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Physiol Genomics [journal]
- CHARACTERIZATION OF BIOLOGICAL PATHWAYS ASSOCIATED WITH A 1.37 MBP GENOMIC REGION PROTECTIVE OF HYPERTENSION IN DAHL S RATS. [JOURNAL ARTICLE]
- Physiol Genomics 2014 Apr 8.
The goal of the present study was to narrow a region of chromosome 13 to only several genes and then apply unbiased statistical approaches to identify molecular networks and biological pathways relevant to blood pressure salt-sensitivity in Dahl salt-sensitive rats. The analysis of 13 overlapping subcongenic strains identified a 1.37 Mbp region on chromosome 13 that influenced the mean arterial blood pressure by at least 25 mmHg in SS rats fed a high salt diet. DNA sequencing and analysis filled genomic gaps and provided identification of five genes in this region, Rfwd2, Fam5b, Astn1, Pappa2, and Tnr. A cross-platform normalization of transcriptome data sets obtained from our previously published Affymetrix GeneChip dataset and newly acquired RNA-seq data from renal outer medullary tissue provided 90 observations for each gene. Two Bayesian methods were used to analyze the data: 1) A linear model analysis to assess 243 biological pathways for their likelihood to discriminate blood pressure levels across experimental groups; 2) A Bayesian graphical modeling of pathways to discover genes with potential relationships to the candidate genes in this region. As none of these five genes are known to be involved in hypertension, this unbiased approach has provided useful clues to be experimentally explored. Of these five genes, Rfwd2, the gene most strongly expressed in the renal outer medulla, was notably associated with pathways that can affect blood pressure via renal transcellular Na(+) and K(+) electrochemical gradients and tubular Na+ transport, mitochondrial TCA cycle and cell energetics, and circadian rhythms.
- Contractile abnormalities of mouse muscles expressing hyperkalemic periodic paralysis mutant NaV1.4 channels do not correlate with Na+ influx or channel content. [JOURNAL ARTICLE]
- Physiol Genomics 2014 Apr 8.
Hyperkalemic periodic paralysis (HyperKPP) is characterized by myotonic discharges that occur between episodic attacks of paralysis. Individuals with HyperKPP rarely suffer respiratory distress even though diaphragm muscle expresses the same defective Na(+) channel isoform (NaV1.4) that causes symptoms in limb muscles. We tested the hypothesis that the extent of the HyperKPP phenotype (low force generation and shift toward oxidative type I and IIA fibers) in muscle is a function of i) the NaV1.4 channel content and ii) the Na(+) influx through the defective channels (i.e., the tetrodotoxin (TTX) sensitive Na(+)influx). We measured NaV1.4 channel protein content, TTX-sensitive Na(+) influx, force generation and myosin isoform expression in four muscles from knock-in mice expressing a NaV1.4 isoform corresponding to the human M1592V mutant. The HyperKPP flexor digitorum brevis (FDB) muscle showed no contractile abnormalities, which correlated well with its low NaV1.4 protein content and by far the lowest TTX-sensitive Na(+) influx. In contrast, diaphragm muscle expressing the HyperKPP mutant contained high levels of NaV1.4 protein and exhibited a TTX-sensitive Na(+) influx that was 22% higher compared to affected extensor digitorum longus (EDL) and soleus muscles. Surprisingly, despite this high burden of Na(+) influx, the contractility phenotype was very mild in mutant diaphragm compared to the robust abnormalities observed in EDL and soleus. This study provides evidence that HyperKPP phenotype does not depend solely on the NaV1.4 content or Na(+) influx and that the diaphragm does not depend solely on Na(+) /K(+) pumps to ameliorate the phenotype.
- Identification of genetic loci associated with different responses to high fat diet induced obesity (DIO) in C57BL/6N and C57BL/6J substrains. [JOURNAL ARTICLE]
- Physiol Genomics 2014 Apr 1.
We have recently demonstrated that C57BL/6NTac and C57BL/6JRj substrains are significantly different in their response to high fat diet induced obesity (DIO). The C57BL/6JRj substrain seems to be protected from DIO and genetic differences between C57BL/6J and C57BL/6N substrains at 11 SNP loci have been identified. To define genetic variants as well as differences in parameters of glucose homeostasis and insulin sensitivity between C57BL/6NTac and C57BL/6JRj substrains which may explain the different response to DIO, we analysed 208 first backcross (BC1) hybrids of C57BL/6NTac and C57BL/6JRj [(C57BL/6NTacxC57BL/6JRj)F1xC57BL/6NTac] mice. Body weight, epigonadal and subcutaneous fat mass, circulating leptin, as well as parameters of glucose metabolism were measured after 10 weeks of high fat diet (HFD). Genetic profiling of BC1 hybrids were performed using TaqMan SNP genotyping assays. Furthermore, to assess if SNP polymorphisms could affect mRNA level, gene expression analysis was carried out in murine liver and subcutaneous adipose tissue samples. Human subcutaneous adipose tissue was used to verify murine data of SNAP29. We identified four gender-specific variants which are associated with the extent of HFD induced weight gain and fat depot mass. BC1 hybrids carrying the combination of risk or beneficial alleles exhibit the phenotypical extremes of the parental strains. Murine and human SC expression analysis revealed Snap29 as strongest candidate. Our data indicate an important role of these loci in responsiveness to HFD induced obesity and suggest genes of the synaptic vesicle release system such as Snap29 being involved in the regulation of high fat DIO.
- UNIQUE TRANSCRIPTOMIC SIGNATURE OF OMENTAL ADIPOSE TISSUE IN OSSAWBAW SWINE: A MODEL OF CHILDHOOD OBESITY. [JOURNAL ARTICLE]
- Physiol Genomics 2014 Mar 18.
To better understand the impact of childhood obesity on intra-abdominal adipose tissue phenotype, a complete transcriptomic analysis using deep RNA-sequencing (RNA-seq) was performed on omental adipose tissue (OMAT) obtained from lean and western diet induced obese juvenile Ossabaw swine. Obese animals had 88% greater body mass, 49% greater body fat content, and a 60% increase in OMAT adipocyte area (all p < 0.05) compared with lean pigs. RNA-seq revealed a 37% increase in the total transcript number in the OMAT of obese pigs. Ingenuity Pathway Analysis showed transcripts in obese OMAT were primarily enriched in the following categories: a) development, b) cellular function and maintenance, and c) connective tissue development and function; while transcripts associated with RNA post-translational modification, lipid metabolism, and small molecule biochemistry were reduced. DAVID and Gene Ontology analyses showed that many of the classically recognized gene pathways associated with adipose tissue dysfunction in obese adults including (hypoxia, inflammation, angiogenesis) were not altered in OMAT in our model. The current study indicates that obesity in juvenile Ossabaw swine is characterized by increases in overall OMAT transcript number and provides novel data describing early transcriptomic alterations that occur in response to excess caloric intake in visceral adipose tissue in a pig model of childhood obesity.
- The liver proteome in hibernating ground squirrels is dominated by metabolic changes associated with the long winter fast. [JOURNAL ARTICLE]
- Physiol Genomics 2014 Mar 18.
Small-bodied hibernators partition the year between active homeothermy and hibernating heterothermy accompanied by fasting. To define molecular events underlying hibernation that are both dependent and independent of fasting, we analyzed the liver proteome among two active and four hibernation states in 13-lined ground squirrels. We also examined fall animals transitioning between fed homeothermy and fasting heterothermy. Significantly enriched pathways differing between activity and hibernation were biased towards metabolic enzymes, concordant with the fuel shifts accompanying fasting physiology. Although metabolic reprogramming to support fasting dominated these data, arousing (rewarming) animals had the most distinct proteome among the hibernation states. Instead of a dominant metabolic enzyme signature, torpor-arousal cycles featured differences in plasma proteins and intracellular membrane traffic and its regulation. Phosphorylated NSFL1C, a membrane regulator, exhibited this torpor-arousal cycle pattern; its role in autophagosome formation may promote utilization of local substrates upon metabolic reactivation in arousal. Fall animals transitioning to hibernation lagged in their proteomic adjustment, indicating that the liver is more responsive than preparatory to the metabolic reprogramming of hibernation. Specifically, torpor use had little impact on the fall liver proteome, consistent with a dominant role of nutritional status. In contrast to our prediction of reprogramming the transition between activity and hibernation by gene expression, and then within-hibernation transitions by posttranslational modification (PTM), we found extremely limited evidence of reversible PTMs within torpor-arousal cycles. Rather, acetylation contributed to seasonal differences, being highest in winter (specifically in torpor), consistent with fasting physiology and decreased abundance of the mitochondrial deacetylase, SIRT3.
- The Influence of the Microenvironment on Cell Fate Determination and Migration. [JOURNAL ARTICLE]
- Physiol Genomics 2014 Mar 11.
Several critical cell functions are influenced by not only internal cellular machinery but also by external mechanical and biochemical cues from the surrounding microenvironment. Slight changes to the microenvironment can result in dramatic changes to the cell's phenotype for example a change in the nutrients or pH of a tumor microenvironment can result in increased tumor metastasis. While cellular fate and the regulators of cell fate have been studied in detail for several decades now, our understanding of the extracellular regulators remains qualitative and far from comprehensive. In this review, we discuss the microenvironment influence on cell fate in terms of adhesion, migration, and differentiation and focus on both developments in experimental and computation tools to analyze cellular fate.
- Serum metabolomics identifies citrulline as a predictor of adverse outcomes in an equine model of gut-derived sepsis. [JOURNAL ARTICLE]
- Physiol Genomics 2014 Mar 11.
Acute laminitis is an inflammatory disease of the equine foot that often occurs secondary to sepsis or systemic inflammation associated with gastrointestinal disease. It has been suggested that laminitis is similar to multiple organ dysfunction syndrome (MODS) in humans, although in horses the weight-bearing laminar epithelium of the foot appears to be the tissue most sensitive to insult and the first "organ" to fail. Metabolomics performed on serum samples collected before (CON) and after (LMN) experimental induction of gastrointestinal-associated sepsis in 6 horses detected 1177 metabolites of both mammalian and bacterial origin in equine serum. Network and correlation analyses suggested a dysregulation of fatty acid metabolism in the LMN group, as well as an accumulation of organic acids such as lactate. Furthermore, concentrations of the amino acid citrulline were decreased in LMN samples from all study animals, suggesting that citrulline might be useful as a biomarker to identify critically ill animals that are at risk of developing laminitis. We therefore established normal ranges of plasma citrulline concentrations in a separate group of horses (n = 36) and tested the ability of citrulline to predict adverse outcomes (laminitis or death) in critically ill horses (n = 23). Plasma citrulline was significantly lower in critically ill horses that went on to experience adverse outcomes (n = 6). Further study is required to accurately determine a diagnostic cutoff, but the present data are suggestive of the predictive value of citrulline as a biomarker for laminar failure in equine sepsis.
- Prepartal dietary energy alters transcriptional adaptations of the liver and subcutaneous adipose tissue of dairy cows during the transition period. [JOURNAL ARTICLE]
- Physiol Genomics 2014 Feb 25.
Overfeeding during the dry period may predispose cows to increased insulin resistance (IR) with enhanced postpartum lipolysis. We studied gene expression in the liver and subcutaneous adipose tissue (SAT) of 16 Finnish Ayrshire dairy cows fed either a controlled energy diet (CON; 99 MJ/d metabolizable energy (ME)) during the last six weeks of the dry period or high energy diet (HIGH; 141 MJ/d ME) for the first three weeks and then gradually decreasing energy allowance during three weeks to 99 MJ/d ME before the expected parturition. Tissue biopsies were collected at -10, 1 and 9 d, and blood samples at -10, 1 and 7 d relative to parturition. Overfed cows had greater dry matter, crude protein and ME intakes and ME balance before parturition. Daily milk yield, live weight and body condition score were not different between treatments. The HIGH cows tended to have greater plasma insulin and lower glucagon/insulin ratio compared with CON cows. No differences in circulating glucose, glucagon, nonesterified fatty acids and β-hydroxybutyrate concentrations, and hepatic triglyceride contents were observed between treatments. Overfeeding compared with CON resulted in lower CPT1A and PCK1, and a tendency for lower G6PC and PC expression in the liver. The HIGH group tended to have lower RETN expression in SAT than CON. No other effects of overfeeding on the expression of genes related to IR in SAT were observed. In conclusion, overfeeding energy prepartum may have compromised hepatic gluconeogenic capacity and slightly affected IR in SAT based on gene expression.
- Basal microRNA expression patterns in reward circuitry of selectively bred high-responder and low-responder rats vary by brain region and genotype. [Journal Article]
- Physiol Genomics 2014 Apr 15; 46(8):290-301.
Mental health disorders involving altered reward, emotionality, and anxiety are thought to result from the interaction of individual predisposition (genetic factors) and personal experience (environmental factors), although the mechanisms that contribute to an individual's vulnerability to these disorders remain poorly understood. We used an animal model of individual variation [inbred high-responder/low-responder (bHR-bLR) rodents] known to vary in reward, anxiety, and emotional processing to examine neuroanatomical expression patterns of microRNAs (miRNAs). Laser capture microdissection was used to dissect the prelimbic cortex and the nucleus accumbens core and shell prior to analysis of basal miRNA expression in bHR and bLR male rats. These studies identified 187 miRNAs differentially expressed by genotype in at least one brain region, 10 of which were validated by qPCR. Four of these 10 qPCR-validated miRNAs demonstrated differential expression across multiple brain regions, and all miRNAs with validated differential expression between genotypes had lower expression in bHR animals compared with bLR animals. microRNA (miR)-484 and miR-128a expression differences between the prelimbic cortex of bHR and bLR animals were validated by semiquantitative in situ hybridization. miRNA expression analysis independent of genotype identified 101 miRNAs differentially expressed by brain region, seven of which validated by qPCR. Dnmt3a mRNA, a validated target of miR-29b, varied in a direction opposite that of miR-29b's differential expression between bHR and bLR animals. These data provide evidence that basal central nervous system miRNA expression varies in the bHR-bLR model, implicating microRNAs as potential epigenetic regulators of key neural circuits and individual differences associated with mental health disorders.
- Ex vivo culture of primary human colonic tissue for studying transcriptional responses to 1α,25(OH)2 and 25(OH) vitamin D. [Journal Article]
- Physiol Genomics 2014 Apr 15; 46(8):302-8.
1α,25-Dihydroxyvitamin D3 [1α,25(OH)2D3] is a steroid hormone derived from circulating 25(OH) vitamin D [25(OH)D] with chemopreventive effects in colorectal cancer. 1α,25(OH)2D3 acts through transcriptional mechanisms; however, our understanding of vitamin D transcriptional responses in the colon is derived from studies in transformed cancer cell lines which may not represent responses in normal healthy tissue. Here, we describe the optimization of an ex vivo culture model using primary colonic biopsy samples for studying short-term transcriptional response induced by 1α,25(OH)2D3 and 25(OH)D treatment. Colon biopsy samples from healthy subjects were maintained in primary culture and treated in parallel with 100 nM 1α,25(OH)2D3 or 62.5 nM 25(OH)D and vehicle control (ethanol). Viability was assessed using histology and enzymatic assays. Genome-wide transcriptional responses to 1α,25(OH)2D3 were assessed and expression of 25(OH)D targets CYP27B1 and CYP24A1 were measured by real time PCR. We show that ex vivo culture of colonic tissue remains viable for up to 8 h. The largest number of differentially expressed genes in response to 1α,25(OH)2D3 was noted after 6 h (n = 120). As proof of concept, the top upregulated gene was CYP24A1, a well-established vitamin D-responsive gene. With 25(OH)D treatment, mRNA expression of CYP27B1 was significantly increased after 1 h, while expression of CYP24A1 was greatest at 8 h. Ex vivo culture can be used to assess short-term transcriptional responses to 1α,25(OH)2D3 and 25(OH)D in primary tissue from human colon. Future studies will address interindividual differences in transcriptional responses.