Saturated fatty acid combined with lipopolysaccharide stimulates a strong inflammatory response in hepatocytes in vivo and in vitro

Abstract

Nonalcoholic fatty liver disease (NAFLD) is the most common chronic liver disease and consumption of high-fat diet (HFD) is a risk factor for NAFLD. The HFD not only increases intake of saturated fatty acid (SFA) but also induces metabolic endotoxemia, an HFD-associated increase in circulating lipopolysaccharide (LPS). Although it is known that SFA or LPS promote hepatic inflammation, a hallmark of NAFLD, it remains unclear how SFA in combination with LPS stimulates host inflammatory response in hepatocytes. In this study, we performed both in vivo and in vitro experiments to investigate the effect of SFA in combination with LPS on proinflammatory gene expression in hepatocytes. Our animal study showed that feeding low-density lipoprotein-deficient mice HFD enriched with SFA and injection of low-dose LPS cooperatively stimulated IL-6 expression in livers. To understand how SFA and LPS interact to promote IL-6 expression, our in vitro studies showed that palmitic acid (PA), a major SFA, and LPS exerted synergistic effect on the expression of IL-6 in hepatocytes. Furthermore, coculture of hepatocytes with macrophages resulted in a greater IL-6 expression than culture of hepatocytes without macrophages in response to the combination of PA and LPS. Finally, we observed that LPS and PA increased ceramide production by cooperatively stimulating ceramide de novo synthesis, which played an essential role in the synergistic stimulation of proinflammatory gene expression by LPS and PA. Taken together, this study showed that SFA in combination with LPS stimulated a strong inflammatory response in hepatocytes in vivo and in vitro.

Keywords: ceramide; fatty acid; hepatocyte; inflammation; lipopolysaccharide.

Fig. 1.

The effect of LPS and diets on hepatic inflammation. After mice were treated with LPS or vehicle PBS in combination with LFD, LP-HFD, or HP-HFD, livers were dissected and subjected to immunohistochemical staining of F4/80 to detect macrophages. Representative photomicrographs of hepatic tissue sections with F4/80 immunostaining for all 6 groups (A) and quantification of F4/80-positive staining area (C) are shown. As negative control, immunostaining using a control IgG (B) is shown. Also, representative photomicrographs of hepatic tissue sections with IL-6 immunostaining (D) and quantification of IL-6 positive staining area (E) are shown. Furthermore, liver tissue sections were stained with oil red O (F) and Sirius red (H). The photomicrographs and quantification of oil red O (G) and Sirius red positive area (I) are shown. The data presented are means ± SD (n = 6–9).

Fig. 2.

LPS in combination with PA increases proinflammatory cytokine expression in hepatocytes. Hepatocytes were treated LPS, PA or LPS plus PA for different times as indicated and IL-6 secreted into the medium (A) and IL-6 mRNA (B) at each time point were quantified using ELISA and real-time PCR, respectively. PA, palmitic acid.

Fig. 3.

Hepatocyte and RAW264.7 macrophages were cultured separately (A and B; E and F) or together (C and G) and exposed to LPS, PA, or LPS plus PA for 24 h. After the treatment, IL-6 (A–C) and TNFα (E–G) in culture medium were quantified using ELISA. The amount of IL-6 (D) and TNFα (H) secretion by hepatocytes, RAW264.7 macrophages or coculture of hepatocytes and RAW264.7 macrophages was compared. Hepatocytes cultured alone or cultured with RAW264.7 macrophages in the coculture system were exposed to LPS, PA, or LPS plus PA for 24 h. After the treatment, IL-6 mRNA was isolated and compared between hepatocytes cultured alone and those in the coculture (I) and between RAW264.7 macrophages cultured alone and those in the coculture (J). The data (mean ± SD) presented are representative of three experiments with similar results. PA, palmitic acid.

Fig. 4.

The hepatic expression of GPR40 and CD36 in vivo and in vitro, and the involvement of GPR40 and CD36 in the stimulation of cytokine secretion by LPS or LPS plus PA. Immunostaining of GPR40 (A) and CD36 (B) in livers of mice fed high-fat diet. Immunoblotting of GPR40 and CD36 in cultured mouse hepatocytes (C). GAPDH was detected as control. Hepatocytes were treated with LPS, PA, or LPS plus PA in the absence or presence of 10 μM of GW1100 (D and F), a selective GPR40 antagonist, or 50 or 100 μM of sulfo-N-succinimidyl oleate (SSO) (E and F), a selective inhibitor of CD36, for 24 h. After the treatment, cytokine secreted into medium was quantified using ELISA. The data (mean ± SD) presented are representative of three experiments with similar results. GPR, G protein-coupled receptor; PA, palmitic acid.

Fig. 5.

The effect of LPS, PA, or LPS plus PA on sphingolipid production in hepatocytes. Hepatocytes were treated with LPS, PA, or LPS plus PA for 12 h, and the cells were harvested for quantification of total CER (A), C16-CER (B), dhC16-CER (C), sphingosine 1 phosphate (D), total sphingomyelin (E), and C16-sphingomyelin (F) using Lipidomics. The data are mean ± SD of triplicate samples. CER, ceramide; PA, palmitic acid.

Fig. 6.

The involvement of the de novo synthesis of CER in IL-6 expression stimulated by LPS, PA or LPS plus PA. A: hepatocytes were treated with LPS, PA, or LPS plus PA in the absence or presence of 5 or 10 μM of myriocin, a selective inhibitor of serine palmitoyltransferase (SPT), for 24 h. After the treatment, IL-6 in culture medium was quantified. B: hepatocytes were treated with LPS, PA, or LPS plus PA in the absence or presence of 10 μM of myriocin for different times as indicated. After the treatment, IL-6 in culture medium was quantified. C: hepatocytes were treated with LPS, PA, or LPS plus PA in the absence or presence of 10 μM of myriocin for 18 or 24 h. After the treatment, IL-6 mRNA was quantified using real-time PCR. D: hepatocytes were treated with 5 or 10 ng/ml of LPS in the absence or presence of 100 μM of PA for 24 h. After the treatment, histone-associated DNA fragments were quantified as described in methods. The data (mean ± SD) presented are representative of three experiments with similar results. CER, ceramide; Ctl, control; PA, palmitic acid.

Fig. 7.

Schematic diagram to show the proposed mechanism involved in the upregulation of proinflammatory gene expression in hepatocytes by LPS and PA. The simultaneous activation of TLR4 signaling and GPR40 or CD36 signaling by LPS and PA, respectively, cooperatively triggers a strong inflammatory response in hepatocytes. Moreover, PA and LPS cooperatively stimulate CER de novo synthesis, which increases CER production and further enhances proinflammatory gene expression. CER, ceramide; GPR, G protein-coupled receptor; PA, palmitic acid; TLR, toll-like receptor.