Journal: Nature metabolism

Article Title: Hyperglycaemia is associated with impaired muscle signalling and aerobic adaptation to exercise.

doi: 10.1038/s42255-020-0240-7

Figure Lengend Snippet: Fig. 1 | Glucose intolerance and hyperglycaemia develop in WD-fed and STZ-injected mice. CD-1 mice were fed a WD, injected with STZ (2 × 40 mg kg−1), or fed a control diet (CON). a,b, After 8 weeks of treatment, all mice underwent GTT (2 g kg−1 glucose) (a) and AUC for glucose over 120 min was calculated (b) (CON n = 52, WD n = 51, STZ n = 52). c, Blood was sampled from unfasted mice under resting conditions during the light (7:00; CON n = 10, WD n = 12, STZ n = 15) and dark (20:00; CON n = 12, WD n = 12, STZ n = 15) cycles to assess the degree of hyperglycaemia. d, Serum insulin was measured by enzyme-linked immunosorbent assay after 12 h of fasting (CON n = 21, WD n = 11, STZ n = 21). e, Body mass was recorded for 8 weeks following initiation of CON (n = 14), WD (n = 12) or STZ (n = 12) treatment in a cohort of mice. f, To determine voluntary wheel running behaviour in CON, WD and STZ groups, running distance was measured daily and expressed as cumulative weekly running distance for 6 weeks (CON n = 25, WD n = 25, STZ n = 26). Two-tailed unpaired t-test with Welch’s correction was used to compare groups in c or by one-way ANOVA followed by Holm–Sidak post hoc testing in all other panels. Data are represented as a point for the result of each individual animal; bars are mean ± s.e.m.

Article Snippet: A second model of glucose intolerance/hyperglycaemia was induced with low-dose STZ (catalogue no. 1621 Tocris Bioscience) dissolved in diluted citrate buffer (114 mM; 0.5 M stock catalogue no. 2034, Boston Bioproducts).

Techniques: Injection, Control, Enzyme-linked Immunosorbent Assay, Two Tailed Test

Journal: Nature metabolism

Article Title: Hyperglycaemia is associated with impaired muscle signalling and aerobic adaptation to exercise.

doi: 10.1038/s42255-020-0240-7

Figure Lengend Snippet: Fig. 2 | Metabolic outcomes in response to exercise training. a, A schematic diagram in outlining the experimental timeline of the aerobic exercise training study. b, Total training distance was recorded from mice with access to wheels after 6 weeks (CON n = 25, WD n = 25, STZ n = 26). c, Skeletal muscle mitochondrial and training markers OXPHOS (complexes I–V), hexokinase II and GLUT4 were assessed by western blotting in gastrocnemius muscle (CON SED n = 12, WD SED n = 12, STZ SED n = 12; CON EXT n = 12, WD EXT n = 12, STZ EXT n = 10). d, Representative blots. e, In the final weeks of exercise training, mice underwent GTT and this shows AUC for glucose over 120 min (CON SED n = 27, WD SED n = 25, STZ SED n = 27; CON EXT n = 25, WD EXT n = 23, STZ EXT n = 25). f,g, Body mass was measured before and after training (f) (CON SED n = 27, WD SED n = 25, STZ SED n = 28; CON EXT n = 25, WD EXT n = 24, STZ EXT n = 26) and body composition was determined in a subset of animals by dual-energy X-ray absorptiometry (g) (CON SED n = 10, WD SED n = 9, STZ SED n = 6; CON EXT n = 10, WD EXT n = 9, STZ EXT n = 6). h, Visceral adipose tissue was collected and stained with H&E and representative images are shown (STZ EXT n = 5; all other groups n = 6). Scale bar, 100 µm. i, Adipocyte size was quantified and expressed as cross-sectional area (STZ EXT n = 5; all other groups n = 6). j, Liver was harvested at dissection, stained with H&E and imaged using bright field microscopy. Scale bar, 50 µm. Images are representative of n = 6 per group. k, Hepatic glycerol was measured by colorimetric assay (CON SED n = 6, WD SED n = 6, STZ SED n = 8; CON EXT n = 6, WD EXT n = 5, STZ EXT n = 9). In cases where a subset of mice was analysed for training effects, exercise-trained mice from each group were matched for training distance. Main effects were determined by one-way ANOVA in b or two-way ANOVA in all other panels, followed by Holm–Sidak post hoc testing. Data are represented as a point for the result of each individual animal; bars are mean ± s.e.m. In a, “Mouse” icon by Iconic, “Test tube” icon by H. Alberto Gongora, “Muscle” icon by P. J. Witt from the Noun Project.

Article Snippet: A second model of glucose intolerance/hyperglycaemia was induced with low-dose STZ (catalogue no. 1621 Tocris Bioscience) dissolved in diluted citrate buffer (114 mM; 0.5 M stock catalogue no. 2034, Boston Bioproducts).

Techniques: Western Blot, Staining, Dissection, Microscopy, Colorimetric Assay

Journal: Nature metabolism

Article Title: Hyperglycaemia is associated with impaired muscle signalling and aerobic adaptation to exercise.

doi: 10.1038/s42255-020-0240-7

Figure Lengend Snippet: Fig. 3 | Exercise capacity and muscle phenotype in response to exercise training. CD-1 mice were fed a control diet (CON), WD or treated with STZ for 8 weeks, and placed in voluntary wheel running cages for a further 8 weeks (exercise-trained). A separate set of mice remained in cages without wheel access to act as controls (sedentary). Aerobic exercise capacity was tested before and after exercise training. a, Individual changes in exercise capacity (Δ) were calculated by subtracting posttraining exercise capacity from pretraining exercise capacity and plotted from lowest to highest response in all trained groups. b,c, Absolute exercise capacity was measured 6 weeks into the training period for exercise-trained and sedentary age-matched controls in distance (b) and time to exhaustion (c). a–c, CON SED n = 17, WD SED n = 16, STZ SED n = 22; CON EXT n = 17, WD EXT n = 16, STZ EXT n = 20). d, Maximal oxygen consumption, VO2peak, was measured in a cohort of sedentary and exercise-trained mice by using a graded treadmill test as an indicator of cardiorespiratory fitness (WD SED n = 4; all other groups n = 5). Gastrocnemius muscles from a subset of animals that were matched for total wheel running distance were sectioned for histology. e, FITC-conjugated Griffonia simplicifolia lectin immunofluorescent staining was used to calculate capillary density in muscle. Scale bar, 100 µm. f, Capillary density (capillaries per mm2) was quantified in FIJI (CON SED n = 5; all other groups n = 6). g, Sections were stained with MHC type I (pink) and MHC type IIA (blue) to calculate oxidative versus glycolytic fibre distribution, and WGA to visualize the ECM (pink). Scale bar, 200 µm. Images are representative of n = 6 per group. h, Combined type I and type IIa fibre proportion was counted and plotted as percentage of total fibres per animal (all groups n = 6). Main effects were determined by one-way ANOVA relative to CON group in a and by two-way ANOVA otherwise, followed by Holm–Sidak post hoc testing. Data are represented as a point for the result of each individual animal, bars are mean ± s.e.m.

Article Snippet: A second model of glucose intolerance/hyperglycaemia was induced with low-dose STZ (catalogue no. 1621 Tocris Bioscience) dissolved in diluted citrate buffer (114 mM; 0.5 M stock catalogue no. 2034, Boston Bioproducts).

Techniques: Control, Muscles, Staining

Journal: Nature metabolism

Article Title: Hyperglycaemia is associated with impaired muscle signalling and aerobic adaptation to exercise.

doi: 10.1038/s42255-020-0240-7

Figure Lengend Snippet: Fig. 4 | Impaired glucose tolerance and hyperglycaemia are associated with muscle ECM accretion. Gastrocnemius muscles from sedentary CON, WD and STZ mice were collected to assess ECM accumulation. a, WGA staining demonstrates a thicker ECM compartment in WD and STZ compared to CON. Images are representative of n = 6 per group. Scale bar, 100 µm. b, Fractional (%) ECM area was calculated (n = 6); boxplot shows mean with error spanning minimum to maximum values. c, The relationship between muscle ECM accumulation in b was correlated with fasting glucose for each animal by Spearman’s correlation (n = 6). d, Hydroxyproline content was measured in gastrocnemius muscle as a marker of skeletal muscle collagen (CON n = 25, WD n = 20, STZ n = 12). e, WGA staining was performed on soleus muscle sections from rats bred for LRT or HRT, demonstrating higher ECM thickness in LRT. Scale bar, 200 µm. f, Relative ECM fractional area was calculated for n = 8 HRT and n = 9 LRT, boxplot shows mean with error spanning minimum to maximum values. g, Hydroxyproline content of LRT and HRT muscles was measured (n = 20 per group). h, Muscle sections were stained for AGE (green) and counterstained with WGA to visualize the ECM (red). i, The glycation signal was quantified (n = 6 per group, three images per individual rat) for pixel intensity and represented as a percentage of LRT; boxplot shows mean with error spanning minimum to maximum values. Scale bar, 200 µm. Main effects were determined by one-way ANOVA in b and d followed by Holm–Sidak post hoc testing, or by two-tailed unpaired t-test with Welch’s correction in remaining panels. Data are represented as a point for the result of each individual animal, bars are mean ± s.e.m.

Article Snippet: A second model of glucose intolerance/hyperglycaemia was induced with low-dose STZ (catalogue no. 1621 Tocris Bioscience) dissolved in diluted citrate buffer (114 mM; 0.5 M stock catalogue no. 2034, Boston Bioproducts).

Techniques: Muscles, Staining, Marker, Two Tailed Test

Journal: Nature metabolism

Article Title: Hyperglycaemia is associated with impaired muscle signalling and aerobic adaptation to exercise.

doi: 10.1038/s42255-020-0240-7

Figure Lengend Snippet: Fig. 5 | In vitro angiogenesis and muscle gene expression changes in response to hyperglycaemia. Cell culture experiments were performed to analyse the effect of hyperglycaemia on endothelial and muscle cell behaviour and gene expression. a, In vitro capillary tube formation was performed in HUVECs to determine the effect of ECM glycation by 0 and 3 mM methylglyoxal (MG) or treatment with glycation inhibitor (aminoguanidine, AG; 100 mM) over 16 h. Images are representative of n = 3 independent experiments. Scale bar, 250 µm. Tube formation was quantified using FIJI and corresponding analysis overlays are shown. b, Quantification for HUVEC total tube length for n = 3 independent experiments. c, Tube formation was measured in HUVECs grown in medium supplemented with 20% serum collected from normoglycemic CON (n = 6) mice or hyperglycemic STZ (n = 5) mice for 16 h. Serum experiments were run in duplicate for each mouse. Representative images from n = 4 mice per group are shown. Scale bar, 500 µm. c, Serum-conditioned HUVEC total tube length was quantified. FIJI representative overlays are shown in c for CON (top) and STZ (bottom). d, Quantification for HUVEC total segment length is shown for CON n = 6 and STZ n = 5 independent animals. e, HUVEC proliferation was measured over 4 d in cells cultured in low (LG, 5 mM) or high (HG, 25 mM) glucose media (n = 6). Fold change is expressed as the increase versus day 1 for each glucose condition. f, MHC and ECM gene expression were measured in C2C12 cells differentiated for 6 d in low (LG; 5 mM, n = 6) or high (HG; 25 mM, n = 6) glucose media. Fold change is expressed relative to the LG mean for each gene. Specific groups were compared via one-way ANOVA in e followed by Holm–Sidak post hoc testing, or by two-tailed unpaired t-test to respective controls with in remaining panels. Data are represented as a point for the result of each independent experiment in b, individual animal d or individual cell culture well in e and f; bars are mean ± s.e.m.

Article Snippet: A second model of glucose intolerance/hyperglycaemia was induced with low-dose STZ (catalogue no. 1621 Tocris Bioscience) dissolved in diluted citrate buffer (114 mM; 0.5 M stock catalogue no. 2034, Boston Bioproducts).

Techniques: In Vitro, Gene Expression, Cell Culture, Two Tailed Test

Journal: Nature metabolism

Article Title: Hyperglycaemia is associated with impaired muscle signalling and aerobic adaptation to exercise.

doi: 10.1038/s42255-020-0240-7

Figure Lengend Snippet: Fig. 6 | Hyperglycaemia is associated with altered muscle signalling with acute exercise. WD-fed, STZ or CON diet-fed mice underwent a bout of moderate-intensity treadmill running for 30 min (AEX). A group of sedentary (SED) mice did not undergo treadmill running and acted as basal controls. Western blotting was used to determine acute exercise-induced signal transduction in gastrocnemius muscles. a,b, Blots are quantified from WD mice (a) and STZ-treated mice (b). Representative images are shown in a and b. In a, CON SED, CON AEX, WD SED, AD EAX n = 6 for all signalling proteins except p-JNK, n = 10 per group. In b, CON SED n = 8 (p-AKT, p-P70S6K n = 4), CON AEX n = 8 (p-MKK4, p-SMAD2L n = 7; p-AKT, p-P38, p-P70S6K n = 4), STZ SED n = 12 (p-AKT, p-P70S6K n = 6), STZ AEX n = 12 (p-AKT, p-MKK4, p-P70S6K n = 6, p-P38 n = 5). C, control diet; S, STZ-treated; SED, resting control; AEX, acute exercise. c, EDL muscles excised from CD-1 mice on control diet were placed in oxygenated Krebs–Henseleit buffer (B, basal) or stretched by 10% of optimal length for 10 min (S, stretch). Muscles were snap frozen for western blotting to determine phosphorylation and activation of mechanosensitive proteins; n = 6 per group. d,e, Mass of gastrocnemius (Gastroc.) (d) and tibialis anterior (TA) muscles were measured (e) (CON n = 10, WD n = 9, STZ n = 6). f, Muscle fibre cross-sectional area was measured in gastrocnemius after WGA staining (CON n = 10, WD n = 6, STZ n = 10). g, JNK activation after 30 min of treadmill running was correlated with blood glucose in a separate cohort of CON, WD, STZ and NOD mice (n = 38). Data in g were analysed using Spearman’s correlation; R and P values are indicated. p-SEK1/MKK4, phosphorylated mitogen-activated protein kinase kinase 4; p-P38, phosphorylated mitogen-activated protein kinase 38; p-JNK, phosphorylated c-JNK; p-SMAD2L, linker region phosphorylated SMAD2 and p-P70S6K T421/S424, phosphorylated P70S6 kinase on residues Thr421/Ser424. Group differences were determined by two-way ANOVA in a and b, or by one-way ANOVA in d, e and f followed by Holm–Sidak post hoc testing. Data are represented as a point for the result of each individual animal; bars are mean ± s.e.m.

Article Snippet: A second model of glucose intolerance/hyperglycaemia was induced with low-dose STZ (catalogue no. 1621 Tocris Bioscience) dissolved in diluted citrate buffer (114 mM; 0.5 M stock catalogue no. 2034, Boston Bioproducts).

Techniques: Western Blot, Transduction, Muscles, Control, Phospho-proteomics, Activation Assay, Staining