Trilobatin, palmitate, DAPI and (2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) amino)-2-deoxyglucose (2NBDG) uptake measurement kits were purchased from Sigma (St Louis, MO, USA). IRS1, p-IRS1 (Ser 612), p-IRS1 (Ser 307), Akt, p-Akt (Ser 473), p-Akt (thr308), Na,K-ATPase, MYH1, MYOD1, and β-actin primary antibodies were bought from Cell Signaling Technology (Danvers, MA, USA). GLUT4 antibody was obtained from Abcam (Cambridge, MA, USA). HRP-conjugated GAPDH primary antibody was purchased from Aksmics (shanghai, China), Specific anti-mouse and anti-rabbit HRP-conjugated second antibodies were obtained from Santa Cruz Biotechnology (Texas, CA, USA). Rat/mouse insulin ELISA kits (EZRMI-13K) and ECL chemiluminescence detection reagent were obtained from Millipore (Billerica, MA, USA). Plasma membrane protein extraction kit, nuclear/cytosolic fractionation kit, RIPA buffer and BCA protein assay kit and other chemicals were purchased from Beyotime (Shanghai, China).
Mouse skeletal muscle cell lines, C2C12 myoblasts, were obtained from American Type Culture Collection (ATCC, Manassas, VA, USA) and maintained in DMEM supplemented with 10% FBS, and 1% penicillin/streptomycin (P/S) at 37°C in a humidified incubator with 5% CO2 atmosphere.
The differentiated C2C12 myotubes were induced as before 19. Generally, to induce the development of myotubes in C2C12 cells, the media was changed with differentiated media, which were prepared with fresh DMEM supplemented with 2% horse serum and 1% P/S, and continued to incubate for 4 days. After that, the cell morphology was observed and marker proteins (MYH1 and MyoD1) were determined by western blot. To induce the insulin resistance in differentiated C2C12 myotubes, the cells were starved in serum-free DMEM for 4 hours, and then incubated with 0.2 mM PA for 24 hours.
Male ob/ob mice (8-10 weeks) and same background C57BL/6 mice were purchased from Tengxin Biotechnology Co. (Chongqing, China), which were allowed ad libitum access to food and water unless otherwise stated, and rooms were maintained at 22°C and 50% humidity on a 12-h light/dark cycle. The mice were administrated with 10 mg/kg trilobatin (intragastric, i.g.) once for each day for continued 4 weeks, and the control mice were treated with same volume of in phosphate-buffered saline (PBS). Before dissection, the mice were euthanatized by CO2 inhalation, and gastrocnemius (the fast-glycolytic) muscles were isolated, froze in liquid nitrogen, and stored at -80°C. All animals’ experiments were carried out in accordance with the principles and guidelines of the Chinese Council Animal Care and also approved by the Institutional Animal Care and Use Committee at Chongqing Science and Technology Committee.
Glucose tolerance test
Glucose tolerance test (GTT) was performed as previously described 20. After the ob/ob mice were treated with 10 mg/kg trilobatin (i. g.) once for each day for continued 4 weeks, the mice were fasted overnight (12 hours), 2 g/kg glucose was administrated intragastrically, blood samples were collected from tail vein at 0, 15, 30, 60 and 120 minutes respectively, and the blood glucose was determined with One Touch Ultra Mini Blood Glucose Monitoring System (Johnson, Life Scan, Inc., Milpitas, CA, USA).
Palmitate solution preparation
BSA-bound palmitate was prepared according to the procedure described previously 21. Palmitate was firstly dissolved in 0.1 M NaOH to a concentration of 75 mM by heating at 70°C in a shaking water bath, and then the stock solution was complexed with 5% fatty acid-free bovine serum albumins (BSA; Ameresco, Solon, Ohio, USA) in PBS at a ratio of 8:1 (v/v) at 56°C in a shaking water bath. After filtration (0.2 μm membrane filter), this solution was stored at -20°C and used within 2 weeks. The same concentration of NaOH mixed with 10% FFA-free BSA was used as a control.
Glucose uptake in differentiated C2C12 myotubes was measured by adding 2-NBDG, a fluorescent D-glucose analog to trace the uptake of glucose directly 22, 23. Generally, after the differentiated C2C12 myotubes were starved in serum-free DMEM for 4 hours，and then treated with 0.2 mM palmitate in the presence or absence of indicated concentrations of trilobatin for 24 hours, the cells were starved in KRBH for 3 h, and then incubated without or with 100 nM insulin for 30 minutes, after that, 2-NBDG with a final concentration as 100 μM was added and continued to incubate for 30 minutes. After incubation, free 2-NBDG was washed out 3 times with PBS, and fluorescence densities in cell monolayers were measured with a fluorescence microplate reader (TECAN, Swiss) at an excitation wavelength of 475 nm and an emission wavelength of 550 nm. The protein concentration of each sample was determined by the BCA method. Results were normalized by mg of total protein.
After washed once with ice-cold PBS, the skeletal muscle tissues or cells were homogenized with hand held homogenizer (Biospec, Bartlesville, OK, USA) for 10 seconds and incubated on ice for 40-60 minutes in a modified RIPA buffer in the presence of 1% protease/phosphatase inhibitor cocktail, and the protein concentrations were measured with BCA protein assay kit from Beyotime (Shanghai, China).
To prepare the membrane and cytoplasm proteins, after the gastrocnemius muscles or cells were washed with PBS, the proteins were obtained by using plasma membrane protein extraction kit and nuclear/cytosolic fractionation kit from Beyotime (Shanghai, China) respectively according to the suggestions from the supplier. Generally, after the gastrocnemius muscles (which were cut into small pieces before use) or cells were incubated with membrane protein extraction reagent A in the ice bath for 10-15 minutes, and homogenized 30-50 times, the lysates were centrifuged for 10 minutes at 700 g and at 4°C to remove the nucleus and unfragmented cells. The cell membrane fragments were precipitated by centrifugation at 14000 × g and at 4°C for 30 minutes, and the supernatant is absorbed as cytoplasmic protein. And then, the precipitate was adding 200 μL membrane protein extraction reagent B and was re-suspended at maximum speed of Vortex for 10 seconds, followed by an ice bath for 15 minutes. The lysates were centrifuged at 14000 × g and at 4°C for 5 minutes, the supernatants were collected and used as the membrane proteins. After the proteins were quantified with BCA protein assay kit from Beyotime (Shanghai, China). Western blot was performed to determine GLUT4 expression on the plasma membrane and cytoplasm. The membrane marker Na, K-ATPase was used as a control in this study.
After the mice were administrated with 10 mg/kg trilobatin for 4 weeks (once for each day), the skeletal muscle tissues were isolated and fixed (10% formalin solution in 0.1 M PBS), frozen at -80°C overnight, and cut into 10 μm sections on a freezing microtome (Leica, Nussloch, Germany). The sections were permeabilized in 0.1% Triton X-100 in PBS and blocked in 1% BSA in PBS before incubation with the primary antibody. After stained with GLUT4 antibody (ab654, Abcam Inc. Cambridge, MA, USA) and together with DAPI nuclear stain (Invitrogen, CA, USA). Fluorescence images were acquired using a confocal microscope (Nikon, Tokio, Japan).
Proteins (20-30 μg) were subjected to 10% SDS-PAGE and then transferred to a PVDF membrane (Immobilon P; Millipore, MA, USA), and the membranes were then blocked for 2 h at room temperature with 5% BSA in TBST (20 mM Tris, 150 mM NaCl, 2.7 mM KCl, 0.1% Tween 20, pH 7.4). The membranes were next immunoblotted with primary antibodies at dilutions of 1:500 to 1:2000. Specific total or phospho-proteins were visualized after subsequent incubation with a 1:10000 dilution of anti-mouse or rabbit IgG conjugated to horseradish peroxidase. Excess antibody was washed off with TBST, and immunoreactivity was detected using ECL western blotting reagent (Millipore, MA, USA). Signal bands were quantified by densitometric analysis using ImageJ software (available from NIH at http://imagej.nih.gov/ij/) after scanning the blotted membrane. Three independent experiments were performed for each condition.
The data are expressed as means ± SD. Statistical comparisons between two groups were carried out using Student’s t-test or one-way analysis of variance (ANOVA), and one-way ANOVA with Bonferroni post-hoc test for multiple comparisons. The differences were considered significant when p < 0.05.