Animals
C57BL/6J mice (Hyochang Science, Daegu, Korea) were housed under standard conditions with a constant room temperature (22 ± 1◦C), relative humidity (50 ± 10%), and a 12 h light/dark cycle (light from 07:00 to 19:00) with an ad libitum access to food (#2014, Harlan Telkad, Madison, USA) and water. All mice were allowed to habituate to the novel environment for 1 week prior to the experiments. All the experimental procedures on the animals were reviewed and approved by the Animal Care and Use Committee of Keimyung University (KM2020-013).
Streptozotocin-induced Type 1 Diabetic Mouse
11-week-old male mice were administered intraperitoneally with 400µl of streptozotocin (50 mg/kg; Sigma-Aldrich, St. Louis, USA; Cat# S0130) or vehicle (0.5M sodium citrate solution; Fisher Scientific, Hampton, NH, USA; Cat# BP-327-1) after 12-h fasting, which is based on the previous report [25]. 10% sucrose water and normal food were provided immediately to prevent death from hypoglycemia. Mice with over 340 mg/dl of blood glucose, after five-day consecutive injection of streptozotocin, were used for further experimentation with an exercise and/or lithium treatment for additional 12 wks.
Lithium Administration And Exercise Training
Diabetic mice were introduced with 10 mg/kg of LiCl (Sigma-Aldrich, Cat# L4408) or saline (300µl) via daily oral gavage one hour before exercise training. The dosage of LiCl was determined based on previous publications [9, 13]. Previous studies have shown that 10 mg/kg of Li administration resulted in 0.3–0.7 mmol/L of Li in blood, which is recommended blood level for the treatment of psychiatric diseases [24, 22, 2]. We also tested toxicity and observed that long-term Li treatment did not induce marked renal or hepatic toxicity in diet-induced obese rats [11]. LiCl was introduced to mice five days per week for 12 weeks of the experiment. Exercise practice on mice was conducted daily between 09:00 a.m. and 12:00 p.m. for 12 weeks on a treadmill (FT-200, Techman Soft, Taoyuan City, Taiwan), and its speed and intensity were decided based on the previous reports [23, 12] with the following modification. The exercise protocol consisted of low intensity (40% VO2 max, 17 m/min, slop 0%) walking on a treadmill for 20 min per day, three days per week for 12 weeks. No electric shocks were used to reduce the stress effect of running on the treadmill during training sessions.
Body weight and food intake were measured daily during the experimental period. After 12 weeks of treatment, mice were rested for 48 h to eliminate the last-bout exercise effect. The mice were fasted overnight, following which they were anesthetized with pentobarbital sodium (5 mg/100 g BW). Blood was collected from the orbital vein using a heparin tube (Micro-Hematocrit capillary tube, #22-362-566, Fisher), and blood glucose was immediately measured using an automatic blood glucose analyzer (YSI 2300, Springfield, USA). The remaining blood was centrifuged (1500 × g, 15 min), and only plasma was stored at − 80°C. The plantaris muscles and liver were snap frozen after dissection and stored at − 80°C until analysis.
Sample Preparation For Western Blotting
Sample Preparation for Western Blotting
The plantaris muscles and liver were homogenized together in an ice-cold RIPA buffer (Tris-HCl pH 7.5 50 mM, SDS 0.1%, Triton X-100 1%, sodium chloride 150 mM, sodium deoxycholate 0.5%, EDTA 2 mM; BIOSOLUTION, Korea) containing protease and phosphatase inhibitors (#1861280, Fisher Scientific, Rockford, IL, USA; #P2850, Sigma-Aldrich, Saint Louis, MO, USA). Homogenization was performed on ice using a Wheaton tissue grinder (#357535 and #357537, DWK Life Sciences, Millville, NJ, USA). Homogenates were subjected to three freeze/thaw cycles and centrifuged for 15 min at 4°C, 1000 x g. The supernatant was separated and stored at − 80°C until assayed.
Western Blotting
Protein concentration was determined using the BSA assay (#5000006, Bio-Rad, Hercules, CA, USA). Aliquots were solubilized in Laemmli buffer (#1610747, Bio-Rad) and subjected to SDS/PAGE. A total of 40 µg of protein was separated by SDS-PAGE gel and transferred to a nitrocellulose membrane. The membrane was blocked for 60 min using 5% non-fat dry milk and Tris-buffered saline with 0.1% Tween 10 (TBST; pH 7.5), washed with TBST, and incubated overnight at 4°C with primary antibodies against the following proteins: calcium/calmodulin-dependent protein kinase kinase 2 (CaMKK2, 1:1000, Cell signaling, #16810), phospho(Thr180/Tyr182)-p38 mitogen-activated protein kinase (MAPK, 1:1000, Cell signaling, #4511), p38 MAPK (1:3000, Cell signaling, #9212), GLUT4 (Santa Cruz Bio, #sc-53566), GSK3β (Santa Cruz Bio, #sc-81462), phospho-GSK3β (Santa Cruz Bio, #sc-373800), glycogen synthase (Cell signaling, #3886), phospho-glycogen synthase (Cell signaling, #47043), PEPCK (Merck Millipore, #ABC1691), PFK-1 (Santa Cruz Bio, #sc-166722), LDHA (Santa Cruz Bio, #sc-137243), Rab10 (Santa Cruz Bio, #sc-101429), phospho-AKT (Cell signaling, #4060), AKT(Cell signaling, #2920), phospho-AMPKα1/2 (Santa Cruz Bio, #sc-33524), AMPKα1/2 (Santa Cruz Bio, #sc-25792), G6Pase (abcam, #ab93857), and β-actin (abcam, #ab106814). After washing with TBST, the samples were treated with the secondary antibody (anti-mouse or anti-rabbit, Santa Cruz Biotechnology, Santa Cruz, CA, USA) for 60 min. The bands were visualized using ECL (Genekhan Scientific, St. Louis, MO, USA), and the relative intensity of the bands was assessed using SigmaGel (Jandel Scientific Corp., Erkrath, Germany).
Immunohistochemistry Of The Pancreatic Islets
The whole frozen pancreas was embedded in FSC22 frozen section media (Leica, Richmond, IL, USA), and these frozen OCT blocks were sectioned to 6-µm thickness. The sectioned pancreas slices were incubated with insulin (Santa Cruz, #sc8033) and glucagon (Abcam, #ab92517) antibodies at 4℃ overnight, followed by washing with PBST (phosphate buffered saline with 0.1% Triton X-100) and subsequently incubating with Alexa-fluor-labeled secondary antibodies (Anti-mouse IgG secondary antibody, Alexa Fluor plus 488 and Anti-rabbit IgG secondary antibody, Alexa Fluor plus 555, Invitrogen) for 3 h at room temperature. Immunofluorescence signals were observed under a fluorescence microscope (Nikon, Tokyo, Japan).
Statistical analysis
Values are means ± standard error of the mean (S.E.M). The significance of the differences between groups was assessed using a one-way analysis of variance (ANOVA) followed by post hoc comparison using the Tukey significant difference method. A p-value < 0.05 was considered statistically significant.