| Diabetes mellitus is currently one of the most important chronic non-communicable diseases worldwide.People with type 2 diabetes mellitus(T2DM)not only have glucose metabolism dysfunction,but also often express dyslipidemia.In recent years,the protective effects of ?-3 polyunsaturated fatty acid(PUFA)on cardiovascular disease have become a hot spot of concern in the medicine and nutrition communities.Dietary?-3 PUFA can be classified into animal-derived and plant-derived ?-3 PUFA.Animal-derived ?-3 PUFA is represented by eicosapentaenoic acid(EPA)and docosahexaenoic acid(DHA).Alpha-linolenic acid(ALA)represents the plant-derived ?-3 PUFA.At present,there are relatively large number of controversial researches in animal-derived ?-3 PUFA,but relatively small number of those in plant-derived ?-3 PUFA regarding T2DM risk and intervention research.Therefore,there is a great need for deeply understanding the effects of ?-3 PUFA from different sources on glucose and lipid metabolism in type 2 diabetic patients with dyslipidemia and its mechanism.This study aimed to determine the effects of fish oil-derived ?-3 PUFA,perilla oil-derived ?-3 PUFA,fish oil mixed with linseed oil-derived ?-3 PUFA on glucolipid metabolism in type 2 diabetic patients with dyslipidemia.Meanwhile,C57BL/6 mice and MyD88-/-mice fed on high fat diet were used to reveal the mechanism of insulin resistance(IR)via TLR4/MyD88 dependent way and TLR4/MyD88 independent way.Part 1:Effects of ?-3 polyunsaturated fatty acids from different sources on glucolipid metabolism in type 2 diabetic patients with dyslipidemiaObjective:(1)To determine the effects of ?-3 polyunsaturated fatty acids from different sources on glucolipid metabolism in type 2 diabetic patients with dyslipidemia.Methods:We recruited participants from the diabetes clinic at the Guanlin hospital in Yixing city,Jiangsu province from February 2017 to March 2017.180 subjects were randomly assigned to 3g/day fish oil(FO,containing 1.14g EPA and 1.44g DHA),3g/day perilla oil(PO,containing 2.04g ALA),or 3g/day fish oil mixed with linseed oil(FLO,containing 0.63g EPA,0.36g DHA and 0.84g ALA)for 6 months.Fasting venous blood sample was obtained from each study subject at baseline,after 3 and 6 months of intervention.Serum glucose,triglyceride(TG),total cholesterol(TC),high density lipoprotein cholesterol(HDL-C),low density lipoprotein cholesterol(LDL-C),apolipoprotein A1(Apo A1),apolipoprotein B(Apo B),Lipoprotein(a)[Lp(a)],free fatty acid(FFA),glutamic-pyruvic transaminase,glutamic-oxalacetic transaminease,creatinine,uric acid,urea nitrogen,total protein,albumin and globulin were determined by automatic biochemical analyzer(Mindray,BS-800).Glycated haemoglobin(HbAlc)was determined by chromatography(Audicom,AC6601).C peptide and insulin were determined by chemiluminescence(Roche,cobas e 602).Results:156 subjects completed the final follow-up after 6 months(FO,54 subjects;PO,52 subjects;FLO,50 subjects).C peptide,insulin,homeostasis model assessment-insulin resistance(HOMA-IR),TG,TC,non HDL-C and Apo A1 in FO,PO and FLO were significantly lower than that at the baseline(P<0.05).Glucose,HbAlc,Apo B and Lp(a)in PO and FLO were significantly lower than that at the baseline(P<0.05).FFA in FLO was significantly lower than that at the baseline(P<0.05).Compared with PO and FLO,TG in FO significantly decreased(P<0.05)after 3 months.There was a trend for HDL-C to increase after fish oil supplementation(P=0.055).Conclusion:The effects of ?-3 PUFA from different sources on glucose metabolism in type 2 diabetic patients with dyslipidemia are similar.TG-lowering effect of animal-derived ?-3 PUFA in type 2 diabetic patients with dyslipidemia shows an advantage over plant-derived ?-3 PUFA and animal mixed with plant-derived ?-3 PUFA.Part 2:Effects of ?-3 polyunsaturated fatty acids from different sources on insulin resistance via TLR4/MyD88 dependent way in C57BL/6 miceObjective:To investigate the effects of ?-3 PUFA from different sources on IR via TLR4/MyD88 dependent way in C57BL/6 mice.Methods:Six-week-old specified pathogens free male C57BL/6 mice were randomly divided into 4 groups(n=10 in each group)after a week of acclimization period according to body weight:(1)normal control(NC)group was fed normal AIN-93M diet accompanied by daily gavage with 0.5g/kg bw 5‰ carboxy methyl cellulose sodium,(2)model control(MC)group was fed high-fat D12451 diet accompanied by daily gavage with 0.5g/kg bw 5‰ carboxy methyl cellulose sodium,(3)fish oil(FO)group was fed high-fat D12451 diet accompanied by daily gavage with 0.5g/kg bw fish oil(38g EPA and 48g DHA per 100g),and(4)perilla oil(PO)group was fed high-fat D12451 diet accompanied by daily gavage with 0.5g/kg bw perilla oil(68g ALA per 100g).After administration of ?-3 PUFAs for 4 weeks,the animals were fasted for 12-14 hours and then sacrificed with sodium pentothal.Blood and epididymal adipose tissue samples were collected for further analyses.Serum glucose,TG,TC,HDL-C and LDL-C were determined by automatic biochemical analyzer(Beckman,DxC800).Serum insulin,monocyte chemotactic protein-1(MCP-1),interleukin-6(IL-6),tumor necrosis factor-a(TNF-a),leptin and resistin were measured using MILLIPLEX(?)map kits(Merck Millipore).The expression of TLR4,MyD88,TRAF6,IKK?,NF-?B p65,RIP1 and IRF3 in epididymal adipose tissue were examined by RT-PCR and western-blot.The epididymal adipose tissue was stained with eosin-hematoxylin for pathological examination.Results:Serum glucose,insulin,TG,TC,HDL-C,LDL-C,non HDL-C,LDL-C/HDL-C,IL-6 TNF-a and leptin were significantly increased in MC than those in NC(P<0.05).However,significantly lower glucose,insulin,TG,TC,HDL-C,LDL-C,non HDL-C and IL-6 were observed in FO and PO,as opposed to MC(P<0.05).The histological examination of epididymal adipose tissue in MC revealed decrease of adipocyte counts(P<0.05)and serious inflammatory cell infiltration.These histopathological changes were ameliorated by fish oil and perilla oil supplementation(P<0.05).Both RT-PCR and western-blot showed that the expression of TLR4,MyD88,TRAF6,IKK?,NF-?B p65,RIP1 and IRF3 were significantly increased in MC than that in NC(P<0.05).However,significantly lower expression of TLR4,MyD88,TRAF6,IKK? and NF-?B p65 were observed in FO and PO,as opposed to MC(P<0.05).Conclusion:These findings indicate that animal-derived ?-3 PUFA and plant-derived ?-3 PUFA could be used to prevent IR and dyslipidemia via TLR4/MyD88 dependent way in C57BL/6 mice.Part 3:Effects of ?-3 polyunsaturated fatty acids from different sources on insulin resistance via TLR4/MyD88 independent way in MyD88-/-miceObjective:To investigate the effects of ?-3 PUFA from different sources on IR via TLR4/MyD88 independent way in MyD88-/-mice.Methods:Eight-week-old specified pathogens free male MyD88-/-mice were randomly divided into 4 groups(n=6 in each group)after a week of acclimization period according to body weight:(1)normal control(NC)group was fed normal AIN-93M diet accompanied by daily gavage with 0.5g/kg bw 5‰ carboxy methyl cellulose sodium,(2)model control(MC)group was fed high-fat D12451 diet accompanied by daily gavage with 0.5g/kg bw 5‰ carboxy methyl cellulose sodium,(3)fish oil(FO)group was fed high-fat D12451 diet accompanied by daily gavage with 0.5g/kg bw fish oil(3 8g EPA and 48g DHA per 100g),and(4)perilla oil(PO)group was fed high-fat D12451 diet accompanied by daily gavage with 0.5g/kg bw perilla oil(68g ALA per 100g).After administration of ?-3 PUFAs for 4 weeks,the animals were fasted for 12-14 hours and then sacrificed with sodium pentothal.Blood and epididymal adipose tissue samples were collected for further analyses.Serum glucose,TG,TC,HDL-C and LDL-C were determined by automatic biochemical analyzer(Beckman,DxC800).Serum insulin,MCP-1,IL-6,TNF-a,leptin and resistin were measured using MILLIPLEX(?)map kits(Merck Millipore).The expression of TLR4,RIP1,IRF3 and NF-?B p65 in epididymal adipose tissue were examined by RT-PCR and western-blot.The epididymal adipose tissue was stained with eosin-hematoxylin for pathological examination.Results:Serum glucose,insulin,TC,HDL-C,leptin and resistin were significantly increased in MC than those in NC(P<0.05).Serum glucose,insulin,TC,HDL-C,MCP-1,IL-6,TNF-a,leptin and resistin were not significantly different in FO and PO,as opposed to MC.The adipocyte counts in MC,FO and PO had no differences from NC.Both RT-PCR and western-blot showed that the expression of TLR4,RIP 1,IRF3 and NF-?B p65 were significantly increased in MC than that in NC(P<0.05).However,the expression of TLR4,MyD88,TRAF6,IK? and NF-?B p65 in FO and PO had no differences from MC.Conclusion:These findings indicate that animal-derived ?-3 PUFA and plant-derived ?-3 PUFA could not be used to prevent IR and dyslipidemia via TLR4/MyD88 independent way in MyD88-/-mice. |
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