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    MathWorks Inc tcacalc
    Palmitate exposure modulates energy metabolism in BV2 cells. BV2 cells were incubated with vehicle (Veh), 200 µmol/L palmitate (PA) for 24 h or 1 µg/mL LPS for 3 h. ( A ) Schematic representation of experiments for OCR measurements depicting the calculated parameters upon addition of oligomycin (1.5 mmol/L), FCCP (0.5 mmol/L), and antimycin A (0.5 mmol/L) plus rotenone (0.5 mmol/L): basal respiration (basal), proton leak-driven respiration (leak), ATP synthesis-linked respiration (ATP), maximal respiration capacity (max), spare respiration capacity (spare), and non-mitochondrial oxygen consumption (NM). ( B-C ) Oxygen consumption rate (OCR) measured for 3 cycles within each respiration state ( B ), and calculated respiration parameters ( C ). ( D ) Representative immunoblotting experiment against the four complexes of the electron transport chain and ATP synthase (complex V), after separation of 30 µg of protein by SDS-PAGE. ( E ) Relative immunoreactivity signal from the 5 complexes in 4 independent experiments. For a given protein, signal within each band was normalized to the average of that in the 3 experimental groups. ( F ) Expression of genes involved in mitochondria biogenesis, fusion and fission. ( G ) Schematic representation of experiments for ECAR measurements depicting the calculated parameters upon addition of oligomycin (1 mmol/L) and 2-deoxy-D-glucose (2DG, 50 mmol/L): basal glycolysis (glyc), glycolytic reserve (res), glycolytic capacity (capac), and non-glycolytic medium acidicitation (NGA). ( H-I ) extracellular medium acidification rate (ECAR) measured for 3 cycles within each respiration state, and calculated glycolytic parameters. ( J ) Relative expression of Slc2a1 gene (GLUT1). ( K ) Representation of 13 C incorporation into glutamate omitting, for simplicity, generation of isotopomers from unlabeled pyruvate/acetyl-CoA, and respective representative multiplets observed in 13 C NMR spectra measured in extracts after metabolizing [1- 13 C]glucose for 24 h. ( L ) Glutamate (Glu) multiplet fractions, and fractional enrichment (FE) of lactate C3 of (Lac) and alanine (Ala). ( M ) Model used in the <t>TCAcalc</t> analysis and relative fluxes, and lactate labeling estimated by fitting glutamate isotopomers, and Ala C3. Abbreviations: CS, citrate synthase; PDH, pyruvate dehydrogenase; Y, flux of anaplerotic substrates through pyruvate carboxylase (Y PC ) or succinyl-CoA (Y S ). Data is shown as mean ± SD of 3–12 independent experiments, represented by the individual symbols. * P < 0.05, ** P < 0.01, *** P < 0.001 depict differences in comparisons following significant effects in ANOVA
    Tcacalc, supplied by MathWorks Inc, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Extracellular vesicles released from microglia after palmitate exposure impact brain function"

    Article Title: Extracellular vesicles released from microglia after palmitate exposure impact brain function

    Journal: Journal of Neuroinflammation

    doi: 10.1186/s12974-024-03168-7

    Palmitate exposure modulates energy metabolism in BV2 cells. BV2 cells were incubated with vehicle (Veh), 200 µmol/L palmitate (PA) for 24 h or 1 µg/mL LPS for 3 h. ( A ) Schematic representation of experiments for OCR measurements depicting the calculated parameters upon addition of oligomycin (1.5 mmol/L), FCCP (0.5 mmol/L), and antimycin A (0.5 mmol/L) plus rotenone (0.5 mmol/L): basal respiration (basal), proton leak-driven respiration (leak), ATP synthesis-linked respiration (ATP), maximal respiration capacity (max), spare respiration capacity (spare), and non-mitochondrial oxygen consumption (NM). ( B-C ) Oxygen consumption rate (OCR) measured for 3 cycles within each respiration state ( B ), and calculated respiration parameters ( C ). ( D ) Representative immunoblotting experiment against the four complexes of the electron transport chain and ATP synthase (complex V), after separation of 30 µg of protein by SDS-PAGE. ( E ) Relative immunoreactivity signal from the 5 complexes in 4 independent experiments. For a given protein, signal within each band was normalized to the average of that in the 3 experimental groups. ( F ) Expression of genes involved in mitochondria biogenesis, fusion and fission. ( G ) Schematic representation of experiments for ECAR measurements depicting the calculated parameters upon addition of oligomycin (1 mmol/L) and 2-deoxy-D-glucose (2DG, 50 mmol/L): basal glycolysis (glyc), glycolytic reserve (res), glycolytic capacity (capac), and non-glycolytic medium acidicitation (NGA). ( H-I ) extracellular medium acidification rate (ECAR) measured for 3 cycles within each respiration state, and calculated glycolytic parameters. ( J ) Relative expression of Slc2a1 gene (GLUT1). ( K ) Representation of 13 C incorporation into glutamate omitting, for simplicity, generation of isotopomers from unlabeled pyruvate/acetyl-CoA, and respective representative multiplets observed in 13 C NMR spectra measured in extracts after metabolizing [1- 13 C]glucose for 24 h. ( L ) Glutamate (Glu) multiplet fractions, and fractional enrichment (FE) of lactate C3 of (Lac) and alanine (Ala). ( M ) Model used in the TCAcalc analysis and relative fluxes, and lactate labeling estimated by fitting glutamate isotopomers, and Ala C3. Abbreviations: CS, citrate synthase; PDH, pyruvate dehydrogenase; Y, flux of anaplerotic substrates through pyruvate carboxylase (Y PC ) or succinyl-CoA (Y S ). Data is shown as mean ± SD of 3–12 independent experiments, represented by the individual symbols. * P < 0.05, ** P < 0.01, *** P < 0.001 depict differences in comparisons following significant effects in ANOVA
    Figure Legend Snippet: Palmitate exposure modulates energy metabolism in BV2 cells. BV2 cells were incubated with vehicle (Veh), 200 µmol/L palmitate (PA) for 24 h or 1 µg/mL LPS for 3 h. ( A ) Schematic representation of experiments for OCR measurements depicting the calculated parameters upon addition of oligomycin (1.5 mmol/L), FCCP (0.5 mmol/L), and antimycin A (0.5 mmol/L) plus rotenone (0.5 mmol/L): basal respiration (basal), proton leak-driven respiration (leak), ATP synthesis-linked respiration (ATP), maximal respiration capacity (max), spare respiration capacity (spare), and non-mitochondrial oxygen consumption (NM). ( B-C ) Oxygen consumption rate (OCR) measured for 3 cycles within each respiration state ( B ), and calculated respiration parameters ( C ). ( D ) Representative immunoblotting experiment against the four complexes of the electron transport chain and ATP synthase (complex V), after separation of 30 µg of protein by SDS-PAGE. ( E ) Relative immunoreactivity signal from the 5 complexes in 4 independent experiments. For a given protein, signal within each band was normalized to the average of that in the 3 experimental groups. ( F ) Expression of genes involved in mitochondria biogenesis, fusion and fission. ( G ) Schematic representation of experiments for ECAR measurements depicting the calculated parameters upon addition of oligomycin (1 mmol/L) and 2-deoxy-D-glucose (2DG, 50 mmol/L): basal glycolysis (glyc), glycolytic reserve (res), glycolytic capacity (capac), and non-glycolytic medium acidicitation (NGA). ( H-I ) extracellular medium acidification rate (ECAR) measured for 3 cycles within each respiration state, and calculated glycolytic parameters. ( J ) Relative expression of Slc2a1 gene (GLUT1). ( K ) Representation of 13 C incorporation into glutamate omitting, for simplicity, generation of isotopomers from unlabeled pyruvate/acetyl-CoA, and respective representative multiplets observed in 13 C NMR spectra measured in extracts after metabolizing [1- 13 C]glucose for 24 h. ( L ) Glutamate (Glu) multiplet fractions, and fractional enrichment (FE) of lactate C3 of (Lac) and alanine (Ala). ( M ) Model used in the TCAcalc analysis and relative fluxes, and lactate labeling estimated by fitting glutamate isotopomers, and Ala C3. Abbreviations: CS, citrate synthase; PDH, pyruvate dehydrogenase; Y, flux of anaplerotic substrates through pyruvate carboxylase (Y PC ) or succinyl-CoA (Y S ). Data is shown as mean ± SD of 3–12 independent experiments, represented by the individual symbols. * P < 0.05, ** P < 0.01, *** P < 0.001 depict differences in comparisons following significant effects in ANOVA

    Techniques Used: Incubation, Western Blot, SDS Page, Expressing, Labeling



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    tcacalc  (MathWorks Inc)
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    MathWorks Inc tcacalc
    Palmitate exposure modulates energy metabolism in BV2 cells. BV2 cells were incubated with vehicle (Veh), 200 µmol/L palmitate (PA) for 24 h or 1 µg/mL LPS for 3 h. ( A ) Schematic representation of experiments for OCR measurements depicting the calculated parameters upon addition of oligomycin (1.5 mmol/L), FCCP (0.5 mmol/L), and antimycin A (0.5 mmol/L) plus rotenone (0.5 mmol/L): basal respiration (basal), proton leak-driven respiration (leak), ATP synthesis-linked respiration (ATP), maximal respiration capacity (max), spare respiration capacity (spare), and non-mitochondrial oxygen consumption (NM). ( B-C ) Oxygen consumption rate (OCR) measured for 3 cycles within each respiration state ( B ), and calculated respiration parameters ( C ). ( D ) Representative immunoblotting experiment against the four complexes of the electron transport chain and ATP synthase (complex V), after separation of 30 µg of protein by SDS-PAGE. ( E ) Relative immunoreactivity signal from the 5 complexes in 4 independent experiments. For a given protein, signal within each band was normalized to the average of that in the 3 experimental groups. ( F ) Expression of genes involved in mitochondria biogenesis, fusion and fission. ( G ) Schematic representation of experiments for ECAR measurements depicting the calculated parameters upon addition of oligomycin (1 mmol/L) and 2-deoxy-D-glucose (2DG, 50 mmol/L): basal glycolysis (glyc), glycolytic reserve (res), glycolytic capacity (capac), and non-glycolytic medium acidicitation (NGA). ( H-I ) extracellular medium acidification rate (ECAR) measured for 3 cycles within each respiration state, and calculated glycolytic parameters. ( J ) Relative expression of Slc2a1 gene (GLUT1). ( K ) Representation of 13 C incorporation into glutamate omitting, for simplicity, generation of isotopomers from unlabeled pyruvate/acetyl-CoA, and respective representative multiplets observed in 13 C NMR spectra measured in extracts after metabolizing [1- 13 C]glucose for 24 h. ( L ) Glutamate (Glu) multiplet fractions, and fractional enrichment (FE) of lactate C3 of (Lac) and alanine (Ala). ( M ) Model used in the <t>TCAcalc</t> analysis and relative fluxes, and lactate labeling estimated by fitting glutamate isotopomers, and Ala C3. Abbreviations: CS, citrate synthase; PDH, pyruvate dehydrogenase; Y, flux of anaplerotic substrates through pyruvate carboxylase (Y PC ) or succinyl-CoA (Y S ). Data is shown as mean ± SD of 3–12 independent experiments, represented by the individual symbols. * P < 0.05, ** P < 0.01, *** P < 0.001 depict differences in comparisons following significant effects in ANOVA
    Tcacalc, supplied by MathWorks Inc, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    tcacalc program  (MathWorks Inc)
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    Expanded 13 C NMR spectra of HEK293 WT and HEK293 MUL1(−/−) cells. (A) The labeling pattern of lactate (C-2), glutamate (C-4), and glutamate (C-2). Lactate-C2 spectra indicating that pyruvate cycled through the pyruvate kinase (PK) flux. The C2D12 and C2D23 represent the [1,2– 13 C]lactate and [2,3– 13 C]lactate isotopomers, respectively, whereas 2Q signals represent [U- 13 C]lactate. S: singlet; D12, D23, and D45: doublet; Q: quartet. (B) Glutamate 13 C signal ratio obtained from 13 C-NMR spectra of HEK293 WT, MUL1(−/−), MUL1(−/−)+Peri, and MUL1(−/−)+CTM cells utilizing the [U- 13 C]glucose. p ≤ 0.05: *, WT vs. MUL1(−/−); #, WT vs. MUL1(−/−)+Peri; ‡, WT vs. MUL1(−/−)+CTM; ¶, MUL1(−/−) vs. MUL1(−/−)+Peri; §, MUL1(−/−) vs. MUL1(−/−)+CTM; £, MUL1(−/−)+Peri vs. MUL1(−/−)+CTM. (C) Metabolic flux model demonstrating the 13 C-labeling pattern of the metabolites derived from [U- 13 C]glucose [U- 13 C]glucose-derived [U- 13 C]pyruvate enters the TCA cycle through YPC (red dots) or PDH flux (green dots). Glucose oxidation through PDH flux labeled C4–C5 of glutamate (green dots) and C2–C3 of glutamate (red dots) through YPC flux. Key enzymatic steps involved in the metabolic flux model are as follows: (1) lactate dehydrogenase (LDH), (2) pyruvate kinase (PK), (3) pyruvate dehydrogenase (PDH), (4) pyruvate carboxylase (YPC), (5) glutamate dehydrogenase (GDH), (6) phosphoenolpyruvate carboxykinase (PEPCK), and (7) anaplerosis via succinyl-CoA (Ys) (note: metabolic model demonstrates the 1/2 turn of the tricarboxylic acid (TCA) cycle). (D) Metabolic flux rates calculated from the 13 C-isotopomers of glutamate observed in the 13 C-NMR spectra. Signal areas obtained from the results of the peak fitting procedure were used as an input to a metabolic model and solved numerically using <t>tcaCALC.</t> All flux rates are referenced to a citrate synthase (CS) flux of 1 and is equivalent to Kreb’s cycle flux. Statistical significance was p ≤ 0.05: *, WT vs. MUL1(−/−); #, WT vs. MUL1(−/−)+Peri; ‡, WT vs. MUL1(−/−)+CTM; ¶, MUL1(−/−) vs. MUL1(−/−)+Peri; §, MUL1(−/−) vs. MUL1(−/−)+CTM; £, MUL1(−/−)+Peri vs. MUL1(−/−)+CTM.
    Tcacalc Program, supplied by MathWorks Inc, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Palmitate exposure modulates energy metabolism in BV2 cells. BV2 cells were incubated with vehicle (Veh), 200 µmol/L palmitate (PA) for 24 h or 1 µg/mL LPS for 3 h. ( A ) Schematic representation of experiments for OCR measurements depicting the calculated parameters upon addition of oligomycin (1.5 mmol/L), FCCP (0.5 mmol/L), and antimycin A (0.5 mmol/L) plus rotenone (0.5 mmol/L): basal respiration (basal), proton leak-driven respiration (leak), ATP synthesis-linked respiration (ATP), maximal respiration capacity (max), spare respiration capacity (spare), and non-mitochondrial oxygen consumption (NM). ( B-C ) Oxygen consumption rate (OCR) measured for 3 cycles within each respiration state ( B ), and calculated respiration parameters ( C ). ( D ) Representative immunoblotting experiment against the four complexes of the electron transport chain and ATP synthase (complex V), after separation of 30 µg of protein by SDS-PAGE. ( E ) Relative immunoreactivity signal from the 5 complexes in 4 independent experiments. For a given protein, signal within each band was normalized to the average of that in the 3 experimental groups. ( F ) Expression of genes involved in mitochondria biogenesis, fusion and fission. ( G ) Schematic representation of experiments for ECAR measurements depicting the calculated parameters upon addition of oligomycin (1 mmol/L) and 2-deoxy-D-glucose (2DG, 50 mmol/L): basal glycolysis (glyc), glycolytic reserve (res), glycolytic capacity (capac), and non-glycolytic medium acidicitation (NGA). ( H-I ) extracellular medium acidification rate (ECAR) measured for 3 cycles within each respiration state, and calculated glycolytic parameters. ( J ) Relative expression of Slc2a1 gene (GLUT1). ( K ) Representation of 13 C incorporation into glutamate omitting, for simplicity, generation of isotopomers from unlabeled pyruvate/acetyl-CoA, and respective representative multiplets observed in 13 C NMR spectra measured in extracts after metabolizing [1- 13 C]glucose for 24 h. ( L ) Glutamate (Glu) multiplet fractions, and fractional enrichment (FE) of lactate C3 of (Lac) and alanine (Ala). ( M ) Model used in the TCAcalc analysis and relative fluxes, and lactate labeling estimated by fitting glutamate isotopomers, and Ala C3. Abbreviations: CS, citrate synthase; PDH, pyruvate dehydrogenase; Y, flux of anaplerotic substrates through pyruvate carboxylase (Y PC ) or succinyl-CoA (Y S ). Data is shown as mean ± SD of 3–12 independent experiments, represented by the individual symbols. * P < 0.05, ** P < 0.01, *** P < 0.001 depict differences in comparisons following significant effects in ANOVA

    Journal: Journal of Neuroinflammation

    Article Title: Extracellular vesicles released from microglia after palmitate exposure impact brain function

    doi: 10.1186/s12974-024-03168-7

    Figure Lengend Snippet: Palmitate exposure modulates energy metabolism in BV2 cells. BV2 cells were incubated with vehicle (Veh), 200 µmol/L palmitate (PA) for 24 h or 1 µg/mL LPS for 3 h. ( A ) Schematic representation of experiments for OCR measurements depicting the calculated parameters upon addition of oligomycin (1.5 mmol/L), FCCP (0.5 mmol/L), and antimycin A (0.5 mmol/L) plus rotenone (0.5 mmol/L): basal respiration (basal), proton leak-driven respiration (leak), ATP synthesis-linked respiration (ATP), maximal respiration capacity (max), spare respiration capacity (spare), and non-mitochondrial oxygen consumption (NM). ( B-C ) Oxygen consumption rate (OCR) measured for 3 cycles within each respiration state ( B ), and calculated respiration parameters ( C ). ( D ) Representative immunoblotting experiment against the four complexes of the electron transport chain and ATP synthase (complex V), after separation of 30 µg of protein by SDS-PAGE. ( E ) Relative immunoreactivity signal from the 5 complexes in 4 independent experiments. For a given protein, signal within each band was normalized to the average of that in the 3 experimental groups. ( F ) Expression of genes involved in mitochondria biogenesis, fusion and fission. ( G ) Schematic representation of experiments for ECAR measurements depicting the calculated parameters upon addition of oligomycin (1 mmol/L) and 2-deoxy-D-glucose (2DG, 50 mmol/L): basal glycolysis (glyc), glycolytic reserve (res), glycolytic capacity (capac), and non-glycolytic medium acidicitation (NGA). ( H-I ) extracellular medium acidification rate (ECAR) measured for 3 cycles within each respiration state, and calculated glycolytic parameters. ( J ) Relative expression of Slc2a1 gene (GLUT1). ( K ) Representation of 13 C incorporation into glutamate omitting, for simplicity, generation of isotopomers from unlabeled pyruvate/acetyl-CoA, and respective representative multiplets observed in 13 C NMR spectra measured in extracts after metabolizing [1- 13 C]glucose for 24 h. ( L ) Glutamate (Glu) multiplet fractions, and fractional enrichment (FE) of lactate C3 of (Lac) and alanine (Ala). ( M ) Model used in the TCAcalc analysis and relative fluxes, and lactate labeling estimated by fitting glutamate isotopomers, and Ala C3. Abbreviations: CS, citrate synthase; PDH, pyruvate dehydrogenase; Y, flux of anaplerotic substrates through pyruvate carboxylase (Y PC ) or succinyl-CoA (Y S ). Data is shown as mean ± SD of 3–12 independent experiments, represented by the individual symbols. * P < 0.05, ** P < 0.01, *** P < 0.001 depict differences in comparisons following significant effects in ANOVA

    Article Snippet: Multiplet fractions from aliphatic carbons of glutamate were group-averaged and used in tcaCALC running on MATLAB 2019a (MathWorks, Natick, MA-USA) to determine rates (relative to citrate synthase) of pyruvate dehydrogenase (PDH), anaplerosis through pyruvate carboxylase (Y PC ), anaplerosis through other pathways such as propionate or glutamine (Y S ), and pyruvate kinase (PK) [ ].

    Techniques: Incubation, Western Blot, SDS Page, Expressing, Labeling

    Expanded 13 C NMR spectra of HEK293 WT and HEK293 MUL1(−/−) cells. (A) The labeling pattern of lactate (C-2), glutamate (C-4), and glutamate (C-2). Lactate-C2 spectra indicating that pyruvate cycled through the pyruvate kinase (PK) flux. The C2D12 and C2D23 represent the [1,2– 13 C]lactate and [2,3– 13 C]lactate isotopomers, respectively, whereas 2Q signals represent [U- 13 C]lactate. S: singlet; D12, D23, and D45: doublet; Q: quartet. (B) Glutamate 13 C signal ratio obtained from 13 C-NMR spectra of HEK293 WT, MUL1(−/−), MUL1(−/−)+Peri, and MUL1(−/−)+CTM cells utilizing the [U- 13 C]glucose. p ≤ 0.05: *, WT vs. MUL1(−/−); #, WT vs. MUL1(−/−)+Peri; ‡, WT vs. MUL1(−/−)+CTM; ¶, MUL1(−/−) vs. MUL1(−/−)+Peri; §, MUL1(−/−) vs. MUL1(−/−)+CTM; £, MUL1(−/−)+Peri vs. MUL1(−/−)+CTM. (C) Metabolic flux model demonstrating the 13 C-labeling pattern of the metabolites derived from [U- 13 C]glucose [U- 13 C]glucose-derived [U- 13 C]pyruvate enters the TCA cycle through YPC (red dots) or PDH flux (green dots). Glucose oxidation through PDH flux labeled C4–C5 of glutamate (green dots) and C2–C3 of glutamate (red dots) through YPC flux. Key enzymatic steps involved in the metabolic flux model are as follows: (1) lactate dehydrogenase (LDH), (2) pyruvate kinase (PK), (3) pyruvate dehydrogenase (PDH), (4) pyruvate carboxylase (YPC), (5) glutamate dehydrogenase (GDH), (6) phosphoenolpyruvate carboxykinase (PEPCK), and (7) anaplerosis via succinyl-CoA (Ys) (note: metabolic model demonstrates the 1/2 turn of the tricarboxylic acid (TCA) cycle). (D) Metabolic flux rates calculated from the 13 C-isotopomers of glutamate observed in the 13 C-NMR spectra. Signal areas obtained from the results of the peak fitting procedure were used as an input to a metabolic model and solved numerically using tcaCALC. All flux rates are referenced to a citrate synthase (CS) flux of 1 and is equivalent to Kreb’s cycle flux. Statistical significance was p ≤ 0.05: *, WT vs. MUL1(−/−); #, WT vs. MUL1(−/−)+Peri; ‡, WT vs. MUL1(−/−)+CTM; ¶, MUL1(−/−) vs. MUL1(−/−)+Peri; §, MUL1(−/−) vs. MUL1(−/−)+CTM; £, MUL1(−/−)+Peri vs. MUL1(−/−)+CTM.

    Journal: Frontiers in Cell and Developmental Biology

    Article Title: Regulation of Metabolism by Mitochondrial MUL1 E3 Ubiquitin Ligase

    doi: 10.3389/fcell.2022.904728

    Figure Lengend Snippet: Expanded 13 C NMR spectra of HEK293 WT and HEK293 MUL1(−/−) cells. (A) The labeling pattern of lactate (C-2), glutamate (C-4), and glutamate (C-2). Lactate-C2 spectra indicating that pyruvate cycled through the pyruvate kinase (PK) flux. The C2D12 and C2D23 represent the [1,2– 13 C]lactate and [2,3– 13 C]lactate isotopomers, respectively, whereas 2Q signals represent [U- 13 C]lactate. S: singlet; D12, D23, and D45: doublet; Q: quartet. (B) Glutamate 13 C signal ratio obtained from 13 C-NMR spectra of HEK293 WT, MUL1(−/−), MUL1(−/−)+Peri, and MUL1(−/−)+CTM cells utilizing the [U- 13 C]glucose. p ≤ 0.05: *, WT vs. MUL1(−/−); #, WT vs. MUL1(−/−)+Peri; ‡, WT vs. MUL1(−/−)+CTM; ¶, MUL1(−/−) vs. MUL1(−/−)+Peri; §, MUL1(−/−) vs. MUL1(−/−)+CTM; £, MUL1(−/−)+Peri vs. MUL1(−/−)+CTM. (C) Metabolic flux model demonstrating the 13 C-labeling pattern of the metabolites derived from [U- 13 C]glucose [U- 13 C]glucose-derived [U- 13 C]pyruvate enters the TCA cycle through YPC (red dots) or PDH flux (green dots). Glucose oxidation through PDH flux labeled C4–C5 of glutamate (green dots) and C2–C3 of glutamate (red dots) through YPC flux. Key enzymatic steps involved in the metabolic flux model are as follows: (1) lactate dehydrogenase (LDH), (2) pyruvate kinase (PK), (3) pyruvate dehydrogenase (PDH), (4) pyruvate carboxylase (YPC), (5) glutamate dehydrogenase (GDH), (6) phosphoenolpyruvate carboxykinase (PEPCK), and (7) anaplerosis via succinyl-CoA (Ys) (note: metabolic model demonstrates the 1/2 turn of the tricarboxylic acid (TCA) cycle). (D) Metabolic flux rates calculated from the 13 C-isotopomers of glutamate observed in the 13 C-NMR spectra. Signal areas obtained from the results of the peak fitting procedure were used as an input to a metabolic model and solved numerically using tcaCALC. All flux rates are referenced to a citrate synthase (CS) flux of 1 and is equivalent to Kreb’s cycle flux. Statistical significance was p ≤ 0.05: *, WT vs. MUL1(−/−); #, WT vs. MUL1(−/−)+Peri; ‡, WT vs. MUL1(−/−)+CTM; ¶, MUL1(−/−) vs. MUL1(−/−)+Peri; §, MUL1(−/−) vs. MUL1(−/−)+CTM; £, MUL1(−/−)+Peri vs. MUL1(−/−)+CTM.

    Article Snippet: The tcaCALC program in MATLAB was utilized to perform an isotopomer analysis to estimate relative pathway fluxes ( ).

    Techniques: Labeling, Derivative Assay

    Akt2 and HIF-1α proteins contribute to support the metabolic phenotype of HEK293 MUL1(−/−) cells. (A) Glycolytic capacity in HEK293 WT and Akt2(−/−) cells before or after treatment with the HIF-1α activator DMOG (100 μM for 4 h). Extracellular acidification rate (ECAR) was measured using the Seahorse analyzer. HEK293 MUL1(−/−) cells were used as a positive control. #, p ≤ 0.035: vs . WT con ; *, p ≤ 0.035: vs . MUL1(−/−) con; ‡, p ≤ 0.045: AKT2(−/−) con vs. AKT2(−/−)+DMOG. (B) Quantification of the glycolysis, glycolytic capacity, and glycolytic reserve obtained from three independent experiments. Data from three separate experiments are presented as means ± SEM. #, p ≤ 0.03: vs . WT con ; *, p ≤ 0.035: vs . MUL1(−/−) con; ‡, p ≤ 0.045: AKT2(−/−) con vs. AKT2(−/−)+DMOG. (C) The effect of HIF-1α activator, DMOG, on the expression levels of HIF-1α, GLUT1, Akt2, P-GSK-3β S9, in Akt2(−/−), or MUL1(−/−) cells was monitored by western blot analysis. (D) Densitometrical analysis of the proteins shown in panel (C), normalized against β-actin. Results shown as means ± SD of three independent experiments. *, p ≤ 0.03 vs . WT con; #, p ≤ 0.025 vs . Akt2(−/−) con. (E) Glutamate 13 C signal ratio obtained from 13 C-NMR spectra of Akt2(−/−) and Akt2(−/−) +DMOG cells utilizing the [U- 13 C]glucose (note: S, D, T, and Q are singlet, doublet, triplet, and quartet, respectively. All signal ratios were calculated with respect to the total area of the corresponding glutamate resonance. Data are represented as mean ± SEM. (F) Metabolic flux rates calculated from the 13 C-isotopomers of glutamate observed in the 13 C-NMR spectra. Signal areas obtained from the results of the peak fitting procedure were used as an input to a metabolic model and solved numerically using tcaCALC. All flux rates are referenced to a citrate synthase (CS) flux of 1 and is equivalent to Kreb’s cycle flux. Statistical significance was p ≤ 0.05: *, for Akt2(−/−) vs. Akt2(−/−)+DMOG.

    Journal: Frontiers in Cell and Developmental Biology

    Article Title: Regulation of Metabolism by Mitochondrial MUL1 E3 Ubiquitin Ligase

    doi: 10.3389/fcell.2022.904728

    Figure Lengend Snippet: Akt2 and HIF-1α proteins contribute to support the metabolic phenotype of HEK293 MUL1(−/−) cells. (A) Glycolytic capacity in HEK293 WT and Akt2(−/−) cells before or after treatment with the HIF-1α activator DMOG (100 μM for 4 h). Extracellular acidification rate (ECAR) was measured using the Seahorse analyzer. HEK293 MUL1(−/−) cells were used as a positive control. #, p ≤ 0.035: vs . WT con ; *, p ≤ 0.035: vs . MUL1(−/−) con; ‡, p ≤ 0.045: AKT2(−/−) con vs. AKT2(−/−)+DMOG. (B) Quantification of the glycolysis, glycolytic capacity, and glycolytic reserve obtained from three independent experiments. Data from three separate experiments are presented as means ± SEM. #, p ≤ 0.03: vs . WT con ; *, p ≤ 0.035: vs . MUL1(−/−) con; ‡, p ≤ 0.045: AKT2(−/−) con vs. AKT2(−/−)+DMOG. (C) The effect of HIF-1α activator, DMOG, on the expression levels of HIF-1α, GLUT1, Akt2, P-GSK-3β S9, in Akt2(−/−), or MUL1(−/−) cells was monitored by western blot analysis. (D) Densitometrical analysis of the proteins shown in panel (C), normalized against β-actin. Results shown as means ± SD of three independent experiments. *, p ≤ 0.03 vs . WT con; #, p ≤ 0.025 vs . Akt2(−/−) con. (E) Glutamate 13 C signal ratio obtained from 13 C-NMR spectra of Akt2(−/−) and Akt2(−/−) +DMOG cells utilizing the [U- 13 C]glucose (note: S, D, T, and Q are singlet, doublet, triplet, and quartet, respectively. All signal ratios were calculated with respect to the total area of the corresponding glutamate resonance. Data are represented as mean ± SEM. (F) Metabolic flux rates calculated from the 13 C-isotopomers of glutamate observed in the 13 C-NMR spectra. Signal areas obtained from the results of the peak fitting procedure were used as an input to a metabolic model and solved numerically using tcaCALC. All flux rates are referenced to a citrate synthase (CS) flux of 1 and is equivalent to Kreb’s cycle flux. Statistical significance was p ≤ 0.05: *, for Akt2(−/−) vs. Akt2(−/−)+DMOG.

    Article Snippet: The tcaCALC program in MATLAB was utilized to perform an isotopomer analysis to estimate relative pathway fluxes ( ).

    Techniques: Positive Control, Expressing, Western Blot