Recent evidence has suggested that dietary polyunsaturated fatty acids (PUFAs) modulate inflammation; however, few studies have focused on the pathobiology of PUFA using isocaloric and isolipidic diets and it is unclear if the associated pathologies are due to dietary PUFA composition, lipid metabolism or obesity, as most studies compare diets fed ad libitum. Our studies used isocaloric and isolipidic liquid diets (35% of calories from fat), with differing compositions of omega (ω)-6 or long chain (Lc) ω-3 PUFA that were pair-fed and assessed hepatic pathology, inflammation and lipid metabolism. Consistent with an isocaloric, pair-fed model we observed no significant difference in diet consumption between the groups. In contrast, the body and liver weight, total lipid level and abdominal fat deposits were significantly higher in mice fed an ω-6 diet. An analysis of the fatty acid profile in plasma and liver showed that mice on the ω-6 diet had significantly more arachidonic acid (AA) in the plasma and liver, whereas, in these mice ω-3 fatty acids such as eicosapentaenoic acid (EPA) were not detected and docosahexaenoic acid (DHA) was significantly lower. Histopathologic analyses documented that mice on the ω-6 diet had a significant increase in macrovesicular steatosis, extramedullary myelopoiesis (EMM), apoptotic hepatocytes and decreased glycogen storage in lobular hepatocytes, and hepatocyte proliferation relative to mice fed the Lc ω-3 diet. Together, these results support PUFA dietary regulation of hepatic pathology and inflammation with implications for enteral feeding regulation of steatosis and other hepatic lesions.

Omega-6 (ω6) and omega-3 (ω3) poly unsaturated fatty acids (PUFA) are pro- and anti-inflammatory respectively. Regulating their dietary ratio provides an approach for cancer prevention and potentially therapy; however, most studies have not separated the inflammatory effects of dietary fatty acids (FA) from that of obesity. Herein we studied the effects of the ω6:ω3 ratio in iso-caloric diets containing 35.5% of calories from fat on mammary tumor growth and metastasis. The ω6:ω3 ratio in ω6 and ω3 based diets were 42:1 and 1:1 respectively (confirmed by gas chromatography-mass spectrometry) using the liquid, Lieber DeCarli diet with fish oil substituting for 70% of olive oil. The ω3 diet contained 3:1 ratio of eicosapentaenoic acid (EPA) and docosahexaenoic (DHA) as major ω3 FA with small amount of linolenic acids. The ω6 diet contained linoleic acid as predominant ω6 FA and small level of linolenic acid as ω3 FA. The diets were pair-fed to avoid the tumor promoting effects of obesity. Two weeks after establishing female BALB/c mice on the diets, they were orthotopically injected with 4T1 mammary tumors. Outcomes included body weight, diet consumption, tumor growth, metastasis and survival. In association with pair feeding there were no significant differences in the diet consumed (ω3 diet used as baseline) and weight gain between animals; however, the time to tumor growth was significantly more rapid in mice fed the ω6 diet versus the ω3 diet cohorts. The ω6 diet fed cohort had significantly higher numbers of pulmonary and hepatic metastases and significantly shortened survival. Further, ω6 diet fed mice had an unusual metastasis profile including an increase in ovarian and renal metastases and an increase in posterior paralysis that in prior studies was associated with demineralization, osteolysis, marrow metastases and spontaneous long bone fractures in mice fed ω6-enriched diet. Histological analysis of tissues supported the observation of more frequent unusual metastasis sites (ovaries, kidneys, heart) along with systemic changes to myeloid cells infiltration in the tissues of mice fed ω6 diet. Further, ω6 and ω3 diets had identical total and fat calories and were pair fed; however, the tumors and livers of ω6 diet fed cohorts had more lipid content in adipocytes. Our results were replicative and also found to be consistent when the tumor growth and survival experiments were compared between young (22 weeks) versus old (58 weeks) mice in independent studies. In summary, our studies demonstrate that the dietary ratio of ω6:ω3 regulates tumor growth and metastasis. Citation Format: Saraswoti Khadge, Geoffrey M. Thiele, Lynell W. Klassen, Graham J. Sharp, Timothy R. McGuire, Michael J. Duryee, Holly C. Britton, Alicia J. Dafferner, Jordan Beck, Paul Black, Concetta DiRusso, James E. Talmadge. Dietary omega-3 suppress mammary tumor growth, metastasis and enhances survival in an iso-caloric pair-fed mice model. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4323.

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Diets containing omega-3 (ω3) PUFAs have health benefits due to their anti-inflammatory activity and lower risk of chronic conditions including cardiac, autoimmune and neoplastic diseases. This contrasts with ω6 PUFA containing Western diets, which are pro-inflammatory. Balancing the dietary ω6:ω3 ratio has been suggested to have cancer preventive and potentially therapeutic activity; however, the majority of these studies have not differentiated dietary PUFA content from obesity. Herein we examined the effects of the ω6:ω3 ratio in iso-caloric diets that were pair fed. These diets had 35.5% of calories from fat with an ω6:ω3 ratio in the ω6 and ω3 based diets of 42:1 and 1:1 respectively (confirmed by gas chromatography-mass spectrometry) using the liquid, Lieber DeCarli diet and fish oil substituting for 70% of olive oil. The ω3 diet contained eicosapentaenoic (EPA) and docosahexaenoic (DHA) acid at a 3:1 ratio, while the ω6 diet contained linoleic acid as the predominant ω6 PUFA and a low level of the ω3 PUFA, linolenic acid. After establishing female BALB/c mice on these diets, weight gain and diet consumption were monitored for 12 weeks, the mice sacrificed and the dietary effects on mammary gland and hepatic histopathology, leukocyte phenotypes and organ and tissue lipidomics determined. In association with pair feeding there were no differences in diet consumed (ω-3 diet used as baseline) and weight gain between cohorts. The cohort on the ω6 diet had depressed numbers of marrow progenitor cells and increased splenic subcapsular extramedullary hematopoiesis consistent with an inflammatory response, increased hepatocyte lipidosis, hypertrophy and multinucleation and increased hepatic vascularity with thicker intima. The ω-6 dietary cohort also had mammary fat pads (MFPs) with increased adipocytes in the tubular epithelium, stromal cellularity and epithelial tissue density. These mammary gland and hepatic histopathologic changes and subclinical inflammation are consistent with pre-neoplastic lesions and were accompanied by organ specific lipodemic changes. This included significant increases in arachidonic acid (AA) in the plasma, spleen and liver, but not MFPs, of the ω-6 cohort, and a significant increase in EPA and DHA levels in the plasma, spleen, MFPs and liver of the ω-3 cohort. In addition, the liver and MFPs had a significant increase in the ω3 PUFA, docosapentaenoic acid. In summary, our studies demonstrate that the dietary ratio of ω6:ω3, independent of obesity can regulate mammary gland and hepatocyte proliferation and subclinical inflammation in association with significant, organ specific increases in AA levels contributing to microenvironmental preneoplastic hyperplasia. Citation Format: Saraswoti Khadge, Paul Black, Concetta DiRusso, Geoff Thiele, Lynell W. Klassen, J Graham Sharp, Timothy R. McGuire, Michael J. Duryee, Holly C. Britton, Alicia Dafferner, Jordan Beck, James E. Talmadge. Preneoplastic activity of dietary poly unsaturated fatty acid (PUFA) regulation of organ and tissue microenvironments in an iso-caloric pair-fed mouse model. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4312.

Adipose tissue is responsible for insulating internal organs, storing energy for times of negative energy balance, and secreting adipokines into circulation. The major cell types found in adipose tissue include undifferentiated adipocytes (pre‐adipocytes), adipocytes, and adipose tissue macrophages macrophages (ATMs). Adipocytes and ATMs are the major sources of adipokines, adipose tissue cytokines with functions ranging from inflammatory mediators to metabolic regulators. Polyunsaturated fatty acids (PUFAs) have been shown to affect the differentiation and lipid droplet formation in 3T3‐L1 adipocytes, but few studies have examined their effects on inflammatory adipokine production from these cells. Adipocyte differentiation, which is marked by lipid droplet (triacylglycerol; TAG) accumulation, is regulated by various transcription activators, including a splice variant of the nuclear receptor PPAR□. PUFAs are major regulators of PPAR□. For example, the n‐3 PUFA EPA has been shown to have an effect on lipid droplet size through effects on lipid catabolism, as well as mediate reductions in pro‐inflammatory adipokine secretion in favor of anti‐inflammatory secretions. The objectives of this experiment were to determine the effects of n‐3 and ‐6 PUFAs on TAG lipolysis, as measured by free fatty acid (FFA) release, and inflammatory cytokine production, as measured by interleukin (IL)‐6 and macrophage inflammatory protein (MIP)‐1α production. In order to examine these effects, the 3T3‐L1 cells were differentiated in media with 100μM of PUFA treatment (α‐linoleic acid [LA], arachidonic acid [ARA], and EPA), followed by a 6 h, 1μg/mL of lipopolysaccharide (LPS) challenge. Fully differentiated 3T3‐L1 adipocytes under these treatments did not release FFA in quantifiable amounts. The LPS+ARA treatment resulted in significantly higher levels of MIP‐1□ (26.3±2.5 pg/ml) and IL‐6 (146.13±17.0 pg/ml) in conditioned media, compared to control (MIP‐1□:0.55±2.5; IL‐6:0.23±17.0), LPS (MIP‐1□:0.06±2.5; IL‐6:16.5±17.0), and LPS+LA (MIP‐1□:7.5±2.5; IL‐6:27.0±17.0). Cells treated with LPS+EPA released similar amounts of MIP‐1□ (14.9±2.5) but significantly lower amounts of IL‐6 (20.3±17.0) compared to LPS+ARA‐treated cells. These data suggest that differentiating 3T3‐L1 adipocytes in the presence of the n‐6 PUFA ARA results in increased release of LPS‐stimulated IL‐6 and MIP‐1□. These results indicate a role of this n‐6 fatty acid on adipocyte‐derived inflammation. Support or Funding Information Laurie Bukovac Hodgson & David Hodgson Endowed Fund, as well as the Denison Summer Science Research Supply Fund