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. Author manuscript; available in PMC: 2026 Jul 19.
Published in final edited form as: Mol Cell Endocrinol. 2025 Jul 19;608:112615. doi: 10.1016/j.mce.2025.112615

Palmitate-induced Downregulation of Lipocalin Prostaglandin D2 Synthase Accompanies Hepatic Lipid Accumulation in HepG2 cells

Rhema Khairnar 1, Md Asrarul Islam 1, Divya K Shetty 1, Vikas V Dukhande 1, Sunil Kumar 1,*
PMCID: PMC12337296  NIHMSID: NIHMS2099886  PMID: 40692003

Abstract:

Metabolic dysfunction-associated steatotic liver disease (MASLD) is associated with multiple metabolic dysfunctions and poses a significant global health challenge. Our prior in vivo studies demonstrated that the absence of lipocalin prostaglandin D2 synthase (L-PGDS) leads to the development of fatty liver disease, and L-PGDS expression significantly decreased when C57BL/6 mice were kept on a high-fat diet. Briefly, L-PGDS belongs to the arachidonic acid pathway and enzymatically isomerizes prostaglandin H2 to prostaglandin D2, which imparts pharmacological effects via two receptors called DP1 and DP2. L-PGDS is an essential key player in fatty liver disease, but its mechanistic regulation still remains unknown. Therefore, we aimed to study the mechanistic regulation of L-PGDS using a palmitate-induced cellular MASLD model. We successfully recapitulated the MASLD phenotype in HepG2 cells with palmitate treatment. Our results showed significant lipid accumulation and increased lipid-associated protein and gene expression, along with palmitate concentration-dependent L-PGDS downregulation. To study the L-PGDS downregulation, we employed MG132, chloroquine, and cycloheximide to assess proteasomal degradation, autophagy, and translational activity, respectively. Our gene and protein expression data suggested the possible reason for L-PGDS downregulation via inhibiting transcription and subsequently translation. Additionally, our autophagy results also showed a role in L-PGDS downregulation. In summary, it can be concluded that palmitate treatment downregulated L-PGDS, possibly involving transcription-translation and/or autophagy pathways. However, further studies are needed to delineate the precise molecular mechanism and apply this knowledge to MASLD pathogenesis and treatment.

Keywords: MASLD, L-PGDS, PGD2, Autophagy, Hepatic Lipid Metabolism

Graphical Abstract

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1. Introduction:

Metabolic dysfunction-associated steatotic liver disease (MASLD) is a liver disease associated with multiple metabolic dysfunctions, including obesity and diabetes. MASLD is considered the most common chronic liver disease worldwide, affecting one-third of the population (1, 2). As part of the spectrum of the disease, triglyceride accumulates in the liver and enhance de novo lipogenesis (3–5), thereby exacerbating hepatic injury in a destructive cycle (6, 7). Research in recent years has significantly advanced our understanding of the etiology behind lipid accumulation in the liver; however, the underlying molecular mechanisms at play are still unknown. Therefore, at present, there is a limited treatment available for fatty liver disease, and patients rely on lifestyle interventions that remain ineffective at advanced stages of the disease (8). Recently, Resmetirom has been approved for the treatment of MASH, but not MAFLD(9), which still remains an area of research to delineate the molecular underpinnings of triglyceride accumulation in MASLD and, thus, to reveal new targets and therapeutic options.

In line with this need, our prior research focused on the role of lipocalin-type prostaglandin D2 synthase (L-PGDS), a protein known to be dysregulated in metabolic disorders like obesity, diabetes, and MASLD. L-PGDS is a highly glycosylated protein and integral to arachidonic acid metabolism. Enzymatically, L-PGDS is responsible for isomerizing prostaglandin H2 (PGH2) to prostaglandin D2 (PGD2) which further leads to formation of numerous prostaglandins, including 15-deoxy-Δ12,14 prostaglandin J2 (15-deoxy-Δ12,14PGJ2), 15-deoxy-Δ12,14PGJ2 is a potent endogenous ligand for peroxisome proliferator-activated receptor gamma (PPARγ), which plays a crucial role in lipid and carbohydrate metabolism to PGD2 (10–12).

Our prior study, where we investigated the role of L-PGDS by performing an extensive study utilizing L-PGDS knockout mice, showed that these mice exhibited an MASLD phenotype accompanied by insulin resistance and weight gain when kept on a high-fat diet for 14 weeks (13). This finding was the first where the role of L-PGDS in MASLD was ever identified, and strongly suggested that a lack of L-PGDS potentially contributes to developing or exacerbating fatty liver disease. In a similar line of this finding, another in vivo study was conducted where C57BL/6 mice were kept on a high-fat diet for 22 weeks to study MASLD. It showed significantly reduced hepatic L-PGDS expression compared to the control group (12). Therefore, a precise research approach is needed to accomplish our long-term research goal of understanding the mechanistic role of L-PGDS in MASLD and applying this knowledge to develop novel therapeutic strategies. To attain this goal, we aimed to understand the mechanistic regulation and metabolic signaling of L-PGDS in MASLD.

Our central hypothesis is that L-PGDS is a key player in regulating glucose and lipid metabolism in the liver, and its dysfunction can contribute to MASLD. Palmitate, being a saturated fatty acid and a key component of triglycerides, palmitate-induced lipid accumulation in HepG2 hepatocytes has been the common model to study the pathogenesis of fatty liver disease (14). Therefore, we hypothesize that palmitate-treated HepG2 cells will show enhanced lipid accumulation and reduced L-PGDS expression in line with prior in vivo findings. Reduced L-PGDS expression is speculated to involve multiple mechanisms, including proteosomal degradation, autophagy, or ubiquitination. Briefly, Ubiquitination is a form of post-translational modification (PTM) in which ubiquitin is attached to a target protein, playing versatile roles in protein degradation and other cellular processes. Ubiquitin is a 76-amino-acid protein that exists in the free form or can be conjugated to a protein as a single ubiquitin (monoubiquitination) or as multiple ubiquitins (polyubiquitination) (15).

To test this hypothesis, we utilized a cellular in vitro steatotic model to recapitulate the human disease condition and investigated the biochemical regulation of L-PGDS in a palmitate-induced in vitro fatty liver disease model.

2. Materials and Methods:

2.1. Cell culture and Sodium Palmitate Treatment:

Human immortalized hepatocellular carcinoma cell line, HepG2, was acquired from ATCC (Manassas, Virginia, USA). The cell line was maintained in Dulbecco’s modified Eagle’s medium (DMEM, Corning, Cat#10013CV), supplemented with 2% fetal bovine serum (FBS; Atlanta Biologics, Georgia, USA) and 1% penicillin-streptomycin (PS; Corning, Cat#30-002-CI). All cells were maintained at 37°C in a humidity-controlled incubator with 5% CO2.

Cells were treated with Sodium Palmitate (Thermo Fisher Scientific, Cat#416700050), and its abbreviated form, palmitate, will be used throughout the manuscript. Palmitate was dissolved in a 50% ethanol + 50% distilled water. After adding Palmitate, the buffer was heated to 70°C for 20-30 minutes to dissolve and prepare the stock solution of 100 mM. The stock solution was stored at -20°C, and the desired concentrations of palmitate solutions were prepared from the stock solution in 1% w/v free fatty acid (FFA) free bovine serum albumin (BSA), and 1% PS supplemented DMEM medium and solution was kept on a shaker for 2 hours at 37 °C for binding before used for the experiment(16).

2.2. MTT Assay:

HepG2 cells were seeded in a 96-well plate at a density of 0.5 × 104 cells/well. The MTT assay was performed to determine the cell viability in various concentrations of Palmitate treatment. Thiazolyl Blue tetrazolium bromide (MTT reagent, Thermo Fisher Scientific, Cat#L1193903) was utilized at a stock concentration of 5 mg/mL and final media concentration of 0.5 mg/mL. After palmitate treatment, both control and treatment groups were incubated with the MTT reagent for 3-4 hrs at 37°C. Subsequently, the media was carefully removed, and 200 μL of DMSO was added and shaken on an orbital shaker for 10 minutes to dissolve the formazan crystals formed by the MTT reagent, and the absorbance of the solution was measured at 570 nm (17).

2.3. Oil Red O Staining:

Oil red O staining was performed to determine lipid accumulation in response to Palmitate treatment. After palmitate treatment, the cells were fixed with 10% formalin. Fixed cells were incubated with 60% isopropanol for 5 minutes, and then the solution was discarded, and the cells were dried. Next, cells were stained with 60% Oil-Red-O (Electron Microscopy Sciences, Cat#26503-02) for 10 minutes and washed with water 3-4 times (until the water came clean). The cells were counter-stained with hematoxylin (18), and pictures were captured at 40X magnification using the EVOS XL Core Imaging System. The Oil-red-O was eluted with 100% isopropyl alcohol, and then the absorbance was measured at 492 nm following a standard published protocol(19).

2.4. ELISA:

Prostaglandin D2 levels in the cell lysate of HepG2 cells in control, vehicle, and palmitate-treated groups were measured using an ELISA kit (Cayman Chemicals, Cat#512031). Data were calculated and analysed following the kit specifications.

2.5. MG132 Treatment:

HepG2 cells were cultured, and when the cells were fully adhered, they were treated with 300 μM palmitate for 24 hours. After 4 hours, all the groups were exposed to 0.03μM of MG132 (APEXBIO, Cat#A2585), a proteasome inhibitor (20) , to determine if the proteasomal degradation is the cause of a decrease in L-PGDS expression. Cells were harvested with RIPA buffer ( Thermo Fisher Scientific, Cat#J63306.AP) supplemented with protease and phosphatase inhibitors (Thermo Fisher Scientific, Cat#A32961). Similarly, for 4, 8, 16, and 20 hours of exposure to MG132, HepG2 cells were seeded, and then treatment started with 300 μM of palmitate; after 4 hours, cells were exposed to MG132 and then harvested after 4, 8, 16, and 20 hours. So, in each treatment group, HepG2 cells were exposed to Palmitate for 8, 12, 20, and 24 hours in total.

2.6. Cycloheximide Treatment:

Cycloheximide (CHX) (ACROS Organics, Cat#357420010), a translation elongation inhibitor (21), was used to inhibit protein translation from mRNA. HepG2 cells were seeded in 6-well plates, and after 24 hours, the control, the vehicle, and 300 μM Palmitate treatment started. After 4 hours, CHX was exposed to all groups at a 50 μg/mL concentration and harvested with RIPA buffer supplemented with protease and phosphatase inhibitors (Thermo Fisher Scientific, Cat#A32961), which is the 0 hours time point. Similarly, for 4, 8, 16, and 20 hours of exposure to CHX, HepG2 cells were seeded, and then treatment started with 300 μM of Palmitate. After 4 hours, cells were exposed to CHX and then harvested after 4, 8, 16, and 20 hours. So, in each treatment group, HepG2 cells were exposed to Palmitate for 8, 12, 20, and 24 hours in total.

2.7. Chloroquine Treatment:

Chloroquine (CQ) (Alfa Aesar, Cat#J64459), an autophagy inhibitor (22) , was used to check the role of autophagy in L-PGDS regulation. HepG2 cells were seeded in 6-well plates, and after 24 hours, the control, the vehicle, and 300 μM Palmitate treatment started. After 4 hours, CQ was exposed to all groups at a 20 μg/mL concentration and harvested with RIPA buffer supplemented with protease and phosphatase inhibitors (Thermo Fisher Scientific, Cat#A32961); this is the 0-hour time point. Similarly, for 4, 8, 16, and 20 hours of exposure to CHX, HepG2 cells were seeded, and then treatment started with 300 μM of Palmitate; after 4 hours, cells were exposed to CQ, and then harvested after 4, 8, 16, and 20 hours. So, HepG2 cells were exposed to Palmitate for 8, 12, 20, and 24 hours in total in each treatment group.

2.8. Immunoprecipitation:

After treatment, the media was aspirated from HepG2 cells, and the cells were washed with DPBS. Radioimmunoprecipitation assay buffer (RIPA) with protease and phosphatase inhibitor cocktail, N-ethylmaleimide (NEM, 5μM) (Sigma, Cat# 04260-5G-F), and MG-132 (5μM) was used to lyse the cells. Each sample was incubated with antibody (10 μL) against LPGDS overnight under gentle rotary agitation at 4 °C. Next, the samples were incubated for 2 hrs at 4 °C with 25 μL of Protein A agarose beads (Cell Signaling, Cat#9863) under gentle rotary agitation. The beads were then washed four times with ice-cold modified RIPA buffer, spun at 5,000g for 5 min., and incubated at 95°C for 10 min. in 100 μL of 2X SDS Page Dye (Novex™ Tris-Glycine SDS Sample Buffer (2X), Cat#LC2676) The samples were spun at 10,000g for 5 min, and the supernatant was collected for western blot analysis, followed by the immunoprecipitation method as explained previously (23).

2.9. Western Blot Analysis:

After palmitate treatment, the cells were lysed using RIPA buffer supplemented with protease and phosphatase inhibitors (Thermo Fisher Scientific, Cat#A32961) to obtain a whole-cell lysate. Subsequently, the protein was extracted from the lysed cells through centrifugation at 14,000 g for 15 minutes. The supernatant was collected, and the total protein content was quantified using a Pierce BCA protein assay kit (Thermo Fisher Scientific, Cat#23227). The protein lysates were subsequently subjected to western blotting using specific primary antibodies (12). After incubation with the primary antibodies (L-PGDS, Abcam, Cat# ab182141; FABP4, Cell Signaling, Cat#2120; PPARγ, Cell Signaling, Cat#2443; SREBP1, Invitrogen, Cat#MA5-16124; SCAP, Abcam, Cat#ab308060, LC3, Sigma, Cat#L8918), the PVDF membrane (Millipore Cat#IPVH00010) were incubated with species-specific peroxidase-conjugated secondary antibodies (Anti-mouse, Cell Signaling, Cat#7076 & Anti-rabbit, Cell Signaling, Cat#7074) at room temperature for 1-2 hours. The membranes were subsequently detected using Clarity™ Western ECL Substrate (Cat#1705060) and visualized using a ChemiDoc imaging system (Bio-Rad). The protein band densities were quantified using ImageJ software (NIH).

2.10. Total RNA extraction and real-time PCR (qPCR):

Total RNA was isolated from the cells using a PureLink RNA mini kit (Cat#12183018A, Thermo Fisher Scientific). cDNA was synthesized using a Reverse Transcriptase kit (Cat#4368814) following the manufacturer’s instructions. Total cDNA was then subsequently used for quantitative real-time PCR analysis. Real-time PCR was performed on an ABI QuantStudio 3 Real-Time PCR System (Applied Biosystems) using TaqMan assay PTGDS, Hs00168748_m1 for L-PGDS, Hs01115513_m1 for PPARγ, Hs01005622_m1 for FASn, and Hs02786624_g1 for GAPDH gene expression. Target gene mRNA expression levels were normalized using the housekeeping gene GAPDH. Gene expression changes were reported as fold change compared to control conditions.

2.11. Statistical Analysis:

Statistical analysis was performed using GraphPad Prism software. Data are presented as means ± SEM. Statistical testing for multiple comparisons between groups was performed using t-tests or a one-way ANOVA, wherever appropriate, followed by recommended corrections with Tukey’s multiple comparisons. Differences were considered statistically significant when P was less than 0.05.

3. Results:

3.1. Concentration-dependent effect of sodium palmitate on cell viability and lipid accumulation in HepG2 cells:

We first examined the concentration-dependent effect of sodium palmitate on cell viability and lipid accumulation in HepG2 hepatocytes. The cells were incubated with 150, 200, 250, 300, 350, 400, 450, and 500 μM of palmitate for 24 hr, and then cell viability was assessed. As shown in Fig. 1A, cells showed no significant toxicity up to 300 μM of palmitate; however, beyond 300 μM, cells showed substantial cell death. Next, we determined the effect of palmitate on lipid accumulation using the oil red O method. Similarly, hepatocytes showed significant lipid accumulation up to 300 μM compared to the control, as shown in Fig. 1B. Lipid accumulation was further confirmed by measuring the absorbance of oil red o solution, as shown in Fig. 1C, which clearly showed significantly increased absorbance at 300 μM palmitate treatment compared to the control.

Figure 1: The effect of Sodium Palmitate on HepG2 cell viability and lipid accumulation:

Figure 1:

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MTT assay using Sodium Palmitate (PA) (150, 200, 250, 300, 350, 400, 450, 500 μM) (1A). Oil-Red-O staining and absorbance (1B-C). Pictures were taken at 40X. Data are presented as mean±SEM; the statistical analysis was performed using one-way ANOVA, and significant p values are indicated by asterisks: **P < 0.01, ***P < 0.001, ****P < 0.0001.

3.2. Lipogenesis-associated protein expression increased with palmitate treatment:

HepG2 cells were treated with 300 μM palmitate for 24 hr, and lipogenesis-associated proteins were measured as shown in Fig. 2. As expected, FABP4 expression was also found to increase significantly in the palmitate group compared to the control group (Fig. 2A–B). Next, we measured PPARγ and found increased expression in the palmitate group compared to the control group, but it was insignificant (Fig. 2C–D). We also measured SREBP, both precursor and cleaved isoforms. Our results showed SREBP1 precursor expression unchanged; however, there was a significant increase in cleaved SREBP1 in palmitate treatment compared to control groups (Fig. E-G). In a similar line of lipogenic protein, SCAP protein expression showed reduced expression in the palmitate-treated group compared to the control group (Fig.H-I). Collectively, the concentration of 300 μM palmitate shows increased lipogenic protein expression corresponding to lipid accumulation data shown in Fig. 1.

Figure 2: Effect of palmitate on Lipid accumulation-related protein expression:

Figure 2:

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HepG2 cells were treated with palmitate for 24 hours. The blots show the protein expression, and the graph shows the relative band density of FABP4 (2A-B), PPARγ (2C-D), SREBP1 (2E-G), and SCAP (2H-I) among C (Control), Veh (Vehicle), and 300 μM of palmitate treatment groups. The density of the bands was measured with ImageJ, and the analysis data were presented as mean ± SEM and n=3. Significant differences between the groups are determined using appropriate one-way ANOVA, and significant p values are indicated by asterisks **P < 0.01.

3.3. Palmitate showed concentration-dependent decrease in L-PGDS expression and PGD2 level in HepG2 cells:

We further determined the concentration-dependent effect of sodium palmitate on L-PGDS expression in HepG2 hepatocytes. Cells were incubated with 150, 200, 250, 300, 350, 400, 450, and 500 μM of palmitate for 24 hr and then subjected to western blot for L-PGDS protein expression. Concentration-dependent L-PGDS protein expression was reduced and reached significance at 250 μM and above concentrations, as shown in Fig. 3A–B. Next, we measured the level of PGD2 using an ELISA kit. Similarly, as mentioned above, cells were treated with different palmitate concentrations for 24 hr, and PGD2 levels were measured using cell lysates. Similar to L-PGDS protein expression, palmitate concentration-dependently, levels of PGD2 were found to be significantly decreased and reached significance at 300 μM (Fig. 3C). Therefore, based on cell viability, lipid accumulation, and L-PGDS protein expression results, further experiments were carried out using 300 μM palmitate for 24-hour treatment in HepG2 cells.

Figure 3: Effect of different concentrations (150, 200, 250, 300, 350, 400, 450, 500 μM) of Sodium Palmitate for 24 hours on Lipocalin-type prostaglandin D synthase (L-PGDS):

Figure 3:

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Western Blots show the protein expression (3A), and the graph shows the relative band density of L-PGDS normalized by β-actin (3B). Effect of different concentrations (150, 200, 250, 300, 350, 400, 450, 500 μM) of palmitate for 24 hours on PGD2 level (3C), here n=4. Data is presented as mean±SEM; the statistical analysis was performed using one-way ANOVA, and significant p values are indicated by asterisks *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

3.4. Palmitate treatment showed significantly decreased L-PGDS protein expression independent of proteasomal degradation but partially dependent on autophagy in HepG2 cells:

Palmitate at 300 μM concentration showed a significant decrease in L-PGDS protein expression. This prompted us to determine the cause of the decrease in L-PGDS expression by performing an experiment using 300 μM palmitate-treated HepG2 cells in the presence or absence of proteasome inhibitor, MG 132, for 24 hours. Our results showed a significant decrease in L-PGDS expression in the palmitate-treated group in the presence or absence of MG 132 compared to the control groups, as shown in Fig. 4A–B. Similarly, an experiment was designed to investigate autophagy using chloroquine as an autophagy inhibitor (24). As expected, there was a decrease in L-PGDS expression in the 300 μM palmitate group in the absence of CQ, but there was a further decrease in L-PGDS expression in the presence of CQ, as shown in Fig. 4C–D. Next, we measured LC3 II expression in the presence or absence of CQ. Our results showed a significant increase in LC3 II expression in the palmitate-treated group compared to the control group in the absence of CQ; however, LC3 II expression remained unchanged in the presence of CQ, as shown in Fig. 4E–F.

Figure 4: Effect of MG132 and Chloroquine (CQ) on L-PGDS protein expression:

Figure 4:

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L-PGDS protein expression in the presence or absence of MG132 (0.03 μM) for C (Control), Veh (Vehicle), and 300 μM of palmitate treatment groups (4A). The graph shows the relative band density of the western blot normalized by β-actin (4B). L-PGDS protein expression in the presence or absence of CQ for C (Control), Veh (Vehicle), and 300 μM of palmitate treatment groups (4C). The graph shows the relative band density of L-PGDS normalized by GAPDH (4D). LC II protein expression for C (Control), Veh (Vehicle), and 300 μM of palmitate treatment groups (4E). The graph shows the relative band density of L-PGDS normalized by GAPDH (4F). The band density was measured with Image J, and the analysis data were presented as mean ± SEM and n=3. Significant differences between the groups are determined using appropriate t-tests, and significant p values are indicated by asterisks: *P < 0.05, **P < 0.0, *** P < 0.001.

3.5. Palmitate treatment showed significantly decreased L-PGDS protein expression independent of translation in HepG2 cells:

Since palmitate at 300 μM concentration showed a significant decrease in L-PGDS protein expression, we wanted to investigate if the decrease in L-PGDS expression is due to a reduction in protein synthesis. Therefore, we decided to perform two experiments, where HepG2 cells were treated with or without 300µM of palmitate for 24 hours, followed by treatment with or without CHX (50 μg/ml). Cells were harvested at 0, 2, 6, and 24 hours post-CHX treatment. As shown in Fig. 5A–B, there was no change in L-PGDS expression in the absence of palmitate and CHX; however, it was significantly decreased in the presence of CHX at 24 hr. However, L-PGDS expression was significantly decreased in the presence of palmitate alone and the combination with CHX at 24 hr treatment, as shown in Fig. 5C–D.

Figure 5: Cycloheximide (CHX) chase assay for LPGDS:

Figure 5:

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HepG2 cells were treated with or without 300µM of palmitate for 24 hours, followed by with or without CHX (50 μg/ml) treatment. Cells were harvested at 0, 2, 6, and 24 hours post-CHX treatment. L-PGDS protein expression was determined. Blot (5A-B) representing the absence of palmitate and (5C-D) in the presence of palmitate, along with their respective relative band density of the western blot normalized by GAPDH. Data are presented as mean ± SEM and n=3. Significant differences between the groups are determined using appropriate t-tests, and significant p values are indicated by asterisks: *P < 0.05, **P < 0.01.

3.6. Time-dependent effect of MG132, Cycloheximide (CHX), and Chloroquine (CQ) exposure on L-PGDS protein expression in palmitate-treated HepG2 cells:

To capture time-bound changes in L-PGDS expression, we further designed an experiment where HepG2 cells were treated with 300 μM palmitate in the presence and absence of MG132, CHX, and CQ at different time points, as shown in Fig. 6. Our results in the 20 and 24-hour treatment groups (Fig. 6G–J) showed a significant decrease in L-PGDS expression compared to their respective control group. However, early time 4, 8, and 16-hour groups showed no change in any L-PGDS expression.

Figure 6: Effect of MG132, Cycloheximide (CHX), and Chloroquine (CQ) exposure for 0 hours, 4 hours, 8 hours, 16 hours, and 20 hours on L-PGDS protein expression with absence and presence of 300µM of palmitate treatment for on HepG2 cells:

Figure 6:

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HepG2 cells were subjected to palmitate treatment for 4 hours followed by addition of MG132, CHX, and CQ and cells were harvested immediately at 0, 4, 8, 16 and 20 hrs post exposures. L-PGDS protein expression was determined, and relative band density was measured as shown at 0 hrs (6A-B), 4 hrs (6C-D), 8 hrs (6E-F), 16 hrs (6G-H), and 20 hrs (6I-J). Data was presented as mean ± SEM (n=3). Significant differences between the groups are determined using appropriate t-tests, and significant p values are indicated by asterisks *P < 0.05, **P < 0.01.

3.7. Effect of palmitate on ubiquitination in HepG2 cells:

To elucidate the regulatory role of ubiquitination in LPGDS protein dynamics, we conducted co-immunoprecipitation (CoIP) assays to analyze ubiquitin modifications, with a specific focus on K48- and K63-linked polyubiquitin chains (Fig. 7A–H). A quantitative assessment revealed modest modulation of ubiquitination patterns under palmitate treatment compared to control conditions. Both the K48-linked polyubiquitination and K63-linked polyubiquitination of LPGDS exhibited no statistical significance. These findings suggest that ubiquitination was not a major factor in the downregulation of L-PGDS expression by palmitate treatment.

Figure 7:

Figure 7:

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(7A) Immunoblot analysis of hepatic expression of LPGDS protein and (7B) band density is shown. Immunoprecipitation (IP) was performed with anti-LPGDS antibody. Immunoblotting (indicated by “IB”) with their respective band density anti-Ub (7C-D), anti-K48 (7E-F), and anti-K63 (7G-H) antibodies was performed to check for changes in ubiquitination patterns between control and palmitate-treated samples. Data was presented as mean ± SEM (n=3).

3.8. Palmitate treatment significantly decreased L-PGDS but increased FAS and PPARγ mRNA expression.

Since palmitate at 300 μM concentration showed a significant decrease in L-PGDS protein expression, we wanted to investigate if the decrease in L-PGDS expression is due to a decrease in transcription. Therefore, we decided to determine mRNA expression, and as shown in Fig. 8A, L-PGDS mRNA expression results showed a significant decrease compared to the control. In parallel, we also determined the mRNA expression of Fatty acid synthase (FASn), Fig. 8B, and Peroxisome proliferator-activated receptor gamma (PPARγ), Fig. 8C, and both showed significantly increased expression compared to the control.

Figure 8: Effect of palmitate on Relative mRNA expression:

Figure 8:

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HepG2 cells were treated with 300µM palmitate for 24 hours. Relative mRNA expression of L-PGDS (8A), FAS (8B), and PPARγ (8C) was done with PCR. The data is presented as mean ± SEM (n=3). Significant differences between the groups are determined using appropriate one-way ANOVA, and significant p values are indicated by asterisks: **P < 0.01, ****P < 0.0001.

4. Discussion:

Obesity has been strongly associated with elevated circulating free fatty acids due to dyslipidemia (25). This cascading effect of lipid metabolism further leads to non-alcoholic fatty liver disease (26). Our previous in vivo study showed dyslipidemia and severe MASLD in an L-PGDS knockout mouse model (27). A subsequent in vivo study showed a significant decrease in hepatic L-PGDS expression when wild C57BL/6 mice were kept on an HFD for 22 weeks (12). These findings strongly suggested the role of L-PGDS in MASLD, which strengthened our previous findings. Since palmitate is the prime substrate in the de novo biosynthesis of ceramides, it plays a crucial role in downstream lipid mediators of elevated FFA effects on inflammation, including obesity and fatty liver diseases (28). Therefore, the current study is designed to recapitulate prior in vivo results investigating L-PGDS regulation using a palmitate-induced cellular MASLD model of HepG2 hepatocytes. First, we determined the palmitate cytotoxicity in HepG2 cells since palmitate is a known lipotoxin (29). Our concentration-dependent effect of palmitate on cell viability showed that up to 300 μM palmitate for 24 hr treatment does not affect cell viability significantly (Fig. 1A) and induces significant lipid accumulation (Fig. 1B–C). Next, we wanted to determine the effect of palmitate on MASLD-associated proteins to confirm palmitate as a cellular MASLD model. A plethora of proteins play an essential role in lipid metabolism; however, FABP4, PPARγ, SREBP1, and SCAP were found to be the most commonly studied in fatty liver disease(30). FABP4 works by fatty acid uptake, transport, and metabolism, and elevated FABP4 levels were associated with MASLD, making it an essential marker for MASLD (31). Interestingly, our results showed significantly increased FABP4 expression (Fig. 2A–B), confirming the previous lipid accumulation results. Since L-PGDS enzymatically isomerises PGH2 to PGD2, which further non-enzymatically metabolizes to 15-deoxy-Δ12,14PGJ2. 15-deoxy-Δ12,14PGJ2 is a potent endogenous ligand for PPARγ, which plays a crucial role in lipid metabolism (9, 24). Our results with 300 μM palmitate treatment showed increased PPARγ expression (Fig. 2C–D). Similarly, SREBP1 is a key regulator of lipid metabolism (32). Overexpression of SREBP1 in the liver is related to increased liver lipid accumulation, which is also evident in patients with MASLD (33). SREBP1 is synthesized as a precursor, which is cleaved to generate a soluble fragment that translocates to the nucleus(34). As expected, SREBP1 (precursor) expression remained unchanged; however, cleaved SREBP1 expression increased significantly (Fig. 2E–G). SCAP (SREBP cleavage-activating protein) plays a crucial role in regulating the expression of SREBP-1, a transcription factor that controls fatty acid synthesis and other lipid metabolism processes (35). It is known that increased SREBP1. And decreased SCAP expression suggests dysregulation of lipid metabolism (36). Therefore, next we measured SCAP expression, and consistent with the literature, our results showed a similar decreased trend of SCAP in the palmitate treatment group (Fig. 2H–I). We established a palmitate-induced cellular MASLD model for further studies based on the results.

Taking the clue from our prior in vivo studies, the prime objective of the project is to determine the effect of palmitate on L-PGDS regulation. Excitingly, our results showed concentration-dependent significant decrease in L-PGDS on palmitate treatment at 300 μM and onwards (Fig. 3A–B). To confirm these results, next we were prompted to measure the level of PGD2 since, enzymatically, L-PGDS functions as a prostaglandin synthase and helps in the production of PGD2 by catalytic isomerization of PGH2 (37). As expected, concentration-dependent PGD2 levels were found to be decreased and reached significance at 300 μM, parallel to the L-PGDS expression (Fig. 3C). Therefore, collectively, based on the obtained results, including cytotoxicity, Oil Red O, and PGD2 level results at 300 μM palmitate for 24-hour treatment, it was considered the most appropriate concentration and exposure time for further studies. The next step was to untangle the puzzle and identify the reason for decreased L-PGDS expression with palmitate treatment. There were multiple approaches to solving this puzzle, and the one we anticipated was this decrease in L-PGDS expression, possibly due to palmitate-induced proteasomal degradation (38). Our results showed no change in L-PGDS expression in the presence and absence of proteasomal inhibitor, MG132 treatment (Fig. 4A–B), suggesting that a decrease in L-PGDS expression with palmitate treatment is independent of proteasomal degradation. Autophagy is another mechanism that may contribute to protein degradation (39). Therefore, we carried out a similar experiment using an autophagy inhibitor, chloroquine (24), and similarly, our results showed significant decreases in L-PGDS expression in the absence of the autophagy inhibitor, chloroquine (Fig. 4C–D). Change in a standard marker for autophagosomes, LC3-II expression, suggests the involvement of autophagy (40). It can be inferred that a significant increase in LC3II expression in the palmitate-treated group compared to the control in the absence of CQ, suggesting autophagy as a contributing player in the L-PGDS downregulation (Fig. 4E–F). Moving forward, we were curious to know whether palmitate has any role in inhibiting translation and blocking L-PGDS protein synthesis. Therefore, we proceeded further and experimented using cycloheximide (CHX), a protein synthesis inhibitor (41). Similarly, HepG2 cells were treated with or without palmitate for 24 hours, followed by the presence or absence of CHX, and cells were harvested at 0, 2, 6, and 24 hrs to capture protein changes in a timely manner. L-PGDS expression in the absence of palmitate and CHX at 24 hr time point remained unchanged. However, interestingly, in the absence of palmitate but in the presence of CHX at 24 hr treatment showed a significant decrease in L-PGDS expression (Fig. 5A–B), suggesting protein synthesis inhibition was at play. Next, we performed a similar experiment in the presence of palmitate but in the absence or presence of CHX. As expected, L-PGDS expression was significantly decreased at 24 hr palmitate treatment in the absence of CHX, but almost disappeared in the presence of CHX (Fig. 5C–D). This finding strongly suggested that palmitate treatment significantly inhibits L-PGDS protein synthesis. To capture time-bound changes in L-PGDS expression, we further designed an experiment where HepG2 cells were treated with 300 μM palmitate in the presence and absence of MG132, CHX, and CQ at different time points, as shown in Fig. 6. Our results in the 20 and 24-hour treatment groups (Fig. 6G–J) showed a significant decrease in L-PGDS expression in palmitate combined groups compared to their respective control group, except the CHX group, as expected. However, the early time 4 hr. (Fig.6A–B), 8 hr. (Fig. 6C–D), and 16 hr. (Fig. 6E–F) groups showed no change in any L-PGDS expression, possibly due to faster protein turnover at early time points, and required almost 20 hrs to capture the changes in protein expression. Next, we investigated the possibility of ubiquitination, as numerous studies have mentioned that palmitate treatment causes insulin resistance and lipid accumulation via the ubiquitination pathway, resulting in protein degradation and loss of function (38). We performed cellular ubiquitination experiments to eliminate the role of the Ubiquitin/Proteasome System (UPS). UPS is a crucial protein degradation system in eukaryotic cells, where it uses ubiquitin, a small protein, to tag proteins for destruction by the proteasome. Attaching ubiquitin, known as ubiquitination, is mediated by a series of ubiquitin ligases. Once a protein is tagged with ubiquitin, iťs recognized by the 26S proteasome, which degrades it (42, 43). Our results showed a decrease in the expression of L-PGDS with palmitate treatment; however, total ubiquitination (Fig. 7C–D)of L-PGDS remains unchanged. Both the K-48 (Fig. 7E–F) and K-63 (Fig. 7G–H) ubiquitination remained unchanged by palmitate treatment. These data suggest ubiquitination is not a major contributor to L-PGDS downregulation (Fig. 7A–H). At last, we measured gene expression, and interestingly, L-PGDS gene expression significantly decreased with palmitate-treatment (Fig. 8A). As expected, fatty acid synthase (FAS) (Fig. 8B) and PPARγ (Fig. 8C) gene expression were found significantly increased. Our gene expression data provided the mechanistic basis for L-PGDS downregulation.

Taken together, it can be interpreted that the decrease in L-PGDS expression with palmitate treatment is not due to any post-translational degradation process involving UPS, suggesting this decrease in L-PGDS expression is due to transcriptional-translational regulatory mechanisms and the autophagy pathway.

In conclusion, we recapitulated the MASLD phenotype successfully in HepG2 cells using palmitate treatment, which induced significant lipid accumulation and lipid-associated protein expression. Interestingly, we observed that lipid changes were accompanied by decreased L-PGDS expression and function. A mechanistic investigation of the downregulation of L-PGDS revealed that transcriptional changes and autophagy are major contributors. However, further studies are needed to delineate the precise molecular mechanism.

Highlights:

  1. Palmitate-induced lipid accumulation in HepG2 hepatocytes establishes a successful cellular MASLD model for mechanistic study

  2. L-PGDS expression significantly decreased with concentration-dependent palmitate treatment.

  3. Decreased L-PGDS protein expression was independent of proteosomal degradation but partially dependent on autophagy.

  4. The reason LPGDS reduced gene and protein expression was possibly due to transcriptional and posttranslational regulation.

Acknowledgment:

The authors would like to thank St. John’s University for its continuous support. The authors would also like to thank BioRender for providing a great platform for creating scientific schematic diagrams for publication.

FUNDING:

Research reported in this publication was supported by the National Institute of General Medical Sciences of the National Institutes of Health under award number R16GM150498.

Footnotes

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Credit authorship contribution statement:

Rhema Khairnar- Original draft, Methodology, Investigation, Formal analysis, Data curation. Md Asrarul Islam- Original draft, Visualization, Validation, Methodology, Investigation, Formal analysis, Data curation. Divya Shetty- Visualization, Data curation, Vikas V. Dukhande- Writing – review and editing, Sunil Kumar- Idea acquisition, Original draft, Visualization, Validation, Methodology, Investigation, Formal analysis, Data curation.

Declaration of competing interest:

The authors declare no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

DISCLOSURE STATEMENT:

All authors agree with the manuscripťs content and declare no conflict of interest.

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