Person:

Kang, Jing

Obejctive: Because of confounding factors, the effects of dietary n-3 polyunsaturated fatty acids (PUFA) on type 1 diabetes remain to be clarified. We therefore evaluated whether fat-1 transgenic mice, a well-controlled experimental model endogenously synthesizing n-3 PUFA, were protected against streptozotocin (STZ)-induced diabetes. We then aimed to elucidate the in vivo response at the pancreatic level. Research Design and Methods: (\beta)-Cell destruction was produced by multiple low-doses STZ (MLD-STZ). Blood glucose level, plasma insulin level, and plasma lipid analysis were then performed. Pancreatic mRNA expression of cytokines, the monocyte chemoattractant protein, and GLUT2 were evaluated as well as pancreas nuclear factor (NF)-(\kappa)B p65 and inhibitor of (\kappa)B (I(\kappa)B) protein expression. Insulin and cleaved caspase-3 immunostaining and lipidomic analysis were performed in the pancreas. Results: STZ-induced fat-1 mice did not develop hyperglycemia compared with wild-type mice, and (\beta)-cell destruction was prevented as evidenced by lack of histological pancreatic damage or reduced insulin level. The prevention of (\beta)-cell destruction was associated with no proinflammatory cytokine induction (tumor necrosis factor-(\alpha), interleukin-1(\beta), inducible nitric oxide synthase) in the pancreas, a decreased NF-(\kappa)B, and increased I(\kappa)B pancreatic protein expression. In the fat-1–treated mice, proinflammatory arachidonic-derived mediators as prostaglandin E(_2) and 12-hydroxyeicosatetraenoic acid were decreased and the anti-inflammatory lipoxin A(_4) was detected. Moreover, the 18-hydroxyeicosapentaenoic acid, precursor of the anti-inflammatory resolvin E1, was highly increased. Conclusions: Collectively, these findings indicate that fat-1 mice were protected against MLD-STZ–induced diabetes and pointed out for the first time in vivo the beneficial effects of n-3 PUFA at the pancreatic level, on each step of the development of the pathology—inflammation, (\beta)-cell damage—through cytokine response and lipid mediator production.

Background: Non–small cell lung cancer is associated with high expression of multidrug resistance (MDR) proteins and low production of reactive oxygen species (ROS). Coptis extract (COP), a Chinese medicinal herb, and its major constituent, berberine (BER), have anticancer properties. This study aims to investigate the effects of COP and BER combined with chemotherapeutic agents, including fluorouracil (5-FU), camptothecin (CPT), and paclitaxel (TAX), on cell proliferation, ROS production, and MDR in A549 human non-small cell lung cancer cells. Methods: A549 cells were treated with different doses of COP and BER, combined with 5-FU, CPT, and TAX. Cell viability was measured by an XTT (2,3-bis-(2-methoxy-4- nitro-5-sulfophenyl)-2 H-tetrazolium-5-carboxanilide) assay. Intracellular ROS levels were determined by measuring the oxidative conversion of cell permeable 2′,7′-dichlorofluorescein diacetate to fluorescent dichlorofluorescein. MDR of A549 cells was assessed by rhodamine 123 retention assay. Results: Both COP and BER significantly inhibited A549 cell growth in a dose-dependent manner. Combinations of COP or BER with chemotherapeutic agents (5-FU, CPT, and TAX) exhibited a stronger inhibitory effect on A549 cell growth. In addition, COP and BER increased ROS production and reduced MDR in A549 cells. Conclusion: As potential adjuvants to chemotherapy for non–small cell lung cancer, COP and BER increase ROS production, reduce MDR, and enhance the inhibitory effects of chemotherapeutic agents on A549 cell growth.

Recent studies have shown that a tumor-supportive microenvironment is characterized by high levels of pro-inflammatory and pro-angiogenic eicosanoids derived from omega-6 (n−6) arachidonic acid (AA). Although the metabolic pathways (COX, LOX, and P450) that generate these n−6 AA eicosanoids have been targeted, the role of endogenous AA production in tumorigenesis remains unexplored. Delta-6 desaturase (D6D) is the rate-limiting enzyme responsible for the synthesis of n−6 AA and increased D6D activity can lead to enhanced n−6 AA production. Here, we show that D6D activity is upregulated during melanoma and lung tumor growth and that suppressing D6D activity, either by RNAi knockdown or a specific D6D inhibitor, dramatically reduces tumor growth. Accordingly, the content of AA and AA-derived tumor-promoting metabolites is significantly decreased. Angiogenesis and inflammatory status are also reduced. These results identify D6D as a key factor for tumor growth and as a potential target for cancer therapy and prevention.

Omega-3 polyunsaturated fatty acids (omega-3 PUFAs) play a role in controlling pathological inflammatory reactions. Endometriosis is characterized by the presence of endometrial tissue on the peritoneum and an exaggerated inflammatory environment around ectopic tissues. Here peritoneal endometriosis was reproduced using a mouse model in which murine endometrial fragments were inoculated into the peritoneal cavity of mice. Fat-1 mice, in which omega-6 can be converted to omega-3 PUFAs, or wild type mice, in which it cannot, were used for the endometriosis model to address the actions of omega-3 PUFAs on the development of endometriotic lesions. The number and weight of cystic endometriotic lesions in fat-1 mice two weeks after inoculation were significantly less than half to those of controls. Mediator lipidomics revealed that cystic endometriotic lesions and peritoneal fluids were abundant in 12/15-hydroxyeicosapentaenoic acid (12/15-HEPE), derived from eicosapentaenoic acid (EPA), and their amount in fat-1 mice was significantly larger than that in controls. 12/15-Lipoxygenase (12/15-LOX)-knockout (KO) and control mice with or without EPA administration were assessed for the endometriosis model. EPA administration decreased the number of lesions in controls but not in 12/15-LOX-KO mice. The peritoneal fluids in EPA-fed 12/15-LOX-KO mice contained reduced levels of EPA metabolites such as 12/15-HEPE and EPA-derived resolvin E3 even after EPA administration. cDNA microarrays of endometriotic lesions revealed that Interleukin-6 (IL-6) expression in fat-1 mice was significantly lower than that in controls. These results suggest that both endogenous and exogenous EPA-derived PUFAs protect against the development of endometriosis through their anti-inflammatory effects and, in particular, the 12/15-LOX-pathway products of EPA may be key mediators to suppress endometriosis.

N-3 polyunsaturated fatty acids (PUFAs) have potential cardiovascular benefit, although the mechanisms underlying this effect remain poorly understood. Fat-1 transgenic mice expressing Caenorhabditis elegans n-3 fatty acid desaturase, which is capable of producing n-3 PUFAs from n-6 PUFAs, exhibited resistance to pressure overload–induced inflammation and fibrosis, as well as reduced cardiac function. Lipidomic analysis revealed selective enrichment of eicosapentaenoic acid (EPA) in fat-1 transgenic bone marrow (BM) cells and EPA-metabolite 18-hydroxyeicosapentaenoic acid (18-HEPE) in fat-1 transgenic macrophages. BM transplantation experiments revealed that fat-1 transgenic BM cells, but not fat-1 transgenic cardiac cells, contributed to the antiremodeling effect and that the 18-HEPE–rich milieu in the fat-1 transgenic heart was generated by BM-derived cells, most likely macrophages. 18-HEPE inhibited macrophage-mediated proinflammatory activation of cardiac fibroblasts in culture, and in vivo administration of 18-HEPE reproduced the fat-1 mice phenotype, including resistance to pressure overload–induced maladaptive cardiac remodeling.

Omega-3 polyunsaturated fatty acids such as eicosapentaenoic acid (EPA) have anti-inflammatory effects. Preterm birth is an important problem in modern obstetrics and one of the main causes is an inflammation. We here showed that abundance of omega-3 fatty acids reduced the incidence of preterm birth induced by LPS with fat-1 mice, capable of converting omega-6 to omega-3 fatty acids. We also indicated that the gene expression of IL-6 and IL-1β in uteruses and the number of cervical infiltrating macrophages were reduced in fat-1 mice. The analyses of lipid metabolomics showed the high level of 18-hydroxyeicosapentaenoate in fat-1 mice, which was derived from EPA and was metabolized to anti-inflammatory product named resolvin E3 (RvE3). We finally showed that the administration of RvE3 to LPS-exposed pregnant wild type mice lowered the incidence of preterm birth. Our data suggest that RvE3 could be a potential new therapeutic for the prevention of preterm birth.

Transgenic mice (named “Omega mice”) were engineered to carry both optimized fat-1 and fat-2 genes from the roundworm Caenorhabditis elegans and are capable of producing essential omega-6 and omega-3 fatty acids from saturated fats or carbohydrates. When maintained on a high-saturated fat diet lacking essential fatty acids or a high-carbohydrate, no-fat diet, the Omega mice exhibit high tissue levels of both omega-6 and omega-3 fatty acids, with a ratio of ∼1∶1. This study thus presents an innovative technology for the production of both omega-6 and omega-3 essential fatty acids, as well as a new animal model for understanding the true impact of fat on human health.

Fat-1 transgenic mice, which endogenously convert n-6 PUFA to n-3 PUFA, are a useful tool in health research; however with this model timing of n-3 PUFA enrichment cannot be directly controlled. To add such capability, the novel Cre-recombinase inducible fat-1 (iFat1) transgenic mouse has been developed. The aim of this study was to characterize the utility of the iFat1 transgene as a model of Cre-inducible endogenous n-3 PUFA enrichment. Functionality of the iFat1 transgene was screened both in vitro and in vivo. In the presence of Cre, the iFat1 transgene resulted in a balancing (p < 0.01) of the n-6/n-3 PUFA ratio within phospholipids in the human embryonic kidney 293T cell line. For in vivo analysis, iFat1 transgenic mice were crossed with the R26-Cre-ERT2 (Tam-Cre) mouse line, a tamoxifen inducible Cre-expression model. Tam-Cre/iFat1 double hybrids were transiently treated with tamoxifen at 6–7 weeks, then terminated 3 weeks later. Tamoxifen treated mice had increased (p < 0.05) tissue n-3 PUFA and ≥two-fold reduction (p < 0.05) in the n-6/n-3 PUFA ratio of liver, kidney and muscle phospholipids relative to vehicle treated controls. Collectively these findings suggest that the iFat1 transgenic mouse may be a promising tool to help elucidate the temporal effects through which n-3 PUFA impacts health related outcomes.

Cancer associated fibroblasts (CAFs) are responsible for tumor growth, angiogenesis, invasion, and metastasis. Matrix metalloproteinase (MMP)-9 secreted from cancer stroma populated by CAFs is a prerequisite for cancer angiogenesis and metastasis. Omega-3 polyunsaturated fatty acids (omega-3 PUFA) have been reported to have anti-tumor effects on diverse types of malignancies. Fat-1 mice, which can convert omega-6 to omega-3 PUFA independent of diet, are useful to investigate the functions of endogenous omega-3 PUFA. To examine the effect of omega-3 PUFA on tumorigenesis, TC-1 cells, a murine epithelial cell line immortalized by human papillomavirus (HPV) oncogenes, were injected subcutaneously into fat-1 or wild type mice. Tumor growth and angiogenesis of the TC-1 tumor were significantly suppressed in fat-1 compared to wild type mice. cDNA microarray of the tumors derived from fat-1 and wild type mice revealed that MMP-9 is downregulated in fat-1 mice. Immunohistochemical study demonstrated immunoreactivity for MMP-9 in the tumor stromal fibroblasts was diffusely positive in wild type whereas focal in fat-1 mice. MMP-9 was expressed in primary cultured fibroblasts isolated from fat-1 and wild type mice but was not expressed in TC-1 cells. Co-culture of fibroblasts with TC-1 cells enhanced the expression and the proteinase activity of MMP-9, although the protease activity of MMP-9 in fat-1-derived fibroblasts was lower than that in wild type fibroblasts. Our data suggests that omega-3 PUFAs suppress MMP-9 induction and tumor angiogenesis. These findings may provide insight into mechanisms by which omega-3 PUFAs exert anti-tumor effects by modulating tumor microenvironment.

A balanced omega-6/omega-3 polyunsaturated fatty acid (PUFA) ratio has been linked to health benefits and the prevention of many chronic diseases. Current dietary intervention studies with different sources of omega-3 fatty acids (omega-3) lack appropriate control diets and carry many other confounding factors derived from genetic and environmental variability. In our study, we used the fat-1 transgenic mouse model as a proxy for long-term omega-3 supplementation to determine, in a well-controlled manner, the molecular phenotype associated with a balanced omega-6/omega-3 ratio. The fat-1 mouse can convert omega-6 to omega-3 PUFAs, which protect against a wide variety of diseases including chronic inflammatory diseases and cancer. Both wild-type (WT) and fat-1 mice were subjected to an identical diet containing 10% corn oil, which has a high omega-6 content similar to that of the Western diet, for a six-month duration. We used a multi-platform lipidomic approach to compare the plasma lipidome between fat-1 and WT mice. In fat-1 mice, an unbiased profiling showed a significant increase in the levels of unesterified eicosapentaenoic acid (EPA), EPA-containing cholesteryl ester, and omega-3 lysophosphospholipids. The increase in omega-3 lipids is accompanied by a significant reduction in omega-6 unesterified docosapentaenoic acid (omega-6 DPA) and DPA-containing cholesteryl ester as well as omega-6 phospholipids and triacylglycerides. Targeted lipidomics profiling highlighted a remarkable increase in EPA-derived diols and epoxides formed via the cytochrome P450 (CYP450) pathway in the plasma of fat-1 mice compared with WT mice. Integration of the results of untargeted and targeted analyses has identified a lipidomic biosignature that may underlie the healthful phenotype associated with a balanced omega-6/omega-3 ratio, and can potentially be used as a circulating biomarker for monitoring the health status and the efficacy of omega-3 intervention in humans.