The Relationship Study Of Toll-like Receptor Family Wtih Hyperlipidemia And Insulin Resistance

Diabetic mellitus has become the worldwide health problem because ofits complications and its association with chronic diseases such as obesity,nonalcoholic fatty liver disease (NAFLD), atherosclerosis, hyperlipidemia,osteoporosis, and so on. The relationship among chronic inflammation,hyperlipidemia and insulin resistance (IR) has been well established; theproposal of “diabetes is glycolipids disease” further clarifies the relationshipbetween diabetes and lipid metabolism disorders. Insulin resistance is thoughtto be the pathological background of metabolism diseases such as diabeticmellitus, obesity, NAFLD, athrosclerosis, etc.Toll-like receptors (TLRs) belong to a class of proteins and a subclass ofpathogen pattern recognition receptors which are broadly expressed in thecells of innate immune system (macrophages, epithelial and endothelial cells,and organ parenchyma cells) and the cells of the adaptive immune system (Bcells, mast cells, T cells, and dendritic cells). Toll-like receptors mediateinfection-induced inflammation by recognizing invading pathogens andactivating downstream signaling pathways that lead to the expression ofdiverse arrays of pro-inflammatory marker gene products. Recent evidencesuggests that certain TLRs can be activated by endogenous molecules derivedfrom tissue injury and saturated fatty acids, so TLRs mediate not onlyinfection-induced inflammation but also sterile inflammation induced byendogenous molecules.In the past decade, researchers are increasingly interested in investigatingthe role of TLR in the pathogenesis of diabetic mellitus. Data from humanstudies as well as animal and cell studies demonstrate that TLR4play a role inthe development of insulin resistance and type2diabetic mellitus and TLR9mainly mediates the development of type1diabetic mellitus. However, whether TLR2play a role in type2diabetic mellitus remain controversial.Meanwhile, studies of other members of this family in the development oftype2diabetes mellitus are limited; the role of TLR in the development ofhyperlipidemia has been farely reported. Therefore, in the present study, wedetected all of the ten receptors include TLR1to TLR10in diabetic andhyperlipidemic individuals.In the animal study section, we use IR andhyperlipidemia model induced by high fat diet. The Gene and proteinexpressions of the ten TLRs in skeletal muscle were detected to explore theassociation of TLRs with IR. The effect of drug intervention on TLR familywas also observed. Through the present study, we hope to find a new target forthe treatment of dyslipidemia and insulin resistance.Part1: The relationship between TLR family gene expression andhyperlipidemiaObjective: To detect the differences of TLR1-10gene expression inperipheral blood mononuclear cells (PBMCs) in individuals withhyperlipidemia.Methods: All of the182hyperlipidemia volunteers aged from26to45years old were selected from medical examination center in General Hospitalof Hebei (aged35.55±4.77;113males and69females). According to theirlipid levels they were assigned to hypertriglyceridemia group (HTG group,43subjects), hypercholesterolemia group (HTC group,84subjects) and mixedhyperlipidemia group (MHL group,55subjects) respectively. Sixty-eighthealthy volunteers were included (aged34.68±4.27;40males and28females)as control group (Con group). Age and sex were matched among differentgroups. Height, weight and waist of all subjects were detected and body massindex (BMI) was calculated. Fasting vein blood samples of every subject werecollected for the detection of triglyceride, cholesterol and other biochemicalindicators. At the same time, fasting blood samples were collected to isolatePBMCs using Ficoll Hypaque centrifugation followed by magnetic separationusing the depletion technique (Miltenyi Biotech, Auburn, CA). Total RNAwas extracted from monocytes using Trizol reagent (Invitrogen, USA) according to the manufacturer’s instructions and the gene expression wasdetected by RT-PCR.Results:1. Basic and biochemical parameters.The HTG group, HTC group and MHL group were similar with Congroup in terms of sex, age and BMI (P>0.05). Waist in MHL group was largerthan that in Con group with no statistic difference (85.29±9.42cm vs82.97±7.52cm, P>0.05). Waists in HTC group and HTG group (82.44±8.36cmand82.44±6.54cm, respectively) were similiar with the Con group. The TGand TC level in HTG group was higher than that in Con group(2.66±0.45mmol/L vs0.87±0.30mmol/L, P<0.05;6.12±0.20mmol/L vs4.46±0.63, P<0.05, respectively). TC and TG levels in MHL group were bothhigher than those in Con group (6.20±0.30mmol/L vs4.46±0.63, P<0.05;2.64±0.49mmol/L vs0.87±0.30mmol/L, P<0.05, respectively).2Comparison of TLR gene expressions in PBMCsThe gene expressions of TLR1and TLR8were significantly higher inMHL and HTC groups in which MHL group had higher levels. For the furthercomparison between hyperlipidemia groups we found that levels of the twogenes in HTG group were significantly lower than MHL group(TLR1:1.53±0.48vs2.13±0.86, P<0.05; TLR8:1.12±0.27vs1.48±0.42,P<0.05). In hyperlipidemia groups, relative gene expressions of TLR2andTLR9were statistically higher than those in Con group, but there were nostatistic difference between the hyperlipidemia groups. TLR3and TLR4werehighly expressed in PBMCs of the three hyperlipidemia groups (P<0.05).HTC group had the highest levels with statistic difference compared withMHL group (TLR3:2.09±0.83vs1.67±0.49, P<0.05; TLR4:2.08±0.51vs1.63±0.41, P<0.05). Gene expressions of TLR5-7were highest in MHL groupcompared with the other three groups (P<0.05). Gene expressions of TLR5-7in HTC group and HTG group were all higher than those in Con group(P<0.05). Gene levels of TLR10gene expression in hyperlipidemia groupswere higher than Con group but there was no statistic significance between MHL group and Con group (1.14±0.63vs1.03±0.30, P>0.05). Also, therewere no statistic differences between hyperlipidemia groups.Conclusions:1Relative TLR1-10gene expressions in PBMCs are higher inhyperlipidemia individuals than in Con group individuals.2Gene expressions of TLR1, TLR3, TLR4and TLR8are mainly relatedwith cholesterol; TLR5-7gene expressions are associated with cholesterol andtriglyceride level. TG and TC levels increase TLR5-7gene expressionssynergically. Gene expressions of TLR2and TLR9are not associated with thefeatures of hyperlipidemia.Part2: The relationship between TLR genes expression and type2diabetic mellitusObjective: To detect the differences of TLR1-10gene expression inperipheral blood mononuclear cells (PBMCs) in individuals with impairedglucose regulation (IGR), type2diabetic mellitus and in healthy people.Methods: All of the104hyperglycemic volunteers include67males and37females aged from26to47years old (34.39±4.78years old) were selectedfrom medical examination center in General Hospital of Hebei. They wereassigned to impaired glucose regulation group (IGR group,37subjects) andtype2diabetic mellitus group (T2DM group,67subjects) respectivelyaccording to their blood glucose levels. Moreover, we selected68healthyvolunteers as control group (Con group). The age and sex were matched incontrol group with hyperglycemic people. Height, weight and waist of allsubjects were detected and body mass index (BMI) was calculated. Fastingvein blood samples of every subject were collected for the assay of fastingblood glucose (FBG), insulin and other biochemical indicators. At the sametime, fasting blood samples were collected to isolate PBMCs by magneticseparation using the depletion technique (Miltenyi Biotech, Auburn, CA).Postprandial blood glucose (PBG) level was tested using ulnar vein bloodsample. Total RNA was extracted from monocytes using TRIzol reagent(Invitrogen, USA) and the genes expression was detect with RT-PCR method. Results:1Clinical and biochemical indicators comparisonThe three groups were matched in terms of sex, age and BMI (P>0.05).Waists of the three groups are not statistically different (T2DM:82.94±6.32cm;IGR:82.78±5.62cm; Con:82.97±7.52cm, P>0.05). Both FBG and PBG inT2DM group and IGR group were significantly higher than Con group. Thechange in insulin resistance index is the similar with change of FBG and PBG;T2DM group had the highest HOMA-IR as1.98±0.33, followed with IGRgroup (1.84±0.42), and the Con group (1.28±0.20).2Comparison of TLR gene expressions in PBMCsTLR1-3, TLR6, TLR8and TLR9were significantly higher in IGR groupand T2DM group than Con group and they were all higher in T2DM groupcompared to IGR group. TLR4and TLR7were both higher in hyperglycemicgroups than Con group, interestingly their levels of T2DM group were lowerthan IGR group (TLR4:1.51±0.34vs1.70±0.41, P<0.05; TLR7:1.57±0.32vs1.71±0.41, P<0.05); TLR5and TLR10had higher expressions in IGR groupand T2DM group compared to Con group. Gene expression of TLR5is higherin T2DM group compared with IGR group with no statistic significance(1.46±0.46vs1.35±0.28, P>0.05). Gene expression level of TLR10is similarin IGR group and T2DM group (1.24±0.30vs1.22±0.45, P>0.05). We foundliner relationships between HOMA-IR and TLR1-9using HOMA-IR asdependent factor and TLRs as independent factor, respectively.Conclusions:1The genetic expressions of TLR1-3, TLR6, TLR8and TLR9inPBMCs are gradually increased in pre-diabetes people and T2DM people.Moreover, the expression levels of the six genes in T2DM group aresignificantly higher compared to IGR group.2Gene expressions of TLR4and TLR7are higher in IGR group anddiabetes group compared with control group. Gene expressions of TLR4andTLR7are higher in IGR group than in diabetes group.3The genetic expressions of TLR5and TLR10in PBMCs are increased in IGR and T2DM group with no difference between T2DM and IGR.4TLR1-9is closely related with insulin resistance.Part3: Expression of TLR in skeletal muscle of high-fat diet rats andinsulin resistanceObjective: To detect whether TLR1-10is highly expressed ininsulin-sensitive tissue-skeletal muscle using a high-fat diet induced insulin-resistant rat model.Methods: Forty-eight male SD rats (120-150g) were randomly dividedinto control group (Con group, n=16) and high-fat diet group (HF group, n=32)after2-week adaption. The basic diet and high-fat-diet raw materials werepurchased from the experimental animal centre of Hebei Medical Univercity.High-fat diet was made according to the formula. Animals in Con group werefed with basic diet which contained65.5%carbohydrate,10.3%fat and24.2%protein; the others in HF group were feed with high-fat feed which contained20.1%protein,20.1%carbohydrate and65.5%fat. All rats were free for foodand water. Six rats were randomly selected from each group at the end of6weeks for hyperinsulinemic euglycemic clamp. Blood samples were kept forthe assay of triglyceride (TG), total cholesterin (TC), high density lipoprotein(HDL), low density lipoprotein (LDL) and very low density lipoprotein(VLDL). Skeletal muscle tissues were frozen for detecting gene and proteinexpression of TLRs. Blood glucose was detected with rapid blood test meter(U.S.A). Gene expression was detected with RT-PCR. Protein contents weredetected by Western blot.Results:1The comparison of weight, blood glucose level and GIRBody weight and fasting blood glucose (FBG) in HF group weresignificantly higher than those of the Con group after6-week high-fat dietintervention (485.99±22.32g vs450.83±25.94g, P<0.05;5.26±0.48mmol/L vs4.28±0.42mmol/L,P<0.05).GIR of HF group was significantly lower thanCon group (14.09±1.40mg/Kg/min vs27.14±0.73mg/Kg/min, P<0.05). TC,TG, LDL and VLDL were higher in HF group than Con group while HDL was lower in HF group. The weight and FBG of HF group were higher than Congroup after8-week additional high-fat-diet intervention. Compared with ratsfed for6weeks, weight and FBG were higher after14-weeks feeding both inHF group and in Con group (P<0.05). LDL and VLDL levels rose up in Congroup after14-weeks feeding (LDL:0.41±0.02mmol/L vs0.35±0.02mmol/L,P<0.05; VLDL:0.16±0.05mmol/L vs0.07±0.02mmol/L, P<0.05). GIR of HFgroup at14weeks was further declined than6weeks (11.72±1.64mg/Kg/minvs14.09±1.40mg/Kg/min, P<0.05) and there was no change in Con group(27.49±1.23mg/Kg/min vs27.14±0.73mg/Kg/min, P>0.05).2Comparison of TLR gene expressions in skeletal muscleThe gene expressions of TLR1-10in skeletal muscle of HF group weresignificantly higher compared to Con group at both6weeks and14weeks.TLR1, TLR5, TLR7and TLR8in HF group had no difference at6weeks and14weeks.TLR2and TLR4were declined at14weeks compared to6weeks inHF group. The gene expressions of TLR3, TLR6, TLR9and TLR10rose up,but the changes are not statistically different (P>0.05).3Comparison of TLR protein expressions in skeletal muscleThe protein levels of TLR1-10in skeletal muscle of HF group were allhigher than those of Con group at both6weeks and14weeks, but the proteinlevel of TLR5is not statistic different between the two groups at6weeks(1.17±0.24vs0.96±0.06, P>0.05). The protein levels of TLR1-3, TLR5,TLR6and TLR10in HF group were higher at14weeks compared to6weeks.Of which, TLR1and TLR6were statistically higher than control group (TLR1:1.75±0.24vs1.34±0.21, P<0.05; TLR6:1.99±0.23vs1.30±0.17, P<0.05).Protein levels of TLR4and TLR7-9at14weeks were declined compared to8weeks in HF group. TLR7and TLR8were also statistically different (TLR7:1.27±0.18vs1.66±0.17, P<0.05; TLR6:1.49±0.19vs1.96±0.21, P<0.05).Conclusions:1Six weeks high-fat diet intervention cause blood lipids increase andGIR decrease, well established insulin resistance experimental rat model.2The gene and protein expression levels of TLR1-10in skeletal muscle of insulin-resistant rats induced by high-fat diet are significantly increased.TLR1and TLR6are closely associated with the extent of insulin resistanceand are increased with longer high-fat feeding.Part4: Effects of drugs on expression of TLR family in skeletal muscle inhigh-fat diet ratsObjective: By improving insulin resistance induced by high-fat diet withdrug intervention, to investigate the relationships between TLR and insulinresistance and to explore the new target for treatment of insulin resistance.Methods: After insulin resistance and hyperlipidemia rat model wereestablished (high-fat diet for6weeks), rats in Con group were given basicfeed as Con group (Con group), high-fat diet group were randomly dividedinto high-fat diet control group (HF gorup), Jin Li Da group (JLD group) andpioglitazone group (Ptz group). Drugs intervention was through intragastricadministration with dose of JLD1.5g/Kg.d and pioglitazone4.5mg/Kg.d. JLDand Ptz were all dissolved with0.5%sodium carboxymethylcellulose and thedrugs concentrations were0.375g/ml and1.125mg/ml respectively. Rats inCon group and HF group were given sodium carboxymethylcellulose4ml/Kg.d through intragastric administration. Two rats lived in one cage andthe light-dark cycle was12hours (6am-6pm), temperature was20℃-23℃.Basic diet and raw materials of high-fat diet were all purchased from theexperiment animal centre of Hebei Medical Univercity. High-fat dietcontained flour100g, soybean meal330g, soy flour200g, fishmeal20g, lard300g, bran20g and bone meal30g per kilometer. Energy ratio of basic feedwas carbohydrates65.5%, fat10.3%and protein24.2%; Energy ratio ofhigh-fat diet was carbohydrates20.1%, protein20.1%and fat59.8%. Allanimals were fed ad libitum with food and water. At the end of8weeks all ratwere given hyperinsulinemic euglycemic clamp study to evaluate insulinsensitivity. Blood samples were collected to test triglyceride (TG), totalcholesterin (TC), high density lipoprotein (HDL), low density lipoprotein(LDL) and very low density lipoprotein (VLDL). Skeletal muscle tissue wasfrozen for detecting gene and protein expression of TLR. Blood glucose was detected with rapid blood test meter (U.S.A). Gene expression was detected byRT-PCR method and protein detected by Western blot.Results:1The comparison of weight, blood glucose level and GIR in different groupsBefore drugs intervention body weight in rats in HF group and drugintervention groups were significantly higher compared to Con group. Therewere no statistic difference between HF group and drug intervention groups inthe term of weight. After8weeks of drug intervention body weight of HFgroup and drug intervention groups were still higher compared to Con group,but body weight of the two drug intervention groups were significantly lowercompared with HF group. Fasting blood glucose (FBG) levels of HF group,JLD group and Ptz group were significantly higher than those of Con group atthe beginning. At the time of14weeks FBG of Con group and HF group, JLDgroup were all higher than at6weeks. On the contrary, FBG of Ptz group at14weeks was decreased. FBG comparison at the time of14weeks revealedthat JLD group and HF group were significantly higher than Con group andthere was no difference between the two groups (6.25±0.19mmol/L vs6.32±0.27mmol/L,P>0.05). FBG of Ptz group was slightly higher at14weekscompared to6weeks without statistic significance (5.15±0.45mmol/L vs4.97±0.25mmol/L, P>0.05). GIR in HF group is than Con group(11.72±1.64mg/Kg/min vs27.49±1.23mg/Kg/min, P<0.05). GIR in JLDgroup was clearly higher than HF group and was not significantly differentwith Con group (28.10±1.77mg/Kg/min vs27.49±1.23mg/Kg/min, P>0.05).Insulin sensitivity was highest in Ptz group with GIR31.27±1.40mg/Kg/min,which was significantly higher than control group (P<0.05).2Comparison of blood lipid after drugs intervention for8weeksLevels of TC, TG, LDL, VLDL were significantly higher in HF groupand HDL level was significantly lower in HF group (P<0.05). Both JLD andPtz improved hyperlipidemia after8weeks intervention. TC, TG, LDL andVLDL levels significantly decreased in JLD group, while HDL level increasedsignificantly. TC, TG, LDL and VLDL levels of Ptz group were also lower than HF group; HDL level higher compared to HF group (0.48±0.02mmol/Lvs0.4±0.02mmol/L, P<0.05). Totally, the improvement effect onhyperlipidemia of Ptz was less than JLD.3Comparison of TLR gene expressions in skeletal muscle after drugintervention for8weeksGene expressions of TLR1-10in skeletal muscle in HF group weresignificantly increased compared with Con group. The gene expressions ofTLR1-10were all decreased in JLD group and Ptz group when compared withthose of the HF group. Gene expression levels of TLR3, TLR4and TLR6inJLD group were higher than Con group with no statistic difference (P>0.05).Gene expressions of TLR1-10in Ptz group were higher than Con group withonly TLR2, TLR7and TLR8statistically different.4Comparison of TLR protein expressions in skeletal muscle after drugsintervention for8weeksTLR protein levels in HF group were higher than those of the Con group,but TLR7was not statistically different after intervention (1.27±0.18vs1.02±0.06, P>0.05). Protein levels of TLR in JLD group and Ptz group wereall declined than HF group, of which TLR5, TLR7and TLR10were notstatistically different. Protein levels of TLR1-4, TLR8and TLR9in Ptz groupwere lower compared to JLD group. Protein levels of TLR6were similarbetween JLD group and Ptz group (1.47±0.16vs1.46±0.33).Conclusions:1Both Ptz and JLD can improve insulin resistance induced byhigh-fat-feeding and improve hyperlipidemia. JLD is more effective than Ptzin improving hyperlipidemia and less effective in insulin resistance.2Ptz and JLD can significantly decrease the gene and proteinexpressions of TLR family (especially TLR1-4and TLR6-8) in skeletalmuscle of high-fat diet rats.