfates of ferulic acid, were also detected at measurable levels by HPLC-ESI-MS/MS in the model system, but only when they were supplied at supra-physiological concentrations (>100 microM). In addition, the transepithelial resistance (TER) of T84 monolayers was measured before and after the addition of polyphenols, with and without short-term exposure to apical sodium caprate (C10), a tight junction (TJ) modulator. Exposure to C10 induced a decrease in TER that was reversible by incubation with polyphenols. However, no increase in paracellular permeability of tested polyphenols was observed after apical C10 exposure, so C10 did not promote fluxes of hydroxycinnamic acids across the monolayers. Further, real-time PCR analysis of the T84 colon cell line showed that ferulic and isoferulic acids induced significant increases in expression of the TJ components zonula occludens-1 (ZO-1) and claudin-4 transcription, but reductions in occludin expression. In contrast, caffeic and p-coumaric acids had no significant effects on the transcription of either ZO-1 or occludin. Our results provide confirmation that T84 cells could be used as model system to simulate the intestinal mucosa, and that polyphenols are able to increase the TER of C10-treated and -untreated T84 monolayers.

Spectral karyotype analysis of colon cancer cell lines of the tumor suppressor and mutator pathway.

Background and aims: Microsatellite instability (MSI) is characterized by the size variation of microsatellites in tumor DNA as compared to matching normal DNA due to defects in the mismatch repair system. To examine the chromosomal differences in microsatellite-stable (MSS) and -unstable (MSI) tumors in detail, we analyzed MSS (Caco-2, Colo-205, SW948) and MSI (HCT-15, HCT-116, LoVo) cell lines by spectral karyotyping (SKY). Methods: SKY is a sensitive method to detect chromosome aberrations by visualizing each chromosome in a different color. Metaphases were hybridized with a SKY probe mixture. Images were visualized with the SpectraCube system and analyzed with the SKYview imaging software. Results: The average number of chromosomes was 49 in LoVo, 45 in HCT-116, 46 in HCT-15, 71 in Colo-205, 89 in Caco-2 and 66 in SW-948. Three aberrant chromosomes were detected in LoVo, three in HCT-116, two in HCT-15, seventeen in Colo-205, fourteen in Caco-2 and nine in SW948. Conclusion: The karyotypes of MSS colon cancer cells displayed complex numerical and structural aberrations. In contrast the chromosomes of MSI colon cancer cells were mostly unaltered but displayed a few isolated numerical and structural aberrations. We speculate that these isolated aberrations may be specifically involved in the pathogenesis of MSI tumors.

LOH and copy neutral LOH (cnLOH) act as alternative mechanism in sporadic colorectal cancers with chromosomal and microsatellite instability

BACKGROUND AND AIMS Tumor suppressor genes are often located in frequently deleted chromosomal regions of colorectal cancers (CRCs). In contrast to microsatellite stable (MSS) tumors, only few loss of heterozygosity (LOH) studies were performed in microsatellite instable (MSI) tumors, because MSI carcinomas are generally considered to be chromosomally stable and classical LOH studies are not feasible due to MSI. The single nucleotide polymorphism (SNP) array technique enables LOH studies also in MSI CRC. The aim of our study was to analyse tissue from MSI and MSS CRC for the existence of (frequently) deleted chromosomal regions and tumor suppressor genes located therein. METHODS AND RESULTS We analyzed tissues from 32 sporadic CRCs and their corresponding normal mucosa (16 MSS and 16 MSI tumors) by means of 50K SNP array analysis. MSS tumors displayed chromosomal instability that resulted in multiple deleted (LOH) and amplified regions and led to the identification of MTUS1 (8p22) as a candidate tumor suppressor gene in this region. Although the MSI tumors were chromosomally stable, we found several copy neutral LOHs (cnLOH) in the MSI tumors; these appear to be instrumental in the inactivation of the tumor suppressor gene hMLH1 and a gene located in chromosomal region 6pter-p22. DISCUSSION Our results suggest that in addition to classical LOH, cnLOH is an important mutational event in relation to the carcinogenesis of MSS and MSI tumors, causing the inactivation of a tumor suppressor gene without copy number alteration of the respective region; this is crucial for the development of MSI tumors and for some chromosomal regions in MSS tumors.

Modulation of Nrf2-dependent gene transcription by bilberry anthocyanins in vivo

In a human pilot intervention study (healthy + ileostomy probands), the questions were addressed whether in vivo consumption of an anthocyanin-rich bilberry (Vaccinium myrtillius L.) pomace extract (BE) affects (i) the transcription of Nrf2-dependent genes in peripheral blood mononuclear cells (PBMC), indicative for systemic effects, and (ii) the level of oxidative DNA damage in these cells. In healthy test subjects transcripts of NAD(P)H quinone oxidoreductase 1 (NQO1) were significantly elevated throughout the observation period (1-8 h), whereas transcription of heme oxygenase 1 (HO-1) and Nrf2 was significantly decreased. NQO1 and HO-1 transcription remained unchanged in the ileostomy probands, whereas Nrf2-transcription was suppressed in both groups. Decrease in oxidative DNA damage was observed 2 h after BE consumption again only in healthy subjects. In vitro studies using a reporter gene approach (CHO) and qPCR (HT29) indicate that not the intact anthocyanins/anthocyanidins are the activating constituents but the intestinal degradation product phloroglucinol aldehyde (PGA). Taken together, consumption of anthocyanin-rich BE was found to modulate Nrf2-dependent gene expression in PBMCs indicative for systemic activity. Limitation of the effect to healthy test subjects suggests a role of colonic processes for bioactivity, supported by the results on Nrf2-activating properties of the intestinal anthocyanin degradation product PGA.

Effects of isomalt consumption on gastrointestinal and metabolic parameters in healthy volunteers

The polyol isomalt (Palatinit) is a well established sugar replacer. The impact of regular isomalt consumption on metabolism and parameters of gut function in nineteen healthy volunteers was examined in a randomised, double-blind, cross-over trial with two 4-week test periods. Volunteers received 30 g isomalt or 30 g sucrose daily as part of a controlled diet. In addition to clinical standard diagnostics, biomarkers and parameters currently discussed as risk factors for CHD, diabetes or obesity were analysed. Urine and stool Ca and phosphate excretions were measured. In addition, mean transit time, defecation frequency, stool consistency and weight were determined. Consumption of test products was affirmed by the urinary excretion of mannitol. Blood lipids were comparable in both phases, especially in volunteers with hyperlipidaemia, apart from lower apo A-1 (P=0.03) for all subjects. Remnant-like particles, oxidised LDL, NEFA, fructosamine and leptin were comparable and not influenced by isomalt. Ca and phosphate homeostasis was not affected. Stool frequency was moderately increased in the isomalt phase (P=0.006) without changes in stool consistency and stool water. This suggests that isomalt is well tolerated and that consumption of isomalt does not impair metabolic function or induce hypercalciuria. In addition, the study data indicate that isomalt could be useful in improving bowel function.

SNP-Array genotyping and spectral karyotyping reveal uniparental disomy as early mutational event in MSS- and MSI-colorectal cancer cell lines

In this study nine colorectal cancer cell lines were analysed by 10K SNP-arrays and spectral karyotyping (SKY). Complex chromosomal alterations and breakpoints of deleted or translocated fragments found by SKY could further be characterized by SNP-array analysis. Interestingly many monoallelic regions identified by SNP-array analysis display no copy number alterations, representing uniparental disomy (UPD). It was demonstrated that UPD seems to be involved in activation of early-acting tumor suppressor genes in MSS- (APC, CDKN2A) and MSI- (MLH1, MSH2, APC, CDKN2A) colorectal cancer cell lines. Genes involved later on in the adenoma-carcinoma sequence (i.e. TP53/SMAD4) were not found to be inactivated by UPD. Furthermore, identified amplified monoallelic regions may include oncogenes activated by allele-specific-amplification (i.e. Cyclin D1). However, at present, the majority of the monoallelic regions located in the present study have not yet been associated with known tumor suppressor genes and oncogenes. Further studies are warranted to identify relevant genes in the respective regions and to further verify the results presented here.

Modulation of HMG-N2 binding to chromatin by butyrate-induced acetylation in human colon adenocarcinoma cells.

Butyrate, a short chain fatty acid (SCFA), is generated by anaerobic fermentation of undigested carbohydrates within the colon. Butyrate enhances acetylation of core histones, a process directly linked to the formation of active chromatin and gene expression. However, additional chromatin components also contribute to the formation of transcriptionally active chromatin. The high mobility group protein N2 (HMG‐N2), a nonhistone protein, is involved in chromatin structure modulation. We examined the effects of butyrate on HMG‐N2 expression, hyperacetylation and chromatin binding. HT29 human adenocarcinoma cells were incubated with butyrate. Levels of HMG‐N2 mRNA and of total or acetylated HMG‐N2 protein were analyzed. Protein dynamics were investigated with transfected cells expressing HMG‐N2‐EGFP fusion proteins. Treatment of HT29 cells with butyrate led to significant hyperacetylation of HMG‐N2. Levels of HMG‐N2 protein remained unchanged. Northern blot analysis revealed a significant reduction in HMG‐N2 mRNA levels after treatment with butyrate. Analysis of HMG‐N2‐EGFP transfected HT29 cells demonstrated that butyrate treatment changes the binding properties of HMG‐N2‐EGFP to chromatin. In addition, butyrate treatment resulted in solubilization of endogenous acetylated HMG‐N2 into the supernatant of permeabilized cells. We demonstrate that butyrate treatment is associated with hyperacetylation of HMG‐N2 protein in HT29 cells. The modulation of this nonhistone chromatin protein resulted in altered binding properties to chromatin. This may represent an additional step in changing chromatin structure and composition with subsequent consequences for transcription and gene expression. Modulation of nonhistone chromatin proteins, like the ubiquitous HMG‐N2 proteins, may be partly responsible for the wide range of butyrate‐associated effects.