Unknown,Transcriptomics,Genomics,Proteomics

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Transcription profiling of mouse small intestine reveals fasting induces a biphasic adaptive metabolic response

ABSTRACT: Background; The gut is a major energy consumer, but a comprehensive overview of the adaptive response to fasting is lacking. Gene-expression profiling, pathway analysis, and immunohistochemistry were therefore carried out on mouse small intestine after 0, 12, 24, and 72 hours of fasting. Results; Intestinal weight declined to 50% of control, but this loss of tissue mass was distributed proportionally among the gutâs structural components, so that the microarraysâ tissue base remained unaffected. Unsupervised hierarchical clustering of the microarrays revealed that the successive time points separated into distinct branches. Pathway analysis depicted a pronounced, but transient early response that peaked at 12 hours, and a late response that became progressively more pronounced with continued fasting. Early changes in gene expression were compatible with a cellular deficiency in glutamine and metabolic adaptations directed at glutamine conservation, inhibition of pyruvate oxidation, stimulation of glutamate catabolism via aspartate and phosphoenolpyruvate to lactate, and enhanced fatty-acid oxidation and ketone-body synthesis. In addition, the expression of key genes involved in cell cycling and apoptosis was suppressed. At 24 hours of fasting, many of the early adaptive changes abated. Major changes upon continued fasting implied the production of glucose rather than lactate from carbohydrate backbones, a downregulation of fatty-acid oxidation and a very strong downregulation of the electron-transport chain. Cell cycling and apoptosis remained suppressed. Conclusion; The changes in gene expression indicate that the small intestine rapidly looses mass during fasting to generate lactate or glucose and ketone bodies. Meanwhile, intestinal architecture is maintained by downregulation of cell turnover. Experiment Overall Design: Male FVB mice (Charles River, Maastricht, The Netherlands) were housed at 20-22Â°C, 50-60% humidity, a 12 hours light/dark cycle, and food and water ad libitum. At 6 weeks of age, mice were fasted by removing chow for up to 72 hours before sacrifice (n = 6 per group). The animals were kept in metabolic cages to prevent the consumption of beddings and were kept warm with an infrared lamp starting at 24h. Experiment Overall Design: All animals were sacrificed between 9:00 and 10:00 a.m. by cervical dislocation. The small intestine was removed quickly in such a way that adherent tissue remained behind. It was opened longitudinally, rinsed in phosphate-buffered saline, blotted, weighed, snap-frozen in liquid N2, and stored at -80Â°C. We opted to use extracts of full-thickness intestine for gene-expression profiling, because the epithelial component of the murine small intestine comprises over 70% of its volume The suitability of this strategy is underscored by a recent microarray study of transporters in the mouse intestine [Anderle P et al., BMC Genomics 2005, 6: 69.]. In addition, isolation of enterocytes is time-consuming and, hence, entails a risk of mRNA degradation, while mucosal scraping harvests villi more efficiently than crypts , whereas many mRNAs are most abundant in the crypts. Experiment Overall Design: Total intestinal RNA was extracted from frozen tissue with guanidiniumthiocyanate [Chomczynski P, Sacchi N: Analytical Biochemistry 1987, 162: 156-159.], followed by cesium-chloride centrifugation [Glisin V, Crkvenjakov R BC: Biochemistry 1974, 13: 2633-2637.] to avoid contamination with mucus. The quality of RNA was assessed with the RNA 6000 Nano LabChipÂ® Kit in an Agilent 2100 bioanalyzer (Agilent Technologies, Palo Alto, USA). Microarray-based quantification of 8 mRNAs with a 1.3-9.2-fold change in expression was validated by qPCR, as described [58]. mRNA concentration was calculated using the LinReg program [59]. In the absence of reverse transcriptase, the signal was < 0.1% of that in its presence for each primer pair (not shown). Experiment Overall Design: Microarrays. The 60-mer Mouse Development (22K) Oligo Microarray G4120A (Agilent) was used. Three arrays per experimental condition were used. Per microarray, 20 Î¼g mRNA, pooled from 2 intestines, was reverse transcribed with Cy3-labelled dCTP (Perkin Elmer, Boston, USA), using the Agilent Fluorescent Direct Label Kit. Cy5-labeled cDNA produced from RNA pooled from 6 fed animals served as the common reference across all arrays (Figure 1A). Hybridized cDNAs were detected with Agilentâs dual-laser microarray slide scanner. The data were retrieved with Agilentâs Feature Extraction software 6.1.1.

ORGANISM(S): Mus musculus

SUBMITTER: Milka Sokolovic

PROVIDER: E-GEOD-8019 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Fasting induces a biphasic adaptive metabolic response in murine small intestine.

Sokolović Milka M Wehkamp Diederik D Sokolović Aleksandar A Vermeulen Jacqueline J Gilhuijs-Pederson Lisa A LA van Haaften Rachel I M RI Nikolsky Yuri Y Evelo Chris T A CT van Kampen Antoine H C AH Hakvoort Theodorus B M TB Lamers Wouter H WH

BMC genomics 20071009

<h4>Background</h4>The gut is a major energy consumer, but a comprehensive overview of the adaptive response to fasting is lacking. Gene-expression profiling, pathway analysis, and immunohistochemistry were therefore carried out on mouse small intestine after 0, 12, 24, and 72 hours of fasting.<h4>Results</h4>Intestinal weight declined to 50% of control, but this loss of tissue mass was distributed proportionally among the gut's structural components, so that the microarrays' tissue base remaine ...[more]

PMID: 17925015

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Project description:Background; In the postabsorptive state, the portal-drained viscera are a major energy consumer, but a comprehensive overview of the adaptive fasting response in the liver is lacking. Hence, gene-expression profiling, pathway, network and gene-set enrichment analysis and immunohistochemistry were carried out on mouse liver after 0, 12, 24, and 72 hours of fasting. Results; Liver weight has fallen to 50% of control after three days of fasting, hepatocyte size was reduced with no apparent increase in apoptosis, while the basic liver structure and metabolic zonnation were preserved. Expression profiling and pathway analysis depicted four master processes, all with response peaking at 24 hours, and all but one decreasing towards 72h. Changes in gene expression were compatible with cellular energy deficiency, and metabolic adaptations were directed at enhanced glucose production, stimulation of lipid catabolism and ketone body synthesis, and strongly augmented ATP production. Consequently, the expression of genes involved in oxidative and endoplasmic reticulum stress response was increased. In addition, urea cycle genes were strongly upregulated during whole duration of fasting, in spite no obvious increase in amino-acid catabolism. Major physiological change upon continued fasting was restoration of glycogen reserves, but with reverse localization, demonstrating utilization of different glycogenic precursor compared to the fed controls. Conclusion; The changes in liver gene expression indicate a rapid onset of generating glucose and ketone bodies during short, and a return to near normal situation in prolonged fasting. The reason apparently lies in glycogen repletion, due to late fasting glucose production by (gut and) kidney. Experiment Overall Design: Male FVB mice (Charles River, Maastricht, The Netherlands) were housed at 20-22Â°C, 50-60% humidity, a 12 hours light/dark cycle, and food and water ad libitum. At 6 weeks of age, mice were fasted by removing chow for up to 72 hours before sacrifice (n = 6 per group). All animals were sacrificed between 9:00 and 10:00 a.m. by cervical dislocation. The liver was removed quickly, weighed, snap-frozen in liquid N2, and stored at -80Â°C. Total intestinal RNA was extracted from frozen tissue using TRIzol (Invitrogen), followed by a cleanup through a RNeasy column (Qiagen). The quality of RNA was assessed with the RNA 6000 Nano LabChipÂ® Kit in an Agilent 2100 bioanalyzer (Agilent Technologies, Palo Alto, USA). The 60-mer Mouse Development (22K) Oligo Microarray G4120A (Agilent) was used. Three arrays per experimental condition were used. Per microarray, 20 Î¼g mRNA, pooled from 2 livers, was reverse transcribed with Cy3-labelled dCTP (Perkin Elmer, Boston, USA), using the Agilent Fluorescent Direct Label Kit. Cy5-labeled cDNA produced from RNA pooled from livers of 6 fed animals served as the common reference across all arrays. Hybridized cDNAs were detected with Agilentâ??s dual-laser microarray slide scanner. The data were retrieved with Agilentâ??s Feature Extraction software 6.1.1.

Project description:Although studies have established that exogenous growth hormone (GH) treatment stimulates growth in fish, its effects on target tissue gene expression are not well characterized. We assessed the effects PosilacÂ® (Monsanto Co., St. Louis, MO), a recombinant bovine somatotropin, on tissue transcript levels. Transcript abundance was measure in liver and muscle using the GRASP 16 K cDNA microarray. A selection of the genes identified as altered with the microarray, and also transcripts for insulin-like growth factors, growth hormone receptors (GHR) and myostatins were measured by realtime PCR in the liver, muscle, brain, kidney, intestine, stomach, gill and heart. In general, transcripts identified as differentially regulated in the muscle on the microarray showed similar direction of expression in the other non-hepatic tissues. Rainbow trout were selected from two high growth rate and two low growth rate families. A total of 113 and 67 transcripts were identified by microarray as differentially expressed with GH treatment across growth rate for muscle and liver respectively. The largest proportion of the transcripts represented novel transcripts, followed by immune and metabolism related genes. The immune related genes were primarily modulated in the liver and indicate activation of a non-specific immune response. The metabolic genes include lipid metabolism, oxidative phosphorylation and one carbon metabolism pathway transcripts. Most notable among the growth axis genes measured by realtime PCR were increases in GHR1 and-2 transcript in liver and muscle. Our results indicate that short-term GH treatment activates the immune system, shifts the metabolic sectors and modulates growth regulating genes. Keywords: Growth Hormone Injection Muscle and Liver Gene Expression Rainbow trout (hatched March 2005) selected for extreme growth rate were obtained from NCCCWA brood stock. Families were selected based on body weight at 7 months of age and thermal growth coefficient for the final month of growth. The two high growth families used in the study were in the top 2% in terms of growth rate, and the low growth families were in the lowest 10% for growth rate. Fish acclimated to the new tanks for two weeks prior to initiation of the treatments. Fish from each family were randomly selected to receive one of three treatments: 1) PosilacÂ® injection (120 mg/kg BW, n = 4 per family); 2) vehicle injection (n = 4 per family); or 3) untouched controls (n = 2 per family). We had determined there was no effect to growth or the GH/IGF-I axis in the vehicle treated fish, and therefore, all of the microarray hybridizations were made between the GH and vehicle injected groups. This study included a total of 16 two-channel arrays designed for the direct comparison of GH treatment levels. That is, for each of the four groups, 1) High Growth Rate Liver; 2) Low Growth Rate Liver; 3) High Growth Rate Muscle; and 4) Low Growth Rate Muscle, four slide were hybridizes using individual RNA samples from individual fish. RNA isolation from each tissue/organ sample was handled separately (without pooling) with the purpose of using biological replications. We hybridized two slides with the GH cDNA labeled with Alexa 555 and vehicle cDNA labeled with Alexa 647; and two slides, using unique RNA samples, for the with GH cDNA labeled with Alexa 647 and tissue from the vehicle injected group labeled with Alexa 647 within each tissue and growth rate. Sixteen slides were used in the current study representing 32 individual tissue samples, meaning a total of four biological replicates for each treatment group.

Project description:Natural Antisense Transcripts (NATs) can regulate gene expression by virtue of their ability to form double-stranded RNA duplexes. To investigate NATs in the maize transcriptome, cDNAs from seedling of two inbred lines (B73 and Mo17) were hybridized to an oligonucleotide microarray designed to validate the expression of in silico detected NATs and to screen for NATs that can anneal to a random set of 3â UTRs and selected repeats found in 3â UTRs regions. Quantitative Real-Time PCR experiments were conducted to determine the minimum detection threshold of microarray experiment and to thereby identify genes for which both sense and antisense transcripts accumulate to detectable levels. Two independent approaches, strand-specific RT-PCR and S1 nuclease assays were conducted to validate the results of the microarray experiment. Based on these conservative assays, NATs accumulate in seedlings that can anneal to over 70% of a random set of maize genes. In addition, both sense and antisense transcripts anneal to more than 80% of a set of maize repeats. Significantly, sense and antisense transcripts exhibit significant different expression patterns between the two genotypes. Based on these findings we hypothesize that interactions between sense and antisense transcripts may contribute to the differential patterns of gene expression in maize hybrids and to heterosis. Keywords: Global antisense transcripts profiling between two maize inbreds To systematically identify NATs in maize, we employed multiple strategies to computationally identify putative antisense transcripts from our partial genome assembly (MAGIs) and a collection of ESTs we sequenced with known sequence orientation. Additionally, we randomly sampled and surveyed maize UTRs which often harbor transposons. Strand-specific oligonucleotides which can hybridize to the antisense strand were designed and spotted with together with the oligonucleotides hybridizing sense strands on a custom, strand-specific oligoarray, providing the first global expression profiling study of NATs in maize UTRs. In total, 10 biological replication were conducted to compare the global gene expression profiles between two maize genotypes (B73 and Mo17).

Project description:Heterosis is the phenomenon whereby the progeny of particular inbred lines have enhanced agronomic performance relative to both parents. Although several hypotheses have been proposed to explain this fundamental biological phenomenon, the responsible molecular mechanisms have not been determined. The maize inbred lines B73 and Mo17 produce a heterotic F1 hybrid. Global patterns of gene expression were compared in seedlings of these three genotypes using a microarray that contains 13,999 cDNAs. Using an estimated 15% false discovery rate as a cut-off, 1,367 ESTs (9.8%) were identified as being significantly differentially expressed among genotypes. All possible modes of gene action were observed, including additivity, high- and low-parent dominance, under-dominance, and over-dominance. The largest proportion of the ESTs (78%, 1,062/1,367) exhibited expression patterns that are not statistically distinguishable from additivity. Even so, 22% of the differentially regulated genes exhibited non-additive modes of gene expression. Classified on the basis of significant pair-wise comparisons of genotype means, 181 of these 305 genes exhibited high-parent dominance and 23 exhibited low-parent dominance. In addition, 44 genes exhibited under- or over-dominant gene action. These findings are consistent with the hypothesis that multiple molecular mechanisms contribute to heterosis, including over-dominance Keywords: Genotype Comparison The inbreds B73 (Schnable Lab Accession #660) and Mo17 (Schnable Lab Accession #2618) used in this study were derived by self-pollination from stocks originally obtained from Don Robertson (Iowa State University) and Mike Lee (Iowa State University), respectively. Mo17 was crossed as a female by B73 to generate the F1. Kernels from three different seed sources (ears) per genotype were used in the experimental design. Prior to microarray analyses genotypes were confirmed by genotyping DNA extracted from each seed source using co-dominant IDP genetic markers that distinguish B73 from Mo17 (Fu et al., in preparation). Ten biological replications of B73, Mo17, and their F1 (Mo17xB73) were grown under highly controlled conditions in a randomized complete block design. For each replication, the B73, Mo17, and F1 samples were hybridized to three two-color cDNA microarrays using a loop design such that each loop included all pairwise comparisons between genotypes. RNA pools for each genotype were alternately labeled providing dye balance within each loop. After hybridization, one biological replicate was removed due to poor quality. The final analysis incorporated 27 microarray slides (3 slides for each of nine high-quality biological replicates).

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Dataset Information

Transcription profiling of mouse small intestine reveals fasting induces a biphasic adaptive metabolic response

Publications

Fasting induces a biphasic adaptive metabolic response in murine small intestine.

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