Characterization of metapopulation of Ellobium chinense through Pleistocene expansions and four covariate COI guanine-hotspots linked to G-quadruplex conformation

Abstract The land snail Ellobium chinense (L. Pfeiffer, 1855) (Eupulmonata, Ellobiida, Ellobiidae), which inhabits the salt marshes along the coastal areas of northwestern Pacific, is an endangered species on the IUCN Red List. Over recent decades, the population size of E. chinense has consistently decreased due to environmental interference caused by natural disasters and human activities. Here, we provide the first assessment of the genetic diversity and population genetic structures of northwestern Pacific E. chinense. The results analyzed with COI and microsatellites revealed that E. chinense population exhibit metapopulation characteristics, retaining under the influence of the Kuroshio warm currents through expansion of the Late-Middle and Late Pleistocene. We also found four phylogenetic groups, regardless of geographical distributions, which were easily distinguishable by four unidirectional and stepwise adenine-to-guanine transitions in COI (sites 207–282–354–420: A–A–A–A, A–A–G–A, G–A–G–A, and G–G–G–G). Additionally, the four COI hotspots were robustly connected with a high degree of covariance between them. We discuss the role of these covariate guanines which link to form four consecutive G-quadruplexes, and their possible beneficial effects under positive selection pressure

Inhibitory effect of spiramycin on expression of adipogenic transcriptional factors and their adipocyte-specific target genes in 3T3-L1 adipocytes.

<p>(A) 3T3-L1 preadipocytes were differentiated into adipocytes in the presence of spiramycin for 6 days. The transcript levels of major adipogenic transcription factors (PPARγ, C/EBPα, and SREBP1) and their adipocyte-specific target genes (aP2, GLUT4, and FAS) were evaluated by qRT-PCR. (B) Western blotting analysis showing the effect of spiramycin on protein levels of major adipogenic transcription factors (PPARγ, C/EBPα, and SREBP1). The numbers at the bottom of the figure indicate the relative intensity of each band (fold-change in comparison with that of the control group), which was estimated using Multi Gauge software version 3.0.</p

Effects of spiramycin on HFD-induced obesity in C57BL/6 mice.

<p>Spiramycin (25 or 50 mg/kg) or orlistat (50 mg/kg) were administered by oral gavage for 8 weeks while the mice were fed the HFD. (A) Experimental outline. (B) Body weight was measured 3 times per week. The group that received HFD alone(■) showed steady body weight gain, while the spiramycin-(▲ or ▼) and orlistat-treated(◆) groups showed significantly attenuated body weight gain. (C) Adipose tissue weight in subcutaneous, epididymal, and mesentery fat. (D) Amount of leptin in plasma measured by ELISA. (E) Liver weight. (F) Plasma levels of GOT and GPT measured using a chemical analyzer. (G) H&E-stained images of liver and subcutaneous adipose tissue samples from the normal diet (control), vehicle-treated, spiramycin-treated, and orlistat-treated groups. The results are expressed as the mean ± SD for each group (n = 8).</p

Anti-Obesity Effects of Spiramycin <i>In Vitro</i> and <i>In Vivo</i>

<div><p>The effects of spiramycin on adipogenesis and high fat diet (HFD)-induced obesity were investigated. Potential mechanisms contributing to these effects were elucidated. The inhibitory effect of spiramycin on adipocyte differentiation was assessed using 3T3-L1 preadipocyte cells, in which several parameters involved in AMPK signal pathways and lipid metabolism were examined. To further investigate the pharmacological effects of spiramycin <i>in vivo</i>, we examined several obesity-related parameters in HFD-induced obese mice. Spiramycin significantly inhibited preadipocyte differentiation by attenuating intracellular lipid accumulation. Spiramycin also reduced the expression of adipogenic master regulators (PPARγ, C/EBPα, and SREBP1c) and their downstream target genes (FAS, aP2, and GLUT4) in 3T3-L1 cells. In addition, AMPK phosphorylation was increased by spiramycin treatment in 3T3-L1 cells during early differentiation. Notably, HFD-induced obese mice administered spiramycin showed substantial decreases in body weight gain, serum leptin levels, adipose tissue mass, and hepatic lipid accumulation. Moreover, the decreased levels of GPT and GOT in the serum indicated that spiramycin attenuated hepatic injury caused by HFD. Taken together, these results demonstrate for the first time that spiramycin effectively attenuates HFD-induced obesity and hepatic steatosis by inhibiting adipogenesis.</p></div

The changes of body and organ weight, and food intake.

<p>The changes of body and organ weight, and food intake.</p

Effect of spiramycin on phosphorylation of AMPK and ACC during 3T3-L1 adipocyte differentiation.

<p>Confluent 3T3-L1 preadipocytes (Day 0) were incubated into DM including MDI with specified concentrations of spiramycin. After 1 h, protein levels of phosphorylated AMPK and ACC (p-AMPK and p-ACC) were analyzed by western blotting. The numbers at the bottom of the figure indicate the relative band intensity normalized to that of the non-phosphorylated protein (fold-change in comparison with that of the control group).</p