All experimental procedures were done in accordance with the institutional guidelines for the care and use of laboratory animals and approved by the University of Kentucky Institutional Animal Care and Use Committee.
Animal care and use
Inducible skeletal muscle-specific Bmal1 knockout mice were generated as previously described . We obtained the skeletal muscle-specific tamoxifen inducible Cre recombinase mouse from the Center for Muscle Biology at the University of Kentucky. This mouse has a Cre recombinase flanked by two mutated estrogen receptors and is driven by a human skeletal actin promoter. Past work confirmed the efficacy of this mouse for muscle-specific gene recombination in adult mice . The tamoxifen inducible Cre recombinase mouse was crossed with the Bmal1-floxed mouse acquired through Jackson Labs (B6, 1294(Cg)-Arntl
tm1Weit/J), to generate the inducible skeletal muscle-specific Bmal1-floxed mouse (iMSBmal1
fl/fl). Activation of Cre recombination was done by intraperitoneal injections of tamoxifen (2 mg/day) for five consecutive days when the mice reached 12 weeks. This age is chosen to eliminate any effects that the lack of Bmal1 might have on skeletal muscle development and the rapid growth that occurs during post-natal maturation. Controls were vehicle (15 % ethanol in sunflower seed oil) treated with Cre+/−:Bmal1
flox/flox mice. Recombination specificity was confirmed and is demonstrated in Hodge et al. . Prior to experimentation, mice were housed in 14:10, light-to-dark conditions. All experiments were performed 3–5 weeks post-treatment. Echo-MRI was also performed at 10–12 weeks post-treatment. Both male and female mice were used in this study. Mice were euthanized prior to glucose uptake experiments and tissue collection. Euthanasia was performed using anesthesia followed by cervical dislocation.
Body composition was quantified in conscious mice at both 3–5 and 10–12 weeks post-treatment using EchoMRI Quantitative Magnetic Resonance Body Composition Analyzer (Echo Medical Systems, Houston, Texas) (mouse numbers n = 6 female iMSBmal1+/+ to n = 7 male iMSBmal1+/+ to n = 8 female iMSBmal1−/−: n = 10 male iMSBmal1−/−).
Glucose tolerance tests and blood work
Glucose tolerance tests [n = 8/group (two females and six males) in both groups] were performed 2 h after lights-off (ZT14), a time at which glucose tolerance is highest . Mice were fasted 6 h prior to the start of the test, and all measurements were done with an AlphaTRAK glucometer (Abbott Animal Health). A measurement was taken prior to injection of a bolus of glucose (0 min). Mice were injected by intraperitoneal injection with 2 mg/kg glucose, and additional measurements were taken at 15, 30, 60, 90, and 120 min post-injection. All fasting blood measures were obtained after a 6-h fast. Fasting blood glucose (n = 8/group (2 females and 6 males in both groups)) was measured with the AlphaTRAK glucometer, and fasting insulin (n = 7/group (3 females and 4 males in both groups)) was measured by collecting blood from the tail vein of the mice and running a colorimetric ELISA (Crystal Chem). Non-fasting blood glucose was measured by measuring blood glucose every 4 h for 24 h and averaged [n = 4 per time point; CT18 iMSBmal1+/+
n = 2 F/2 M; iMSBmal1−/−
n = 2 F/2 M; CT22 iMSBmal1+/+
n = 4 M; iMSBmal1−/−
n = 2 F/2 M; CT26 iMSBmal1+/+
n = 2 F/2 M; iMSBmal1−/−
n = 1 F/3 M; CT30 iMSBmal1+/+
n = 1 F/3 M; iMSBmal1−/−
n = 1 F/3 M; CT34 iMSBmal1+/+
n = 1 F/3 M; iMSBmal1−/−
n = 1 F/3 M]
Glucose uptake experiments
All solutions in this experiment were bubbled with 95 % O2/5 % CO2 and kept at 35 °C. Extensor digitorum longus (EDL) muscles were excised from each leg of the mice [n = 8 (2 F/6 M)/group for insulin stimulation; n = 5 all females/group for 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) stimulation] and placed in a recovery media (Krebs Henseleit Buffer (KHB) with 0.1 % bovine serum albumin, 2 mM sodium pyruvate, 6 mM mannitol) with or without 2000 μU/mL insulin or 2 mmol/L AICAR. After 30 min, muscles were transferred to an incubation buffer (KHB with 0.1 % bovine serum albumin, 2 mM sodium pyruvate, 6 mM mannitol, 1 mM 2-deoxy-d-glucose, 2.25 μL/mL [3H]-2-deoxyglucose, 2 μL/mL [14C]-mannitol) with or without 2000 μU/mL insulin or 2 mmol/L AICAR (consistent with what the muscle was exposed to in recovery media) for exactly 20 (insulin) or 40 (AICAR) min. Following this incubation, tissues were flash frozen. Tissue lysates were prepared, and 100 μL of each sample was added to a scintillation vial with 5 mL of scintillation fluid followed by radioactivity measurement in a scintillation counter.
Real-time PCR (RT-PCR)
RNA was isolated from gastrocnemius (GTN) samples of iMSBmal1
+/+ and iMSBmal1
−/− mice [n = 7 (3 F/4 M)/group]. Briefly, 50–100 mg of GTN tissue was homogenized in 1 mL Trizol (Invitrogen). Phase separation, RNA precipitation and RNA washes, and RNA resuspension were carried out as per manufacturer’s instructions. RNA was quantified through spectrophotometrically (λ = 260 nm). Total RNA was then used to synthesize cDNA using a mixture of oligo(dT) primer and random hexamers in SuperScript III First-Strand Synthesis SuperMix (Invitrogen, Waltham, MA, USA) kit. Expression of glucose transporter type 4 (Glut4), hexokinase 2 (Hk2), and phosphofructokinase 1 (Pfk1) was performed using cDNA and the following Taqman primers (Applied Biosystems): Mm01245502_m1, Mm00443385_m1, Mm01309576_m1, Mm99999915_g1, and Mm02343715_g1. RPL26 or glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were used as the internal calibration controls. The ∆∆CT method was used for the quantification of real-time PCR data. Gene expression in each sample was shown as the relative value compared to the mean vehicle value in that tissue (GTN).
Western blot analysis
Tissue lysates were made from GTN muscles of iMSBmal1
+/+ and iMSBmal1
−/− mice [n = 7 (3 F/4 M)/group]. Proteins were separated by SDS-PAGE using 4–15 % Tris-HCl precast gels (BioRad), transferred, and immunoblotted using routine methods. GLUT4 was detected using a primary monoclonal GLUT4 antibody (Cell Signaling, #2213) and an AlexaFluor680 goat anti-rabbit secondary antibody (Invitrogen, #A-21109).
Hexokinase activity assay
Hexokinase activity was measure following as previously described . GTN tissue [n = 7 (3 F/4 M)/group] was homogenized in a buffer (1:10, weight to volume) containing 150 mM KCl, 10 mM MgCl2, 5 mM EDTA, and 5 mM β-mercaptoethanol. Samples were centrifuged at 15,000×g for 1 h while experimental solutions A (47 mM Tris (pH 7.4), 10 mM MgCl2, 0.8 mM NADP, 0.5 mM glucose, 5.0 mM mercaptoethanol, 0.1 units glucose 6-phosphate dehydrogenase) and B were prepared(47 mM Tris (pH 7.4), 10 mM MgCl2, 0.8 mM NADP, 0.5 mM glucose, 5.0 mM mercaptoethanol, 0.1 units glucose 6-phosphate dehydrogenase, 5 mM ATP, 0.27 mM phosphoglyceric acid). An Eppendorf tube containing 2.45 mL of A or B was made for each tissue sample. After centrifugation was complete, 0.05 mL of tissue sample was added to each tube. Absorbance was measured at 30 °C and 340 nM every 2 s for 10 min. Samples were measured in duplicates. As the glycolytic reaction takes place, NADP is oxidized to form NADPH which has an absorbance at 340 nm.
Phosphofructokinase activity assay
The same tissue samples [n = 7 (3 F/4 M)/group] used for the hexokinase activity assay were used for the phosphofructokinase assay. The reaction mixture for this assay contained 50 mM Tris-HCl (pH 8), 1 mM EDTA, 6 mM MgCl2, 2.5 mM dithiothreitol, 0.16 mM NADH, 1 mM ATP, 1 mM fructose-6-phosphate, 0.4 units aldolase, 2.4 units triose-phosphate isomerase, and 0.4 units α–glycero-phosphate dehydrogenase. Five microliters of tissue sample was added to 295 μL of reaction mixture, and absorbance was read at 25 °C and 340 nM for 10 min. As the glycolytic reaction takes place, NADH, which is detectable at 340 nm, is reduced to NAD+ and absorbance decreases.
GTN muscles were dissected and freeze clamped in situ at ZT14 from anesthetized iMSBmal1
+/+ and iMSBmal1
−/− mice [n = 7 (3 F/4 M)/group] at 5 weeks post-treatment. Flash-frozen samples were sent to the University of Michigan Metabolomics Core Services for targeted metabolomics of glycolysis/tricarboxylic acid (TCA)/nucleotides involved in central metabolism. Twenty milligrams of skeletal muscle tissue was extracted first exposing the tissue to liquid nitrogen to harden the sample, then grinding the tissue in a pre-chilled mortar and pestle until the tissue was deemed adequately disrupted. The ground tissue was then transferred to a microtube, and metabolites were extracted with 0.5 mL of a mixture of methanol, chloroform, and water (8:1:1) containing isotope-labeled internal standards using a probe sonicator at 40 % output power, 20 % duty cycle for 20 s. Samples were allowed to rest at 4 °C for 10 min and then centrifuged at 4 °C, 14,000 rpm for 10 min. The extracts were removed and placed into an autosampler vial for mass spec analysis. Ten microliters of each sample was removed and pooled in a separate autosampler vial for quality control purposes. A series of calibration standards were prepared along with samples to quantify metabolites.
LC-MS analysis was performed on an Agilent system consisting of a 1260 UPLC module coupled with an 6520 Quadrupole-Time-of-flight (QTOF) mass spectrometer (Agilent Technologies, CA.) Metabolites were separated on a 150 × 1 mm Luna NH2 hydrophilic interaction chromatography column (Phenomenex, CA) using 10 mM ammonium acetate in water, adjusted to pH 9.9 with ammonium hydroxide, as mobile phase A, and acetonitrile as mobile phase B. The flow rate was 0.075 mL/min, and the gradient was linear 20 to 100 % A over 15 min, followed by isocratic elution at 100 % A for 5 min. The system was returned to starting conditions (20 % A) in 0.1 min and held there for 10 min to allow for column re-equilibration before injecting another sample. The mass spectrometer was operated in ESI-mode with the following conditions: gas temperature 350 °C, drying gas 10 L/min, nebulizer 20 psi.
Data were processed using MassHunter Quantitative analysis version B.07.00. Metabolites in the glycolysis/tricarboxylic acid (TCA)/ppp pathways were normalized to the nearest isotope-labeled internal standard and quantitated using two replicated injections of five standards to create a linear calibration curve with accuracy better than 80 % for each standard. Other compounds in the analysis were normalized to the nearest internal standard, and the peak areas were used for differential analysis between groups. Results from this analysis were used to identify enriched pathways using the online software Metaboanalyst 3.0, a comprehensive tool for metabolomic data analysis supported by The Metabolomics Innovation Center (TMIC), a Genome Canada-funded core facility.
Telemetry (Data Sciences International (DSI)) was used to evaluate changes in cage activity after vehicle/tamoxifen injection. Mice were anesthetized with isoflurane, and transmitter units (PhysioTel PA-C10; DSI) were implanted. Mice were housed singly and allowed to recover for 1 week. Data were recorded for 24 h/day, for 3–4 days weekly up to 10 weeks after vehicle or tamoxifen treatment. Data were collected and analyzed with DSI Dataquest ART4.1 telemetry software.
For statistics on blood concentrations, glucose tolerance, RT-PCR, body composition, behavior and enzyme activities, a Student’s t test was utilized. Two-way ANOVA was used to sex differences in body weight and body composition and data from glucose uptake experiments (comparing time exposed to insulin and genotype). Statistical analysis of metabolites was largely performed using Student’s t test to address differences between genotypes. To address whether metabolites within the glycolytic pathway were changed we used Hotelling’s T-squared test to determine if metabolites were changed between iMSBmal1
+/+ and iMSBmal1