Journal: Physiological Reports

Article Title: High metabolic substrate load induces mitochondrial dysfunction in rat skeletal muscle microvascular endothelial cells

doi: 10.14814/phy2.14855

Figure Lengend Snippet: Overview of the isolation procedure and cell experiment. Upper panel show the isolation procedure (elaborated in the text) and bottom panel show the cell experiment protocol and mitochondrial measurement. (a) Depicts rat skeletal muscles from hind‐ and forelimbs were minced with scissors in Dulbecco's modified Eagle medium (DMEM) containing 1% Pen Strep and digested with 0.2% collagenase type II 0.2% collagenase, 0.01% DNase, and 0.25% trypsin. (b) Shows the single cell solution seeded out onto 110‐mm dishes for 5 days before the dynabeading procedure was proceeded. (c) Anticoated magnetic dynabeads added to the single cell solution to isolate endothelial cells. (d) A magnet was applied to collect the bound microvascular endothelial cells. (e) Cells were counted and seeded onto 35‐mm dishes. (f) Cells were confluent and ready for experiments after 4–5 days. (g) The cells were used for determination of mitochondrial respiration and H 2 O 2 emission in acute and longterm metabolic substrate levels. (h) Overview of the diffrent acute and longterm metabolic interventions.

Article Snippet: Skeletal muscle microvascular endothelial cell cultures were prepared from male Sprague–Dawley Rats (Taconic M&B A/S).

Techniques: Isolation, Muscles, Modification

Journal: Physiological Reports

Article Title: High metabolic substrate load induces mitochondrial dysfunction in rat skeletal muscle microvascular endothelial cells

doi: 10.14814/phy2.14855

Figure Lengend Snippet: Representative micrograph showing immunohistochemical identification of microvascular endothelial cell suspension used for respirometry measurements. Arrows shows Griffonia Simplifolica lectin (red) staining for identification of endothelial cells and DAPI (blue) staining for cell nuclei. Remaining antibody‐coated magnetic dynabeads appear as circle shapes. Overall, staining procedures identified that approximately 95% of the cells were positive for the lectin

Article Snippet: Skeletal muscle microvascular endothelial cell cultures were prepared from male Sprague–Dawley Rats (Taconic M&B A/S).

Techniques: Immunohistochemical staining, Suspension, Staining

Journal: Physiological Reports

Article Title: High metabolic substrate load induces mitochondrial dysfunction in rat skeletal muscle microvascular endothelial cells

doi: 10.14814/phy2.14855

Figure Lengend Snippet: SUIT protocol of mitochondrial respiration and H 2 O 2 /O 2 emission in microvascular endothelial cells in the presence of 5 mM glucose. (a) Mitochondrial respiration and (b) mitochondrial H 2 O 2 /O 2 emission in intact and permeabilized cell ( n = 10 in each state). Data was analyzed by one‐way ANOVA and Tukey multiple pairwise‐comparison. *Significant difference between states in the SUIT protocol in presence of glucose (5 mM). ** Denotes significant difference p < 0.01. *** Denotes significant difference p < 0.001. All data are presented as mean ± SEM. LEAK CI; Leak state with substrates feeding complex I (glutamate and malate), LEAK CI + II; Leak state with complex I and complex II substrates (2 mM succinate), LEAK CI + CII (10 mM); Leak state using 10 mM succinate complex I and complex II leak respiration, OXPHOS; Maximal respiration with 5 mM ADP (state 3), LEAK; Leak state with oligomycin (state 4o), ETS; Electron transfer system by uncoupling oxidative phosphorylation with FCCP, ETS CII; Complex II flux with rotenone (shutdown of complex I)

Article Snippet: Skeletal muscle microvascular endothelial cell cultures were prepared from male Sprague–Dawley Rats (Taconic M&B A/S).

Techniques: Comparison, Phospho-proteomics

Journal: Physiological Reports

Article Title: High metabolic substrate load induces mitochondrial dysfunction in rat skeletal muscle microvascular endothelial cells

doi: 10.14814/phy2.14855

Figure Lengend Snippet: Mitochondrial respiration and hydrogen peroxide (H 2 O 2 /O 2 ) emission in in intact and permeabilized microvascular endothelial cells in the presence of acute substrate. (a) Depicts mitochondrial respiration in presence of 1.7 mM palmitic acid (PA) with or without 5 mM glucose, and (b) mitochondrial H 2 O 2 /O 2 emission in presence of 1.7 mM palmitic acid (PA) with or without 5 mM glucose ( n = 10 in glucose intervention, n = 8 in glucose + PA intervention, and n = 5 in PA intervention). Data were analyzed with linear mixed model. *Significantly different from glucose (5 mM), p < 0.05. ** Denotes significantly different ( p < 0.01) from glucose. *** Denotes significantly different ( p < 0.001) from glucose. †† Denotes significantly different ( p < 0.01) from no added substrate. ††† Denotes significantly different ( p < 0.001) from no added substrate. ‡ Significantly different from PA (1.7 mM). ‡‡‡ Denotes significantly different ( p < 0.001) from PA. LEAKCI + CII (10 mM); Leak state with complex I and complex II substrates (10 mM succinate) the maximal complex I and complex II leak respiration. All data are presented as mean ± SEM

Article Snippet: Skeletal muscle microvascular endothelial cell cultures were prepared from male Sprague–Dawley Rats (Taconic M&B A/S).

Techniques:

Journal: Physiological Reports

Article Title: High metabolic substrate load induces mitochondrial dysfunction in rat skeletal muscle microvascular endothelial cells

doi: 10.14814/phy2.14855

Figure Lengend Snippet: Mitochondrial respiration and hydrogen peroxide (H 2 O 2 /O 2 ) emission in intact and permeabilized microvascular endothelial cells in the presence of acute glucose intervention. (a) Mitochondrial respiration in presence of regular (5 mM) ( n = 6) or elevated (11 mM) glucose levels ( n = 7), and (b) Mitochondrial H 2 O 2 /O 2 emission in intact and permeabilized cells (LEAK CI + CII) in the presence of regular (5 mM) ( n = 6) or elevated (11 mM) glucose levels ( n = 7). Data were analyzed with linear mixed model. LEAKCI + CII (10 mM); Leak state with complex I and complex II substrates (10 mM succinate) the maximal complex I and complex II leak respiration. All data are presented as mean ± SEM

Article Snippet: Skeletal muscle microvascular endothelial cell cultures were prepared from male Sprague–Dawley Rats (Taconic M&B A/S).

Techniques:

Journal: Physiological Reports

Article Title: High metabolic substrate load induces mitochondrial dysfunction in rat skeletal muscle microvascular endothelial cells

doi: 10.14814/phy2.14855

Figure Lengend Snippet: Mitochondrial respiration and hydrogen peroxide (H 2 O 2 /O 2 ) emission in intact and permeabilized microvascular endothelial cells in the presence of acute palmitic acid intervention. (a) Mitochondrial respiration in the presence of regular (1.7 mM) or elevated (2.2 mM) PA concentrations and regular (5 mM) glucose, and (b) mitochondrial H 2 O 2 /O 2 emission in intact and permeabilized cells (LEAK CI + CII) ( n = 8 in regular PA and glucose, and n = 9 in elevated PA and glucose). Data were analyzed with linear mixed model. †† Denotes significantly different from regular PA p < 0.01. Leak state with complex I and complex II substrates (10 mM succinate) the maximal complex I and complex II leak respiration. All data are presented as mean ± SEM

Article Snippet: Skeletal muscle microvascular endothelial cell cultures were prepared from male Sprague–Dawley Rats (Taconic M&B A/S).

Techniques:

Journal: Physiological Reports

Article Title: High metabolic substrate load induces mitochondrial dysfunction in rat skeletal muscle microvascular endothelial cells

doi: 10.14814/phy2.14855

Figure Lengend Snippet: Mitochondrial respiration and hydrogen peroxide (H 2 O 2 /O 2 ) emission after 3‐days prolonged glucose and palmitic acid incubation of microvascular endothelial cells. (a) Mitochondrial respiration in the presence of regular (5 mM) glucose or regular glucose and regular (1.7 mM) palmitic acid (PA), and (b) mitochondrial H 2 O 2 /O 2 emission in intact with 5 mM glucose concentration and permeabilized cells (LEAK CI + CII) with regular (5 mM) glucose or regular glucose and regular (1.7 mM) PA incubations ( n = 10 in glucose intervention and n = 6 in PA intervention). Data were analyzed with linear mixed model. *Significantly different from regular glucose (5 mM) p < 0.05. ** shows p < 0.01 and *** shows p < 0.001. All data are presented as mean ± SEM. LEAKCI + CII; Leak state with complex I and complex II substrates with 10 mM succinate

Article Snippet: Skeletal muscle microvascular endothelial cell cultures were prepared from male Sprague–Dawley Rats (Taconic M&B A/S).

Techniques: Incubation, Concentration Assay

Journal: Physiological Reports

Article Title: High metabolic substrate load induces mitochondrial dysfunction in rat skeletal muscle microvascular endothelial cells

doi: 10.14814/phy2.14855

Figure Lengend Snippet: CS activity in microvascular endothelial cells after 3‐day incubation with regular and elevated glucose or regular and elevated palmitic acid (PA) with 5 mM glucose concentrations ( n = 6 in each group). Data were analyzed with linear mixed model. All data are presented as mean ± SEM. PA; palmitic acid, Maximal oxidation is achieved using FCCP, which means maximal uncoupled conditions

Article Snippet: Skeletal muscle microvascular endothelial cell cultures were prepared from male Sprague–Dawley Rats (Taconic M&B A/S).

Techniques: Activity Assay, Incubation

Journal: Physiological Reports

Article Title: High metabolic substrate load induces mitochondrial dysfunction in rat skeletal muscle microvascular endothelial cells

doi: 10.14814/phy2.14855

Figure Lengend Snippet: Intrinsic mitochondrial respiration in microvascular endothelial cells after 3‐day incubation with regular and elevated glucose or regular and elevated palmitic acid (PA) with 5 mM glucose concentrations. (a) Mitochondrial respiration divided by CS activity in microvascular endothelial cells after 3 days of incubation with regular glucose or regular palmitic acid levels ( n = 6 in each group), (b) Intrinsic mitochondrial respiration after 3 days incubation with regular or elevated glucose levels ( n = 6 in each group), and (c) Intrinsic mitochondrial respiration after 3 days incubation with regular or elevated PA levels with 5 mM glucose ( n = 6 in each group). Data were analyzed with linear mixed model. ** Denotes a significantly different from regular (5 mM) glucose p < 0.01. *** Denotes significantly different from regular (5 mM) glucose p < 0.001. All data are presented as mean ± SEM. PA, palmitic acid; Maximal oxidation is achieved using FCCP, representing maximal uncoupled conditions

Article Snippet: Skeletal muscle microvascular endothelial cell cultures were prepared from male Sprague–Dawley Rats (Taconic M&B A/S).

Techniques: Incubation, Activity Assay

Journal: Physiological Reports

Article Title: High metabolic substrate load induces mitochondrial dysfunction in rat skeletal muscle microvascular endothelial cells

doi: 10.14814/phy2.14855

Figure Lengend Snippet: Mitochondrial respiration and hydrogen peroxide (H 2 O 2 /O 2 ) emission after 3‐days prolonged glucose incubation of microvascular endothelial cells. (a) Mitochondrial respiration after 3‐days of incubation of microvascular endothelial cells with regular (5 mM) or elevated (11 mM) glucose concentrations, and (b) Mitochondrial H 2 O 2 /O 2 emission in intact with 5 mM glucose concentration and permeabilized cells in regular and elevated glucose (LEAK CI + CII) ( n = 10 regular‐ and n = 8 in elevated glucose concentration). Data were analyzed with linear mixed model. * Significantly different from regular glucose (5 mM) p < 0.05. All data are presented as mean ± SEM. LEAKCI + CII; Leak state with complex I and complex II substrates with 10 mM succinate

Article Snippet: Skeletal muscle microvascular endothelial cell cultures were prepared from male Sprague–Dawley Rats (Taconic M&B A/S).

Techniques: Incubation, Concentration Assay

Journal: Physiological Reports

Article Title: High metabolic substrate load induces mitochondrial dysfunction in rat skeletal muscle microvascular endothelial cells

doi: 10.14814/phy2.14855

Figure Lengend Snippet: Mitochondrial respiration and hydrogen peroxide (H 2 O 2 /O 2 ) emission after 3‐days prolonged palmitic acids incubation of microvascular endothelial cells. (a) Mitochondrial respiration after 3‐days incubation of microvascular endothelial cells with regular (1.7 mM) or elevated (2.2 mM) PA concentrations and regular glucose (5 mM) ( n = 6 in each group), and (b) Mitochondrial H 2 O 2 /O 2 emission in intact with 5 mM glucose concentration and permeabilized cells (LEAK CI + CII) in regular and elevated PA concentrations ( n = 6 in each group). Data were analyzed with linear mixed model. All data are presented as mean ± SEM. LEAKCI + CII; Leak state with complex I and complex II substrates with 10 mM succinate

Article Snippet: Skeletal muscle microvascular endothelial cell cultures were prepared from male Sprague–Dawley Rats (Taconic M&B A/S).

Techniques: Incubation, Concentration Assay

Journal: Cells

Article Title: Integrated ATAC-seq and RNA-seq Analysis of In Vitro Cultured Skeletal Muscle Satellite Cells to Understand Changes in Cell Proliferation

doi: 10.3390/cells13121031

Figure Lengend Snippet: The cell proliferation potential and distribution of cell cycle phases of skeletal muscle satellite cells cultured in vitro vary across different passages. ( A ) Images of third-passage (F3), fifth-passage (F5), and seventh-passage (F7) pig skeletal muscle cells stained with 5-ethynyl-2′-deoxyuridine (EdU). Images were captured at 100× magnification. ( B ) Statistical analysis of the EdU positivity rate in cells from each passage. Data are displayed as mean ± SD, n = 9 independent experiments. *** p < 0.001. ( C ) Distribution of cell cycle phases among F3 (left), F5 (middle), and F7 (right) cells. ( D ) Quantification of the cell cycle phase distribution for each passage. Data are displayed as mean ± SD, n = 3 independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: The cell culture medium utilized in this study was sourced from iCell Bioscience Inc. (Shanghai, China) and comprised a basal medium for primary skeletal muscle cells, fetal bovine serum (FBS), antibiotics (penicillin/streptomycin, P/S), and a supplement specifically designed for primary skeletal muscle cell culture.

Techniques: Cell Culture, In Vitro, Staining