LXRB-SREBP1 axis regulating lipogenic homeostasis by controlling the synthesis of polyunsaturated fatty acids in goat mammary epithelial cells

doi:10.21203/rs.3.rs-1605048/v1

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LXRB-SREBP1 axis regulating lipogenic homeostasis by controlling the synthesis of polyunsaturated fatty acids in goat mammary epithelial cells

https://doi.org/10.21203/rs.3.rs-1605048/v1

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Background: Liver X receptor beta (LXRB) is the predominant LXR subtype in ruminant mammary cells. Whether and how LXRB plays a role in the synthesis of polyunsaturated fatty acids (PUFA) in the mammary gland remains to be determined. In the current study, we used overexpression and knockdown of LXRB in goat primary mammary epithelial cells (GMEC) to evaluate abundance of lipogenic enzymes, fatty acid profiles, content of lipid stores and activity of the stearoyl-CoA desaturase (SCD1) promoter.

Results: Overexpression of LXRB markedly upregulated the protein abundance of LXRB while incubation with siRNA targeting LXRB markedly decreased abundance of LXRB protein. Overexpression of LXRB plus ligand T0901317 (T09) dramatically upregulated genes encoding proteins related to PUFA synthesis. Compared with the control, cells overexpressing LXRB plus T09 had greater concentrations of C16:0, 16:1, 18:1n7,18:1n9 and C18:2 as well as desaturation and elongation indices of C16:0. Furthermore, LXRB overexpressing cells incubated with T09 had greater levels of triacylglycerol and cholesterol. Knockdown of LXRB in cells incubated with T09 led to downregulation of genes encoding elongases and desaturases. In contrast, knockdown of LXRB attenuated the increase in TAG and cholesterol that was induced by T09. In cells treated with DMSO, knockdown of LXRB increased the concentration of C16:0 at the expense of C18:0, while a significant decrease in C18:2 was observed in cells incubated with both siLXRB and T09. The abundance of sterol regulatory element binding transcription factor 1 (pSREBP1) and its mature fragment (nSREBP1) was upregulated by T09, but not LXRB overexpression. In the cells cultured with T09, knockdown of LXRB downregulated the abundance for pSREBP and nSREBP1. Luciferase reporter assays with site directed mutagenesis of SREBP1 response elements (SRE) revealed that that the SCD1 promoter with the wild type or SRE mutation decreased markedly when LXRB was knocked down. Activity of the SCD1 promoter that was induced by T09 was impaired when the SRE mutation was introduced.

Conclusion: There is an important role of LXRB-SREBP1 axis in the synthesis of PUFA via the regulation of genes encoding elongases and desaturases. Thus, this network elicits broad effects on lipid homeostasis in ruminant mammary gland.

liver X receptor

PUFA synthesis

elongase

lipid homeostasis

Lipids are one of the most-valuable components in dairy products, especially the polyunsaturated fatty acids (PUFA) [1, 2]. Although ruminant products contain low amounts of PUFA relative to non-ruminants, the fact that endogenous PUFA impact multiple pathways regulating hepatic lipid metabolism illustrates their potential role in the cellular hemostasis of important organs of the animal [3, 4].

In non-ruminants, the activity of fatty acid desaturases and elongases is the rate-limiting step for the synthesis of PUFA [5]. The functional roles of fatty acid desaturases and elongases, e.g. stearoyl-CoA desaturase 1 (SCD1), fatty acid desaturase (FADS) and elongation of very long chain fatty acid-like fatty acid elongases (ELOVL) in ruminant mammary gland have been investigated [6–9]. It is evident that sterol regulatory element binding transcription factor 1 and liver X receptor (LXR) play a crucial role in regulating the expression of SCD1 [10, 11]. Although some evidence suggested that ELOVLs were targets of SREBP1 in rodents [12, 13] and goat [14], knowledge of how the synthesis of PUFA is regulated in the ruminant mammary gland is unclear.

The inactive SREBP1 (pSREBP1) protein complex is moved to the Golgi leading to maturation and formation of nuclear SREBP1 (nSREBP1) activating genes involved in biosynthesis of fatty acids [15–17]. Data in rodents underscored an important role for SREBP1 in altering elongation of long chain fatty acids [18, 19]. For instance, in mouse [12] and goat [14], ELOVL6 is suggested to be a target of SREBP1 because its promoter contains SREBP response elements. Besides SREBP-1, at least in nonruminants, the transcription factor liver X receptor (LXR) is also involved in lipogenesis in an SREBP-dependent and -independent manner [20]. Upon activation by oxysterols or synthetic agonists in humans and rodents, LXR binds to LXR response elements (LXRE) in the promoters of target genes as a heterodimeric complex with retinoid X receptor, leading to transcription of lipogenic enzymes [21]. Studies have identified LXRE in the SREBP1c promoter in goats [22], cows [23], and mice [24]. Along with the fact that the mouse ELOVL5 promoter is regulated directly by LXR-SREBP1c [25], these data indicated that the LXR pathway might play an important role in regulating PUFA synthesis.

To date, 2 protein subtypes of LXR (LXRA and LXRB) have been identified. Evidence in humans and rodents supports a different functional role for these two subtypes [26, 27]. LXRA is required for the control of cholesterol metabolism in the liver [28] and mammary gland [29], while LXRB is a major regulator of fat storage in muscle and WAT [30]. However, these data are challenged by in vitro dose-response studies with macrophages from LXRA^−/− and LXRB^−/− mice suggesting an equivalent role for LXRA and LXRB in the regulation of lipogenic genes [31]. These inconsistencies suggested that roles for LXR subtypes are specific across tissues. Because of the specific response of the SREBP1c promoter upon LXRB activation, recent data in goat mammary cells suggested that LXRB is the predominant subtype that regulates lipogenic genes [32]. Although recent data in goat mammary cells confirmed the existence of SRE sites in the ELOVL6 promoter [14], whether and how LXRB-SREBP1 axis alters PUFA profiles in ruminant mammary cells remains undetermined.

The main goal of the current study was to assess the transcriptional regulatory mechanisms in the LXRB-SREBP1c axis that could alter PUFA composition in goat mammary epithelial cells (GMEC). To address this, we used in vitro culture of primary GMEC. The protein abundance of LXRB was altered through adenovirus or small interference RNA in cells incubated with its pharmacological agonist (T0901317, T09). The data provided novel insights on the role of the LXRB-SREBP1c axis in lipid hemostasis via controlling the synthesis of PUFA.

Cell culture and treatment

The GMEC were isolated from lactating Saanen dairy goats as described previously [33]. Details of cell culture are described elsewhere [34]. Culture medium was composed of DMEM/F12 (Hyclone, USA), hydrocortisone (5 mg/L, Sigma, USA), insulin (5mg/L, Sigma, USA) and penicillin/streptomycin (10k unit/L, Harbin Pharmaceutical Group, China).

The LXRβwas overexpressed by adenoviral vector recombination with a flag tag epitope (Ad-LXRB) and was knockdown via RNA interference (siLXRB) as described previously [32]. For adenoviral infection, the GMEC cultured in six–well plates were incubated with adenovirus medium Ad-GFP or Ad-LXRB, respectively. For RNA interference, the GMEC cultured in six–well plates were transfected with siLXRB or control (siNC), respectivel, using Lipofectamine® RNAiMAX according to the manufacturer’s protocol (Invitrogen, USA). Treated GMEC were cultured with 1 µM T09 (final concentration, diluted in dimethyl sulfoxide, DMSO, Sigma, St. Louis, MO, USA) or control (DMSO, Sigma, St. Louis, MO) after 24 h of initial culture, and then harvested at 48 h (24 h later) for RNA extraction, lipid analysis and protein collection.

RNA extraction and qPCR

Total RNA from GMEC was extracted using the RNA Prep pure cell Kit (Tiangen Biotech Co. Ltd., Beijing, China). Synthesis of cDNA from 1 µg RNA was conducted using the PrimeScript™ RT kit (Takara Bio Inc., Otsu, Japan) according to the manufacturer’s instructions. Quantitative real-time PCR (qPCR) was performed according to the manufacturer’s instructions using a kit (SYBR® Premix Ex Taq™ II, Perfect Real Time, Takara Bio Inc.).

Genes studied include those involved in elongation (ELOVL5, ELOVL6, ELOVL 7), lipid droplet formation (PLIN2), fatty acid desaturase (SCD1, FADS1 and FADS2) triacylglycerol (DGAT2) and cholesterol synthesis (ABCA1). All qPCR reactions were performed in an ABI7500 (Thermo fisher) sequence detector. Ubiquitously expressed transcript (UXT) was used as internal controls. Primer sequences are reported in STable 1 (Supplementary file).

Lipid analysis

Total cellular TAG was extracted according to the protocol of the GPO-Trinder triglyceride assay kit (Applygen Technologies, China). The mass of TAG was determined according to the manufacturer's instructions on a micro-titer plate reader (BioTek, USA). Quantification of total cellular TAG was normalized to protein concentration determined in each well using a BCA protein assay (Pierce, USA) according to the manufacturer's instructions.

Oil-Red-O staining was performed on GMEC according to a method described previously [35]. The images were captured using a Leica fluorescent microscope (DMI4000B, Germany).

For assay of fatty acid profiles, collected GMEC were scraped off the culture dish using a 2-mL aliquot of 2.5% (vol/vol) vitriol:methanol. Then, total lipid extraction and methylation were performed according to Shi et al. [36]. Methylated lipid samples were analyzed using a Gas Chromatography-Mass Spectrometer (Agilent Technologies, Santa Clara, CA) installed with an HP-88 column (100 m × 0.25 mm i.d. × 0.25 µm film thickness, Agilent Technologies) following a published procedure [8]. The relative proportion of each fatty acid was determined as the ratio of the FA peak to the total peaks in each run. Data for each fatty acid were analyzed as a proportion of the total fatty acids detected.

Luciferase analysis

The fragments of wild type SCD1 promoter (SCD1-wild) and the SRE site mutation (SCD1-SRE-mut) were constructed into the pGL3-basic vector as described previously [10]. For the luciferase assay, the cells cultured in 48-well plates at 80–90% confluent were co-transfected with 300 ng of the vector (SCD1-wild or SCD1-SRE-mut) plus 10 ng of control vector (renilla luciferase) per well using Lipofactamine 2000 reagent (Invitrogen, USA). After 12 h of initial culture, transfected GMEC were incubated with siRNA using Lipofectamine® RNAiMAX. The transfected GMEC were treated with 1 µM T09 (final concentration, diluted in DMSO, Sigma, USA) or control (DMSO, Sigma, St. Louis, MO) after 24 h of initial culture, and then harvested at 48 h. The dual-Luciferase Reporter assay kit (Promega, Madison, USA) was used to measure luciferase activity on a Fluoroskan Ascent apparatus (Thermo Scientific, USA) according to the kit manufacturer’s protocol. The relative luciferase activity was calculated as the ratio of firefly luciferase compared with renilla luciferase activity.

Western blotting

The blots were incubated with antibodies against SREBP1 (ab3259, Abcam, Cambridge, UK; 1:500), LXRB (ER1912-56, Huabio, Hangzhou, China; 1:300), β-actin (CW0096, CW Biotech, Beijing, China; 1:1,000) and flag (80010-1-RR, proteintech, Wuhan, China; 1:1000). Secondary antibody was used IgG with HRP (Proteintech, Wuhan, China 1:1000). Signals were detected using a chemiluminescent ECL system (Pierce, USA) and visualized by autoradiography with a cold CCD camera (Bio-Rad, USA).

Statistical analysis

Treatments were replicated at least 3 times, and results are expressed as means ± standard error of the means (SEM). Data from qPCR were analyzed using the 2^−ΔΔCt method and normalized to the respective control (siNC + DMSO or Ad-GFP + DMSO). Densitometry values of western blott were measured using Image J software (http://imagej.nih.gov/ij/). Protein abundance was normalized to β-actin. All data are reported as means ± SEM. Statistical differences were determined with a one-way ANOVA using SPSS 19.0 (SPSS, Inc. /IBM Corp., Chicago, IL). Significance was declared at P < 0.05.

LXRB activation increased expression of genes related to PUFA synthesis

As shown in SFigure 1 (Supplementary file), the expression of LXRB mRNA was significantly upregulated compared with the control (Ad-GFP + DMSO). Consistent with the mRNA level, the protein abundance of LXRB increased markedly when the GMEC were incubated with Ad-LXRB (Fig. 1A). Compared with control (Ad-GFP + DMSO), T09 treatment (Ad-GFP + T09) significantly enhanced the mRNA level of ELOVL7 (P = 0.01), SCD1(P = 0.02) and ABCA1 (P < 0.01) (Fig. 1C and D) while LXRB overexpression alone increased mRNA expression of ELOVL6 (P = 0.02), ELOVL7 (P = 0.01), FADS1(P = 0.03), PLIN2 (P = 0.01) and DGAT2 (P < 0.01). No significant changes (P > 0.05) were observed for ELOVL5, SCD1, FADS2 and ABCA1 (Fig. 1B, C and D). Addition of T09 to cultures overexpressing LXRB (Ad-LXRB + T09) upregulated the mRNA abundance of ELOVL6 (P = 0.02), ELOVL7 (P < 0.01), SCD1 (P = 0.04), FADS1 (P = 0.02), PLIN2 (P = 0.02), DGAT2 (P < 0.01) and ABCA1 (P = 0.02), respectively, relative to the control (Ad-GFP + T09) (Fig. 1B, C and D).

LXRB enhanced cellular concentrations of TAG and cholesterol

Oil-Red-O staining revealed that T09 increased cellular lipid accumulation in the presence or absence of Ad-LXRB (Fig. 2A). Consistent with the data of Oil-Red-O staining, TAG analysis confirmed that cells incubated with T09 had higher TAG concentration (P = 0.02) compared with the control (Ad-GFP + DMSO) (Fig. 1B). In addition, LXRB overexpression further enhanced the concentration of TAG in cells incubated with T09 (P = 0.01, Fig. 1B). Overexpression of LXRB alone had no significant change for concentration of cholesterol (Fig. 1C). Compared with the control (Ad-GFP + DMSO), a lower level of cholesterol was observed in cells incubated with T09. A significant increase in cholesterol was also observed (P = 0.02) when LXRB was overexpressed in the cells incubated with T09.

LXRB increased desaturation and elongation of FA

Compared with the control (Ad-GFP + DMSO group), incubation with T09 upregulated the ratio of C16:1(P = 0.01) and C18:1n7 (P = 0.01) (Fig. 3A) while overexpression of LXRB alone (Ad-LXRB + DMSO) had no effect (P > 0.05) on cellular fatty acid profiles. Overexpressing LXRB in cells plus T09 increased significantly the ratio of C16:0 (P = 0.02), C16:1 (P < 0.01), C18:1n9 (P = 0.01) and C18:2 (P = 0.03) compared with the control (Ad-GFP + DMSO). However, no significant changes (P > 0.05) for the concentrations of C18:0, C20:4, C22:6 and C20:5 were observed regardless of treatment with Ad-LXRB or T09. Consistent with the alteration of fatty acid profiles, an increase in desaturation index of C16:0 (P < 0.01) and C18:0 (P = 0.04) was observed when treated with T09. The desaturation index of C16:0 was further enhanced by treatment with T09. LXRB overexpression significantly upregulated desaturation index of C18:0 (P = 0.01) and the elongation index of C16:0 (P < 0.01) in the cells incubated with T09.

Knockdown of LXRB decreased the expression of genes related to PUFA synthesis

Consistent with the marked decrease in mRNA abundance of LXRB (SFigure 1B, Supplementary file), protein abundance of LXRB was significantly increased when incubated with T09 and was markedly reduced by the siLXRB treatment (Fig. 4A). Compared with the control (siNC + DMSO), LXRB knockdown (siLXRB + DMSO) did not significantly alter the mRNA abundance of ELOVL5, SCD1, FADS2, PLIN2, DGAT2 and ABCA1. However, it significantly decreased ELOVL6 (P = 0.02), ELOVL7 (P = 0.04) and FADS1 (P = 0.02) (Fig. 4B, C and D). In the cells incubated with T09, knockdown of LXRB significantly decreased the abundance of ELOVL5 (P = 0.04), ELOVL6 (P = 0.02), ELOVL7(P = 0.04), SCD1(P = 0.03), FADS1(P = 0.03) and DGAT2 (P = 0.04) (Fig. 4B, C and D).

Knockdown of LXRB altered the concentrations of TAG and cholesterol

As shown with the Oil-red staining and the TAG assay, knockdown of LXRB alone had no significant effect on lipid accumulation compared with the control group (Ad-GFP + DMSO) (Fig. 5A, B and C). The addition of T09 to cultures with knockdown of LXRB decreased the accumulation of TAG (P = 0.04, Fig. 5A and B). Compared with the control (siNC + DMSO), the addition of T09 led to a lower level of cholesterol (P = 0.03), while this effect was impaired in cells incubated with siLXRB (P = 0.07).

Knockdown of LXRB altered the FA profiles

Compared with control (siNC + DMSO), knockdown of LXRB alone had no significant effect on the ratio of C16:1, C18:2. C18:1n9, C18:1n7, C20:4, C22:6 and C20:5. An increase in C16:0 (P = 0.03) at the expense of C18:0 (P = 0.04) was observed in the cells incubated with siLXRB alone. In the cells containing T09 and incubated with siLXRB the ratio of C18:2 (P = 0.04) decreased, but there was no effect on the remaining fatty acids (siLXRB + T09 group vs siNC + T09 group). Incubation with siLXRB alone had no effect on desaturation of C16:0 (P = 0.98) while an increase in desaturation of C18:0 (P < 0.001) was observed compared with the control (siNC + DMSO). In the cells containing T09, siLXRB incubation had no significant effect on desaturation of C16:0 (P = 0.64), but decreased desaturation of C18:0 (P < 0.01). A decrease in elongation of C16:0 was observed in the cells incubated with siLXRB alone (P < 0.01), and was impaired by the addition of T09.

Full LXRB activation is dependent on SREBP1 protein

Compared with the control (Ad-GFP + DMSO), LXR activated by T09 significantly upregulated the abundance of pSREBP (P < 0.01) and nSREBP1(P = 0.01) (Figure A, C and D). The GMEC incubated with Ad-LXRB or siLXRB alone had no significant effect (P > 0.05) on the protein level of pSREBP and nSREBP1. In the cells incubated with T09, knockdown of LXRB significant downregulated the abundance of pSREBP (P = 0.04) and nSREBP1(P < 0.01), respectively (Figure B, E and F).

As shown in Fig. 8, T09 treatment increased the activity of SCD1-wild (P = 0.02) compared with control (cells treated with DMSO). However, knockdown of LXRB impaired the induction of T09 on SCD1-wild activity (P > 0.05). T09 treatment alone had no significant effect on SCD1-SREM activity (P > 0.05) while a lower activity of SCD1-SREM (P = 0.03) was observed in the cells incubated siLXRB alone compared with control (siNC + DMSO). In the cells plus T09, knockdown of LXRB resulted in a lower activity of SCD1-SREM (P = 0.01) (Fig. 4C).

Although previous evidence suggested that the FADS enzyme family, SCD1 and ELOVLs are associated with the content of milk PUFA [37–39], the mechanism whereby synthesis of PUFA is controlled remains to be addressed. In humans and rodents, LXR is a transcription factor regulating the desaturation and elongation of endogenous PUFA [31, 40]. A role of LXR in regulating the expression of desaturases (SCD1) was also reported in dairy goats [11] and cow [41, 42]. As the predominant subtype of LXR in ruminant mammary gland [32], however, whether LXRB plays a role in controlling the synthesis of PUFA in ruminant mammary cells has not been addressed. The present study is novel in that we studied the genes associated with PUFA synthesis and FA profiles through specific overexpressing or knockdown of LXRB protein abundance. Our data underscore an important role for LXRB in PUFA synthesis via controlling the processes of elongation and desaturation.

The ELOVL family is essential for fatty acid elongation and synthesis of key saturated and PUFA in humans and rodents [38, 43]. In the current study, the higher levels of ELOVL5-7 in the cells incubated with T09 when LXRB was overexpressed and the lower levels of ELOVL5-7 when LXRB was knockdown suggested that they are targets of LXRB. The alteration of ELOVL5 and ELOVL6 in the current study agree with the data in the LXRB null mouse [30]. Together with the fact that the gene promoters of ELOVL5-6 contain an LXRE site [14, 25], the pattern of expression for ELOVL5-6 with LXRB further confirmed that they are the direct targets of LXRB in the mammary gland. In the physiological context of the role of ELOVLs and its production in the insulin sensitive [4, 44], our data also suggest a central role of LXRB in cellular homeostasis via broadly regulating aspects of lipogenesis (Fig. 8B). This idea agrees with the observation in mouse liver that the binding of LXRs to hepatic genes has broad effects on the transcriptional landscape beyond its canonical function as an activator of lipid metabolism genes [20].

Consistent with the pattern expression of the ELOVL5-7 after overexpressing or knockdown of LXRB, the increased elongation index of C16 agrees with the findings in goats that ELOVL6 plays a role in the elongation of long-chain SFA (C16:0 to C18:0) while ELOVL5 and ELOVL7 are involved in the elongation of UFA containing 16 and 18 carbons [5, 7, 8]. In the cells containing T09, compared with the control, an increase of C18:2 after LXRB overexpression and a decrease of C18:2 after LXRB knockdown underscores a role of LXRB in enhancing the synthesis of PUFA. This finding agrees with data in goat [45] and humans [31]. Although evidence in macrophages suggested an increase of C20:4 and C20:5 upon activation of LXR [31], minor changes were observed for these PUFA in the present study. We speculate that this response was likely due to the limited substrate availability in GMEC culture. Another possible explanation for this inconsistent effect is the specific tissue differences for LXR activation, an idea supported by the different roles of LXR subtypes on the regulation of SREBP1expression in macrophages and mammary cells [31, 32]. The lack of goat-specific antibodies for ELOVLs clearly are a limiting factor in the present study. Despite being unable to measure the protein level of ELOVL5-7, the present data illustrates that LXRB activation increased cellular PUFA (at least, C18:2) through controlling the activity of ELOVL5-7 (Fig. 8B).

The desaturases work in concert with elongases during the endogenous biosynthesis of PUFA and in mouse are regulated by the activation of LXR [18]. The significant upregulation of SCD1 level and its promoter activity after LXRB overexpression or activation by T09 agree with previous data in goats [10]. The upregulation of SCD1 mRNA in the present study is consistent with the increased concentration of C16:1 and C18:1n9 when LXRB was activated. Along with the lack of alteration of SCD1 after knockdown of LXRA [10], the lower promoter activity of SCD1 in cells incubated with LXRB supports a predominant role of this subtype in regulating FA desaturation.

The FADS family catalyzes desaturation reactions at positions 5 and 6 of the fatty acyl chain during PUFA synthesis. The finding that a significant change for FADS1 and a minor change for FADS2 were observed in the current study suggests a specific regulatory role of LXRB in FADS1 to control PUFA synthesis. The lack of effect of LXRB on FADS2 in the present study agreed with the lack of change in concentrations of 22-carbon fatty acids. This idea is supported by previous work in mouse [46]. Combined with the fact that the products of SCD1 or FADS1 can serve to activate various signaling pathways [47], the alterations in fatty acid composition in the current study suggest that LXRB plays a role in the cellular homeostasis via controlling cellular synthesis of PUFA (Fig. 8B). The idea is supported by the fact that repression of LXRs restricts macrophage biosynthesis of insulin-sensitizing Omega 3 fatty acids [46].

The accumulation of C16:0 would result in endoplasmic reticulum stress and apoptosis (Green and Olson, 2011). In the current study, the activation of LXRB in the mammary cells promoting the elongation and desaturation of C16:0 and C18:0 might serve to protect mammary cells from lipotoxicity. In ruminants, the DGAT2 coding enzyme is involved in synthesis of TAG and is upregulated during lactation [48]. The upregulation of PLIN2 and DGAT2 when LXRB was overexpressed agrees with the accumulation of TAG in the present study. In the physiological context of PLIN2 for the formation and secretion of lipid droplets [49], the inducible increase of PLIN2 in the present study suggested that LXRB activation would facilitate milk secretion. This idea was supported by the accumulation of TAG when LXRB was expressed.

ABCA1 is important in eliminating excess cholesterol from cells and, thus, contributes to cellular homeostasis [50]. ABCA1 is induced by LXR in mouse liver [28]. The markedly high expression of ABCA1 in the cells incubated with T09 suggests it is a target gene of LXR in the goat mammary gland. The lower level of cholesterol in the cells incubated with T09 agrees with the role of ABCA1 in eliminating excess cholesterol [50]. It is worth noting that overexpression of LXRB upon T09 increased the level of cholesterol, suggesting a role of LXRB promoting cholesterol synthesis in the ruminant mammary gland (Fig. 8B). This idea agrees with data in mouse liver [21, 27]. The modest change when LXRB was knockdown could have been due to LXRA eliciting a compensatory effect when LXRB expression is reduced. This idea is supported by data demonstrating that LXRA is prone to activate cholesterol metabolism [27]. The data in the current study suggests that LXRB is a key transcription factor that controls lipogenic homeostasis in the mammary gland.

Evidence from the promoter analysis suggests that LXR regulates several downstream lipogenic genes directly or in an SREBP1-dependent manner [11, 14, 32]. In the current study, the increases of SREBP1 and nSREBP1 protein abundance in the cells treated with T09 are consistent with the upregulation of SREBP1 mRNA level [32]. Combined with the fact that the promoter of SREBP1 is mainly responsive to LXRB activation [32], the observation in cells containing T09 that LXRB knockdown decreased the abundance of SREBP1 and nSREBP1 suggests a direct mechanism of LXRB on SREBP1 at a transcriptional level.

To further assess the mechanism whereby LXRB controls its downstream genes, SCD1 promoter was used as a model in the current study. The lower activity of SCD1-LXRE-mut supports a crucial role for SREBP1 in regulating SCD1 in the goat [11]. Along with the observation that activation of LXR by T09 had no significant effect for the SCD-SREM, the current data suggests that the full function of LXR in regulating lipogenesis is in an SREBP1-dependent manner in the goat mammary gland. Along with the finding that LXRA interference had a weak effect on SCD1 promoter activity [10], the observation that knockdown of LXRB significantly decreased the level of SCD1 promoter activity regardless of T09 treatment further illustrates that LXRB is the predominant subtype regulating lipogenesis in the ruminant mammary gland. Collectively, the data in the current study highlight an important role of LXRB-SREBP1 axis in PUFA synthesis through the genes encoding elongases and desaturases, and, thus, regulating lipid homeostasis in the mammary gland.

The increase of PUFA content and genes encoding elongases and desaturases by LXRB activation demonstrates its important role in the synthesis of PUFA. The full lipogenic response elicited by LXRB is in an SREBP1-dependent fashion. In addition, the LXRB-SREBP1 axis has a broad effect on cellular homeostasis via altering lipid droplet formation and cholesterol efflux. Although the present study cannot explain the full mechanisms of LXRB-SREBP1 axis action in the lactating mammary gland, it seems closely related to the synthesis of PUFA. These endogenous PUFA would act a natural ligand participating in milk synthesis and secretion. Therefore, controlling the activation of LXRB-SREBP1 axis in the lactating mammary gland may provide possible strategies to improve the quality and production of milk.

ABCA1

ATP binding cassette subfamily a member 1

Ad-LXRB

recombinant adenovirus expressing LXRB protein with a flag tag epitope

Ad-GFP

recombinant adenovirus expressing green fluorescent protein

DGAT2

diacylglycerol O-acyltransferase 2

LXRB

Liver X receptor alfa

LXRB

Liver X receptor beta

PLIN2

adipose differentiation-related protein

fatty acid

ELOVL5

elongation of very long chain fatty acids protein 5

ELOVL6

elongation of very long chain fatty acids protein 6, ELOVL 7:elongation of very long chain fatty acids protein 7

FADS1

fatty acid desaturase 1

FADS2

fatty acid desaturase 2

GMEC

goat mammary epithelial cells

pRL-TK

Renilla vector

PUFA

polyunsaturated fatty acids

qPCR

quantitative real-time PCR

SCD1

Stearoyl-CoA desaturase 1

SCD1-wild

SCD1 promoter

SCD1-SRE-mut

SCD1 promoter with SREBP1 response element mutation

siLXRB

small interference RNA targeted LXRB

siNC

negative control for the small interference RNA

SREBP1

sterol regulatory element binding transcription factor 1

UXT

ubiquitously expressed transcript

TAG

triacylglycerol.

Ethics approval and consent to participate

All animal experiments and procedures were approved by the Animal Use and Care Committee of Zhejiang University (Hangzhou, China).

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

Authors’ contributions

Hengbo Shi, Jianxin Liu and Jun Luo conceived and supervised the study; Changhui Zhang, Wenying Zhang and Huifen Xu performed experiments; Changhui Zhang and Hengbo Shi analyzed the data; Hengbo Shi and Wenying Zhang wrote the draft of manuscript; Juan Loor interpreted data, revised and edited the manuscript. The author(s) read and approved the final manuscript.

Funding

This research was jointly supported by the National Natural Science Foundation of China (31702090), Key R&D program of Zhejiang Province (2022C04017), Zhejiang Provincial Major Science and Technology Projects on Agricultural New Varieties Selection and Breeding (2021C02068-6) and Opening fund in Key Laboratory of Molecular Animal Nutrition (Zhejiang University, KLMAN202103).

Availability of data and materials

All data measured or analyzed during this work are available from the corresponding author upon reasonable request.

Shingfield KJ, Chilliard Y, Toivonen V, Kairenius P, Givens DI. Trans fatty acids and bioactive lipids in ruminant milk. Adv Exp Med Biol. 2008;606:3–65.
Han S, Schroeder EA, Silva-Garcia CG, Hebestreit K, Mair WB, Brunet A. Mono-unsaturated fatty acids link H3K4me3 modifiers to C. elegans lifespan. Nature. 2017;544(7649):185–90.
Wang Y, Torres-Gonzalez M, Tripathy S, Botolin D, Christian B, Jump DB. Elevated hepatic fatty acid elongase-5 activity affects multiple pathways controlling hepatic lipid and carbohydrate composition. J Lipid Res. 2008;49(7):1538–52.
Tripathy S, Torres-Gonzalez M, Jump DB. Elevated hepatic fatty acid elongase-5 activity corrects dietary fat-induced hyperglycemia in obese C57BL/6J mice. J Lipid Res. 2010;51(9):2642–54.
Shi HB, Du Y, Zhang CH, Sun C, He YL, Wu YH, Liu JX, Luo J, Loor JJ. Fatty acid elongase 5 (ELOVL5) alters the synthesis of long-chain unsaturated fatty acids in goat mammary epithelial cells. J Dairy Sci. 2018;101(1):4586–94.
Yao D, Luo J, He Q, Shi H, Li J, Wang H, Xu H, Chen Z, Yi Y, Loor JJ. SCD1 alters long-chain fatty acid (LCFA) composition and its expression is directly regulated by SREBP-1 and PPARgamma 1 in dairy goat mammary cells. J Cell Physiol. 2017;232(3):635–49.
Shi HB, Wu M, Zhu JJ, Zhang CH, Yao DW, Luo J, Loor JJ. Fatty acid elongase 6 plays a role in the synthesis of long-chain fatty acids in goat mammary epithelial cells. J Dairy Sci. 2017;100(6):4987–95.
Shi W, Luo L, Loor L. Fatty acid elongase 7 (ELOVL7) plays a role in the synthesis of long-chain unsaturated fatty acids in goat mammary epithelial cells. Animals. 2019;9(6):389.
Tian H, Luo J, Zhang Z, Wu J, Zhang T, Busato S, Huang L, Song N, Bionaz M. CRISPR/Cas9-mediated stearoyl-coA desaturase 1 (SCD1) deficiency affects fatty acid metabolism in goat mammary epithelial cells. J Agric Food Chem. 2018;66(38):10041–52.
Yao DW, Luo J, He QY, Li J, Wang H, Shi HB, Xu HF, Wang M, Loor JJ. Characterization of the liver X receptor-dependent regulatory mechanism of goat stearoyl-coenzyme A desaturase 1 gene by linoleic acid. J Dairy Sci. 2016;99(5):3945–57.
Yao DW, Luo J, He QY, Xu HF, Li J, Shi HB, Wang H, Chen Z, Loor JJ. Liver X receptor α promotes the synthesis of monounsaturated fatty acids in goat mammary epithelial cells via the control of stearoyl-coenzyme A desaturase 1 in an SREBP-1-dependent manner. J Dairy Sci. 2016;99(8):6391–402.
Kumadaki S, Matsuzaka T, Kato T, Yahagi N, Yamamoto T, Okada S, Kobayashi K, Takahashi A, Yatoh S, Suzuki H, et al. Mouse Elovl-6 promoter is an SREBP target. Biochem Biophys Res Commun. 2008;368(2):261–6.
Bae J-S, Oh A-R, Lee H-J, Ahn Y-h, Cha J-Y. Hepatic Elovl6 gene expression is regulated by the synergistic action of ChREBP and SREBP-1c. Biochem Biophys Res Commun. 2016;478(3):1060–6.
He Q, Luo J, Wu J, Li Z, Yao W, Zang S, Niu H. ELOVL6 promoter binding sites directly targeted by sterol regulatory element binding protein 1 in fatty acid synthesis of goat mammary epithelial cells. J Dairy Sci. 2021;104(5):6253–66.
DeBose-Boyd RA, Ye J. SREBPs in Lipid Metabolism, Insulin Signaling, and Beyond. Trends Biochem Sci. 2018;43(5):358–68.
Harvatine KJ, Boisclair YR, Bauman DE. Recent advances in the regulation of milk fat synthesis. Animal. 2009;3(1):40–54.
Shi HB, Tai DM, Wang C, Liu JX, Loor JJ, Liu HY. Short communication: The antilipogenic effect of trans-10,cis-12 conjugated linoleic acid in bovine mammary epithelial cells is associated with proteasome activity and ATP production. J Dairy Sci. 2020;103(10):9096–101.
Jalil A, Bourgeois T, Menegaut L, Lagrost L, Thomas C, Masson D. Revisiting the Role of LXRs in PUFA Metabolism and Phospholipid Homeostasis. Int J Mol Sci 2019, 20(15).
Oishi Y, Spann NJ, Link VM, Muse ED, Strid T, Edillor C, Kolar MJ, Matsuzaka T, Hayakawa S, Tao J, et al. SREBP1 contributes to resolution of pro-inflammatory TLR4 signaling by reprogramming fatty acid metabolism. Cell Metab. 2017;25(2):412–27.
Bideyan L, Fan W, Kaczor-Urbanowicz KE, Priest C, Casero D, Tontonoz P. Integrative analysis reveals multiple modes of LXR transcriptional regulation in liver. Proc Natl Acad Sci U S A 2022, 119(7).
Ramón-Vázquez A, de la Rosa JV, Tabraue C, Lopez F, Díaz-Chico BN, Bosca L, Tontonoz P, Alemany S, Castrillo A. Common and differential transcriptional actions of nuclear receptors liver X receptors α and β in macrophages. Mol Cell Biol. 2019;39(5):e00376-00318.
Xu HF, Luo J, Wang HP, Wang H, Zhang TY, Tian HB, Yao DW, Loor JJ. Sterol regulatory element binding protein-1 (SREBP-1)c promoter: characterization and transcriptional regulation by mature SREBP-1 and liver X receptor alpha in goat mammary epithelial cells. J Dairy Sci. 2016;99(2):1595–604.
Lengi AJ, Corl BA. Short communication: Identification of the bovine sterol regulatory element binding protein-1c promoter and its activation by liver X receptor. J Dairy Sci. 2010;93(12):5831–6.
Repa JJ, Liang G, Ou J, Bashmakov Y, Lobaccaro J-MA, Shimomura I, Shan B, Brown MS, Goldstein JL, Mangelsdorf DJ. Regulation of mouse sterol regulatory element-binding protein-1c gene (SREBP-1c) by oxysterol receptors, LXRα and LXRβ. Genes Dev. 2000;14(22):2819–30.
Qin Y, Dalen KT, Gustafsson JA, Nebb HI. Regulation of hepatic fatty acid elongase 5 by LXRalpha-SREBP-1c. Biochim Biophys Acta. 2009;1791(2):140–7.
Ishibashi M, Filomenko R, Rébé C, Chevriaux A, Varin A, Derangère V, Bessède G, Gambert P, Lagrost L, Masson D. Knock-down of the oxysterol receptor LXRα impairs cholesterol efflux in human primary macrophages: Lack of compensation by LXRβ activation. Biochem Pharmacol. 2013;86(1):122–9.
Quinet EM, Savio DA, Halpern AR, Chen L, Schuster GU, Gustafsson J-Å, Basso MD, Nambi P: Liver X. receptor (LXR)-β regulation in LXRα-deficient mice: implications for therapeutic Targeting. Mol Pharmacol. 2006;70(4):1340–9.
Endo-Umeda K, Makishima M. Liver X receptors regulate cholesterol metabolism and immunity in hepatic nonparenchymal cells. Int J Mol Sci 2019, 20(20).
Grinman DY, Careaga VP, Wellberg EA, Dansey MV, Kordon EC, Anderson SM, Maier MS, Burton G, MacLean PS, Rudolph MC, et al. Liver X receptor-α activation enhances cholesterol secretion in lactating mammary epithelium. Am J Physiol Endocrinol Metab. 2019;316(6):E1136–45.
Korach-Andre M, Parini P, Larsson L, Arner A, Steffensen KR, Gustafsson JA. Separate and overlapping metabolic functions of LXRalpha and LXRbeta in C57Bl/6 female mice. Am J Physiol Endocrinol Metab. 2010;298(2):E167–78.
Varin A, Thomas C, Ishibashi M, Ménégaut L, Gautier T, Trousson A, Bergas V, de Barros JPP, Narce M, Lobaccaro JMA, et al. Liver X receptor activation promotes polyunsaturated fatty acid synthesis in macrophages: relevance in the context of atherosclerosis. Arterioscler Thromb Vasc Biol. 2015;35(6):1357–65.
Shi HB, Zhang CH, Xu ZA, Xu XF, Lv ZB, Luo J, Loor JJ. Nuclear receptor subfamily 1 group H member 2 (LXRB) is the predominant liver X receptor subtype regulating transcription of 2 major lipogenic genes in goat primary mammary epithelial cells. J Dairy Sci. 2017;100(8):6743–52.
Shi HB, Zhang CH, Zhao W, Luo J, Loor JJ. Peroxisome proliferator-activated receptor delta facilitates lipid secretion and catabolism of fatty acids in dairy goat mammary epithelial cells. J Dairy Sci. 2017;100(1):797–806.
Shi HB, Zhang CH, Xu ZA, Lou GG, Liu JX, Luo J, Loor JJ. Peroxisome proliferator activated receptor delta regulates lipid droplet formation and transport in goat mammary epithelial cells. J Dairy Sci. 2018;101(1):2641–9.
Mehlem A, Hagberg CE, Muhl L, Eriksson U, Falkevall A. Imaging of neutral lipids by oil red O for analyzing the metabolic status in health and disease. Nat Protocols. 2013;8(6):1149–54.
Shi HB, Luo J, Yao DW, Zhu JJ, Xu HF, Shi HP, Loor JJ. Peroxisome proliferator-activated receptor-Y stimulates the synthesis of monounsaturated fatty acids in dairy goat mammary epithelial cells via the control of stearoyl-coenzyme A desaturase. J Dairy Sci. 2013;96(12):7844–53.
Green CD, Ozguden-Akkoc CG, Wang Y, Jump DB, Olson LK. Role of fatty acid elongases in determination of de novo synthesized monounsaturated fatty acid species. J Lipid Res. 2010;51(7):1871–7.
Kabeya N, Yamamoto Y, Cummins SF, Elizur A, Yazawa R, Takeuchi Y, Haga Y, Satoh S, Yoshizaki G. Polyunsaturated fatty acid metabolism in a marine teleost, Nibe croaker Nibea mitsukurii: Functional characterization of Fads2 desaturase and Elovl5 and Elovl4 elongases. Comp Biochem Physiol B Biochem Mol Biol. 2015;188:37–45.
Kuah MK, Jaya-Ram A, Shu-Chien AC. The capacity for long-chain polyunsaturated fatty acid synthesis in a carnivorous vertebrate: Functional characterisation and nutritional regulation of a Fads2 fatty acyl desaturase with Delta4 activity and an Elovl5 elongase in striped snakehead (Channa striata). Biochim Biophys Acta. 2015;1851(3):248–60.
Courtney R, Landreth GE. LXR Regulation of Brain Cholesterol: From Development to Disease. Trends Endocrinol Metab. 2016;27(6):404–14.
McFadden JW, Corl BA. Activation of liver X receptor (LXR) enhances de novo fatty acid synthesis in bovine mammary epithelial cells. J Dairy Sci. 2010;93(10):4651–8.
Oppi-Williams C, Suagee JK, Corl BA. Regulation of lipid synthesis by liver X receptor alpha and sterol regulatory element-binding protein 1 in mammary epithelial cells. J Dairy Sci. 2013;96(1):112–21.
Ohno Y, Suto S, Yamanaka M, Mizutani Y, Mitsutake S, Igarashi Y, Sassa T, Kihara A. ELOVL1 production of C24 acyl-CoAs is linked to C24 sphingolipid synthesis. Proc Natl Acad Sci U S A. 2010;107(43):18439–44.
Matsuzaka T, Shimano H, Yahagi N, Kato T, Atsumi A, Yamamoto T, Inoue N, Ishikawa M, Okada S, Ishigaki N, et al. Crucial role of a long-chain fatty acid elongase, Elovl6, in obesity-induced insulin resistance. Nat Med. 2007;13(10):1193–202.
Wang W, Luo J, Zhong Y, Lin X, Shi H, Zhu J, Li J, Sun Y, Zhao W: Goat liver X receptor α, molecular cloning, functional characterization and regulating fatty acid synthesis in epithelial cells of goat mammary glands. Gene 2012, 505(2012):114–120.
Li P, Spann NJ, Kaikkonen MU, Lu M, Oh DY, Fox JN, Bandyopadhyay G, Talukdar S, Xu J, Lagakos WS, et al. NCoR repression of LXRs restricts macrophage biosynthesis of insulin-sensitizing omega 3 fatty acids. Cell. 2013;155(1):200–14.
Zhang Y, Zheng Y, Wang X, Qiu J, Liang C, Cheng G, Wang H, Zhao C, Yang W, Zan L, et al. Bovine Stearoyl-CoA Desaturase 1 Promotes Adipogenesis by Activating the PPARgamma Receptor. J Agric Food Chem. 2020;68(43):12058–66.
Bionaz M, Loor J. Gene networks driving bovine milk fat synthesis during the lactation cycle. BMC Genomics. 2008;9(1):366.
Shi HB, Yu K, Luo J, Li J, Tian HB, Zhu JJ, Sun YT, Yao DW, Xu HF, Shi HP, et al. Adipocyte differentiation-related protein promotes lipid accumulation in goat mammary epithelial cells. J Dairy Sci. 2015;98(10):6954–64.
Matsuo M. ABCA1 and ABCG1 as potential therapeutic targets for the prevention of atherosclerosis. J Pharmacol Sci. 2022;148(2):197–203.

Sfile.docx
Supplementary informationAdditional file 1: STable 1: Name, accession number, sequences, amplicon length of primer pairs used in the present experiment, efficiency of amplification of PCR, and references. SFigure 1: Relative mRNA expression level in the goat mammary epithelial cells incubated with adenovirus expression liver X receptor beta (LXRB). SFigure 2: Relative mRNA expression level in the goat mammary epithelial cells incubated siRNA targeted liver X receptor beta (LXRB).

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Editorial decision: Major revision
07 Jun, 2022
Reviewers agreed at journal
23 May, 2022
Reviews received at journal
10 May, 2022
Reviewers invited by journal
05 May, 2022
Editor assigned by journal
02 May, 2022
First submitted to journal
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LXRB-SREBP1 axis regulating lipogenic homeostasis by controlling the synthesis of polyunsaturated fatty acids in goat mammary epithelial cells

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Abstract

Figures

Introduction

Materials And Methods

Cell culture and treatment

RNA extraction and qPCR

Lipid analysis

Luciferase analysis

Western blotting

Statistical analysis

Results

LXRB activation increased expression of genes related to PUFA synthesis

LXRB enhanced cellular concentrations of TAG and cholesterol

LXRB increased desaturation and elongation of FA

Knockdown of LXRB decreased the expression of genes related to PUFA synthesis

Knockdown of LXRB altered the concentrations of TAG and cholesterol

Knockdown of LXRB altered the FA profiles

Full LXRB activation is dependent on SREBP1 protein

Discussion

Conclusion

Abbreviations

Declarations

References

Supplementary Files

Status:

Version 1