Search

The insulin-induced genes (INSIG1and INSIG2) have been demonstrated to play a vital role in regulating lipid metabolism in mammals, however the function and regulation mechanism of them remains unknown in poultry. In this study, firstly the phylogenetic trees of INSIGsamong various species were constructed and their subcellular locations were mapped in chicken LMH. Then the spatiotemporal expression profiles, over-expression and knockdown assays of chicken INSIGswere conducted. Furthermore, conservation of potential miRNA binding sites in INSIGsamong species were analyzed, and the miRNA biological function and regulatory role were verified. The results showed that chicken INSIGslocated in cellular endoplasmic reticulum, and were originated from the common ancestors of their mammalian counterparts. The INSIGswere widely expressed in all detected tissues, and their expression levels in the liver of chicken at 30 wk were significantly higher than that at 20 wk (P< 0.01). Over-expression of INSIGsled no significant increase in mRNA abundance of lipid metabolism-related genes and the contents of triacylglycerol (TG) and cholesterol (TC) in LMH cells. Knockdown of INSIG1led to the decreased expressions of ACSL1, MTTP-L, ApoB, ApoVLDLIIgenes and TG, TC contents (P< 0.05). Knockdown of INSIG2 could significantly decrease the contents of TG and TC, and expressions of key genes related to the lipid metabolism (P< 0.05). Moreover, INSIG1was directly targeted by both miR-130b-3p and miR-218-5p, and INSIG2was directly targeted by miR-130b-3p. MiR-130b-3p mimic and miR-218-5p mimic treatment could significant decrease the mRNA and protein levels of INSIGs, mRNA levels of genes related to lipid metabolism, and the contents of TG and TC in LMH cells. The inhibition of miR-130b-3p and miR-218-5p on TG and TC contents could be restored by the overexpression of INSIGs, respectively. No significant alteration in expressions of sterol regulatory element binding protein (SREBPs) and SREBP cleavage-activating protein (SCAP) were observed when INSIGs were over-expressed. SCAPwas down-regulated when INSIG1was knocked down, while SREBP1was down-regulated when INSIG2was knocked down. Taken together, these results highlight the role of INSIG1and INSIG2in lipid metabolism and their regulatory mechanism in chicken.

miR-19b-3pis reported to undertake various biological role, while its function and action mechanism in chicken hepatic lipid metabolism is unclear. Conservation analysis and tissue expression pattern of miR-19b-3pand its target gene were evaluated, respectively. Dual luciferase reporter system and western blot technologies were adopted to validate miR-19b-3ptarget gene. Overexpression and knockdown assays were done to explore the biological functions of miR-19b-3pand target gene in Leghorn Male Hepatoma cell line (LMH). Regulatory approaches of estrogen on miR-19b-3pand target gene expressions are analyzed through site-directed mutation combined with estrogen receptors antagonist treatment assays. The results showed that, chicken miR-19b-3pmature sequences are highly conserved among Capra hircus, Columba livia, Rattus norvegicus, Mus musculus, Cricetulus griseus, Danio rerio, Danio novaehollandiae, Orycodylus porosus, Crocodylus porosus, Gadus morhua, and widely expressed in lung, ovary, spleen, duodenum, kidney, heart, liver, leg muscle and pectoral muscle tissues. miR-19b-3pcould significantly increase intracellular triglyceride (TG) content and decrease intracellular cholesterol (TC) content via targeting methylsterol monooxygenase 1 (MSMO1) and elongase of very long chain fatty acids 5 (ELOVL5), which are highly conserved among species, in both mRNA and protein levels. Estrogen could inhibit miR-19b-3pexpression, but directly promoted MSMO1transcription via estrogen receptor α (ERα) and indirectly regulated ELOVL5expression at the transcription level. Meanwhile, estrogen could also up-regulate MSMO1and ELOVL5expression through inhibiting miR-19b-3pexpression at the post-transcription level. Taken together, these results highlight the role and regulatory mechanism of miR-19b-3pin hepatic lipid metabolism in chicken, and might produce useful comparative information for human obesity studies and biomedical research.