The cancer-inhibitory activity of weight loss by calorie restriction and/or exercise has been well documented in many animal models, including the skin carcinogenic model (5). A relationship between reduced dietary energy intake and decreased tumor rates has been established in rodents (6). The molecular targets in response to energy balance for prevention of skin carcinogenesis has also been suggested (7). Michna et al. demonstrated that voluntary wheel running exercise inhibited UVB-induced skin tumorigenesis in mice (8). The exercise-induced skin cancer inhibition was further associated with enhanced apoptosis in the epidermis via a p53-independent mechanism (9). Most studies from animal models suggest physical activity inhibits carcinogenesis; however, the exercise-induced inhibition appears less consistent or less potent than calorie restriction (10). Research by the Hursting group suggested it is negative energy balance rather than exercise alone that inhibited the development of intestinal polyps in APC^Min mice (11). By means of dietary regimens in both of high fat and calorie restricted diet, the groups of DiGiovanni and Hursting recently concluded that the dietary energy balance might modulate IGF-1 and IGF-1 receptor signaling (12). This reduction is due to both reduced total levels of IGF-1 and a reduction in bioavailability due to an increase in levels of IGF-1 binding proteins. Similarly, our previous studies found that exercise with pair feeding, but not exercise alone, was effective in controlling body weight and selectively abrogating the tumor promoter-induced PI3K-Akt pathway in the skin epidermis, resulting in enhanced apoptosis and reduced proliferation (13).

Many studies have investigated the molecular mechanism of cancer prevention by physical activity. However, the underlying mechanisms of this phenomenon are not clear. Physical activity has been proposed to prevent cancer through deletion of reactive oxygen species and an increase in antioxidant status (14), reduction of sex hormone levels (15), reduction of the metabolic hormones insulin, IGF-1 and/or leptin (16–18), and improvement in immune function (19). Hormone-related signaling alterations, especially of IGF-1, seem to be an important factor in weight-control for cancer prevention. Phosphatidylinostiol 3-kinase (PI3K) is one of the targets activated by IGF-1. Activated PI3K usually phosphorylates phosphatidylinositols (PIs)¹ in the cell membrane to produce PI phosphates or phosphotinositides. PI(3, 4, 5)P₃, a major product of PI3K, is able to bind and activate Akt, therefore activating many downstream signaling proteins that regulate cell survival and cell cycle progress. Elevated levels of PI(3, 4, 5)P₃ and upregulation of Akt are found to be oncogenic and promote the transition to malignancy (20–22). Therefore, in addition to being membrane structural building blocks, PIs and their derivatives phosphoinositides, have been found to play an important role in cellular signaling and intracellular trafficking as well as in the cancer disease process (23).

Among the phospholipids, some lysophosphatidylcholines (LysoPC) may activate phospholipase C, leading to the production of diacyglycerols and inositol triphosphate, activation of protein kinase C, release of and intracellular Ca²⁺, and activation of MAP kinase signaling (24). In addition to the signaling, processes involving specific lipid head group classes, alterations in the membrane lipid fatty acid composition may also be involved in cancer progression. For example, breast cancer patients were found to have a higher risk of early occurrence of visceral metastasis when they had a lower level of polyunsaturated fatty acids in phosphatidylethanolamines (PE) (25). Decreased levels of stearic acid in phosphatidylcholine (PC) were found in breast cancer patients with metastasis compared to those who remained metastasis-free (26). Recent studies by the Pougnoux group showed that a lipid profile, rather than a single fatty acid, could be important; they showed that decreased linoleic acid, increased cis-monounsaturated fatty acids, and a low ω6/ω3 fatty acid ratio, was associated with lower risk of breast cancer (27). Very long chain ω-3 fatty acids, such as docosahexaenoic acid (DHA), are well known to have a preventive effect on cardiovascular disease and cancer and may be involved in alterations of membrane structure and function, eicosanoid metabolism, gene expression, and inhibition of lipid peroxidation (28).

Physical activity has been found to affect membrane phospholipids. For example, it is reported that particular types of exercise increase sphinganine and sphingosine in rat skeletal muscle (29) and reduce total content of ceramide in rat heart muscle (30). Regular exercise was also found to significantly increase oleic acid 18:1 (ω-9) and DHA 22:6 (ω-3) in human muscle (31). However, previous lipid composition studies related to cancer were usually focused on total cholesterol, lipoproteins, and triglyceride (32–33). The overview of membrane phospholipid profile and the response to exercise-induced weight control has not been studied yet.

With the development of recent lipid analysis, phospholipid compositional profiles can be determined by electrospray ionization tandem mass spectrometry (ESI/MS-MS). The technique is highly sensitive, accurate, and reproducible (34–36).

The results demonstrated that exercise with ad libitum feeding did not effectively decrease body weight and body fat. Similar results are also observed by Mehl et al. in APC^Min mice and Michna et al., in SKH mice (39, 8). A moderate increase of food intake from 3.43±0.28 g/day for the control to 3.67±0.18 g/day for Ex+AL mice may compensate, at least in part, the exercise-induced energy expenditure. Furthermore, the dietary energy increase in Ex+AL might not necessarily match the treadmill exercise-induced energy expenditure, since the energy expenditure could have been altered due to the changed spontaneous activity in the cage and/or resting energy metabolism. In addition to physical activity, Badman and Flier (40) have suggested that the total energy expenditure should also consist of adaptive thermogenesis and obligatory energy expenditure. It is interesting to compare our results with Huffman’s study (41). The exercise training protocol appears a little bit different between two studies. In our experiment, the mice run treadmill at zero grade for 13 weeks, but Huffman’s mice were exercised at 8% grade for 24 weeks. The most significant difference is due to strain- and diet-related phenomena. We used a lean mouse model that fed a normal AIN-93 diet (5% of calories from fat), but Huffman et al. (41) used C57BL/6 strain, a classic high-fat diet-induced obese model. To induce weight gain, Huffman’s mice were fed a moderately high-fat diet (35% of calories from fat). Therefore, the average of body weights for the control mice in Haffman’s study was about 30 g in the beginning of experiments and 46 g in Week 14. In contrast, the average of body weight for our control mice is ~26 g in the beginning and 32 g in Week 14. It is no doubt that the moderately high-fat diet-induced overweight C57BL/6 strain in Haffman’s study should be much more susceptible to exercise-induced weight loss than our lean CD-1 strain. Actually, our data from the lean CD-1 strain are consistent with what we observed in another lean SENCAR strain (Xie et al., 2007). When compared with Haffman’s study, our contradictory data may provide a diverse phenomenon for a lean strain model with normal diet treatment.

When we adjusted the food consumption of the exercised mice to that of sedentary control mice, significant effects on body weight and body fat were observed. Studies in the Hursting lab found that voluntary wheel running mice with restricted food consumption, a pair-feeding strategy similar to ours, had a significantly lower body weight and less intestinal polyp development (12). Overall, this study indicates that a negative calorie balance via both increasing energy expenditure and limiting calorie intake seems most effective in preventing body weight gain. It should be noted that we may not be able to differentiate body weight control from caloric balance. Although exercise alone with ad libitum feeding was not sufficient to decrease body weight due to, at least in part, the corresponding increase in dietary intake, the lack of an exercise effect in AL+Exe mice on the body weight/fat and various phospholipids might be in part due to the insufficient magnitude of the calorie deficit. Since the average food intake for three treatment groups are comparable, we did not find any significant correlation of calorie intake with specific phospholipids, and the caloric deficit via exercise was not matched with the diet intake, and thus the results should be interpreted accordingly.

Although it wonders whether the turnover rate of skin would make it a better indicator of subtle changes than other tissues, previous studies by ours and others have demonstrated a cancer-inhibitory activity of weight loss by dietary calorie restriction and/or exercise in animal skin cancer model (5–8). Furthermore, exercise-induced skin cancer inhibition has been linked to apoptosis induction and anti- proliferation in the skin epidermis (9, 13). To further evaluate the impact of weight control, lipidomics analysis for all the phospholipids in skin tissues 2 hours after TPA treatment was performed. First of all, we did not find any significant differences of phospholipids between TPA and acetone-vehicle control. The reason related to a lack of a significant impact of TPA on the phospholipid profile may be due to a short time exposure to TPA treatment in vivo. The selection of 2-hr period for TPA treatment is based on the previous observation that was adequate for a significant activation of Ras and ERK activities in skin epidermis (13).

Furthermore, the finding that the major molecular specie of PI was 38:4 is consistent with the typical pattern of PIs found in mammals, and in particular in mouse tissues where this major specie has demonstrated to be 1–18:0, 2–20:4 PI (42). The observed decrease of the most PI species in the Ex+PF mice led us to measure expression of PI3K, a key kinase required for various signaling cascades for cancer-related cellular function including cell growth, proliferation, differentiation, motility, survival, and intracellular trafficking (13). As expected, we found the decreased PI levels corresponded to lowered levels of PI3K protein in the skin epidermis of Ex+PF mice. Skin cancer development is usually associated with uncontrolled proliferation of epidermal cells (43), so the lower protein expression of class I PI3K in epidermal cells as measured by immunohistochemistry may result in less proliferation as found in our previous report (13). Furthermore, the increased levels of PI3K staining observed in TPA-treated sedentary control when compared with acetone-vehicle control. When compared with TPA-treated sedentary control (Fig 5B) but not acetone-vehicle sedentary control (Fig 5A), no significant difference was found in Ex+AL group. However, TPA-induced increase of PI3K protein expression was significantly suppressed by exercise with pair-feeding, suggesting the direct product of PI3K, the second messenger PI(3, 4, 5)P₃, might be reduced. It is well recognized that PI(3, 4, 5)P₃ recruits some signaling enzymes with pleckstrin homology domains, such as protein serine-threonine kinases, including Akt and adaptor proteins, to the membrane. The activation of these enzymes impacts protein synthesis, cell cycle entry, and cell survival function, etc. (20). Decreased PIs and down regulation of PI3K expression observed in this study may imply that body weight control through exercise with controlled dietary intake may prevent against TPA-induced cancer risk. In addition, many studies by us and others have found that weight control was associated with reduced levels of circulating growth hormones or factors such as IGF-1 (44). Considering the requirement of PI3K activation by IGF-1-dependent signaling, the down-regulation of PI3K protein expression, and the reduced PI3K-related PI substrates in the exercised but pair-fed mice might be caused by a decrease in plasma IGF-1 levels. In our studies, IGF-1 was restored in the exercised and pair-fed mice by either intraperitoneal injection at 10 µg/g body weight twice per wk (13) or via osmotic minipumps (unpublished data). We have found the reduction of PI3K protein expression and the PI species were partially reversed by IGF-1 restoration.

In addition to PIs, we also found most of the ePCs and LysoPCs were significantly reduced in the exercised and pair-fed mice, while 22:6 lysoPE was increased in Ex+PF group. The lower levels of ePCs and lysoPCs may prevent cancer by reducing cellular damage and proliferation, since ePCs are required for the formation of platelet-activating-factor and lysoPCs are produced during LDL oxidation within atherosclerotic plaques for atherosclerotic lesion development (45–48).

The impact of weight control via exercise on the PCs and PEs is interesting. When compared with the sedentary control, exercise with ad libitum-fed did not change the profile of PCs and PEs. However, there are significant changes observed in the exercised and pair-fed mice. As some short chain fatty acids of PCs were decreased, the long chain polyunsaturated fatty acids, i.e., 40:5 and 40:6, increased significantly in Ex+PF mice in comparison with either control or Ex+AL group. A similar impact was found on PEs (data not shown). By means of product ion analysis, the increased polyunsaturated 40:5 and 40:6 fatty acids in PCs and PEs were further discovered to contain a combination of either 18:0–22:5 or 18:0–22:6. The 22:6 fatty acid is undoubtedly DHA. The 22:5 fatty acid, i.e., docosapentaenoic acid (DPA), could be either ω-6 adrenic acid or ω-3 clupanodonic acid. As one of the three major ω-3 long chain polyunsaturated fatty acids, clupanodonic acid could be intermediary between eicosapentaenoic acid (EPA) and DHA (49). It should be noted that ω-3 22:6 DHA was found to be elevated significantly in the exercised and pair-fed mice not only for PCs and PEs, but also for lysoPEs. It is well known that the mammals can make DHA and EPA through desaturation and elongation of essential ω3-linolenic acid (50–51). Our microarray data further confirmed that elongation of (very) long chain fatty acid-like elongase gene 1 (Elvol1) was expressed significantly more in Ex+PF as compared to the Ex+AL group. Elevation of DHA by exercise has been reported by others in human studies (31, 52). Considering the general health benefits of ω-3 fatty acids (53–56) and a specific inhibitory role in TPA-induced signaling activation (28, 57), the increase of ω-3 fatty acids found in this study may provide a novel approach to understand the mechanisms by which exercise with controlled calorie intake may protect against cancer.

To overview the effects of exercise with or without consideration of diet intake upon the phospholipid profiling, we applied a discriminant function analysis to the 57 significantly changed phospholipids. Twenty-five of the total 57 phospholipids were able to distinguish the treatment groups with 92% classification efficiency. These 25 phospholipids are possible candidates for biomarkers to distinguish the effects of diet regiments and exercise in mice. It should be noted that the most 25 phospholipids selected are PCs, ePC, or lysiPCs. The functional impact of these PC-related species changes and how such changes might afford protection from cancer warrant further studies.