Increased glycolysis in skeletal muscle coordinates with adipose tissue in systemic metabolic homeostasis

Abstract Insulin‐independent glucose metabolism, including anaerobic glycolysis that is promoted in resistance training, plays critical roles in glucose disposal and systemic metabolic regulation. However, the underlying mechanisms are not completely understood. In this study, through genetically manipulating the glycolytic process by overexpressing human glucose transporter 1 (GLUT1), hexokinase 2 (HK2) and 6‐phosphofructo‐2‐kinase‐fructose‐2,6‐biphosphatase 3 (PFKFB3) in mouse skeletal muscle, we examined the impact of enhanced glycolysis in metabolic homeostasis. Enhanced glycolysis in skeletal muscle promoted accelerated glucose disposal, a lean phenotype and a high metabolic rate in mice despite attenuated lipid metabolism in muscle, even under High‐Fat diet (HFD). Further study revealed that the glucose metabolite sensor carbohydrate‐response element‐binding protein (ChREBP) was activated in the highly glycolytic muscle and stimulated the elevation of plasma fibroblast growth factor 21 (FGF21), possibly mediating enhanced lipid oxidation in adipose tissue and contributing to a systemic effect. PFKFB3 was critically involved in promoting the glucose‐sensing mechanism in myocytes. Thus, a high level of glycolysis in skeletal muscle may be intrinsically coupled to distal lipid metabolism through intracellular glucose sensing. This study provides novel insights for the benefit of resistance training and for manipulating insulin‐independent glucose metabolism.

with SYBR Green reagents (Roche) on an ABI Prism Step-One bio-analyzer. Sequences of primers are listed in Table S2.
Expression data were normalized to β-actin mRNA expression. Expression changes were calculated using the ∆∆Ct method and expressed as fold change over control. Cells and tissues were harvested, homogenized in ice-cold RIPA buffer with Protease Inhibitor Cocktail Tablets (Roche). Supernatant protein concentration was determined by Pierce TM BCA protein assay kit (Thermo). Proteins were electrophoretically separated and immunoblotted onto polyvinylidene fluoride membranes. Membranes were incubated at 4 °C for 16 h with indicated primary antibodies and subsequently at room temperature for 2 h with horseradish-peroxidase-conjugated secondary antibodies. Detection was carried out with Highsig ECL (Tanon), and signals were visualized by a gel documentation system (Syngene, UK). Protein bands were quantified by densitometry using Image J. The antibodies are listed in Table S3. For immunoprecipitation, tissues were collected and lysed with ice-cold pre-lysis buffer (50 mM Tris-HCl, pH 8.0, 300 mM NaCl, 2 mM EDTA, 1% NP40 with protease inhibitor cocktail). Tissue lysates were incubated with Lamtor1 antibodies (#8975, Cell Signaling) overnight at 4℃ on rotation. Protein A/G PLUS-Agarose (sc-2003, Santa Crzu) were then added to the overnight protein aggregates and mixed for another 1h at 4 ℃ on rotation. The supernatant was collected by centrifugation of the mix at 4 °C, 3000 rpm for 3 min as lysates. The beads were washed with 2x10 minutes with 600 ul of low-salt wash buffer (25 mM Tris-HCl, pH 7.4 and 150 mM NaCl) at 4 °C and another 10 minutes with 600 ul of high-salt wash buffer (25 mM Tris-HCl, pH 7.4 and 500 mM NaCl) at 4 °C. Then the beads were mixed with an equal volume of 2×SDS sample buffer for immunoblotting.

Measurement of oxygen consumption in cells and tissues
Both the ECAR and OCR of MEFs were measured using the XF e extracellular flux analyzer (Seahorse Bioscience) as follows: 20,000 cells were plated per well into an XF24 cell culture microplate in quintuplicate (5 wells each). For ECAR analysis, final concentrations of 10 mM glucose, 1 μM oligomycin, 100 mM 2-DG (101706-100, Seahorse Bioscience) were applied. And 1 μM oligomycin, 0.5 μM FCCP, 1 μM rotenone and antimycin (102194-100, Seahorse Bioscience) were applied in OCR. Mitochondrial respiration rates were measured in saponin-permeabilized EDL muscle and white fat tissue with indicated substrates as described previously. 4

Muscle glucose uptake and lipid uptake and oxidation ex vivo
Muscle glucose uptake and lipid uptake and oxidation was carried out in isolated soleus or EDL muscles as previously described. 5,6 Intact soleus and EDL muscles were isolated from female mice for glucose uptake, and soleus isolated from male mice were applied for lipid uptake and oxidation. Briefly, for glucose uptake, intact soleus and EDL muscles were isolated from mice fasted for 16 hours and incubated with or without insulin in Krebs-Ringer-bicarbonate (KRB) buffer at 37 °C for 50 min. The insulin concentration was 0.1 mU/ml for soleus stimulation and 50 mU/ml for EDL. After incubation, muscles were used for glucose uptake in KRB buffer containing 2-deoxy-D-[1,2-3 H(N)] glucose and D-[1-14 C] Mannitol for another 10 min in 30 °C with or without insulin. After incubation, the process was terminated in ice-cold KRB buffer containing cytochalasin B, and muscles were blotted dry in liquid nitrogen, weighed, lysed by 1 M NaOH solution at 80 °C, and neutralized by 1 M HCl followed by adding dinonyl-phthalate oil for scintillation counting. 3 H and 14 C radioisotopes in muscle lysates were measured using a Tri-Carb 2800TR scintillation counter (PerkinElmer) for calculation of muscle glucose uptake. For lipid uptake and oxidation, isolated soleus was stimulated with or without insulin for 30 min and then incubated in KRB buffer (with/without insulin) containing 14 C-palmitic acid for another 50 min. After incubation, muscles were blotted dry and lysed for measurements of radioisotopes using a Tri-Carb 2800TR scintillation counter. Gaseous 14 CO2 was evolved from incubation media with 0.6 M perchloric acid (311421, Sigma-Aldrich) and trapped in benzethonium hydroxide-soaked filters (B2156, Sigma-Aldrich). Radioactivity in trapped 14 CO2 was measured to determine muscle lipid oxidation. The sum of radioactivity in muscle and gaseous 14 CO2 was calculated to indicate lipid uptake in muscle.
Tissue samples were obtained and frozen in liquid nitrogen and then stored at -80 °C until measurement.
Tissue TG content was measured following the manufacturer's instructions (E1013, Applygen). Muscle and cell G-6-P concentrations were determined following the manufacturer's instructions (MAK014, Sigma).
The ATP levels were examined in muscle using a commercially available kit (S0026, Beyotime, China) following the manufacturer's instructions.

Glucose, insulin, and oral lipid tolerance tests (GTT, ITT, and OLTT)
After withdrawal of food for 16 h (for GTT) or 4 h (for ITT and OLTT), mice were intraperitoneally injected with a bolus of glucose (2 mg glucose per g of body weight) for IPGTT, insulin (0.75 mU insulin per g of body weight) for ITT, or orally administered via gavage with a bolus of olive oil (6 ul olive oil per g of body weight) for OLTT. Blood was collected from tail veins at the indicated times. Blood glucose, plasma free fatty acid and triglyceride levels were determined using Breeze 2 glucometer (Bayer), LabAssay NEFA kit (294-63601), and LabAssay Triglyceride kit (290-63701), respectively.

Assessment of body composition and treadmill endurance
Body weight was measured weekly. Body composition was determined via dual-energy X-ray absorptiometry using a Lunar PIXImus II densitometer (GE Healthcare) following the manufacturer's instructions. Mice were acclimated (run for 9 minutes at 10 meters (m)/minute followed by 1 minute at 20 m/minute) to the treadmill for 2 consecutive days prior to the experimental protocol. For low intensity (endurance) exercise studies, fed mice were run for 10 minutes at 10 m/minute followed by a constant speed of 20 m/minute until exhaustion.

Indirect calorimetry
Mice were housed individually in metabolic cages at a 12-h light and dark cycle with free access to food and water using the Comprehensive Lab Animal Monitoring System (CLAMS, Columbus Instruments). Mice were acclimated in the metabolic cage for 1 day prior to the recording according to the instructions of the manufacturer. Food, energy expenditure, physical activity, VO2 and VCO2 were assessed simultaneously. For co-culture studies, myotubes were washed with PBS and incubated with DMEM for 2 days. CM were then collected, centrifuged at 1,300 g for 5 min, and stored at -80 °C for future use. Primary preadipocytes were isolated and differentiated as previously described. 7

Statistics
Data were expressed as mean ± SEM. Comparisons were performed via unpaired two-tailed student's ttest for two groups or via two-way ANOVA followed by Bonferroni's post hoc test for multiple groups using GraphPad Prism 9.0.0 software (GraphPad, San Diego, CA, USA). Comparison of VCO2, VO2, and EE were carried out by ANCOVA with the covariate of body weight using the web-based tool (CalR; https://CalRapp.org/). 8,9 All p values less than 0.05 were considered statistically significant. Generally, * means p < 0.05, ** means p < 0.01 and *** means p < 0.001. For animal experiments, animal numbers were kept as small as possible, and yet still resulted in statistically meaningful data.