Anti-diabetic effects and mechanisms of action of a Chinese herbal medicine preparation JQ-R in vitro and in diabetic KKAy mice

Refined-JQ(JQ-R) is a mixture of refined extracts from Coptis chinensis(Ranunculaceae),Astragalus membranaceus(Leguminosae) and Lonicera japonica(Caprifoliaceae), the three major herbs of Jin Qi-Jiang Tang 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 KK^(Ay)mice 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 KK^(Ay)mice. 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-κB p65), inhibitor of NF-κB(IκBα), 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 KK^(Ay)mice. 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.

Microbial transformation of lovastatin by Beauveria bassiana

Microbial transformation of lovastatin(1)by resting cells of a filamentous fungus,Beauveria bassiana AS 3.4270,yielded five metabolites(2-6),which were unambiguously characterized by various spectroscopic data analyses.The occurred reactions included hydroxyla-tion,lactone hydrolysis and methylation.The inhibitory effects of all metabolites on the 3-hydroxy-3-methylglutaryl coenzyme A(HMG-CoA)reductase were evaluated.Of the five compounds,4 ex hibited comparable inhibitory effect to lovastatin on the HMG-CoA reductase with ICso value of 2.2μM,and inhibitory rate of 81.4%.

Flavanols from the Camellia sinensis var.assamica and their hypoglycemic and hypolipidemic activities

α-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-O-(Z)-coumarate(1), and 16 known analogs(2–17) 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 α-glucosidases and lipases in vitro;compound 15 showed moderate inhibitory effect against sucrase with an IC_(50) value of 32.5 μmol/L and significant inhibitory effect against maltase with an IC_(50) value of 1.3 μmol/L. Compounds 8, 10, 11 and15 displayed moderate activity against a lipase with IC_(50) values of 16.0, 13.6, 19.8, and 13.3 μmol/L,respectively.

The application of 2-NBDG as a fluorescent tracer for assessing hepatic glucose production in mice during hyperinsulinemic euglycemic clamp

Methods of performing insulin clamps vary between laboratories.Here we present a protocol of insulin clamping in conscious mice,with the significant advantage of avoiding multiple surgical catheterizations and non-physiologic metabolism during the induction of anesthesia.Using this technique we also established a new method for measuring hepa tic glucose production(HGP)using a fuorescent D-glucose analog,2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-2-deoxyglu-cose(2-NBDG).To prove the reliability and feasibility of this method,whole-body insulin sensitivity was compared between conscious normal ICR mice and diabetic KK^(Ay) mice using the insulin clamp.Basal and clamp HGP was compared between normal C57 mice and diabetic db/db mice by using the modified clamp with 2-NBDG as a tracer.The glucose infusion rate(GIR),an index of insulin sensitivity,was significantly lower in KKAy mice than normal ICR mice.(6.2±1.3 mg/kg/min vs.31.3±2.9 mg/kg/min,P<0.001).The db/db mice also showed higher basal hepatic glucose production(25.8±2.2 mg/kg/min vs.16.7±2.5 mg/kg/min,P<0.05),higher clamp HGP after insulin suppression(7.3±1.0 mg/kg/min vs.0 mg/kg/min,P<0.001),and lower GIR(71.6±2.8 mg/kg/min vs.15.2±1.6 mg/kg/min,P<0.001)than that obtained with normal C57 mice.In conclusion,this is the first report of the application of 2-NBDG,rather than isotopic tracers,for the determination of HGP in vivo.

Establishment of a selective evaluation method for DPP4 inhibitors based on recombinant human DPP8 and DPP9 proteins

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.

A cell-based fluorescent glucose transporter assay for SGLT2 inhibitor discovery

The sodium/glucose cotransporter 2(SGLT2)is responsible for the majority of glucose reabsorption in the kidney,and currently,SGLT2 inhibitors are considered as promising hypoglycemic agents for the treatment of type 2 diabetes mellitus.By constructing CHO cell lines that stably express the human SGLT2 transmembrane protein,along with a fluorescent glucose transporter assay that uses 2-[N-(7-nitrobenz-2-oxa-l,3-diazol-4-yl)amino]2-deoxyglucose(2-NBDG)as a glucose analog,we have developed a nonradioactive,cell-based assay for the discovery and characterization of SGLT2 inhibitors.

Novel phenyl-urea derivatives as dual-target ligands that can activate both GK and PPARγ

A series of novel phenyl-urea derivatives which can simultaneously activate gluco kinase(GK)and peroxisome proliferator-activated receptorγ(PPARγ)was designed and prepared,and their activation of GK and PPARγ was evaluated.The structure--activity relationships of these compounds are also described.Three compounds showed potent ability to activate both GK and PPARγ.The possible binding mode of one of these compounds with GK and PPARγ were predicted by molecular docking simulation.