2013 Annual Report
1a.Objectives (from AD-416):
The overall goal of the project is to elucidate the molecular and cellular mechanisms that respond to selected health promoting food components to reduce the risk of chronic diseases such as cancers and obesity. A secondary aim is to explore the utility of a porcine model to test the effect of health maintenance via diet and identify resulting biomarkers that reflect health status. Objective 1. Elucidate biological activities of health promoting phytochemicals from grape, soy, and cruciferous vegetables against development of breast and prostate cancer. Objective 2. Identify molecular targets and mechanisms of action of health promoting food components in animal or in vitro models of cancer and obesity. Objective 3. Ascertain the effects of specific probiotic strains in appropriate animal models of obesity. Objective 4. Identify plant polyphenols and probiotics that affect adipocyte numbers, size, and fat accumulation, and the regulation of proinflammatory mRNA stability by tristetraprolin. Objective 5. Tie together obesity, inflammation, and cancer mechanistically in appropriate animal or in vitro models.
1b.Approach (from AD-416):
Studies will evaluate if phytoalexins structurally similar to resveratrol exerts similar anti-prostate cancer effects; if soy phytoalexin glyceollins exert anti-prostate cancer effects; if phytochemicals modulate LXR-mediated pathways in prostate epithelial cells and modulate LXR-mediated pathways in macrophage. Other studies will determine if probiotic bacterial strains differ in their protective effects against chronic diseases related to obesity; regulate adipocyte numbers, size, and fat accumulation associated with the anti-inflammatory protein tristetraprolin (TTP); if obesity alters the macrophage phenotype and function in adipose tissue, colon, breast, and prostate following increased localized inflammation; and if broccoli-derived phytochemicals modulate LXR-responsive pathways in vivo. The studies will involve in vitro cell culture approaches confirmed by rodent and pig animal models.
In vitro and in vivo models have been used to identify biologically active compounds from the diet. Using a prostate cancer cell model in-vitro, we observed that soy glyceollins inhibited LNCaP prostate cancer cell growth similar to that of the soy isoflavone genistein. The growth inhibitory effects of the glyceollins appeared to be due to an inhibition of cell cycle progression. In addition, glyceollin treatments led to down-regulated mRNA levels for androgen responsive genes. We began to characterize Liver X receptors (LXR)-dependent pathways in-vitro by cancer protective phytochemicals. Expression of LXR-responsive genes is being used as markers for potential modulation of the pathway. We also initiated a study to examine whether cancer protective effects of broccoli-derived phytochemicals can be synergistic or additive.
LXR regulate cholesterol metabolism and inflammatory responses. High fat (HF) diets and microbial infection antagonize the LXR pathway leading to accumulation of cholesteryl-esters (CE) and increased expression of pro-inflammatory mediators in macrophages. Alveolar macrophages (AM) isolated from Ossabaw pigs fed an HF diet had significantly higher concentrations of CE compared to AM from pigs fed a control (C) basal diet, but not in pigs fed a HF diet and given the probiotic L. paracasei (HFPB). Ex vivo stimulation of AM with LPS significantly opposed LXR agonist-mediated transcription of cholesterol metabolism genes in pigs. This transcriptional effect was abrogated in AM from pigs given L. paracasei. Protein analysis of cell culture supernatants revealed that AM from HFPB-fed pigs had significantly lower LPS-induced protein expression of IL-1ß than AM from HF-fed pigs. These data demonstrated a role for L. paracasei in modulating AM cholesterol metabolism and the response to LPS.
The anti-inflammatory protein tristetraprolin (TTP) was analyzed and we did not observe significant changes related to regulation of adipocyte numbers, size or fat accumulation by probiotics. Hence, a large-scale non-targeted metabolomic approach was used to reveal metabolite patterns from obesity-related metabolic disturbances in a pig model. Feeding a high energy/high fat diet influenced metabolic homeostasis of Ossabaw pigs throughout all tissues and biofluids examined. Metabolite profiling involving multiple sample types was shown to be a feasible method to provide information on metabolic pathways affected by the dietary treatment.
Proanthocyanidins, which are amongst the most bioactive polyphenols in the diet, are catabolized by intestinal microbiota. The effect of cranberry extract containing ~7% proanthocyanidins (ECPA), on weight gain and composition of intestinal microbiota was evaluated in pig model. Young pigs fed ECPA added to a basal or high fat diet for 12 weeks were compared to pigs fed similar diets without ECPA. DNA from fecal samples, collected at day 0 and weeks 2, 4, 6, 10, and contents from proximal and distal colon collected at necropsy were processed. Molecular and metabolic analyses are currently underway.
Conducted a large scale metabolomic analysis of pigs on a high energy/high fat diet. Typical analyses of body fluids from human dietary interventions do not provide adequate information about diet-induced metabolic changes taking place in tissues. We used a large-scale, non-targeted metabolomic approach to reveal metabolite patterns from obesity-related metabolic disturbances produced in a specialized pig model. Feeding a high energy/high fat diet influenced metabolic homeostasis of Ossabaw pigs throughout all tissues and biofluids including plasma, urine, bile, liver, pancreas, brain cortex, intestinal jejunum, and proximal colon. Metabolite profiling involving multiple sample types was shown to be a feasible methodology to provide information on metabolic pathways affected by the dietary treatment. This study provides a more comprehensive evaluation of the effect of diet on metabolism of the whole animal that can be used to better understand the role of diet in health.
Daily feeding of probiotics can improve the activity of immune cells in the lungs.
High fat diets and microbial infection can antagonize important pathways in inflammatory cells in the lung called macrophages that normally accumulate cholesterol from the blood and release pro-inflammatory molecules to control infection. Feeding the probiotic bacteria Lactobacillus paracasei can lower blood cholesterol and modulate protective immune function. We used this information to demonstrate that daily feeding of L. paracasei to a unique variety of Ossabaw pigs that become obese when fed a high fat diet could modulate cholesterol metabolism and improve macrophages immune capacity. This is one of the few demonstrations of the beneficial effects of adding probiotics to the diet to improve metabolism and immune function in the lung.
Infection with parasitic worms can improve metabolic function in obese mice.
Obesity is associated with a chronic low-grade inflammation and increased levels of pro-inflammatory molecules that can disrupt metabolic homeostasis. Parasitic worm infection has been shown to reduce the intensity of certain inflammatory diseases. We examined the effects of worm infection in a mouse model of obesity and associated metabolic dysfunction to explore the mechanism of action and determine if features of the interaction could be used to control obesity related inflammation. The infection reduced body weight and fat tissue mass in mice fed a high fat diet and improved glucose metabolism along with healthy changes in the profile of metabolic hormones. The results show that parasitic worms have both preventive and therapeutic effects against the development of obesity and associated features of metabolic dysfunction in mice. Although not directly useful as a treatment strategy in humans, it does provide a model to look for the molecules produced by worms that could be applied in humans to reduce inflammatory diseases.
Identify plant bioactive components and their mechanisms of action. The anti-cancer effects of a broccoli-derived compound indole-3-carbinol and diindolylmethane was examined using an in-vitro model of prostate cancer. These compounds were found to indirectly affect monocyte/macrophage migration by regulating the production of chemo-attractants of monocyte/macrophage by prostate cancer cells. Inflammation is an important promoter of tumor cell invasion and metastasis. This information provides mechanistic support for the potential beneficial effects of cruciferous vegetables on prostate cancer development.
Hanhineva, K., Barri, T., Kolemainen, M., Pekkinen, J., Pihlajamaki, J., Vesterbacka, A., Solano Aguilar, G., Mykkänen, H., Dragsted, L., Urban Jr, J.F., Poutanen, K. 2013. Comparative non-targeted metabolite profiling of metabolic changes in tissues and bio-fluids in high-fat diet fed Ossabaw pig. Journal of Proteome Research. DOI: 10.1021/pr400257d.
Yang, Z., Grinchuk, V., Smith, A.D., Qin, B., Bohl, J.A., Sun, R., Notari, L., Zhang, Z., Sesaki, H., Urban Jr, J.F., Shea-Donahue, T., Zhao, A. 2013. Parasitic nematode-induced modulation of body weight and associated metabolic dysfunction in mouse models of obesity. Infection and Immunity. 81(6):1905-1914.
Huang, H., Fletcher, A., Niu, Y., Wang, T.T., Yu, L. 2012. Characterization of lipopolysaccharide-stimulated cytokine expression in macrophages and monocytes. Inflammation Research. 61(12):1329-1338.
Kim, E.K., Kim, Y.S., Milner, J.A., Wang, T.T. 2013. Indole-3-carbinol and 3’, 3’-diindolylmethane modulate androgen effect up-regulation on C-C chemokine ligand 2 and monocyte attraction to prostate cancer cells. Cancer Prevention Research. 6(6):519-529.
Wang, T.T., Edwards, A.J., Clevidence, B.A. 2013. Strong and weak plasma response to dietary carotenoids identified by cluster analysis and linked to beta-carotene 15,15'-monooxygenase 1 single nucleotide polymorphisms. Journal of Nutritional Biochemistry. 24(8):1538-1546.
Lou, Y., Wang, T.T., Teng, Z., Chen, P., Sun, J., Wang, Q. 2013. Encapsulation of indole-3-carbinol and 3'3'-diindolylmethane in zein/carboxymethyl chitosan nanoparticles with controlled release property and improved stability. Food Chemistry. 139(1-4):224-30.