TLR2-Deficiency Promotes Prenatal LPS Exposure-Induced Offspring Hyperlipidemia

Abstract

Toll-like receptor 2 (TLR2), which recognizes several lipopeptides and transduces inflammatory signaling, promotes the pathogenesis of diet-induced dyslipidemia and obesity. TLR2-deficient mice were shown to have improved insulin sensitivity and reduced diet-induced metabolic syndrome. Previous studies demonstrated that prenatal lipopolysaccharide (LPS) exposure causes dyslipidemia accompanied by increased body weight and insulin resistance in offspring. To determine whether TLRs are involved in this complex abnormal phenotype, we analyzed TLR2 and TLR4 expression levels in adipose tissues from offspring with prenatal LPS-exposure (offspring-pLPS) and compared these levels to those of control offspring with prenatal saline-exposure (offspring-pSaline). TLR2 expression was specifically upregulated in the adipose tissue of offspring-pLPS mice. However, unexpectedly, TLR2-deficient offspring-pLPS mice not only presented with an abnormal phenotype comparable to that of wild-type offspring-pLPS mice but also exhibited significantly more severe hyperlipidemia. Our further analyses revealed a dramatic upregulation of TLR4 expression and overactivation of the TLR4/Myd88 signaling pathway in TLR2-deficient offspring-pLPS adipose tissue. Our finding suggests a compensatory genetic interaction between TLR2 and TLR4 in the context of prenatal inflammatory stimulation, and this interaction likely contributes to the prenatal inflammation-induced hyperlipidemia and lipid overload-induced obesity, thus providing a potential mechanism for the fetal origin of adult metabolic diseases.

Keywords: TLR2; TLR4; VLDLR; hyperlipidemia; prenatal lipopolysaccharide stimulation.

FIGURE 1

Toll-like receptor 2 (TLR2) expression is elevated in offspring with prenatal Lipopolysaccharide (LPS) exposure. Relative mRNA (A) and protein (B) expression of TLR2 and TLR4 detected by qRT-PCR and Western blot (WB) from 12-week-old offspring adipose tissue. pSaline: offspring from prenatal saline-treated mothers; pLPS: offspring from prenatal LPS-treated mothers. All data are presented as the mean ± SEM and were obtained from three independent experiments.

FIGURE 2

TLR2 deficiency does not prevent prenatal LPS exposure-induced obesity and insulin resistance. (A) Schematic diagram of experimental design. (B) Bodyweight of 12-week-old offspring of the indicated group. (C) The 12-week-old offspring relative adipose tissue weight was calculated as: (wet adipose tissue weight/body weight) × 1000. (D) Fat distribution was detected by micro-CT and data were processed with Analyze 12.0. (E) Representative adipose tissue H&E staining. Adipocyte size was calculated as described in the Methods section. (F,G) Intraperitoneal Glucose Tolerance Test (IPGTT) and Intraperitoneal Insulin Tolerance Test (IPITT) were performed on indicated mice. (H) Protein expression of AKT, IRS-1, p-AKT^ser473, and p-IRS-1^Ser307 were detected by WB. All data are presented as the mean ± SEM and were obtained from three independent experiments. pSaline: offspring from prenatal saline-treated mother; pLPS: offspring from prenatal LPS-treated mother; TLR2^–/– + pLPS: offspring from prenatal LPS treatment homozygous mated TLR2^–/– mother; TLR2^–/– + pSaline: offspring from prenatal Saline treatment homozygous mated TLR2^–/– mother.

FIGURE 3

TLR2 deficiency does not prevent high-fat induced obesity and insulin resistance in offspring with prenatal LPS exposure fed a high-fat diet. (A) Schematic diagram of experimental design on diet intervention. (B) Bodyweight of 12-week-old offspring after diet intervention. (C) The 12-week-old diet intervened offspring relative adipose tissue weight was calculated as wet adipose tissue weight in % body weight. (D) IPGTT was performed, and the area under the curve (AUC) for glucose was calculated for 12-week-old diet intervened offspring. All data are presented as the mean ± SEM.

FIGURE 4

TLR2 deficiency promotes hyperlipidemia induced by prenatal LPS exposure. Measurement of serum content of TCH (A), TG (B), HDL-c (C) and LDL-c (D) in 12-week-old offspring. All data are presented as the mean ± SEM.

FIGURE 5

Compensatory TLR4 activation in TLR2-knockout offspring with prenatal LPS exposure. (A) qRT-PCR analyzing relative mRNA expression of TLR4, Myd88 TRAF6, TNF-α, and IL-6 in adipose tissue of 12-week-old offspring under different treatments. Serum TNF-α (B) and IL-6 (C) concentration were determined. (D) WB analyzing relative protein expression of TLR4, Myd88, p-Myd88^Tyr257, and TRAF6 in adipose tissue of 12-week-old offspring. All data are presented as the mean ± SEM.

FIGURE 6

Upregulated HMGB-1 expression in TLR2-deficient offspring with prenatal LPS exposure. (A) qRT-PCR analyzing relative mRNA expression of HMGB-1 and RAGE in adipose tissue of 12-week-old offspring. (B) Western blot analyzing relative protein expression of HMGB-1 in adipose tissue of 12-week-old offspring. All data are presented as the mean ± SEM.

FIGURE 7

Very Low Density Lipoprotein Receptor (VLDLR) expression in TLR2-deficient mice with prenatal LPS exposure. (A) qRT-PCR analyzing relative expression of Cholesteryl Ester Transfer Protein (CETP), VLDLR, ABCG1,CD36, SR-BI, LDLR, LRP1b, LRP6, LRP10, and LRP12 in adipose tissue of 12-week-old offspring. (B) Western blot analyzing relative expression of CETP, VLDLR, ABCG1, and SR-BI in adipose tissue of 12-week-old offspring. All data are presented as the mean ± SEM.