TLR2-deficiency Promotes Prenatal LPS Exposure-induced Offspring Hyperlipidemia And Mechanism Research

Currently,the number of obese patients around the world is increasing.In this situation,the global prevalence of obesity will reach 18%for men and 21%for women by 2025.Metabolically unhealthy people are showed to be imbalance in energy,especially energy intake exceeds energy expenditure.However,the imbalance can lead to weight gain and obesity.Obesity has been proven to be related to genetics,epigenetics,diet and lifestyle.To some extent,obesity improvement and prevent metabolic changes can be achieved by managing diet and adjusting patient`s lifestyles.Moderate progressive weight loss causes dose-dependent changes in major fat-related pathways and improves metabolic function to varying degrees.On the one hand,this method can`t be suit for each patient;in the other hand,the compliance of patients also should to be considered.Approaches such as surgical treatment or drug treatment for obese patients are not flawless as well.Despite various treatments and lifestyle interventions,the global incidence of obesity continues to increase gradually,suggesting undiscovered mechanisms are involved in the development of obesity.Gradually increasing epidemiological and animal researches indicate that early environmental in uterine effects are important in fetal origin of health and disease.The physiological system of fetuses is stimulated by the environment and creates compensatory regulation aimed at strengthening fetus,ability of adapting to the postnatal environment.However,these compensatory changes may bring adverse consequences in adulthood.Previous studies by us have also shown that stimulated with LPS during pregnancy can significantly cause obesity in rats and mice,and LPS stimulation during pregnancy promotes fat adipocyte differentiation and magnifies adipocyte volume,or lead to obesity and hyperlipidemia in rat offspring.During the second trimester during rats,pregnancy,pyrrolidine dithiocarbamate?PDTC,a selective inhibitor of NF-?B?treatment can alleviate the symptoms of obesity,hyperlipidemia and hypertension caused by LPS stimulation during pregnancy.Toll-like receptor?TLRs?has been proved to be closely related with the occurrence of various diseases.TLRs are one of the pattern recognition receptors,which consist of an extramembranous domain containing multiple leucine-rich repeats?recognizing and binding ligands?,a single transmembrane domain?determining receptor location?and cytoplasmic Toll/The IL-1 receptor domain?recognized signal adaptor molecule?.Not only external signals such as lipoproteins,LPS,flagellin and viral nucleic acids,but also endogenous ligands or self-molecules derived from the host can be recognized,also known as damage-related molecular patterns?DAMPS?.After TLR activation,the adaptor comprising a TIR domain,such as myeloid differentiation factor 88?Myd88?or TRIF,is recruited to initiate downstream signal transduction and ultimately maintain or eliminate inflammation,Whether TLR plays a key role in the progeny obesity model induced by LPS during pregnancy is remains unknown.Two metabolism related TLRs?TLR2/TLR4?were elected in this study,we sought specifically to determine the role of TLRs in insulin resistance and obesity in offspring with prenatal LPS exposure.Method:1.AnimalsTLR2-deficient mice?C57BL/6 background?were maintained with C57BL/6 for heterozygous breeders.All mice were maintained in specific-pathogen-free?SPF?conditions and given free access to a standard laboratory mouse chow diet and tap water.Virgin heterozygous female?8-to-12-weeks?mice were time-mated with heterozygous males,and the day on which a vaginal plug was detected in the mated female was defined as 0.5 days post coitum?dpc?.At 11.5 dpc,female mice with a weight gain of 2 grams were considered pregnant and were subsequently intraperitoneally injected with either saline?100?L?or LPS?75?g/kg?.The pups were raised with the same lactating mother without LPS exposure to weaning age?4 weeks of age?,at which time they were transferred to cages containing three or four pups.Only wild-type and homozygous mutant male mice were used in the study.2.Dietary interventionMale offspring of C57BL/6 WT mice and congenic TLR2-deficient littermates received normal chow diet?NCD?or HFD starting at the age of 4 weeks.Based on genotypes,the treatment of the mother,and the types of chow consumed,the offspring were divided into 8 groups:pSaline,pLPS,TLR2-deficient+pSaline,TLR2-deficient+pLPS,pSaline+HFD,pLPS+HFD,TLR2-deficient+pSaline+HFD,TLR2-deficient+pLPS+HFD.3.Weighing and sample collectionAt the age of 12 weeks,the mice were sacrificed,and blood samples were collected.The serum samples were analyzed in the Department of Clinical Laboratory,Xinqiao Hospital,Chongqing,China.The epididymal white adipose tissue was dissected,weighed,and immediately frozen in liquid nitrogen or fixed in 4%paraformaldehyde for further biochemical or histological analyses.4 Calculation of relative fat coefficient and determination of fat sizeRelative adipose tissue weight=Total weight of adipose tissue g/Body weight of mice g׉After fixation,adipose tissue underwent embedding,staining,dehydration,transparency and sealing.Finally,the size of fat cells was observed under microscope.5.Determination of relative fat content in mice with Micro-CTThe mice were fixed on the support platform of small animal tomography equipment after anesthesia with isoflurane,and were given respiratory support throughout the experiment.Fat analysis was performed using the analysis 12.0?Analyzedirect?software specified in the micro-CT device instructions.6.Intraperitoneal glucose tolerance test?IPGTT?Groups of mice were 12 weeks to carry on the glucose tolerance test,groups of mice fasting after 6 hours by intraperitoneal injection of 2 g/Kg dose configured aseptic glucose solution,injection postscript 0 min and through the tail vein of mice on the way to take a small amount of blood stained with the blood sugar test paper,reading blood sugar from the blood glucose meter,and then 30 min after injection,60 min,120 min,respectively,repeat the blood sugar testing operation,calculate the area under the curve?AUC?.7.Intraperitoneal insulin resistance test?IPITT?An intraperitoneal injection of 0.75 U/kg insulin?Eli Lilly?was administered after 6hours of fasting,and blood glucose levels were measured at 0,15,30 and 60 min using a glucose meter.The AUC was calculated for fasting blood glucose.8.Cytokine assaySerum concentrations of Il-6 and TNF-?were determined by ELISA kits.9.Western BlotTotal protein of adipose tissue was extracted and quantified by BCA method.Protein was isolated by SDS-page electrophoresis.Then the protein was transferred to 0.22m?PVDF membrane,and the membrane was incubated with primary antibody and corresponding secondary antibody after blocking.After several times of washing by TBST,detects the corresponding signal with the chemiluminescence detection system.10 Real-time PCRAfter tissue RNA was extracted,total RNA was synthesized into cDNA by reverse transcription with cDNA kit.The obtained DNA was mixed with primers and amplified by40 cycles.Result1.TLR2-deficiency does not prevent obesity and insulin resistance in offspring-pLPSTLR2 mRNA level was significantly elevated in the adipose tissue isolated from offspring-pLPS mice compared to the offspring-pSaline controls,while the mRNA levels of TLR4 remained unchanged between the two groups.Western Blot analysis further confirmed this finding that TLR2 was significantly elevated in the adipose tissue of offspring-pLPS.The offspring of both wild-type and TLR2-deficient mice prenatally treated with LPS developed similar obese phenotypes compared to control offspring prenatally treated with saline?offspring-pSaline?of either wild-type or TLR2-deficient mice at 12-weeks of age.We also found a similar trend in glucose and insulin resistance,as indicated by IPGTT,IPITT and protein expression of phosphorylated IRS-1^Ser307 and AKT^ser473,in offspring-pLPS mice of both wild-type and TLR2-deficient genotypes when compared to the offspring-pSaline mice of both wild-type and TLR2-deficient genotypes2.TLR2-deficiency does not prevent high-fat induced obesity and insulin resistance in offspring-pLPS.The wild-type offspring-pLPS mice fed a HFD had significantly higher body weights than HFD-fed offspring-pSaline control mice.Surprisingly,TLR2-deficiency did not alter the phenotype,as the HFD-fed TLR2-deficient offspring-pLPS mice had a similarly increased level of body weight compared to HFD-fed wild type offspring-pLPS mice.Consistent with this finding,all HFD-fed offspring-pLPS of wild-type and TLR2-deficient mice presented with a similarly elevated level of relative adipose tissue weight and impaired glucose tolerance curve compared to HFD-fed pSaline controls of either genotype.3.TLR2-deficiency promotes hyperlipidemia in offspring-pLPSWe analyzed serum lipid content of TLR2-deficient offspring-pLPS.Unexpectedly,our data demonstrated more severely elevated serum levels of TCH,TG and LDL-c in TLR2-deficient offspring-pLPS compared to saline and LPS treated wild-type control mice.TLR2-deficiency alone had no effect on blood lipid levels.The elevation of HDL-c appeared only relevant to prenatal LPS exposure,suggesting a protective feedback in response to dyslipidemia induced by prenatal inflammatory exposure.4.Compensatory TLR4 activation in TLR2-deficient offspring with prenatal LPS exposureData showed that TLR4 expression was dramatically increased in TLR2-deficient offspring-pLPS when compared to wild-type offspring-pLPS and TLR2-deficient offspring-pSaline mice.Consistent with the upregulation of TLR4,the Myd88 transcript was highly elevated in TLR2-deficient offspring-pLPS.Additionally,the serum concentration of cytokines for TLR4 activation,such as TNF-?and IL-6,were also significantly elevated in TLR2-deficient offspring-pLPS.While the expression of phospho-Myd88tyr257 was proportionally increased when normalized to total Myd88protein levels in the TLR2-deficient offspring-pLPS adipose tissue5.Increased high-mobility group box 1?HMGB-1?expression in TLR2-deficient offspring-pLPSBoth qRT-PCR and Western Blot analyses demonstrated that HMGB-1 expression levels were significantly elevated in the TLR2-deficient offspring-pLPS adipose tissue.RAGE expression was not affected in these mice?Fig.6A?,suggesting that HMGB/TLR4 is the major signaling component of inflammation in TLR2-deficient offspring-pLPS.6.VLDLR expression in TLR2-deficient offspring-pLPSWe found significantly downregulated CETP expression in offspring-pLPS independent of the TLR2 genotype,which might partly contribute to the dyslipidemia phenotype.We observed a specific upregulation of VLDLR at both the mRNA and protein levels in offspring-pLPS,and its expression was further elevated in TLR2-deficient offspring-pLPS adipose tissue.Conclusion1.TLR2-deficiency promotes hyperlipidemia in offspring-pLPS.2.TLR2-deficiency does not prevent obesity and insulin resistance in offspring-pLPS,as well as in offspring-HFD-pLPS.3.The promoted prenatal lipopolysaccharide stimulation induced hyperlipidemia caused by TLR2-deficient is partly due to increased expression of high-mobility group box1?HMGB-1?and TLR4 signal.