Jenifer I. Fenton

Dietary Fish Oil Alters T Lymphocyte Cell Populations and Exacerbates Disease in a Mouse Model of Inflammatory Colitis

Inflammatory bowel diseases (IBD) increase the risk of developing colorectal cancer. Dietary components that reduce inflammation are associated with lower cancer risk. The long-chain omega-3 fatty acid docosahexaenoic acid (DHA) is present in fish oil and has potent anti-inflammatory properties. The objective of this study is to determine whether dietary fish oil enriched with DHA (DFO) could reduce experimentally induced colitis and colon cancer risk in a mouse model. When SMAD3-/- mice are exposed to Helicobacter hepaticus, mild colitis is observed 4 weeks postinfection. Mice were fed isocaloric diets modified to include corn oil, safflower oil, or DFO (doses ranging from 0.75% to 6.00%) as the fatty acid source for 8 weeks. Mice were gavaged with H. hepaticus; DFO feeding was continued; and mice were sacrificed 4 weeks after infection. The colon and cecum were collected for histopathology. Spleens and mesenteric lymph nodes were collected and analyzed for T-cell populations using flow cytometry. Contrary to expectations, DFO induced severe colitis and adenocarcinoma formation. DFO consumption was associated with decreased CD8(+) cell frequency and diminished CD69 expression on CD4(+) and CD8(+) T-cell populations. Mice consuming DFO also exhibited higher FoxP3(+) CD25(+) CD4(+) T regulatory cell frequency, FoxP3 expression, and altered L-selectin expression during infection. We concluded that DFO-fed mice may be less equipped to mount a successful response to H. hepaticus infection, increasing colon cancer risk. These results support the need to establish a tolerable upper limit for DHA intake particularly in the context of chronic inflammatory conditions such as IBD.

Leptin, Insulin-Like Growth Factor-1, and Insulin-Like Growth Factor-2 Are Mitogens in ApcMin/+ but not Apc+/+ Colonic Epithelial Cell Lines

The obese state is associated with elevated circulating levels of insulin, insulin-like growth factors (IGF), and leptin. Research is contradictory regarding the role of these elevated growth factors in colon cancer risk. We hypothesized that colonic epithelial cells that were Apc deficient (ApcMin/+) but not those expressing wild-type Apc (Apc+/+) would experience a hyperproliferative and antiapoptotic phenotype when exposed to these growth factors. This hypothesis was addressed using two nontumorigenic murine colonic epithelial cell lines with distinct Apc genotypes: Apc+/+ YAMC cells and ApcMin/+ IMCE cells. Cells were treated for 48 hours with various concentrations of leptin (0.001-50 ng/mL), IGF-1 (0.1-200 ng/mL), or IGF-2 (0.1-600 ng/mL). In YAMC cells, leptin caused a significant decrease in cell proliferation (P < 0.01) compared with controls due to induction of caspase activity and apoptosis. In contrast, in the IMCE cells, leptin induced a 75% increase in cell proliferation compared with controls (P < 0.0001). IGF-1 and IGF-2 also induced 50% greater proliferation in the IMCE cells (P < 0.001) compared with controls. Cotreatment of IMCE cells with leptin and either IGF-1 or IGF-2 induced greater proliferation than either growth factor alone (P < 0.0001). IMCE cell proliferation caused by leptin only treatment was associated with activation of p42/44 mitogen-activated protein kinase (MAPK), p38 MAPK, and nuclear factor-κB nuclear translocation but not with MAPK kinase or Janus-activated kinase/signal transducers and activators of transcription activation. These data provide the first evidence that leptin may interact with IGFs to promote survival and expansion of colonic epithelial cells that were Apc deficient (ApcMin/+) but not those expressing wild-type Apc (Apc+/+).

Adiponectin blocks multiple signaling cascades associated with leptin-induced cell proliferation in ApcMin/+ colon epithelial cells

We previously demonstrated that leptin, an adipose‐derived hormone, induces cell proliferation in a model of preneoplastic (IMCE (ApcMin/+), but not normal (YAMC (Apc+/+), colon epithelial cells by inducing autocrine IL‐6 production and trans‐IL‐6 signaling. Low serum adiponectin is associated with colon, prostate and breast cancer. Adiponectin is secreted by white adipose tissue; the levels of adiponectin in the blood decrease as body mass index (and leptin) increases. In our study, we tested whether murine recombinant globular adiponectin (gArcp30) could modulate leptin‐induced cell proliferation, autocrine IL‐6 production, trans‐IL‐6 signaling and other leptin‐induced cell signaling events previously observed in IMCE cells but not YAMC cells. Under serum‐free conditions, adiponectin (1 μg/ml) inhibited leptin‐induced autocrine IL‐6 production, soluble IL‐6 receptor shedding, trans‐IL‐6 signaling and subsequent STAT3 phosphorylation in IMCE cells. Adiponectin inhibited leptin‐induced cell proliferation in the IMCE cells and this inhibition was associated with IκB‐α phosphorylation, IκB‐α degradation and decreased NF‐κB p65 DNA activation and binding. These data indicate that adiponectin acts on preneoplastic colon epithelial cells to regulate cell growth via 2 distinct pathways inhibiting leptin‐induced NF‐κB‐dependent autocrine IL‐6 production and trans‐IL‐6 signaling. We hypothesize that adiponectin may be an important regulator of colon epithelial cell homeostasis by linking the observed reduced risk for cancer in populations with high serum adiponectin concentrations to specific mechanisms of cell number homeostasis in a model of preneoplastic colon epithelial cells. These data may have broad implications for diet and lifestyle strategies for the prevention and treatment of obesity‐associated cancers.

Novel mechanism for obesity-induced colon cancer progression

Adipose tissue secretes factors linked to colon cancer risk including leptin. A hallmark of cancer is sustained angiogenesis. While leptin promotes angiogenesis in adipose tissue, it is unknown whether leptin can induce epithelial cells to produce factors that may drive angiogenesis, vascular development and therefore cancer progression. The purpose of this study was to compare the effects of leptin-stimulated colon epithelial cells differing in adenomatous polyposis coli (Apc) genotype (gatekeeper tumor suppressor gene for colon cancer) on angiogenesis. We employed novel colonic epithelial cell lines derived from the Immorto mouse [young adult mouse colon (YAMC)] and the Immorto-Min mouse [Immorto-Min colonic epithelial cell (IMCE)], which carries the Apc Min mutation, to study the effects of leptin-stimulated colon epithelial cells on angiogenesis. We utilized ex vivo rat mesenteric capillary bioassay and human umbilical vein endothelial cell (HUVEC) models to study angiogenesis. IMCE cells stimulated with leptin produced significantly more vascular endothelial growth factor (VEGF) than YAMC (268 +/- 18 versus 124 +/- 8 pg/ml; P < 0.01) cells. Leptin treatment induced dose-dependent increases in VEGF only in IMCE cells. Conditioned media from leptin (50 ng/ml)-treated IMCE cells induced significant capillary formation compared with control, which was blocked by the addition of a neutralizing antibody against VEGF. Conditioned media from leptin-treated IMCE cells also induced HUVEC cell proliferation, chemotaxis, upregulation of adhesion proteins and cell-signaling activation resulting in nuclear factor kappa B nuclear translocation and DNA binding due to VEGF. This is the first study demonstrating that leptin can induce preneoplastic colon epithelial cells to orchestrate VEGF-driven angiogenesis and vascular development, thus providing a specific mechanism and potential target for obesity-associated cancer.

Diet‐induced adiposity alters the serum profile of inflammation in C57BL/6N mice as measured by antibody array

Morbid obesity is considered a systemic inflammatory state. The objective of this project was to characterize the adipokine, cytokine and chemokine protein profile in serum from control, lean and obese mice. We hypothesized that chemokines and cytokines are altered by caloric restriction and diet‐induced obesity as a function of changes in body composition. Six‐week‐old female C57BL/6N mice (n = 12 per group) were randomized to one of three diets: control (fed ad libitum); lean (30% calorie‐restricted regimen relative to control) and diet‐induced obese (DIO; high calorie diet, fed ad libitum). Body weight, body composition and food intake were monitored throughout the study. After 10 weeks on the diets, blood samples were collected, and adipokine/cytokine/chemokine serum profiles were measured by antibody array. Lean mice, relative to the control group, displayed increased concentrations of insulin‐like growth factor (IGF) binding protein‐3, ‐5 and ‐6 and adiponectin and decreased IGF‐1. These mice also showed increased concentrations of interleukin (IL)‐10, IL‐12 p40/p70, eotaxin, monocyte chemoattractant protein‐5 and SDF‐1. In contrast, DIO mice displayed increased leptin, IL‐6 and LPS‐induced chemokine and decreased concentrations of all chemokines/cytokines measured relative to control mice. As such, these data indicate that DIO may lead to an inflammatory state characterized as a shift towards a T helper lymphocyte type 1–skewed responsiveness. The demonstration of differential adipokine, cytokine and chemokine protein profile in control, lean and DIO mice may have implications for immune responsiveness and risk of disease.

Adipokine regulation of colon cancer: adiponectin attenuates interleukin-6-induced colon carcinoma cell proliferation via STAT-3.

Obesity results in increased circulating levels of specific adipokines, which are associated with colon cancer risk. The disease state is associated with increased leptin, insulin, IGF‐1, and IL‐6. Conversely, adiponectin levels are decreased in obese individuals. Previously, we demonstrated adipokine‐enhanced cell proliferation in preneoplastic, but not normal, colon epithelial cells, demonstrating a differential effect of adipokines on colon cancer progression in vitro. Using a model of late stage carcinoma cancer cell, namely murine MC‐38 colon carcinoma cells, we compared the effect of obesity‐associated adipokines (leptin, insulin, IGF‐1, and IL‐6) on MC‐38 cell proliferation and determined whether adiponectin (full length or globular) could modulate adipokine‐induced cell proliferation. We show that insulin and IL‐6, but not leptin and IGF‐1, induce proliferation in MC‐38 cells. Adiponectin treatment of MC‐38 cells did not inhibit insulin‐induced cell proliferation but did inhibit IL‐6‐induced cell proliferation by decreasing STAT‐3 phosphorylation and activation. Nitric oxide (NO) production was increased in MC‐38 cells treated with IL‐6; co‐treatment with adiponectin blocked IL‐6‐induced iNOS and subsequent NO production. These data are compared to previously reported findings from our laboratory using the YAMC (model normal colon epithelial cells) and IMCE (model preneoplastic) cells. The cell lines are utilized to construct a model summarizing the hormonal consequences of obesity and the impact on the differential regulation of colon epithelial cells along the continuum to carcinoma. These data, taken together, highlight mechanisms involved in obesity‐associated cancers and may lead to potential‐targeted therapies.

Immunomodulation by dietary long chain omega-3 fatty acids and the potential for adverse health outcomes

Recommendations to consume fish for prevention of cardiovascular disease (CVD), along with the U.S. Food and Drug Administration-approved generally recognized as safe (GRAS) status for long chain omega-3 fatty acids, may have had the unanticipated consequence of encouraging long-chain omega-3 (ω-3) fatty acid [(eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA)] supplementation and fortification practices. While there is evidence supporting a protective role for EPA/DHA supplementation in reducing sudden cardiac events, the safety and efficacy of supplementation with LCω-3PUFA in the context of other disease outcomes is unclear. Recent studies of bacterial, viral, and fungal infections in animal models of infectious disease demonstrate that LCω-3PUFA intake dampens immunity and alters pathogen clearance and can result in reduced survival. The same physiological properties of EPA/DHA that are responsible for the amelioration of inflammation associated with chronic cardiovascular pathology or autoimmune states, may impair pathogen clearance during acute infections by decreasing host resistance or interfere with tumor surveillance resulting in adverse health outcomes. Recent observations that high serum LCω-3PUFA levels are associated with higher risk of prostate cancer and atrial fibrillation raise concern for adverse outcomes. Given the widespread use of supplements and fortification of common food items with LCω-3PUFA, this review focuses on the immunomodulatory effects of the dietary LCω-3PUFAs, EPA and DHA, the mechanistic basis for potential negative health outcomes, and calls for biomarker development and validation as rational first steps towards setting recommended dietary intake levels.

DHA-enriched fish oil targets B cell lipid microdomains and enhances ex vivo and in vivo B cell function

DHA is a n‐3 LCPUFA in fish oil that generally suppresses T lymphocyte function. However, the effect of fish oil on B cell function remains relatively understudied. Given the important role of B cells in gut immunity and increasing human fish oil supplementation, we sought to determine whether DFO leads to enhanced B cell activation in the SMAD−/− colitis‐prone mouse model, similar to that observed with C57BL/6 mice. This study tested the hypothesis that DHA from fish oil is incorporated into the B cell membrane to alter lipid microdomain clustering and enhance B cell function. Purified, splenic B cells from DFO‐fed mice displayed increased DHA levels and diminished GM1 microdomain clustering. DFO enhanced LPS‐induced B cell secretion of IL‐6 and TNF‐α and increased CD40 expression ex vivo compared with CON. Despite increased MHCII expression in the unstimulated ex vivo B cells from DFO‐fed mice, we observed no difference in ex vivo OVA‐FITC uptake in B cells from DFO or CON mice. In vivo, DFO increased lymphoid tissue B cell populations and surface markers of activation compared with CON. Finally, we investigated whether these ex vivo and in vivo observations were consistent with systemic changes. Indeed, DFO‐fed mice had significantly higher plasma IL‐5, IL‐13, and IL‐9 (Th2‐biasing cytokines) and cecal IgA compared with CON. These results support the hypothesis and an emerging concept that fish oil enhances B cell function in vivo.

Adipokines and obesity are associated with colorectal polyps in adult males: A cross-sectional study

Background Obesity increases the risk of colon cancer. It is also known that most colorectal cancers develop from adenomatous polyps. However, the effects of obesity and adipokines on colonic polyp formation are unknown. Methods To determine if BMI, waist circumference or adipokines are associated with colon polyps in males, 126 asymptomatic men (48–65 yr) were recruited at time of colonoscopy, and anthropometric measures as well as blood were collected. Odds ratios were determined using polytomous logistic regression for polyp number (0 or ≥3) and polyp type (no polyp, hyperplastic polyp, tubular adenoma). Results 41% of the men in our study were obese (BMI ≥30). The odds of an obese individual having ≥3 polyps was 6.5 (CI: 1.3–33.0) times greater than those of a lean (BMI<25) individual. Additionally, relative to lean individuals, obese individuals were 7.8 (CI: 2.0–30.8) times more likely to have a tubular adenoma than no polyp. As BMI category increased, participants were 2.9 (CI: 1.5–5.4) times more likely to have a tubular adenoma than no polyps. Serum leptin, IP-10 and TNF-α were significantly associated with tubular adenoma presence. Serum leptin and IP-10 were significantly associated with increased likelihood of ≥3 polyps, and TNF-α showed a trend (p = 0.09). Conclusions Obese men are more likely to have at least three polyps and adenomas. This cross-sectional study provides evidence that colonoscopy should be recommended for obese, white males.

Energy Balance Modulates Colon Tumor Growth: Interactive Roles of Insulin and Estrogen

Obesity increases colorectal cancer (CRC) risk and progression. However, the impact of obesity on CRC in women is dependent on ovarian hormone status. The purpose of this study was to determine the interactive roles of obesity and ovarian hormones on serum markers of inflammation, cell signaling, and transplanted colon tumor growth. Female C57BL/6 mice (6 wk) were either ovariectomized (OVX) or ovaries left intact (nonovariectomized, NOVX) and randomized to receive a (1) control, (2) 30% calorie‐restricted (CR), or (3) diet‐induced obese (DIO) diet regimen for 20 wk to induce differing levels of adiposity. Serum was collected and inflammatory and metabolic markers were measured using an antibody array (62 proteins) and ELISAs. Mice were subcutaneously injected with syngeneic MC38 colon cancer cells after 20 wk and sacrificed 4 wk later. CR mice had the smallest tumors irrespective of hormone status, whereas the largest tumors were observed in DIO‐OVX mice. Glucose tolerance was impaired in OVX mice, being most severe in the DIO‐OVX group. Cytokine arrays suggested that in CR animals, inhibition of tumor growth paralleled insulin sensitivity and associated changes in leptin, adiponectin, and IGF‐BPs. Conversely, in DIO‐OVX animals, tumor growth was associated with insulin and leptin resistance as well as higher levels of pro‐inflammatory proteins. In vitro, leptin and adiponectin had no effect, whereas insulin induced MC38 cell proliferation and MAPK activation. Co‐treatment with estrogen blocked the stimulatory effects of insulin. Thus, our in vitro and in vivo data indicate female reproductive hormones have a modulating effect on obesity‐induced insulin resistance and inflammation, which may directly or indirectly influence CRC progression. ©2010 Wiley‐Liss, Inc.