In most mammalian cells the majority of cholesterol resides in the plasma membrane (PM), yet feedback regulation of cholesterol synthesis and metabolism of cholesterol happens at intracellular membranes of the endoplasmic reticulum (ER), mitochondria, and nucleus. Therefore, the trafficking of PM cholesterol to intracellular organelles is tightly controlled to maintain cellular homeostasis. A recently identified family of ER-resident proteins called Asters (Aster-A, -B, and -C) form membrane-membrane contact sites to facilitate the transfer cholesterol away from the PM (Sandhu et al., 2018; Naito et al., 2019; Ferrari et al., 2020; Ferrari et al., 2023; Xiao et al., 2023). Aster-B was first described as a liver X receptor (LXR)-stimulated cholesterol transporter that facilitates PM-to-ER sterol transfer for cholesterol ester (CE) storage in the adrenal gland (Sandhu et al., 2018). More recently, Aster-C was found to be a direct transcriptional target gene for the farnesoid X receptor (FXR) in the liver where it plays a role in reverse cholesterol transport (RCT) (Xiao et al., 2023). Although there is emerging evidence of functional redundancy in some tissues, all three Asters (A, B, and C) have been implicated in both intestinal and hepatic cholesterol trafficking under certain conditions of fasting and re-feeding (Ferrari et al., 2023; Xiao et al., 2023). However, the specific individual role for each Aster protein in regulating whole body cholesterol balance under conditions of excess dietary cholesterol is not well understood.

Although only 5 years have passed since their original discovery as sterol transporters by Tontonoz and colleagues (Sandhu et al., 2018), there has been rapid progress to understand the relative and tissue-specific roles each ASTER family member plays in cholesterol transport. Each Aster has a cholesterol-binding pocket that is near the N-terminal GRAM domain and are tethered to the ER via a transmembrane domain at the C terminus. The GRAM domain plays a central role in binding the plasma membrane (PM) at PM-ER contact sites where Asters can facilitate nonvesicular transfer of cholesterol to the ER for esterification or feedback regulation of de novo cholesterol synthesis via the sterol regulatory element-binding protein cleavage-activating protein (SCAP-SREBP) system (Sandhu et al., 2018). In addition to their roles in cellular cholesterol transport, Aster proteins have also been shown to transport carotenoid lipids (Bandara et al., 2022). Aster-C specifically has also been implicated in regulating autophagosome biogenesis and mitochondrial bioenergetics (Ng et al., 2022; Charsou et al., 2023), but how the lipid transporting function of Aster-C facilitates autophagy is still incompletely understood. One potential clue is that Aster-C has been reported to activate mTORC1 (Zhang et al., 2020), which is a master regulator of nutrient sensing and autophagy. Several recent reports have linked differential expression of GRAMD1C to diverse cancers including hepatocellular carcinoma, clear cell renal carcinoma, and breast cancer (Hao et al., 2019; Li et al., 2022; Fan et al., 2023; Gong et al., 2023). Although there is emerging evidence that Aster-C can facilitate sterol transport and potentially signal transduction, the relative role that Aster-C plays in tissue and whole body sterol homeostasis has yet to be defined. Here we address this gap by studying sterol balance in Gramd1c-deficient mice under conditions of limited versus excess dietary cholesterol.

Gramd1c knockout mice were obtained from MMRRC UC Davis, strain 047990-UCD, C57BL/6N-Atm1Brd Gramd1ctm1a(KOMP)Wtsi/JMmucd. A breeding colony was developed at the Cleveland Clinic after the mice were genotype confirmed using a commercial partner (Transnetyx, Inc., Cordova, TN, United States). Heterozygous (Gramd1c+/−) mice were interbred to generate littermate controls. At 8 weeks of age, female wild type (Gramd1c+/+) or Gramd1c knockout mice (Gramd1c−/−) were switched from standard rodent chow to one of two experimental synthetic diets containing low (0.02%, wt/wt) or high (0.2%, wt/wt) levels of dietary cholesterol. These experimental diets were synthesized by Envigo—Teklad Diets (Madison, WI, United States) and additional information can be found for each of these diets by referencing the following diet numbers: TD.130,104 (low cholesterol diet) and TD.160,514 (high cholesterol diet) from our previous work (Pathak et al., 2020). Mice were maintained on these diets over a period 4 consecutive weeks, and phenotyped as described below for sterol and bile acid homeostasis. In a subset of male mice, we studied liver X receptor-driven alterations in sterol balance. For LXR agonist studies, the LXR agonist GW3965 was suspended in a vehicle containing 1.0% carboxymethylcellulose (CMC) and 0.1% Tween 80. Mice were gavaged with either vehicle or 40 mg/kg GW3965 once daily for a period of seven consecutive days. All mice were maintained in an Association for the Assessment and Accreditation of Laboratory Animal Care, International-approved animal facility and all experimental protocols were approved by the Institutional Animal Care and Use Committee (IACUC) of the Cleveland Clinic (IACUC protocol # 00002499 and 00003201).

Quantification of individual plasma bile acid species was conducted using a quantitative stable isotope dilution LC-MS/MS analytical method as recently described (Choucair et al., 2020). Briefly, stable isotope labeled internal standards (IS) included were: D4-glycolithocholic acid, D4-glycoursodeoxycholic acid, D4-glycodeoxycholic acid, D4-glycocholic acid, D4-taurolithocholic acid, D4-tauroursodeoxycholic acid, D4-taurochenodeoxycholic acid, D4-taurodeoxycholic acid, and D4-taurocholic acid; D4-lithocholic acid, D4-chenodeoxycholic acid, D4-deoxycholic acid, and D4-cholic acid, and D4-glycochenodeoxycholic acid. Mouse plasma samples were mixed with ice-cold methanolic IS working solution of internal standard, and were vortexed for 10 min and centrifuged (14,000 g, 20 min, 4°C). The supernatant was transferred to glass HPLC vials for LC/MS/MS analysis using a 4000 Q-Trap triple quadrupole tandem mass spectrometer (AB SCIEX, MA, United States) equipped with an electrospray ionization source operating in negative ion mode. Mass spectrometry parameters were as follows: ions spray voltage—4200 V, ion source heater temperature 500°C, source gas 1: 35 psi, source gas 2: 45 psi, and curtain gas 35 psi. Nitrogen gas was used for the nebulizer, curtain and collision gas. Analyses were performed using electrospray ionization in negative-ion mode with multiple reaction monitoring (MRM) of precursor and characteristic product ions specific for each monitored bile acid. The HPLC system consisted of four binary pumps (LC-20 AD), autosampler operating at 10°C (Nexera X2 SIL-30AC), controller (CBM-20A) (Shimadzu Scientific Instruments, Inc., MD, United States) and a dual column switching valve system Rheodyne (IDEX Health and Science, MA, United States). Chromatographic separations were performed on a reverse phase columns (Kinetix C18, 2.6 µm, 150 mm × 4.6 mm ID; catalog # 00F-4462-E0; Phenomenex, Torrance, CA). Mobile phase A was 1 mM ammonium acetate and 0.1% acetic acid in methnol:acetonitrile:water (1:1:3; v/v/v) and mobile phase B was 0.1% acetic acid in methanol:acetonitrile:2-propanol (4.5:4.5:1; v/v/v). Samples were injected onto columns equilibrated in 100% A, and separated using a gradient as follows: 0–2 min 0% B; 2–20 min 0%–100% B; 20–28.5 min 100% B. Flow rate was programmed as follows: 0.3 mL/min from 0–20 min, and 0.5 mL/min from 20–28 min. Samples are introduced to the mass spec for analysis from 9–28 min. To eliminate carry over, an extensive washing step alternating between mobile phase A and B was added at the end of each run as follows: 100% A for 28–35 min, then directly switched to 100% B from 36–46 min, and equilibration step of 100% A from 46–60 min. To increase sample throughput 2-fold, a dual chromatographic system was used. At 28 min of the gradient on the first column, the next sample was injected into a second column; thus, during the first column washing and equilibration, the second column is used for BAs separation and diversion to the mass spectrometer for analysis. Calibration curves were built by fitting each analyte concentration (10 different points) to peak area ratios (analyte/internal standard). The limit of detection (LOD) was defined as the lowest concentration of analyte in sample matrix (e.g., serum) that generated a signal-to-noise ratio of ≥3. The limit of quantification (LOQ) was defined as the lowest concentration of analyte in sample matrix that generated a signal-to-noise ratio of ≥10. Recovery was tested by comparing area of deuterated standards added to pooled human serum (mix of ≥10 equal serum aliquots from healthy normal subjects) versus methanol, and calculated according to the following formula: % recovery = (average area spiked in serum pool/average area spiked in methanol) x100.

A targeted steroid hormone panel from plasma including extraction was performed by the West Coast Metabolomics Center at the University of California—Davis (Director—Dr. Oliver Fiehn). The extraction protocol was adapted from Pedersen and Newman. 2018. Plasma sample was vortexed and 50 µL of Surrogate Standards of the 3 lipid classes was added along with CDU and vortexed again for 30sec. Samples were centrifuged at 6°C for 5 min at 15,000 g and the supernatant (∼240 µL) was transferred into new Eppendorf tube/filter plate Spin Filter (0.1 um) and stored in −20°C until analysis. The samples were run using Thermo Scientific Vanquish Horizon UPLC/SciEx QTrap 6,500+.

Lipids were extracted by addition of 2 mL hexane/isopropanol (3:2, v/v) or chloroform/methanol (2:1, v/v) from the cell monolayers and liver tissue, respectively. 24–25- and 27- hydroxy cholesterol levels were quantified by isotope dilution mass spectrometry as previously described (Dzeletovic et al., 1995). Oxysterol levels were normalized for the total protein content measured using the RC DC™ Protein Assay (BioRad Laboratories Inc., United States) in liver tissue after digestion with NaOH (0.25 mol/L).

The AbsoluteIDQ® p400 HR kit from Biocrates Life Sciences AG was used to obtain targeted quantitative metabolomics data as we have previously described (Osborn et al., 2019). Liver tissue was collected at time of necropsy and samples were prepared according to manufacturer protocol. Briefly, at least 30 mg of each tissue sample was cut and weighed, and kept frozen throughout this protocol. The tissue samples were homogenized and centrifuged at 10,000 × g for 5 min. The supernatant was then collected, 10 μL of which per sample was loaded onto a 96-well plate containing stable isotope-labeled standards, and processed according to manufacturer protocol. The LCMS analysis was done using specific parameters (both Tune and LCMS methods) per kit manufacturer’s recommendations. The assay was performed on a Q-Exactive HF (operated only in positive ESI mode) coupled with a Vanquish UHPLC + focused liquid chromatography as detailed per assay instructions. The manufacturer-provided software, MetIDQ (Biocrates, Life Science AG), was used to provide the peak identification.

Tissue RNA extraction and quantitative polymerase chain reaction (qPCR) analyses of relative mRNA abundance were conducted as previously described (Helsley et al., 2019; Pathak et al., 2020). mRNA expression levels were calculated based on the ΔΔ-CT method using Cyclophilin A as a housekeeping gene. qPCR was conducted using SYBR Fast reagents (AB#4385612) on the Applied Biosystems 7,500 Real-Time PCR System. Primers used for qPCR are CycloA F - GCG​GCA​GGT​CCA​TCT​ACG, CycloA R - GCC​ATC​CAG​CCA​TTC​AGT​C, Gramd1a F - CAT​GCA​CAC​CTC​AGG​TTC​CC, Gramd1a R - ACG​ATG​AGG​ACA​ATG​CTG​ATG, Gramd1b F - GCT​GGT​TAT​CAG​CTG​TGT​TCT​G, Gramd1b R - GTG​AGG​GTC​TGG​GTG​GTG​TA, Gramd1c F -CAG​TTA​TGA​CAC​CGC​CCT​TAT Gramd1c R - CTG​GGT​AGC​GTG​TTC​TAT​CTT​T. RNA extraction, library preparation, sequencing and analysis was conducted at Azenta Life Sciences (South Plainfield, NJ, United States) as follows: Extraction: Total RNA was extracted using Qiagen Rneasy Plus Universal Mini kit following manufacturer’s instructions (Qiagen, Hilden, Germany). Library Preparation with PolyA selection and Illumina Sequencing: RNA samples were quantified using Qubit 2.0 Fluorometer (Life Technologies, Carlsbad, CA, United States) and RNA integrity was checked using Agilent TapeStation 4,200 (Agilent Technologies, Palo Alto, CA, United States). RNA sequencing libraries were prepared using the NEBNext Ultra RNA Library Prep Kit for Illumina using manufacturer’s instructions (NEB, Ipswich, MA, United States). Briefly, mRNAs were initially enriched with Oligod(T) beads. Enriched mRNAs were fragmented for 15 min at 94°C. First strand and second strand cDNA were subsequently synthesized. cDNA fragments were end repaired and adenylated at 3′ends, and universal adapters were ligated to cDNA fragments, followed by index addition and library enrichment by PCR with limited cycles. The sequencing library was validated on the Agilent TapeStation (Agilent Technologies, Palo Alto, CA, United States), and quantified by using Qubit 2.0 Fluorometer (Invitrogen, Carlsbad, CA) as well as by quantitative PCR (KAPA Biosystems, Wilmington, MA, United States). The sequencing libraries were multiplexed and clustered onto a flowcell on the Illumina NovaSeq instrument according to manufacturer’s instructions. The samples were sequenced using a 2 × 150 bp Paired End (PE) configuration. Image analysis and base calling were conducted by the NovaSeq Control Software (NCS). Raw sequence data (.bcl files) generated from Illumina NovaSeq was converted into fastq files and de-multiplexed using Illumina bcl2fastq 2.20 software. One mis-match was allowed for index sequence identification. Transcript per million (TPM) values for each differentially expressed gene were plotted using Prism Software and the statistical analysis was done using JMP software.

Whole liver homogenates were made from tissues in a modified RIPA buffer (Abcam #156034) as previously described (Helsley et al., 2019; Pathak et al., 2020), and protein was quantified using the bicinchoninic (BCA) assay (Pierce). Proteins were separated by 4%–12% SDS-PAGE, transferred to polyvinylidene difluoride (PVDF) membranes (ThermoSci #88518), and proteins were detected after incubation with an antibody recognizing mouse ASTER-C (generated here) or β-actin (Cell Signaling Technologies product # 4970S). Given the paucity of available antibodies, a custom rabbit polyclonal antibody was generated in collaboration with Thermofisher. The antigen used was the N-terminus 34 kDa mouse Aster-C protein (Uniprot Q8CI52; amino acids 1–300). A 72 days injection protocol with a primary immunization and three subsequent boosters was followed, and the terminal 72 days crude anti-sera sample (50 mL) was affinity purified (expressed protein conjugated to the affinity column) by ThermoFisher. Affinity-purified antibodies were eluted using a step-wise pH gradient and collected in neutralizing buffer. Purified antibodies were then concentrated and final concentration is measured by BCA. Membrane was blocked with 5% milk in Tris buffered saline containing 0.2% Tween 20.1:500 dilution of this Gramd1c antibody in Blocking buffer was used for Western blotting and the bands were detected using an anti-rabbit-HRP conjugated secondary antibody.

CRISPR-Cas9 genome editing was accomplished using methods previously described (Ran et al., 2013). Gramd1c sgRNAs were designed by an online tool (https://www.benchling.com/) and cloned into the Lenti-CRISPER v2 vector (Addgene (Ran et al., 2013) with D10A nickase version of Cas9 (Cas9n)). Gramd1c KO cell lines were generated following lentiviral transduction of the Lenti-CRISPER v2-Cas9 D10A- Gramd1c sgRNA in RAW264.7cells. Gramd1c KO cells were validated by analyzing the expression of GRAMD1c by Western blot. Primers used for gene editing were: GRAMD1c-E5-Nick-5F: 5′-CAC​CGC​AGA​GCA​CCC​TCC​AAG​TCA​C-3′, GRAMD1c-E5-Nick-5R: 5′-AAA​CGT​GAC​TTG​GAG​GGT​GCT​CTG​C-3′, GRAMD1c-E5-Nick-3F: 5′- CAC​CGC​TCT​TTT​CTG​AAG​CTA​GCG​A-3′, and GRAMD1c-E5-Nick-3R: 5′- AAA​CTC​GCT​AGC​TTC​AGA​AAA​GAG​C-3’.

To examine whether Aster-C can alter systemic cholesterol metabolism under normal physiologic conditions, we fed young wild type (Gramd1c+/+) or global Gramd1c knockout mice (Gramd1c−/−) one of two experimental diets containing low (0.02%, wt/wt) or high (0.2%, wt/wt) levels of dietary cholesterol for a period up to 4 weeks. Deletion of Gramd1c was confirmed in the liver, where both messenger RNA (Figure 1A) and protein (Figure 1B) were significantly reduced in Gramd1c−/− mice compared to littermate controls (Gramd1c+/+). Gramd1c−/− mice were born at normal Mendelian ratios and were indistinguishable from wild type littermates. Irrespective of dietary cholesterol level, Gramd1c−/− mice maintained similar body weights compared to wild type mice throughout the study (Figure 1C). When we examined either the total, unesterified (free), or esterified levels of cholesterol in the plasma, Gramd1c−/− mice were not significantly different when compared to wild type controls (Figures 2B–D). Likewise, the cholesterol levels in very low-density (VLDL), low-density (LDL), or high-density lipoproteins (HDL) were similar when comparing Gramd1c+/+ to Gramd1c−/− mice (Figures 2A,E,G). Given plasma cholesterol levels were unchanged in Gramd1c−/− mice (Figure 2), we wanted to look more broadly at other potential sterol substrates including several phytosterols and biosynthetic intermediates generated during the de novo synthesis of cholesterol. It is important to note that Gramd1c−/− mice had normal circulating levels of 7-dehydrocholesterol, desmosterol, lanosterol, lathosterol, β-sitosterol, campesterol, brassicasterol, and stigmasterol (Figure 3). These data show that Aster-C alone does not play a quantitatively important role in determining the circulating levels of cholesterol or other diverse sterols of plant or mammalian origin under conditions of low and excess dietary cholesterol.

Figure 1. Selective loss of Gramd1c does not alter body weight. At 8 weeks of age, female wild type (Gramd1c+/+) or Gramd1c knockout mice (Gramd1c−/−) were switched from standard rodent chow to one of two experimental synthetic diets containing low (0.02%, wt/wt) or high (0.2%, wt/wt) levels of dietary cholesterol. Mice were maintained on these diets over a 4-week period of study. (A) The expression of Gramd1a, Gramd1b, and Gramd1c in the liver was quantified via qPCR. (B) Western blot in liver lysates from n = 3 mice per group. (C) Body weight curves over the 4-week feeding period. Data are presented as mean ± SEM from n = three to six mice per group. There were no statistically significant differences between Gramd1c+/+ and Gramd1c−/− mice on either the low or high cholesterol diets.

Figure 2. Global deletion of Gramd1c does not significantly alter circulating lipoprotein cholesterol levels. At 8 weeks of age, female wild type (Gramd1c+/+) or Gramd1c knockout mice (Gramd1c−/−) were switched from standard rodent chow to one of two experimental synthetic diets containing low (0.02%, wt/wt) or high (0.2%, wt/wt) levels of dietary cholesterol. Mice were maintained on these diets over a 4-week period of study. (A) Plasma was subjected to size exclusion chromatography to examine total cholesterol distribution across lipoprotein fractions. (B–D) Total plasma cholesterol (B), total plasma free cholesterol (C), and total plasma esterified cholesterol (D) levels were determined by gas chromatography-tandem mass spectrometry (GC-MS/MS). (E–G) Plasma very low-density lipoprotein (VLDL), low-density lipoprotein (LDL), and high-density lipoprotein (HDL) cholesterol levels. Data are presented as mean ± SEM from n = three to six mice per group. There were no statistically significant differences between Gramd1c+/+ and Gramd1c−/− mice on either the low or high cholesterol diets.

Figure 3. Global deletion of Gramd1c does not significantly alter circulating levels of various sterols. At 8 weeks of age, female wild type (Gramd1c+/+) or Gramd1c knockout mice (Gramd1c−/−) were switched from standard rodent chow to one of two experimental synthetic diets containing low (0.02%, wt/wt) or high (0.2%, wt/wt) levels of dietary cholesterol. Mice were maintained on these diets over a 4-week period of study. The total and free levels of a variety of sterols in the liver were quantified by gas chromatography tandem mass spectrometry (GC-MS/MS) in samples with or without alkaline hydrolysis. Data are presented as mean ± SEM from n = five to six mice per group. There were no statistically significant differences between Gramd1c+/+ and Gramd1c−/− mice on either the low or high cholesterol diets.

We next turned our attention to test whether Aster-C plays a role in sterol homeostasis in the liver, given the central role the liver plays in whole body cholesterol balance. Here, we applied several complimentary and broad targeted lipidomic methods to quantify diverse sterols, but also major classes of glycerophospholipids and neutral lipids. Similar to findings in plasma (Figure 2), compared to wild type mice, Gramd1c−/− mice have unaltered levels of total, free, and esterified cholesterol in the liver (Figures 4A–C). Gramd1c+/+ and Gramd1c−/− mice also had similar hepatic levels of 7-dehydrocholesterol, desmosterol, lanosterol, lathosterol, β-sitosterol, campesterol, brassicasterol, and stigmasterol (Figure 5). We next examined the hepatic levels of the major oxysterol species that are generated from cholesterol substrate (24-hydroxycholesterol and 27-hydroxycholesterol), and found similar levels in Gramd1c+/+ and Gramd1c−/− mice (Figures 4D,E). Finally, we performed a broad lipidomic approach to examine a variety of glycerophospholipid and neutral lipids in the liver. Gramd1c+/+ and Gramd1c−/− mice had similar levels of all molecular species of glycerophospholipids detected (data not shown). However, this broader method was able to uncover a modest increase in cholesterol esters containing a 17:2 fatty acyl chain in high cholesterol diet-fed mice (Figure 4F). Yet, all other molecular species of cholesteryl esters were unchanged in Gramd1c−/− mice (Figure 4G). Furthermore, under low dietary cholesterol conditions, one specific molecular species of triacylglycerol (TG 58:8) was increased in Gramd1c−/− mice (Figure 4H), but all other detected species of triacylglycerol were similar to wild to mice (Figures 4I,J).

Figure 4. The hepatic lipidome is modestly impacted by global deletion of Gramd1c. At 8 weeks of age, female wild type (Gramd1c+/+) or Gramd1c knockout mice (Gramd1c−/−) were switched from standard rodent chow to one of two experimental synthetic diets containing low (0.02%, wt/wt) or high (0.2%, wt/wt) levels of dietary cholesterol. Mice were maintained on these diets over a 4-week period of study. A variety of lipids were quantified by targeted mass spectrometry assays including (A) total cholesterol, (B) free cholesterol, (C) total esterified cholesterol, (D) 24-hydoxycholesterol (24-OH Chol.), (E) 27-hydroxycholesterol (27-OH Chol.) (F) 17:2 cholesteryl ester (CE 17:2), (G) 18:2 cholesteryl ester (CE 18:2), (H) 58:8 triacylglycerol (TG 58:8), (I) 56:7 triacylglycerol (TG 56:7), and (J) 52:7 triacylglycerol (TG 52:7). Data are presented as mean ± SEM from n = 6 mice per group. * = significantly different (p ≤ 0.05) when comparing Gramd1c+/+ and Gramd1c−/− mice within each diet group.

Figure 5. Global deletion of Gramd1c does not significantly alter levels of various sterols in the liver. At 8 weeks of age, female wild type (Gramd1c+/+) or Gramd1c knockout mice (Gramd1c−/−) were switched from standard rodent chow to one of two experimental synthetic diets containing low (0.02%, wt/wt) or high (0.2%, wt/wt) levels of dietary cholesterol. Mice were maintained on these diets over a 4-week period of study. The total and free levels of a variety of sterols in the liver were quantified by gas chromatography tandem mass spectrometry (GC-MS/MS) in samples with or without alkaline hydrolysis. Data are presented as mean ± SEM from n = 6 mice per group. There were no statistically significant differences between Gramd1c+/+ and Gramd1c−/− mice on either the low or high cholesterol diets.

A recent report by Xiao and colleagues reported that mice lacking both Aster-A and Aster-C selectively in hepatocytes have impaired reverse cholesterol transport (RCT) (Xiao et al., 2023). This important work showed that Aster-A/Aster-C double knockout mice have reduced movement of cholesterol radiotracer (14C-cholesterol) into the feces when either derived from LDL or HDL sources (Xiao et al., 2023). However, they did not test whether Aster-C alone can impact the mass amount of cholesterol excreted in the feces. When we examined the quantitative fecal excretion of cholesterol in global Gramd1c−/− mice fed low or high cholesterol, there was no significant difference when compared to wild type littermates (Figure 6). Likewise, the fecal excretion of other phytosterols and sterol intermediates such as 7-dehydrocholesterol, desmosterol, lanosterol, lathosterol, β-sitosterol, campesterol, brassicasterol, and stigmasterol were similar in Gramd1c+/+ and Gramd1c−/− mice (Figure 6). Another way the liver can facilitate RCT is via the enzymatic conversion of cholesterol into bile acids, which can further contribute to removal of cholesterol from the body. Although the majority of plasma bile acid species were unchanged in Gramd1c−/− mice, levels of deoxycholic acid were significantly reduced in Gramd1c−/− mice fed a low cholesterol diet (Figure 7). Given the clear role that Aster-B plays in adrenal gland cholesterol homeostasis (Sandhu et al., 2018), we also wanted to understand whether Aster-C may play a role in cholesterol-derived hormone production. Only under low dietary cholesterol conditions, Gramd1c−/− mice have elevated plasma cortisol compared to wild type controls (Figure 8A). However, all other steroid hormones measured including corticosterone, progesterone, 17-α-hydroxyprogesterone, allo-pregnanolone, β-pregnanolone, dehydroepiandrosterone (DHEA) sulfate, cortexolone, androstenedione, free DHEA, dihydrotestosterone, and aldosterone were unaltered in Gramd1c−/− mice (Figures 8B–L).

Figure 6. Global deletion of Gramd1c does not significantly alter fecal sterol loss. At 8 weeks of age, female wild type (Gramd1c+/+) or Gramd1c knockout mice (Gramd1c−/−) were switched from standard rodent chow to one of two experimental synthetic diets containing low (0.02%, wt/wt) or high (0.2%, wt/wt) levels of dietary cholesterol. Mice were maintained on these diets over a 4-week period of study, and feces were quantitatively collected over the final 72 h for fecal sterol analyses via gas chromatography tandem mass spectrometry (GC-MS/MS). Data are presented as mean ± SEM from n = 6 mice per group. There were no statistically significant differences between Gramd1c+/+ and Gramd1c−/− mice on either the low or high cholesterol diets.

Figure 7. The impact of Gramd1c deficiency on plasma bile acids. At 8 weeks of age, female wild type (Gramd1c+/+) or Gramd1c knockout mice (Gramd1c−/−) were switched from standard rodent chow to one of two experimental synthetic diets containing low (0.02%, wt/wt) or high (0.2%, wt/wt) levels of dietary cholesterol. Mice were maintained on these diets over a 4-week period of study. The levels of various bile acids including (A) deoxycholic acid (DCA), (B) lithocholic acid (LCA), (C) β-muricholic acid (β-MCA), (D) ω/α-muricholic acid (ω/α-MCA), (E) taurodeoxycholic acid (Taura-DCA), (F) taurochenodeoxycholic acid (Tauro-CDCA), (G) tauro-β-muricholic acid (Taura-β-MCA), and (H) tauro-ω/α-muricholic acid (Tauro-ω/α-MCA) were quantified by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Data are presented as mean ± SEM from n = three to four mice per group. * = significantly different (p ≤ 0.05) when comparing Gramd1c+/+ and Gramd1c−/− mice within each diet group.

Figure 8. The impact of Gramd1c deficiency on plasma steroid hormones. At 8 weeks of age, female wild type (Gramd1c+/+) or Gramd1c knockout mice (Gramd1c−/−) were switched from standard rodent chow to one of two experimental synthetic diets containing low (0.02%, wt/wt) or high (0.2%, wt/wt) levels of dietary cholesterol. Mice were maintained on these diets over a 4-week period of study. The plasma levels of various steroid hormones including (A) cortisol, (B) corticosterone, (C) progesterone, (D) 17-α-hydroxyprogesterone, (E) allo-pregnanolone, (F) β-pregnanolone, (G) dehydroepiandrosterone (DHEA) sulfate, (H) cortexolone, (I) androstenedione, (J) dehydroepiandrosterone (DHEA), (K) dihydrotestosterone, and (L) aldosterone were measured semi-quantitatively by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Data are presented as mean ± SEM from n = 5 mice per group.). * = significantly different (p ≤ 0.05) when comparing Gramd1c+/+ and Gramd1c−/− mice within the low cholesterol diet.

We next performed bulk RNA sequencing to understand the effects of Gramd1c deletion on global gene expression in the liver (Figure 9). The most differentially expressed gene was Gramd1c, which was markedly reduced in Gramd1c−/− mice compared to wild type Gramd1c+/+ controls in both low and high cholesterol diet conditions (Figure 9). It is important to note that several sterol-responsive genes including proprotein convertase subtilisin/kexin type 9 (Pcsk9), squalene epoxidase (Sqle), low density lipoprotein receptor (Ldlr), 3-hydroxy-3-methylglutaryl-CoA synthase 1 (Hmgcs1), and 7-dehydrocholesterol reductase (Dhcr7) were significantly repressed under high dietary cholesterol conditions, but the dietary cholesterol-driven suppression of these genes was similar in both Gramd1c+/+ and Gramd1c−/− mice (Figure 9). Unexpectedly, the expression of two mitochondrially-encoded genes, mitochondria encoded tRNA-Ile (AUU/C) (Mt-Ti) and mitochondrially-encoded tRNA-Gln (CAA/G) (Mt-Tq), were elevated in Gramd1c−/− mice only under low dietary cholesterol conditions (Figure 9). In parallel, only under low dietary cholesterol conditions, the expression of several members of the cytochrome P450 family 3 subfamily A genes (Cyp3a41a, Cyp3a41b, Cyp3a44, and Cyp3a16) were significantly lower in Gramd1c−/− mice when compared to Gramd1c+/+mice (Figure 9). Also, the hepatic expression of several genes encoding major urinary proteins (Mup7, Mup11, Mup12, and Mup20) were elevated in Gramd1c−/− mice, only under low dietary cholesterol conditions (Figure 9). Other genes altered in low cholesterol-fed Gramd1c−/− mice includes solute transport genes (Slc22a28 and Slc41a2), sulfotransferase family 3A, member 2 (Sult3a2), and sushi domain-containing 4 (Susd4). These data show that, particularly under low dietary cholesterol conditions, Gramd1c deficiency is associated with reduced expression of several genes involved in xenobiotic metabolism (Cyp3a41a, Cyp3a41b, Cyp3a44, Cyp3a16, and Sult3a2) as well as increased expression of major urinary protein genes (Mup7, Mup11, Mup12, and Mup20) that encode lipid-binding proteins within the lipocalin family.

Figure 9. Bulk RNA sequencing in the liver of Gramd1c−/− mice identifies alterations in a small subset of genes. At 8 weeks of age, female wild type (Gramd1c+/+) or Gramd1c knockout mice (Gramd1c−/−) were switched from standard rodent chow to one of two experimental synthetic diets containing low (0.02%, wt/wt) or high (0.2%, wt/wt) levels of dietary cholesterol. Mice were maintained on these diets over a 4-week period of study, and thereafter bulk RNA sequencing was performed to identify differentially-expressed genes. Data are presented as mean ± SEM from n = four to five mice per group. * = significantly different (p ≤ 0.05) when comparing Gramd1c+/+ and Gramd1c−/− mice within each diet group.

Given previous reports (Sandhu et al., 2018) have shown that Gramd1b can be a direct transcriptional target of the nuclear hormone receptor liver X receptor (LXR), we next treated male Gramd1c+/+ and Gramd1c−/− mice with a pharmacologic LXR agonist (Figure 10). Much like in female mice (Figures 1–9), males lacking Gramd1c−/− had normal plasma and liver cholesterol levels both under vehicle and GW3965-treated conditions (Figures 10A–G). Although there were some modest trends, Gramd1c−/− mice also did not have statistically significant differences in hepatic oxysterol levels (Figures 10H,I). Although Gramd1c−/− mice did not have altered steroid hormone levels under vehicle-treated conditions (Figures 10J–L), upon LXR agonist treatment Gramd1c−/− mice had slightly elevated plasma cortisol levels and reduced levels of corticosterone (Figure 10K) compared to Gramd1c+/+ mice. All other steroid hormones measured were not altered in Gramd1c−/− mice (Figure 10L).

Figure 10. Global deletion of Gramd1c does not significantly alter circulating or hepatic cholesterol levels in male mice. At 8 weeks of age, male wild type (Gramd1c+/+) or Gramd1c knockout mice (Gramd1c−/−) were maintained on a standard chow diet in the absence (vehicle) or presence of the LXR agonist GW3965 (40 mg/kg per day) for 7 consecutive days. (A) Total plasma cholesterol levels were measure by gas chromatography-tandem mass spectrometry (GC-MS/MS). (B–D) Plasma was subjected to size exclusion chromatography to examine total cholesterol distribution across very low density (VLDL), low density (LDL), or high density (HDL) lipoprotein fractions. In addition, a variety of hepatic lipids were quantified by targeted mass spectrometry assays including (E) liver total cholesterol, (F) liver free cholesterol, (G) liver esterified cholesterol, (H) 24-hydoxycholesterol (24-OH Chol.), and (I) 27-hydroxycholesterol (27-OH Chol.) The plasma levels of various steroid hormones including (J) cortisol, (L) corticosterone, and (L) dehydroepiandrosterone (DHEA) were measured semi-quantitatively by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Data are presented as mean ± SEM from n = four to six mice per group. *(p ≤ 0.05), **(p ≤ 0.01) = significantly different when comparing Gramd1c+/+ and Gramd1c−/− mice within each drug treatment group.

It has been nearly 5 years since the original discovery of the Aster protein family as novel regulators of non-vesicular transport of PM cholesterol to the ER (Sandhu et al., 2018). This seminal report unequivocally established that Aster-B plays an essential roles in cholesterol balance and steroid hormone production, given that Gramd1b−/− mice have very low levels of cholesteryl ester storage in the adrenal gland (Sandhu et al., 2018). However, the cell autonomous and systemic roles of Aster-A and Aster-C in cholesterol uptake and metabolism have been more elusive. Given the tissue-specific expression patterns of Aster proteins, and unique nuclear hormone receptor-driven transcriptional control (i.e., LXR-driven transcription of Gramd1b and FXR-driven transcription of Gramd1c) (Sandhu et al., 2018; Xiao et al., 2023; Ferrari et al., 2023), it is logical to assume that each Aster protein may play unique roles in choleste