Evaluation of the antidiabetic effect of APE in OZR
Animals
Fourteen week old, obese male OZR were obtained from Charles Rivers Laboratories (Orleans Cedex, France). They were individually housed under a 12-h cycle of light and dark at 21 °C. All experimental procedures were carried out according to the ethical guidelines for animal experimentation provided by the Spanish National Research Council (RD 1201/2005 October 10).
Acute effect of APE on the postprandial glycemic response during the time course of a Meal Tolerance Test (MTT)
Twenty rats were assigned into two groups: Control (n = 10) and APE groups (n = 10). The control group was orally treated with a solution containing maltodextrin (MD, DE 21.5, Cerestar, Spain) at the dose of 1 g/kg body weight (bw). The APE group was given an oral dose of MD containing APE at the dose of 150 mg APE/kg bw. APE was purchased from Exxentia (Spain) and the polyphenolic concentration of this extract was 57.5 %, underscoring the amount of phloridzin (9.9 %), chlorogenic acid (15.8 %) and quercetin (0.4 %). Blood samples were obtained via tail vein at baseline and at 30, 60, 90 and 120 min postprandial for glucose and insulin analysis.
In order to investigate the second-meal effect after the ingestion of APE at breakfast, a second meal tolerance test was performed by administering only MD at the dose of 1 g/kg bw after 270 min from the first gavage. Blood samples were also collected at baseline and 30, 60, 90, 120, 180 and 240 min for glucose and insulin analysis.
Ex-vivo α-glucosidase activity of APE
α-glucosidase inhibitory properties of APE were assayed in intestinal mucosa isolated from OZR following Dahlqvist methodology [12]. Maltase, isomaltase and sucrase activities were determined using maltose, isomaltose and sucrose as substrates. Results were expressed as the concentration of APE required to achieve 50 % of inhibition (IC50).
Chronic effect of APE extract on insulin sensitivity
The effect of APE on whole body insulin sensitivity was performed on 15 week old OZR. Twenty OZR were weighed and divided into two experimental groups (Control and APE) of ten animals. The control group received the standard purified rodent diet (AIN-93 M) [13] ad libitum for five weeks. The APE group received the standard diet supplemented with APE (3 g/kg diet) during the same period. Individual body weights were recorded at the beginning of the study and at weekly intervals thereafter. The food consumption of each rat was determined twice per week.
Four weeks prior to the end of the feeding study, an additional MTT assay was performed on both study groups using MD in fasting conditions.
The insulin sensitizing effect of APE was determined one week later using a modified euglycemic-hyperinsulinemic clamp technique developed by De Fronzo et al. [14]. In brief, overnight-fasted rats were anesthetized with sodium pentobarbital (50 mg/kg bw) (Pentothal, Abbott Laboratories, Madrid, Spain). Two catheters were placed in the right jugular vein for insulin and glucose infusion. Another catheter was placed in the left carotid artery for blood sampling. A tracheotomy was performed to allow for tracheal clearing. After approximately 40 min of surgery, arterial blood samples were collected for determination of glucose and insulin basal concentrations. At time 0, human insulin (Humulin R; Eli Lilly,Indianapolis, IN) was infused at a concentration of 15 mU/kg per minute. Blood samples were subsequently drawn at five-minute intervals for determination of blood glucose (Precision G Medisense, Bedford, Massachusetts, USA). An infusion of 30 % glucose was adjusted to maintain blood glucose at 100 mg/dL. Steady state was ascertained when a fixed glucose infusion rate (GIR) maintained blood glucose measurements constant for at least 30 min.
Analytical procedures
Blood glucose was determined using Precision PCx glucose meter equipment (Abbott Laboratories). Serum insulin concentration was measured by enzyme immunoassay (Mercodia AB, Uppsala, Sweden) using rat insulin as standard. Serum biochemical markers were determined using an Alcyon C-3500 autoanalyzer (Abbott Laboratories).
Evaluation of the insulin sensitivity mechanisms of APE in L6 myotubes
Cell culture
L6.C11 rat skeletal muscle myoblast line (ECACC n° 92102119) and CHO-k1 (ATCC no. CCL-61) cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) containing 10 % (v/v) fetal bovine serum (FBS), 2 mM glutamine plus 100 units/mL penicillin and 0.1 mg/mL streptomycin (Sigma, St. Louis, MO, USA) in an atmosphere of air/CO2 (95:5) and maintained at subconfluent densities in growth media. L6 myoblasts were differentiated into myotubes by exchanging the growth medium with a differentiation medium consisting of DMEM containing 2 % (v/v) fetal bovine serum for 5–6 days (>50 % fusion into multinucleated myotubes).
2-Deoxy-[3H]D-glucose (2-DG) uptake
Cells were grown in 48-well plates (Corning, NY, USA). They were differentiated into myotubes and then incubated in serum-free medium for 18–21 h. Treatments were performed in serum-free medium unless otherwise indicated. Triplicate measurements of 2-DG (Perkin Elmer, Waltham, MA, USA) uptake were taken after 10 min of incubation following the method described by Yonemitsu et al. [15].
Subcellular fractionation
Membrane fractions from myotubes were prepared as described [15, 16]. 5′-nucleotidase and cytochrome c reductase activities were assayed as marker enzymes for plasma membranes and low-density microsomes, respectively [17].
Glucose transporter 4 (GLUT4) protein analyses
L6 myotubes membrane fraction proteins were electrophoresed in 10 % (w/v) SDS-PAGE and processed for western blot with anti-GLUT4 antibodies (Biogenesis, Poole, UK). Immunoreactive bands were visualized by chemiluminescence and quantified with NIH Image Software. Results were normalized with the band intensity of actin (Developmental Studies Hybridoma Bank, Iowa City, IA, USA) or caveolin-1 (Cell Signaling Technology, Beverly, MA, USA).
Signaling pathways analyses
L6 myotubes were preincubated with different inhibitors implicated in distinct signaling pathways leading to GLUT4 translocation: wortmannin (100 nmol/L) (an inhibitor of the PI3K), PD 98059 (30 μmol/L) (an inhibitor of the ERK1/2 phosphorylation) and GW9662 (10 μmol/L) (an inhibitor of PPARγ). The inhibitors were added 30 min before the incubation with APE (25 μg/mL) and maintained during the treatments (2 h).
Protein phosphorylation analysis were carried out as described in [16]. Briefly, L6 myotubes were incubated in FBS-free medium for 18–24 h and then treated with APE (25 μg/mL) or rosiglitazone (10 μmol/L) in serum-free medium. After treatment, plates were flash frozen in liquid nitrogen and scraped with 50 μL/60 mm/plate of cold 30 mmol/L Tris-HCl pH 7.4, 25 mmol/L NaCl, 1 % (v/v) Triton X-100, 0.1 % SDS, 10 mmol/L sodium fluoride, 10 mmol/L sodium pyrophosphate, 1 mmol/L sodium orthovanadate, 1 mmol/L EGTA, 20 nmol/L okadaic acid, 10 μg/mL aprotinin, 10 μg/mL leupeptin, 10 μg/mL pepstatin. After 10 min on ice, extracts were centrifuged at 13,000 ×g for 10 min at 4 °C. For the study of PPARγ expression, nuclear extracts were obtained in accordance with Giron et al. 2008.
For the western blots analysis, proteins (25 μg) were separated by SDS-PAGE and immunoblotted with antibodies against PPAR-γ (Santa Cruz Biotechnology, Santa Cruz, CA, USA) and histone H3 (Epitomics, Burlingame, CA, USA). The immunoblots were developed by chemiluminescence detection.
The expression plasmids pSV SPORT PPARγ1 and pSV SPORT PPARγ2 and the PPAR responsive element-driven luciferase reporter vector PPRE X3-TK-Luc PPRE X3-TK-Luc were kindly provided by Dr. Bruce M. Spiegelman (Dana-Farber Cancer Institute, Harvard Medical School, Addgene plasmids 8886, 8862 and 1015). CHO-k1 cells were transiently transfected with either pSV SPORT PPARγ1 or pSV SPORT PPARγ2, the PPRE X3-TK-Luc and the control plasmid pRL-TK. After 24 h, cells were treated with rosiglitazone (10 μmol/L) or APE (25 μg/mL) and cultured for 4 h in DMEM containing 2 % (v/v) Charcoal/dextran stripped FBS (Sigma). The cells were then used for dual luciferase reporter gene assay.
Statistical analyses
Data are presented as mean ± standard error of the mean (SEM). In the case of the in vivo study, two-way repeated measurements ANOVA was used to analyze the data for body weight, intake, glycemic and insulinemic responses. A posteriori comparisons were done using the Bonferroni test. The remaining data was analyzed using the unpaired Student t test. All statistical analyses were performed using GraphPad 4.0 software.
Statistical analysis for in vitro studies was performed by one way ANOVA followed by the Tukey test as appropriate. P < 0.05 was considered statistically significant.