Allosteric modulation on specific gene mutated Vitamin D receptor by essential PUFAs and its distinct molecular effects on type 2 diabetes mellitus

A serious health threat affecting the T2DM group is evident more cases T2DM are diagnosed. In this research, we choose to research into all of this possible mechanism of 3T3-L1 Cell lines and Molecular Docking studies Schrodinger software identified Vitamin D, Omega-3, and 6 PUFAs (EPA DHA & AA) Compounds of hydrophilic and hydrophobic pocket throughout molecular modeling besides T2DM. A group of three analog VDRs is being developed for discovery treatment with T2DM. Its use as it was agreed to run a molecular cell culture and docking study. Recognize the binding method involving the compound in T2DM through ADME prediction. The molecular dynamics simulation was enhanced by confirmation of the strength of the possible composite binding. Based on the computational results, the Omega-3 and 6 PUFAs compound encourages energy interaction. The composite contains an in vitro anti-diabetic activity; the compounds have clearly shown that they are active on T2DM. Our studies provide vital information on the findings of the bimolecular T2DM inhibitors.

Rutin Promotes Browning of 3T3-L1 Preadipocytes and Its Mechanism / 中国实验方剂学杂志

ObjectiveTo investigate the effect of rutin on the browning of 3T3-L1 preadipocytes and the mechanism. MethodCell counting kit-8 （CCK-8） assay was used to detect the effect of different concentration of rutin （3.125， 6.25， 12.5， 25， 50， 100， 200 μmol·L-1） on 3T3-L1 cell activity， and Western blot to examine the effect of rutin （12.5， 25， 50 μmol·L-1） on the expression of thermogenesis-associated proteins uncoupling protein 1 （UCP1）， PR domain containing 16 （PRDM16） and peroxisome proliferator-activated receptor γ coactivator-1α （PGC-1α） in adipocytes. After the optimal concentration of rutin was determined， the effect of rutin on lipid droplet formation in adipocytes was observed based on oil red O staining， and the expression of nuclear respiratory factor 1 （NRF1）， nuclear respiratory factor 2 （NRF2） and mitochondrial transcription factor A （TFAM）， which were the landmark proteins of mitochondrial biosynthesis， was detected by Western blot. ResultCompared with the blank group， 200 μmol·L-1 rutin inhibited 3T3-L1 cell activity （P<0.01）. Compared with the blank group， at the concentration of 12.5， 25， 50 μmol·L-1 rutin significantly promoted the expression of thermogenesis-associated proteins （UCP1， PRDM16， and PGC-1α） （P<0.01）， which was determined as the optimal concentration. Compared with the blank group， 50 μmol·L-1 rutin significantly increased the immunofluorescence intensity of mitochondrial UCP1 protein in 3T3-L1 cells （P<0.01） and the expression of the markers of mitochondrial biosynthesis （NRF1， NRF2， and TFAM） （P<0.01）. In addition， 50 μmol·L-1 rutin significantly inhibited lipid droplet formation of 3T3-L1 adipocytes （P<0.01）. ConclusionRutin inhibited lipid droplet deposition in 3T3-L1 adipocytes and increased the expression of thermogenesis-related proteins （UCP1， PRDM16， and PGC-1α） and markers of mitochondrial biosynthesis （NRF1， NRF2， and TFAM）， thereby inducing the browning of 3T3-L1 adipocytes. This lays a basis for the development of drugs that safely regulate the browning of white cells.

Effect of Daizongfang on Insulin Resistance of 3T3-L1 Adipocytes Induced by Different Methods / 中国实验方剂学杂志

Objective:To investigate the effects of Daizongfang （DZF） on insulin resistance （IR） of adipocytes induced by different methods. Method:The cocktail induction method was adopted to induce the differentiation and maturity of 3T3-L1 preadipocytes. An IR model in mature adipocytes was established by the induction of palmitic acid （PA）， high-concentration glucose （HG）， and dexamethasone （DEX）. DZF extracts at different concentrations （2.0， 0.5， 0.1 g·L^-1） intervened for 24 hours. A model group， a rosiglitazone （RSG） group， and a blank control group were set up at the same time. The glucose concentration in the culture supernatant was measured by the glucose oxidase-peroxidase （GOD-POD） method. Glucose consumptions under basic conditions （G_Basic） and insulin stimulation （G_Ins） were calculated to evaluate the insulin sensitivity index （ISI）. The mRNA expression of glucose transporter 4 （GLUT4） was detected by the real-time polymerase chain reaction （PCR）. Result:Compared with the model group， the DZF （2.0 g·L^-1） showed increased G_Basic， G_Ins， and ISI in three IR models （<italic>P</italic><0.05， <italic>P</italic><0.01）. In addition， for the PA-induced IR model， G_Basic and G_Ins in the DZF （0.5 g·L^-1） group were elevated （<italic>P</italic><0.01）， and G_Basic， G_Ins， and ISI in the RSG group increased （<italic>P</italic><0.05， <italic>P</italic><0.01）. For the HG-induced IR model， G_Ins and ISI increased in the DZF （0.5 g·L^-1） group （<italic>P</italic><0.05）， and G_Basic， G_Ins， and ISI were elevated in the RSG group （<italic>P</italic><0.01）. For the DEX-induced IR model， G_Ins and ISI increased in the RSG group （<italic>P</italic><0.01）. In the three models， there were differences among groups with different doses. G_Basic， G_Ins， and ISI in the high-dose DZF group increased in varying degrees compared with those in the medium- and low-dose DZF groups （<italic>P</italic><0.05）. In the three models， the DZF （2.0 g·L^-1） group and the RSG group both increased GLUT4 mRNA expression （<italic>P</italic><0.05）. Conclusion:DZF can reduce IR of adipocytes induced by HG， DEX， or PA in a dose-dependent manner and increase glucose uptake in an insulin-independent manner， which may be related to the increase in GLUT4 expression.

Fermentation of mulberry leaves with Cordyceps militaris enhanced anti-adipogenesis activity in 3T3-L1 cells through down-regulation of PPAR-? pathway signaling

Objective: To establish an efficacious and efficient fermentation method of enhancing the anti-adipogenesis effect of mulberry (Morus alba) leaves using Cordyceps militais. Methods: Dried mulberry leaves, dried mulberry leaves with 50% raw silkworm pupa and raw silkworm pupa were fermented with Cordyceps militais for 4 weeks at 25 °C, after which the dried mulberry leaves and fermented product were extracted with 70% ethanol and subjected to high performance liquid chromatography (HPLC). The contents of cordycepin, pelargonidin, chlorogenic acid, iso-quercetin and caffeic acid were determined. We then used the 3T3-L1 cells to investigate whether extracts of fermentation enhanced anti-adipogenesis activity in vitro. Results: HPLC showed that fermentation changed the contents of cordycepin, pelargonidin, chlorogenic acid, iso-quercetin and caffeic acid. Furthermore, fermented dried mulberry leaves with 50% raw silkworm pupa had a better efficacy of anti-adipogenesis than dried mulberry leaves, fermented dried mulberry leaves and fermented silkworm pupa and inhibited triglycerides accumulation and glucose consumption. Additionally, fermented dried mulberry leaves with 50% raw silkworm pupa inhibited PPAR-? signaling. Conclusions: Fermentation with Cordyceps militaris enhanced anti-adipogenesis efficacy of mulberry leaves.

Effect of protease inhibitor from Agaricus bisporus on glucose uptake and oxidative stress in 3T3-L1 adipocytes

Objective: To explore the effect of the protease inhibitor from Agaricus bisporus (J.E. Lange) Imbach (AbPI) on glucose uptake and oxidative stress in 3T3-L1 adipocytes. Methods: Adipocytes were differentiated and stained with Oil-Red-O staining to confirm adipogenesis. The toxic/protective effect of AbPI on the adipocytes was determined by MTT assay, intracellular reactive oxygen species generation through flow cytometry, and morphologically through confocal microscopy using propidium iodide, 4,6-diamino-2-phenylindol dihydrochloride, and 2',7'-dichlorofluorescein diacetate dyes. The uptake of fluorescent glucose analog, 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) amino]-2-deoxy-d-glucose by adipocytes was also studied through confocal microscopy. Results: MTT assay showed that the cell survival rate was (28.00±3.00)％, (92.33±2.60)％, and (71.34±2.10)％ in the presence of 2 mM H2O2, AbPI alone, and AbPI and H2O2 both, respectively, in comparison to the control. Oil-Red-O staining indicated that AbPI enhanced adipogenesis. AbPI stimulated the glucose uptake by adipocytes similar to the drug rosiglitazone, and showed insulin-sensitizing effect in the presence of insulin, but failed to stimulate the uptake in the absence of insulin. Intracellular reactive oxygen species generation was reduced in differentiating adipocytes upon AbPI treatment. Confocal microscopy showed that the damaged cell population rose to 3.50％, 117.84％, and 261.50％ in the presence of AbPI alone, AbPI with H2O2, and H2O2 alone, respectively. Conclusions: The protease inhibitor enhances glucose uptake by adipocytes and exhibits a cytoprotective effect on them.

Fermentation of mulberry leaves with Cordyceps militaris enhanced anti-adipogenesis activity in 3T3-L1 cells through down-regulation of PPAR-? pathway signaling

Objective: To establish an efficacious and efficient fermentation method of enhancing the anti-adipogenesis effect of mulberry (Morus alba) leaves using Cordyceps militais. Methods: Dried mulberry leaves, dried mulberry leaves with 50% raw silkworm pupa and raw silkworm pupa were fermented with Cordyceps militais for 4 weeks at 25 °C, after which the dried mulberry leaves and fermented product were extracted with 70% ethanol and subjected to high performance liquid chromatography (HPLC). The contents of cordycepin, pelargonidin, chlorogenic acid, iso-quercetin and caffeic acid were determined. We then used the 3T3-L1 cells to investigate whether extracts of fermentation enhanced anti-adipogenesis activity in vitro. Results: HPLC showed that fermentation changed the contents of cordycepin, pelargonidin, chlorogenic acid, iso-quercetin and caffeic acid. Furthermore, fermented dried mulberry leaves with 50% raw silkworm pupa had a better efficacy of anti-adipogenesis than dried mulberry leaves, fermented dried mulberry leaves and fermented silkworm pupa and inhibited triglycerides accumulation and glucose consumption. Additionally, fermented dried mulberry leaves with 50% raw silkworm pupa inhibited PPAR-? signaling. Conclusions: Fermentation with Cordyceps militaris enhanced anti-adipogenesis efficacy of mulberry leaves.

Effect of protease inhibitor from Agaricus bisporus on glucose uptake and oxidative stress in 3T3-L1 adipocytes

Objective: To explore the effect of the protease inhibitor from Agaricus bisporus (J.E. Lange) Imbach (AbPI) on glucose uptake and oxidative stress in 3T3-L1 adipocytes. Methods: Adipocytes were differentiated and stained with Oil-Red-O staining to confirm adipogenesis. The toxic/protective effect of AbPI on the adipocytes was determined by MTT assay, intracellular reactive oxygen species generation through flow cytometry, and morphologically through confocal microscopy using propidium iodide, 4,6-diamino-2-phenylindol dihydrochloride, and 2',7'-dichlorofluorescein diacetate dyes. The uptake of fluorescent glucose analog, 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) amino]-2-deoxy-d-glucose by adipocytes was also studied through confocal microscopy. Results: MTT assay showed that the cell survival rate was (28.00±3.00)%, (92.33±2.60)%, and (71.34±2.10)% in the presence of 2 mM H

Caulerpa okamurae extract attenuates inflammatory interaction, regulates glucose metabolism and increases insulin sensitivity in 3T3-L1 adipocytes and RAW 264.7 macrophages / 中西医结合学报

OBJECTIVE@#To examine whether Caulerpa okamurae ethanolic extract (COE) could inhibit obesity-mediated inflammation, improve glucose metabolism and increase insulin sensitivity, using in vitro cell models of RAW 264.7 macrophages and 3T3-L1 adipocytes.@*METHODS@#We cocultured 3T3-L1 adipocytes in direct contact with lipopolysaccharide-stimulated RAW 264.7 macrophages and induced insulin resistance in 3T3-L1 adipocytes with tumor necrosis factor-α (TNF-α) in the presence or absence of 250 µg/mL of COE. We investigated various markers of inflammation, glucose regulation and insulin sensitivity in these models using Griess reagent to measure nitric oxide (NO) production, 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) amino]-2-deoxyglucose to measure glucose uptake, Western blot analysis to quantify protein expression and reverse transcriptase-polymerase chain reaction to evaluate mRNA expression.@*RESULTS@#We found that COE (250 µg/mL) significantly inhibited the lipopolysaccharide-induced inflammatory response in RAW 264.7 macrophages by downregulating NO production, nitric oxide synthase 2 expression and nuclear translocation of nuclear factor-κB. COE also showed similar anti-inflammatory activity in coculture, along with decreased TNF-α, interleukin-6 and monocyte chemoattractant protein mRNA expression. In addition, COE also improved glucose uptake in coculture by upregulating glucose transporter-4 (GLUT-4) and adiponectin and reducing serine phosphorylation of insulin receptor substrate-1 (IRS1). In the TNF-α-induced insulin resistance model of 3T3-L1 adipocytes, COE significantly improved both basal and insulin-stimulated glucose uptake, accompanied by phosphorylation of IRS1 at tyrosine 632, phospho-5' adenosine monophosphate-activated protein kinase α and glycogen synthase kinase-3β (Ser9) as well as upregulation of GLUT-4.@*CONCLUSION@#Together, these findings suggest that COE has potential to treat or prevent obesity-induced metabolic disorders.

Flavonoids kaempferide and 4,2'-dihydroxy-4',5',6'-trimethoxychalcone inhibit mitotic clonal expansion and induce apoptosis during the early phase of adipogenesis in 3T3-L1 cells / 中西医结合学报

OBJECTIVE@#Kaempferide and 4,2'-dihydroxy-4',5',6'-trimethoxychalcone (DTMC) are two major flavonoids found in Chromolaena odorata Linn. leaf extract. The aim of this study was to elucidate the mechanism by which these two flavonoids exerted their effect on adipogenesis. The inhibitory effect of kaempferide and DTMC on adipocyte differentiation and their mechanisms involving mitotic clonal expansion (MCE) and apoptosis during the early stage of adipogenesis were investigated.@*METHODS@#Confluent 3T3-L1 preadipocytes were induced to differentiate and exposed to the flavonoids during various phases of differentiation. Intracellular lipid accumulation, cell density and expression of the transcription factors peroxisome proliferator-activated receptor γ and CCAAT/enhancer-binding proteins α were assessed using AdipoRed, Oil red O and Western blot assays. Effects of both flavonoids on cell proliferation and apoptosis were also determined by carboxyfluorescein diacetate succinimidyl ester and annexin V-fluorescein isothiocyanate/propidium iodide-staining assays, respectively.@*RESULTS@#Kaempferide and DTMC showed significant, concentration-dependent anti-adipogenic activity and effect on cell density in the early phase of adipogenesis. The expression of the transcription factors seemed to be reduced when the treatment was prolonged or in the early phase of adipogenesis. These flavonoids interrupted MCE via inhibition of preadipocyte proliferation and induction of apoptosis. DTMC was nearly three times more potent than kaempferide in inducing apoptosis.@*CONCLUSION@#Kaempferide and DTMC exerted their anti-adipogenic activity through inhibition of MCE, either by suppressing cell proliferation or by inducing apoptosis during the early phase of differentiation.

Role of Ad36 in inducing browning of 3T3-L1 cells / 中华内分泌代谢杂志

Objective@#To investigate the role of adenovirus type 36 (Ad36) in the browning of 3T3-L1 cells.@*Methods@#BODIPY staining was performed on the 0, 2nd, 4th, 6th and 8th days of the cocktail induction (control) group and the cocktail plus Ad36 induction (experimental) group to observe the adipogenesis of 3T3-L1 cells.The mRNA and protein expressions of uncoupling protein-1(Ucp1), ATP synthase, H+ transporting, mitochondrial F1 complex, O subunit (Atp5o), cytochrome c oxidase subunit 5B(Cox5b), and perilipin were detected by real-time PCR and Western-blot.@*Results@#The results of BODIPY staining showed that the lipid droplets in the control group gradually became larger with the differentiation of the cells, while the lipid droplets in the experimental group firstly became larger, and then appeared smaller after Ad36 was added on the fourth day. Compared with the control group, the mRNA and protein expression levels of Ucp1, Atp5o, and Cox5b in the experimental group were significantly increased while the expression level of perilipin was significantly decreased (all P<0.05).@*Conclusion@#During the differentiation of 3T3-L1 cells, Ad36 promotes its browning via increasing the expressions of Ucp1, Atp5o, and Cox5b.

Role of ad36 in inducing browning of 3T3-L1 cells / 中华内分泌代谢杂志

Objective To investigate the role of adenovirus type 36 ( Ad36) in the browning of 3T3-L1 cells. Methods BODIPY staining was performed on the 0, 2nd, 4th, 6th and 8th days of the cocktail induction (control) group and the cocktail plus Ad36 induction ( experimental) group to observe the adipogenesis of 3T3-L1 cells. The mRNA and protein expressions of uncoupling protein-1 ( Ucp1 ), ATP synthase, H+ transporting, mitochondrial F1 complex, O subunit ( Atp5o), cytochrome c oxidase subunit 5B ( Cox5b), and perilipin were detected by real-time PCR and Western-blot. Results The results of BODIPY staining showed that the lipid droplets in the control group gradually became larger with the differentiation of the cells, while the lipid droplets in the experimental group firstly became larger, and then appeared smaller after Ad36 was added on the fourth day. Compared with the control group, the mRNA and protein expression levels of Ucp1, Atp5o, and Cox5b in the experimental group were significantly increased while the expression level of perilipin was significantly decreased ( all P＜0. 05 ). Conclusion During the differentiation of 3T3-L1 cells, Ad36 promotes its browning via increasing the expressions of Ucp1, Atp5o, and Cox5b.

Influence of alisol B 23-acetate on blood glucose in type 2 diabetic mice / 中国药理学通报

Aim To explore whether alisol B 23-acetate possesses the therapeutic potential for treatment of type 2 diabetes mellitus ( T2DM ). Methods T2DM mouse model was established by combined administration of streptozotocin and nicotinamide. After three weeks of oral administration of rosiglitazone or alisol B 23-acetate, the blood glucose of type 2 diabetic mice was measured. Oral glucose tolerance test(OGTT) was carried out the next day. Rosiglitazone was chosen as positive drug. 2-[N-(7-nitrobenz-2-oxa-l, 3-diazol-4-yl) amino]-2-deoxy-D-glucose (2-NBDG) uptake assay in adipocytes was adopted to test whether alisol B 23-acetate had effect on glucose uptake in cells. 3T3-Ll pre-adipocytes differentiation model was performed to evaluate whether alisol B 23-acetate promoted adipo-genesis. Results Mice exhibited significantly higher blood glucose concentration after intraperitoneal injection of streptozotocin and nicotinamide for three weeks, as examined by blood glucose concentration on day 21 and OGTT on day 22, compared with normal mice in blank control group. After orally administrating alisol B 23-acetate at dose of 5 mg • kg-1 , 10 mg • kg-1 ,20 mg • kg-1 daily for three weeks, respectively, or orally administered rosiglitazone at dose of 10 mg • kg-1 daily for three weeks, blood glucose greatly decreased in type 2 diabetic mice. Moreover, insulin resistance was also improved to a certain degree during OGTT. Furthermore, alisol B 23-acetate not only increased insulin-induced glucose uptake in adipocytes at the concentration of 30 mmol • L-1 glucose, but also accelerated 3T3-L1 pre-adipocytes differentiation process at concentration of 1 μmol • L-1 and 10 μmol • L-1 . Conclusions Alisol B 23-acetate reduces blood glucose of type 2 diabetic mice, promotes pre-adipocyte differentiation and increases glucose uptake in adipocytes; however, the mechanism of action needs further exploration.

Hypoglycemic effect of alismoxide in type 2 diabetic mice / 中国药理学通报

Aim To investigate the hypoglycemic effect of alismoxide. Methods Type 2 diabetes mellitus (DM) mouse model induced by combined administration of streptozotocin and nicotinamide was adopted. Three weeks later, blood glucose of blank control group and type 2 diabetic mouse model group was measured on day 21 , and oral glucose tolerance test(OGTT) was carried out on day 2 2 , respectively. After type 2 diabetic mouse model was successfully established, rosiglitazorie was chosen as positive drug. Oral administration of rosiglitazone at dose of 10 mgk g-1 daily was performed for three weeks in positive group. Oral administration of alismoxide at dose of 5 , 10 and 20 mg kg"1 daily for three weeks was carried out in alismoxide different dose group, respectively. Furthermore, influence of alismoxide on differentiation was investigated in 3T3-L1 pre-adipocytes, and Oil red 0 staining was adopted. Results Not only blood glucose was decreased by alismoxide in type 2 DM mice, but also hypoglycemic trend was exhibited during OGTT. Furthermore, at concentration of 0. 5 and 1 fimol L " 1 , alismoxide promoted 3T3-L1 pre-adipocyte differentiation. Conclusions It suggests that alismoxide might possess hypoglycemic property and accelerate pre-adipocyte differentiation; however, the mechanism involved needs further study.

Chemical constituents of Pteridium aquilinum supercritical extract and its antiadipogenic effect on 3T3L1 preadipocytes / 中国药理学通报

Aim To identify chemical constituents of supercritical extract from Pteridium aquilinum (Linn.) Kuhn (SFEPA) and investigate its antiadipogenic effect and underlying mechanism. Methods The chemical constituents of SFEPA were identified by GCMS. The differentiation of 3T3L1 preadipocytes was induced to establish cell model. MTT assay was employed to detect cell viability. Oil Red 0 staining was used to analyze the effect of SFEPA on the differentiation of 3T3L1 preadipocytes. qRTPCR was performed to examine the expression of genes related with adipogenesis and lipogenesis. Results Ten compounds of SFEPA were identified. The major constituents were 4, 4, 5, 7, 8pentamethyldihydrocoumarin (36. 0 7 %), ßsitosterol (25. 6 6 %), 4amino7diethylaminochromen2one (8 . 2 6 %) and palmitic acid (5. 0 5 %). SFEPA suppressed 3T3L1 adipocyte differentiation and reduced fat accumulation in a dosedependent manner. Treatment of SFEPA significantly downregulated the mRNA expression of PPARy, C/ EBPa, SREBPlc, as well as FAS and ACC. Conclusions SFEPA can inhibit the adipogenesis and differentiation of 3T3L1 cells, and its mechanism may be closed related to its inhibition of genes expression regulating adipogenesis and lipogenesis. Phenols and alcohols might be potential bioactive components.

Adipogenic and Lipolytic Effects of Ascorbic Acid in Ovariectomized Rats

PURPOSE: Ascorbic acid has been reported to have an adipogenic effect on 3T3-L1 preadipocytes, while evidence also suggests that ascorbic acid reduces body weight in humans. In this study, we tested the effects of ascorbic acid on adipogenesis and the balance of lipid accumulation in ovariectomized rats, in addition to long-term culture of differentiated 3T3-L1 adipocytes. MATERIALS AND METHODS: Murine 3T3-L1 fibroblasts and ovariectomized rats were treated with ascorbic acid at various time points. In vitro adipogenesis was analyzed by Oil Red O staining, and in vivo body fat was measured by a body composition analyzer using nuclear magnetic resonance. RESULTS: When ascorbic acid was applied during an early time point in 3T3-L1 preadipocyte differentiation and after bilateral ovariectomy (OVX) in rats, adipogenesis and fat mass gain significantly increased, respectively. However, lipid accumulation in well-differentiated 3T3-L1 adipocytes showed a significant reduction when ascorbic acid was applied after differentiation (10 days after induction). Also, oral ascorbic acid administration 4 weeks after OVX in rats significantly reduced both body weight and subcutaneous fat layer. In comparison to the results of ascorbic acid, which is a well-known cofactor for an enzyme of collagen synthesis, and the antioxidant ramalin, a potent antioxidant but not a cofactor, showed only a lipolytic effect in well-differentiated 3T3-L1 adipocytes, not an adipogenic effect. CONCLUSION: Taking these results into account, we concluded that ascorbic acid has both an adipogenic effect as a cofactor of an enzymatic process and a lipolytic effect as an antioxidant.

Jatrorrhizine regulates GLUTs with multiple manners for hypoglycemic effect in insulin-resistance 3T3-L1 adipocytes / 中国中药杂志

This paper aimed to investigate the hypoglycemic effect and relative mechanism of jatrorrhizine in insulin-resistance (IR)-3T3-L1 adipocytes. The 3T3-L1 preadipocytes were used to induce mature adipocytes, then the stable IR model was established with 1 μmol·L⁻¹ dexamethasone. The adipocytes were divided into normal group, IR model group, rosiglitazone positive group and jatrorrhizine group (0.5, 1, 5, 10, 20 μmol·L⁻¹). After different time points (12, 24, 30, 36, 48 h) treatment, glucose content of 3T3-L1 adipocytes was detected by the glucose oxidase peroxidase method and TG content was measured by glycerol phosphate oxidase method, whereas cell viability was detected by CCK-8 assay. Furthermore, the protein expression levels of insulin receptor substrate 2 (IRS2), phosphinositide-3-kinase regulatory subunit 1(PI3KR1), phosphorylated protein B [p-AKT (Ser473)], phosph-AMP-activated protein [p-AMPK (Thr172)], and glucose transporter type 4/1/2 (GLUT4/1/2) were detected by Western blot assay. The results showed that as compared with the normal group, the glucose consumptionwas significantly decreased in IR model group(<0.01); whereas 0.5, 1, 5, 10, 20 μmol·L⁻¹ jatrorrhizine and rosiglitazone group elevated IR-3T3-L1 cells glucose consumption (<0.01) at 36 h and 48 h administration as compared with IR group. The optimal administration time was 48 h for jatrorrhizine. 1, 5, 10, 20 μmol·L⁻¹ of jatrorrhizine decreased the TG content in 3T3-L1 adipocytes for 48 h administration (<0.05). The protein expression levels of IRS2, PI3KR1, p-AKT (Ser473), p-AMPK (Thr172), GLUT4/1/2 were significantly up-regulated by different concentrations of jatrorrhizine and rosiglitazone (<0.01). The results showed that jatrorrhizine increased glucose uptake with elevated glucose consumption, whereas reduced intracellular TG content in IR-3T3-L1 adipocytes. Moreover, it intervened classic insulin signal pathway IRS2/PI3KR1/p-AKT/GLUT4 and increase AMPK protein phosphorylation level for the activation of GLUT1/4 for insulin sensibility. Thus, jatrorrhizine could effectively regulate the GLUTs with multiple manners for hypoglycemic effect.

Effect and mechanism of Vaspin on insulin resistance of 3T3-L1 adipocytes induced by palmitic acid / 中华临床营养杂志

Objective To investigate the effects of vaspin on insulin resistants of 3T3-L1 adipocyte through the insulin receptor substrates (IRS) /phosphatidylinositol 3-kinase (PI3K) /protein kinase B (Akt) /glucose transporter (Glut) signaling pathway.Methods 3T3-L1 cells cultured by palmitic acid (PA) were used to establish insulin resistance models,which were divided into PA group,PA + 100 ng/ml vaspin group,PA+200 ng/ml vaspin group,PA+400 ng/ml vaspin group and PA+400 ng/ml vaspin+wortmannin (PI3K inhibitor) group.Glucose uptake and consumption were assessed by 2-deoxy H3-D-glucose incorporation and glucose oxidase-peroxidase respectively.IRS/PI3K/Akt/Glut signaling pathway was evaluated using reverse transcription polymerase chain reaction and Western blot analysis.Results Compared with PA group,glucose uptake and consumption increased gradually with the increasing of vaspin concentration in other groups (P ＜ 0.05).mRNA levels of IRS-1,Akt and Glut 4 increased gradually as vaspin concentration increasing (P＜0.05),and the ratios of p-IRS-1 to IRS-1,p-Akt to Akt and Glut 4 protein level also showed the same trends (P＜0.05).However,glucose uptake and consumption in PA+400 ng/ml vaspin+wortmannin group were less than that of PA +400 ng/ml vaspin group (P＜0.05).PA+400 ng/ml vaspin+wortmannin group showed lower mRNA and protein phosphorylation levels of IRS-1,Akt and Glut 4 (P＜0.05),and that the ratios of p-IRS-1 to IRS-1,p-Akt to Akt and Glut 4 protein levels decreased (P＜0.05).Conclusions Vaspin can improve the insulin sensitivity of 3T3-L1 adipocyte by activating IRS/PI3K/Akt/Glut signaling pathway.

Antidiabetic effect of combination of fractionated-extracts of Andrographis paniculata and Centella asiatica: In vitro study

Objective: To examine the effect of combination of Andrographis paniculata herb fraction (AHF) and Centella asiatica herb fraction (CHF) on PPARγ and GLUT4 mRNA expressions in 3T3-L1 adipocyte, and its effect on insulin-stimulated glucose uptake. Methods: 3T3-L1 adipocyte cells were used to investigate gene expression of PPARγ and GLUT4 proteins by reverse transcription-polymerase chain reaction method. The adipocyte cells were differentiated by using insulin, dexamethasone and 3-isobutyl-1-methylxanthine from 3T3-L1 cells. Pioglitazone, AHF, CHF and the combination of both herbs were evaluated on glucose uptake activity, PPARγ and GLUT4 mRNA expressions in 3T3-L1 adipocyte. Results: The results showed that combination of AHF at 30 μg/mL and CHF at 10 μg/mL could enhance insulin-stimulated glucose uptake. The combination also increased PPARγ and GLUT4 mRNA expressions significantly in comparison to those of negative control (DMSO). These effects were equal in comparison to those of pioglitazone (0.02 μM) and its single extracts. Conclusions: The combination of AHF and CHF can increase glucose uptake and insulin sensitivity through up-regulation of PPARγ and GLUT4 mRNA expressions in 3T3-L1 adipocyte.

miR-202 promotes the differentiation of 3T3-L1 preadipocyte via inhibiting PGC1β expression / 基础医学与临床

Objective To explore the effect and molecular mechanism of miR-202 on the differentiation of 3T3-L1 preadipocyte .Methods Through lentivirus infected with 3T3-L1 preadipocytes , we set up the AMO-miR-202 group and the random control group , then, these cells were induced to differentiate , nine days later, differentiation was assessed by Oil Red O staining and we examined the mRNA expression of PPARγ2 and aP2 by RT-PCR method. We examined the mRNA expression of PPARγ2,aP2 and PGC1βby Western blot method .Results After packa-ging lentivirus with AMO-miR-202 and random sequence control miRNA through cell line 293T, 80%-90%cells with fluorescence were found under fluorescence microscope; After these two lentivirus respectively infected with 3T3-L1 preadipocytes, About 70%-80%cells with fluorescence were found under fluorescence microscope .Oil Red O staining test showed that these cells with Oil Red O stained bright red fat droplets of AMO-miR-202 group and PPARγ2 and aP2 mRNA expression in the AMO-miR-202 group significantly lower than control groups (P<0.05). Western blot assay showed that the protein expression of PGC 1βin the AMO-miRNA-202 group was significantly increased(P<0.05), but the expression of aP 2 and PPARγ2 was significantly decreased (P<0.01).However, the random control group and the adipocyte group had no significant effect on the above indexes .Conclusions miR-202 can promote the differentiation of 3T3-L1 preadipocyte by inhibiting the protein expression of PGC 1βand im-proving the protein expression of PPARγ2 and aP2.

Antidiabetic effect of combination of fractionated-extracts of Andrographis paniculata and Centella asiatica: In vitro study

Objective: To examine the effect of combination of Andrographis paniculata herb fraction (AHF) and Centella asiatica herb fraction (CHF) on PPARγ and GLUT4 mRNA expressions in 3T3-L1 adipocyte, and its effect on insulin-stimulated glucose uptake. Methods: 3T3-L1 adipocyte cells were used to investigate gene expression of PPAR γ and GLUT4 proteins by reverse transcription-polymerase chain reaction method. The adipocyte cells were differentiated by using insulin, dexamethasone and 3-isobutyl-1-methylxanthine from 3T3-L1 cells. Pioglitazone, AHF, CHF and the combination of both herbs were evaluated on glucose uptake activity, PPARγ and GLUT4 mRNA expressions in 3T3-L1 adipocyte. Results: The results showed that combination of AHF at 30 μg/mL and CHF at 10 μg/mL could enhance insulin-stimulated glucose uptake. The combination also increased PPARγ and GLUT4 mRNA expressions significantly in comparison to those of negative control (DMSO). These effects were equal in comparison to those of pioglitazone (0.02 μM) and its single extracts. Conclusions: The combination of AHF and CHF can increase glucose uptake and insulin sensitivity through up-regulation of PPARγ and GLUT4 mRNA expressions in 3T3-L1 adipocyte.