Nalin Siriwardhana

Eicosapentaenoic Acid Prevents and Reverses Insulin Resistance in High-Fat Diet-Induced Obese Mice via Modulation of Adipose Tissue Inflammation

We investigated the effects of eicosapentaenoic acid (EPA) on prevention (P) and reversal (R) of high saturated-fat (HF) diet-induced obesity and glucose-insulin homeostasis. Male C57BL/6J mice were fed low-fat (LF; 10% energy from fat), HF (45% energy from fat), or a HF-EPA-P (45% energy from fat; 36 g/kg EPA) diet for 11 wk. A 4th group was initially fed HF for 6 wk followed by the HF-EPA-R diet for 5 wk. As expected, mice fed the HF diet developed obesity and glucose intolerance. In contrast, mice fed the HF-EPA-P diet maintained normal glucose tolerance despite weight gain compared with the LF group. Whereas the HF group developed hyperglycemia and hyperinsulinemia, both HF-EPA groups (P and R) exhibited normal glycemia and insulinemia. Further, plasma adiponectin concentration was lower in the HF group but was comparable in the LF and HF-EPA groups, suggesting a role of EPA in preventing and improving insulin resistance induced by HF feeding. Further analysis of adipose tissue adipokine levels and proteomic studies in cultured adipocytes indicated that dietary EPA supplementation of HF diets was associated with reduced adipose inflammation and lipogenesis and elevated markers of fatty acid oxidation. In C57BL/6J mice, EPA minimized saturated fat-induced insulin resistance and this is in part mediated by its effects on fatty acid oxidation and inflammation.

Health benefits of n-3 polyunsaturated fatty acids: eicosapentaenoic acid and docosahexaenoic acid.

Marine-based fish and fish oil are the most popular and well-known sources of n-3 polyunsaturated fatty acids (PUFAs), namely, eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). These n-3 PUFAs are known to have variety of health benefits against cardiovascular diseases (CVDs) including well-established hypotriglyceridemic and anti-inflammatory effects. Also, various studies indicate promising antihypertensive, anticancer, antioxidant, antidepression, antiaging, and antiarthritis effects. Moreover, recent studies also indicate anti-inflammatory and insulin-sensitizing effects of these fatty acids in metabolic disorders. Classically, n-3 PUFAs mediate some of these effects by antagonizing n-6 PUFA (arachidonic acid)-induced proinflammatory prostaglandin E₂ (PGE₂) formation. Another well-known mechanism by which n-3 PUFAs impart their anti-inflammatory effects is via reduction of nuclear factor-κB activation. This transcription factor is a potent inducer of proinflammatory cytokine production, including interleukin 6 and tumor necrosis factor-α, both of which are decreased by EPA and DHA. Other evidence also demonstrates that n-3 PUFAs repress lipogenesis and increase resolvins and protectin generation, ultimately leading to reduced inflammation. Finally, beneficial effects of EPA and DHA in insulin resistance include their ability to increase secretion of adiponectin, an anti-inflammatory adipokine. In summary, n-3 PUFAs have multiple health benefits mediated at least in part by their anti-inflammatory actions; thus their consumption, especially from dietary sources, should be encouraged.

n-3 and n-6 polyunsaturated fatty acids differentially regulate adipose angiotensinogen and other inflammatory adipokines in part via NF-κB-dependent mechanisms

Excessive secretion of proinflammatory adipokines has been linked to metabolic disorders. We have previously documented anti-inflammatory effects of n-3 polyunsaturated fatty acids (n-3 PUFAs) in adipose tissue; however, the mechanisms by which these fatty acids regulate adipokine secretion remain unclear. Here, we determined differential effects of eicosapentaenoic acid (EPA, n-3 PUFA) vs. arachidonic acid (AA, n-6 PUFA) on expression and secretion of angiotensinogen (Agt), interleukin 6 (IL-6) and monocyte chemotactic protein (MCP-1) in 3T3-L1 adipocytes. While both PUFAs increased intracellular Agt protein and mRNA expression, Agt secretion into culture media was increased only by AA treatment, which in turn was prevented by co-treatment with EPA. At various AA/EPA ratios, increasing AA concentrations significantly increased secretion of the above three adipokines, whereas increasing EPA dose-dependently, while lowering AA, decreased their secretion. Moreover, IL-6 and MCP-1 were more significantly reduced by EPA treatment compared to Agt (IL-6>MCP>Agt). Next, we tested whether nuclear factor-κB (NF-κB), a major proinflammatory transcription factor, was involved in regulation of these adipokines by PUFAs. EPA significantly inhibited NF-κB activation compared to control or AA treatments. Moreover, EPA attenuated tumor necrosis factor-α-induced MCP-1 and further reduced its secretion in the presence of an NF-κB inhibitor. Taken together, we reported here novel beneficial effects of EPA in adipocytes. We demonstrated direct anti-inflammatory effects of EPA, which are at least in part due to the inhibitory effects of this n-3 PUFA on the NF-κB pathway in adipocytes. In conclusion, these studies further support beneficial effects of n-3 PUFAs in adipocyte inflammation and metabolic disorders.

Grape Seed Proanthocyanidin Suppression of Breast Cell Carcinogenesis Induced by Chronic Exposure to Combined 4-(Methylnitrosamino)-1-(3-Pyridyl)-1-Butanone and Benzo[a]Pyrene

Breast cancer is the most common type of cancer among women in northern America and northern Europe; dietary prevention is a cost‐efficient strategy to reduce the risk of this disease. To identify dietary components for the prevention of human breast cancer associated with long‐term exposure to environmental carcinogens, we studied the activity of grape seed proanthocyanidin extract (GSPE) in suppression of cellular carcinogenesis induced by repeated exposures to low doses of environmental carcinogens. We used combined carcinogens 4‐(methylnitrosamino)‐1‐(3‐pyridyl)‐1‐butanone (NNK) and benzo[a]pyrene (B[a]P), at picomolar concentrations, to repeatedly treat noncancerous, human breast epithelial MCF10A cells to induce cellular acquisition of cancer‐related properties of reduced dependence on growth factors, anchorage‐independent growth, and acinar‐conformational disruption. Using these properties as biological target endpoints, we verified the ability of GSPE to suppress combined NNK‐ and B[a]P‐induced precancerous cellular carcinogenesis and identified the minimal, noncytotoxic concentration of GSPE required for suppressing precancerous cellular carcinogenesis. We also identified that hydroxysteroid‐11‐beta‐dehydrogenase 2 (HSD11B2) may play a role in NNK‐ and B[a]P‐induced precancerous cellular carcinogenesis, and its expression may act as a molecular target endpoint in GSPEs suppression of precancerous cellular carcinogenesis. And, the ability of GSPE to reduce gene expression of cytochrome‐P450 enzymes CYP1A1 and CYP1B1, which can bioactivate NNK and B[a]P, possibly contributes to the preventive mechanism for GSPE in suppression of precancerous cellular carcinogenesis. Our model system with biological and molecular target endpoints verified the value of GSPE for the prevention of human breast cell carcinogenesis induced by repeated exposures to low doses of multiple environmental carcinogens.

Precancerous carcinogenesis of human breast epithelial cells by chronic exposure to benzo[a]pyrene

To understand carcinogenesis of human breast epithelial cells induced by chronic exposure to environmental pollutants, we studied biological and molecular changes in progression of cellular carcinogenesis induced by accumulated exposures to the potent environmental carcinogen benzo[a]pyrene (B[a]P). Increasing exposures of human breast epithelial MCF10A cells to B[a]P at picomolar concentrations resulted in cellular transformation from a noncancerous stage to precancerous substages, in which cells acquired the cancerous abilities of a reduced dependence on growth factors, anchorage‐independent growth, and disruption in acini formation on reconstituted basement membranes. Using cDNA microarrays, we detected seven upregulated genes related to human cancers in B[a]P‐transformed MCF10A cells. Using this model, we verified that green tea catechin significantly (P < 0.05) suppressed B[a]P‐induced carcinogenesis. Our studies indicate that this cellular model may serve as a cost‐efficient, in vitro system, mimicking the chronic carcinogenesis of breast cells that likely occurs in early stages of carcinogenesis in vivo, to identify agents that inhibit cellular carcinogenesis.

Angiotensinogen Gene Silencing Reduces Markers of Lipid Accumulation and Inflammation in Cultured Adipocytes

Inflammatory adipokines secreted from adipose tissue are major contributors to obesity-associated inflammation and other metabolic dysfunctions. We and others have recently documented the contribution of adipose tissue renin-angiotensin system to the pathogenesis of obesity, inflammation, and insulin resistance. We hypothesized that adipocyte-derived angiotensinogen (Agt) plays a critical role in adipogenesis and/or lipogenesis as well as inflammation. This was tested using 3T3-L1 adipocytes, stably transfected with Agt-shRNA or scrambled Sc-shRNA as a control. Transfected preadipocytes were differentiated and used to investigate the role of adipose Agt through microarray and PCR analyses and adipokine profiling. As expected, Agt gene silencing significantly reduced the expression of Agt and its hormone product angiotensin II (Ang II), as well as lipid accumulation in 3T3-L1 adipocytes. Microarray studies identified several genes involved in lipid metabolism and inflammatory pathways which were down-regulated by Agt gene inactivation, such as glycerol-3-phosphate dehydrogenase 1 (Gpd1), serum amyloid A 3 (Saa3), nucleotide-binding oligomerization domain containing 1 (Nod1), and signal transducer and activator of transcription 1 (Stat1). Mouse adipogenesis PCR arrays revealed lower expression levels of adipogenic/lipogenic genes such as peroxisome proliferator activated receptor gamma (PPARγ), sterol regulatory element binding transcription factor 1 (Srebf1), adipogenin (Adig), and fatty acid binding protein 4 (Fabp4). Further, silencing of Agt gene significantly lowered expression of pro-inflammatory adipokines including interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-α), and monocyte chemotactic protein-1 (MCP-1). In conclusion, this study directly demonstrates critical effects of Agt in adipocyte metabolism and inflammation and further support a potential role for adipose Agt in the pathogenesis of obesity-associated metabolic alterations.

BCL-2 family protein, BAD is down-regulated in breast cancer and inhibits cell invasion.

We have previously demonstrated that the anti-apoptotic protein BAD is expressed in normal human breast tissue and shown that BAD inhibits expression of cyclin D1 to delay cell-cycle progression in breast cancer cells. Herein, expression of proteins in breast tissues was studied by immunohistochemistry and results were analyzed statistically to obtain semi-quantitative data. Biochemical and functional changes in BAD-overexpressing MCF7 breast cancer cells were evaluated using PCR, reporter assays, western blotting, ELISA and extracellular matrix invasion assays. Compared to normal tissues, Grade II breast cancers expressed low total/phosphorylated forms of BAD in both cytoplasmic and nuclear compartments. BAD overexpression decreased the expression of β-catenin, Sp1, and phosphorylation of STATs. BAD inhibited Ras/MEK/ERK and JNK signaling pathways, without affecting the p38 signaling pathway. Expression of the metastasis-related proteins, MMP10, VEGF, SNAIL, CXCR4, E-cadherin and TlMP2 was regulated by BAD with concomitant inhibition of extracellular matrix invasion. Inhibition of BAD by siRNA increased invasion and Akt/p-Akt levels. Clinical data and the results herein suggest that in addition to the effect on apoptosis, BAD conveys anti-metastatic effects and is a valuable prognostic marker in breast cancer.

Inflammatory Cytokines Link Obesity and Breast Cancer

The risk of postmenopausal breast cancer is significantly increased by obesity. Further, low grade chronic inflammation, a hallmark of obesity, can contribute to detrimental health effects including high cancer incidence. Our goal is to understand the molecular basis for obesity-breast cancer interactions and dissect the role of inflammatory mediators secreted by adipocytes and breast cancer cells. Accordingly, we developed a three-dimensional (3D) coculture system to facilitate direct cell-cell interactions and also performed media transfer from adipocyte cultures to growing breast cell cultures. The co-culture system will facilitate both adipocyte and breast cell growth in an environment closely mimicking in-vivo tumor microenvironment. Co-cultures of human primary adipocytes obtained from lean and obese women with MCF10A, MCF7 and MDAMB231 breast cells (non-cancerous epithelial cells, cancerous and invasive breast cancer cells, respectively), led to cell type specific changes in secretion of several pro-inflammatory cytokines, such as IL-6 and TNFα, compared to monocultures. Regulation of cytokine secretion of breast cells by adipocytes and vice versa indicates the two-way communication between breast cells and adipocytes. Further, 3D co-culture and adipocyte conditioned media transfer experiments demonstrated that obese adipocytederived conditioned media can promote higher growth of breast cancer cells compared to that from lean adipocytes. Moreover, obese adipocyte conditioned media increased the activation of nuclear factor-κB (NF-κB) family members in breast cells, compared to lean adipocyte-derived media. Our results provide a novel model system to study adipocytebreast cancer cell interactions which may underlie the link between breast cancer and obesity and also demonstrate that inflammatory cytokines and other secreted factors are important in this interaction.

Abstract 3111: Normal breast epithelial architecture and breast-cancer cell metabolism are altered by adipocytes and inflammatory fatty acids: Implications for postmenopausal breast cancer

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Alteration of organized breast epithelial ductal architecture accompanies neoplastic transformation. Obesity and chronic inflammation are associated with increased risk of postmenopausal breast cancer (BC). Further, omega 6 arachodonic acid (AA) significantly increases the secretion of pro-inflammatory cytokines by adipocytes compared to omega 3 eicosapentaenoic acid (EPA), therefore, we compared the effect of adipocytes and these two fatty acids on primary breast epithelial cell growth and formation of acini-like spheroid architecture. We used three dimensional (3D) MatrigelTM cultures of both basal and luminal primary breast cells and a novel 3D co-culture system with primary breast epithelial cells (basal/luminal) and adipocytes from lean or obese women. Effects of adipocytes as well as fatty acids on cellular architecture were assessed. Culture of basal or luminal epithelial cells with obese adipocytes/AA showed a significant increase in the irregularity of acini-like spheroid formation compared to normal spheroid formation in the absence of adipocytes or AA. Conditioned media transfer from lean/obese adipocytes into growing breast cell cultures showed remarkably different effects on LKB1/AMPK/mTOR metabolic signaling systems specifically in invasive MDA MB 321 cells. Obese adipocyte media decreased the activation of LKB1, AMPK, Acetyl-CoA carboxylase (ACC) and mTOR levels compared to lean adipocyte media. Further the expression of LKB1, pLKB1, pACC and mTOR were significantly higher in MDA MB 231 cells compared to less invasive MCF7 or epithelial MCF10A cells. Our data highlights the regulatory influence of adipocytes and inflammatory fatty acids on breast cellular architecture as well as metabolism and such alterations may underlie the effects of obesity on transformation of normal breast cells to BC cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3111. doi:10.1158/1538-7445.AM2011-3111

Abstract 4376: Biological, biochemical, and molecular targets for dietary catechin-induced suppression of breast cell chronic carcinogenesis

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC This study is to identify biological, biochemical, and molecular targets for dietary catechins in early prevention of breast cell carcinogenesis induced chronically by environmental carcinogens. Breast cell carcinogenesis is a multiyear, multistep, and multipath disease process and is the most common type of cancer among women in northern America and northern Europe. Epidemiologic and experimental evidence suggests that dietary constituents in fruits and vegetables prevent human cancers; dietary prevention is a cost-efficient strategy to reduce the risk of this disease. However, the mechanisms for dietary prevention of cellular carcinogenesis induced by chronic exposure to environmental carcinogens remain to be elucidated. To identify dietary components for prevention of chronic human breast cell carcinogenesis, we studied biological, biochemical, and molecular target endpoints that were induced by low doses of carcinogens and suppressed by dietary catechins. Immortalized, non-cancerous human breast epithelial MCF10A cells were repeatedly exposed to carcinogens 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and/or benzo[α]pyrene (B[α]P), each at picomolar concentrations, to induce cancer-associated biological, biochemical, and molecular properties in cells. We then used these changes as target endpoints to reveal the activity of various dietary catechins in suppression of NNK- and B[α]P-induced cellular carcinogenesis. We identified that cells acquiring the biological properties of reduced dependence on growth factors, anchorage-independent growth, and acinar-conformational disruption were reduced by treatment with dietary catechins. We identified that hydroxysteroid (11-beta) dehydrogenase 2 (HSD11B2) played a novel role in NNK- and B[α]P-induced cellular carcinogenesis, and its expression acted as a molecular target endpoint in catechin-induced suppression of cellular carcinogenesis. The ability of catechins to reduce gene expression of cytochrome P450 enzymes CYP1A1 and CYP1B1, which can bioactivate NNK and B[α]P, may be involved in the preventive mechanism for catechins in suppression of cellular carcinogenesis. Our model system with biological, biochemical, and molecular target endpoints verified the value of dietary catechins for prevention of human breast cell carcinogenesis induced by repeated exposures to low doses of multiple environmental carcinogens. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4376.