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. 2024 Mar 6;13(5):463.
doi: 10.3390/cells13050463.

Neutral Sphingomyelinase 2 Inhibition Limits Hepatic Steatosis and Inflammation

Fatema Al-Rashed  1 , Hossein Arefanian  1 , Ashraf Al Madhoun  2 , Fatemah Bahman  1 , Sardar Sindhu  2 , Halemah AlSaeed  1 , Texy Jacob  1 , Reeby Thomas  1 , Areej Al-Roub  1 , Fawaz Alzaid  3 , M D Zubbair Malik  4 , Rasheeba Nizam  4 , Thangavel Alphonse Thanaraj  4 , Fahd Al-Mulla  4 , Yusuf A Hannun  5 , Rasheed Ahmad  1
Affiliations

Neutral Sphingomyelinase 2 Inhibition Limits Hepatic Steatosis and Inflammation

Fatema Al-Rashed et al. Cells. .

Abstract

Non-alcoholic fatty liver disease (NAFLD) is manifested by hepatic steatosis, insulin resistance, hepatocyte death, and systemic inflammation. Obesity induces steatosis and chronic inflammation in the liver. However, the precise mechanism underlying hepatic steatosis in the setting of obesity remains unclear. Here, we report studies that address this question. After 14 weeks on a high-fat diet (HFD) with high sucrose, C57BL/6 mice revealed a phenotype of liver steatosis. Transcriptional profiling analysis of the liver tissues was performed using RNA sequencing (RNA-seq). Our RNA-seq data revealed 692 differentially expressed genes involved in processes of lipid metabolism, oxidative stress, immune responses, and cell proliferation. Notably, the gene encoding neutral sphingomyelinase, SMPD3, was predominantly upregulated in the liver tissues of the mice displaying a phenotype of steatosis. Moreover, nSMase2 activity was elevated in these tissues of the liver. Pharmacological and genetic inhibition of nSMase2 prevented intracellular lipid accumulation and TNFα-induced inflammation in in-vitro HepG2-steatosis cellular model. Furthermore, nSMase2 inhibition ameliorates oxidative damage by rescuing PPARα and preventing cell death associated with high glucose/oleic acid-induced fat accumulation in HepG2 cells. Collectively, our findings highlight the prominent role of nSMase2 in hepatic steatosis, which could serve as a potential therapeutic target for NAFLD and other hepatic steatosis-linked disorders.

Keywords: NAFLD; Smpd3; inflammation; lipotoxicity; nSmase2; steatosis.

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Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Figure 1
Figure 1
High-fat diet (HFD) feeding induces chronic liver injury and a NAFLD phenotype in mice. C57BL/6 male mice were fed HFD or chow diet (CD), 5 each, for 14 weeks, and the livers were collected after euthanasia at the end of feeding. (A) Body weights (g) over time are shown for HFD and CD groups. (B) Gross appearance of the representative livers from HFD- and CD-fed mice. (C) Liver weights compared between CD and HFD groups at the end of 14 week feeding. (D) Representative H&E stainings showing histopathological changes in the livers of HFD and CD mice. (E) Macrovascular and microvascular steatosis and lobular inflammation scores related to the livers of CD and HFD mice. (F) Representative immunohistochemical (IHC) stainings showing macrophage infiltration in the livers of HFD and CD mice. (G) F4/80 staining (% area) related to the livers of HFD and CD mice. (H) Representative Oil Red O (ORO) Staining showing intracellular lipid accumulation (I) ORO staining (%area) related to the liver of HFD and CD mice. (J) Quantification of triglyceride levels in the liver tissue. For histopathological analysis, 10 random fields/specimen were counted and viewed at 20× or 40× magnification. All data are expressed as mean ± SEM. * p ≤ 0.5, ** p ≤ 0.1, *** p ≤ 0.001.
Figure 2
Figure 2
Transcriptomic and bioinformatic analyses identify the enhanced Smpd3 expression in the steatotic livers of high-fat diet (HFD)-fed mice. RNA-seq was used to identify potential gene target(s) in the livers of mice fed with a HFD, compared to those fed with a chow diet (CD), 5 mice per group. (A) Principal component analysis (PCA) shows differential clustering patterns in the livers of HFD and CD mice. (B) Volcano plot distribution highlights the differentially expressed genes (DEGs) in the livers of HFD and CD mice. Orange and cyan color dots denote the up- (510) and downregulated (672) significantly DEGs, respectively, while the gray color dots represent non-significant DEGs between CD- and HFD-fed mice. (C,D) Gene Ontology (GO) enrichment analysis based bar plots represent the biological processes that are significantly altered in relation to up- and downregulated DEGs in the liver samples from CD- and HFD-fed mice. X-axes represent the Fold Enrichment, with statistical significance indicated by a numerical value and a color code as shown. (E,F) KEGG pathway analysis of the significantly up- and down-regulated DEGs respectively, in the livers of HFD and CD mice. Dot size indicates the count which represents the number of genes associated with each pathway. Dot color denotes the statistical significance (red being the highest), and the x-axis represents the fold enrichment. (G) Major gene networks interconnectivity including lipid, cholesterol, and fatty acid metabolic processes, apoptosis, oxidative stress by ROS, and ATP response. Orange and cyan color dots represent the up- and downregulated, respectively, DEGs in the livers of HFD and CD mice. (H) Chord plot showing the relationship between genes and top significant GO terms including cholesterol, lipid, and fatty acid metabolism. Genes involved in the enrichment are arranged (on the left) in order of their expression level (−log of fold change). (I) Increased nSMase2 activity level in in the livers of HFD mice, compared to CD mice. *** p ˂ 0.001.
Figure 3
Figure 3
Increased Smpd3 expression and nSMase2 activity in HepG2 cell model of steatosis. HepG2 cells were cultured in normoglycemic (5.6 mmol D-glucose) or hyperglycemic (25.2 mmol D-glucose) conditions, in the presence or absence of oleic acid (OA) stimulation. Gene expression of SGMS1 and SGMS2 was determined using RT-qPCR; nSMase2 and aSMase activities were determined using commercial kits as described in Materials and Methods. (A) Schematic representation of the main sphingomyelin (SM) pathway. Reduced gene expression of (B) SGMS1 and (C) SGMS2 in HepG2 cells under glucolipotoxic conditions. (D) Increased Smpd3 gene expression in HepG2 cells under glucolipotoxic conditions. (E) Smpd1 gene expression in HepG2 cells differed non-significantly between glucolipotoxic and normal conditions. (F) Increased nSMase activity in HepG2 cells under glucolipotoxic conditions. (G) aSMase activity in HepG2 cells differed non-significantly between glucolipotoxic and normal conditions. All data are expressed as mean ± SEM. * p ≤ 0.05, ** p ≤ 0.01, **** p ≤ 0.0001, ns: non-significant.
Figure 4
Figure 4
Effect of nSMase2 inhibition in HepG2 cell model of steatosis. HepG2 cells were pre-treated with nSMase 2 inhibitor GW4869 (10 μM), aSMase inhibitor Imipramine (10 μM) or with nSMase 2 agonist Daunorubicin (1 μM) for 1 h, followed by overnight stimulation with oleic acid (OA; 150 μM). Activities of nSMase2 and aSMase were measured using commercial kits as described in Materials and Methods. (A) Changes in nSMase2 activity are shown in HepG2 cell model of steatosis. (B) Changes in aSMase activity are shown in HepG2 cell model of steatosis. (C) Changes in tricellular lipids (BODIPY 493/503). (D) Representative data from three independent determinations with similar results. (E) Representative confocal microscopy images of Nile red fluorescence staining of HepG2 cells, treated with steatosis inducing or basal (normal) conditions, obtained from three independent experiments with similar results. (F) ACACA gene expression in response to pre-treatments under steatosis condition. (G) FASN gene expression in response to pre-treatments under steatosis condition. (H) CPT1α gene expression are in response to pre-treatments under steatosis condition. (I) CPT2 gene expression are in response to pre-treatments under steatosis condition. Data are expressed as mean ± SEM. * p ≤ 0.05, ** p ≤ 0.01, **** p ≤ 0.0001, ns: non-significant. Images are shown at 20× magnification. Scale bar = 50 μm.
Figure 5
Figure 5
nSMase2 inhibition suppresses inflammation, oxidative stress, and apoptosis in HepG2 cell model of steatosis. HepG2 cells were exposed to steatosis-inducing vs. control conditions, with or without pre-treatments including nSMase2 inhibitor GW4869, aSMase inhibitor imipramine, and nSMase 2 agonist daunorubicin as described in Materials and Methods. Target gene expression was determined using RT-qPCR and protein expression using immunoblotting. Cell survival was determined using MTT assay. (A) Changes in TNF-α gene expression are shown in response to pre-treatments under steatosis condition. (B) Changes in TNF-α protein expression are shown in response to pre-treatments under steatosis condition. (C) TNF-α secreted protein levels in HepG2 culture supernatants are shown after pre-treatments under steatosis condition. (D) Changes in IL-1β gene expression are shown in response to pre-treatments under steatosis condition. (E) Changes in IL-6 gene expression are shown in response to pre-treatments under steatosis condition. (F) Changes in PPARα gene expression are shown in response to pre-treatments under steatosis condition. (G) Changes in PPARα protein expression are shown in response to pre-treatments under steatosis condition. (H) Representative Immunoblot for PPARα protein expression. (I) Changes in Cyp2E1 gene expression are shown in response to pre-treatments under steatosis condition. (J) Changes in DITT3 gene expression are shown in response to pre-treatments under steatosis condition. (K) Changes in SREBP gene expression are shown in response to pre-treatments under steatosis condition. (L) Changes in cell viability are shown in relation to pre-treatments under steatosis condition. All data are expressed as mean ± SEM with n = 4. * p≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001, ns: non-significant.
Figure 6
Figure 6
nSMase2 deficiency improves steatosis-associated pathologic changes in HepG2 cells. nSMase2 was genetically suppressed by transfecting HepG2 cells with Smpd3-specific siRNA, and controls were transfected with scrambled siRNA. Target gene expression was determined using RT-qPCR, protein expression by western blotting or flow cytometry, intracellular lipids by BODIPY 493/503 flow cytometry, apoptosis by annexin-V/PI staining, and cell viability using MTT assay as described in Materials and Methods. (A) Genetic suppression of Smpd3 is shown in HepG2 cells transfected with specific and scrambled siRNAs. (B) TNF-α gene expression is significantly reduced in Smpd3 siRNA-transfected cells under steatosis-inducing conditions, compared to controls. (C) TNF-α protein expression is significantly reduced in Smpd3 siRNA-transfected cells under steatosis-inducing conditions, compared to controls. (D) PPARα gene expression is significantly upregulated in Smpd3 siRNA-transfected HepG2 cells under steatosis-inducing conditions, compared to controls. (E) PPARα protein expression is significantly increased in Smpd3 siRNA-transfected cells under steatosis-inducing conditions, compared to controls. (F) Cyp2E1 gene expression is significantly reduced in Smpd3 siRNA-transfected HepG2 cells under steatosis-inducing conditions, compared to controls. (G) Representative changes in intracellular lipid accumulation, from three independent determinations with similar results, are shown. (H) Representative changes in apoptosis, from three independent experiments with similar results, are shown. (I) Bar graph of median fluorescence intensity (MFI) representing Annexin V-PI staining, from three independent determinations with similar results are shown. (J) Bar graph of cell viability (% survival) is shown for HepG2 cells transfected with Smpd3-specific and scrambled siRNAs. Similar data were obtained from three independent determinations. All data are expressed as mean ± SEM. * p ≤ 0.05, ** p ≤ 0.01, **** p ≤ 0.0001, ns: non-significant.
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