| BackgroundObesity and type 2 diabetes both are metabolic disease which closely related to life-style.Obesity and diabetes are increasing at an alarming rate worldwide,but the strategies for the prevention and treatment of these disorders remain inadequate.According to related statistic,there are about one billion people are overweight or obese in the world.Insulin resistance is an important feature of obesity and type 2 diabetes mellitus.Adipose tissue dysfunction plays an important role in the development of insulin resistance.Therefore,to explore the deep mechanism of insulin resistance in adipose tissue of obesity and type 2 diabetes mellitus and to establish a mechanism-oriented treatment measures has become a hotspot in the prevention and treatment of diabetes mellitus in recent years.Adipose tissue is closely associated with insulin resistance.Adipose tissue can not only regulate the body’s energy metabolism,but also be an important secretory organ.Adipose tissue can produce a variety of adipose factors,thus affecting the metabolic state of the body.Obese patients;especially central obese patients have more visceral adipose tissue,the more visceral adipose tissue they have,the more serious is the insulin resistance,because visceral adipose tissue is metabolically active,large amount of free fatty acids(FFA)will be produced when the adipose tissue dysfunction.Overmuch FFA leads to ectopic deposition of FFA in insulin target organs such as liver,muscle,et al;and plays an effect of fatty toxicity in the ectopic deposition organs.Therefore,the dysfunction of adipose tissue plays an immeasurable role in the occurrence and development of obesity and diabetes.AMPK plays an important role in adipocyte dysfunction.AMPK is abundant in adipose tissue,liver tissue,skeletal muscle and pancreas.The AMPK activation process is mainly realized by the activation of the metabolic function and the closure of the synthetic metabolic function,so as to keep the metabolic and energy balance in a stable state.Therefore,AMPK is widely regarded as the "master switch" to regulate the energy metabolism balance of the body.Existing studies have shown that AMPK is widely involved in the metabolic regulation process of major glycolipid metabolic tissues of the body,and it is likely to be a new target for the prevention and treatment of obesity and type 2 diabetes.TRB3 is an important suppressor of AMPK.Tribble3(TRB3)is discovered by Gossans in files.TRB3 is a pseudokinase which can inhibit the mitosis.TRBs genes include TRB1,TRB2,and TRB3.TRB3 in files is mammal homologue,so TRB3 gene has drawn great attention over the recent years.TRB3 has broad biological activity.It has been demonstrated to be involved in lipid metabolism and in impairment of insulin exocytosis.Among the various biological activities of TRB3,the most widely concerned is its irole in the formation of insulin resistance.A large number of studies have shown that TRB3 can directly regulate AMPK and is a key regulatory protein in the AMPK pathway.Therefore,we speculated that TRB3 may aggravate the dysfunction of glucose and lipid metabolism in adipose tissue by inhibiting AMPK activity,thus leading to insulin resistance in the whole body of fat diabetes mellitus.In this study,we inducted insulin-resistant adipocytes modle.To further elucidate the role of TRB3/AMPK signaling pathway in insulin-resistant adipocytes,we used TRB3 gene silencing in vitro to inhibit TRB3 expression.We explored the changes in adipocyte dysfunction after TRB3 gene silencing by red-oil staining and molecular techniques and then explored the role of TRB3/AMPK signaling pathway in insulin resistance of adipocytes and its possible molecular mechanism.ObjectivesTo further elucidate the role of TRB3/AMPK signaling pathway in obesity and DM,we used TRB3 gene silencing in vitro to explore the mechanisms of TRB3/AMPK signaling pathway in obesity and DM as a potential target for treatment.Methods1.Cell culture and induction of insulin resistance(IR)Murine 3T3-L1 pre-adipocytes,purchased from American Type Culture Collection(ATCC),were cultured in DMEM containing 10%FBS,and differentiated in a cocktail containing insulin,3-isobutyl-1-methylxanthine and dexamethason based on a standard protocol.The fully differentiated 3T3-L1 adipocytes were pre-incubated for 24h in serum-free and low-glucose DMEM,and randomly divided into two groups:control group(Control)and insulin resistance(IR)group,control group was incubated with low-glucose DMEM containing 5.5mM glucose and 10%FBS,IR was induced by incubating differentiated 3T3-L1 adipocytes in high-glucose DMEM containing 25mM glucose,10%FBS and 10nM insulin for 24h.Another two groups are:control group(Control)and free fatty acids(FFA)group,FFA group was incubated with medium containing low-glucose DMEM,FBS,BSA and 0.75 mM palmitate for 24h as previously,2.Oil Red O stainingThe fully differentiated 3T3-L1 adipocytes were washed twice with PBS and fixed with 4%paraformaldehyde for 15min.Then,cells were washed with water and stained for 30 min with Oil Red O solution(60%isopropanol,40%water).Excess stain was removed by washing with PBS,and then microscopic images were recorded.3.Triglyceride assayThe fully differentiated 3T3-L1 adipocytes triglycerides were extracted,and measured using a Triglyceride Quantification Kit(Abcam),following the manufacturer’s instructions for colorimetric assay.4.Glucose uptakeFor experiments,all culture medium was removed from each well and replaced with low-glucose DMEM containing 10%FBS,1%2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-2-deoxyglucose(2-NBDG)(Invitrogen,Carls’bad,CA,USA)and 1nM insulin for 30min.Removed the medium,cells were washed and resuspended with PBS quickly,and then cells were moved at 1×104/well to 96-well plates.Fluorescence retained was measured using a microplate reader set at an excitation wave-length of 485 nm and an emission wavelength of 535 nm.5.Gene silence of TRB3 in vitroThe fully differentiated 3T3-L1 adipocytes were randomized to receive TRB3 adenovirus transfection or vehicle treatment.6.Real Time-PCRFresh mature adipocytes were collected and mRNA was extracted by Trizol.Real Time-PCR technique was used to detect the relative mRNA expression of TRB3 in adipocytes of each group.7.Western blotAcquire fresh mature adipocytes and extract adipocyte proteins.Western blot was used to detect the relative protein expression of GLUT4,IRS-1,p-AMPK,AMPK,and TRB3 in adipocytes of each group.Results1.Establishment of insulin resistant adipocye modleCompared with control group,the expressions of IRS-1 and GLUT4 were significantly decreased in IR group(P<0.001).Compared with control group,the glucose uptake of adipocyte was significantly decreased in IR group(P<0.001).Compared with control group,the expressions of IRS-1 and GLUT4 were significantly decreased in FFA group(P<0.001).Compared with control group,the glucose uptake of adipocyte was significantly decreased in FFA group(P<0.001).These results suggest that high glucose and high insulin or FFA could induce:inusulin resistance in adipocyte.2.Triglyceride quantification of insulin resistant adipocyteOil red O staining:Compared with control group,the volume and quantities of lipid droplets were significantly increased in IR group.Compared with control group,the triglyceride content level was significantly increased in IR group(P<0.001).Oil red O staining:Compared with control group,the volume and quantities of lipid droplets were significantly increased in FFA group.Compared with control group,the triglyceride content level was significantly increased in FFA group(P<0.001).These results suggest that high glucose and high insulin or FFA could increase lipid droplets formation in adipocytes.3.Infulence on TRB3/AMPK signaling pathway of insulin resistanceCompared with control group,adipocytes in IR group had higher level of TRB3(P<0.001),but lower level of the phosphorylation of AMPK(P<0.001).Compared with control group,adipocytes in FFA group had higher level of TRB3(P<0.001),but lower level of the phosphorylation of AMPK(P<0.001).These results suggest that high glucose and high insulin or FFA activates TRB3 signaling pathway and phosphorylation of AMPK is inhibited.4.TRB3siRNA transfection inhibits TRB3 expression in adipocyeCompared with vehicle group,TRB3 mRNA level was significantly decreased in TRB3 siRNA group(P<0.001).Compared with vehicle group,the TRB3 protein level was significantly decreased in TRB3 siRNA group(P<0.01).These results suggest that TRB3 adenovirus transfection is effective.5.TRB3 gene silencing improved insulin resistanceCompared with IR+vehicle group,the expression of IRS-1 and GLUT4 was significantly increased in IR+TRB3 siRNA group(P<0.001).Compared with FFA+vehicle group,the expression of IRS-1 and GLUT4 was significantly increased in FFA+TRB3 siRNA group(P<0.001).The results suggest that TRB3 gene silencing alleviated insulin resistance.6.TRB3 gene silencing increased AMPK activityCompared with IR+vehicle group,the expression of P-AMPK was significantly increased in IR+TRB3 siRNA group(P<0.001).Compared with FFA+vehicle group,the expression of P-AMPK was significantly increased in FFA+TRB3 siRNA group(P<0.01).The results suggest that TRB3 gene silencing increases phosphorylation of AMPK,and then alleviates adipocyte insulin resistance.Conclusions:1.In the high glucose+high insulin medium or palmitic acid medium incubated adipocyte,the expression of IRS-1 and GLUT4 was significantly decreased,the glucose uptake of adipocytes was significantly decreased,but the triglyceride level was significantly increased.It was proved that we successfully established insulin-resistant adipocyte modle.2.The expression of TRB3 significantly increased in insulin-resistant adipocytes;Silence of TRB3 increased the phosphorylation of AMPK,improved metabolism disturbance and alleviated insulin resistance in adipocytes.BackgroundObesity can cause insulin resistance,which is an important cause of type 2diabetes.Adipose tissue,as the initial site of insulin resistance,plays an important role in the occurrence and development of insulin resistance.Adipose tissue is not only a place to store free fatty acids,but also has complexendocrine functions.It produces a large number of hormones and cytokines,which play an important role in the regulation of glycolipid metabolism,and is closely related to the occurrence and development of metabolic syndrome,diabetes mellitus and vascular diseases.In obesity and diabetes,dysfunctional adipose tissue is a rich source of proinflammatory factors,which can directly cause vascular injury,insulin resistance and atherosclerosis.However,the specific molecular mechanism of regulating adipose tissue dysfunction remains unclear.Adipose tissue is closely associated with insulin resistance.Adipose tissue is divided into visceral adipose tissue,subcutaneous adipose tissue and brown adipose tissue.Previous studies have focused on the effects of various types of adipose tissue on insulin resistance,and few have compared the effects of different types of fat on insulin resistance.Studies have shown that increased visceral adipose tissue is more closely associated with insulin resistance than subcutaneous adipose tissue.Brown adipose tissue is also associated with insulin resistance.Insulin resistance in adipose tissue is related to adipose tissue dysfunction,but the mechanism of adipose tissue dysfunction is not clear.TRB3 has been demonstrated to be involved in lipid metabolism and in impairment of insulin exocytosis.Epidemiological studies recognized that TRB3 was increased in obese humans with IR and in patients with T2 DM.Our previous study showed that TRB3 was increased in adipose tissue of fructose-fed rats compared with the controls.Therefore,TRB3 could be a promising candidate for investigating IR and related clinical disorders.The role of TRB3 in adipose tissue dysfanction has not been studied and therefore remains to be elucidated.TRB3 is an important suppressor of AMPK via directly binding to it.What is clear is that the consequences of dysfunctional AMPK include an increased risk of insulin resistance,hypertension and cardiovascul^ur disease(CVD)and possibly a predilection to certain cancers.However,the TRB3/AMPK signaling pathway has not been investigated in vivo on adipose tissues directly.Thus,we hypothesized that TRB3/AMPK signaling pathway was an attractive strategy for the treatment of obesity and type 2 DM.First,we induced obese and type2 DM rat modle.To further elucidate the role of TRB3/AMPK signaling pathway in obesity and DM? we used TRB3 gene silencing in vivo to explore the mechanisms of TRB3/AMPK signaling pathway in obesity and DM as a potential target for treatment.ObjectivesTo further elucidate the role of TRB3/AMPK signaling pathway in obesity and DM,we used TRB3 gene silencing in vivo to explore the mechanisms of TRB3/AMPK signaling pathway in obesity and DM as a potential target for treatment.Methods1.Animal modleForty male SD rats were all fed high-fat(HF)diet,and randomly divided into four groups: HF+Vehicle,HF+TRB3siRNA,diabetes(DM)+Vehicle and DM+TRB3siRNA.Type 2 diabetic rat modle was induced by high-fat diet and small dose of strqjtozotocin(STZ).We used TRB3 gene silencing in vivo 12 weeks after onset of diabetes.At the end of the treatment,TRB3 expression was detected inadipose tissue and liver.The fasting blood glucose,insulin,serum lipid levels were assayed,and the insulin sensitivity index [ISI =ln(FBGxfasting insulin)"1] wascalculated.The histopathologic analyses in adipose tissues and liver were evaluated.Triglycercide and glycogen of adipose tissues were assayed.2.Gene silence of TRB3 in vivoForty rats were randomized to receive TRB3 small interfering RNA(siRNA)or vehicle treatment.After 12 weeks of diabetes,animals were then injected via the jugular vein with 2.5xl〇10 plaque-forming units of an adenovirus harboring TRB3gene(TRB3-siRNA)or a control empty virus(vehicle).Adenovirus transfer was repeated in 2 weeks.According to our present and previous studies,Four weeks afterthe first adenovirus injection,rats were killed.The adipose tissue and liver was excised and weighed.3.Blood analysesAfter rats fasted overnight,we collected jugular blood.Total cholesterol,triglyceride levels,and FBG were analyzed with use of the Bayer 1650 blood chemistry analyzer(Bayer,Tarrytown,NY).Free fatty acid(FFA)concentrations were measured using an enzymatic test kit(CSB-E08770r;HuaMei BIO-TECH,Wuhan,China).Fasting insulin level was measured by enzyme-linked immunosorbent assay.ISI was calculated.4.Western blot analysisTissue were harvested in ice-cold lysis buffer,Protein concentrations were determined with the Bradford method.We used antibodies against TRB3(Calbiochem),p-AMPK/AMPK,and p-Akt/Akt(Cell Signaling Technology),followed by anti-IgG horseradish peroxidase—conjugated secondary antibody.TRB3 protein level was normalized to that of(3-actin? as an internal control and phosphospecific proteins to that of total protein.5.Adipose tissue triglyceride and glycogen content assayAdipose tissue triglyceride content was measured using a Triglyceride Quantification Kit(Abeam),following the manufacturer’s instructions for colorimetric assay,Adipose tissue glycogen content was measured using a Glucose(HK)assay kit(Sigma-Aldrich,St.Louis,MO).6. Histology and morphometric analysisParaformaldehyde(4%)-fixed adipose tissue was embedded in paraffin,and cut into 5-(am sections.A single adipocyte cell was measured with images captured from hematoxylin and eosin-stained sections.The adipocyte cross-sectional area was assessed under 3400 magnification within the adipose tissue,and a mean was obtained by quantitative morphometry with automated image analysis(Image-Pro Plus,Version 5.0;Media Cybematics,Houston,TX).Paraformaldehyde(4%)-fixed liver was embedded in paraffin,and cut into 5-|im sections for HE staining.Liver frozen sections(5-jim)were stained with Oil Red O(Sigma,St.Louis,MO).A Nikon microscope(Nikon? Melville,NY)was used to capture the Oil Red O-stained tissuesections for semiquantification of Oil Red O staining.Results1.General characteristics of diabetic ratsCompared with HF+Vehicle group,the values of water intake,food intake and urine volume were significantly increased in DM+Vehicle group(P < 0.01-P <0.001).2.Biochemical measurement of diabetic ratsCompared with HF+Vehicle group,the serum FBG level were significantly increased in DM+Vehicle group(P < 0.01).Compared with HF+Vehicle group,the ISI were significantly decreased in DM+Vehicle group(P< 0.01).3.TRB3 gene silencing improved metabolism in vivoTRB3 gene silencing in this type 2 DM modle led to a significant decrease in water intake,urine volume,and FBG in DM+TRB3siRNA group compared with DM+Vehicle group(P<0.05?P<0.01).ISI was markedly increased in DM +TRB3siRNA group compared with DM +Vehicle group(P<0.05).There was no significant difference in body weight and food intake between experimental animals.4.TRB3 expression in adipose tissue after TRB3 gene silencingCompared with HF+vehicle group,the expression of TRB3 in adipose tissue was significantly decreased in HF+TRB3 siRNA group(P < 0.001).Compared with DM+vehicle group,the expression of TRB3 in adipose tissue was significantly decreased in DM+TRB3 siRNA group(P < 0.001).5.Changes of TRB3/AMPK signaling pathway in adipose tissue after TRB3 gene silencingCompared with HF+vehicle group,the expression of p-AMPK in EAT was significantly increased in HF+TRB3 siRNA group(P < 0.001).Compared with DM+vehicle group,the expression of p-AMPK in EAT was significantly increased in DM+TRB3 siRNA group(P < 0.001).Compared with HF+vehicle group,the expression of p-AMPK in SAT was significantly increased in HF+TRB3 siRNA group(P < 0.05).Compared with DM+vehicle group,the expression of p-AMPK in SAT was no significant statistical difference in DM+TRB3 siRNA group.Compared with HF+vehicle group,the expression of p-AMPK in BAT was significantly increased in HF+TRB3 siRNA group(P < 0.001).Compared with DM+vehicle group,the expression of p-AMPK in BAT was significantly increased in DM+TRB3 siRNA group(P< 0.001).6.TRB3 gene silencing alleviated adipose tissue remodeling in vivoSilence of TRB3 decreased the adipocyte size of epididymal and subcutaneous adipose tissues in obesity and diabetic rats,adipocyte aligned more evenly and orderly than control.TRB3 gene silencing alleviate the infiltration of white lipid droplet in brown adipose tissue.7.Effect of TRB3 gene silencing on the triglyceride and glycogen in adipose tissueTRB3 gene silencing decreases triglyceride content level and increase glycogen content level in diabetic epididymal and brown adipose tissues in DM+TRB3siRNA group(P<0.05 ?P<0.01).8.TRB3 gene silencing alleviated abberrant lipid accumulation in liverCompared with HF+vehicle group,oil red O-stained area in liver wassignificantly decreased in HF+TRB3 siRNA group(P < 0.01).Compared with DM+vehicle group,oil red O-stained area in liver was significantly decreased in DM+TRB3 siRNA group(P< 0.001).9. TRB3 expression in liver after TRB3 gene silencingConpared with HF+vehicle group,the expression of TRB3 in liver was significantly decreased in HF+TRB3 siRNA group(P < 0.001).Compared with DM+vehicle group,the expression of TRB3 in liver was significantly decreased in DM+TRB3 siRNA group(P< 0.001).10.Changes ofp-Akt in liver after TRB3 gene silencingCompared with HF+vehicle group,the expression of p-Akt in liver was significantly increased in HF+TRB3 siRNA group(P < 0.001).Compared with DM+vehicle group,the expression of p-Akt in liver was significantly increased in DM+TRB3 siRNA group(P < 0.001).Conclusions:1.TRB3 gene silencing effectively improved glucose and lipid metabolism ? and further improved insulin resistance,effectively reduced risk factors related to obesity and T2 DM.2.TRB3 gene silencing alleviated adipose tissue of obese and diabetic rats remodeling5 and alleviated the infiltration of white lipid droplet in brown adipose tissue3fijrtha: improved systemic metabolic disorders.3.TRB3 gene silencing remarkably increased AMPK activity in epididymal,subcutaneous and brown adipose tissues of obese rats.TRB3 gene silencing significantly decreased triglyceride,increased glycogen simultaneously in diabetic epididymal and brown adipose tissues,remarkably increased AMPK activity in epididymal and brown adipose tissues of diabetic rats.Consistently,TRB3/AMPK signaling pathway was closely associated with glucose and lipid metabolism disorder of adipose tissues. |
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