Common Food Additive Carrageenan Research Articles

Common food additive carrageenan inhibits proglucagon expression and GLP-1 secretion by human enteroendocrine L-cells

Proglucagon mRNA expression and GLP-1 secretion by cultured human L-cells (NCI-H716) were inhibited following exposure to λ-carrageenan, a commonly used additive in processed foods. Carrageenan is composed of sulfated or unsulfated galactose residues linked in alternating alpha-1,3 and beta-1,4 bonds and resembles the endogenous sulfated glycosaminoglycans. However, carrageenan has unusual alpha-1,3-galactosidic bonds, which are not innate to human cells and are implicated in immune responses. Exposure to carrageenan predictably causes inflammation, and carrageenan impairs glucose tolerance and contributes to insulin resistance. When cultured human L-cells were deprived overnight of glucose and serum and then exposed to high glucose, 10% FBS, and λ-carrageenan (1 µg/ml) for 10 minutes, 1 h, and 24 h, mRNA expression of proglucagon and secretion of GLP-1 were significantly reduced, compared to control cells not exposed to carrageenan. mRNA expression of proglucagon by mouse L-cells (STC-1) was also significantly reduced and supports the findings in the human cells. Exposure of co-cultured human intestinal epithelial cells (LS174T) to the spent media of the carrageenan-treated L-cells led to a decline in mRNA expression of GLUT-2 at 24 h. These findings suggest that ingestion of carrageenan-containing processed foods may impair the production of GLP-1, counteract the effect of GLP-1 receptor agonists and induce secondary effects on intestinal epithelial cells.

Distinct Effects of Carrageenan and High-Fat Consumption on the Mechanisms of Insulin Resistance in Nonobese and Obese Models of Type 2 Diabetes.

Exposure to low concentration of the common food additive carrageenan (10 mg/L) for only six days led to glucose intolerance and insulin resistance in the C57BL/6J mouse. Longer exposure produced fasting hyperglycemia but with no increase in weight, in contrast to the HFD. Glucose intolerance was attributable to carrageenan-induced inflammation and to increased expression of GRB10. Both HFD and carrageenan increased p(Ser32)-IκBα and p(Ser307)-IRS1, and the increases were greater following the combined exposure. The effects of carrageenan were inhibited by the combination of the free radical inhibitor Tempol and BCL10 siRNA, which had no impact on the HFD-mediated increase. In contrast, the PKC inhibitor sotrastaurin blocked the HFD-induced increases, without an effect on the carrageenan-mediated effects. HFD had no impact on the expression of GRB10. Both carrageenan and high fat increased hepatic infiltration by F4/80-positive macrophages. Serum galectin-3 and galectin-3 binding to the insulin receptor increased by carrageenan and by HFD. Tyrosine phosphorylation of the insulin receptor declined following either exposure and was further reduced by their combination. Carrageenan reduced the activity of the enzyme N-acetylgalactosamine-4-sulfatase (ARSB; arylsulfatase B), which was unchanged following HFD. Dietary exposure to both high fat and carrageenan can impair insulin signaling through both similar and distinct mechanisms.

Increase in Galectin-3 Contributes to Glucose Intolerance and Insulin Resistance following Exposure to the Common Food Additive Carrageenan

Exposure to low concentration of the common food additive carrageenan (CGN) leads to impaired glucose tolerance and insulin resistance in C57BL/6J mice, due to CGN’s pro-inflammatory effects and changes in phospho-IRS1 phosphorylation. CGN is a sulfated polysaccharide with α-1,3 and β-1,4 galactosidic bonds. Other studies showed: 1) CGN reduced activity of arylsulfatase B, which removes sulfate groups from N-acetylgalactosamine 4-sulfate residues of chondroitin 4-sulfate (C4S) and dermatan sulfate; and 2) galectin-3 binds less with C4S when ARSB is reduced. Increase in galectin-3, which binds to β-galactosides, was reported to inhibit insulin signaling (Li P et al) by effects on the insulin receptor. This background led to the current assessment of effects of the no-CGN diet on galectin-3 levels in serum and galectin-3 mRNA in circulating mononuclear cells. Blood samples were obtained from adults with prediabetes before and after dietary intervention. Baseline serum galectin-3 values averaged 8.94 ng/ml (range 4.06-12.64). In 7 patients on a no-CGN diet for 12 weeks, the mean level declined to 7.25 ng/ml (p=0.006, paired t-test). In contrast, serum galectin-3 was unchanged in participants on the CGN-containing control diet and increased by over 2 ng/ml in subjects on their regular diet. mRNA expression of galectin-3 (NGALS3) in circulating mononuclear cells of patients on the no-CGN diet declined by 69%, and increased by 36% in the CGN-containing diet group and by 98% in the regular diet group (p=0.0002, 1-way ANOVA). Also, serum galectin-3 levels increased to 1.72 times baseline in C57BL/6J mice given CGN in their water supply for 12 weeks. In HepG2 cells, exogenous galectin-3 (25 µg/ml) inhibited insulin-induced glucose uptake by 39%, and was highly correlated with decline in tyrosine phosphorylation of IRS-1. These findings suggest that CGN contributes to glucose intolerance and insulin resistance, in part, by effects on galectin-3. Disclosure J.K. Tobacman: None. S. Bhattacharyya: None. L. Feferman: None.

Impact of the No-Carrageenan Diet on Glucose Tolerance and Insulin Resistance in Adults with Prediabetes

Previous animal and cell-based studies indicated that exposure to the common food additive carrageenan impaired glucose tolerance and increased insulin resistance. Carrageenan is widely used in processed foods to improve solubility, but predictably causes inflammation, which contributes to insulin resistance by inhibition of insulin signaling. To test if removal of carrageenan from the diet could reduce insulin resistance, we initiated a double-blinded, controlled feeding study in individuals with prediabetes, defined by Hemoglobin A1c values of 5.7%-6.4%. All procedures were approved by the University of Illinois at Chicago IRB. Participants were provided study diets for 12 weeks, which either contained or did not contain carrageenan, and had similar composition of protein, carbohydrate, and fat. An additional group was comprised of individuals who participated in study tests, but ate their usual diet. Subjects on study diets picked up their food weekly at UIC and had blood tests, including 2h glucose tolerance tests performed at entry, 6, and 12 weeks. Inflammatory parameters, including fecal calprotectin, were measured at entry and conclusion. Preliminary study results from 17 participants indicate that the carrageenan-free diet was associated with significant decline in hemoglobin A1c (p=0.004, unpaired t-test, two-tailed), HOMA-IR (homeostatic model for insulin resistance) score (p=0.0064), insulin resistance (p=0.02), and leukocyte phospho-serine IRS-1 (p=0.003) and NF-kappaB, compared to study participants on the control study diet or their regular diet. Study findings indicate significant decline in insulin resistance by removal of carrageenan from the diet. Additional studies of inflammatory parameters, immune cell subsets, and fecal microbiome are pending. Disclosure J.K. Tobacman: None.

Carrageenan Inhibits Insulin Signaling through GRB10-mediated Decrease in Tyr(P)-IRS1 and through Inflammation-induced Increase in Ser(P)307-IRS1

Inflammation induced by exposure to the common food additive carrageenan leads to insulin resistance by increase in Ser(P)(307)-insulin receptor substrate 1 (IRS1) and subsequent decline in the insulin-stimulated increase in Ser(P)(473)-AKT. Inhibition of carrageenan-induced inflammation reversed the increase in Ser(P)(307)-IRS1 but did not completely reverse the carrageenan-induced decline in Ser(P)(473)-AKT. To identify the additional mechanism responsible for the decrease in Ser(P)(473)-AKT, studies were performed in human HepG2 cells and in C57BL/6J mice. Following carrageenan, expression of GRB10 (growth factor receptor-bound 10 protein), an adaptor protein that binds to the insulin receptor and inhibits insulin signaling, increased significantly. GRB10 silencing blocked the carrageenan-induced reduction of the insulin-stimulated increase in Tyr(P)-IRS1 and partially reversed the decline in Ser(P)(473)-AKT. The combination of GRB10 silencing with BCL10 silencing and the reactive oxygen species inhibitor Tempol completely reversed the decline in Ser(P)(473)-AKT. After carrageenan, GRB10 promoter activity was enhanced because of activation by GATA2. A direct correlation between Ser(P)(473)-AKT and Ser(P)(401)-GATA2 was evident, and inhibition of AKT phosphorylation by the PI3K inhibitor LY294002 blocked Ser(401)-GATA2 phosphorylation and the increase in GRB10 expression. Studies indicated that carrageenan inhibited insulin signaling by two mechanisms: through the inflammation-mediated increase in Ser(P)(307)-IRS1, a negative regulator of insulin signaling, and through a transcriptional mechanism leading to increase in GRB10 expression and GRB10-inhibition of Tyr(P)-IRS1, a positive regulator of insulin signaling. These mechanisms converge to inhibit the insulin-induced increase in Ser(P)(473)-AKT. They provide internal feedback, mediated by Ser(P)(473)-AKT, Ser(P)(401)-GATA2, and nuclear GATA2, which links the opposing effects of serine and tyrosine phosphorylations of IRS1 and can modulate insulin responsiveness.

The common food additive carrageenan is not a ligand for Toll-Like- Receptor 4 (TLR4) in an HEK293-TLR4 reporter cell-line model

Carrageenan (CGN) is widely used in the food manufacturing industry as an additive that stabilizes and thickens food products. Standard animal safety studies in which CGN was administered in diet showed no adverse effects. However, several in vitro studies have reported that intestinal inflammation is caused by CGN and that this effect is mediated through Toll-Like-Receptor 4 (TLR4). The purpose of this study was to evaluate the ability of different types of CGN to bind and activate TLR4 signaling. To accomplish this a TLR4/MD-2/CD14/NFκB/SEAP reporter construct in a HEK293 cell line was used. The reporter molecule, secretable alkaline phosphatase (SEAP), was measured as an indicator of TLR4 activation. The test compounds were exposed to this system at concentrations of 0.1, 1, 10, 50, 100, 500, 1000, and 5000 ng/mL for 24 h. Cytotoxicity was evaluated following the 24 h exposure period by LDH leakage and ATP. CGN binding to serum proteins was characterized by Toluidine Blue. The results show that CGN does not bind to TLR4 and is not cytotoxic to the HEK293 cells at the concentrations and experimental conditions tested and that CGN binds tightly to serum proteins.

Exposure to common food additive carrageenan alone leads to fasting hyperglycemia and in combination with high fat diet exacerbates glucose intolerance and hyperlipidemia without effect on weight.

Aims. Major aims were to determine whether exposure to the commonly used food additive carrageenan could induce fasting hyperglycemia and could increase the effects of a high fat diet on glucose intolerance and dyslipidemia. Methods. C57BL/6J mice were exposed to either carrageenan, high fat diet, or the combination of high fat diet and carrageenan, or untreated, for one year. Effects on fasting blood glucose, glucose tolerance, lipid parameters, weight, glycogen stores, and inflammation were compared. Results. Exposure to carrageenan led to glucose intolerance by six days and produced elevated fasting blood glucose by 23 weeks. Effects of carrageenan on glucose tolerance were more severe than from high fat alone. Carrageenan in combination with high fat produced earlier onset of fasting hyperglycemia and higher glucose levels in glucose tolerance tests and exacerbated dyslipidemia. In contrast to high fat, carrageenan did not lead to weight gain. In hyperinsulinemic, euglycemic clamp studies, the carrageenan-exposed mice had higher early glucose levels and lower glucose infusion rate and longer interval to achieve the steady-state. Conclusions. Carrageenan in the Western diet may contribute to the development of diabetes and the effects of high fat consumption. Carrageenan may be useful as a nonobese model of diabetes in the mouse.

Increased Expression of Colonic Wnt9A through Sp1-mediated Transcriptional Effects involving Arylsulfatase B, Chondroitin 4-Sulfate, and Galectin-3

In cultured human colonic epithelial cells and mouse colonic tissue, exposure to the common food additive carrageenan leads to inflammation, activation of Wnt signaling, increased Wnt9A expression, and decline in the activity of the enzyme arylsulfatase B (ARSB; N-acetylgalactosamine-4-sulfatase). In this study, the novel transcriptional mechanism by which carrageenan and decline in ARSB increase Wnt9A expression in NCM460 and HT-29 human colonic epithelial cells and in mouse colon is presented. Increased expression of Wnt9A has been associated with multiple malignancies, including colon carcinoma, and with ectodermal and mesoendodermal morphogenesis. When ARSB activity was reduced by siRNA or by exposure to carrageenan (1 μg/ml for 24 h), degradation of chondroitin 4-sulfate (C4S) was inhibited, leading to accumulation of more highly sulfated C4S, which binds less galectin-3, a β-galactoside-binding protein. Nuclear galectin-3 increased and mediated increased binding of Sp1 to the Sp1 consensus sequence in the Wnt9A promoter, shown by oligonucleotide-binding assay and by chromatin immunoprecipitation assay. When galectin-3 was silenced, the increases in Sp1 binding to the Wnt9A promoter and in Wnt9A expression, which followed carrageenan or ARSB silencing, were inhibited. Mithramycin A, a specific inhibitor of Sp1 oligonucleotide binding, and Sp1 siRNA blocked the carrageenan- and ARSB siRNA-induced increases in Wnt9A expression. These studies reveal how carrageenan exposure can lead to transcriptional events in colonic epithelial cells through decline in arylsulfatase B activity, with subsequent impact on C4S, galectin-3, Sp1, and Wnt9A and can exert significant effects on Wnt-initiated signaling and related vital cell processes.

Common Food Additive Carrageenan Stimulates Wnt/ β-Catenin Signaling in Colonic Epithelium by Inhibition of Nucleoredoxin Reduction

Exposure to the common food additive carrageenan was previously associated with increased Wnt9A expression and increased cytoplasmic β-catenin in human colonic epithelial cells. In this report, exposure of human colonic epithelial cells in culture and of mouse colonic epithelium in vivo to low concentrations of carrageenan is shown to activate the Wnt/β-catenin signaling pathway, leading to increases in nuclear β-catenin, T-cell factor/lymphoid enhancer factor activation, and cyclin D1 expression and decline in bone morphogenetic protein-4. These effects are mediated through carrageenan-induced reactive oxygen species (ROS), and inhibited by the ROS scavenger Tempol. Carrageenan exposure and ROS production inhibited thioredoxin reductase activity and increased oxidation of nucleoredoxin, a member of the thioredoxin family of redox proteins. When oxidized, nucleoredoxin co-immunoprecipitation with dishevelled (DVL) declined, enabling DVL to interact with and inhibit the cytoplasmic β-catenin destruction complex, and facilitating nuclear translocation of β-catenin. Both nucleoredoxin silencing and carrageenan exposure produced similar declines in thioredoxin reductase activity. In addition to activation of Wnt signaling, carrageenan exposure also increased Wnt9A mRNA expression in the mouse colonic epithelium and the human colonic epithelial cells, thereby potentially permitting ongoing stimulation of the Wnt/β-catenin pathway. These findings suggest how a common dietary ingredient can contribute to colon carcinogenesis by effects on Wnt signaling and Wnt expression.

Sa1684 Common Food Additive Carrageenan Stimulates Wnt/β-Catenin Signaling Pathway in Colonic Epithelium by Inhibition of Nucleoredoxin Reduction

Sa1684 Common Food Additive Carrageenan Stimulates Wnt/β-Catenin Signaling Pathway in Colonic Epithelium by Inhibition of Nucleoredoxin Reduction

Carrageenan-Induced Colonic Inflammation Is Reduced in Bcl10 Null Mice and Increased in IL-10-Deficient Mice

The common food additive carrageenan is a known activator of inflammation in mammalian tissues and stimulates both the canonical and noncanonical pathways of NF-κB activation. Exposure to low concentrations of carrageenan (10 μg/mL in the water supply) has produced glucose intolerance, insulin resistance, and impaired insulin signaling in C57BL/6 mice. B-cell leukemia/lymphoma 10 (Bcl10) is a mediator of inflammatory signals from Toll-like receptor (TLR) 4 in myeloid and epithelial cells. Since the TLR4 signaling pathway is activated in diabetes and by carrageenan, we addressed systemic and intestinal inflammatory responses following carrageenan exposure in Bcl10 wild type, heterozygous, and null mice. Fecal calprotectin and circulating keratinocyte chemokine (KC), nuclear RelA and RelB, phospho(Thr559)-NF-κB-inducing kinase (NIK), and phospho(Ser36)-IκBα in the colonic epithelial cells were significantly less (P < 0.001) in the carrageenan-treated Bcl10 null mice than in controls. IL-10-deficient mice exposed to carrageenan in a germ-free environment showed an increase in activation of the canonical pathway of NF-κB (RelA) activation, but without increase in RelB or phospho-Bcl10, and exogenous IL-10 inhibited only the canonical pathway of NF-κB activation in cultured colonic cells. These findings demonstrate a Bcl10 requirement for maximum development of carrageenan-induced inflammation and lack of complete suppression by IL-10 of carrageenan-induced inflammation.

Abstract 163: Common food additive carrageenan activates wnt signaling pathway in mouse colon and human colonic epithelial cells

Abstract The Wnt-β-catenin signaling pathway plays a pivotal role in development and disease. It is crucial for basic developmental processes, such as cell-fate specification, progenitor-cell proliferation and the control of asymmetric cell division. Recent studies suggest that self-renewal of stem/progenitor cells by the canonical Wnt cascade is subverted in cancer cells to allow malignant proliferation in intestine. Carrageenans are highly sulfated polysaccharides, which are widely used as food additives to improve the texture of processed foods, and are consumed at levels of about 250 mg/day in the average diet. Reports of many investigators have demonstrated that carrageenan induces inflammation, colonic ulcerations, polyps, and colorectal tumors. We have reported distinct pathways by which carrageenan induces the activation of NF-κB and secretion of IL-8, mediated through TLR4-BCL10 or reactive oxygen species (ROS) in colonic epithelial cells. Our previous work also indicated that carrageenan exposure caused activation of the Wnt-β-catenin pathway in colonic epithelial cells. This investigation was undertaken to elucidate the Wnt signaling mechanisms activated by carrageenan exposure, using NCM460 (INCELL) human colonic epithelial cells exposed to carrageenan (1 μg/ml) x 24 h and colonic tissue from ten-week old C57BL/6J mice exposed to carrageenan (10 - 100 μg/ml) in their water for four weeks. Nuclear β-catenin in NCM460 cells and mouse colonic tissue was determined by Western blot and ELISA and increased to two times the baseline level following carrageenan. This increase in nuclear β-catenin in NCM460 cells was inhibited by the ROS quencher Tempol, but not by TLR4 or BCL10 siRNA. TCF/Lef-1 reporter assay demonstrated increased nuclear binding post carrageenan treatment in the mouse colon (to 4x baseline) and in the NCM460 cells (to 6.3x baseline). Also, mRNA expression of cyclin D1 and wnt9a was increased more than two-fold, and BMP4 expression was decreased. In the NCM460 cells, these responses to carrageenan were inhibited by Tempol, but not BCL10 silencing. Also, carrageenan-induced increases in ROS and lipid peroxidation were inhibited by Tempol. Since Wnt signaling is affected by free dishevelled 2 (dsh2), and dsh2 binding to nucleoredoxin (NRX) is greater when NRX is in its reduced state, we tested whether carrageenan exposure could impact upon the Wnt signaling pathway by modifying the redox status of NRX. The ratio of free NRX to NRX bound to dsh2 increased to six times baseline following carrageenan, and was reversed by Tempol. These profound effects of carrageenan on the Wnt signaling pathway in colonic cells can help to explain how diet can contribute to development of colonic neoplasia. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 163. doi:1538-7445.AM2012-163

Exposure to common food additive carrageenan leads to reduced sulfatase activity and increase in sulfated glycosaminoglycans in human epithelial cells

The commonly used food additive carrageenan, including lambda (λ), kappa (κ) and iota (ι) forms, is composed of galactose disaccharides linked in alpha-1,3 and beta-1,4 glycosidic bonds with up to three sulfate groups per disaccharide residue. Carrageenan closely resembles the endogenous galactose or N-acetylgalactosamine-containing glycosaminoglycans (GAGs), chondroitin sulfate (CS), dermatan sulfate (DS), and keratan sulfate. However, these GAGs have beta-1,3 and beta-1,4 glycosidic bonds, in contrast to the unusual alpha-1,3 glycosidic bond in carrageenan. Since sulfatase activity is inhibited by sulfate, and carrageenan is so highly sulfated, we tested the effect of carrageenan exposure on sulfatase activity in human intestinal and mammary epithelial cell lines and found that carrageenan exposure significantly reduced the activity of sulfatases, including N-acetylgalactosamine-4-sulfatase, galactose-6-sulfatase, iduronate sulfatase, steroid sulfatase, arylsulfatase A, SULF-1,2, and heparan sulfamidase. Consistent with the inhibition of sulfatase activity, following exposure to carrageenan, GAG content increased significantly and showed marked differences in disaccharide composition. Specific changes in CS disaccharides included increases in di-sulfated disaccharide components of CSD (2S6S) and CS-E (4S6S), with declines in CS-A (4S) and CS-C (6S). Specific changes in heparin-heparan sulfate disaccharides included increases in 6S disaccharides, as well as increases in NS and 2S6S disaccharides. Study results suggest that carrageenan inhibition of sulfatase activity leads to re-distribution of the cellular GAG composition with increase in di-sulfated CS and with potential consequences for cell structure and function.

Exposure to the common food additive carrageenan leads to glucose intolerance, insulin resistance and inhibition of insulin signalling in HepG2 cells and C57BL/6J mice

The aim of this study was to determine the impact of the common food additive carrageenan (E407) on glucose tolerance, insulin sensitivity and insulin signalling in a mouse model and human hepatic cells, since carrageenan is known to cause inflammation through interaction with toll-like receptor (TLR)4, which is associated with inflammation in diabetes. Male C57BL/6J mice were given carrageenan (10 mg/l) in their drinking water, and underwent a glucose tolerance test (GTT), an insulin tolerance test (ITT) and an ante-mortem intraperitoneal insulin injection. HepG2 cells were exposed to carrageenan (1 mg/l × 24 h) and insulin. Levels of phospho(Ser473)-protein kinase B (Akt), phospho(Ser307)-IRS1, phosphoinositide 3-kinase (PI3K) activity and phospho(Ser32)-inhibitor of κB (IκBα) were determined by western blotting and ELISA. Glucose tolerance was significantly impaired in carrageenan-treated 12-week-old mice compared with untreated controls at all time points (n = 12; p < 0.0001). Baseline insulin and insulin levels at 30 min after taking glucose during the GTT were significantly higher following carrageenan treatment. During the ITT, glucose levels declined by more than 80% in controls, but not in carrageenan-treated mice. Carrageenan exposure completely inhibited insulin-induced increases in phospho-(Ser473)-Akt and PI3K activity in vivo in mouse liver and in human HepG2 cells. Carrageenan increased phospho(Ser307)-IRS1 levels, and this was blocked when carrageenan-induced inflammation was inhibited. This is the first report of the impact of carrageenan on glucose tolerance and indicates that carrageenan impairs glucose tolerance, increases insulin resistance and inhibits insulin signalling in vivo in mouse liver and human HepG2 cells. These effects may result from carrageenan-induced inflammation. The results demonstrate extra-colonic manifestations of ingested carrageenan and suggest that carrageenan in the human diet may contribute to the development of diabetes.

Common Food Additive Carrageenan Activates Polyp Formation Pathways by Reactive Oxygen Species, Not by TLR4-BCL10 Inflammatory Cascade

Common Food Additive Carrageenan Activates Polyp Formation Pathways by Reactive Oxygen Species, Not by TLR4-BCL10 Inflammatory Cascade

Abstract 1022: TNFα-induced apoptosis is reduced, but inflammation is increased by combined exposure with common food additive carrageenan

Abstract Tumor necrosis factor (TNF)-α, a homotrimeric, pleiotropic cytokine, mediates both apoptosis and inflammation. Following exposure to TNF-α, activation of caspases leads to apoptotic cell death and stimulation of an inflammatory cascade through the non-canonical pathway of NF-κB activation leads to increased nuclear RelB and p52. In contrast, the common food additive carrageenan (CGN), which contains sulfated galactose residues, stimulates inflammation through both the canonical and non-canonical pathways of NF-κB activation. CGN-induced inflammation utilizes B-cell leukemia/lymphoma (BCL)-10 to activate a pathway of innate immunity that is initiated by interaction with toll-like receptor (TLR)-4. CGN also stimulates inflammation through a reactive oxygen species-mediated pathway. In a series of cell-based experiments, colonic epithelial cells and mouse embryonic fibroblasts were treated with TNF-α and CGN, in order to simulate the possible effects of exposure to dietary CGN in the setting of TNF-α-mediated effects. Marked increase in secretion of Interleukin (IL)-8 occurred, attributable to synergistic effects on phospho-NIK in the non-canonical pathway. TNF-α induced the ubiquitination of TNF receptor-associated factor (TRAF)-2 which interacts with NFκB-inducing kinase (NIK), and CGN induced phosphorylation of BCL10, which is required for NIK-phosphorylation. These results suggest that TNF-α and CGN in combination act to increase NIK phosphorylation, thereby increasing activation of the non-canonical pathway of NF-κB activation. In contrast, the apoptotic effects of TNF-α, including activation of caspase-8 and poly [ADP-ribose] polymerase (PARP)-1 fragmentation, were markedly reduced in the presence of CGN. CGN exposure caused reduction in Fas by cDNA microarray and by ELISA. The study results demonstrate that exposure to CGN drives TNF-α stimulated cells toward inflammation, rather than toward apoptotic cell death. The findings suggest that CGN exposure may compromise the effectiveness of anti-TNF-α therapy. Average consumption of CGN in the typical Western diet is reported to be 250 mg/day, in contrast to therapeutic glycosides, such as digoxin that are prescribed in doses of ∼0.1 mg/day. Ingestion of CGN-containing foods has the potential to both inhibit the pro-apoptotic effect and to increase the pro-inflammatory effect of TNF-α and may contribute to malignant transformation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1022. doi:10.1158/1538-7445.AM2011-1022

Tumor Necrosis Factor α-induced Inflammation Is Increased but Apoptosis Is Inhibited by Common Food Additive Carrageenan

Tumor necrosis factor (TNF)-α, a homotrimeric, pleiotropic cytokine, is secreted in response to inflammatory stimuli in diseases such as rheumatoid arthritis and inflammatory bowel disease. TNF-α mediates both apoptosis and inflammation, stimulating an inflammatory cascade through the non-canonical pathway of NF-κB activation, leading to increased nuclear RelB and p52. In contrast, the common food additive carrageenan (CGN) stimulates inflammation through both the canonical and non-canonical pathways of NF-κB activation and utilizes the adaptor molecule BCL10 (B-cell leukemia/lymphoma 10). In a series of experiments, colonic epithelial cells and mouse embryonic fibroblasts were treated with TNF-α and carrageenan in order to simulate the possible effects of exposure to dietary CGN in the setting of a TNF-α-mediated inflammatory disease process. A marked increase in secretion of IL-8 occurred, attributable to synergistic effects on phosphorylated NF-κB-inducing kinase (NIK) in the non-canonical pathway. TNF-α induced the ubiquitination of TRAF2 (TNF receptor-associated factor 2), which interacts with NIK, and CGN induced phosphorylation of BCL10, leading to increased NIK phosphorylation. These results suggest that TNF-α and CGN in combination act to increase NIK phosphorylation, thereby increasing activation of the non-canonical pathway of NF-κB activation. In contrast, the apoptotic effects of TNF-α, including activation of caspase-8 and PARP-1 (poly(ADP-ribose) polymerase 1) fragmentation, were markedly reduced in the presence of CGN, and CGN caused reduced expression of Fas. These findings demonstrate that exposure to CGN drives TNF-α-stimulated cells toward inflammation rather than toward apoptotic cell death and suggest that CGN exposure may compromise the effectiveness of anti-TNF-α therapy.

Abstract 5734: Prolonged activation of NFκB (RelA and RelB), BCL10, and IL-8 following exposure to common food additive carrageenan in human colonic epithelial cells may be attributable to NFκB binding site in BCL10 promoter

Abstract The common food additive carrageenan (CGN) is a sulfated polysaccharide that resembles the naturally occurring sulfated glycosaminoglycans (sGAG), particularly chondroitin sulfate and keratan sulfate. Unlike the naturally occurring sGAG, CGN contains alpha-1,3-galactosidic linkages that are immunogenic in humans. Previously, we reported that CGN activation of a TLR4-MyD88 mediated inflammatory cascade proceeds through BCL10 and NFκB (p65), leading to increased secretion of IL-8 in human colonic epithelial cells. Translocations involving BCL10 and MALT1 are found in mucosa-associated lymphoid tissue (MALT) lymphomas, associated with constitutive activation of NFκB. Since CGN has been widely used to induce colitis in animal models of inflammatory bowel disease, we addressed whether CGN exposure can lead to sustained activation of BCL10 and NFκB, consistent with a model of constitutive activation of inflammation and possible pre-malignant changes. Experiments were performed with NCM460 cells, a human colonic epithelial cell line derived from normal colon, in M3:10A medium (Incell) with λCGN (1μg/ml). Triplicate cell preparations and spent media from untreated control cell preparations, cells treated with CGN for only the initial 12 hour time interval, and cells treated for six 12-hour periods were harvested every 12 hours over 72 hours, and media, with or without CGN was replenished. IL-8 in the spent media, and BCL10 in the cytosolic preparations and NFκB [RelA (p65) and RelB] in the nuclear preparations were measured at successive 12-hour periods. IL-8 remained significantly increased for an additional 36 hours following withdrawal of CGN. BCL10 and RelA remained significantly increased for an additional 24 hours, and RelB was significantly increased for an additional 12 hours. These findings are consistent with previous data that demonstrate that CGN activates both canonical and non-canonical pathways of NFκB. The presence of a sequence with high homology to the established NFκB consensus oligonucleotide sequence in the BCL10 promoter may be responsible for an ongoing process of activation following withdrawal of the CGN exposure. In vitro testing of binding of RelA and RelB to the NFκB consensus oligonucleotide, the homologous BCL10 promoter sequence, a mutated BCL10 promoter sequence, and a scrambled NFκB consensus sequence indicates over 50% binding of either RelA or RelB to the BCL10 promoter sequence, compared to 100% to the established NFκB consensus sequence. Hence, the study findings demonstrate that an environmental exposure to a common food additive (CGN) can trigger prolonged activation of an inflammatory loop involving BCL10, RelA, RelB, and IL-8 in human colonic epithelial cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5734.

Carrageenan-induced innate immune response is modified by enzymes that hydrolyze distinct galactosidic bonds

The common food additive carrageenan (CGN) predictably induces intestinal inflammation in animal models. Mechanisms of CGN-induced nuclear factor κB and interleukin-8 (IL-8) stimulation include an immune-mediated pathway involving toll-like receptor 4 (TLR4) and B-cell lymphoma/leukemia 10 (BCL10) and a reactive oxygen species (ROS)-mediated pathway. To determine how the structure of CGN contributes to its initiation of inflammation through these two distinct mechanisms, we treated CGNs with galactosidases and carrageenases (CGNases) and determined the impact on IL-8 secretion and BCL10 production. Hydrolysis of CGN by the enzyme α-1→(3,6)-galactosidase significantly reduced increases in IL-8 and BCL10, but other galactosidases tested, including α-1→6-galactosidase, β-1→4-galactosidase and β-1→3,6-galactosidase, had no effect. In contrast, specific κ-CGNases or ι-CGNases, which hydrolyze β-1,4-galactosidic bonds, produced increases in IL-8 and BCL10 attributable to increased exposure of the immunogenic α-1→3-galactosidic epitope of CGN to TLR4. These results were consistent with induction of innate immune response by an interaction of TLR4 with the unusual α- d-Gal-(1→3)- d-Gal epitope present in CGN. Activation of the ROS-mediated pathway was unaffected by treatment of κ-CGN with either κ-CGNase (3 mg/L), α-1→(3,6)-galactosidase (20 mU/ml) or these enzymes in combination, indicating that changes in IL-8 production were attributable to the effects of induction of inflammation on the TLR4–BCL10-mediated innate immune pathway. These findings provide new information about the specificity of carbohydrate–protein interaction between CGN and TLR4 and may help to devise treatments that modify the immune reactivity induced by carbohydrate antigens.