Obesity is a major risk factor for colon cancer development in rodents and humans 
. Chronic high fat consumption is a primary cause of obesity and insulin resistance; however, dietary proteins and carbohydrates also have an impact on resultant metabolic and endocrine consequences and thus, potentially on colon cancer risk 
. Previous work using rodent models found inhibitory effects of dietary SPI on: a) colon and mammary cancers, b) hepatosteatosis, c) adipose tissue deposition, and d) mammary and liver ME1 expression 
. These published data suggested a linkage between metabolism, adiposity and tumorigenic status, possibly involving ME1. The present study is the first to evaluate the individual and combined effects of anti-obesogenic soy protein isolate intake and ME1 deficiency on systemic and tissue parameters that are known to affect propensity for colon cancer development. A particular focus was on whether ME1 mediates any of the effects of SPI.
We used male mice for our studies since the obesity-colon cancer relationship (i.e., relative risk) in humans is stronger for men than women 
. Our results showed that the ME1 gene as well as SPI diet, significantly impact the endocrine profile of mice when challenged with obesogenic diet, and which potentially affects colon cancer propensity. Both consumption of SPI and the absence of ME1 protein (MOD-1 mice) conferred protection against development of high fat diet-induced adiposity and its attendant putative pro-tumorigenic endocrine environment. This was reflected in the observed reductions in body weights, liver and fat tissue weights, serum insulin and leptin concentrations, leptin/adiponectin ratios, hepatosteatosis, and white adipose cell size, relative to WT-CAS-HF-fed mice. These data extend previous research on the anti-obesogenic effects of soy-based diets in rodent models 
and the MOD-1 mouse phenotype 
Systemic hormonal factors are presumed to be important for tumor development by virtue of their induction/repression of genes and proteins associated with cell survival, proliferation, apoptosis and autophagy 
. Our analysis of FASN, ME1, mTOR, Cyclin D1 and Ki67 gene expression revealed significant changes associated with dietary protein type and presence or absence of ME1, but within specific tissue contexts. These responses manifested regional variations (colon vs.
jejunum) which most likely reflected differential responses to the local and systemic environments, as well as differences in specific tissue architecture and cell physiology. mTOR is an important signal transducer downstream of the PI3K/AKT pathway that mediates the effects of multiple growth factors, is up-regulated and highly active in multiple cancers including those of the colon, and is a drug target for cancer therapeutics 
. FASN is robustly expressed in many solid tumors and early tumorigenic lesions in the colon and supports tumor cell proliferation and survival through enhanced lipogenesis and anti-apoptosis 
. Given that FASN and mTOR are mediators of insulin action 
and ME1 has been proposed to augment insulin secretion 
, the decrease in proliferation-associated gene expression with dietary SPI and loss of ME1 is consistent with attenuated insulin action in distal colon and jejunum. The observed reduction in colon mTOR gene expression with dietary SPI also may also be related to levels of leptin, adiponectin and/or other serum factors; alternatively, it may reflect inhibition by as yet unknown bioactive component(s) in SPI 
. With regard to the above, leptin is known to induce/activate mTOR via the PI3K/AKT pathway 
, whereas adiponectin has been shown to suppress this gene through the AMPK pathway 
. Soy isoflavones, at high levels, can induce apoptosis and growth arrest of cells through direct inhibition of mTOR in certain cancer cell lines 
. Taken together, results suggest that the endocrine status associated with dietary SPI and/or ME1 null genotype exerts considerable influence on the expression of key proliferation and lipogenic genes in the colon and small intestine.
Serum insulin and its more stable surrogate biomarker, C-peptide, are significant risk factors for colon cancer in both men and women 
. Exogenous insulin increased tumor incidence and tumor multiplicity in rats given the intestinal carcinogen azoxymethane 
. In a previous study from our laboratory, lifetime consumption of SPI lowered: tumor incidence, body weight accretion and serum insulin and leptin levels in a non-obese, rat model of colon cancer 
. Serum leptin is a significant risk factor for human colon cancer 
and leptin enhanced colon tumor cell proliferation in obese mouse models 
. Adiponectin, in contrast, is a suppressor of intestinal tumorigenesis and intestinal epithelial cell proliferation in rat and mouse models 
. A high leptin:adiponectin ratio has been correlated with increased risk for developing insulin resistance in obese and non-obese individuals 
. The decrease in leptin:adiponectin ratios in MOD-1 and SPI-HF-fed WT mice, compared to CAS-HF- fed WT mice, further substantiates the linkage between the cancer-preventive effects of SPI consumption and reduction in ME1 expression. However, dietary SPI did not mimic the effects of loss of ME1 on all serum and tissue parameters we evaluated in MOD-1 mice. For example, we did not observe reductions in ME1 expression in jejunum and distal colon of WT mice fed HF-SPI, relative to WT mice fed CAS-HF. Rather, SPI caused a reduction in ME1 expression only in retroperitoneal fat, indicating a tissue-specific effect of SPI. This reduced ME1 expression was correlated with lower FASN expression and adipocyte size, consistent with a previous publication 
. Similarly, absence of ME1 in MOD-1 mice conferred a state of reduced lipogenesis in fat, as inferred from diminished FASN expression and cell size. Interestingly, SPI induced ME1 expression in livers of WT mice, similar to what we previously reported in genetically obese rats 
. However, this induction was not associated with increased FASN gene expression or hepatosteatosis, which suggests that the anti-steatotic effect of SPI is via another pathway(s) (such as fatty acid oxidation) or gene. Nonetheless, MOD-1 mice had significant reductions in lipogenic enzyme gene expression and steatosis in the liver, suggesting direct or indirect ME1 action. In contrast, colon ME1 is likely not a direct target of SPI (and its associated endocrine milieu); rather, SPI may act indirectly, most probably through adipose-expressed ME1 to influence adiposity and growth and tumorigenic potential of the distal colon (model in ).
A proposed integrative model for how dietary soy protein and ME1 null mutation may affect colon cancer risk/propensity.
MOD-1 mice fed either diet had shallower crypts than WT mice. This points to a direct or indirect stimulatory effect of ME1 on colon stem-progenitor cell proliferation, although these possibilities remain speculative at present. Recently, O’Brien et al. reported that the Drosophila intestine adapts to feeding conditions, by altered intestinal stem cell dynamics as a consequence of perturbations in gastrointestinal insulin levels 
. Overfeeding increased local insulin production, which led to a significant increase in gut dimensions, whereas fasting reduced insulin levels and gut dimensions. Although MOD-1 mice had a marked reduction in serum insulin levels, this can only partially explain the differences in crypt depth, since SPI, which significantly lowered serum insulin levels in WT mice, had no effect on colon crypt depth. Given the higher IRS1 and IRS2 mRNA expression in white adipose tissue of MOD-1 mice, indicative of increased insulin sensitivity 
, in MOD-1 mice, but not in that of WT mice fed SPI-HF diet, our data suggest that enhanced insulin sensitivity may lead to reduced steady-state insulin levels and subsequently, attenuated proliferative state within the GI tract with loss of ME1.
In summary, the absence of functional ME1 protein affects body weight, adiposity, endocrine profile and colon crypt depth of male mice fed a high fat diet. Dietary soy protein consumption elicited favorable physiological effects including reduced body weights and retroperitoneal adiposity. Both the absence of ME1 as well as SPI consumption led to reduced lipogenic gene expression in retroperitoneal adipose tissue and which undoubtedly contributed to the changes in adiposity. We speculate that colon and jejunum are tissue targets of these favorable effects. However, many of the associations observed here are correlative and thus, require experimental confirmation. In this regard, future studies using tissue-specific null alleles of ME1 may provide a way to confirm or refute various aspects of the integrative model presented in . The collective data suggest that dietary intervention by soy-based diet, and/or pharmacologic targeting of ME1 to prevent excessive adipose tissue deposition and improve endocrine parameters and insulin sensitivity also may provide protection against colonic epithelial cell transformation. The inability of dietary SPI to recapitulate all of the effects of ME1 null mutation may reflect in part, the total absence of ME1 in MOD-1 mice (extreme case) as opposed to the reduced ME1 expression in SPI-HF diet-fed WT animals. Our observations that SPI did not further enhance effects of MOD-1 mutation on physiologic indices, is consistent with ME1 as a major component in the SPI-HF diet response.