Project description:Gene expression studies in peripheral blood mononuclear cells (PBMC) can provide knowledge that would be difficult to obtain using other types of biological samples, as these cells can reflect overall response of the body to a specific stimulus, such as diet. Here, we aimed to study the impact of sustained intake of isocaloric diets with an unbalanced macronutrient proportion (rich in fat or protein) on PBMC gene expression in rats using transcriptomics, to better understand the effects of these diets on metabolism and health. Macronutrient diet composition (especially increased protein content) affected PBMC gene expression. An important effect was observed in immune response-related genes. High protein (HP) diet produced an activation of genes mainly related to antigen recognition/presentation, whereas the high fat (HF) diet was more related to a decreased expression of these genes. Key energy homeostasis genes (mainly involved in lipid metabolism) were also affected, reflecting a nutritional adaptive response to the diets. Decreased Dhrs3 and increased Pref-1/Dlk1 expression observed in HP-fed animals reflects a lower lipogenic capacity. In addition, HF diet affected the expression of genes related to insulin resistance/signaling (Msra/Slc9a6, Asb6), hypoxia (Akap12), cardiovascular problems (Ethe1, Lr11), cognitive impairment (Tmcc2) and tumorigenesis (Ddx17, Phb, Rnasen/Drosha, and Tsc2). HP diet also impaired the expression of genes related to glucose metabolism and insulinemia (Glut-4/Slc2a4, Sfxn5, Slc16a1), but positively affected genes involved in liver function (Clec4f and St6galnac3) and cardiovascular protection (Sort1). In conclusion, gene expression analysis in PBMC can provide information that could be useful to detect metabolic deviations and health consequences of diets with an unbalanced macronutrient composition. 2 months-old male Wistar rats received a low fat control diet, or a high fat (HF) diet, or a high protein (HP) diet for 4 months, respectively. At the end of the intervention, prior to the sacrifice of the animals, peripheral blood samples (1.5-2.5 mL) were collected from the safena vein, using heparin in NaCl (0.9%) as anticoagulant. Immediately after blood collection, peripheral blood mononuclear cells (PBMC) were isolated by Ficoll gradient separation. Total RNA was isolated from PBMC, quantified and qualified, and subsequently used for global gene expression profiling using Agilent 4x44K microarrays.

Project description:The present study was constructed to confirm previous findings that mice on a high fat diet (HFD) treated by subcutaneous injection with exenatide (EXE) at 3µg/kg once daily for 6 weeks develop exocrine pancreatic injury (Rouse et al. 2014). The present study included 12 weeks of EXE exposure at multiple concentrations (3, 10, or 30 Œºg/kg) with multiple endpoints (histopathology evaluations, immunoassay for cytokines, immunostaining of the pancreas, serum chemistries and measurement of trypsin, amylase, and, lipase, and gene expression profiles). Time- and dose-dependent exocrine pancreatic injury was observed in mice associated with EXE exposure in a HFD environment. The time- and dose-dependent morphological changes identified in the pancreas involved acinar cell injury and death (autophagy, apoptosis, necrosis, and atrophy), cell adaptations (hypertrophy and hyperplasia), and cell survival (regeneration) accompanied with varying degrees of inflammatory response leading to secondary injury in pancreatic blood vessels, ducts, and adipose tissues. Gene expression profiles supported the presence of increased signaling for cell survival and altered lipid metabolism. The potential for EXE to cause acute or early chronic pancreatic injury was identified in a HFD environment. In human disease, the influence of pancreatitis risk factors or pre-existing chronic pancreatitis on this injury potential requires further investigation. Male C57BL/6 mice 6 to 8 weeks of age were purchased from Harlan Laboratories (Frederick, MD). Mice were housed individually in an environmentally controlled room (18C-21C, 40%-70% relative humidity) with a twelve-hour light/dark cycle. Mice were initially fed Certified Purina Rodent Chow #5002 (Ralston Purina Co., St. Louis, MO) providing 63% of calories through carbohydrates, 24% from protein, and 13% from fat. Per the experimental design, mice were subsequently placed on a Teklad Custom Research Diet, TD.06415 (Harlan, Frederick, MD), providing 45% of calories through fats (36% saturated, 47% monounsaturated, 17% polyunsaturated), 36% from carbohydrates, and 19% from protein. Water and food were available ad libitum. Thorough out the experiment all mice were weighed weekly and dosing adjusted accordingly for each mouse. A cohort of eighty 6 to 8 week-old male C57BL6 mice were received from Harlan Laboratories. The cohort represented experimental time point of 12 weeks. The cohort was further divided into 4 exenatide (EXE) treatment groups (0, 3, 10, or 30 ug/kg) of 20 mice each. Initially, all mice were placed on HFD for six weeks without additional treatment and were then maintained on HFD for an additional 12 weeks while receiving daily subcutaneous injections of saline or EXE (Creative Peptides, Shirley, NY; 3, 10, or 30 ug/kg). At the 12 week timepoint, twenty-four hours following their final dose, the mice from the treatment cohort were anesthetized with isoflurane and euthanized by exsanguination with collection of blood for serum harvest. The pancreas from 12 mice in each cohort was preserved in 10% formalin for histology processing. The pancreas from 5 mice in each cohort was placed in RNAlater (Life Technologies, Grand Island, NY) for subsequent RNA extraction and the pancreas from 3 mice in each cohort was immediately frozen in liquid nitrogen and then stored at -80C. Pancreas tissue was resected, preserved in RNALater, and stored at -80 C. After thawing, 2.5 mg of each sample were processed using miRNeasy Mini Kits (Cat # 217004) (Qiagen, Valencia, CA). Samples were homogenized in 700 uL of Qiazol Lysis Reagent (Qiagen), for 5 minutes at 50 hz in a TissueLyser LT (Qiagen), and processed using the automated purification of total RNA on a Qiacube (Qiagen), using the 'Purification of total RNA, including small RNAs, from animal tissues & cells (aqueous phase), version 2 (April 2010)' standard protocol, as described in the miRNeasy Mini Handbook (1073008 07/2012) & http://www.qiagen.com/QIAcube/Standard/ProtocolView.aspx?StandardProtocolID=847.Mean yield per 2.5 mg of pancreas tissue was approximately 35 ug, determined by NanoDrop spectrophotometer (Thermo Scientific, Wilmington, DE). Average RIN values were approximately 5.4 +/- 1.4, as assayed by a 2100 Bioanalyzer Instrument (Agilent Technologies, Santa Clara, CA), using an Agilent RNA 6000 Nano Kit (cat # 5067-1511). This RIN mean was consistent with our previous experiences in RNA extraction from murine pancreatic tissue. The isolated RNA was labeled and gene expression analysis performed by Expression Analysis, Inc. (Durham, NC). The 3 samples with the highest RIN from each treatment group were used to probe treatment effect on gene expression. Total RNA samples (100 ng) were converted into cDNA using Ovation WGA FFPE System (NuGEN, Part No. 6200). The second strand cDNA is then purified with Agencourt RNAClean beads and followed by SPIA amplification. Amplified SPIA cDNA product is purified using Agencourt RNAClean beads and quantitated using a NanoDrop ND-8000 spectrophotometer. Target preparation was performed using 4 ug of amplified cDNA and the NuGEN FL-Ovation cDNA Biotin Module V2. Fragmented and biotin-labeled target was hybridized to Affymetrix GeneChip Mouse Genome 430 2.0 microarrays. Raw gene expression data were first analyzed using ArrayTrack bioinformatics tool with robust multichip average algorithm and quantile normalization. Differentially expressed genes were defined by p < 0.05 and fold change > 1.3 in Welch t-test comparing treatment and control groups. Expression data were then imported to Ingenuity Pathway Analysis (IPA) tool for further analyses of pathway, biofunction, and toxicity using general and pancreas-specific knowledge bases.

Project description:Background: This study examines the impact of dietary fatty acids on regulation of gene expression in the mammary epithelial cells before and during puberty. Methods: The diets primarily consisted of n-9 monounsaturated fatty acids (olive oil), n-6 polyunsaturated fatty acids (safflower), saturated acids (butter), and the reference AIN-93G diet (soy oil). The dietary regimen mimics the repetitive nature of fatty acid exposure in Western diets. Dietary-induced changes in gene expression were examined in the LCM (Laser Capture Microdissected) captured mammary ductal epithelial cells at day of weaning (21 days) and at the end of puberty (50 days after birth). PCNA immunohistochemistry analysis was used to compare proliferation rates between diets. Results: Genes differentially expressed between each of the test diets and the reference diet in both age groups were significantly enriched by cell cycle genes. Some of these genes were involved in the activation of the cell cycle pathway or the G2/M check point pathway. Although there were some differences in the level of differential expression, all diets showed qualitatively same pattern of differential expression compared to the reference diet. Cluster analysis identified an expanded set of cell cycle as well as immunity and sterol metabolism related clusters of differentially expressed genes. Conclusion: Fatty acid-enriched diets significantly up-regulated proliferation above the normal physiological level at day 50. The higher cellular proliferation during puberty caused by enriched fatty acid diets pose a potential increase risk of mammary cancer in later life. The human homologs of 27 of 62 cell cycle cluster of rat genes are included in a human breast cancer cluster of 45 cell cycle related genes further emphasizing the importance of our findings in the rat model. Female, virgin Spraque-Dawley rats were obtained from Taconic Farms (Germantown, NY) at approximately 7 weeks of age and placed on one of 9 pelleted purified diets for one month. After one month of diet exposure, female rats were bred with male Sprague-Dawley rats of approximately 3 months of age. Litters were weighed and monitored throughout gestational period. Pups had access to dam’s milk as well as tap water and food throughout gestation. Pups were weaned at DOL 21 and individually housed with tap water and diets ad libitum. Rats were killed by CO2 asphyxiation and decapitated at the following two ages, DOW (DOL21) and DOL 50 with no other treatments.

Project description:The current study was designed to investigate the circulating miRNA expression signature of dairy cows during several key time points around calving, and under different fatty acids (FA) treatments to get insights into different aspects of metabolic adaptations and their interaction with administrated FA. In this trial, 32 transition Holstein dairy cows were randomly assigned to one of the following treatments: 1-CTRL (n = 8; coconut oil, Bio-Kokosöl #665, Kräuterhaus Sanct Bernhard, KG, Bad Ditzenbach, Germany; 38 and 76 g/d in antepartum (AP) and postpartum (PP)), 2- Essential fatty acids (EFA, n=8, a combination of linseed oil (DERBY® Leinöl #4026921003087, DERBY Spezialfutter GmbH, Münster, Germany; 39 and 78 g/d in AP and PP) and safflower oil (GEFRO Distelöl, GEFRO Reformversand Frommlet KG, Memmingen, Germany; 2 and 4 g/d in AP and PP), 3- conjugated linoleic acid (CLA, n = 8, Lutalin® ; cis-9, trans-11, 10 g/d trans- 10, cis-12 CLA, BASF SE, Lampertheim, Germany; 19 and 38 g/d in AP and PP), and 4- EFA+CLA, a combination of linseed oil, safflower oil and Lutalin® from day 63 AP to day 63 PP. From each cow, four blood samples were collected individually on days -21, +1, +28, and +63 relative to calving, and the plasma fraction was used for RNA extraction. Total RNA was extracted from 128 plasma samples using the QIAGEN miRNeasy serum/plasma kit (Qiagen GmbH, Hilden, Germany), and checked for yield and purity using the small RNA kit on a 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). We used the QIAseq miRNA library kit (QIAGEN, Hilden, Germany) for library preparation and Caliper (PerkinElmer, Waltham, MA) for quality check. NGS was performed on the libraries using the NovaSeq 6000 (Illumina, San Diego, CA) setting on single-end 100 bp mode on the respective platform of IGA Technology Services, Udine, Italy (https://igatechnology.com/). Raw-sequencing data was checked for quality (format conversion to FASTQ, demultiplexing, Adapter Trimming, and UMI Remova) using the Illumina Bcl2fastq2 Conversion Software (version 2.20). Detected sequences were analyzed with the miRDeep2 (miRDeep2.pl) software package (version 2.0.0.5) to detect known miRNA and predict putative novel miRNA (using human, ovine, and goat miRNA datasets). MiRDeep2 was fed with the Bos taurus miRNA collection available at miRBase (www.mirbase.org). Read counts for each known and novel miRNA were compiled using the HTSeq-count script. The quantified counts were normalized using the TMM method via the edgeR (version 3.12.0) package in R software (Version 4.0). Only miRNA with at least one count per million over at least 2 samples were taken into consideration for the analysis. Differentially expressed miRNA (DEM) during the time points were identified by performing generalized linear model (GLM) likelihood ratio tests (glmLRT) using the GLM approach in edgeR. The DEMs have been interpreted separately I-during different time points, and also II-between treatments, to gain insight into metabolic and immune adaptation, as well as the role of fatty acids at each time point. A time-affected DEM identified key signaling pathways that are involved in initiating and regulating metabolic shifts during the transition from gestation (non-lactation) to lactation. Although, fatty acid treatments had only minor effects on miRNA expression (insufficient DEM to perform pathway analysis).

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Effects of diets high in animal or plant protein on oxidative stress in individuals with type 2 diabetes: a randomized clinical trial

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