Liver receptor homolog-1 (LRH-1/NR5A2) is a representative of the nuclear receptor 5A subfamily of orphan nuclear receptors, mainly expressed in the liver, pancreas, ovary, and intestine (1). It is the principal regulator of glucose, bile acid, and cholesterol metabolism with varied biological roles extending from regulation of the cell cycle to the maintenance of steroid homeostasis (2, 3). In the pancreas, LRH-1 with pancreas transcription factor stimulates the expression of genes encoding pancreatic digestive enzymes and secretory proteins (4). Moreover, LRH-1 regulates the maturation of ovarian follicles and ovulation in the ovary (5) and is responsible for mitochondrial function by regulating cytochrome p450, family 11, subfamily a, polypeptide 1 and cytochrome p450, family 11, subfamily b, polypeptide 1 in the intestinal epithelium (6). In the liver, LRH-1 is involved in mitochondrial biogenesis and β-oxidation through the regulation of peroxisome proliferator-activated receptor gamma coactivator 1-alpha gene expression (PGC-1α) (7). It is also involved in maintaining the pool of arachidonoyl phospholipids, which are important for normal lipid homeostasis in the liver (8). In addition, LRH-1 liver-specific knockout (Lrh-1LKO) mice show endoplasmic reticulum stress-induced fatty liver, indicating that LRH-1 plays a major role in triglyceride (TG) accumulation in the liver (9).
Excess TGs in the liver are mainly reserved within lipid droplets (LDs) (10). LDs are used to balance lipid storage and utilization and are strongly regulated in a cell type-specific manner (11). As LDs modulate low intracellular free fatty acid levels, they play a crucial role in protecting the liver from lipotoxicity caused by excess fatty acids in nutritional stress conditions (10, 12). In addition, the mobilization of LDs reflects the metabolic state of cells and also indicates changes in the LD-related proteins that contribute to the regulation of lipid metabolism and lipid homeostasis (13).
The perilipin (PLIN) protein family, which attaches to LDs, is a representative group of LD-associated proteins that utilizes the stored lipids of LDs via lipolysis (10, 11). It is composed of five members, named PLIN1–5 and is classified based on stability in a free state. PLIN1 and PLIN2 rapidly degrade in a free state; however, they exist when bound to LDs. The remaining proteins from the family, PLIN3, PLIN4, and PLIN5, are found either free in the cytosol or lining the LDs (14). PLIN1 is found in white and brown adipose tissue (WAT and BAT), whereas PLIN2 and PLIN3 are distributed in many cell types, and PLIN2 is especially observed in hepatocytes. PLIN4 is expressed in cardiomyocytes, adipocytes, and myocytes, and PLIN5 is usually confined within tissues or cells with high oxidative capacity, namely the liver, heart, BAT, and muscle (11, 14, 15).
Among the PLIN family, PLIN5 has emerged as indispensable for adjusting lipid abundance. It is highly active upon fatty acid treatment in cultured cells, with a high-fat diet, and upon prolonged fasting (10). Prolonged fasting is also known to increase TGs accumulation in the liver (16, 17) due to increased adipose tissue lipolysis (18). Therefore, PLIN5 is also regarded as the regulator of TGs metabolism (19). The overexpression of PLIN5 in cells enhances the expression of genes encoding proteins involved in aerobic catabolism and promotes both TGs storage and fatty acid oxidation. Thus, PLIN5 rapidly mobilizes energy by sensing the nutrient demand (20).
The nuclear receptor LRH-1 is activated during nutritional stress and is involved in protecting the liver from lipid overload by increasing the β-oxidation. Even though LRH-1 regulates mitochondrial biogenesis and lipid metabolism, its regulatory mechanism and function under fasting conditions has not been completely addressed. Therefore, in this study, the regulatory function of LRH-1 was investigated during fasting state by utilizing Lrh-1f/f and Lrh-1LKO mice to verify the function of LRH-1 in cellular energy demands and lipid overloading state.
To understand the function of LRH-1 in regulating TGs during fasting, Lrh-1f/f and Lrh-1LKO mice were either fed or starved for 24 h, and the liver (Fig. 1A) and serum were isolated to examine hepatic and serum TGs levels. Initially, serum β-hydroxybutyrate levels was measured in both Lrh-1f/f and Lrh-1LKO mice to confirm the fasting condition. As expected, the fasting condition has increased serum β-hydroxybutyrate levels but did not show significant differences between the genotypes (Supplementary Fig. 2A). The livers from Lrh-1LKO mice starved for 24 h displayed higher accumulation of lipids compared to that in livers of starved Lrh-1f/f mice in Oil red-O staining (Fig. 1B, C), implying the necessity for further analysis of lipid levels in the liver and serum. Interestingly, fasted Lrh-1LKO mice exhibited remarkably escalated hepatic TGs levels compared to those in fasted Lrh-1f/f mice. However, there was no significant difference between the fed mice of either genotype (Fig. 1D). In contrast, hepatic cholesterol was not altered between the genotypes in either fed or starved conditions (Fig. 1E). Surprisingly, Lrh-1LKO mice either fed or starved showed a notable decrease in serum TGs levels compared to those in Lrh-1f/f mice (Fig. 1F). However, serum cholesterol and non-esterified fatty acids (NEFA) levels were not altered significantly (Fig. 1G, H). These findings suggest that the loss of LRH-1 results in a buildup of lipids in the liver, indicating that LRH-1 might be a key regulator in balancing the hepatic lipid content.
To discover new potent target genes of LRH-1 involved in lipid metabolism, the mRNA and protein expression of various genes was examined in liver samples. The expression levels of
In the liver, PLIN2 and PLIN5 are highly expressed (21) therefore, mRNA expression was measured. However,
Additionally, TGs and β-oxidation are strongly interconnected with lipid metabolism. To understand the different liver phenotypes in Lrh-1f/f and Lrh-1LKO mice, the expression of genes involved in fatty acid β-oxidation was measured. Carnitine palmitoyltransferase-1 alpha (
To determine whether PLIN5 is regulated by an LRH-1 agonist, human cultured HepG2 cells were treated with 100 μM DLPC for 24 h and the gene expression and protein levels of PLIN5 were measured. In the presence of DLPC,
To discover potent LRH-1-regulated genes, putative LRE in the
To confirm whether LRH-1 controls
This finding shows that −1620/−1614 in the
To evaluate the regulation of LDs via LRH-1, primary hepatocytes were isolated from Lrh-1f/f and Lrh-1LKO mice to perform BODIPY staining. Primary hepatocytes were grown either in complete (fed) or fasting media to understand the regulatory mechanism of LRH-1 during fasting in the liver. In the fasting media, PLIN5 surrounding the LDs was more abundant in the Lrh-1f/f hepatocytes than in the Lrh-1LKO hepatocytes. Moreover, fasting condition increased LDs number in Lrh-1LKO hepatocytes relative to that in Lrh-1f/f hepatocytes. Nevertheless, the sizes of the LDs in Lrh-1f/f hepatocytes were found to be distinct and increased compared to that in Lrh-1LKO hepatocytes (Fig. 4A, B). Additionally, in fasting condition, the co-localization of PLIN5 in LDs was increased in Lrh-1f/f hepatocytes compared to that in complete media (Fig. 4C).
Next, to verify alterations in the lipid quantity in the liver and serum, the mRNA expression of microsomal triglyceride transfer protein (
Hepatic LRH-1 is a transcriptional regulator of glucose metabolism and bile acid homeostasis (26). This study discovered PLIN5 as a direct target of LRH-1 and explored the function of LRH-1 in the liver during fasting. In this study, fasting increased the accumulation of liver TGs more readily in the Lrh-1LKO livers compared to that in Lrh-1f/f livers. The elevation of hepatic TGs in the liver might be due to a decrease in β-oxidation. As expected, livers of Lrh-1LKO mice either fed or starved showed the markedly diminished expression of key genes responsible for β-oxidation, as well as their enhancer gene, indicating the possible accumulation of lipids in the liver. Recent studies on Lrh-1LKO mice revealed that LRH-1 promotes β-oxidation and mitochondrial biogenesis (7). Incidentally, this result coincides with the previous findings in PLIN5LKO mice, demonstrating elevated hepatic TGs levels and a reduction in fatty acid oxidation in the liver (10). However, these findings were in contrast with the reported research performed by Wang
Interestingly, starved Lrh-1LKO mice exhibited a decrease in serum TGs levels compared to that in Lrh-1f/f mice. Altered liver and serum TGs levels between the genotypes were also observed, which might be due to a decrease in TGs secretion from the liver (28). Therefore, the genes involved in VLDL secretion from the liver were measured. The gene expression of
Transcription factors often bind and sense lipid molecules (29). LRH-1 binds to the −1620/−1614 binding sequence in the
PLIN5 balances fatty acid requirements to meet cellular needs, protecting mitochondria during extreme fatty acid flux with low-energy demands and encouraging fatty acid mobilization and oxidation with high-energy demands (31). Based on BODIPY staining, starved Lrh-1f/f hepatocytes demonstrated the utilization of LDs, whereas lipids were accumulated in the fasted Lrh-1LKO hepatocytes. Furthermore, PLIN5 and BODIPY staining clearly resulted in more intense red and green fluorescence, respectively, implying the co-localization of PLIN5 in LDs during starvation in Lrh-1f/f hepatocytes. This indicates that the loss of LRH-1 decreases PLIN5 co-localization in LDs and increases the lipid content. Surprisingly, in the starved Lrh-1f/f hepatocytes, the size of the LDs was increased and distinct compared to that in starved Lrh-1LKO hepatocytes. However, the quantity of lipids was increased in the fasted Lrh-1LKO hepatocytes. A previous study reported a decrease in the amount and size of LDs in the whole body PLIN5-KO mice (27). Nevertheless, in this study, the number of LDs increased in the Lrh-1LKO mice. PLIN5 regulates both the storage and usage of TGs and is regarded as metabolically protective (32). In fasted Lrh-1f/f hepatocytes, LRH-1 regulates PLIN5 to protect the liver by increasing the influx of TGs within the LDs. As a result, this increases the size of LDs. In addition, it promotes fatty acid oxidation to meet cellular energy demands during starvation, resulting in fewer LDs. In contrast, the lack of LRH-1 in the Lrh-1LKO mice attenuated PLIN5 expression, resulting in small-sized LDs and increasing their numbers (Fig. 4F). This might be the mechanism underlying the changes in the size and quantity of LDs, respectively. Furthermore, the overexpression of PLIN5 decreased the fasting-induced TGs accumulation in the Lrh-1LKO liver, which confirmed the significant role of PLIN5 expression in regulating TGs metabolism. Overall, these observations indicate that LRH-1 regulates PLIN5 to mobilize LDs and balances hepatic lipid contents.
In summary, this study uncovered the function of LRH-1 in the liver during fasting and presents a novel target of LRH-1 in the liver. Results further suggest the necessity of LRH-1 in lipid management to protect the liver from lipid accumulation. In the liver, LRH-1 regulates PLIN5 to mobilize lipids and maintains this balance during fasting conditions. Additionally, LRH-1 regulates PLIN5 to equilibrate the cellular needs and storage of lipids, thus protecting the liver from metabolic diseases associated with a fatty liver. Although LRH-1 is involved in managing the lipid content in the liver, further studies are required to assess the possible targeting of this molecule for the treatment of non-alcoholic steatohepatitis. Thus, this study might be a platform to elucidate the mechanism underlying the treatment of non-alcoholic steatohepatitis and could be beneficial for the pro-tection of the liver.
The detailed methods are described in the “Supplementary Information”.
We acknowledge Dr. Timothy F. Osborne at Johns Hopkins University School of Medicine for kindly providing Lrh-1f/f mice. This study was supported by grants of the Korea Research Foundation, an NRF grant funded by the Korea Government (MSIP) (2019R1A2C2085302, NRF-2021R1A4A1029238) and KMPC (2013M3A9D5072550).
The authors have no conflicting interests.