Ethical approval
All experimental protocols were approved by the Animal Experimental Committee of The University of Tokyo (No. 28–6).
Animals
Six-week-old male ICR mice were obtained from CLEA Japan Inc. (Tokyo, Japan). All mice were housed in an environment maintained at 23 °C with a 12/12 h light–dark cycle (dark: 7:00–19:00) and were provided with water and standard chow (3.59 kcal/g; 55.3% carbohydrates, 23.1% protein, 5.1% fat, 5.8% ash, 2.8% fiber, and 7.9% moisture, MF diet) (Oriental Yeast, Tokyo, Japan) ad libitum. They were acclimated for 1 week and were familiarized with the treadmill running exercise at a speed of 15–20 m/min for 10 min for 2 days before starting the experiment.
Materials
D-glucose and U-13C6 glucose were obtained (Fujifilm Wako Chemical Corporation) (D-glucose; 047–31,161, U-13C6 glucose; 574–69,731, Osaka, Japan) and diluted with water to a concentration of 6%.
Experimental protocols
Figure 1 shows a schematic overview of the experimental procedures.
Experiment 1: The effect of different carbohydrate intake frequencies on blood substrate and post-exercise glycogen recovery was investigated. Mice were randomly divided into bolus ingestion group (1.2 mg/g of BW glucose treatment immediately after exercise) and pulse ingestion group (1.2 mg/g BW glucose treatment every 15 min × 4 times). After 120 min of fasting, the mice were subjected to treadmill running exercise (20 m/min for 60 min) to decrease the levels of muscle and liver glycogen. The mice were then administered glucose in bolus or pulse form, and blood samples were collected from the tail vein at 0, 15, 30, 45, and 60 min. Blood samples were then centrifuged (4 °C, 5000×g, 10 min), and the plasma fraction was rapidly frozen in liquid nitrogen and stored at − 80 °C until further analysis. After a 1-week washout period, experiments were performed using a similar protocol. Mice were anaesthetized using isoflurane and euthanized by blood collection at 60 min from the portal vein and inferior vena cava. After collecting 50 μL of blood from the portal vein, the remaining blood samples were collected from the inferior vena cava. The plantaris muscle, soleus muscle, and liver were removed, rapidly frozen in liquid nitrogen, and stored at − 80 °C until further analysis.
Experiment 2: The effects of different ingestion methods on signaling pathways related to glycogen recovery were examined. After performing the same protocol as in experiment 1, the mice were anaesthetized using isoflurane and euthanized by inferior vena cava blood collection. Then, the plantaris muscle and liver were removed, rapidly frozen in liquid nitrogen, and stored at − 80 °C until further analysis. The sampling timings were 15 and 60 min after the first glucose administration in each group.
Experiment 3: The effects of expended recovery time on glycogen content were examined. After 120 min of fasting, mice were subjected to treadmill exercise (20 m/min for 60 min) and glucose was administered in bolus or pulse form. The mice were then allowed to rest for 120 min, were anaesthetized using isoflurane, and euthanized by inferior vena cava blood collection. The plantaris muscle, soleus muscle, and liver were removed, rapidly frozen in liquid nitrogen, and stored at − 80 °C until further analysis.
Experiment 4: To measure exogenous glucose utilization during the recovery period, an exhaled gas analysis using 13C glucose was performed. After 120 min of fasting, the mice were subjected to treadmill exercise (20 m/min for 60 min) and administered U-13C6 stable isotope-labelled glucose (1.2 mg/g BW) in bolus or pulse form. The mice were then placed in a sealed metabolic chamber (MK-680AT, Muromachi Kikai Co. Ltd., Japan). Their exhaled gas was collected (200 mL/min) in the sampling bag using an air pump (Compact Air Station Suction Cas-1, AS ONE, Japan) at 0, 60, and 120 min. Baseline breath samples were collected before the treadmill exercise. The 13CO2 concentration in the exhaled gas was measured using an infrared spectrophotometer (POCone, Otsuka Electronics Co., Ltd., Japan), following the methods of a previous study [14]. The measured values are presented as ∆13CO2 (‰). In addition, ∆13CO2 incremental area under the curve (iAUC) was calculated by summing the area of the increase from the pre-treatment value.
Blood and plasma substrate concentrations
Blood glucose collected from the tail vein was measured using an auto analyzer (Glutest Ace, Arkray Inc., Kyoto, Japan). Plasma insulin concentration was measured using a Mouse Insulin Enzyme-Linked Immunosorbent Assay Kit (M1102, Morinaga Institute of Biological Science, Inc., Kanagawa, Japan). Portal plasma glucose concentration was measured using a Glucose CII Test Wako Kit (439–90,901, Fujifilm Wako Chemical Corporation, Osaka, Japan). Blood glucose and plasma insulin iAUC was calculated by summing the area of the increase from the pre-treatment value.
Liver and muscle glycogen concentrations
Glycogen levels in the liver, plantaris muscle, and soleus muscle were measured using the phenol–sulfuric acid method, as described previously [14]. Tissues were weighed and added to 300 μL of 30% KOH with Na2SO4 to completely dissolve the tissue. The homogenized solutions were mixed with 360 μL ethanol and placed on ice for 30 min, followed by centrifugation (4 °C, 5000×g, 15 min), and the supernatants were removed. The glycogen-containing precipitate was dissolved in distilled water. Phenol and sulfuric acid were added to the solution, the mixture was allowed to react for 15 min, and the absorbance was measured at 490 nm.
Western blot analysis
The plantaris muscles were homogenized using radioimmunoprecipitation assay lysis buffer (20–188, Millipore, MA, USA) containing a protease inhibitor (1,183,617,001, Complete Mini EDTA-free, Roche Life Science, Indianapolis, IN, USA) and a phosphatase inhibitor (04906837001, PhosSTOP phosphatase inhibitor cocktail, Roche Life Science). The homogenates were placed on ice for 60 min and centrifuged (4 °C, 1500×g, 20 min). The total protein content of the samples was determined using a BCA Protein Assay Kit (23,227, Pierce, Rockford, IL, USA). The proteins (10 μg of each sample) were separated by sodium dodecyl sulphate–polyacrylamide gel electrophoresis. The proteins were then transferred to polyvinylidene difluoride membranes before being blocked for 60 min with 5% [w/v] bovine serum albumin in Tris-buffered saline with 0.1% [v/v] Tween 20 (TBST). Membranes were incubated overnight at 4 °C with the following primary antibodies: phospho-protein kinase B (Akt) (p-Akt Thr308, 9275, Cell Signaling Technology [CST], Tokyo, Japan); phospho-Akt (p-Akt Ser473, 9271, CST); total-Akt (t-Akt, 9272, CST); p-Akt substrate of 160 kDa (AS160) (p-AS160, Thr642, 8881, CST); total-AS160 (t-AS160, 2670, CST); phospho-AMP-activated protein kinase (AMPK) (p-AMPK Thr172, 2535, CST); total-AMPK (t-AMPK, 5832, CST); phospho-glycogen synthase (GS) (p-GS, Ser641, 3891, CST); total-GS (t-GS, 3893, CST); glucose transporter 4 (GLUT4, 07–1404, Merck, Tokyo, Japan); and glyceraldehyde 3-phosphate dehydrogenase (GAPDH, 14C10, 2118, CST). After incubation, the membranes were washed in TBST, incubated for 1 h at room temperature with secondary antibodies (A102PT, American Qualex, CA, USA), and washed again in TBST. Chemiluminescent reagents (RPN 2232 and RPN 2109, GE Healthcare Japan, Tokyo, Japan) were used for blot detection. The blots were scanned and quantified using a ChemiDoc XRS (170–8071, Bio-Rad, Hercules, CA, USA) and Quantity One software (170–9600, Bio-Rad, Hercules, CA, USA).
Statistical analysis
All data are expressed as the mean ± standard error of the mean. Student’s unpaired t-test and two-way analysis of variance (time × treatment) were performed. The Tukey–Kramer multiple comparisons test was used for post-hoc analysis. The relationship between liver glycogen content and blood substrate concentrations at 120 min after glucose ingestion was assessed by calculating the Pearson’s correlation coefficient. Significant differences were defined as p-values < 0.05.