Hepatic Proteomics Analysis of Nonalcoholic Fatty Liver Disease Obese Rat Model After Short- and Long-Term Soy Protein Isolate Feeding.

To identify possible mechanisms involved in the development and progression of nonalcoholic fatty liver disease (NAFLD), we conducted shotgun proteomics analysis on liver of obese Zucker rats fed either casein (CAS) or soy protein isolate (SPI) for 8 and 16 weeks. Rats (7 weeks old, n = 8-9/group) were randomly assigned to either a CAS-based or an SPI-based diet. Rats were killed after 8 or 16 weeks of feeding and livers were stored at -80°C. Ingenuity Pathway Analysis (IPA) software was used to facilitate interpretation of proteomics data. Predictions of activation or inhibition of molecules in the data were made based on activation z-score and P value of overlap (P < .05). Activation z-scores ≥2.0 indicate that a molecule is predicted to be activated, whereas activation z-scores of less than or equal to -2.0 indicate that a target molecule is predicted to be inhibited. Upstream regulator analysis with IPA revealed Neuregulin 1 (NRG1) to be the top activated protein in (z-score = 2.48, P < .05), and MKNK1 as the top inhibited protein (z-score = -2.83, P < .05) in SPI diet compared with CAS diet after both 8 and 16 weeks of SPI feeding. Regulator effects analysis also predicted that some proteins would be participating, directly or indirectly, in the inhibition of immune response functions (such as leukocyte migration) and lipid metabolism (such as synthesis of lipids) in SPI-fed rats relative to CAS-fed rats. Our results suggest that SPI diet modifies the expression of proteins that could be involved in the reduction of NAFLD.

Research Note: Modified serum fluorescein isothiocyanate dextran (FITC-d) assay procedure to determine intestinal permeability in poultry fed diets high in natural or synthetic pigments.

Oral administration of fluorescein isothiocyanate dextran (FITC-d) has been used as an indicator for intestinal permeability in poultry research for several years. Under healthy conditions, tight junctions in the intestinal wall will not allow the 4-6kDa FITC-d to enter the bloodstream. Detection of FITC-d in serum (1-hour post-oral administration of FITC-d) has proven to be a reliable indicator of leaky gut syndrome (increased intestinal inflammation and disruption of tight junctions). Administration of supplementary phytobiotics in feed, particularly products with high beta-carotene levels or other pigments, has resulted in strong serum background fluorescence, which can render this assay unreliable. To account for this increase in background autofluorescence, the FITC-d assay procedure has been modified to accommodate these particular serum samples by including pre-administration serum collection from each treatment group to remove background fluorescence. The modified FITC-d procedure detailed will allow for analysis of intestinal permeability in pigmented serum.

A Comparison of Short- and Long-Term Soy Protein Isolate Intake and Its Ability to Reduce Liver Steatosis in Obese Zucker Rats Through Modifications of Genes Involved in Inflammation and Lipid Transport.

Obesity can lead to several health disorders including nonalcoholic fatty liver disease (NAFLD), the aggregation of lipids within hepatocytes, and consequent inflammation of the liver tissue. Previously, we reported that feeding obese Zucker rats with soy protein isolate (SPI) can reduce liver steatosis. To understand how SPI reduced liver steatosis, we conducted global gene expression analysis on liver samples obtained from these rats after short- (8 weeks) and long-term SPI feeding (16 weeks). We compared and contrasted these data using Ingenuity Pathway Analysis (IPA) software. This study focused mainly on target molecules that could be participating in inflammation processes and lipid metabolism that are well-known components of NAFLD. Inflammatory response was predicted to be inhibited in animals fed the SPI diet at both 8 and 16 weeks of experiment. This general prediction was based on negative activation z scores obtained through IPA (z score < -2.0, P < .00001) for eight aspects of immune function/inflammatory response. Lipid metabolism was predicted to be strongly enhanced in rats fed the SPI diet for 16 weeks than for 8 weeks. This prediction was based on positive activation z scores (z scores >2.0, P < .00001) of eight functions involved in lipid transport and metabolism. We observed that the longer the rats were fed the SPI diet, the more beneficial it resulted against NAFLD. Based on our findings, the predicted reductions in inflammatory mechanisms while enhancing lipid transport out of the liver could be the reasons behind the reduction of liver steatosis.

Effect of Morinda citrifolia (Noni)-Enriched Diet on Hepatic Heat Shock Protein and Lipid Metabolism-Related Genes in Heat Stressed Broiler Chickens.

Heat stress (HS) has been reported to alter fat deposition in broilers, however the underlying molecular mechanisms are not well-defined. The objectives of the current study were, therefore: (1) to determine the effects of acute (2 h) and chronic (3 weeks) HS on the expression of key molecular signatures involved in hepatic lipogenic and lipolytic programs, and (2) to assess if diet supplementation with dried Noni medicinal plant (0.2% of the diet) modulates these effects. Broilers (480 males, 1 d) were randomly assigned to 12 environmental chambers, subjected to two environmental conditions (heat stress, HS, 35°C vs. thermoneutral condition, TN, 24°C) and fed two diets (control vs. Noni) in a 2 × 2 factorial design. Feed intake and body weights were recorded, and blood and liver samples were collected at 2 h and 3 weeks post-heat exposure. HS depressed feed intake, reduced body weight, and up regulated the hepatic expression of heat shock protein HSP60, HSP70, HSP90 as well as key lipogenic proteins (fatty acid synthase, FASN; acetyl co-A carboxylase alpha, ACCα and ATP citrate lyase, ACLY). HS down regulated the hepatic expression of lipoprotein lipase (LPL) and hepatic triacylglycerol lipase (LIPC), but up-regulated ATGL. Although it did not affect growth performance, Noni supplementation regulated the hepatic expression of lipogenic proteins in a time- and gene-specific manner. Prior to HS, Noni increased ACLY and FASN in the acute and chronic experimental conditions, respectively. During acute HS, Noni increased ACCα, but reduced FASN and ACLY expression. Under chronic HS, Noni up regulated ACCα and FASN but it down regulated ACLY. In vitro studies, using chicken hepatocyte cell lines, showed that HS down-regulated the expression of ACCα, FASN, and ACLY. Treatment with quercetin, one bioactive ingredient in Noni, up-regulated the expression of ACCα, FASN, and ACLY under TN conditions, but it appeared to down-regulate ACCα and increase ACLY levels under HS exposure. In conclusion, our findings indicate that HS induces hepatic lipogenesis in chickens and this effect is probably mediated via HSPs. The modulation of hepatic HSP expression suggest also that Noni might be involved in modulating the stress response in chicken liver.

Noni (Morinda citrifolia) Modulates the Hypothalamic Expression of Stress- and Metabolic-Related Genes in Broilers Exposed to Acute Heat Stress.

Heat stress (HS) adversely affects growth performance and inflicts heavy economic losses to the poultry industry. There is, therefore, a critical need to identify new alternative strategies to alleviate the negative effects induced by HS. The tropic medicinal plant, Morinda citrifolia (Noni), is being used in livestock nutrition, however the literature is limited and conflicting for its impact on growth performance. The present study aimed to determine the effect of Noni on feeding and drinking behavior as well as on the hypothalamic expression of stress- and metabolic-related genes in broiler chickens exposed to acute HS. A total of 480 1 day-old male broiler chicks were randomly assigned to 12 controlled environmental chambers. Birds were subjected to two environmental conditions (TN, 25°C vs. HS, 35°C for 2 h) and fed two diets (control vs. 0.2% Noni) in a 2 × 2 factorial design. Feed intake and core body temperature (BT) were recorded during HS period. Blood was collected and hypothalamic tissues were harvested for target gene and protein analyses. Acute HS-broilers exhibited higher BT (~1°C), spent less time eating with a significant decrease in feed intake, and spent more time drinking along with higher drinking frequency compared to those maintained under TN conditions. Although Noni supplementation did not improve feed intake, it significantly delayed (~30 min) and reduced the BT-induced by HS. At molecular levels and under HS conditions, Noni supplementation down regulated the hypothalamic expression of HSP90 and its related transcription factors HSF1, 2, and 4, increased orexin mRNA levels, and decreased the phosphorylation levels of AMPKα1/2Thr172 and mTORSer2481. Together, these data indicated that Noni supplementation might modulate HS response in broilers through central orexin-AMPK-mTOR pathways.

Leucine supplementation at the onset of high-fat feeding does not prevent weight gain or improve glycemic regulation in male Sprague-Dawley rats

Obesity is a major public health concern and it is essential to identify effective treatments and preventative strategies to stop continued increases in obesity rates. The potential functional roles of the branched chain amino acid leucine make this amino acid an attractive candidate for the treatment and/or prevention of obesity. The objective of this study was to determine if long-term leucine supplementation could prevent the development of obesity and reduce the risk factors for chronic disease in rats fed a high-fat (60 % fat) diet. Male Sprague-Dawley rats (n = 30 per dietary treatment) were meal-fed (3 meals/day) either a control, low-fat diet (LF), control + leucine (LFL), high-fat (HF), or high-fat + leucine (HFL) for 42 days. On day 42, rats were sacrificed at 0, 30, or 90 min postprandial. Animals fed the HF and HFL diets had higher (P < 0.05) final body weights and weight gain compared to animals fed the LF and LFL diets. Leucine supplementation increased epididymal fat mass (P < 0.05) and decreased muscle mass (P < 0.05). There was no effect of leucine supplementation on postprandial glucose or insulin response. However, there was a significant effect (P < 0.05) of diet and time on free fatty acid concentrations. There was no effect of leucine on muscle markers of protein synthesis (4E-BP1, p70S6K) or energy metabolism (Akt, AMPK). Leucine supplementation decreased (P < 0.05) PGC1α expression and increased (P < 0.05) PPARγ expression in skeletal muscle. In conclusion, long-term leucine supplementation does not prevent weight gain, improve body composition, or improve glycemic control in rats fed a high-fat diet (AU)

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Leucine supplementation at the onset of high-fat feeding does not prevent weight gain or improve glycemic regulation in male Sprague-Dawley rats.

Obesity is a major public health concern and it is essential to identify effective treatments and preventative strategies to stop continued increases in obesity rates. The potential functional roles of the branched chain amino acid leucine make this amino acid an attractive candidate for the treatment and/or prevention of obesity. The objective of this study was to determine if long-term leucine supplementation could prevent the development of obesity and reduce the risk factors for chronic disease in rats fed a high-fat (60 % fat) diet. Male Sprague-Dawley rats (n = 30 per dietary treatment) were meal-fed (3 meals/day) either a control, low-fat diet (LF), control + leucine (LFL), high-fat (HF), or high-fat + leucine (HFL) for 42 days. On day 42, rats were sacrificed at 0, 30, or 90 min postprandial. Animals fed the HF and HFL diets had higher (P < 0.05) final body weights and weight gain compared to animals fed the LF and LFL diets. Leucine supplementation increased epididymal fat mass (P < 0.05) and decreased muscle mass (P < 0.05). There was no effect of leucine supplementation on postprandial glucose or insulin response. However, there was a significant effect (P < 0.05) of diet and time on free fatty acid concentrations. There was no effect of leucine on muscle markers of protein synthesis (4E-BP1, p70S6K) or energy metabolism (Akt, AMPK). Leucine supplementation decreased (P < 0.05) PGC1α expression and increased (P < 0.05) PPARγ expression in skeletal muscle. In conclusion, long-term leucine supplementation does not prevent weight gain, improve body composition, or improve glycemic control in rats fed a high-fat diet.

Differential expression of feeding-related hypothalamic neuropeptides in the first generation of quails divergently selected for low or high feed efficiency.

Livestock and poultry sectors are facing a combination of challenges, including a substantial increase in global demand for high quality animal protein, general droughts and steady rise in animal feed cost. Thus feed efficiency (FE), which defines the animal's ability to convert feed into body weight, is a vital economic and agricultural trait. Genetic selection for FE has been largely used in chickens and has been applied without knowledge of the underlying molecular mechanisms. Although it has made tremendous progress (breast yield, growth rate, egg production), there have been a number of undesirable changes such as metabolic disorders. In the present study we divergently selected male and female quail for high and low FE and we aimed to characterize the molecular basis of these differences at the central level, with the long-term goal of maximizing FE and avoiding the unfavorable consequences. The FE phenotype in first generation quails seemed to be achieved by reduced feed intake in female and increased body weight gain in males. At the molecular level, we found that the expression of feeding-related hypothalamic genes is gender- and line-dependent. Indeed, the expression of NPY, POMC, CART, CRH, melanocortin system (MC1R, MC2R, MC4R, MC5R), ORX, mTOR and ACCα was significantly decreased, however ORXR1/2, AMPKα1, S6K1 and STAT1, 5 and 6 were increased in high compared to low FE males (P<0.05). These genes did not differ between the two female lines. ADPN gene expression was higher and its receptor Adip-R1 was lower in LFE compared to HFE females (P<0.05). In male however, although there was no difference in ADPN gene expression between the genotypes, Adip-R1 and Adip-R2 mRNA abundances were higher in the LFE compared to HFE line (P<0.05). This study identified several key central feeding-related genes that are differentially expressed between low and high FE male and female quails which might explain the differences in feed intake/body weight gain observed between the two lines. Of particular interest, we provided novel insights into central AMPK-mTOR-ACC transcriptional differences between low and high FE quail which may open new research avenues on their roles in the regulation of energy balance and FE in poultry and livestock species.

Chronic Mild Cold Conditioning Modulates the Expression of Hypothalamic Neuropeptide and Intermediary Metabolic-Related Genes and Improves Growth Performances in Young Chicks.

BACKGROUND: Low environmental temperatures are among the most challenging stressors in poultry industries. Although landmark studies using acute severe cold exposure have been conducted, still the molecular mechanisms underlying cold-stress responses in birds are not completely defined. In the present study we determine the effect of chronic mild cold conditioning (CMCC) on growth performances and on the expression of key metabolic-related genes in three metabolically important tissues: brain (main site for feed intake control), liver (main site for lipogenesis) and muscle (main site for thermogenesis). METHODS: 80 one-day old male broiler chicks were divided into two weight-matched groups and maintained in two different temperature floor pen rooms (40 birds/room). The temperature of control room was 32°C, while the cold room temperature started at 26.7°C and gradually reduced every day (1°C/day) to reach 19.7°C at the seventh day of the experiment. At day 7, growth performances were recorded (from all birds) and blood samples and tissues were collected (n = 10). The rest of birds were maintained at the same standard environmental condition for two more weeks and growth performances were measured. RESULTS: Although feed intake remained unchanged, body weight gain was significantly increased in CMCC compared to the control chicks resulting in a significant low feed conversion ratio (FCR). Circulating cholesterol and creatine kinase levels were higher in CMCC chicks compared to the control group (P<0.05). CMCC significantly decreased the expression of both the hypothalamic orexigenic neuropeptide Y (NPY) and anorexigenic cocaine and amphetamine regulated transcript (CART) in chick brain which may explain the similar feed intake between the two groups. Compared to the control condition, CMCC increased the mRNA abundance of AMPKα1/α2 and decreased mTOR gene expression (P<0.05), the master energy and nutrient sensors, respectively. It also significantly decreased the expression of fatty acid synthase (FAS) gene in chick brain compared to the control. Although their roles are still unknown in avian species, adiponectin (Adpn) and its related receptors (AdipoR1 and 2) were down regulated in the brain of CMCC compared to control chicks (P<0.05). In the liver, CMCC significantly down regulated the expression of lipogenic genes namely FAS, acetyl-CoA carboxylase alpha (ACCα) and malic enzyme (ME) and their related transcription factors sterol regulatory element binding protein 1/2 (SREBP-1 and 2). Hepatic mTOR mRNA levels and phosphorylated mTOR at Ser2448 were down regulated (P<0.05), however phosphorylated ACCαSer79 (inactivation) was up regulated (P<0.05) in CMCC compared to control chicks, indicating that CMCC switch hepatic catabolism on and inhibits hepatic lipogenesis. In the muscle however, CMCC significantly up regulated the expression of carnitine palmitoyltransferase 1 (CPT-1) gene and the mRNA and phosphorylated protein levels of mTOR compared to the control chicks, indicating that CMCC enhanced muscle fatty acid ß-oxidation. CONCLUSIONS: In conclusion, this is the first report indicating that CMCC may regulate AMPK-mTOR expression in a tissue specific manner and identifying AMPK-mTOR as a potential molecular signature that controls cellular fatty acid utilization (inhibition of hepatic lipogenesis and induction of muscle fatty acid ß-oxidation) to enhance growth performance during mild cold acclimation.