Casein-based Diet Research Articles

Rice Endosperm Protein Improves the Anti-Inflammatory Effects of High-Density Lipoprotein and Produces Lower Atherosclerotic Lesion Accelerated by the Renal Mass Reduction than Casein in a Mouse Model

Background Chronic kidney disease (CKD) impairs the anti-inflammatory effects of high-density lipoprotein (HDL) and increases cardiovascular mortality. Though the potential role of dietary interventions to manage HDL is well studied, the clinical trials aimed to increase HDL levels have failed to reduce cardiovascular risk, rendering HDL function to be explored as a more relevant clinical parameter. This study investigates the effects of rice endosperm protein (REP), a plant-based protein, on the anti-inflammatory properties of HDL and renal injury-driven atherosclerosis in comparison with casein, an animal protein. Materials and methods Ten-week-old apolipoprotein E-deficient hyperlipidemic mice underwent uninephrectomy. The mice (n = 6 each) were pair-fed a normal casein-based diet or a REP-based diet (both with 20.0% protein content) for seven weeks. Atherosclerotic lesions were detected by en face Sudan IV staining of the aorta. Results The number and sizes of the atherosclerotic lesions were significantly lower in the REP-based diet-fed group than the casein-based diet-fed group (p = 0.038). However, the REP-based diet neither elicited an ameliorative effect on kidney function or histology nor impacted the cholesterol profiles. Furthermore, HDL from the REP-based diet-fed mice significantly suppressed the inflammatory cytokine response of human umbilical vein endothelial cells than that from the casein-based diet-fed mice (MCP-1, p = 0.010; IL-6, p = 0.011; IL-1β, p = 0.028). Conclusions The REP-based diet has a higher potential to lessen the atherosclerotic lesions accelerated by renal mass reduction than a casein-based diet, which could be associated with the anti-inflammatory effects of HDL.

Dietary protein source contributes to the risk of developing maternal syndrome in the Dahl salt-sensitive rat.

Preeclampsia (PE) is a disorder of pregnancy, which is categorized by hypertension and proteinuria or signs of end-organ damage. Though PE is the leading cause of maternal and fetal morbidity and mortality, the mechanisms leading to PE remain unclear. The present study examined the contribution of dietary protein source (casein versus wheat gluten) to the risk of developing maternal syndrome utilizing two colonies of Dahl salt-sensitive (SS/JrHsdMcwi) rats. While the only difference between the colonies is the diet, the colonies exhibit profound differences in the pregnancy phenotypes. The SS rats maintained on the wheat gluten (SSWG) chow are protected from developing maternal syndrome; however, approximately half of the SS rats fed a casein-based diet (SSC) exhibit maternal syndrome. Those SSC rats that develop pregnancy-specific increases in blood pressure and proteinuria have no observable differences in renal or placental immune profiles compared to the protected SS rats. A gene profile array of placental tissue revealed a downregulation in Nos3 and Cyp26a1 in the SSC rats that develop maternal syndrome accompanied with increases in uterine artery resistance index suggesting the source of this phenotype could be linked to inadequate remodeling within the placenta. Investigations into the effects of multiple pregnancies on maternal health replicated similar findings. The SSC colony displayed an exacerbation in proteinuria, renal hypertrophy and renal immune cell infiltration associated with an increased mortality rate while the SSWG colony were protected highlighting how dietary protein source could have beneficial effects in PE.

Comparative Effects of Pyrroloquinoline Quinone (PQQ) and Resveratrol (RSV) on Mitochondriogenesis in Mice

Objectives and Hypothesis Many phytochemicals and biofactors influence mitochondrial function, lipid deposition, and oxidative metabolism. Examples include phytoalexins, flavonoids, and quinones, such as PQQ. Herein, PQQ is compared to RSV (a phytoalexin). Animal tissues retain nM concentrations of PQQ, which promote NAD+ production and sirtuin-related mitochondriogenesis. For example, PQQ promotes mitochondriogenesis in animal models when fed at 0.5 mg PQQ or more/Kg of diet. Such effects elicited by RSV often require 100X or more those amounts. Accordingly, we hypothesize that many of the responses ascribed to RSV may be due to compounds like PQQ or PQQ-derivatives. (cf. Chowanadisai et al., J of Biol Chem 285, 2010:142-52). Methods Two experiments were performed. (Exp. 1) Swiss-derived CD-1 mice express excessive weight gain and fat deposition when exposed to high-fat diets. A goal was to determine if PQQ can attenuate such increases in weight or fat deposition. The mice were maintained in keeping with the FASEB Principles for the Use of Animals in Research and Education. Groups of mice were fed either a low-fat (LFD, 5%) or high-fat (HFD, 20%) casein-based diet with or without PQQ addition at 10 mg/Kg diet for 4 weeks. (Exp.2) Adult C-57BL mice (4-6 months old) were used with or without PQQ addition at 10 mg/Kg diet or RSV at 400 mg/Kg diet. Liver, brown fat, and muscle were examined. The relative numbers of mitochondria were estimated using real-time PCR methods to assess mtDNA/nuclearDNA expression to compare PQQ vs. RSV responses. PQQ in plasma was measured using chemical and mass spectrometric methods. Results (Exp 1) PQQ attenuated the increase in body weight and fat in mice fed the high-fat diet (p<0.05). Mice fed the high-fat diet (-PQQ) weighed 40+/-3 g. Those receiving the high-fat diet (+PQQ) or those containing 5% fat weighed 27 to 32 g. PQQ exposure reduced body fat by 50 % or more**. The relative mtDNA/DNA concentrations in skeletal muscle for CD-1 mice were: 10.8 ± 3.3 (HFD-PQQ) vs. 15.1±1.2 (HFD+PQQ)*, and 18.5 ± 3.0 (LFD-PQQ) vs. 20. 8 ± 3.9 (LFD+PQQ). (Exp.2) PQQ at 1/40th the dietary dose as RSV promoted mtDNA stimulation in liver, brown fat, and skeletal muscle. As a percentage of control values, the mtDNA/DNA ratios, although variable, were increased by 113% and 111% following RSV and PQQ exposures, respectively. For brown fat, the values were 115% and 129% for RSV (NS) and PQQ**, respectively. For skeletal muscle, the values were 104% and 113% for RSV (NS) and PQQ**, respectively. The addition of PQQ to diets markedly enhanced plasma PQQ levels (3±2 pmol PQQ/mL, control; 17±4 pmol PQQ/mL, PQQ suppl.). Of interest, exposure to RSV also increased plasma PQQ (6.5±2 pmol PQQ/mL). [NS=not significant, *=p<0.05, **=0.01] Conclusions and Comments PQQ promotes mitochondriogenesis at lower dietary intakes compared to RSV. For the current observations, 10 mg of PQQ/Kg of diet promoted mtDNA changes equivalent to doses of 400 mg of RSV/ Kg of diet. The data also indicate that PQQ at low levels of exposure normalizes body weight and fat in suspectable mice exposed to a high-fat diet.

Dietary influences on the Dahl SS rat gut microbiota and its effects on salt-sensitive hypertension and renal damage.

Our previous studies have demonstrated the importance of dietary factors in the determination of hypertension in Dahl salt-sensitive (SS) rats. Since the gut microbiota has been implicated in chronic diseases like hypertension, we hypothesized that dietary alterations shift the microbiota to mediate the development of salt-sensitive hypertension and renal disease. This study utilized SS rats from the Medical College of Wisconsin (SS/MCW) maintained on a purified, casein-based diet (0.4% NaCl AIN-76A, Dyets) and from Charles River Laboratories (SS/CRL) fed a whole grain diet (0.75% NaCl 5L79, LabDiet). Faecal 16S rDNA sequencing was used to phenotype the gut microbiota. Directly examining the contribution of the gut microbiota, SS/CRL rats were administered faecal microbiota transfer (FMT) experiments with either SS/MCW stool or vehicle (Vehl) in conjunction with the HS AIN-76A diet. SS/MCW rats exhibit renal damage and inflammation when fed high salt (HS, 4.0% NaCl AIN-76A), which is significantly attenuated in SS/CRL. Gut microbiota phenotyping revealed distinct profiles that correlate with disease severity. SS/MCW FMT worsened the SS/CRL response to HS, evidenced by increased albuminuria (67.4±6.9 vs 113.7±25.0mg/day, Vehl vs FMT, P=.007), systolic arterial pressure (158.6±5.8 vs 177.8±8.9mmHg, Vehl vs FMT, P=.09) and renal T-cell infiltration (1.9-fold). Amplicon sequence variant (ASV)-based analysis of faecal 16S rDNA sequencing data revealed taxa that significantly shifted with FMT: Erysipelotrichaceae_2, Parabacteroides gordonii, Streptococcus alactolyticus, Bacteroidales_1, Desulfovibrionaceae_2, Ruminococcus albus. These data demonstrate that dietary modulation of the gut microbiota directly contributes to the development of Dahl SS hypertension and renal injury.

Measurement of Endogenous Phosphorus Losses in Broiler Chickens.

The experiment reported herein was conducted to estimate the ileal and excreta endogenous phosphorus (P) losses in broiler chickens. Three purified diets, namely a P-free diet, a gelatin-based diet containing negligible amounts of P, and a casein-based diet with 100% available P, were formulated. Test diets were offered ad libitum from day 25 to 28 post hatch and ileal digesta were collected. Excreta samples were also collected to estimate total tract endogenous P losses. Ileal endogenous P losses in birds fed the casein-based diet were higher (P<0.05) than those in birds fed P-free and gelatin-based diets. The ileal endogenous losses of P in birds fed P-free, gelatin-based, and casein-based diets were 25, 104 and 438 mg/kg dry matter intake, respectively. The endogenous P loss values estimated at the excreta level were 830, 560 and 372 mg/kg dry matter intake, respectively. Ileal and excreta endogenous losses of P in birds fed a casein-based diet were similar (P>0.05), but ileal losses were lower (P<0.05) than the excreta values in birds fed P-free and gelatin-based diets, resulting in a significant (P<0.001) assay diet by site of measurement interaction. The present data demonstrate that values determined for endogenous P losses in broiler chickens vary widely depending on the assay diet used.

Typical gut indigenous bacteria in ICR mice fed a soy protein-based normal or low-protein diet

For patients with inflammatory bowel disease, cow’s milk allergy, and lactose intolerance, soymilk is a potential alternative to cow’s milk. In this study, we aimed to identify the effects of a soy protein-based low-protein diet on the body and organ weights and the gut microbiome of six-week-old mice fed a diet containing 20% (SP) or 5% (LP) soy protein for 14 days via 16S rRNA (V4) amplicon sequencing. Body weight gain (growth) and liver, spleen, and fat tissue weight were significantly suppressed by the LP diet. Operational taxonomic unit numbers and α-diversity were lower in the LP group than in the SP group. A principal coordinate analysis revealed differences in the gut microbiome compositions of SP and LP mice. The abundances of caecal Roseburia sp., Alistipes sp., and bacteria from the family Muribaculaceae were lower in the LP group than in the SP group. In contrast, the abundance of Desulfovibrionaceae, which is positively correlated with inflammation, was higher in the LP group than in the SP group. These results differed from the effects of a milk casein-based low-protein diet (reported previously). Based on these findings, we conclude that the undesirable effects of a low-protein diet and/or protein deficiency are related to changes in the gut microbiome composition and may differ depending on the kind of proteins used.

Whole egg consumption increases gene expression within the glutathione pathway in the liver of Zucker Diabetic Fatty rats.

Nutrigenomic evidence supports the idea that Type 2 Diabetes Mellitus (T2DM) arises due to the interactions between the transcriptome, individual genetic profiles, lifestyle, and diet. Since eggs are a nutrient dense food containing bioactive ingredients that modify gene expression, our goal was to examine the role of whole egg consumption on the transcriptome during T2DM. We analyzed whether whole egg consumption in Zucker Diabetic Fatty (ZDF) rats alters microRNA and mRNA expression across the adipose, liver, kidney, and prefrontal cortex tissue. Male ZDF (fa/fa) rats (n = 12) and their lean controls (fa/+) (n = 12) were obtained at 6 wk of age. Rats had ad libitum access to water and were randomly assigned to a modified semi-purified AIN93G casein-based diet or a whole egg-based diet, both providing 20% protein (w/w). TotalRNA libraries were prepared using QuantSeq 3' mRNA-Seq and Lexogen smallRNA library prep kits and were further sequenced on an Illumina HighSeq3000. Differential gene expression was conducted using DESeq2 in R and Benjamini-Hochberg adjusted P-values controlling for false discovery rate at 5%. We identified 9 microRNAs and 583 genes that were differentially expressed in response to 8 wk of consuming whole egg-based diets. Kyto Encyclopedia of Genes and Genomes/Gene ontology pathway analyses demonstrated that 12 genes in the glutathione metabolism pathway were upregulated in the liver and kidney of ZDF rats fed whole egg. Whole egg consumption primarily altered glutathione pathways such as conjugation, methylation, glucuronidation, and detoxification of reactive oxygen species. These pathways are often negatively affected during T2DM, therefore this data provides unique insight into the nutrigenomic response of dietary whole egg consumption during the progression of T2DM.

Abstract 4642: Effects of soy protein diet on plasma isoflavones levels in obese DMBA-induced mammary tumor rat model

Abstract Obesity epidemic in the US and world grows as the number of overweight and obese adults continues to rise. Obese women have higher mortality rates from all cancers including breast cancer. Several studies have reported the protective effects of soy intake on breast cancer development, while other studies report the adverse effects of soy protein diet on breast cancer development. Soybeans contain isoflavones genistein, daidzein and glycitein, known as isoflavonoids, which are structurally similar to mammalian estrogens, have estrogenic properties and are potential anticarcinogens. Gut flora metabolizes daidzein into equol, a non-steroidal estrogen and has cancer chemopreventive property. The objective of this study was to determine the effects of obesity and a high isoflavones soy protein diet on plasma isoflavones; genistein, daidzein, glycitein, equol and O-Desmethylangolensin (O-MDA) levels using 7,12-dimethylbenz(a)anthracene (DMBA)-induced mammary tumor model. Five-week-old female Zucker rats (45 obese fa/fa and 54 lean) were used. After one week of acclimation (age 42 days), rats were randomly assigned to the following diet groups; 1) Lean, casein; 2) Obese, casein; 3) Lean, soy and 4) Obese, soy. Rats had ad lib access to water and diet with either casein or soy protein isolate as source of protein. All rats were orally gavaged at age 50 days with 65 mg/kg DMBA. Rats were weighed and palpated twice weekly and sacrificed 21 weeks post-DMBA treatment. Plasma was collected to measure genistein, daidzein, glycitein, equol and O-MDA levels using liquid chromatography−tandem mass spectrometry (LC-MS/MS). At end of the experiment, 69% of the Lean casein rats developed mammary tumors compared to 50% in lean soy group (p= 0.176). In the obese group, 76% of the soy-fed rats developed mammary tumors compared to 15% (P&amp;lt;0.001) of obese casein- fed rats. Plasma daidzein and genistein concentration were not significantly different in lean vs obese soy-fed rats. However, O-MDA and equol concentration were significantly lower (P&amp;lt;0.05) in obese soy-fed rats compared to lean soy rats. These data suggests that obese rats fed soy protein diet containing high isoflavone levels promoted DMBA-induced mammary tumor development compared to a casein-based diet. Also, obese soy-fed rats exhibited significantly lower plasma equol concentrations than those observed in lean soy-fed rats and this reduction may aid in explaining soy's impaired cancer chemopreventative effects in obese breast cancer model. Citation Format: Reza Hakkak, Soheila Korourian. Effects of soy protein diet on plasma isoflavones levels in obese DMBA-induced mammary tumor rat model [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4642.

Whole Egg Consumption Decreases Cumulative Weight Gain in Diet-Induced Obese Rats

Whole egg (WE) consumption has been demonstrated to attenuate body weight (BW) gain and adiposity in genetic animal models of type 2 diabetes (T2D). This finding was accompanied by increased food consumption. This study aimed to examine the effects of long-term WE intake on BW gain, fat distribution, and food intake in a rat model of diet-induced obesity (DIO). Male Sprague Dawley rats (n=24) were obtained at 5 wk of age and were randomly weight-matched across 1 of 4 dietary intervention groups (6 rats per group): a casein-based diet (CAS), a high-fat high-sucrose CAS diet (HFHS CAS), a whole egg-based diet (EGG), or a high-fat high-sucrose EGG diet (HFHS EGG). All diets provided 20% (w/w) protein and were provided for 33 wk. HFHS diets provided ∼61% of kilocalories from fat and 10% from sucrose. Daily weight gain and food intake were recorded, biochemical parameters were measured via ELISA, and epididymal fat pad weights were recorded at the end of the study. At 33 wk, cumulative BW gain in DIO rats fed HFHS EGG resulted in 23% lower weight gain compared with DIO rats fed HFHS CAS (P <0.0001), but no significant differences in BW gain were observed between the HFHS EGG group and the control EGG and CAS groups (P=0.71 and P=0.61, respectively). Relative food intake (grams per kilogram BW) was 23% lower (P<0.0001) in rats fed HFHS CAS compared with CAS, whereas there was no difference in food intake within the EGG dietary groups. DIO rats fed HFHS EGG exhibited a 22% decrease in epididymal fat weight compared with their counterparts fed the HFHS CAS. Our data demonstrate that consumption of a WE-based diet reduced BW gain and visceral fat in the DIO rat, similar to our previous findings in a genetic rat model with T2D.

Restriction of essential amino acids dictates the systemic metabolic response to dietary protein dilution

Dietary protein dilution (DPD) promotes metabolic-remodelling and -health but the precise nutritional components driving this response remain elusive. Here, by mimicking amino acid (AA) supply from a casein-based diet, we demonstrate that restriction of dietary essential AA (EAA), but not non-EAA, drives the systemic metabolic response to total AA deprivation; independent from dietary carbohydrate supply. Furthermore, systemic deprivation of threonine and tryptophan, independent of total AA supply, are both adequate and necessary to confer the systemic metabolic response to both diet, and genetic AA-transport loss, driven AA restriction. Dietary threonine restriction (DTR) retards the development of obesity-associated metabolic dysfunction. Liver-derived fibroblast growth factor 21 is required for the metabolic remodelling with DTR. Strikingly, hepatocyte-selective establishment of threonine biosynthetic capacity reverses the systemic metabolic response to DTR. Taken together, our studies of mice demonstrate that the restriction of EAA are sufficient and necessary to confer the systemic metabolic effects of DPD.

Dose Dependent Effects of Whole Blueberry on Cartilage Health and Pain in a Monosodium Iodoacetate (MIA) Induced Rat Model of Osteoarthritis

Abstract Objectives The purpose of this study was to examine the dose-dependent effects of whole blueberry in a monosodium iodoacetate (MIA) induced rat model of Osteoarthritis (OA). There is no known cure for OA and the present pharmacological treatment options are limited and associated with adverse side effects. We hypothesize that the addition of whole blueberry into the diets of rats with MIA induced OA will reduce pain behavior and improve cartilage health. Methods A total of forty, 45-day-old female CD rats were used for this study. Thirty rats were injected with MIA to induce joint destruction associated with OA. Ten rats served as control without induction of joint destruction. The MIA induced rats were randomized into three groups consisting of 10 animals. All groups were fed a casein-based diet with two of the MIA induced groups receiving an addition of whole blueberry powder at 5% and 10%, respectively. All groups were fed the respective diets for 46 days. The animals were weighted weekly throughout the study period and food intake monitored and recorded. Fasted blood specimens and other tissues of interest were collected after euthanasia for analysis. Mechanical allodynia was assessed at four time points throughout the study to evaluate changes in pain behaviors. Results There were no difference in body weight between the four groups at baseline. After 46 days of feeding there was no significant change in body weight in either of the blueberry fed groups in comparison to the MIA rats. Plasma hyaluronic acid levels were higher in the MIA group in comparison to the blueberry treated groups. We observed a dose dependent decrease in hyaluronic acid levels with blueberry treatment, respectively. At baseline, the MIA group exhibited significant (P ≤ 0.05) mechanical allodynia compared to the control group. Mechanical allodynia was significantly (P ≤ 0.05) reduced after 40 days in the 10% whole blueberry treated group. Conclusions Incorporation of whole blueberry into the diets of rats with MIA-induced OA reduced hyaluronic acid concentration and pain sensitivity. These results suggest that whole blueberry may be an effective therapeutic alternative for improving joint health and alleviating pain associated with OA. Funding Sources Research Enhancement Program, Texas Woman's University.

SUN-351 Sex Is a Strong Variable in the Mineral Metabolism Defects and Endocrine Dysfunction Associated with the Murine Adenine Diet Model of Chronic Kidney Disease (CKD)

The adenine diet is widely used in animal models to produce a tubulointerstitial fibrosis and inflammation that mimics human CKD in many aspects. These include the biochemical manifestations hyperphosphatemia and anemia, as well as endocrine dysfunction with elevated FGF23 and hyperparathyroidism. Male rodents are known to be less tolerant of adenine diet regimen than females, however the underlying mechanisms driving the sex differences remain unclear. Additionally, much of the data for adenine studies arises from rats, whereas mice are more commonly used in laboratory settings and are far easier to manipulate genetically. To this end, as part of a larger study to test the effects of iron-handling in CKD, we assessed the biochemical, molecular, and physical differences between male and female mice receiving an adenine diet to induce CKD. Flox-Fgf23 mice (8 weeks of age, n=4-6/group; mice were Cre negative, thus phenotypically wild type) were placed on a 0.2% adenine-containing diet (CKD); a matching casein-based diet served as control. After 6 weeks, mice were euthanized, and blood and tissues were collected for analysis. As expected, body weight at baseline was initially higher in males than in females, however males lost significantly more weight. Serum BUN was also elevated in both sexes receiving adenine, although males were higher (1.2 fold; p<0.01). Males also had elevated creatinine and lower total serum iron from baseline whereas females had no significant changes. FGF23 was elevated in all mice, with no significant differences between sexes. Kidney fibrosis and inflammation markers were elevated in the CKD mice, with males having higher expression of Col1a1 and -3a1 versus females (3.5/1.5 fold; p<0.001) and TNFα mRNA (2 fold; p<0.001). Renal expression of the anabolic vitamin D metabolizing enzyme Cyp27b1 (1α-hydroyxlase) and early growth response 1 (Egr1) were increased in CKD mice, with males having higher expression over females. Conversely, CKD males had lower kidney Klotho mRNA expression, and both sexes fed adenine expressed significantly lower NPT2a (sodium- phosphate co-transporter2a) mRNA. Liver expression of ferritin (Fth1) was elevated in male CKD mice compared to diet controls, whereas female mice had no differences. Elevated FGF23 has been linked to ventricular hypertrophy, and CKD males had significantly higher heart weight to femur ratio at completion of the study. Our results support that male mice succumb more rapidly than females to adenine diet mediated CKD phenotypes, potentially enhanced by fibrosis and inflammation. It remains to be determined whether the more rapid onset of defects in iron handling parameters accelerate the severe male CKD phenotype.

Effect of partial replacement of fat in sausage diet with different flours at 5%, 10% and 15% level of inclusion on performance, hematological and serum biochemistry of wistar albino rat

The demand for functional meats with reduced Animal Fat (AF) has increased globally. Chronic health disorders are often associated with high levels of dietary AF in meat products such as beef sausage (BS). Functional beef sausage (FBS) could be produced with alternative flours as replacer for AF. Effect of partial replacement of fat in sausage diet with different flours at 5%, 10% and 15% level of inclusion on performance, hematological and serum biochemistry of Wister albino rat was evaluated. The BS (g/100g: beef 65.0, lard 20.0, sodium chloride (SC) 2.0, others 13.0) was used. The FBS were developed using Wheat flour (WF), Irish potato flour (IPF) or corn flour (CF) at 5%, 10% and 15% to replace fat (lard) in beef sausage recipe. Functional properties of the flour were carried out using standard procedure. 60 weaned male albino rats (Wister strain) weighing between 60 - 62g were purchased. The rats were allotted to 12 dietary treatments with 6 rats per treatment in a completely randomised design. The study lasted 21 days and changes in their body weight were recorded. Feed Intake (FI), Feed Conversion Ratio (FCR), Protein Intake (PI) and Protein Efficiency Ratio (PER) were calculated. At the end of the feeding period, the animals were fasted of feed for 12 hours before blood samples were collected for hematological and serum biochemical analysis. Data were analysed using descriptive statistics and ANOVA at α0.05.Irish potato flour was significantly higher in bulk density, oil retention, foaming capacity, oil absorption capacity and emulsion activities compared to other flours. Corn flour was statistically higher (P&lt;0.05) in water retention, foam stability and water absorption capacity. Significant differences (P&lt;0.05) were observed in all the hematological indices except for red blood cell which had similar values (P&gt;0.05). However, there were no consistent trend observed in white blood cell count and its differential counts, which were rather fluctuating. Rats fed Irish potato based sausage had the highest Alanine Amino transferase (ALT) value while rats fed wheat based sausage diet had the lowest ALT of 11.87i.u/L. The Aspartate Aminotransferase (AST) assay of rats reveals that rats fed casein-based diet and lard-based sausage had the highest AST values of 7.30i.u/L each with least AST in rats fed protein deficient diet. The urea value for rats on the casein-based diet was the highest (45.29 mg/dL) with least value in rats fed corn-based sausage diet. Rats fed wheat-based sausage had the highest cholesterol (107.97mg/dL), while corn-based sausage had the lowest cholesterol value of 56.16 mg/dL. Corn based breakfast sausage diet was higher (P&lt;0.05) in protein efficiency ratio with least score in protein deficient diet. Corn-based sausage had the highest biological value (95.68) with protein deficient diet having the least value (15.49).It was observed that rats fed corn-based breakfast sausage had the highest gained weight of 27g, while rats fed non-protein diet had the lowest weight change of -11g. Replacement of animal fat with corn flour at 10% enhanced better growth performance, hematological and serum biochemistry.

Clinical Implications

Clinical Implications

Dietary supplementation with ovine serum immunoglobulin modulates correlations between mucin, microbiota and immunity proteins in the growing rat.

The objective of this study was to evaluate the relationship among the number of bacteria, number of goblet cells, gut mucin gene expression, mucin protein and immunity protein levels of rats fed a diet containing freeze-dried ovine Ig (FD). Sprague Dawley male rats were used in a 21-days study and were fed a basal control diet (BD; no Ig) and a test diet containing freeze-dried ovine Ig (FD). Diets were isocaloric and contained the same amount of the first limiting amino acids, methionine plus cysteine. Pearson's correlation analysis was conducted on the data (stomach, ileum and colon) obtained from individual rats (n=10) fed either casein-based diet (BD) or ovine serum Ig (FD) to evaluate the relationship between number of bacteria, number of goblet cells, gut mucin gene expression and gut mucin protein levels. Pearson's correlation analysis was then conducted with the data from the FD fed rats to evaluate the relationship among the above said variables. In the stomach content, a significant (p<.05) correlation was found between the Muc5Ac gene expression and mucosal mucin protein. In the ileum and colon, a significant (p<.05) correlation was observed among the mRNA levels of mucin (Muc2 and Muc4) genes. There was also evidence of a strong relationship (p<.05) between digesta mucin and mucosal mucin protein concentrations. A negative correlation of mucosal IgA protein concentration with total Lactobacillus (in ileum and colon) and total bacteria (in the ileum) was not evident with FD fed rats when compared to the results obtained using both BD and FD fed rats. In conclusion, this study suggests that feeding freeze-dried ovine Ig in growing rats results in a strong correlation between the number of bacteria, mucin and immunity proteins.

A Short-Term Feeding of Dietary Casein Increases Abundance of Lactococcus lactis and Upregulates Gene Expression Involving Obesity Prevention in Cecum of Young Rats Compared With Dietary Chicken Protein.

Casein and chicken are assessed to contain high quality proteins, which are essential for human health. Studies have shown that ingestion of the two dietary proteins resulted in distinct effects on physiology, liver transcriptome and gut microbiota. However, its underlying mechanism is not fully understood, in particular for a crosstalk between gut microbiota and host under a specific diet intervention. We fed young rats with a casein or a chicken protein-based diet (CHPD) for 7 days, and characterized cecal microbiota composition and cecal gene expression. We found that a short-term intervention with a casein-based diet (CAD) induced a higher relative abundance of beneficial bacterium Lactococcus lactis as well as Bifidobacterium pseudolongum, which upregulated galactose metabolism of the microbiome compared with a CHPD. The CAD also upregulated gene expression involved in obesity associated pathways (e.g., Adipoq and Irs1) in cecal tissue of rats. These genes and the bacterial taxon were reported to play an important role in protecting development of obesity. Furthermore, the differentially represented bacterial taxon L. lactis was positively associated with these differentially expressed genes in the gut tissue. Our results provide a new insight into the crosstalk between gut microbiota and host in response to dietary proteins, indicating a potential mechanism of obesity prevention function by casein.

Abstract 152: Downregulation of Placental Genes are Associated With the Development of Maternal Syndrome in Dahl Salt-Sensitive Rats

The Dahl Salt-Sensitive rat maintained on a low salt, casein-based diet (SSC) develops maternal syndrome (hypertension and proteinuria) compared to virgin controls. Our group has previously demonstrated that a substitution of the casein protein for a wheat gluten protein in the rat chow (SSWG) protects the Dahl SS rat from the development of maternal syndrome. This protection is observed in both the blood pressure (141±3 vs 114±4 mmHg, p &lt;0.001, SSC vs. SSWG, respectively) and proteinuria (98±16 vs 52±12 mg/day, p &lt;0.01). It is also associated with an improvement in uterine artery resistance index (0.9±0.02 vs 0.5±0.04, p&lt; 0.01) suggesting dietary protein source influences pregnancy in Dahl SS rats. While the exact mechanisms responsible for the development of maternal syndrome are unclear, it is thought that poor placentation is the initiating step in the disease process. Since a substitution of the protein source in the rat chow greatly impacted the phenotype in the Dahl SS rat, we tested the hypothesis that there was differential gene expression in the placental tissue between the SSC rats prone to maternal syndrome compared to the protected SSWG rats. To investigate these phenotypic differences, a PCR array approach was utilized to explore gene expression in placental tissue of both SSC and SSWG rats. Placental tissue from both strains at gestational day 20 was homogenized and RNA isolated to investigate 89 genes related to rat pregnancy and preeclampsia. Using a commercially available PCR array approach identified 4 genes that were differentially expressed between the SSC and SSWG placentas. There was a significant downregulation in expression of Atp1b1 (0.0018-fold change, p &lt;0.01) , Nos3 (0.13-fold change, p &lt;0.005) , Cdh13 (0.42-fold change, p &lt;0.05) and Cyp26a1 (0.48-fold change, p &lt;0.05) in the SSC compared to the SSWG rats. All four of these genes have been demonstrated to play roles in the placentation phase of gestation in other animal models. These results suggest that downregulation of these genes in placentas of the SSC rats, as a result of alterations in dietary protein intake, contributes to the maternal syndrome phenotype and provide avenues for future studies.

Dietary Egg Protein Prevents Hyperhomocysteinemia via Upregulation of Hepatic Betaine-Homocysteine S-Methyltransferase Activity in Folate-Restricted Rats

Hyperhomocysteinemia is associated with increased cardiovascular disease risk. Whole eggs contain several nutrients known to affect homocysteine regulation, including sulfur amino acids, choline, and B vitamins. The aim of this study was to determine the effect of whole eggs and egg components (i.e., egg protein and choline) with respect to 1) homocysteine balance and 2) the hepatic expression and activity of betaine-homocysteine S-methyltransferase (BHMT) and cystathionine β-synthase (CBS) in a folate-restricted (FR) rat model of hyperhomocysteinemia. Male Sprague Dawley rats (n=48; 6 wk of age) were randomly assigned to a casein-based diet (C; n=12), a casein-based diet supplemented with choline (C+Cho; 1.3%, wt:wt; n=12), an egg protein-based diet (EP; n=12), or a whole egg-based diet (WE; n=12). At week 2, half of the rats in each of the 4 dietary groups were provided an FR (0 g folic acid/kg) diet and half continued on the folate-sufficient (FS; 0.2 g folic acid/kg) diet for an additional 6 wk. All diets contained 20% (wt:wt) total protein. Serum homocysteine was measured by HPLC and BHMT and CBS expression and activity were evaluated using real-time quantitative polymerase chain reaction, Western blot, and enzyme activity. A 2-factor ANOVA was used for statistical comparisons. Rats fed FR-C exhibited a 53% increase in circulating homocysteine concentrations compared with rats fed FS-C (P<0.001). In contrast, serum homocysteine did not differ between rats fed FS-C and FR-EP (P=0.078). Hepatic BHMT activity was increased by 45% and 40% by the EP (P<0.001) and WE (P=0.002) diets compared with the C diets, respectively. Dietary intervention with egg protein prevented elevated circulating homocysteine concentrations in a rat model of hyperhomocysteinemia, due in part to upregulation of hepatic BHMT. These data may support the inclusion of egg protein for dietary recommendations targeting hyperhomocysteinemia prevention.

Acute Effects of Whole Egg Consumption on Circulating MicroRNAs in Sprague Dawley Animals (P15-001-19)

ObjectivesMicroRNAs, small ∼22 nt non-coding RNAs, have a functional role in post-transcriptional regulation of gene expression. We have shown that whole egg-based diets reduce weight gain despite increased food intake compared to casein-based diets in Zucker-Diabetic Fatty (ZDF) rats. We hypothesized that the microRNA content of whole egg may underlie the differential impact on weight gain in ZDF rats.Our first objective was to characterize the circulating microRNA content in response to feeding whole egg-based diets over an 8 hour period. Our second objective was to identify and characterize the microRNAs present in whole egg- and casein-based diets. MethodsMale Sprague Dawley rats (n = 12; 6 weeks of age) were meal-trained to consume 4 grams of feed in one hour. After one week of meal-training, animals were fasted overnight for 10 hours (h) with water ad libitum followed by controlled feeding of either an AIN93G casein-based diet or isocaloric AIN93G diet containing whole egg (20% egg protein, w/w). At 8 weeks of age, serum was collected via the tail vein at 0, 2, 4, 6, and 8 h following refeeding. Serum microRNA was extracted using a RNA SPLIT kit and prepared for sequencing on an Illumina HiSeq 3000 using a smallRNA Library Prep. Five RNA extraction methods were tested on 6 samples of each diet: 1) Trizol (Invitrogen), 2) Trizol + Directzol (Zymogen), 3) Trizol followed by a second extraction using a fatty acid RNA extraction kit (Norgen), 4) mirPremier (Sigma-Aldrich), 5) ISOLATE II RNA (Bioline). RNA 260/280 ratios were assessed via Nanodrop and RNA integrity was assessed on an Agilent Bioanalyzer. ResultsThe greatest total RNA recovery (ng/μL) and most accurate 260/280 absorbance ratios were achieved using Trizol. The following concentrations of extracted RNA and their respective ratios (means ± SD) were achieved for each dietary component: casein diet (460.9 ± 528.1; 1.56 ± 0.12) and whole egg diet (394.3 ± 292.8; 1.55 ± 0.16), whole egg (73.4 ± 20.3; 1.15 ± 0.23), corn oil (67.5 ± 20.0; 1.41 ± 0.04), and cornstarch (247.12 ± 110.34; 1.64 ± 0.13). Total RNA recovered from the diets had an RNA integrity number (RIN) of 0 and microRNA concentrations were undetected. ConclusionsDespite successful total RNA recovery from the diets, all samples had a RIN value of 0, which is not optimal for downstream sequencing. Currently, microRNAs from plasma are being sequenced to determine if varying diets had differential effects on circulating microRNA expression. Funding SourcesPresidential Interdisciplinary Research Seed Grant Program, Iowa State University.

Whole Egg Consumption in Zucker Diabetic Fatty Rats Display a Dose-dependent Reduction in Weight Gain and Total Body Fat, Accompanied by an Increase in Lean Body Mass (P21-054-19)

ObjectivesThe objective of this study was to determine the lowest dose of whole egg-based diets to effectively attenuate the obese phenotype in type 2 diabetic (T2D) rats using a dose-response experimental design. MethodsMale Zucker diabetic fatty (ZDF) rats (n = 8) and their lean controls (n = 8) were obtained at 6 weeks of age. Following one week of acclimation, animals were randomly assigned to one of 5 treatment groups: a casein-based diet (20% protein, w/w) or a whole-egg based diet provided at either 20, 10, 5, or 2.5% egg protein (w/w). Animals were fed their respective diets for 8 weeks with weight gain and food intake measured daily. At 14 weeks of age, body composition was analyzed by dual X-ray absorptiometry and statistical differences were measured between groups using a 2-way ANOVA at P < 0.05. ResultsWhole egg-based diets exerted a dose-dependent decrease in cumulative body weight gain and final body weight; increased in food intake; decreased total body fat; and increased lean body mass. Interestingly, the 20% whole egg protein diet decreased body fat and increased lean body mass in the ZDF rats and their lean controls. ConclusionsTogether, these data support the hypothesis that dietary consumption of whole eggs may decrease weight gain, reduce body fat, and increase lean body mass in a dose-dependent manner in ZDF rats. These results suggest the need to modify dietary recommendations during T2D and obesity to potentially consume more whole egg. Funding SourcesThis work was supported by the Egg Nutrition Center and in part by a National Science Foundation Graduate Research Fellowship. Supporting Tables, Images and/or Graphs▪▪▪