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Acta Physico-Chimica Sinca  2018, Vol. 34 Issue (1): 73-80    DOI: 10.3866/PKU.WHXB201707043
ARTICLE     
Microcalorimetric Analysis of Isolated Rat Liver Mitochondrial Metabolism under Different Conditions
Lian YUAN1,Yu-Jiao LIU1,Huan HE1,Feng-Lei JIANG1,Hui-Rong LI2,Yi LIU1,*()
1 College of Chemistry and Molecular Sciences, Wuhan University, Wuhan 430072, P. R. China
2 College of Life Science and Chemistry, Wuhan Donghu University, Wuhan 430212, P. R. China
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Abstract  

Isolated rat liver mitochondria were proposed as a model to monitor real-time heat metabolism.A high-throughput and sensitive thermal activity monitor Ⅲ (TAM Ⅲ) was used to detect the P-t curves of mitochondria under different conditions, including different mitochondrial concentrations, different substrates, different buffers, respiratory inhibitors, Ca2+, and CsA.We determined the thermokinetic parameters through calculation.The results showed that:(1) higher concentration of mitochondria led to faster energetic metabolism; (2) when succinate was the direct respiratory substrate, it promoted mitochondrial metabolism, in contrast to the condition when an indirect substrate, pyruvate, was used; (3) high concentration of Ca2+(2.5 mmol·L-1) stimulated mitochondrial metabolism, however CsA, an inhibitor of mitochondrial permeability transition pores, could not reverse the Ca2+-induced mitochondrial alteration; (4) mitochondria in various buffers displayed different rates of heat metabolism, because of the different composition of the buffers; (5) mitochondrial metabolism was inhibited by respiratory inhibitors, especially NaN3, which is an inhibitor of Complex Ⅳ and which completely stopped the mitochondrial heat release.



Key wordsMitochondria      Microcalorimetry      Metabolic rate      Respiratory inhibitor      Mitochondrial permeability transition     
Received: 13 June 2017      Published: 04 July 2017
MSC2000:  O642.3  
Fund:  the National Natural Science Foundation of China(21673166);Natural Science Foundation of Hubei Province, China(2015CFC892)
Corresponding Authors: Yi LIU     E-mail: yiliuchem@whu.edu.cn
Cite this article:

Lian YUAN,Yu-Jiao LIU,Huan HE,Feng-Lei JIANG,Hui-Rong LI,Yi LIU. Microcalorimetric Analysis of Isolated Rat Liver Mitochondrial Metabolism under Different Conditions. Acta Physico-Chimica Sinca, 2018, 34(1): 73-80.

URL:

http://www.whxb.pku.edu.cn/10.3866/PKU.WHXB201707043     OR     http://www.whxb.pku.edu.cn/Y2018/V34/I1/73

BufferComponents
Buffer A220 mmol·L-1 mannitol, 70 mmol·L-1 sucrose, 20 mmol·L-1 HEPES, 2 mmol·L-1 Tris-HCl, 1 mmol·L-1 EDTA, pH 7.4
Buffer B220 mmol·L-1 mannitol, 70 mmol·L-1 sucrose, 5 mmol·L-1 Tris-HCl, 1 mmol·L-1 EDTA, pH 7.4
Media C220 mmol·L-1 mannitol, 70 mmol·L-1 sucrose, 1 mmol·L-1 EDTA, pH 7.4
MRB100 mmol·L-1 sucrose, 10 mmol·L-1 Tris, 10 mmol·L-1 MOPS, 2 mmol·L-1 MgCl2,
10 mmol·L-1 KH2PO4, 50 mmol·L-1 KCl, 10 mmol·L-1 EDTA, 2 mmol·L-1 rotenone, pH 7.4
MAB200 mmol·L-1 sucrose, 10 mmol·L-1 Tris, 10 mmol·L-1 MOPS, 1 mmol·L-1 Na3PO4,
5 mmol·L-1 succinate, 10 mmol·L-1 EGTA, 3 mg·mL-1 oligomycin, 2 mmol·L-1 rotenone, pH 7.4
Buffer H135 mmol·L-1 KAc, 5 mmol·L-1 HEPES, 10 mmol·L-1 EGTA, 10 mmol·L-1 EDTA,
2 mmol·L-1 rotenone, 1 mg·mL-1 valinomycin, pH 7.1
Buffer K135 mmol·L-1 KNO3, 5 mmol·L-1 HEPES, 10 mmol·L-1 EGTA, 10 mmol·L-1 EDTA, 2 mmol·L-1 rotenone, pH 7.1
Table 1 The components of 7 buffers
Fig 1 Thermogenic curves of mitochondria with 15 mmol?L?1 of succinate as respiratory substrate. The concentrations of mitochondria were 7.0 mg protein?mL?1 and 3.5 mg protein?mL?1.
SamplesQ/Jt1/hP1/μWt2/hP2/μWk1/h-1R12k2/h-1R22k3/h-1R32
a200 mito + 15 mmol?L-1 succinate11.4328.83126.8837.37231.790.2560.99880.0990.9982-0.8440.9874
100 mito + 15 mmol?L-1 succinate11.0729.2165.1748.28144.270.2380.99890.0510.9800-0.7060.9796
b200 mito12.1121.9966.6359.2584.520.3150.98880.0120.9958-0.6720.9936
200 mito + 15 mmol?L-1 succinate11.4328.83126.8837.37231.790.2560.99880.0990.9982-0.8440.9874
200 mito + 15 mmol?L-1pyruvate11.0221.0053.9144.25158.140.3490.99890.0700.9988-0.6700.9897
ccontrol (succinate)10.4321.22127.3631.13201.950.5100.99610.0500.9952-1.3410.9635
0.1 mmol?L-1 Ca2+9.8320.83110.1430.21208.210.1960.99890.0740.9932-1.9570.9501
2.5 mmol?L-1 Ca2+16.1519.39417.280.7420.9984-0.3210.9991
10 μmol?L-1 CsA8.8620.45115.2329.56163.410.87240.99020.0540.9928-1.4670.9428
CsA + 0.1 mmol?L-1 Ca2+9.8919.79134.2728.78189.860.4520.99360.0520.9956-1.9020.9506
CsA + 2.5 mmol?L-1 Ca2+16.5019.01423.740.8030.9988-0.2930.9994
dMRB12.4722.3163.6748.28173.680.3330.99450.0470.9950-0.9810.9801
MAB13.5418.3769.1847.19218.420.2440.97900.0720.9854-0.4780.9866
H0.00--
K10.8627.3341.6476.6777.780.3250.99300.0440.9956-0.6070.9733
C11.9924.0569.1857.45100.570.3530.99730.0260.9954-0.6760.9884
PBS11.5226.2457.4167.06111.580.3010.99570.0350.9939-0.9250.9142
econtrol10.8333.34235.000.4360.9953-0.8800.9675
0.03 mmol?L-1 NaN310.9735.26244.110.2490.9943-0.7560.9945
0.3 mmol?L-1 NaN30.00--
3 mmol?L-1 NaN30.00--
40 μmol·L?1 rotenone9.8734.53264.640.2100.9896?0.9070.9964
8 mmol·L?1 malonate10.4737.27198.6622.6370.350.5160.96760.0870.9987?1.0720.9917
Table 2 Thermokinetic parameters of mitochondrial metabolism in different conditions.
Fig 2 Thermogenic curves of mitochondria with different substrates.
Fig 3 Thermogenic curves of mitochondria with Ca2+ and CsA.
Fig 4 Thermogenic curves of mitochondria with different buffers.
Fig 5 Thermogenic curves of mitochondria with several respiration inhibitors (rotenone, malonate and NaN3).
1 Said J. ; Walker M. ; Parsons D. ; Stapleton P. ; Beezer A. E. ; Gaisford S. Methods 2015, 76, 32.
2 Tsenga J. M. ; Shu C. M. Thermochim. Acta 2010, 507, 45.
3 Haman N. ; Romano A. ; Asaduzzaman M. ; Ferrentino G. ; Biasioli F. ; Scampicchio M. Talanta 2017, 164 (1), 407.
4 Liu S. H. ; Hou H.Y. ; Shu C. M. Thermochim. Acta 2015, 605 (10), 68.
5 Smith R. A. J. ; Hartley R. C. ; Cochemé ; H. M. ; Murphy M. P. Trends Pharmacol. Sci. 2012, 33 (6), 341.
6 Murphy M. P. ; Brand M. D. Eur. J. Biochem. 1988, 173 (3), 637.
7 Stock D. ; Leslie A. G. ; Walker J. E. Science 1999, 286, 1700.
8 Stepien G. ; Torroni A. ; Chung A. B. ; Hodge J. A. ; Wallace D. C. J. Biol. Chem. 1992, 267 (21), 14592.
9 Schaefer A. M. ; Taylor R. W. ; Turnbull D. M. ; Chinnery P. F. Biochim. Biophys. Acta 2004, 1659, 115.
10 Liu X. R. ; Beugelsdijk A. ; Chen J. Biophys. J. 2015, 109 (5), 1049.
11 Zulian A. ; Schiavone M. ; Giorgio V. ; Bernardi P. Pharmacol. Res. 2016, 113 (Pt A), 563.
12 Neuzil J. ; Dong L. F. ; Rohlena J. ; Truksa J. ; Ralph S. J. Mitochondrion 2013, 13 (3), 199.
13 Shiau C. W. ; Huang J. W. ; Wang D. S. ; Weng J. R. ; Yang C. C. ; Lin C. H. ; Li C. ; Chen C. S. J. Biol. Chem. 2006, 281 (17), 11819.
14 Fantin V. R. ; Berardi M. J. ; Scorrano L. ; Korsmeyer S. J. ; Leder P. Cancer Cell 2002, 2 (1), 29.
15 He H. ; Li D. W. ; Yang L. Y. ; Fu L. ; Zhu X. J. ; Wong W. K. ; Jiang F. L. ; Liu Y. Sci. Rep. 2015, 5, 13543.
16 Wang J. ; Fan X. Y. ; Yang L. Y. ; He H. ; Huang R. ; Jiang F. L. ; Liu Y. Med. Chem. Comm. 2016, 7 (10), 2016.
17 Yuan L. ; Gao T. ; He H. ; Jiang F. L. ; Liu Y. Toxicol. Res. 2017.
18 Monteiro J. P. ; Oliveira P. J. ; Moreno A. J. ; Jurado A. S. Chemosphere 2008, 72 (9), 1347.
19 Gornall A. G. ; Bardawill C. J. ; David M. M. J. Biol. Chem. 1949, 177 (2), 751.
20 Hall D. O. ; Hawkins S. E. aboratory Manual of Cell Biology English University Press: London, 1975, Chap.2.
21 Mannella C. A. J. Bioenerg. Biomembr. 2000, 32 (1), 1.
22 Nelson, D. L.; Cox, M. M. Lehninger's Principle of Biochemisotry, 5th ed.; Worth Publisher: INC., 2008; p. 523.
23 Baines C. P. J. Mol. Cell Cardiol. 2009, 46 (6), 850.
24 Halestrap A. P. Biochem. Soc. Trans. 2010, 38 (4), 841.
25 Zhivotovsky B. ; Galluzzi L. ; Kepp O. ; Kroemer G. Cell Death Differ. 2009, 16 (11), 1419.
26 Hoppe U. C. FEBS Letters 2010, 584 (10), 1975.
27 Zoratti M. ; Szabò ; I . ; De Marchi U. Biochim. Biophys. Acta 2005, 1706 (1–2), 40.
28 Fernandes M. A. ; Custó ; dio J. B. ; Santos M. S. ; Moreno A. J. ; Vicente J. A. Mitochondrion 2006, 6 (4), 176.
29 Vicente J. A. ; Santos M. S. ; Vercesi A. E. ; Madeira V. M. C. Pestic. Sci. 1998, 54, 43.
30 Yarana C. ; Sripetchwandee J. ; Sanit J. ; Chattipakorn S. ; Chattipakorn N. Arch. Med. Res. 2012, 43 (5), 333.
31 Corbet C. ; Feron O. Biochim. Biophys. Acta 2017, 1868 (1), 7.
32 Li N. ; Ragheb K. ; Lawler G. ; Sturgis J. ; Rajwa B. ; Melendez J. A. ; Robinson J. P. J. Biol. Chem. 2003, 278 (10), 8516.
33 Stobiecka M. ; Jakiela S. ; Chalupa A. ; Bednarczyk P. ; Dworakowska B. Biosens. Bioelectron. 2017, 88, 114.
34 Giannattasio S. ; Atlante A. ; Antonacci L. ; Guaragnella N. ; Lattanzio P. ; Passarella S. ; Marra E. FEBS Letters 2008, 582 (10), 1519.
35 Tsesin N. ; Khalfin B. ; Nathan I. ; Parola A. H. Chem. Phys. Lipids 2014, 183, 159.
36 Lykhmus O. ; Gergalova G. ; Koval L. ; Zhmak M. ; Komisarenko S. ; Skok M. Int. J. Biochem. Cell Biol. 2014, 53, 246.
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