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. 2012;7(6):e39286.
doi: 10.1371/journal.pone.0039286. Epub 2012 Jun 29.

Palmitoleate induces hepatic steatosis but suppresses liver inflammatory response in mice

Xin Guo  1 , Honggui Li, Hang Xu, Vera Halim, Weiyu Zhang, Huan Wang, Kuok Teong Ong, Shih-Lung Woo, Rosemary L Walzem, Douglas G Mashek, Hui Dong, Fuer Lu, Lai Wei, Yuqing Huo, Chaodong Wu
Affiliations

Palmitoleate induces hepatic steatosis but suppresses liver inflammatory response in mice

Xin Guo et al. PLoS One. 2012.

Abstract

The interaction between fat deposition and inflammation during obesity contributes to the development of non-alcoholic fatty liver disease (NAFLD). The present study examined the effects of palmitoleate, a monounsaturated fatty acid (16:1n7), on liver metabolic and inflammatory responses, and investigated the mechanisms by which palmitoleate increases hepatocyte fatty acid synthase (FAS) expression. Male wild-type C57BL/6J mice were supplemented with palmitoleate and subjected to the assays to analyze hepatic steatosis and liver inflammatory response. Additionally, mouse primary hepatocytes were treated with palmitoleate and used to analyze fat deposition, the inflammatory response, and sterol regulatory element-binding protein 1c (SREBP1c) activation. Compared with controls, palmitoleate supplementation increased the circulating levels of palmitoleate and improved systemic insulin sensitivity. Locally, hepatic fat deposition and SREBP1c and FAS expression were significantly increased in palmitoleate-supplemented mice. These pro-lipogenic events were accompanied by improvement of liver insulin signaling. In addition, palmitoleate supplementation reduced the numbers of macrophages/Kupffer cells in livers of the treated mice. Consistently, supplementation of palmitoleate decreased the phosphorylation of nuclear factor kappa B (NF-κB, p65) and the expression of proinflammatory cytokines. These results were recapitulated in primary mouse hepatocytes. In terms of regulating FAS expression, treatment of palmitoleate increased the transcription activity of SREBP1c and enhanced the binding of SREBP1c to FAS promoter. Palmitoleate also decreased the phosphorylation of NF-κB p65 and the expression of proinflammatory cytokines in cultured macrophages. Together, these results suggest that palmitoleate acts through dissociating liver inflammatory response from hepatic steatosis to play a unique role in NAFLD.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Improvement of systemic insulin sensitivity in LFD-fed mice.
Male C57BL/6J mice, at 5–6 weeks of age, were fed a low-fat diet for 12 weeks and supplemented with palmitoleate (PO), oleate (Ole), or bovine serum albumin (BSA) for the last 4 weeks. (A) Plasma lipid profile. (B) Insulin tolerance tests (ITT). (C) Area under curve (AUC) was calculated based on ITT. (D) Glucose tolerance tests (GTT). (E) AUC was calculated based on GTT. For B and D, mice were fasted for 4 hrs and received an intraperitoneal injection of insulin (0.5 U/kg) (B) or glucose (2 g/kg) (D). Data are means ± SE, n = 4–6. †, P<0.05 palmitoleate vs. BSA (in A – E) or oleate for the same time point (in B and D); *, P<0.05 and **, P<0.01 palmitoleate vs. oleate (in A, C, and E); ‡, P<0.05 oleate vs. BSA (in A).
Figure 2
Figure 2. Induction of hepatic steatosis while improving liver insulin sensitivity and decreasing liver inflammatory response in LFD-fed mice.
Male C57BL/6J mice, at 5–6 weeks of age, were fed a low-fat diet for 12 weeks and supplemented with palmitoleate (600 mg/kg/d), oleate (600 mg/kg/d), or bovine serum albumin (BSA) via oral gavages for the last 4 weeks. (A) Liver fat deposition. Right panels, liver sections were stained with H&E. Left panel, hepatic triglyceride levels. (B) Liver gene expression. SREBP1c, sterol-regulatory element-binding protein 1c; FAS, fatty acid synthase; CPT1a, carnitine palmitoyltransferase 1a; VLDLr, very low density lipoprotein receptor; GK, glucokinase; and G6Pase, glucose-6-phosphatase. (C) Liver insulin signaling. Livers of the treated mice were collected at 5 min after a bolus injection of insulin (1 U/kg) or PBS into the portal vein. Left panels, Akt and phospho-Akt (Ser473) were examined using Western blot analyses; right panel, quantification of P-Akt/Akt. AU, arbitrary unit. (D) Liver macrophages/Kupffer cells (left two panels, F4/80 staining; right panel, F4/80+ cell fraction). (E) Liver NF-κB p65 and phospho-p65 (Ser468). (F) Liver cytokine expression. For A – D and F, numeric data are means ± SE, n = 4–6. †, P<0.05 and ††, P<0.01 palmitoleate vs. BSA (in A and D) for the same gene (in B and F) under the same condition (in C).
Figure 3
Figure 3. Improvement of systemic insulin sensitivity.
Male C57BL/6J mice, at 5–6 weeks of age, were fed a high-fat diet for 12 weeks and supplemented with palmitoleate (PO) or bovine serum albumin (BSA) via oral gavages for the last 4 weeks. (A) Insulin tolerance tests (ITT). (B) Area under curve (AUC) was calculated based on ITT. (C) Glucose tolerance tests (GTT). (D) AUC was calculated based on GTT. For A and C, mice were fasted for 4 hrs and received an intraperitoneal injection of insulin (1 U/kg) (A) or glucose (2 g/kg) (C). (E) Hepatic levels of triglycerides. (F) Liver NF-κB p65 and phospho-p65 (Ser468). For A – E, data are means ± SE, n = 4–6. †, P<0.05 and ††, P<0.01 palmitoleate vs. BSA (in B, D, and E) for the same time point (in A and C).
Figure 4
Figure 4. Induction of hepatocyte fat deposition while improving insulin signaling.
(A) Hepatocyte fat deposition. Mouse primary hepatocytes were treated with palmitoleate (50 µM), oleate (200 µM), or BSA (in PBS) for 48 hrs in the presence or absence of palmitate (250 µM) for the last 24 hrs and stained with Oil-Red-O or 1 hr. (B) Hepatocyte gene expression. Mouse primary hepatocytes were treated with palmitoleate (50 µM) or BSA (in PBS) for 48 hrs. The expression of genes related to lipid metabolism was analyzed using real-time RT-PCR. ChREBP, carbohydrate responsive element-binding protein; ACC1, acetyl-CoA carboxylase 1. (C) Hepatocyte SREBP1c transcription activity. Mouse primary hepatocytes were transfected with a plasmid containing firefly luciferase reporter driven by SRE sequence on FAS gene (FAS-SRE-luc), or a control plasmid (pGL3-luc) for 24 hrs. After transfection, the cells were treated with palmitoleate (50 µM) or BSA for 48 hrs in the presence or absence of insulin (100 nM) for the last 24 hrs. (D) Hepatocyte ChIP assay. Mouse primary hepatocytes were treated with palmitoleate (50 µM), oleate (200 µM), or BSA for 48 hrs and subjected to the ChIP assay using antibodies against SREBP1c. The resultant DNA was analyzed by PCR with primers amplifying SRE-1 on FAS promoter. The input (control) contained 10% of each of the immunoprecipitants. (E) Hepatocyte insulin signaling. Cells were treated as described in (B). Prior to harvest, the cells were treated with or without insulin (100 nM) for 30 min. Top two panels, the levels and phosphorylation state of Akt (Ser473); bottom panel, quantification of P-Akt/Akt. AU, arbitrary unit. For B, C, and E, numeric data are means ± SE. All experiments were performed at least in quadruplicate. †, P<0.05 and ††, P<0.01 palmitoleate vs. BSA for the same gene (B) under the same condition (C and E).
Figure 5
Figure 5. Suppression of inflammatory responses.
Mouse primary hepatocytes and RAW macrophages were treated with palmitoleate (50 µM) or BSA (in PBS) for 48 hrs. (A) Hepatocyte NF-κB p65 and phospho-p65 (Ser468). (B) Hepatocyte expression of proinflammatory cytokines. (C) RAW macrophage NF-κB p65 and phospho-p65 (Ser468). (D) RAW macrophage expression of proinflammatory cytokines. For B and D, data are means ± SE. All experiments were performed in quadruplicate. †, P<0.05 and ††, P<0.01 palmitoleate vs. BSA for the same gene.
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