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Mitochondria are key organelles maintaining cellular bioenergetics and integrity, and their regulation of {[}Ca2+](i) homeostasis has been investigated in many cell types. We investigated the short-term Ca-SANDOZ (R) treatment on brown adipocyte mitochondria, using imaging and molecular biology techniques. Two-month-old male Wistar rats were divided into two groups: Ca-SANDOZ (R) drinking or tap water (control) drinking for three days. Alizarin Red S staining showed increased Ca2+ level in the brown adipocytes of treated rats, and potassium pyroantimonate staining localized electron-dense regions in the cytoplasm, mitochondria and around lipid droplets. Ca-SANDOZ (R) decreased mitochondrial number, but increased their size and mitochondrial cristae volume. Transmission electron microscopy revealed numerous enlarged and fusioned-like mitochondria in the Ca-SANDOZ (R) treated group compared to the control, and megamitochondria in some brown adipocytes. The Ca2+ diet affected mitochondrial fusion as mitofusin 1 (MFN1) and mitofusin 2 (MFN2) were increased, and mitochondrial fission as dynamin related protein 1 (DRP1) was decreased. Confocal microscopy showed a higher colocalization rate between functional mitochondria and endoplasmic reticulum (ER). The level of uncoupling protein-1 (UCP1) was elevated, which was confirmed by immunohistochemistry and Western blot analysis. These results suggest that Ca-SANDOZ (R) stimulates mitochondrial fusion, increases mitochondrial-ER contacts and the thermogenic capacity of brown adipocytes. Serbian Ministry of Education, Science and Technological Development {[}173055, 173054]

The aim of the present study was to investigate whether hyperinsulinaemia, which frequently precedes insulin resistance syndrome (obesity, diabetes), induces apoptosis of endothelial cells (ECs) in brown adipose tissue (BAT) and causes BAT atrophy and also, to investigate the possible mechanisms underlying ECs death. In order to induce hyperinsulinaemia, adult male rats of Wistar strain were treated with high dose of insulin (4 U/kg, intraperitonealy) for one or three days. Examinations at ultrastructural level showed apoptotic changes of ECs, allowing us to point out that changes mainly but not exclusively, occur in nuclei. Besides different stages of condensation and alterations of the chromatin, nuclear fragmentation was also observed. Higher number of ECs apoptotic nuclei in the BAT of hyperinsulinaemic rats was also confirmed by propidium iodide staining. Immunohistochemical localization of tumor necrosis factor-alpha (TNF-α) revealed increased expression in ECs of BAT of hyperinsulinaemic animals, indicating its possible role in insulin-induced apoptotic changes. These results suggest that BAT atrophy in hyperinsulinaemia is a result of endothelial and adipocyte apoptosis combined, rather than any of functional components alone.