Tamaoki K et al. (2016), Morphological, biochemical, transcriptional and...

XB-ART-51789

Cell Biosci 2016 Jan 21;6:2. doi: 10.1186/s13578-016-0067-9.

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Morphological, biochemical, transcriptional and epigenetic responses to fasting and refeeding in intestine of Xenopus laevis.

Tamaoki K , Okada R , Ishihara A , Shiojiri N , Mochizuki K , Goda T , Yamauchi K .

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BACKGROUND: Amphibians are able to survive for several months without food. However, it is unclear what molecular mechanisms underlie their survival. To characterize the intestinal responses to fasting and refeeding, we investigated morphological, biochemical, transcriptional and epigenetic changes in the intestine from adult male Xenopus laevis. RESULTS: Frogs were fed for 22 days, fasted for 22 days, or fasted for 21 days and refed for 1 day. Fasting reduced, and refeeding recovered partially or fully, morphological parameters (wet weight of the intestine, circumference of the epithelial layer and number of troughs in a villus-trough unit), activities of digestive enzymes and plasma biochemical parameters (glucose, triglycerides, cholesterol and free fatty acids). Reverse transcription-quantitative polymerase chain reaction analysis revealed overall suppression of the transcript levels by fasting, with various recovery rates on refeeding. Chromatin immunoprecipitation assays on the selected genes whose transcript levels declined with fasting and recovered quickly with refeeding, showed several euchromatin marks in histone (acetylation and methylation) and RNA polymerase II modifications (phosphorylation) with fasting, and returned to the feeding levels by refeeding. The mRNA levels of these genes responded to fasting and refeeding to greater extents than did the pre-mRNA levels, suggesting the involvement of post-transcriptional regulation. CONCLUSIONS: Our results demonstrate that the X. laevis intestine may undergo overall metabolic suppression at least at the transcriptional level to save energy during fasting and quickly recovered to moderate nutritional deficiency by refeeding, and suggest that these dietary responses of the intestine are epigenetically and post-transcriptionally regulated.

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Species referenced: Xenopus laevis
Genes referenced: cdx2 fabp1 fabp2 nr1h4 rpl8

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	Fig. 1. Morphology and a cell proliferation marker in the intestines of fed, fasted, and refed X. laevis. A Histology of representative intestines of fed, fasted, and refed X. laevis stained with hematoxylin and eosin. The frogs were fed for 22 days (a), fasted for 22 days (b), or fasted for 21 days and then refed for 1 day (c), and fasting for 5 months (d). e Schematically illustrated structure of the X. laevis intestine with morphological parameters measured (see Table 1). The box in each panels (a–d) is enlarged image. el epitherial layer, gc goblet cell, lu lumen, me muscularis externa, tr trough, vi villus. Bar 500 μm in panels a, b, c and d, and 100 μm in boxes. These experiments were repeated at least three times, with similar results. B Protein expression of proliferating cell nuclear antigen (PCNA) in the intestines from fed, fasted, and refed X. laevis. Intestine homogenates (60 μg protein; two samples/each group) were analyzed by SDS-PAGE, followed by Western blotting. Band intensities were analyzed and expressed relative to α-tubulin, and values are expressed relative to the value of the fed frog (left) that was set to 1.00. (a) PCNA; (b) α-tubulin
	Fig. 2. The enzyme activities in the intestines of fed, fasted and refed X. laevis. Enzyme assays were conducted using the extracts obtained from the intestines of the frogs that were fed for 22 days (fed), fasted for 22 days (fasted), or fasted for 21 days and then refed for 1 day (refed). The activities were expressed as μmol hydrolysed substrates or generated products/min/g protein. A alkaline phosphatase; B aminopeptidase; C glucoamylase; D maltase. Values presented are mean ± SEM (n = 8). Different letters indicate significant differences between groups (p < 0.05). These experiments were repeated at least three times, with similar results
	Fig. 3. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) analysis of gene transcripts in the intestines of fed, fasted and refed X. laevis. RNAs were prepared from the intestines of the frogs that were fed for 22 days, fasted for 22 days (fasted), or fasted for 21 days and then refed for 1 day (refed), followed by RT-qPCR (n = 8). Transcripts of 74 genes were divided to the following categories: digestion or absorption (22 genes), apoptosis (7 genes), proliferation (8 genes), regulation of gene expression (19 genes), metabolism (16 genes) and others (2 genes). Color scale indicated fold changes of gene expression. The data of the full name of the genes tested, the exact fold changes, SEM and statistic analysis are shown in Additional file 2: Table S1. These experiments were repeated at least two times, with similar results
	Fig. 4. Epigenetic modifications on fabp1, fabp2, cdx2 and fxr genes in the intestines of fed, fasted and refed X. laevis. Chromatin samples were prepared from the intestines of the frogs that were fed for 22 days (fed), fasted for 22 days (fasted), or fasted for 21 days and then refed for 1 day (refed). Signals of ChIP on fabp1 (A, F, K, P and U), fabp2 (B, G, L, Q and V), cdx2 (C, H, M, R and W), fxr (D, I, N, S and X) and rpl8 (E, J, O, T and Y) genes were detected by qPCR following immunoprecipitation with antibodies against H3K9ac (A–E), H4ac (F–J), H3K4me1 (K–O), RNAPII (P–T) and RNAPIIS5P (U–Y). Primers used in qPCR are shown in Additional file 6: Table S3. Each value is the mean ± SEM (n = 8). Distinct letters denote significantly different means (p < 0.05). These experiments were repeated at least two times, with similar results
	Fig. 5. Epigenetic modifications on fabp1, fabp2, cdx2 and fxr genes in the intestines of fed, fasted and refed X. laevis. Chromatin samples were prepared from the intestines from the frogs that were fed for 22 days (fed), fasted for 22 days (fasted), or fasted for 21 days and then refed for 1 day (refed). Signals of ChIP on fabp1 (A, F, K and P ), fabp2 (B, G, L and Q), cdx2 (C, H, M and P), fxr (D, I, N and S) and rpl8 (E, J, O and T) genes were detected by qPCR following immunoprecipitation with antibodies against H3K36me1 (A–E), H3K36me2 (F–J), H3K36me3 (K–O), and RNAPIIS2P (P–T). Primers used in qPCR are shown in Additional file 6: Table S3. Each value is the mean ± SEM (n = 8). Distinct letters denote significantly different means (p < 0.05). These experiments were repeated at least two times, with similar results
	Fig. 6. Post-transcriptional regulation of diet-response genes in the small intestine of X. laevis. A Comparison of pre-mRNA levels with matured mRNA levels of four fasting/refeeding-response genes. RNAs were prepared as shown in Fig. 3, and RT-qPCR (n = 8) was conducted using intron specific primers (a–d) and exon-specific primers (e–h) for fabp1 (a and e), fabp2 (b and f), cdx2 (c and g) and fxr (d and h) genes. Primers used in qPCR are shown in Additional file 6: Table S3. Each value is the mean ± SEM (n = 8). Distinct letters denote significantly different means (p < 0.05). B RT-qPCR of the cdx2 mRNA expression after treatment with actinomycin D. mRNAs were extracted from the primary tissue culture of the fed (circle), fasted (triangle) or refed (square) animals, as described in “Methods”. mRNA amounts at time 0 were set at 100 %. Depicted half lives were calculated using exponential regression. These experiments were repeated at least two times, with similar results

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