This study establishes the importance of visceral adiposity in obesity-associated intestinal tumorigenesis in the Apc1638/N+
mouse model. Numerous epidemiologic and pre-clinical studies, including a prior effort in this model (31
), have linked the obese state to increased colon cancer risk and/or mortality. However, obesity is a complex phenotype, characterized not only by excess weight gain and adiposity, but also by dietary factors and a sedentary lifestyle (32
). Collectively, this has made isolating the individual contribution of adipose tissue to cancer risk challenging for the field. Here, using a surgical approach to deplete VF, we have circumvented these issues, providing causal evidence linking visceral adiposity per se
to intestinal tumorigenesis, independent of other important confounders related to energy balance. Similarly, Lu et al found that partial removal of VF protected female SKH-1 mice against UVB-induced skin carcinogenesis (33
). Importantly, these data provide yet another line of evidence directly linking VF to the etiology of aging (25
) and age-related diseases (23
Remarkably, we found that VF removal was not only effective at attenuating macroadenoma development, but this reduction was comparable to that observed in CR mice. This was accompanied by improved survival in CR mice, with a similar, albeit non-significant trend observed for increased survival in VF- mice, as compared to AL mice. However, we also observed a clear effect of sex differences in the efficacy of these interventions on tumor initiation, promotion and survival. Perhaps most striking was the marked protection conferred by VF removal for development of macroadenomas in females, but not in males. While it is possible that the inability of VF removal to protect against macroadenomas in males was simply due to inherent sex differences, we also observed a marked shift in fat distribution among VF- males that could also explain this difference. Indeed, when we evaluated mesenteric fat mass in females () and males (), we observed a distinct increase in mesenteric fat in VF- males, but not in VF- females. Given the hazardous nature of the mesenteric fat depot, coupled with anatomic location of this depot lying in close proximity to the intestine, it is plausible that mesenteric fat accretion abrogated the benefits of removing the epididymal and perinephric fat depots via endocrine and/or paracrine mechanisms.
Figure 4 Mesenteric fat mass in male and female AL, VF- and CR mice. Mesenteric fat is believed to be the most closely related VF depot to humans, both anatomically and due to its portal access. However, unlike other VF depots, this fat pad cannot be surgically (more ...)
Interestingly, whereas VF removal conferred protection against macroadenoma development in female mice, it was unexpectedly accompanied by a greater incidence of microadenomas. We believe the most likely reason for this finding is that VF removal led to a systemic change in factor(s) that blocked the progression of tumor development at the microadenoma-macroadenoma transition. In contrast, CR was associated with increased dysplasia in males, but was protective against the development of both micro- and macroadenomas. However, CR had no apparent effect on tumorigenesis in females. This suggests that energy availability may play a unique role in tumor initiation and early promotion in male mice, but CR was also beneficial for females, perhaps at later stages, as demonstrated by their improved survival (see ). Future studies are needed to explore the mechanisms whereby abdominal obesity and nutrient availability act independently during stages of initiation, promotion and progression, and how these interactions are modulated by gender.
We also noted that while both AL males (24.3% fat) and females (26.3% fat) were obese, due to consuming a 45% high-fat diet, AL females developed fewer tumors (P
<0.05) and had improved survival (P
<0.05), as compared to AL males. Indeed, consistent evidence from human studies shows a stronger risk posed by obesity and visceral obesity to colon cancer incidence and mortality in men, as compared to women (9
). The reason for this sex difference is not clear, but may be related to reproductive hormone status, as indicated by protection conferred from oral contraceptive use in women (35
), and evidence that ovariectomized female mice injected with colon cancer cells have increased fat mass, insulin resistance and tumor growth (30
). Females in our study also had approximately 50% lower insulin levels than males, and nearly two-fold greater adiponectin levels, which may also have also contributed to these differences.
Another important observation from this study was the observed hyperinsulinemia, hyperleptinemia, and reduced adiponectin levels with VF removal in females, which appears to be at odds with the reduction in macroadenomas. Indeed, the modest, but significant increase in fasting insulin was unexpected given that our group has previously shown improved hepatic insulin action with VF removal in chow-fed male rats (24
), while Shi et. al
) showed that removing just a single VF depot (periovarian) was sufficient to improve glucose tolerance in high-fat fed female mice. It should be pointed out however, that any potential difference in insulin action here between AL and VF-female mice was likely limited to basal conditions as we did not detect any difference in response to an insulin challenge (ITTs).
We also observed that VF removal in males and females resulted in an ~21% reduction in adiponectin, which was not unexpected, given that a significant amount of fat tissue was removed. While there is in vitro
and in vivo
evidence to support a protective role for increased adiponectin levels in colon cancer (16
), rodent studies linking low adiponectin levels with colon cancer risk are mostly derived from models of constitutive adiponectin deficiency (adiponectin knock-out mice) (36
). Thus, a much more modest 21% reduction in adiponectin may not be sufficient to predispose to tumorigenesis, at least in females, in which levels are two-fold higher than in male mice. However, an adverse effect in males cannot be ruled out and along with the compensatory increase in mesenteric fat, may explain why VF removal was ineffective in male mice.
Leptin has also been widely implicated in linking obesity to colon as well as other cancers (32
), and we observed that VF- females had greater leptin levels, which may be indicative of leptin resistance in these mice. However, this may be a complex effect since evidence for leptin as a stimulator of proliferation is clear only for colon cancer cells in vitro
). We also measured several other inflammatory mediators in serum and observed a reduction in the chemokine, CXCL-1, which has been linked to angiogenesis and metastasis, in both male and female CR mice, but no other consistent patterns emerged. It is important to note that Apc1638/N+
mice presented with splenomegaly, which is commonly observed in these mice (38
), and corresponded with elevated cytokine levels in some animals, making it difficult to distinguish the contribution of adipose tissue to the pro-inflammatory milieu.
The evidence implicating obesity and its related sequelae to cancer risk spans a wide array of models and systems. Yet, efforts to elucidate mechanism(s) linking the obese state to site-specific cancers such as breast, colon, prostate and skin, particularly in vivo
, have revealed a more complex biology then perhaps was initially anticipated. For example, studies utilizing the fatless A-ZIP/F-1 mouse model demonstrated that adipokines are not absolutely required for tumor development. Indeed, despite the absence of adipose tissue and adipose-derived peptides such as leptin, these mice present with several other features of the obese phenotype, including hyperglycemia, hyperinsulinemia, and elevated levels of pro-inflammatory cytokines. When subjected to a two-stage skin carcinogenesis procedure, these mice develop more skin papillomas (39
), but this effect is not seen in the severely obese, ob/ob
mouse model (40
A-ZIP/F-1 mice also have accelerated development of mammary tumors when crossed with C3(1)/T-Ag transgenic mice (39
), while a similar effect was observed when MKR mice, which are lean but diabetic, were crossed with MMTV-PyVmT mice (41
). In contrast, MMTV-TGFα/db/db
, which are obese and insulin resistant, but leptin-receptor deficient, fail to develop mammary tumors (20
), but have increased incidence of intestinal neoplasms (31
). Thus, our finding that protection conferred from intestinal tumors in VF- females occurs despite a failure to produce favorable changes in factors suggested to play an important role in obesity-associated tumor growth is not without precedent. Collectively, these data demonstrate the complexity of the obesity-cancer interface and emphasize the need for continued efforts to delineate how specific perturbations to endocrine and other factors (43
) by obesity, contribute to site-specific cancer risk.
In summary, these data provide causal evidence linking VF to intestinal cancer risk. The protection conferred by VF removal was preferentially seen in female mice, despite a lack of favorable changes in leptin, insulin, adiponectin, and several pro-inflammatory cytokines and chemokines, suggesting that other unknown mechanisms may underlie the obesity-colon cancer link. However, since the genetic model used here develops tumors predominantly in the small intestine, rather than the colon, further work on the underlying mechanisms will need to focus on a model in which the colon is the principal site of tumor development. Given that VF accrual and subcutaneous fat depletion represent a common hallmark of aging (44
), it is tempting to conclude that the nearly logarithmic increase in cancer incidence and mortality with age is driven in part by these unfavorable changes in fat re-distribution. Therefore, strategies designed to deplete VF stores in abdominally-obese individuals, including pharmacologic and/or behavioral strategies, such as diet and exercise, may be an important cancer prevention strategy as well as an adjuvant therapy for improving outcomes following a cancer diagnosis.