Open for submissionsPeer reviewed, fully open access, and a low article processing charge.Submit manuscript

Research ArticleOpen AccessGoogle Scholar indexed

A Statistical Evaluation of Uncoupling Protein 1 in the Limited Area of Brown Adipose Tissue by Immunoelectron Microscopy

,
,
,
  1. 1 Department of Anatomy, Faculty of Medicine, Oita University, Oita, Japan
  2. 2 Emeritus Professor, Faculty of Medicine, Oita University, Oita, Japan
Computational Chemistry·Volume 10, Issue 3·Pages 121–137·Published 15 August 2022·DOI10.4236/cc.2022.103006
Download PDF

Abstract

Uncoupling protein 1 (UCP1) expressed by the brown adipose tissue (BAT) in the mitochondrial crista acts as a homeostatic thermogenerator of eutherians. The evaluation of UCP1 expression in the BAT offers significant scientific insight, especially in studies targeting limited areas such as the periarterial and pericardial regions of small experimental mammals. However, the negligible amount of this adipose tissue would render the general quantitative evaluation of the protein unreliable because of lipid contamination and low protein concentration. To address this problem, we quantitatively evaluated UCP1 expression in the mitochondrion of the mouse interscapular BAT using immunoelectron microscopy and immunohistochemical studies using a combination of primary and secondary antibodies in scheme A (rabbit anti-UCP1 IgG/gold particle-conjugated goat anti-rabbit IgG), B (rabbit IgG/gold particle-conjugated goat anti-rabbit IgG), C (rabbit anti-UCP1 IgG/gold particle-unconjugated goat anti-rabbit IgG), and D (rabbit IgG/gold particle-unconjugated goat anti-rabbit IgG). Scheme A shows the immunopositive reaction of obvious gold particles in the mitochondrial area, whereas other procedures revealed less distinctive reactions. The distinctive gold particle immunoreaction comprised electrical high-density spots with a mean diameter of >5 nm. However, in scheme B, the electrical high-density spots were scattered outside the mitochondrion and were significantly smaller than 4 nm; schemes C and D demonstrated few immunoreactions. Logistic regression analysis between schemes A and B showed that the threshold diameter of the electrical high-density spots measuring >5 nm indicated a true positive immunoreaction to anti-UCP1 antibody specifically in the mitochondrial area. Minor statistical difference was observed in the primary anti-UCP1 antibody between polyclonal IgG and monoclonal antibodies. Therefore, immunoelectron microscopy might be useful for evaluating negligible protein expression in some limited areas, such as UCP1 expression in the BAT of small experimental animals.

KeywordsUncoupling Protein 1 (UCP1)Brown AdiposeImmunoelectron MicroscopyImmunohistochemical StainingLogistic Regression Analysis
  1. Shinde, A.B., Song, A. and Wang, Q.A. (2021) Brown Adipose Tissue Heterogeneity, Energy Metabolism, and Beyond. Frontiers in Endocrinology (Lausanne), 12, Article ID: 651763. https://doi.org/10.3389/fendo.2021.651763
  2. Acin-Perez, R., Petcherski, A., Veliova, M., Benador, I.Y., Assali, E.A., Colleluori, G., Cinti, S., Brownstein, A.J., Baghdasarian, S., Livhits, M.J., Yeh, M.W., Krishnan, K.C., Vergnes, L., Winn, N.C., Padilla, J., Liesa, M., Sacks, H.S. and Shirihai, O.S. (2021) Recruitment and Remodeling of Peridroplet Mitochondria in Human Adipose Tissue. Redox Biology, 46, Article ID: 102087. https://doi.org/10.1016/j.redox.2021.102087
  3. Ruan, H.B. (2020) Developmental and Functional Heterogeneity of Thermogenic Adipose Tissue. Journal of Molecular Cell Biology, 12, 775-784. https://doi.org/10.1093/jmcb/mjaa029
  4. Wei, H., Chiba, S., Moriwaki, C., Kitamura, H., Ina, K., Aosa, T., Tomonari, K., Gotoh, K., Masaki, T., Katsuragi, I., Noguchi, H., Kakuma, T., Hamaguchi, K., Shimada, T., Fujikura, Y. and Shibata, H. (2015) A Clinical Approach to Brown Adipose Tissue in the Para-Aortic Area of the Human Thorax. PLOS ONE, 10, e0122594. https://doi.org/10.1371/journal.pone.0122594
  5. Chouchani, E.T., Kazak, L., Jedrychowski, M.P., Lu, G.Z., Erickson, B.K., Szpyt, J., Pierce, K.A., Laznik-Bogoslavski, D., Vetrivelan, R., Clish, C.B., Robinson, A.J., Gygi, S.P. and Spiegelman, B.M. (2016) Mitochondrial ROS Regulate Thermogenic Energy Expenditure and Sulfenylation of UCP1. Nature, 532, 112-116. https://doi.org/10.1038/nature17399
  6. Pinckard, K.M. and Stanford, K.I. (2022) The Heartwarming Effect of Brown Adipose Tissue. Molecular Pharmacology, 102, 460-471. https://doi.org/10.1124/molpharm.121.000328
  7. Hou, D., Fu, H., Zheng, Y., Lu, D., Ma, Y., Yin, Y., Zhang, L. and Bao, D. (2022) Uncoupling Protein 1 Knockout Aggravates Isoproterenol-Induced Acute Myocardial Ischemia via AMPK/mTOR/PPARalpha Pathways in Rats. Transgenic Research, 31, 107-118. https://doi.org/10.1007/s11248-021-00289-0
  8. Moreno-Santos, I., Macias-Gonzalez, M., Porras-Martin, C., Castellano-Castillo, D., Sanchez-Espin, G., Gomez-Doblas, J.J., de Teresa-Galvan, E. and Jimenez-Navarro, M. (2019) Role of Epicardial Adipose Tissue NPR-C in Acute Coronary Syndrome. Atherosclerosis, 286, 79-87. https://doi.org/10.1016/j.atherosclerosis.2019.05.010
  9. Kortelainen, M.L., Pelletier, G., Ricquier, D. and Bukowiecki, L.J. (1993) Immunohistochemical Detection of Human Brown Adipose Tissue Uncoupling Protein in an Autopsy Series. Journal of Histochemistry & Cytochemistry, 41, 759-764. https://doi.org/10.1177/41.5.8468458
  10. An, Y.A. and Scherer, P.E. (2020) Mouse Adipose Tissue Protein Extraction. Bio-Protocol, 10, e3631. https://doi.org/10.21769/BioProtoc.3631
  11. Diaz Marin, R., Crespo-Garcia, S., Wilson, A.M. and Sapieha, P. (2019) RELi Protocol: Optimization for Protein Extraction from White, Brown and Beige Adipose Tissues. MethodsX, 6, 918-928. https://doi.org/10.1016/j.mex.2019.04.010
  12. Cinti, S., Cancello, R., Zingaretti, M.C., Ceresi, E., De Matteis, R., Giordano, A., Himms-Hagen, J. and Ricquier, D. (2002) CL316,243 and Cold Stress Induce Heterogeneous Expression of UCP1 mRNA and Protein in Rodent Brown Adipocytes. Journal of Histochemistry & Cytochemistry, 50, 21-31. https://doi.org/10.1177/002215540205000103
  13. Cilingir-Kaya, O.T., Moore, C., Meshul, C.K., Gursoy, D., Onat, F. and Sirvanci, S. (2021) Neurogenesis Is Enhanced in Young Rats with Genetic Absence Epilepsy: An Immuno-Electron Microscopic Study. Turkish Neurosurgery, 31, 623-633. https://doi.org/10.5137/1019-5149.JTN.31996-20.2
  14. Alfaro-Ruiz, R., Aguado, C., Martin-Belmonte, A., Moreno-Martinez, A.E. and Lujan, R. (2020) Cellular and Subcellular Localisation of Kv4-Associated KChIP Proteins in the Rat Cerebellum. International Journal of Molecular Sciences, 21, Article 6403. https://doi.org/10.3390/ijms21176403
  15. Martin-Belmonte, A., Aguado, C., Alfaro-Ruiz, R., Albasanz, J. L., Martin, M., Moreno-Martinez, A.E., Fukazawa, Y. and Lujan, R. (2021) The Density of Group I mGlu5 Receptors Is Reduced along the Neuronal Surface of Hippocampal Cells in a Mouse Model of Alzheimer’s Disease. International Journal of Molecular Sciences, 22, Article 5867. https://doi.org/10.3390/ijms22115867
  16. Yang, F., Yang, L., Teng, L., Zhang, H. and Katayama, I. (2021) Morphological Alterations and Increased S100B Expression in Epidermal Langerhans Cells Detected in Skin from Patients with Progressive Vitiligo. Life (Basel), 11, Article 579. https://doi.org/10.3390/life11060579
  17. Zhou, M., Tanaka, O., Sekiguchi, M., He, H.J., Yasuoka, Y., Itoh, H., Kawahara, K. and Abe, H. (2005) ATP-Sensitive K+-Channel Subunits on the Mitochondria and Endoplasmic Reticulum of Rat Cardiomyocytes. Journal of Histochemistry & Cytochemistry, 53, 1491-1500. https://doi.org/10.1369/jhc.5A6736.2005
  18. Karnovsky, M.J. (1961) Simple Methods for “Staining with Lead” at High pH in Electron Microscopy. The Journal of Biophysical and Biochemical Cytology, 11, 729-732. https://doi.org/10.1083/jcb.11.3.729
  19. Nagai, K., Noguchi, T., Fujiwara, S., Kawahara, K. and Shimada, T. (2005) Distribution of Langerhans Cells in the Human Esophagus, as Revealed by Immunohistochemistry. Acta Histochemica et Cytochemica, 38, 115-119. https://doi.org/10.1267/ahc.38.115
  20. Reynes, B., Garcia-Ruiz, E., Oliver, P. and Palou, A. (2015) Gene Expression of Peripheral Blood Mononuclear Cells Is Affected by Cold Exposure. The American Journal of Physiology-Regulatory, Integrative and Comparative Physiology, 309, R824-S834. https://doi.org/10.1152/ajpregu.00221.2015
  21. Golic, I., Kalezic, A., Jankovic, A., Jonic, S., Korac, B. and Korac, A. (2020) Insulin Modulates the Bioenergetic and Thermogenic Capacity of Rat Brown Adipocytes in Vivo by Modulating Mitochondrial Mosaicism. International Journal of Molecular Sciences, 21, Article 9204. https://doi.org/10.3390/ijms21239204
  22. Nagai, K., Noguchi, T., Fujiwara, S., Kawahara, K. and Shimada, T. (2005) Distribution of Langerhans Cells in the Human Esophagus, as Revealed by Immunohistochemistry. Acta Histochemica et Cytochemica, 38, 115-119. https://doi.org/10.1267/ahc.38.115
  23. Karnovsky, M.J. (1961) Simple Methods for “Staining with Lead” at High pH in Electron Microscopy. The Journal of Biophysical and Biochemical Cytology, 11, 729-732. https://doi.org/10.1083/jcb.11.3.729
  24. Ina, K., Kitamura, H., Tatsukawa, S. and Fujikura, Y. (2011) Significance of Alpha-SMA in Myofibroblasts Emerging in Renal Tubulointerstitial Fibrosis. Histology & Histopathology, 26, 855-866.