RORα fine-tunes the circadian control of hepatic triglyceride synthesis and gluconeogenesis

Monnier, Chloé; Ganbold, Munkhzul; Auclair, Martine; Roblot, Natacha; Boutin, Andréas Barnabé; Boutin, Paul Ketil; Fève, Bruno; Antoine, Bénédicte

doi:10.1038/s41598-025-95228-y

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Published: 26 March 2025

RORα fine-tunes the circadian control of hepatic triglyceride synthesis and gluconeogenesis

Chloé Monnier¹,
Munkhzul Ganbold¹,
Martine Auclair¹,
Natacha Roblot¹,
Andréas Barnabé Boutin¹,
Paul Ketil Boutin¹,
Bruno Fève^1,2 &
…
Bénédicte Antoine¹

Scientific Reports volume 15, Article number: 10464 (2025) Cite this article

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Abstract

Circadian rhythms play a fundamental role in hepatic metabolism, orchestrating lipid synthesis and glucose homeostasis. RORα, a nuclear receptor involved in circadian regulation, has been implicated in fine-tuning these metabolic processes. We previously showed a therapeutic potential of antagonizing RORα to reduce body fat in mice. Our current aim is to investigate the impact of the whole-body RORα deletion on hepatic lipid metabolism over a complete circadian cycle. Using RORα-knockout (staggerer) mice, this study reveals a time-dependent disruption in hepatic triglyceride synthesis, with reduced lipogenesis during the light-phase and altered transcriptional regulation of key metabolic genes, including Srebp1c and Insigs. Despite increased Srebp1c transcription at night, the anticipated rise in lipid synthesis was prevented by phase-shifted Insig expression, modulating precursor maturation. Moreover, core clock genes rhythmic expression was attenuated and phase-shifted for Reverbα. Pharmacological inhibition of RORα using an inverse agonist (SR3335) mirrored the metabolic effects observed in staggerer mice, further supporting the role of RORα as a crucial regulator of lipid and glucose homeostasis in mice fed a chow diet. These findings highlight the intricate interaction between the circadian clock and hepatic metabolism, situating RORα as a promising target to prevent metabolic disorders such as obesity and dyslipidemia.

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Introduction

A large body of experimental and clinical observations supports the strong relationship between metabolic processes and circadian clocks¹. The molecular circadian clock machinery is composed of a master clock located in cells of the supra-chiasmatic nucleus (SCN). This master clock paces peripheral clocks throughout the body of mammalian species². This ensures optimal coordination of active and rest phases, feeding and other anticipated and repetitive daily tasks, to peak expression of metabolism genes involved in those activities¹. Some organs—including the liver- are not paced exclusively by the SCN master clock, but also by feeding³.

The molecular circadian clock machinery includes 4 master genes that compose its core loop (Clock, Bmal1, Per2 and Cry1), which is regulated by an additional feedback loop. The nuclear receptors RORs and Reverbs are involved in this feedback loop by binding to RORE sequences⁴ on some target clock genes (Clock, Bmal1, Cry1), RORs being activators⁵ and Reverbs repressors⁶. RORα, RORγ and Reverbα have been implicated in the regulation of glucose and lipid metabolism and in energy homeostasis⁷. The whole-body RORα-KO or RORγ-KO mouse models share similarities (hypoglycemia, hypotriglyceridemia)^8,9, while Reverbα-KO exhibit a trend towards an opposite phenotype¹⁰. Despite some redundancy between RORα and RORγ in target genes¹¹, they also have specific functions. Indeed, the stagerrer (sg/sg) mice, a spontaneous mutant RORα-KO, are characterized by slenderness and improved metabolic phenotype. Exhibiting lower plasma levels of triglycerides (TG), cholesterol, insulin and glucose^8,9,12, sg/sg mice resist to hypercaloric diet-induced dysfunctions, in spite of relatively higher food consumption¹³. They display reduced mass in fat depots,—with down-regulation in inflammatory gene expression^9,14, higher insulin sensitivity and lower fatty acid re-esterification via glyceroneogenesis¹⁵. This lean phenotype has been linked to the beiging of their white adipose depots that increases both thermogenesis and overall energy expenditure^16,17. At the liver level, a lower rate of gluconeogenesis is observed¹⁵, and a decreased TG content associated with a down-regulation of the genes involved in lipid synthesis is reported by most studies^8,9.

Considering the worldwide challenge in the prevention and management of metabolic diseases, the improved metabolic phenotype of the sg/sg mouse hints at the relevance of this model to identify new molecular mechanisms underlying whole-body regulation of energy storage and utilization. There was an interest in designing therapies that could modulate RORα activity, and natural or chemical ligands were identified¹⁸. RORα agonist and inverse-agonists modulated physiological PCK1-dependent pathways in adipose tissue and liver^15,19. First used as in vitro tools on isolated organs, such compounds regulate glyceroneogenesis in adipose tissue and gluconeogenesis in the liver¹⁵. We also showed that a selective RORα inverse-agonist (SR3335) was an inducer of fat browning²⁰, before being administrated in vivo in mice, where it was confirmed to increase adaptive thermogenesis and decrease fat mass¹⁷. This, combined with its hypoglycemic effect¹⁹, made SR3335 an attractive compound to prevent obesity and related metabolic disorders¹⁷. Further emphasis was placed on identifying potential targets, modulated through dampening RORα signaling pathways in other organs, and particularly in the liver.

Indeed, whilst the whole-body RORα-KO model (sg/sg) presented reduced liver TG content compared to WT mice regardless of their food intake^8,9, this was not the case for liver-specific-RORα-KO (L-RORα-KO) mice on chow diet^21,22 or western diet²¹. This suggested that the hepatic lipid metabolism per se did not seem to be a major autonomous target of RORα. It also hinted that multiple organs contribute to the phenotype of sg/sg mice. Of note, the liver-specific depletion of both RORα and RORγ (L-RORαγ-KO) has been shown to display up-regulated expression of lipogenic genes during the night (active period) exclusively, with a trend to increased hepatic TG in the night on chow diet²³. This highlighted autonomous metabolic role of RORs in the liver, dependent on the circadian rhythm. Unfortunately, most of the metabolic studies performed on sg/sg mice did not take into account circadian rhythms, with analysis only performed during the day, which is not adequate for exploring all metabolic pathways. This represents a major issue when investigating the role of RORα, a transcription factor that directly interacts with several major effectors of the core clock. Further work would help distinguish between autonomous and non-autonomous role of RORα in liver metabolism.

The aim of this study was to explore the lipid metabolism of the liver in the whole-body RORα-KO mice (sg/sg) over a full circadian cycle (i.e. throughout a 24 h period). We focused on the metabolic pathways involved in TG synthesis, that have been investigated at the functional level and at different periods of a day, according to the circadian rhythmic expression of related metabolic proteins and transcripts. We also analyzed the impact of an absence of RORα on its core clock partners expression pattern. Finally, we tested, in WT mice, the in vivo effect of a RORα inverse-agonist (SR3335) on hepatic TG synthesis, and on gluconeogenesis, in order to mimic the metabolic daytime-dependent phenotype of the sg/sg mouse with a pharmacological approach.

Results and discussion

Metabolic blood parameters and hepatic TG content are improved throughout the day in the RORα-KO (sg/sg) mice

We examined the role of RORα in lipid and glucose homeostasis throughout the day by collecting samples every 4 h over a period of 24 h -mice exposed to light for 12 h, from ZT0 to ZT12, and in darkness for 12 h, from ZT12 to ZT24 (Fig. 1). The circadian fluctuations of these parameters in WT mice corroborated those previously described²⁴. In sg/sg mice, only experiments performed in the light period have been published and reported lower levels of hepatic TG content, plasma TG and glucose concentrations when compared to WT mice^8,9,12. Our data confirmed such pattern during the light period and illustrated, for the first time, that the same profile persisted during the night period. Overall, the circadian variation in lipid homeostasis seemed to be preserved in the sg/sg mice, but with lower amplitude than in WT mice. Since L-RORαγ-KO revealed time-dependent impact of RORs on lipogenic gene expression and TG content²³, we focused our analysis on pathways involved in hepatic TG synthesis over a full circadian cycle.

Triglyceride synthesis was altered in the liver of sg/sg mice, in a time-dependent way

Hepatic TG synthesis includes free fatty acid (FFA) esterification into TG, and FFA synthesis from hexoses (de novo lipogenesis). The first process requires the synthesis of glycerol-3-phosphate (G3P) from various substrates according their availability: pyruvate and glycerol during the fasting period, and glucose when insulinemia increases compared to cAMP. Lipogenesis includes glycolysis as a first step, and de novo lipogenesis producing FFA from acetyl-CoA. The lipogenic pathway was studied at a functional level (Fig. 2a), and compared with the expression of related proteins (Fig. 2b) and transcripts (Fig. 2c).

Glyceroneogenesis (GNG) is a truncated pathway of gluconeogenesis which provides G3P from lactate and pyruvate, and phosphoenolpyruvate carboxykinase (PEPCKc) is its rate-limiting enzyme. We previously showed that GNG was functionally reduced in adipocytes of sg/sg mice, due to the combined decrease of the PCK1 gene expression, PEPCKc protein levels and its specific activity¹⁵. Given that GNG also significantly takes place in the liver²⁵, we functionally assessed hepatic GNG by measuring the incorporation of ¹⁴C₁ pyruvate into TG in freshly prepared liver samples. GNG was decreased in the sg/sg compared to WT mice at ZT10, i.e. in the light and fasting period, but not at ZT0, i.e.at the end of the night and fed period (Fig. 2a, left panel). Quantification of the relative amount of PEPCKc confirmed a rather constant decrease in the protein expression all along the 24 h period, with a conserved circadian rhythm (Fig. 2b, left panel); the PCK1 transcript cycled accordingly, with lower amplitude mostly in the light period (Fig. 2c, left panel). This data confirms that RORα is a physiological trans-activator of PCK1¹⁹, required for maximal gene expression, but not for its cycling in the liver.

Glycerol, derived from TG lipolysis in adipocytes, is another substrate used for synthesis after its phosphorylation by glycerol kinase (GyK). Analysis of U-¹⁴C glycerol incorporation into TG showed a significant increase at ZT0, that was no more significant at ZT10, in the liver of sg/sg compared to WT mice (Fig. 2a, left panel). Accordingly, the amount of GyK proteins level was slightly enhanced in the liver of the sg/sg mice in the light period (Fig. 2b, left panel). By contrast, the GyK transcript abundance appeared equal or lower in sg/sg compared to WT mice (Fig. 2c, left panel), suggesting some stabilization of the protein in the liver of the sg/sg mice. Finally, glycerol was used about 3-times less than pyruvate for supplying G3P at ZT10 (Fig. 2a); this confirms GNG as the major pathway for G3P synthesis in the rest period of mice²⁵.

Glucose is a shared substrate for both G3P and FFA synthesis through glycolysis. We compared the incorporation of U-¹⁴C glucose into TG in the liver of both sg/sg and WT genotypes at three time points over a 24 h period (Fig. 2a, mid panel). We detected a sharp reduction of glucose incorporation into TG at ZT4 in the liver of sg/sg compared to WT mice, still noticeable at ZT12 although not significant. Western blot analysis of the glycolysis limiting enzyme, L-Pyruvate kinase (L-PK), confirmed a marked decrease of the protein amount in sg/sg vs WT mice, in particular during the light period (Fig. 2b, mid panel). The circadian rhythm of the PKLR transcript seemed to be preserved in the absence of RORα with a peak at ZT14, similar to that of WT mice²⁶, although with a dampened amplitude (Fig. 2c, mid panel).

Finally, to by-pass the glycolytic step and investigate the impact of RORα on de novo lipogenenesis only, we tested the incorporation of U-¹⁴C acetate as a precursor of FFA synthesis in the light and the dark periods. In sg/sg mice, we observed a sharp decrease of acetate incorporation into TG at ZT6 and no difference between the genotypes at ZT18 (Fig. 2a, right panel). In WT mice, de novo lipogenesis was found to be higher at ZT6 than at ZT18; this corroborates data showing that the peak of TG accumulation in the liver occurs around ZT8²⁷. Western blot analysis of two major actors of lipogenesis—Acetyl-coenzyme A-carboxylase (ACC) and fatty acid-synthase (FAS)—revealed mild rhythmic variation of these proteins throughout the day, with the highest amount occurring at ZT22 in WT mice (Fig. 2b, right panel). The pattern in sg/sg mice was rather similar to that of the WT counterparts except at ZT6, where ACC and FAS levels were reduced. This lower level of ACC and FAS protein content in sg/sg mice did correspond to the nadir of their transcript at ZT6 (Fig. 2c, right panel and⁸.

Our functional approach demonstrated for the first time that the whole body RORα-KO leads to an altered hepatic TG synthesis that is restricted to the light period and impacts several pathways: glycolysis and de novo lipogenesis for the synthesis of FFA, and GNG for their subsequent esterification into TG. Such data corroborated some reported decrease of PCK1, PKLR and FAS transcripts in the liver of sg/sg mice¹⁵, presumably performed at light^8,11. Given that RORα and/or RORγ are recruited to ROREs in several key genes involved in glucose homeostasis and lipid metabolism, including PCK1, PKLR and FAS¹¹, one possibility to explain their decreased transcription in the liver of sg/sg mice in the light period is as a direct consequence of the local loss of RORα transcriptional activity. RORα would then be an effector of the cycling of these pathways. However, FAS and ACC transcripts did not vary in two studies of L-RORα-KO mice fed a chow diet^21,22—with one showing a trend to decrease, and no mention made of the timing of the analysis in the other²². This does not point to a liver-autonomous mechanism.

Given RORα belongs to the core clock machinery, his absence had potential consequences on the circadian rhythm and/or expression of its clock partners. This was important to delineate as clock genes can themselves control some metabolic genes²⁴.

RORα is necessary for robust circadian rhythm of core clock gene expression and its loss of function led to rhythmic disruption in Reverbα protein amount

Amongst the core clock genes family, Bmal1, Clock and Cry1 are direct target genes of RORs, but not Per2²⁸. Accordingly, our data depicted their dampened transcript level in sg/sg mice, with no alteration in their rhythmicity (excepted a 4 h-delayed peak of Bmal1 transcript) while Per2 expression was not affected (Fig. 3a). Consequently, Bmal1 and Clock protein amounts were decreased at ZT6 (Fig. 3b), as expected for true targets of RORα. We also assessed the ROR family, both at the transcript (Fig. 3a, right panel) and the protein (Fig. 3b) levels over a 24 h period. The complete lack of the RORα protein in sg/sg mice was confirmed by western blot (Fig. 3b). Our data further revealed that, in WT mice, RORα protein content exhibited a marked rhythmic expression with a peak at ZT22 (Fig. 3b), though it remained arrhythmic at the transcript level (Fig. 3a) and⁵. The RORγ transcript showed a robust oscillatory pattern with a peak during the night in the two genotypes, with a weakly extended peak (significant increase at ZT22 to ZT2) in sg/sg compared to WT mice (Fig. 3a), as previously suggested in various RORα-KO models^9,21,23. Accordingly, the RORγ protein amount was increased (by 1.3-fold) in sg/sg mice with higher levels at night (Fig. 3b). The Reverbα transcript and protein exhibited marked cyclic expression in WT mice, peaking at ZT6 (Fig. 3a) and ZT10 (Fig. 3b), respectively⁶. By contrast, the RORα deletion led to a 4 h-shift of the transcription of Reverbα (Fig. 3a), followed by a 4 h-delay and dampening of its protein expression profile during the night (Fig. 3b). A mild decrease of Reverbα transcript level at ZT4 in sg/sg mice²⁹ and in L-RORα-KO²¹ were already noted, but its circadian protein pattern in the liver of RORα-KO mice was never published, although Reverbα is a well-known RORα-target³⁰.

Finally, the circadian pattern of the ROR family members was compared within each genotype (Fig. 3c). In WT mice, RORα and RORγ protein peaked in phase and were anti-phasic with their trans-dominant negative partner, Reverbα. These protein profiles perfectly fit their DNA binding activity—between ZT22 and ZT6 for RORs and at ZT10 for Reverbα³¹. That depicts how some RORE-target genes are cycled over the 24 h period: activated by RORs in the light period then repressed by Reverbα in the night. By contrast, in sg/sg mice, the Reverbα protein profile was abnormally in phase with RORγ (Fig. 3c, right panel), thus potentially competing for shared RORE-binding sites on target genes³² and neutralizing their action in the night. Such a scenario could contribute to the failure of RORγ to replace RORα in sg/sg mice, at least with regards to common target genes.

In summary, our data confirm a cyclic profile of the RORα protein, as previously documented³³, reinforcing the notion that RORα is a meaningful actor in the hepatic circadian control of gene expression. Indeed, its deletion in sg/sg mice lead to a 4 h-delay in peaks of Bmal1 and Reverbα transcripts and a modest RORγ over-expression. This suggests that RORα is more efficient than RORγ in promoting the transcription of these two target genes^29,30. The modified protein pattern of Bmal1 could alter those of PKLR and Reverbα, which are direct targets of Bmal1^6,26. Subsequently, the loss in the counter-regulation exerted by Reverbα in a suitable timeframe could up-regulate RORγ³⁴ and possibly have also some effect on lipogenesis, since Reverbα controls the diurnal rhythm of de novo lipogenesis in hepatocytes³⁵.

Disturbances in rhythmic expression of transcription factors involved in lipogenesis in sg/sg mice.

Given the increased lipogenesis observed at night in L-RORαγ-KO mice²³, we focused on the molecular pathways that regulate lipogenesis according the time of the day in sg/sg mice. We first checked the circadian expression profile of three major transcription factors governing lipogenesis (Fig. 4a). Liver-X receptor (LXRα) and carbohydrate response element-binding protein (Chrebpα) are both critical regulators of sterol regulatory element-binding transcription factor (Srebp1c), that further mediates the insulin response to lipogenic gene transcription.

In accordance with the decreased lipogenic capacity observed in sg/sg mice during the light period (Fig. 2a), Srebp1c and LXRα gene expression decreased at this period in sg/sg mice (Fig. 4a). Given RORα binds the Srebp1c promoter⁸, the reduced expression of Srebp1c transcript at this period could be a direct consequence to the lack of RORα in sg/sg mice. Accordingly, a direct Srebp1c target gene, SCD1, was decreased in one L-RORα-KO model²², suggesting a cell-autonomous process of regulation.

By contrast, at night, despite similar lipogenic capacities between the two genotypes (Fig. 2a), an early peak of Chrebpα mRNA was noted in sg/sg mice, possibly linked to their relatively early daily food intake¹³, as well as some transient over-expression of Srebp1c and LXRα transcripts at ZT14 (Fig. 4a). Physiologically, LXRα gene expression is not cycling²⁷ but activated by metabolic ligands³⁶. The LXRα increased transcription in sg/sg mice is explicable since RORα is able to interrupt the autoregulatory loop of LXRα³⁷. The fact that this increase was never seen before is probably due to the timing of previous analyses^8,38. The increase of Srebp1c transcript at ZT14 in sg/sg mice could therefore result from LXRα increase, since the promoter for Srebp1c contains LXR-binding sites³⁹. Srebp1c transcription is also driven by clock genes⁴⁰. In particular, Reverbα binds Srebp1c promoter under physiological conditions⁴¹. Thus, the unusual lack of Reverbα at the end of the light period in sg/sg mice (Fig. 3b) could result in a loss of Srebp1c inhibition at that time.

In summary, our data confirms that RORα and Reverbα take part in the rhythmic control of Srebp1c gene expression, as activator (during the day) and inhibitor (at night), respectively, according their alternative circadian expression. Moreover, RORα seems necessary to interrupt the autoregulatory loop of LXRα, and its unusual transactivation at night in sg/sg mice could further increase Srebp1c transcription.

To investigate the discrepancy between the levels of Srebp1c transcript and those of its target genes at night in sg/sg mice, we assessed the levels of both its precursor (pSrebp1c) and its nuclear-cleaved form (mSrebp1c) by western blot over the 24 h period. Indeed, Srebp1c is synthesized as an inactive precursor and remains in the endoplasmic reticulum by the intervention of “Insulin-induced genes” (Insigs) that exert a negative feedback control on lipogenesis. The complex of pSrebp1c-Insig prevents the migration of pSrebp1c to the Golgi apparatus, where the necessary proteases for its proteolytic activation into mSrebp1c are located. mSrebp1c is then able to translocate to the nucleus (Fig. 4f). According to the pattern of Srebp1c transcript (Fig. 4a), pSrebp1c accumulated more in the liver of sg/sg than in WT mice during the night period (Fig. 4b). However, although mSrebp1c expectedly rose at ZT18 in WT mice, its amount stayed rather stable in sg/sg mice from ZT6 to ZT22, excepted at ZT2 where it was decreased compared to WT mice. The illustration of their respective amounts in each genotype (Fig. 4c) hinted at an alteration of the processing of pSrebp1c into its mature form in sg/sg mice during the night.

There are two Insig isoforms that are rhythmically expressed in the liver, but they are distinctive by their regulation and half-lives⁴². In sg/sg mice, the peaks of Insig1 and Insig2 proteins were early (by 8 h and 4 h, respectively) compared to WT mice (Fig. 4d). Therefore, the increased amount of both Insigs at night in sg/sg mice possibly contributes to prevent the maturation of pSREBP1c into mSREBP1c at that time, as a counter-regulatory process on de novo lipogenesis. Their altered expression in sg/sg mice would be, at least partly, due to transcriptional events. Indeed, sg/sg mice exhibited both an early increase of Insig1 transcript, as well as a relative increase of Insig2b transcript at ZT14 (Fig. 4e).

Insig1 is closely associated with lipogenic genes⁴³; its transcription requires mSREBP1c⁴⁴ and, accordingly, was delayed by about 8 h, compared to that of mSREBP1c in both genotypes. Insig2 could be translated from two transcripts, Insig2a being liver-specific and Insig2b ubiquitous⁴². Our data showed that the increase of the Insig2 protein at night in sg/sg mice results from the increase of Insig2b transcript (Fig. 4e). The observation of some over-expression of Insig2b in L-RORαγ-KO mice²³ suggests a liver-autonomous mechanism due the lack of RORα. Insig2a is a specific RORγ target³⁸ and inhibited by Reverbα⁴⁵. Unexpectedly, Insig2a transcript was decreased at ZT2 in sg/sg compared to WT mice (Fig. 4e). This could reflect the competition between RORγ and Reverbα that were found to be abnormally co-expressed at ZT22 in sg/sg mice (Fig. 3c), leading to the decrease of Insig2a transcript at ZT2 (Fig. 4e), and then of its protein at ZT2 to 6 (Fig. 4d).

In summary, disruptions in the rhythmic expression of transcription factors involved in hepatic lipogenesis were noted in the whole-body RORα-KO mice. LXRα and Srebp1c transcription profiles were more cyclic in sg/sg than in WT mice (decreased in the light period, and increased at the beginning of the night period). However, the subsequent pSREBP1c over-expression at night did not drive any increased lipogenesis in sg/sg mice because Insig1 and 2 proteins increased simultaneously, prohibiting its processing into mSREBP1c (see Fig. 4f for a schematic representation). The increase of both Srebp1c and Insig2b gene transcription was also observed in L-RORαγ-KO mice²³, suggesting a liver autonomous mechanism of regulation by RORα.

Pharmacological antagonism of RORα in WT mice mimics the time-dependent metabolic variations observed in the liver of sg/sg mice

Given the whole-body RORα-deletion decreased hepatic TG synthesis in the day period, we decided to pharmacologically modulate RORα in WT mice, expecting to mimic the benefits observed in the lipid metabolism of sg/sg mice during the day period. We already used the selective RORα inverse agonist SR3335 to increase adaptive thermogenesis and decrease fat mass in C57Bl/6 J mice in vivo¹⁷. We started by assessing relevant parameters in these C57Bl/6 J mice treated with four consecutive intra-peritoneal doses of SR3335. The RORα inverse agonist decreased their TG content in the liver (Fig. 5a) and plasma (Fig. 5b), and increased plasma glycerol concentration (Fig. 5b), suggesting an activation of adipose lipolysis.

To explain the causes of the loss of liver TG content in response to SR3335, hepatic lipogenesis was functionally tested through the analysis of U-¹⁴C glucose incorporation into TG in freshly prepared liver slices from SR3335- or DMSO-treated sg/sg and WT mice around ZT4-6. Figure 5c shows that one single SR3335 injection at ZT0 (around the daily peak of RORα protein amount, see Fig. 3b) was sufficient to cause a decrease, 28–30 h later, in the hepatic lipogenesis of WT mice. This took place to a level comparable to that observed in sg/sg mice. This downregulation did not occur in the SR3335-treated sg/sg mice and thus was RORα-specific. The liver samples from these mice were analyzed by western blot to further delineate the proteins influenced by SR3335. As anticipated, SR3335 led to a reduction in the level of mSREBP1c by half in WT and not in sg/sg mice around ZT4-6 (Fig. 5d) A similar reduction in FAS and L-PK in WT mice in response to SR3335 was observed, with no significant change in sg/sg mice. We also assessed the impact of SR3335 on hepatic TG content and lipogenic actors at ZT22 (Supplementary Fig. 1). As expected, SR3335 did not alter the lipogenic protein amount (mSrebp1c, FAS and L-PK), nor the liver TG content in WT mice during the night.

Gluconeogenesis was previously shown to be decreased in male sg/sg compared to WT mice, with a lower rise in glycemia in response to a pyruvate injection (pyruvate tolerance test, PTT)¹⁵. Here, we obtained similar results with female sg/sg mice, in which glycemia was comparatively lower than in WT mice from T0 (overnight fast) until one hour after the pyruvate injection (Fig. 6a). We further examined the effect of SR3335 in vivo on PTT results. Figure 6a shows that one single SR3335 injection was able to decrease both the glycemia of WT mice after an overnight fast (T0), and their capacity to de novo synthesize glucose from pyruvate for at least 30 min. As expected, SR3335 injection had no effect on PTT results in sg/sg mice. Furthermore, we investigated the capacity of SR3335-treated mice to produce glucose from liver explants ex vivo. The hepatic glucose production was significantly decreased in WT mice in response to SR3335, but not in sg/sg mice (Fig. 6b). At the same time, the PEPCKc protein amount was decreased in the liver of SR3335-treated WT mice, while SR3335 had no effect on this protein in sg/sg animals (Fig. 6c).

Given antagonizing RORα lead to a decrease in both gluconeogenesis and lipogenesis in the day period, we posited that activating RORα should exert an opposite effect. We tested a ROR-agonist, SR1078, on these pathways (Supplementary Fig. 2). A single SR1078 injection increased glucose neo-production (by 1.3-fold) and glucose incorporation into TG (by 1.5-fold) in liver explants from WT mice at ZT10, without any effect on sg/sg mice. Alongside this, their liver samples exhibited a two-fold increase of FAS and L-PK protein expression in WT but not in sg/sg mice.

Taken together, this data illustrates how the RORα-inverse agonist SR3335 can mimic in vivo the impact of the full RORα deletion both on lipid and glucose hepatic metabolic pathways. This translates to reduced rates of lipogenesis and gluconeogenesis in the day period of SR3335-treated WT mice, without any variation at night. The noted opposite effect obtained with a ROR-agonist further confirms how RORα acts as an important physiological cyclic modulator of liver function.

Conclusion

Our functional approach demonstrated how the whole-body RORα-KO decreases liver TG synthesis (through decreased glyceroneogenesis, glycolysis and de novo lipogenesis) only during the light period, and without an increase in the dark period of mice fed ad libitum. Moreover, the observation that this time-dependent scenario was pharmacologically reproducible by the RORα-inverse agonist SR3335 in WT mice and not in sg/sg mice, supported a RORα-related mechanism. Together with the opposite observations made with a ROR-agonist, this points towards RORα contributing in a cell-autonomous manner to the circadian control of Srebp1c, FAS, ACC, PK-L and PEPCKc gene transcription, especially as these genes were identified as bound to, or controlled by RORs^8,11. However, the transcription level of some of these genes, having been analyzed in L-RORα-KO models (Srebp1c, FAS, ACC), was not changed or only mildly diminished in the light period^21,22. One could point to inappropriate timing of assessments, but these L-RORα-KO mice do not exhibit lower TG content^21,22, or improved PTT²¹. They do not possess the same liver phenotype as whole-body RORα-KO (sg/sg) mice. Our impression is that these metabolic genes are true molecular targets of the couple RORα/Reverbα in the liver, but their cyclic control by clock genes could be meaningful only in specific situations, such as a low FFA availability and/or a misfeeding (increased daily food intake) which are only observed in the whole-body RORα-KO mice. Indeed, sg/sg mice exhibit increased FFA oxidation in fat and muscle^12,16,20, thereby modifying the strong temporal correlation of FFA across tissues⁴⁶. Furthermore, the inhibitory control of Reverbα against certain fluctuations of lipogenesis^35,45 only became evident in one study, in a context of mistimed feeding during the day time (food intake from ZT4 to 8)⁴⁷. Collectively, this suggests that RORα fines-tune lipogenesis, with an up-regulation in the day, and a down-regulation at night.

Mechanistically, we demonstrated a strong circadian cycling of the RORα protein in the liver. Its deletion induced a phase-shifted expression of its targets Bmal1 and Reverbα, as suggested in RORαγ-KO mice models – both in whole body²⁹ and liver-specific^21,23 fashion. The phase-shifting of Reverbα in particular, would further deregulate the Srebp1c/Insigs pathway, as observed in Reverbα-KO mice⁴⁵. However, in sg/sg mice, the increased Srebp1c transcription at night was counteracted because of the early overexpression of both Insigs (see Supplementary Fig. 3). Finally, our data confirmed a role of RORα in the inhibition of LXRα transcription³⁷, which could further control Srebp1c expression and lipogenesis at night. We hypothesized that the increased TG liver content observed at night in L-RORαγ-KO was related to the combined consequence of the lack of RORα (which caused increased Srebp1c transcription) and the lack of RORγ (which decreased Insig2a transcription), thereby leading to its maturation into mSrebp1c (see Supplementary Fig. 3). The LXRα increase at night in RORα-KO mice possibly explains why the L-RORα-KO mice develop steatosis after high fat diet²². This is not the case in global RORα-KO⁸, because of simultaneous increased FFA oxidation in others organs^12,16,20. Taken together, this illustrates how it might be more metabolically beneficial to reduce RORα expression throughout the whole body rather than just in the liver. This supports the rational use of a pharmacological RORα-blockade to prevent pathologies affecting metabolic pathways. This can be achieved by natural RORs inverse-agonists, such as vitamin D hydroxyderivatives^48,49. However, this mediation could be restricted to subjects benefiting from a rather healthy diet and without proven steatosis. Indeed, in a model of obesity or western-diet feeding background, it seemed most relevant to focus on RORα activation (and not inhibition). Firstly, liver-specific RORα-deficient mice develop hepatic steatosis only when challenged by a high fat diet²². Secondly, nobiletin, a natural flavonoid acting as a ROR-agonist, improves liver steatosis in obese mice but without any effect on mice fed a standard chow diet⁵⁰. In such pathologic circumstances, some of the benefits mediated by an activation of RORα-mediated pathways could result from its anti-inflammatory properties⁵¹. We, themselves, have previously described an anti-inflammatory role for RORα in response to a Western Diet feeding, but not in chow diet-fed mice¹⁴.

Methods

Animals and tissue collection

The staggerer (RORα sg/sg) mutant mouse was maintained on a C57BL/6 J genetic background. Heterozygous sg/ + have been kindly provided by Prof. J. Mariani (Institut Gustave Roussy, Villejuif, France) and were referred to as B6.C3 (Cg-RORα ^sg/J) in the Jackson laboratory (Bar Harbor, ME). Generation of sg/sg mice were obtained from crossing heterozygous sg/ + mice and compared with their + / + littermates. All animals care and use procedures were conducted according to the European Communities Council Directive (2010/63/UE) and approved by the Regional Animal Care and Use Committee (Ile-de-France, Paris, no5; agreement number APAFIS#26,094–2,020,061,808,514,224 v1). We confirmed that the study was carried out in compliance with the ARRIVE guidelines, all methods being in accordance with relevant guidelines and regulations. Mice were housed in pathogen-free, temperature-controlled environment, scheduled with 12–12 h light–dark (LD) cycles (lights on from 8:00AM/ZT0) to 8:00PM/ZT12) and maintained with free access to food (A03, UAR, Epinay-sur-Orge, France) and water. Sg/sg mice were kept with their mother throughout their life and fed softened and moistened food directly placed on the bottom of the cage, as soon as one week before weaning, in order to improving their survival to similar of that of sg/ + and + / + mice. Male mice (4–6 months-old) were sacrificed by cervical dislocation at 4 h intervals over a 24 h period with four to six mice for each time point; when older and/or female mice have been used, it was specified in the figure legend. Livers were pulverized under liquid nitrogen (to exclude differences linked to the zonation of hepatocytes) and stored at − 80 °C before RNA extraction or protein lysates preparation. Blood samples were collected via cardiac puncture, under EDTA, centrifuged and plasma samples were aliquoted and snap-frozen. For in vivo treatment, mice were intra-peritoneally (IP) injected with SR3335 or SR1078 (10 mg/kg) or with the excipient [10% dimethyl sulfoxide (DMSO) and 10% Tween 80 solution].

Pyruvate tolerance test

This was performed by intra-peritoneally injecting 16 h-fasted mice with a 2 g/kg buffered pyruvate solution. Blood glucose levels were then checked every 15 min from the tail vein by using a glucometer (OneTouch, LifeScan, Issy-les-moulineaux, France).

Transcript and protein analysis

Hepatic mRNAs were extracted using the RNeasy Tissue Mini Kit (Qiagen, Courtaboeuf, France), then reverse-transcribed using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems Carlsbad, CA, USA). Quantitative PCR of the genes of interest was performed using SYBR Green and a Light Cycler 480 Real-Time PCR System (Roche Diagnostics, Meylan, France) with specific primers listed in Table 1. Gene expression was normalized to the mean of three housekeeping mRNAs (GAPDH, 36B4 and HPRT) and data analysis was based on the DDCt method.

Table 1 Primer sequences and antibodies used in the present work.

Full size table

Proteins from mouse liver were isolated on ice using a Tissue Protein Lysis Buffer (Invitrogen, Villebon-sur-Yvette, France) then centrifuged at 12,000 × g, 4 °C for 10 min to remove insoluble material. Protein concentration was measured with BCA reagent (Thermoscientific, Waltham, MA USA). Immuno-blot analysis was performed on gradient SDS-PAGE. The antibodies used are listed in Table 1. Protein detection and semi-quantitative analysis of western blot compared to actin were performed using iBright CL1500 imaging system (Invitrogen, Villebon-sur-Yvette, France) and the software Image J (NIH, USA), respectively. All compared samples were run on the same gel. Original blots were included in supplementary data.

Blood and tissue biochemical analysis

Frozen liver tissue (40 mg) was homogenized in 800 μl of HPLC-grade acetone. After incubation with agitation at room temperature overnight, aliquots of acetone-extract lipid suspension were used for the determination of hepatic TG content by using the kit TR0100 (Sigma-Aldrich, St louis MO, USA). Glycerol and glucose concentration were analyzed with the Free glycerol reagent F-6428 and the assay kit GAGO-20, respectively, from Sigma Aldrich. Non esterified FFA were measured with Free fatty acids, half micro test (Roche Diagnostic, Meylan, France).

Functional incorporation of radio-labelled substrate into TG

Liver was minced systematically into very small pieces and samples (200 mg) were pre-incubated in cell culture inserts (Thermoscientific, Waltham, MA USA) in multi-well plates containing glucose-free DMEM with 0.5% BSA for 2 h, then in Krebs–Henseleit bicarbonate buffer (KRBH) containing 0.5% fatty acid-free BSA, ¹⁴C₁ pyruvate (2 μCi/ml), 0.5 mM pyruvate and 1 μM (-) isoproterenol. After 2 h at 37 °C under constant agitation, the explants were rinsed in PBS, precisely weighted and frozen in liquid nitrogen before undergoing lipid extraction by the simplified method of Bligh and Dyer⁵². The incorporation of ¹⁴C₁ pyruvate into the lipid moiety was estimated by counting the radioactivity associated with this fraction. ¹⁴C₁ pyruvate was chosen because, in contrast to C₂ or C₃-labeled molecules, only the C₁ carbon of pyruvate is conserved in the G3P moiety of newly synthesized TG, and thus, is a marker for GNG. When the glycerol incorporation into TG was tested, U-¹⁴C _-glycerol (2 μCi/ml) and 0.25 mM glycerol were added to the KRBH, and the experiment was performed without isoproterenol. Glucose incorporation was analyzed using U-¹⁴C _-glucose (2 μCi/ml), 1 mM glucose and 100 nM insulin. When acetate was concerned, we used U-¹⁴C _-acetate (2 μCi/ml) and 3 mM acetate.

Glucose neo-production by liver slices

Liver slices were prepared from 4 h-fasted mice as described above, then incubated in KRBH containing 0.5% BSA, 1 mM lactate and 0.1 mM pyruvate for 2 h at 37 °C with constant gentle agitation. The incubation medium was then assessed for glucose concentration.

RORα chemical ligands

SR3338 and SR1078 were respectively purchased from Cayman (Interchim, Montigny le Bretonneux, France) and from Calbiochem (Merck Chemicals, Nottingham, UK).

Statistical analysis

Values are presented as means + /− SD. Statistical analysis was performed by one-way or two-way ANOVA or by unpaired Student ‘s t test as mentioned in the figures caption. p < 0.05 was considered the limit for statistical significance.

Data availability

The authors declare that all data supporting the findings of this study are available in the article or from the corresponding author upon reasonable request.

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Acknowledgements

We greatly thank Tatiana Ledent and Laetitia Dinard for their help in the sg/sg mice-housing facility. B.A. is a CNRS researcher.

Funding

Our funding is from CNRS, Inserm, Sorbonne University and “la Fondation pour la Recherche Médicale” (EQU201903007868).

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Authors and Affiliations

UMR_S938, Centre de Recherche Saint-Antoine, IHU ICAN, Faculté de Médecine Site Saint-Antoine, Sorbonne Université-Inserm, 27 Rue Chaligny, 75012, Paris, France
Chloé Monnier, Munkhzul Ganbold, Martine Auclair, Natacha Roblot, Andréas Barnabé Boutin, Paul Ketil Boutin, Bruno Fève & Bénédicte Antoine
APHP, Department of Endocrinology and CRMR PRISIS, Saint-Antoine Hospital, Paris, France
Bruno Fève

Authors

Chloé Monnier
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Munkhzul Ganbold
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Martine Auclair
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Natacha Roblot
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Andréas Barnabé Boutin
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Paul Ketil Boutin
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Bruno Fève
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Bénédicte Antoine
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Contributions

Conceptualization: B.A.; Methodology: C.M., M.G., M.A., N.R., A.B.B., P.K.B. and B.A.; Validation: C.M., M.G., M.A., N.R., A.B.B., P.K.B., B.F. and B.A.; Formal analysis: C.M., M.G., B.F. and B.A.; Resources: B.F. and B.A.; Writing-original draft preparation: C.M., B.F. and B.A.; Visualization: C.M., M.G., M.A., N.R., A.B.B., P.K.B., B.F. and B.A; Supervision: B.F.; Project administration: B.A.

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Correspondence to Bénédicte Antoine.

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Monnier, C., Ganbold, M., Auclair, M. et al. RORα fine-tunes the circadian control of hepatic triglyceride synthesis and gluconeogenesis. Sci Rep 15, 10464 (2025). https://doi.org/10.1038/s41598-025-95228-y

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Received: 12 August 2024
Accepted: 18 March 2025
Published: 26 March 2025
DOI: https://doi.org/10.1038/s41598-025-95228-y