Exploring the Putative Involvement of MALAT1 in Mediating the Beneficial Effect of Exendin-4 on Oleic Acid-Induced Lipid Accumulation in HepG2 Cells

Figures

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  Figure 1. HepG2 cell culturing, MALAT1 transfection, and oleic acid and Exendin-4 treatment protocols. HepG2 cells were starved for 6 h and then treated with 400 µM of oleic acid for 16 h. Cells were then treated with 200 nM of Exendin-4 for 3 h. β-actin was used as a housekeeping gene during the qPCR. Significance levels are denoted by * p < 0.05, ** p < 0.01.

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  Figure 2. The impact of Exendin-4 on mitigating oleic acid-induced lipid accumulation in HepG2 cells. HepG2 cells were subjected to a 6 h starvation period, followed by treatment with oleic acid (OA; 400 μM) for 16 h, and subsequently treated for 3 h with OA with or without Exendin-4 (Ex-4; 200 nM). (A) Quantifications of mRNA expression levels of perilipin 1 (PLIN1), perilipin 2 (PLIN2), and perilipin 3 (PLIN3). The expression levels were normalized to the level of β-actin. (B) Exendin-4 significantly reduced the oleic acid-induced elevation in triglyceride (TG) contents within HepG2 cells. All data are presented as the mean ± standard error (SE) with n = 4. Significance levels are denoted by * p < 0.05, ** p < 0.01.

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  Figure 3. Silencing of MALAT1 counteracts the influence of oleic acid (OA) on HepG2 cells. (A) HepG2 cells were exposed to various concentrations of OA (200, 300, 400, 500 μM) for 24 h, after which the MALAT1 levels were measured, with white bars representing cells treated with OA and black bars representing control cells. (B) HepG2 cells were transfected with scrambled si-RNA or si-MALAT1 for 48 h, followed by the quantification of MALAT1 expression using qPCR. (C) MALAT1 knockdown did not alter the mRNA expression of the PLIN1, PLIN2, and PLIN3 genes in untreated HepG2 cells. (D–F) The mRNA expression of PLIN1, PLIN2, and PLIN3, respectively, in HepG2 cells transfected with scrambled si-RNA and si-MALAT1 for 24 h, starved for 6 h, and subsequently treated with oleic acid (OA; 400 μM) for 16 h. (G) MALAT1 expression in HepG2 cells with OA, OA+Ex-4, and Ex-4-alone treatment. The gene expressions of PLIN1, PLIN2, and PLIN3 were then quantified under MALAT-1 silencing. The results are depicted as the mean ± standard error (SE) based on six replicates. Statistical significance is denoted by ** p < 0.01, *** p < 0.001, **** p < 0.0001, and ns: not significant.

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  Figure 4. MALAT1 silencing does not affect de novo lipogenesis genes. The quantification of de novo lipogenesis gene expression was conducted using qRT-PCR, and the results were normalized to the level of β-actin. (A) MALAT1 transfection did not alter the mRNA expression of PPARγ and SREBP-1 genes in untreated HepG2 cells (control cells). (B,C) The expression levels of PPARγ (peroxisome proliferator-activated receptor gamma) and SREBP-1 (sterol regulatory element-binding transcription factor 1), respectively, transfected with either scrambled MALAT1 si-RNA or MALAT1 siRNA at a concentration of 20 nM. All data are expressed as the mean ± standard error (SE) with n = 6. Statistical significance is indicated by * p < 0.05, ** p < 0.01, and ns: not significant.

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  Figure 5. MALAT1 suppression alters the expression of lipogenesis-related genes in HepG2 cells. All genes were transfected with either scrambled MALAT1 si-RNA or MALAT1 siRNA for 24 h. Quantifications of the mRNA expression levels for DGAT1 (Diacylglycerol O-acyltransferase 1), DGAT2 (Diacylglycerol O-acyltransferase 2), FAS (fatty acid synthase), ACADL (Acyl-CoA Dehydrogenase Long Chain), CPT1A (Carnitine Palmitoyl Transferase 1A), SCD1 (Stearoyl-CoA desaturase 1), ACC (Acetyl-CoA carboxylase alpha), SCL2 (Collagen-like protein 2), FABP4 (fatty acid-binding protein 4), NR1H2 (nuclear receptor subfamily 1 group H member 2), and MTTP (Microsomal Triglyceride Transfer Protein) were performed using qPCR after treatment with OA alone or OA combined with Ex-4 (A–J). Data are presented as the mean ± standard error (SE) with n = 6. Statistical significance is indicated by * p < 0.05, ** p < 0.01, and ns: not significant.

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  Figure 6. Western blotting and quantification of the following transcription factors: stearoyl-CoA desaturase 1 (SCD1), sterol regulatory element-binding protein-1 (SREBP1), the precursor and nuclear forms of SREBP1, peroxisome proliferator-activated receptor gamma (PPAR-γ), and FAS (fatty acid synthetase (A–E). Proteins were normalized against β-actin. All values are expressed as the mean SE. (n = 3). ns: not significant, * p < 0.05. Full-length blots are displayed in Data S1.

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  Figure 7. Proposed simplified signaling pathway elucidating the mechanism underlying the amelioration of steatosis induced by Exendin-4. At rest, when the levels of extracellular circulating FFAs are low, the uptake of these FFAs is low and the expression of MALAT1 and many lipid metabolism genes is downregulated. Following stimulation with OA, mimicking an increase in extracellular FFA levels, the expression of MALAT1 leads to the modulation of the expression of several genes, including SREBP-1, SCD1, FAS, ACC, DGAT1, DGTA2, MTTP, and NR1H2. In the presence of Ex-4 treatment, a downregulation of MALAT1 is associated with reversal of the modulations of numerous genes induced by OA, leading to a reduction in steatosis. Predictive upstream and downstream targets are highlighted. Upward and downward arrows indicate upregulation and downregulation, respectively.