c-Jun NH 2 -terminal kinase (JNK) activation plays a major role in acetaminophen (APAP)-induced hepatotoxicity. However, the exact mechanism of APAP-induced JNK activation is incompletely understood. It has been established that apoptosis signal-regulating kinase 1 (ASK1) regulates the late phase of APAP-induced JNK activation, but the mitogen-activated protein kinase kinase kinase that mediates the initial phase of APAP-induced JNK activation has not been identified. Oxidative stress produced during APAP metabolism causes JNK activation, which promotes mitochondrial dysfunction and results in the amplification of oxidative stress. Therefore, inhibition of the initial phase of JNK activation may be key to protection against APAP-induced liver injury. The goal of this study was to determine whether mixed-lineage kinase 3 (MLK3) mediates the initial, ASK1-independent phase of APAP-induced JNK activation and thus promotes drug-induced hepatotoxicity. We found that MLK3 was activated by oxidative stress and was required for JNK activation in response to oxidative stress. Loss of MLK3 attenuated APAP-induced JNK activation and hepatocyte death in vitro, independent of receptor-interacting protein 1. Moreover, JNK and glycogen synthase kinase 3␤ activation was significantly attenuated, and Mcl-1 degradation was inhibited in APAP-treated MLK3-knockout mice. Furthermore, we showed that loss of MLK3 increased expression of glutamate cysteine ligase, accelerated hepatic GSH recovery, and decreased production of reactive oxygen species after APAP treatment. MLK3-deficient mice were significantly protected from APAP-induced liver injury, compared with wildtype mice. Together, these studies establish a novel role for MLK3 in APAP-induced JNK activation and hepatotoxicity, and they suggest MLK3 as a possible target in the treatment of APAP-induced liver injury.

The effectiveness of whey protein isolate (WPI) coatings incorporated with grape seed extract (GSE), nisin (N), malic acid (MA), and ethylenediamine tetraacetic acid (EDTA) and their combinations to inhibit the growth of Listeria monocytogenes, E. coli O157:H7, and Salmonella typhimurium were evaluated in a turkey frankfurter system through surface inoculation (approximately 10(6) CFU/g) of pathogens. The inoculated frankfurters were dipped into WPI film forming solutions both with and without the addition of antimicrobial agents (GSE, MA, or N and EDTA, or combinations). Samples were stored at 4 degrees C for 28 d. The L. monocytogenes population (5.5 log/g) decreased to 2.3 log/g after 28 d at 4 degrees C in the samples containing nisin (6000 IU/g) combined with GSE (0.5%) and MA (1.0%). The S. typhimurium population (6.0 log/g) was decreased to approximately 1 log cycles after 28 d at 4 degrees C in the samples coated with WPI containing a combination of N, MA, GSE, and EDTA. The E. coli O157:H7 population (6.15 log/g) was decreased by 4.6 log cycles after 28 d in samples containing WPI coating incorporated with N, MA, and EDTA. These findings demonstrated that the use of an edible film coating containing nisin, organic acids, and natural extracts is a promising means of controlling the growth and recontamination of L. monocytogenes, S. typhimurium, and E. coli O157:H7 in ready-to-eat poultry products.

The objective of this research was to determine whether commercial proteases are capable of hydrolyzing denatured poultry by-product proteins that have gone through a rendering process. The material used for this research was low-ash poultry meal obtained from a local poultry processor. Samples of poultry meal were treated with the commercial proteases Alcalase, Flavourzyme, Protamex, and Liquipanol that were added individually or as a combination of 2 proteases, which were incorporated either simultaneously or sequentially. Temperature was controlled during the reaction to the optimal level for each enzyme, and pH was initially adjusted to the most favorable level for each enzyme and was maintained during the course of the reaction by addition of NaOH at established intervals. Consumption of NaOH was used to calculate the degree of hydrolysis. At the end of the hydrolysis, the molecular weight of selected hydrolysates was determined by size exclusion chromatography and by gel electrophoresis. In addition, amino acid analysis was performed on selected hydrolysates. Results show that the sequential treatment with Alcalase and Flavourzyme served best for the preparation of poultry meal hydrolysates with a maximum degree of hydrolysis of 11.1% and the highest hydrolyzable material recovered at 58%, which is attributed to the combined nature of the endo- and exocatalytic action of Alcalase and Flavourzyme, respectively. Hydrolysate with Flavourzyme or the combination of Flavourzyme and Alcalase were both good methods to produce significant amounts of free amino acids. This research shows the feasibility of hydrolyzing poultry by-products that went through a rendering process using different brands of commercial proteases. Findings from this research are important in the preparation of palatants, in which relatively inexpensive hydrolyzed poultry meal could be used to improve the flavor of companion animal food products.

The objective of this study was to examine the extent to which bitter melon seed (BMS) alleviates the symptoms associated with metabolic syndrome and elucidate the mechanism by which BMS exerts beneficial effects. Three-month-old female Zucker rats were assigned to following groups: lean control (L-Ctrl), obese control (O-Ctrl), and obese + BMS (O-BMS). The control groups were fed AIN-93M purified rodent diet, and the O-BMS group was fed AIN-93M diet modified to contain 3.0% (wt/wt) ground BMS for 100 days. After 100 days of treatment, BMS supplementation in the obese rats lowered the total serum cholesterol by 38% and low-density lipoprotein-cholesterol levels by about 52% and increased the ratio of serum high-density lipoprotein-cholesterol to total cholesterol compared to the O-Ctrl group. The percentage of total liver lipids was about 32% lower and serum triglyceride levels were 71% higher in the O-BMS group compared to the O-Ctrl group. Serum glucose levels were significantly lowered partly because of the increase in the serum insulin levels in the BMS-based diet groups. BMS supplementation increased the expression of peroxisome proliferator-activated receptor-γ (PPAR-γ) in the white adipose tissue of the obese rats significantly (P < .05) and down-regulated the expression of PPAR-γ, nuclear factor-κB (NF-κB), and interferon-γ mRNA in heart tissue of the obese rats. The findings of this study suggest that BMS improves the serum and liver lipid profiles and serum glucose levels by modulating PPAR-γ gene expression. To our knowledge, this study for the first time shows that BMS exerts cardioprotective effects by down-regulating the NF-κB inflammatory pathway.

MLK3) is a mitogenactivated protein kinase kinase kinase (MAP3K) that mediates JNK activation in response to saturated fatty acids in vitro; however, the exact mechanism for diet-induced JNK activation in vivo is not known. Here, we have used MLK3-deficient mice to examine the role of MLK3 in a saturated-fat diet model of obesity. MLK3-KO mice fed a high-fat diet enriched in medium-chain saturated fatty acids for 16 wk had decreased body fat compared with wild-type (WT) mice due to increased energy expenditure independently of food consumption and physical activity. Moreover, MLK3 deficiency attenuated palmitate-induced JNK activation and M1 polarization in bone marrowderived macrophages in vitro, and obesity induced JNK activation, macrophage infiltration into adipose tissue, and expression of proinflammatory cytokines in vivo. In addition, loss of MLK3 improved insulin resistance and decreased hepatic steatosis. Together, these data demonstrate that MLK3 promotes saturated fatty acid-induced JNK activation in vivo and diet-induced metabolic dysfunction. mixed-lineage kinase 3; saturated fatty acids; insulin resistance; inflammation

Objective Mitogen Activated Protein Kinase (MAPK) pathways play an important role in neointima formation secondary to vascular injury, in part by promoting proliferation of vascular smooth muscle cells (VSMC). Mixed-lineage kinase 3 (MLK3) is a MAP kinase kinase kinase (MAP3K) that activates multiple MAPK pathways, and has been implicated in regulating proliferation in several cell types. However, the role of MLK3 in VSMC proliferation and neointima formation is unknown. The aim of this study was to determine the function of MLK3 in the development of neointimal hyperplasia, and to elucidate the underlying mechanisms. Approach and Results Neointima formation was analyzed after endothelial denudation of carotid arteries from wild-type (WT) and Mlk3−/− mice. MLK3 deficiency promoted injury-induced neointima formation and increased proliferation of primary VSMC derived from aortas isolated from Mlk3−/− mice compared to WT mice. Furthermore, MLK3 deficiency increased activation of p63RhoGEF, RhoA and Rho-kinase (ROCK) in VSMC, a pathway known to promote neointimal hyperplasia, and reconstitution of MLK3 expression attenuated ROCK activation. Moreover, JNK activation was decreased in MLK3 deficient VSMC and proliferation of WT but not MLK3 KO cells treated with a JNK inhibitor was attenuated. Conclusions We demonstrate that MLK3 limits RhoA activation and injury-induced neointima formation, by binding to and inhibiting activation of p63RhoGEF, a RhoA activator. In MLK3 deficient cells, activation of p63RhoGEF proceeds in an unchecked manner, leading to a net increase in RhoA pathway activation. Reconstitution of MLK3 expression restores MLK3/p63RhoGEF interaction, which is attenuated by feedback from activated JNK.

Conjugated linoleic acid (CLA) is a collection of octadecadienoic fatty acids that have been shown to possess numerous health benefits. The CLA used in our study was produced by the photoisomerization of soybean oil and consists of about 20% CLA; this CLA consists of 75% trans-trans (a mixture of t8,t10; t9,t11; t10,t12) isomers. This method could be readily used to increase the CLA content of all soybean oil used as a food ingredient. The objective of this study was to determine the effects of trans-trans CLA-rich soy oil, fed as a dietary supplement, on body composition, dyslipidemia, hepatic steatosis, and markers of glucose control and liver function of obese fa/fa Zucker rats. The trans-trans CLA-rich soy oil lowered the serum cholesterol and low density lipoprotein-cholesterol levels by 41 and 50%, respectively, when compared to obese controls. Trans-trans CLA-rich soy oil supplementation also lowered the liver lipid content significantly (P < 0.05) with a concomitant decrease in the liver weight in the obese rats. In addition, glycated hemoglobin values were improved in the group receiving CLA-enriched soybean oil in comparison to the obese control. PPAR-γ expression in white adipose tissue was unchanged. In conclusion, trans-trans CLA-rich soy oil was effective in lowering total liver lipids and serum cholesterol.

Background: One in five adults in the United States is obese as defined by a body mass index of 30 kg/m2. Obesity is associated with metabolic syndrome, a combination of medical conditions including cardiovascular disease, type 2 diabetes, hypertension, hypercholesterolemia, and hypertriglyceridemia. These conditions present challenges to the medical care system and require a multifaceted approach through a variety of interventions. This study investigated the effects of fructooligosaccharides (FOS) at the level of 5 % (w/w) in alleviating the complications associated with metabolic syndrome.Methods: The study was carried out using thirty-six, three-month old female lean and obese Zucker rats housed in an environmentally controlled laboratory. The Zucker rats were divided into three groups (N=12): Lean (L-CTRL) and obese controls (O-CTRL) and obese-FOS (O-FOS). The controls received AIN-93M purified rodent diet and the animals in the O-FOS group were fed AIN-93M diet modified to contain 5.0% FOS (w/w). After 100 days of treatment, the rats were fasted for 12 hours and sacrificed. Tissue and organs of interest, and blood were collected for analysis. Serum concentrations of the following were determined: glucose, glycosylated hemoglobin (HbA1c), total cholesterol (TC), low-density lipoprotein-cholesterol (LDL-C), high-density lipoprotein-cholesterol (HDL-C), triglycerides (TG), and insulin. Gravimetric quantification of liver lipids was performed and peroxisome proliferator-activated receptor-γ (PPAR-γ) gene expression was determined in white adipose tissue by qRT-PCR.Results: No significant differences were observed in the serum lipids, fasting blood glucose, HbA1c and PPAR-γ gene expression in white adipose tissue of O-FOS group compared to O-CTRL group. FOS supplementation significantly lowered the percent total liver lipids by 12% with a subsequent reduction in the liver weights compared to O-CTRL rats. Serum insulin concentrations were lowered 3.6 fold in O-FOS group compared to O-CTRL (P < 0.05). Conclusion: Based on these findings we conclude that dietary supplementation of 5% FOS (w/w) may reduce hepatic steatosis and the risk for non-alcoholic fatty liver disease (NAFLD) associated with insulin resistance without changes in blood lipids and glucose levels.Key words: Dietary fiber, Fructooligosaccharide, Hepatosteatosis, Liver lipids, Non-alcoholic fatty liver disease (NAFLD), Zucker.