MicroRNAs, as a class of small noncoding RNA molecules, can bind to the 3’UTRs of the mRNAs of their target genes, thus inhibit translation of the genes and/or promote destabilization and degradation of the mRNAs . As a key posttranscriptional repressor of gene expression, microRNAs are implicated in almost all physiological and pathological processes . For example, miR-33, embedded in the 16th intron of sterol response element binding transcription factor (SREBF) gene , is co-transcribed with SREBF and can target a number of genes including CROT, HADHB, NPC1, CPT1A and AMPK, etc. . CROT is required for the transport of medium and long chain acyl CoA molecules from the peroxisome to the cytoplasm and mitochondria. It mainly exists in the peroxisome and mitochondria, and functions as an auxiliary enzyme in fatty acid metabolism . HADHB encodes the beta subunit of mitochondrial trifunctional protein, which catalyzes the last three steps of long-chain fatty acid β-oxidation in mitochondria . Therefore, induction of CROT and HADHB expression may promote fatty acid oxidation, while inhibition of their expression may promote fatty acid accumulation. Indeed, miR-33 knockdown induces the expression of CROT, CPT1A, HADHB and AMPK, and thus promotes fatty acid oxidation . In contrary, overexpression of miR-33 can inhibit fatty acid oxidation in liver cells . These effects of miR-33 may be mediated by regulating the expression of its target genes, including CROT and HADHB.
Although miR-33 is implicated in fatty acid metabolism, it is unclear whether miR-33 participates in the development of goose fatty liver via CROT gene. Goose fatty liver is characterized by deposition of large amount of fat, which sometimes accounts for about 60% of liver weight. The weight of fatty liver can reach 8–10 times that of normal liver in a short period of time (3–4 wk) . These features reflect the marvelous capacity of goose liver in lipid accumulation. Revealing the mechanism underlying this capacity may help improve the production of goose fatty liver (or foie gras), as well as provide new ideas to develop therapeutic approach to NAFLD in human and other domestic animals. Considering the role of miR-33 and CROT genes in lipid metabolism, we speculate that miR-33 and CROT genes contribute to this unique mechanism that goose liver owns. In this study, data showed that the expression of miR-33 in the liver, muscle and fat tissues of the overfed geese was significantly upregulated compared with the normally fed control geese. The targeting regulatory relationship between miR-33 and CROT genes was also subsequently validated in goose liver cells. As expected, the mRNA and protein expressions of CROT gene were significantly downregulated in goose fatty liver vs. normal liver, which is contrary to the expression of miR-33. These findings suggest that, although CROT gene expression could be regulated by multiple factors, miR-33 is a major factor contributing to the downregulation of CROT gene in the development of goose fatty liver. As miR-33 is an intronic sequence of the major regulator of lipid metabolism, SREBF, it may enhance the role of SREBF by being co-transcribed with SREBF and suppressing its target genes including CROT. Interestingly, upregulation of miR-33 expression also occurred in muscle and fat tissues of the overfed geese vs. control geese, thus lipid accumulation in these tissues may be partially attributed to the upregulation of miR-33. Consistently, miR-33 mimic promoted lipid deposition in goose primary hepatocytes. However, it seemed conflicting that CROT overexpression increased lipid deposition and reduced lipid peroxidation in goose primary hepatocytes. One explanation is that the function of CROT is to mobilize fatty acids by promoting β-oxidation of medium- and long-chain fatty acids and transport of the product of β-oxidation from peroxisome to the cytosol. Whether CROT promotes or inhibits lipid deposition, it depends on the context where other genes join and decide the fate of fatty acids, i.e., entering mitochondria to be further degraded or forming triacylgycerols in the cytosol. In other words, lipid deposition in the cell is not only determined by CROT, but also is subjected to regulation by multiple proteins and processes, which may be applicable to the regulation of lipid deposition by miR-33 in goose fatty liver. In line with this explanation, lipid peroxidation inhibited by CROT overexpression in goose primary hepatocytes suggested that fatty acid oxidation in mitochondria was somehow suppressed. Lipid peroxidation is mainly caused by mitochondria-derived reactive oxygen species. Recent studies indicate that CROT gene may participate in the regulation of fatty acid composition [27–28], thus it is worthwhile to determine if CROT gene has dual functions (i.e., involving in β-oxidation of fatty acids, and modulating fatty acid composition) in the development of goose fatty liver.
In addition, previous study shows that CROT overexpression in HepG2 cells induces CRAT expression, a gene playing a key role in fatty acid oxidation . Consistently, this study showed that CROT overexpression in goose primary hepatocytes could induce the expression of PEX5,EHHADH༌CAT and ACOT8 genes in addition to CRAT gene. It is known that all the genes are associated with fatty acid oxidation. PEX5 (peroxisome biogenic factor 5) plays an important role in peroxisome protein input by binding to the C-terminal PTS1 type tripeptide peroxisome targeting signal (SKL type) and thus is necessary for protein assembly of functional peroxisome. EHHADH (enol COA hydratase and 3-hydroxyacyl CoA dehydrogenase) is one of the four enzymes involved in peroxisomal β-oxidation pathway as its N-terminal contains enol CoA hydratase activity and C-terminal contains 3-hydroxyacyl CoA dehydrogenase activity . ACOT8 is a peroxisomal thioesterase, which is involved in the oxidation of fatty acids. As peroxisomes play a key role in fatty acid oxidation and the genes are involved in assembly and function of peroxisome, CROT gene may promote fatty acid oxidation via these genes. As upregulation of fatty acid oxidation increases release of reactive oxidative species, it is reasonable that CROT gene overexpression induced the expression of CAT gene, a key antioxidant enzyme in the cell, which provides an explanation why CROT overexpression inhibited lipid peroxidation in goose primary hepatocytes. Similarly, the induction of these genes only indicates mobilization but not degradation of fatty acids as suggested by CROT overexpression assay. Induction of CRAT, PEX5, EHHADH and ACOT8 genes may be due to the increased level of product generated by CROT. It is found that significant reduction in PEX5 expression was concomitant with the inhibition of CROT expression in goose fatty liver vs. normal liver. This is probably due to the regulation of other factors on the expression of these genes during the development of goose fatty liver, which needs to be further investigated.
Finally, this study showed that high level of glucose could inhibit miR-33 expression and induce CROT expression in goose primary hepatocytes, whereas high level of insulin could induce miR-33 expression without changing CROT expression. As the development of fatty liver is usually accompanied with hyperglycemia and hyperinsulinemia, the results suggest that hyperglycemia and hyperinsulinemia are involved in the regulation of miR-33 in the development of goose fatty liver, but only hyperglycemia is involved in the expression of CROT gene in goose fatty liver. It is, however, unknown how miR-33 expression is regulated by glucose and insulin. It is likely that glucose and insulin regulate the expression of miR-33 by regulating the expression of SREBP as previous studies have shown that glucose and insulin can regulate the expression of SREBF [18 28]. Moreover, other factors may also regulate the expression of miR-33 by regulating the expression of SREBF, such as thyroid hormone, which is known to be able to stimulate the expression of SREBF . These speculations warrant further investigation.
It is noteworthy that, although the regulatory relationship between goose miR-33 and CROT has been published (Zheng et al., 2015), the relationship is demonstrated only in CHO cells (from Chinese hamster ovary) other than goose liver cells. This study provides some new insights into the relationship between miR-33 and CROT and the functions of the genes in goose liver cells, including confirmation of the reciprocal relationship between miR-33 and CROT in goose fatty liver and primary hepatocytes, the regulation of miR-33 and CROT expression by insulin and glucose, and the induction of lipid deposition by miR-33 mimics in goose primary hepatocytes.