Effects of Dietary Fiber on Short Chain Fatty Acid Receptor mRNA in Microglia and Serotonergic Neurons in the Mouse Brain.

Short-chain fatty acids (SCFAs) are bioactive lipids that are released into the colon as a metabolite of bacterial fermentation of dietary fibers. Beyond their function in the gastrointestinal tract, SCFAs can also have effects inthe brain, as a part of the gut-brain axis. Recent investigations into potential therapeutic interventions via the manipulation of the gut microbiome-and thus their SCFA metabolites-has been emerging as a new branch of personalized medicine,especially for mental health conditions. The current study sought to measure and localize SCFA receptors in the mouse brain. Two cell types have been implicated in the gut-brain axis: microglia and serotonergic neurons. We used fluorescentin situhybridization in brain sections from mice fed diets with different compositions of fat and fiber to quantify the mRNA levels of known gene markers of these two cell types and colocalize each with mRNA for free fatty acid receptors that bind SCFAs. We focused onmicroglia in the hippocampus and the serotonergic neurons of the dorsal raphe. We found high colocalization of SCFA receptors in both microglia and serotonergic neurons and discovered that SCFA receptor expression in the dorsal raphe is driven by fiber solubility, while SCFA receptor expression in the hippocampus is driven by fiber amount. Higher dietary fiber was associated with decreased tyrosine hydroxylase expression. Thus, our results indicate that the amount and solubility of dietary fiber can change gene expression in the brain's microglia and serotonin neurons, potentially via sensitivity to circulating levels of SCFAs produced in the gut.

Basal Diet Fed to Recipient Mice Was the Driving Factor for Colitis and Colon Tumorigenesis, despite Fecal Microbiota Transfer from Mice with Severe or Mild Disease.

Consumption of the total Western diet (TWD) in mice has been shown to increase gut inflammation, promote colon tumorigenesis, and alter fecal microbiome composition when compared to mice fed a healthy diet, i.e., AIN93G (AIN). However, it is unclear whether the gut microbiome contributes directly to colitis-associated CRC in this model. The objective of this study was to determine whether dynamic fecal microbiota transfer (FMT) from donor mice fed either the AIN basal diet or the TWD would alter colitis symptoms or colitis-associated CRC in recipient mice, which were fed either the AIN diet or the TWD, using a 2 × 2 factorial experiment design. Time-matched FMT from the donor mice fed the TWD did not significantly enhance symptoms of colitis, colon epithelial inflammation, mucosal injury, or colon tumor burden in the recipient mice fed the AIN diet. Conversely, FMT from the AIN-fed donors did not impart a protective effect on the recipient mice fed the TWD. Likewise, the composition of fecal microbiomes of the recipient mice was also affected to a much greater extent by the diet they consumed than by the source of FMT. In summary, FMT from the donor mice fed either basal diet with differing colitis or tumor outcomes did not shift colitis symptoms or colon tumorigenesis in the recipient mice, regardless of the basal diet they consumed. These observations suggest that the gut microbiome may not contribute directly to the development of disease in this animal model.

Dietary Supplementation with Black Raspberries Altered the Gut Microbiome Composition in a Mouse Model of Colitis-Associated Colorectal Cancer, although with Differing Effects for a Healthy versus a Western Basal Diet.

Black raspberries (BRB) are rich in anthocyanins with purported anti-inflammatory properties. However, it is not known whether dietary supplementation would ameliorate Western-diet enhanced gut inflammation and colon tumorigenesis. We employed a mouse model of colitis-associated colorectal cancer (CAC) to determine the effects of dietary supplementation with 5 to 10% (w/w) whole, freeze-dried BRB in male C57BL/6J mice fed either a standard healthy diet (AIN93G) or the total Western diet (TWD). In a pilot study, BRB suppressed colitis and colon tumorigenesis while also shifting the composition of the fecal microbiome in favor of taxa with purported health benefits, including Bifidobacterium pseudolongum. In a follow-up experiment using a 2 × 2 factorial design with AIN and TWD basal diets with and without 10% (w/w) BRB, supplementation with BRB reduced tumor multiplicity and increased colon length, irrespective of the basal diet, but it did not apparently affect colitis symptoms, colon inflammation or mucosal injury based on histopathological findings. However, BRB intake increased alpha diversity, altered beta diversity and changed the relative abundance of Erysipelotrichaceae, Bifidobacteriaceae, Streptococcaceae, Rikenellaceae, Ruminococcaceae and Akkermansiaceae, among others, of the fecal microbiome. Notably, changes in microbiome profiles were inconsistent with respect to the basal diet consumed. Overall, these studies provide equivocal evidence for in vivo anti-inflammatory effects of BRB on colitis and colon tumorigenesis; yet, BRB supplementation led to dynamic changes in the fecal microbiome composition over the course of disease development.

Resistant Potato Starch Alters the Cecal Microbiome and Gene Expression in Mice Fed a Western Diet Based on NHANES Data.

Several studies indicate that the four major types of resistant starch (RS1-4) are fermented in the cecum and colon to produce short-chain fatty acids (SCFAs) and can alter the microbiome and host physiology. However, nearly all these studies were conducted in rodents fed with a diet that does not approximate what is typically consumed by humans. To address this, mice were fed a Total Western Diet (TWD) based on National Health and Nutrition Examination Survey (NHANES) data that mimics the macro and micronutrient composition of a typical American diet for 6 weeks and then supplemented with 0, 2, 5, or 10% of the RS2, resistant potato starch (RPS), for an additional 3 weeks. The cecal microbiome was analyzed by 16S sequencing. The alpha-diversity of the microbiome decreased with increasing consumption of RPS while a beta-diversity plot showed four discreet groupings based on the RPS level in the diet. The relative abundance of various genera was altered by feeding increasing levels of RPS. In particular, the genus Lachnospiraceae NK4A136 group was markedly increased. Cecal, proximal, and distal colon tissue mRNA abundance was analyzed by RNASeq. The cecal mRNA abundance principal component analysis showed clear segregation of the four dietary groups whose separation decreased in the proximal and distal colon. Differential expression of the genes was highest in the cecum, but substantially decreased in the proximal colon (PC) and distal colon (DC). Most differentially expressed genes were unique to each tissue with little overlap in between. The pattern of the observed gene expression suggests that RPS, likely through metabolic changes secondary to differences in microbial composition, appears to prime the host to respond to a range of pathogens, including viruses, bacteria, and parasites. In summary, consumption of dietary RPS led to significant changes to the microbiome and gene expression in the cecum and to a lesser extent in the proximal and distal colon.

Excess Vitamins or Imbalance of Folic Acid and Choline in the Gestational Diet Alter the Gut Microbiota and Obesogenic Effects in Wistar Rat Offspring.

Excess vitamin intake during pregnancy leads to obesogenic phenotypes, and folic acid accounts for many of these effects in male, but not in female, offspring. These outcomes may be modulated by another methyl nutrient choline and attributed to the gut microbiota. Pregnant Wistar rats were fed an AIN-93G diet with recommended vitamin (RV), high 10-fold multivitamin (HV), high 10-fold folic acid with recommended choline (HFol) or high 10-fold folic acid without choline (HFol-C) content. Male and female offspring were weaned to a high-fat RV diet for 12 weeks post-weaning. Removing choline from the HFol gestational diet resulted in obesogenic phenotypes that resembled more closely to HV in male and female offspring with higher body weight, food intake, glucose response to a glucose load and body fat percentage with altered activity, concentrations of short-chain fatty acids and gut microbiota composition. Gestational diet and sex of the offspring predicted the gut microbiota differences. Differentially abundant microbes may be important contributors to obesogenic outcomes across diet and sex. In conclusion, a gestational diet high in vitamins or imbalanced folic acid and choline content contributes to the gut microbiota alterations consistent with the obesogenic phenotypes of in male and female offspring.

The Western Dietary Pattern Combined with Vancomycin-Mediated Changes to the Gut Microbiome Exacerbates Colitis Severity and Colon Tumorigenesis.

Previous work by our group using a mouse model of inflammation-associated colorectal cancer (CAC) showed that the total Western diet (TWD) promoted colon tumor development. Others have also shown that vancomycin-mediated changes to the gut microbiome increased colorectal cancer (CRC). Therefore, the objective of this study was to determine the impact of vancomycin on colon tumorigenesis in the context of a standard mouse diet or the TWD. A 2 × 2 factorial design was used, in which C57Bl/6J mice were fed either the standard AIN93G diet or TWD and with vancomycin in the drinking water or not. While both the TWD and vancomycin treatments independently increased parameters associated with gut inflammation and tumorigenesis compared to AIN93G and plain water controls, mice fed the TWD and treated with vancomycin had significantly increased tumor multiplicity and burden relative to all other treatments. Vancomycin treatment significantly decreased alpha diversity and changed the abundance of several taxa at the phylum, family, and genus levels. Conversely, basal diet had relatively minor effects on the gut microbiome composition. These results support our previous research that the TWD promotes colon tumorigenesis and suggest that vancomycin-induced changes to the gut microbiome are associated with higher tumor rates.

Effect of supplemental prebiotics, probiotics and bioactive proteins on the microbiome composition and fecal calprotectin in C57BL6/j mice.

The composition and metabolic activity of the microbiome affect many aspects of health, and there is current interest in dietary constituents that may affect this system. The purpose of this study was to evaluate the effects of a mix of probiotics, a mix of prebiotics and a bioactive protein fraction on the microbiome, when fed to mice alone and in combination at physiologically relevant doses. Mice were fed the total western diet (TWD) supplemented with prebiotics, probiotics, and bioactive proteins individually and in combination for four weeks. Subsequently, effects on the composition of the gut microbiome, gut short-chain fatty acids (SCFAs) concentration, and gut inflammation were measured. Ruminococcus gnavus was increased in mice gut microbiome after feeding prebiotics. Bifidobacterium longum was increased after feeding probiotics. The treatments significantly affected beta-diversity with minor treatment effects on cecal or fecal SCFAs levels, and the treatments did not affect gut inflammation as measured by fecal calprotectin.

Food matrix and the microbiome: considerations for preclinical chronic disease studies.

Animal models of chronic disease are continuously being refined and have evolved with the goal of increasing the translation of results to human populations. Examples of this progress include transgenic models and germ-free animals conventionalized with human microbiota. The gut microbiome is involved in the etiology of several chronic diseases. Therefore, consideration of the experimental conditions that may affect the gut microbiome in preclinical disease is very important. Of note, diet plays a large role in shaping the gut microbiome and can be a source of variation between animal models and human populations. Traditionally, nutrition researchers have focused on manipulating the macronutrient profile of experimental diets to model diseases such as metabolic syndrome. However, other dietary components found in human foods, but not in animal diets, can have sizable effects on the composition and metabolic capacity of the gut microbiome and, as a consequence, manifestation of the chronic disease being modeled. The purpose of this review is to describe how food matrix food components, including diverse fiber sources, oxidation products from cooking, and dietary fat emulsifiers, shape the composition of the gut microbiome and influence gut health.

Consumption of the Total Western Diet Promotes Colitis and Inflammation-Associated Colorectal Cancer in Mice.

Consumption of a Western type diet is a known risk factor for colorectal cancer. Our group previously developed the total Western diet (TWD) for rodents with energy and nutrient profiles that emulate a typical Western diet. In this study, we tested the hypothesis that consumption of the TWD would enhance colitis, delay recovery from gut injury and promote colon tumorigenesis. In multiple experiments using the azoxymethane + dextran sodium sulfate or ApcMin/+ mouse models of colitis-associated colorectal carcinogenesis (CAC), we determined that mice fed TWD experienced more severe and more prolonged colitis compared to their counterparts fed the standard AIN93G diet, ultimately leading to markedly enhanced colon tumorigenesis. Additionally, this increased tumor response was attributed to the micronutrient fraction of the TWD, and restoration of calcium and vitamin D to standard amounts ameliorated the tumor-promoting effects of TWD. Finally, exposure to the TWD elicited large scale, dynamic changes in mRNA signatures of colon mucosa associated with interferon (IFN) response, inflammation, innate immunity, adaptive immunity, and antigen processing pathways, among others. Taken together, these observations indicate that consumption of the TWD markedly enhanced colitis, delayed recovery from gut injury, and enhanced colon tumorigenesis likely via extensive changes in expression of immune-related genes in the colon mucosa.

Basal Diet Determined Long-Term Composition of the Gut Microbiome and Mouse Phenotype to a Greater Extent than Fecal Microbiome Transfer from Lean or Obese Human Donors.

The Western dietary pattern can alter the gut microbiome and cause obesity and metabolic disorders. To examine the interactions between diet, the microbiome, and obesity, we transplanted gut microbiota from lean or obese human donors into mice fed one of three diets for 22 weeks: (1) a control AIN93G diet; (2) the total Western diet (TWD), which mimics the American diet; or (3) a 45% high-fat diet-induced obesity (DIO) diet. We hypothesized that a fecal microbiome transfer (FMT) from obese donors would lead to an obese phenotype and aberrant glucose metabolism in recipient mice that would be exacerbated by consumption of the TWD or DIO diets. Prior to the FMT, the native microbiome was depleted using an established broad-spectrum antibiotic protocol. Interestingly, the human donor body type microbiome did not significantly affect final body weight or body composition in mice fed any of the experimental diets. Beta diversity analysis and linear discriminant analysis with effect size (LEfSe) showed that mice that received an FMT from obese donors had a significantly different microbiome compared to mice that received an FMT from lean donors. However, after 22 weeks, diet influenced the microbiome composition irrespective of donor body type, suggesting that diet is a key variable in the shaping of the gut microbiome after FMT.

Modeling the Western Diet for Preclinical Investigations.

Rodent models have been invaluable for biomedical research. Preclinical investigations with rodents allow researchers to investigate diseases by using study designs that are not suitable for human subjects. The primary criticism of preclinical animal models is that results are not always translatable to humans. Some of this lack of translation is due to inherent differences between species. However, rodent models have been refined over time, and translatability to humans has improved. Transgenic animals have greatly aided our understanding of interactions between genes and disease and have narrowed the translation gap between humans and model animals. Despite the technological innovations of animal models through advances in genetics, relatively little attention has been given to animal diets. Namely, developing diets that replicate what humans eat will help make animal models more relevant to human populations. This review focuses on commonly used rodent diets that are used to emulate the Western dietary pattern in preclinical studies of obesity and type 2 diabetes, nonalcoholic liver disease, maternal nutrition, and colorectal cancer.

Consumption of the total Western diet differentially affects the response to green tea in rodent models of chronic disease compared to the AIN93G diet.

SCOPE: In pre-clinical studies investigating bioactive components, the efficacy of the bioactive is likely influenced by the basal diet provided to rodents. In this study, we hypothesized that a model bioactive, green tea extract (GTE), would have different effects on colon carcinogenesis, body composition, and lipid metabolism in mice fed a basal diet formulated to promote animal health and growth (AIN93G) as compared to a Western diet that emulates typical American intakes of micro- and macronutrients, the total Western diet (TWD). METHODS AND RESULTS: Mice were fed either AIN93G or TWD, with or without GTE added to drinking water for 18 weeks. Aberrant crypt foci (ACF) in azoxymethane-initiated mice was nearly three times greater in mice fed TWD compared to AIN93G. Consumption of GTE suppressed ACF development only in mice fed the TWD. Similarly, supplementation with GTE suppressed weight gain and fasted glucose only in mice fed TWD, while GTE suppressed fat mass in mice fed either diet. Irrespective of diet, GTE supplementation increased cecum weight and decreased cecal SCFA concentration. CONCLUSION: Collectively, these observations indicate that the TWD influences the bioactivity of GTE in rodent models of obesity, metabolism, and carcinogenesis.

The new total Western diet for rodents does not induce an overweight phenotype or alter parameters of metabolic syndrome in mice.

In this study, we determined the impact of the total Western diet (TWD) for rodents and its macro- and micronutrient components on weight gain and biomarkers of metabolic function in mice compared to a 45% fat diet-induced obesity (DIO) diet and the standard AIN93G diet. We hypothesized that mice fed the TWD would have increased body fat with indicators of metabolic syndrome similar to mice consuming the DIO diet. As expected, DIO-fed mice acquired a metabolic syndrome phenotype typified by increased energy intake, increased body weight gain, increased fat mass, higher fasting glucose, impaired glucose tolerance, and higher plasma leptin relative to the AIN93G diet. Mice fed a macronutrient-modified (MM) diet (with standard vitamin and mineral composition) had a similar response, albeit to a lesser degree than mice fed the DIO diet. Mice fed a vitamin- and mineral-modified diet (with standard macronutrient composition) were not different from mice fed the AIN93G diet. Surprisingly, the TWD (with modified macronutrients, vitamins and minerals) did not significantly affect any of these parameters, despite the fact that the TWD macronutrient profile was identical to the MM diet. These data suggest that, in the context of the TWD, vitamin and mineral intakes in mice that reflect a Western dietary pattern inhibit the hyperphagia and resulting increased weight gain associated with the higher fat content of the TWD. In conclusion, these observations underscore the need to consider the influence of micronutrient intakes in pre-clinical models of obesity and metabolic syndrome.

Broad scope method for creating humanized animal models for animal health and disease research through antibiotic treatment and human fecal transfer.

Traditionally, mouse humanization studies have used human fecal transfer to germ-free animals. This practice requires gnotobiotic facilities and is restricted to gnotobiotic mouse lines, which limits humanized mouse research. We have developed a generalizable method to humanize non germ-free mice using antibiotic treatment and human fecal transfer. The method involves depleting resident intestinal microbiota with broad-spectrum antibiotics, introducing human microbiota from frozen fecal samples by weekly gavage, and maintaining mice in HEPA-filtered microisolator cages. Pyrosequencing cecal microbiota 16S rRNA genes showed that recipient mice adopt a humanized microbiota profile analogous to their human donors, and distinct from mice treated with only antibiotics (no fecal transfer) or untreated control mice. In the humanized mice, 75% of the sequence mass was observed in their respective human donor and conversely, 68% of the donor sequence mass was recovered in the recipient mice. Principal component analyses of GC- and HPLC-separated cecal metabolites were performed to determine effects of transplanted microbiota on the metabolome. Cecal metabolite profiles of mice treated with only antibiotics (no fecal transfer) and control mice were dissimilar from each other and from humanized mice. Metabolite profiles for mice humanized from different donor samples clustered near each other, yet were sufficiently distinct that separate clusters were apparent for each donor. Also, cecal concentrations of 57 metabolites were significantly different between humanization treatments. These data demonstrate that our protocol can be used to humanize non germ-free mice and is sufficiently robust to generate metabolomic differences between mice humanized from different human donors.

Dietary fat composition influences tissue lipid profile and gene expression in Fischer-344 rats.

The AIN-76A diet causes fatty liver in rodents when fed for long periods of time. The aim of this study was to utilize fatty acid analysis and transcriptomics to investigate the effects of different fat sources in the AIN-76A diet on tissue lipid profiles and gene expression in male, weanling Fischer-344 rats. Animals were fed isocaloric diets that differed only in the fat source: (1) corn oil (CO) (2) anhydrous milk fat (AMF), and (3) AMF supplemented with 10% phospholipids from the milk fat globule membrane (AMF-MFGM). There were no differences in food intake, body weight, growth rate, or body fat composition among the groups, and the fatty acid compositions of red blood cells (RBC), plasma, muscle, and visceral adipose tissues reflected the dietary fat sources. Modifying the fat source resulted in 293 genes differentially regulated in skeletal muscle, 1,124 in adipose, and 831 in liver as determined by analysis of variance (ANOVA). Although tissue fatty acid profiles mostly reflected the diet, there were several quantitative differences in lipid classes in the liver and plasma. The AMF diet resulted in the highest level of hepatic triacylglycerols, but the lowest level in plasma. The CO diet resulted in significant accumulation of hepatic unesterified fatty acids and decreased DGAT expression and activity, a potential trigger for steatohepatitis. These results indicate that the fatty acid composition and presence of polar lipids in the AIN-76A diets have significant effects on lipid partitioning, gene expression, and potentially the development of liver pathology.

Role of the 1,25D3-MARRS receptor in the 1,25(OH)2D3-stimulated uptake of calcium and phosphate in intestinal cells.

We have used mice with a targeted knockout (KO) of the 1,25D(3)-MARRS receptor (ERp57/PDIA3) in intestine to study rapid responses to 1,25-dihydroxyvitamin D(3) [1,25D(3)] with regards to calcium or phosphate uptake. Western analyses indicated the presence of the 1,25D(3)-MARRS receptor in littermate (LM) mice, but not KO mice. Saturation analyses for [(3)H]1,25D(3) binding revealed comparable affinities for the hormone in lysates from female and male LM, but a reduced B(max) in females. Binding in lysates from KO mice was absent or severely reduced. Enterocytes from KO mice failed to respond to hormone with regard to either ion uptake, while cells from LM mice exhibited an increase in uptake. For calcium uptake, the protein kinase (PK) A pathway mediated the response to 1,25D(3). Enterocytes from LM mice responded to 1,25D(3) with enhanced PKA activity, while cells from KO mice did not, although both cell types responded to forskolin. Calcium transport in LM mice in vivo was greater than in KO mice. Cells from LM and KO mice had cell surface VDR; however, anti-VDR antibodies had no effect on ion uptake. Unlike chicks, the PKC pathway was not involved in phosphate uptake. As in chicks and rats, intestinal cells from adult male mice lost the ability to respond to 1,25D(3) with enhanced phosphate uptake, whereas in female mice, uptake in cells from adults was greater than that observed in young mice. Finally, when we tested phosphate uptake in vivo, we found that young female mice had a much greater rate of transport than young male mice.

Formulation of the Total Western Diet (TWD) as a basal diet for rodent cancer studies.

Rodent cancer studies typically use defined diets with nutrient profiles optimized for rodent health. However, a defined rodent diet that represents typical American nutrition in all aspects, including calorie sources and macro- and micronutrient composition, is not yet available. Thus, a nutrient density approach was used to formulate the new Total Western Diet (TWD) based on NHANES data for macro- and micronutrient intakes. The TWD has fewer calories from protein and carbohydrate sources and twice that from fat as compared to the AIN-93 diet. The new diet contains more saturated and monounsaturated fats, less polyunsaturated fat, fewer complex carbohydrates, and twice the level of simple sugars. The TWD includes less calcium, copper, folate, thiamin, and vitamins B6, B12, D, and E, but much more sodium. This newly devised diet that better represents typical American nutrition will be highly useful for studies employing animal models of human disease, including cancer.

Hepcidin: A Critical Regulator of Iron Metabolism during Hypoxia.

Iron status affects cognitive and physical performance in humans. Recent evidence indicates that iron balance is a tightly regulated process affected by a series of factors other than diet, to include hypoxia. Hypoxia has profound effects on iron absorption and results in increased iron acquisition and erythropoiesis when humans move from sea level to altitude. The effects of hypoxia on iron balance have been attributed to hepcidin, a central regulator of iron homeostasis. This paper will focus on the molecular mechanisms by which hypoxia affects hepcidin expression, to include a review of the hypoxia inducible factor (HIF)/hypoxia response element (HRE) system, as well as recent evidence indicating that localized adipose hypoxia due to obesity may affect hepcidin signaling and organismal iron metabolism.

Antioxidants in foods: state of the science important to the food industry.

Antioxidant foods and ingredients are an important component of the food industry. In the past, antioxidants were used primarily to control oxidation and retard spoilage, but today many are used because of putative health benefits. However, the traditional message that oxidative stress, which involves the production of reactive oxygen species (ROS), is the basis for chronic diseases and aging is being reexamined. Accumulating evidence suggests that ROS exert essential metabolic functions and that removal of too many ROS can upset cell signaling pathways and actually increase the risk of chronic disease. It is imperative that the food industry be aware of progress in this field to present the science relative to foods in a forthright and clear manner. This may mean reexamining the health implications of adding large amounts of antioxidants to foods.

Dietary milk fat globule membrane reduces the incidence of aberrant crypt foci in Fischer-344 rats.

Milk fat globule membrane (MFGM) is a biopolymer composed primarily of membrane proteins and lipids that surround the fat globules in milk. Although it is considered to have potential as a bioactive ingredient, few feeding studies have been conducted to measure its potential benefits. The aim of this investigation was to determine if dietary MFGM confers protection against colon carcinogenesis compared to diets containing corn oil (CO) or anhydrous milk fat (AMF). Male, weanling Fischer-344 rats were randomly assigned to one of three dietary treatments that differed only in the fat source: (1) AIN-76A diet, corn oil; (2) AIN-76A diet, AMF; and (3) AIN-76A diet, 50% MFGM, 50% AMF. Each diet contained 50 g/kg diet of fat. With the exception of the fat source, diets were formulated to be identical in macro and micro nutrient content. Animals were injected with 1,2-dimethylhydrazine once per week at weeks 3 and 4, and fed experimental diets for a total of 13 weeks. Over the course of the study dietary treatment did not affect food consumption, weight gain or body composition. After 13 weeks animals were sacrificed, colons were removed and aberrant crypt foci (ACF) were counted by microscopy. Rats fed the MFGM diet (n = 16) had significantly fewer ACF (20.9 +/- 5.7) compared to rats fed corn oil (n = 17) or AMF (n = 16) diets (31.3 +/- 9.5 and 29.8 +/- 11.4 respectively; P < 0.05). Gene expression analysis of colonic mucosa did not reveal differential expression of candidate colon cancer genes, and the sphingolipid profile of the colonic mucosa was not affected by diet. While there were notable and significant differences in plasma and red blood cell lipids, there was no relationship to the cancer protection. These results support previous findings that dietary sphingolipids are protective against colon carcinogenesis yet extend this finding to MFGM, a milk fat fraction available as a food ingredient.