Background and aims:
Mitochondria are dynamic organelles that frequently undergo fusion and fission. Mitochondrial dynamics has been shown to be essential for a variety of cellular functions and its abrogation has been associated with several diseases. However, the role fusion, fission and architecture play in mitochondrial bioenergetics is still not well understood. Brown adipose tissue (BAT) is a mitochondria dense organ that converts cellular fuels into heat by mitochondrial uncoupling. When activated adrenergically, BAT shows a unique increase in mitochondrial respiratory activity. Therefore, BAT may be a good model to study the interdependence between mitochondrial morphology, dynamics and function. To date, mitochondrial dynamics was not studied in BAT. In this study we set out to examine mitochondrial morphology in BAT and test the hypothesis that mitochondrial morphology is of importance for BAT function.
Materials and methods:
BAT was harvested from 3 to 4-week-old wild-type male C57BL6/J mice and from 5-8 day old pups (Mitofusin2 knockout). Brown adipocytes were differentiated in vitro. A LSM 710 laser scanning confocal microscopy (Zeiss) was used for imaging of mitochondrial morphology using several fluorescent dyes and proteins. Oxygen consumption was measured using the XF24 platform (Seahorse Bioscience). The pro fusion protein Mfn2 was knocked out under the AP2 promoter and the pro fission protein Drp1 was inhibited with adenoviral expression of its dominant negative form.
Mitochondria were found to be highly networked and dependent on mitochondrial dynamics proteins. When stimulating cells with a combination of norepinephrine and free fatty acids we found a synergistic response that included a marked increase in oxygen consumption rates and mitochondrial membrane potential (Δψm) depolarization. Somewhat unexpectedly it was also found that mitochondria in parallel underwent a distinct fragmentation. The fragmented mitochondria appeared sphere-like and had dampened fusion; however cells regained normal function as well as mitochondrial morphology and Δψm within 24h. Interestingly, Δψm depolarized and mitochondria fragmented in a wave-like fashion where depolarization preceded fragmentation. Inhibition of the pro-fission protein Drp1 was found to inhibit the synergistic response, while knock-out of the pro-fusion protein Mfn2 did not. Thus, mitochondrial fission appeared essential for proper BAT function. Finally, we found the synergistic response to go through the β-adrenergic pathway and be dependent on reactive oxygen species but not on Ca++, permeability transition pore or uncoupling protein 1 expression levels.
Taken together, these findings suggest that mitochondrial morphology in general and mitochondrial fission in particular may play an important physiological role in BA. Future studies will reveal if this may represent a therapeutic target for manipulating BAT activity.