ABSTRACT
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.
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-Glucosidase and lipase inhibitors play important roles in the treatment of hyperglycaemia and dyslipidemia. To identify novel naturally occurring inhibitors, a bioactivity-guided phytochemical research was performed on the pu-erh tea. One new flavanol, named (-)-epicatechin-3---coumarate (), andknown analogs (-) were isolated from the aqueous extract of the pu-erh tea. Their structures were determined by spectroscopic and chemical methods. Furthermore, the water extract of pu-erh tea and its fractions exhibited inhibitory activities against-glucosidases and lipases; compoundshowed moderate inhibitory effect against sucrase with an ICvalue of 32.5 μmol/L and significant inhibitory effect against maltase with an ICvalue of 1.3 μmol/L. Compounds,,anddisplayed moderate activity against a lipase with ICvalues of 16.0, 13.6, 19.8, and 13.3 μmol/L, respectively.
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Refined-JQ (JQ-R) is a mixture of refined extracts from(Ranunculaceae),(Leguminosae) and(Caprifoliaceae), the three major herbs of JinQi-JiangTang tablet, a traditional Chinese medicine (TCM) formula. The mechanisms by which JQ-R regulates glucose metabolism and improves insulin sensitivity were studied in type 2 diabetic KKmice and insulin-resistant L6 myotubes. To investigate the mechanisms by which JQ-R improves insulin sensitivity, a model of insulin-resistant cells induced with palmitic acid (PA) was established in L6 myotubes. Glucose uptake and expression of factors involved in insulin signaling, stress, and inflammatory pathways were detected by immunoblotting. JQ-R showed beneficial effects on glucose homeostasis and insulin resistance in a euglycemic clamp experiment and decreased fasting insulin levels in diabetic KKmice. JQ-R also improved the plasma lipid profiles. JQ-R directly increased the activity of superoxide dismutase (SOD) and decreased malondialdehyde (MDA) as well as inducible nitric oxide synthase (iNOS) levels in insulin-resistant L6 cells, and elevated the insulin-stimulated glucose uptake with upregulated phosphorylation of AKT. The phosphorylation levels of nuclear factor kappa B (NF-B p65), inhibitor of NF-B (IB), c-Jun N-terminal kinase (JNK1/2) and extracellular-signal-regulated kinases (ERK1/2) were also changed after JQ-R treatment compared with the control group. Together these findings suggest that JQ-R improved glucose and lipid metabolism in diabetic KKmice. JQ-R directly enhanced insulin-stimulated glucose uptake in insulin-resistant myotubes with improved insulin signalling and inflammatory response and oxidative stress. JQ-R could be a candidate to achieve improved glucose metabolism and insulin sensitivity in type 2 diabetes mellitus.
ABSTRACT
The high and continuing soaring incidence of diabetes may become a huge obstacle to China's development. The antidiabetic drug development is one way to solve the problem. Animal model is a powerful tool for drug development. This paper compares and analyzes the three kinds of animal models for antidiabetic drug development in replicating principle, methods and characteristic, then summarized the application in the research of traditional Chinese medicine. At the same time, the analysis of the market, application and clinical advantages of hypoglycemic medicine from traditional Chinese medicine, is given in this paper, based on the literature analysis. From the point of the clinic advantage embodiment and new drug development, this paper will provide advisory and assistance support for the anti-diabetic fighting with traditional Chinese medicine.
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This study aims to establish a method to determine the serum acetaminophen concentration based on diazo reaction, and apply it in the gastric emptying evaluation. Theoretically, acetaminophen could take hydrolysis reaction in hydrochloric acid solution to produce p-aminophenol, which could then take diazo reaction resulting in a product with special absorption peak at 312 nm. Then the serum acetaminophen concentration and recovery rate were calculated according to the standard curve drawn with absorbance at 312 nm. ICR mice were given a dose of acetaminophen (500 mg x kg(-1)) by gavage and the serum acetaminophen was dynamically measured through the diazo reaction. Besides, ICR mice were subcutaneously injected with the long-acting GLP-1 analog GW002 before the gavage of acetaminophen, and serum acetaminophen concentration was measured as above to study how GW002 could influence the gastric emptying. The data showed acetaminophen ranging from 0 to 160 μg x mL(-1) could take diazo reaction with excellent linear relationship, and the regression equation was y = 0.0181 x +0.0104, R2 = 0.9997. The serum acetaminophen was also measured with good linear relationship (y = 0.0045 x + 0.0462, R = 0.9982) and the recovery rate was 97.4%-116.7%. The serum concentration of acetaminophen reached peak at about 0.5 h after gavage, and then gradually decreased. GW002 could significantly lower the serum acetaminophen concentration and make the area under the concentration-time curve (AUC) decrease by 28.4%. In conclusion, a method for the determination of serum acetaminophen based on the diazo reaction was established with good accuracy and could be used in the evaluation of gastric emptying.
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Ongoing study on the chemical constituents of the roots of Macleaya microcarpa led to the isolation of eight compounds of derivatives of triterpenes and organic acids in addition to some previously identified benzophenanthridines. The eight compounds were identified by spectroscopic methods as well as comparison with literature values as 1-oxo-2, 22 (30)-hopandien-29-oic acid (1), 3-oxo-12-oleanen-30-oic acid (2), 3α-hydroxy-12-oleanen-30-oic acid (3), 3β-hydroxy-12-oleanen-30-oic acid (4), ferulic acid (5), ferulic acid 4-O-β-D-glucoside (6), 3-O-feruloylquinic acid (7), and methyl 3-O-feruloylquinate (8). Of which, 1 is a new triterpenoid of hopanes and 2-8 are isolated from M microcarpa for the first time. In order to discover natural active compounds as potential agents of anti-ulcerative colitis (UC), an in vitro drug high-throughput screening model targeted x-box-binding protein 1 (xbp1) was employed to evaluate the activity of the major chemical constituents of M microcarpa. The result confirmed that two dihydrobenzophenanthridines, dihydrosanguinarine (9) and dihydrochelerythrine (10), showed a certain activity on activating the transcription of xbpl, a transcription factor (TF) associated with the occurrence, development, and potential treatment of UC, with their relative activating ratios being 1.76 and 1.77 times, respectively, as compared with control group.
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As a member of nuclear receptor superfamily, farnesoid X receptor (FXR) has been shown to regulate numerous metabolic pathways, which include playing an important role in bile acid metabolism, maintaining lipid and glucose homeostasis when FXR is activated. With the prevalence of the glucose and lipids disorder, FXR attracts increasing attention. It may be a potential target for the treatment of type 2 diabetes mellitus and lipid disorders.
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In-vitro assay methods were established to evaluate transactivation and binding activity of compounds on peroxisome proliferator-activated receptor y (PPARγ). Firstly, plasmids were constructed for transactivation assay of PPARγ response element (PPRE) triggered reporter gene expression, and for cell-based binding activity assay of the chimeric receptor, which was fused with PPARγ ligand binding domain (LBD) and yeast transcriptional activator Gal4. Secondly, by using PPARy competitive binding assay based on time resolved-fluorescence resonance energy transfer (TR-FRET), affinities of compounds and drugs to PPARγ were evaluated. In application of these above methods, the PPARγ activating potency and characteristics of different compounds were evaluated, and a novel benzeneselfonamide derivative, ZLJ01, was found to have comparable binding activity and affinity with the well-known PPARy agonist, but lack of PPRE mediated transactivation activity. In preliminary study on in-vitro hypoglycemic activity, ZLJ1 was found to promote insulin-stimulated glucose uptake by liver cells. Therefore, we believe that combining transactivation and binding activity as well as affinity evaluation, the system could be used to screen non-agonist PPARγ ligand as anovel PPARγ modulator
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This study is to evaluate the effects of the metformin (Met) on β cell function of diabetic KKAy mice. Female diabetic KKAy mice selected by insulin tolerance test (ITT) were divided randomly into two groups. Con group was orally administered by gavage with water, Met group with metformin hydrochloride at a dose of 0.2 g x kg(-1) for about 12 weeks. ITT and glucose tolerance tests (OGTT) were determined. Beta cell function was assessed by hyperglycemic clamp. Pancreatic biochemical indicators were tested. The changes of gene and protein expression in the pancreas and islets were also analyzed by Real-Time-PCR and immunostaining. Met significantly improved glucose intolerance and insulin resistance in KKAy mice. Fasting plasma glucose and insulin levels were also decreased. In addition, Met markedly increased glucose infusion rate (GIR) and elevated the Ist phase and maximum insulin secretion during clamp. It showed that Met decreased TG content and iNOS activities and increased Ca(2+) -Mg(2+)-ATPase activity in pancreas. Islets periphery was improved, and down-regulation of glucagon and up-regulated insulin protein expressions were found after Met treatment. Pancreatic mRNA expressions of inflammation factors including TLR4, NF-κB, JNK, IL-6 and TNF-α were down-regulated, p-NF-κB p65 protein levels also down-regulated by Met. And mRNA expressions of ion homeostasis involved in insulin secretion including SERCA2 and Kir6.2 were up-regulated by Met. Met increased SIRT5 expression level in pancreas of KKAy mice under the hyperglycemic clamp. These results indicated that chronic administration of Met regulated pancreatic inflammation generation, ion and hormone homeostasis and improved β cell function of diabetic KKAy mice.
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Dipeptidyl peptidase 4 (DPP4) is recognised as an attractive anti-diabetic drug target, and several DPP4 inhibitors are already on the market. As members of the same gene family, dipeptidyl peptidase 8 (DPP8) and dipeptidyl peptidase 9 (DPP9) share high sequence and structural homology as well as functional activity with DPP4. However, the inhibition of their activities was reported to cause severe toxicities. Thus, the development of DPP4 inhibitors that do not have DPP8 and DPP9 inhibitory activity is critical for safe anti-diabetic therapy. To achieve this goal, we established a selective evaluation method for DPP4 inhibitors based on recombinant human DPP8 and DPP9 proteins expressed by Rosetta cells. In this method, we used purified recombinant 120 kDa DPP8 or DPP9 protein from the Rosetta expression system. The optimum concentrations of the recombinant DPP8 and DPP9 proteins were 30 ng/mL and 20 ng/mL, respectively, and the corresponding concentrations of their substrates were both 0.2 mmol/L. This method was highly reproducible and reliable for the evaluation of the DPP8 and DPP9 selectivity for DPP4 inhibitor candidates, which would provide valuable guidance in the development of safe DPP4 inhibitors.
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Three cyclotides were isolated from the whole plant of Viola yedoensis in this study. The two, vary peptide E and cycloviolacin Y5, were previously reported, and a novel cycloviolacin VY1 was characterized according to the interpretation of MS/MS fragmentation of peptides which were produced from the reduced and alkylated parent peptide with the digestion of Endo Lys-C, trypsin and chymotrypsin, separately. The stability of remarkable resistance to proteolytic degradation by trypsin and chymotrypsin, and that of thermal denaturation was confirmed again. Besides, the IC50 value of cycloviolacin VYI against influenza A H1N1 virus was (2.27 +/- 0.20) microg x mL(-1). It is the first cyclotide reported with anti-influenza A H1N1 virus activity in vitro assay.
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A series of novel tetrahydrocarboline derivatives was designed and synthesized in order to discover more potent peroxisome proliferator-activated receptor (PPAR) alpha/gamma dual regulators. The structures of these compounds were confirmed by 1H NMR and HR-MS; their PPAR-regulating activities were evaluated in vitro. Compounds 6h, 6n, 6p and 6q exhibited more potent PPARalpha agonistic activities than the control drug WY14643, while compounds 60, 6g, 6i and 6q exhibited more potent PPARgamma agonistic activities than the control drug rosiglitazone. Compound 6q was discovered as a potent PPARalpha/gamma dual agonist and deserves further investigation.
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Glucokinase (GK) is a new target for the treatment of type II diabetes mellitus (T2DM). In order to find a structure-simplified small molecule GK activator, 19 salicylic acid derivatives were designed and synthesized based on new lead compound (1). Experimental results showed that the potency of compound 8h is superior to control RO-28-0450 in GK activation.
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This study is to evaluate the anti-diabetic effects of the alpha-glucosidase inhibitor valibose in a streptozotocin (STZ)-induced type 1 diabetes rat model. Diabetes was induced by a single dose of STZ (58 mg x kg(-1), ip) in SD rats, rats with elevated fasting blood glucose levels (250-450 mg x dL(-1)) were selected and divided into five groups (n = 10 in each). Another ten normal SD rats were chosen as normal group. Valibose mixed with the high sucrose diets (0.4, 1.0 and 2.5 mg 100 g(-1) diets) or acarbose (30 mg x 100 g(-1) diets) was administrated in the diabetic rats for about 5 weeks. In all groups, fasting and postprandial plasma glucose, plasma lipids, glycosylated serum protein, N-acetyl-beta-D-glucosaminidase (NAG), creatinine (Cre), blood urea nitrogen (BUN) and urine sugar levels were determined during the treatment. At the end of the experiment, the morphological alterations in kidney were evaluated by hematoxylin-eosin (HE) staining. After 3-weeks administration, valibose significantly decreased postprandial and fasting blood glucose, urine glucose, and reduced the levels of serum fructosamine. Valibose also decreased plasma triglyceride and cholesterol levels after 4 weeks treatment. These results indicated that valibose ameliorated metabolic disturbance of glucose and lipids in STZ-induced diabetic rats. In addition, valibose markedly reduced level of serum NAG and BUN, and decreased the weight index of kidney. HE staining showed reduced kidney pathological changes after valibose treatment. The findings of the present study indicate that valibose may be a novel alpha-glucosidase inhibitor for the prevention from hyperglycemia in STZ-induced type 1 diabetes rats. And valibose might have a potential role for protecting against diabetic nephropathy during hyperglycemia.
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The goal of treatment of metabolic syndrome is the prevention of diabetes and cardiovascular events. A series of novel tetrahydrocoptisine quaternary ammonium compounds were prepared to evaluate their action of hypoglycemia and hypolipidemia for finding the therapeutic agents of metabolic syndrome. Starting from the coptisine hydrochloride (2), fifteen target compounds were synthesized by reduction and substitution of the 7-N position. All of the target compounds were characterized by 1H NMR and HR-MS. Their hypoglycemic activities were evaluated in HepG2 cell and hypolipidemic activities of compounds with better hypoglycemic activity were tested further in vivo. Results indicated that compounds 5, 7, 8 and 9 exhibited better hypoglycemic activities in vitro and compounds 5 and 8 exhibited good hypolipidemic activities in high-fat-diet (HFD) induced hyperlipidemia mice and (or) hamsters. However, the activity is not as good as simvastatin.
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The aim of this study is to establish a simple and stable model like poloxamer 407 (P-407)-induced dyslipidemia of golden hamster model, and investigate the mechanism of lipid metabolism disturbance in this model. PPARalpha agonist and HMG-CoA reductase inhibitor were administrated to validate the efficacy on regulating lipid metabolism in the dyslipidemia golden hamster model. Six weeks male golden hamsters were chosen to inject P-407 intraperitoneally at a bolus dose of 300 mg x kg(-1), an intermittent injection at a dose of 200 mg x kg(-1) every 72 hours after the bolus. The results showed that P-407-induced golden hamster model characterized as increased serum triglyceride (TG), total cholesterol (TC), free cholesterol (free-CHO), cholesteryl ester (CE), free fatty acids (FFA) and apoB levels, and the hyperlipidemia state maintained at a stable level persistently. Meanwhile, augmented malondialdehyde (MDA) and nitric oxide (NO) level was observed. LCAT and SR-B I mRNA levels in liver of model group were down-regulated (expression ratio is 0.426; 0.783), while HMG-CoA reductase mRNA level was up-regulated (expression ratio is 1.493) compared with those of the normal group. The serum cholesterol and triglyceride levels were significantly lower in P-407-induced dyslipidemia hamster model after treated with atorvastatin (Ato) at a dose of 50 mg x kg(1) or fenofibrate (Fen) at 100 mg x kg(-1) for two weeks. These findings suggest that serum lipid distribution in dyslipidemia golden hamster is similar to that of human, and which may be relevant to the disturbance of the enzymes expression involved in lipid metabolism in liver. Results obtained from this study support the concept that dyslipidemia golden hamster may be an adequate animal model to evaluate the efficacy of lipid-lowering agents.
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A series of tetrahydroisoquinoline derivatives were prepared and their peroxisome proliferator-activated receptor (PPAR) alpha/gamma agonistic activities were evaluated to obtain more potent PPAR agonist. All of them were new compounds, and their structures were confirmed by 1H NMR and HR-MS. Three compounds exhibited higher agonistic activities of PPARgamma than that of the comparison, six compounds exhibited higher agonistic activities of PPARalpha than that of the comparison, and compound 8a was discovered as a highly potent PPARalpha/gamma agonist that is much more active than that of WY14643 and rosiglitazone. The development of potent PPAR agonists may offer a new choice for the treatment of diabetes.
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The blood insulin levels of insulin resistant monosodium glutamate induced mice (IR-MSG)and hyperglycemic monosodium glutamate mice (HG-MSG) markedly increased ( P<0. 01 ), and the weight index of pancreas significantly decreased ( P<0. 01 ), accompanied with overt inflammatory infiltration in the exocrine part of pancreas. Besides, insulin and glucagon in the islets also markedly increased with irregular distribution ( P<0.01 ). Compared to IR-MSG mice, HG-MSG mice showed overt hyperglycemia and much lower blood insulin. Moreover, the weight of pancreas in HG-MSG mice markedly decreased ( P < 0.05 ), along with less insulin and more glucagon in the lessened and contracted islets, suggesting that the injury of pancreas in the HGMSG mice might be more severe, which may result in hyperglycemia.
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Berberine, an isoquinoline alkaloid isolated from some Chinese medicinal herbs such as Coptidis rhizoma, has been used for the treatment of diarrhea and other gastrointestinal infections as an antibacterial drug in Chinese medicine. In recent years, it was reported to have beneficial effects on the metabolism disorders states of diabetes. The mechanisms involve many aspects of the diabetes, including regulating the blood cholesterol and triglyceride, lowering blood glucose, ameliorating the insulin resistant state and influencing the function of the pancreatic beta cell.
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To research the structure-activity relationship (SAR) of glycinamide-bearing compounds that used as inhibitors of dipeptidyl peptidase IV (DPP-IV), P32/98 and compound A were chosen as the leading compounds, heterocycles containing nitrogen atom were introduced to form amide, and different residues on a-position of carbonyl were designed. The nineteen designed compounds were synthesized by a simple route and were evaluated as inhibitors of DPP-IV. All of the structures were characterized by 1H NMR and HRMS. The preliminary SAR result was obtained.