Low Utilization of Circulating Glucose after Food Withdrawal in Snell Dwarf Mice

Glucose metabolism is altered in long-lived people and mice. Although it is clear that there is an association between altered glucose metabolism and longevity, it is not known whether this link is causal or not. Our current hypothesis is that decreased fasting glucose utilization may increase longevity by reducing oxygen radical production, a potential cause of aging. We observed that whole body fasting glucose utilization was lower in the Snell dwarf, a long-lived mutant mouse. Whole body fasting glucose utilization may be reduced by a decrease in the production of circulating glucose. Our isotope labeling analysis indicated both gluconeogenesis and glycogenolysis were suppressed in Snell dwarfs. Elevated circulating adiponectin may contribute to the reduction of glucose production in Snell dwarfs. Adiponectin lowered the appearance of glucose in the media over hepatoma cells by suppressing gluconeogenesis and glycogenolysis. The suppression of glucose production by adiponectin in vitro depended on AMP-activated protein kinase, a cell mediator of fatty acid oxidation. Elevated fatty acid oxidation was indicated in Snell dwarfs by increased utilization of circulating oleic acid, reduced intracellular triglyceride content, and increased phosphorylation of acetyl-CoA carboxylase. Finally, protein carbonyl content, a marker of oxygen radical damage, was decreased in Snell dwarfs. The correlation between high glucose utilization and elevated oxygen radical production was also observed in vitro by altering the concentrations of glucose and fatty acids in the media or pharmacologic inhibition of glucose and fatty acid oxidation with 4-hydroxycyanocinnamic acid and etomoxir, respectively

Intronic Cis-Regulatory Modules Mediate Tissue-Specific and Microbial Control of angptl4/fiaf Transcription

The intestinal microbiota enhances dietary energy harvest leading to increased fat storage in adipose tissues. This effect is caused in part by the microbial suppression of intestinal epithelial expression of a circulating inhibitor of lipoprotein lipase called Angiopoietin-like 4 (Angptl4/Fiaf). To define the cis-regulatory mechanisms underlying intestine-specific and microbial control of Angptl4 transcription, we utilized the zebrafish system in which host regulatory DNA can be rapidly analyzed in a live, transparent, and gnotobiotic vertebrate. We found that zebrafish angptl4 is transcribed in multiple tissues including the liver, pancreatic islet, and intestinal epithelium, which is similar to its mammalian homologs. Zebrafish angptl4 is also specifically suppressed in the intestinal epithelium upon colonization with a microbiota. In vivo transgenic reporter assays identified discrete tissue-specific regulatory modules within angptl4 intron 3 sufficient to drive expression in the liver, pancreatic islet β-cells, or intestinal enterocytes. Comparative sequence analyses and heterologous functional assays of angptl4 intron 3 sequences from 12 teleost fish species revealed differential evolution of the islet and intestinal regulatory modules. High-resolution functional mapping and site-directed mutagenesis defined the minimal set of regulatory sequences required for intestinal activity. Strikingly, the microbiota suppressed the transcriptional activity of the intestine-specific regulatory module similar to the endogenous angptl4 gene. These results suggest that the microbiota might regulate host intestinal Angptl4 protein expression and peripheral fat storage by suppressing the activity of an intestine-specific transcriptional enhancer. This study provides a useful paradigm for understanding how microbial signals interact with tissue-specific regulatory networks to control the activity and evolution of host gene transcription

Ontogeny and nutritional control of adipogenesis in zebrafish (Danio rerio)

The global obesity epidemic demands an improved understanding of the developmental and environmental factors regulating fat storage. Adipocytes serve as major sites of fat storage and as regulators of energy balance and inflammation. The optical transparency of developing zebrafish provides new opportunities to investigate mechanisms governing adipocyte biology, however zebrafish adipocytes remain uncharacterized. We have developed methods for visualizing zebrafish adipocytes in vivo by labeling neutral lipid droplets with Nile Red. Our results establish that neutral lipid droplets first accumulate in visceral adipocytes during larval stages and increase in number and distribution as zebrafish grow. We show that the cellular anatomy of zebrafish adipocytes is similar to mammalian white adipocytes and identify peroxisome-proliferator activated receptor γ and fatty acid binding protein 11a as markers of the zebrafish adipocyte lineage. By monitoring adipocyte development prior to neutral lipid deposition, we find that the first visceral preadipocytes appear in association with the pancreas shortly after initiation of exogenous nutrition. Zebrafish reared in the absence of food fail to form visceral preadipocytes, indicating that exogenous nutrition is required for adipocyte development. These results reveal homologies between zebrafish and mammalian adipocytes and establish the zebrafish as a new model for adipocyte research

The intestinal module in3.4 recapitulates microbial suppression of <i>angptl4</i>.

<p>(A) Semi-quantitative whole mount <i>in situ</i> hybridization of <i>angptl4</i> mRNA in 6 dpf germ-free (GF) and conventionalized (CONVD) animals. Arrowheads mark intestinal expression. Note that the background staining in the gills (arrows) is similar in GF and CONVD fish. Transverse sections show that microbial suppression of <i>angptl4</i> mRNA is specific to the intestinal epithelium. (B) Quantitative RT-PCR of <i>angptl4</i> and <i>GFP</i> mRNA levels in 6 dpf GF and CONVD <i>Tg(in3.4-Mmu.Fos:GFP)</i> animals. GF and CONVD animals were derived from the same <i>Tg(in3.4-Mmu.Fos:GFP)</i> stable line. <i>GFP</i> and <i>angptl4</i> mRNA were normalized to <i>18S</i> rRNA levels and are shown as fold difference compared to GF controls averaged across 3 experimental replicates ± SEM (2 biological replicate groups of 10 larvae per condition per experiment). Similar results were attained when normalized to <i>ribosomal protein L32</i> (<i>rpl32</i>) rRNA levels. Asterisks denote P-value<.01 from unpaired T-test between GF and CONVD conditions for each gene. See also Figure S8.</p

Tissue-specific expression of zebrafish <i>angptl4</i> mRNA.

<p>(A) Distance phylogram of Angptl4 protein from zebrafish (<i>Dr</i>, <i>Danio rerio</i>), catfish (<i>Ip</i>, <i>Ictalurus punctatus</i>), medaka (<i>Ol</i>, <i>Oryzias latipes</i>), tetraodon (<i>Tn</i>, <i>Tetraodoan nigroviridis</i>), fugu (<i>Tr</i>, <i>Takifugu rubipres</i>), xenopus (<i>Xt</i>, <i>Xenopus tropicalis</i>), chicken (<i>Gg</i>, <i>Gallus gallus</i>), mouse (<i>Mm</i>, Mus <i>musculus</i>), human (<i>Hs</i>, <i>Homo sapiens</i>), dog (<i>Cf</i>, <i>Canis familiaris</i>), pig (<i>Ss</i>, <i>Sus scrofa</i>), cow (<i>Bt</i>, <i>Bos taurus</i>). All nodes are significant (>700/1000 bootstrap replicates) except those marked with an asterisk (*). Scale bar indicates phylogenetic distance, in number of amino acid substitutions per site. We found that the genomes of zebrafish, channel catfish (<i>Ictaluris punctatus</i>), and medaka (<i>Oryzias latipes</i>) encode a single ortholog of mammalian Angptl4, whereas two pufferfish species (<i>Takifugu rubripes</i> and <i>Tetraodon nigroviridis</i>) encode two Angptl4 paralogs. See also <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002585#pgen.1002585.s001" target="_blank">Figure S1</a>. (B–G) Whole-mount <i>in situ</i> hybridization (WISH) using a riboprobe targeting <i>angptl4</i> mRNA during various stages in zebrafish development reveals dynamic spatiotemporal gene expression patterns. (B) At 1 day post fertilization (dpf) embryos exhibit ubiquitous expression of <i>angptl4</i>. (C–D) By 4 dpf, marked expression is observed in the intestinal epithelium (in, black arrowhead), but by 6 dpf, robust expression becomes largely localized to the intestine (black arrowhead) and pancreatic islet (not shown). The black arrow marks the boundary between the anterior intestine (segment 1) and mid-intestine (segment 2). Scale bars = 500 µm. (E–F) Transverse sections of 6 dpf and 8 dpf animals confirm expression in the intestinal epithelium (E, in, black arrowhead) and pancreatic islet (F, is, black triangle). Scale bars = 50 µm. (G–H) At 17 dpf, strong expression is observed in the liver (li, white arrowhead, dotted line outlines the liver). G, Scale bar = 250 µm; H, Scale bar = 50 µm.</p

Functional evolution of the islet and intestinal regulatory modules in 12 fish species.

<p>(A) Unscaled phylogram based on information from <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002585#pgen.1002585-Hedges1" target="_blank">[58]</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002585#pgen.1002585-Peng1" target="_blank">[59]</a> showing images and relative relationships of 12 fish for which intronic sequences were analyzed. <i>Danio rerio</i> (<i>Dr</i>, zebrafish), <i>Danio nigrofasciatus</i> (<i>Dn</i>), <i>Danio albolineatus</i> (<i>Dalb</i>), <i>Danio choprae</i> (<i>Dc</i>), <i>Danio feegradei</i> (<i>Df</i>), <i>Devario aequipinnatus</i> (<i>Daeq</i>, giant danio), <i>Carassius auratus</i> (<i>Ca</i>, goldfish), <i>Cyprinus carpio</i> (<i>Cc</i>, carp), <i>Puntius conchonius</i> (<i>Pc</i>, rosy barb), <i>Chromobotia macracanthus</i> (<i>Cm</i>, clown loach), <i>Ictalurus punctatus</i> (<i>Ip</i>, channel catfish), <i>Oryzias latipes</i> (<i>Ol</i>, medaka). (B) VISTA plot displaying the global pairwise alignment of orthologous in3.2 regions from each species anchored to zebrafish (<i>Dr</i>) in3.2. Orange peaks correspond to regions in the alignment that correspond to <i>Dr</i> in3.3 (islet module). Blue peaks correspond to regions in the alignment that correspond to <i>Dr</i> in3.4 (intestine module). Percent identity is calculated from pairwise alignments of each module with zebrafish (VISTA parameters: 25 bp sliding window, LAGAN alignment). (C) Representative islet and intestinal images from injections of each orthologous in3.2 module. Orange or blue arrowheads mark positive islet or intestine expression, respectively. The absence of arrowheads denotes negative expression in each tissue. (D) Summary of mosaic expression for each species. Ratios of islet or intestine positive fish versus total fish expressing gfp are shown. Orange or blue (+) denotes that the construct was sufficient to confer expression in the islet or intestine, respectively. Black (−) denotes insufficiency. Note that <i>Dalb</i> and <i>Cm</i> sequences were not tested (nt) in this heterologous functional assay. See also <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002585#pgen.1002585.s005" target="_blank">Figures S5</a> and <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002585#pgen.1002585.s006" target="_blank">S6</a>.</p

Summary of functional conservation and mapping of islet and intestinal regulatory information.

<p>(A) Conservation plots, module truncations, and predicted transcription factor binding sites (TFBS) in islet CRM in3.3 are overlayed and annotated to scale. The grey shaded box represents the region that is present in all positive truncations and has strong conservation in islet-positive species. (B) Conservation plots, module truncations, SDM data, and predicted transcription factor binding sites (TFBS) in intestinal CRM in3.4 are overlayed and annotated to scale. Two grey shaded boxes represent regions that are present in all positive truncations, are required for intestinal expression, and have strong conservation in intestine-positive species. Dotted boxes in panels A and B represent highly conserved regions from each (A) islet-positive or (B) intestine-positive species used to predict common TFBS (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002585#pgen.1002585.s005" target="_blank">Figures S5</a> and <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002585#pgen.1002585.s006" target="_blank">S6</a>, and <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002585#s4" target="_blank">Materials and Methods</a>).</p

Multiple-species alignments reveal conservation in <i>angptl4</i> gene structure and location of conserved non-coding regions.

<p>(A) VISTA plot displaying the global pairwise alignment of the zebrafish <i>angptl4</i> locus with the orthologous medaka, tetraodon, and fugu regions and (B) human <i>ANGPTL4</i> locus with the orthologous mouse and dog regions. Purple conservation peaks correspond to exonic sequences, and green conservation peaks represent non-coding sequences. The zebrafish and human gene structure are denoted by purple boxes above the corresponding VISTA plot (VISTA parameters: 100 bp sliding window, LAGAN alignment). Note that the concentration of conservation peaks within intron 3 of both teleost and mammalian <i>angptl4</i> genes.</p