Estrogen- and Satiety State-Dependent Metabolic Lateralization in the Hypothalamus of Female Rats

Hypothalamus is the highest center and the main crossroad of numerous homeostatic regulatory pathways including reproduction and energy metabolism. Previous reports indicate that some of these functions may be driven by the synchronized but distinct functioning of the left and right hypothalamic sides. However, the nature of interplay between the hemispheres with regard to distinct hypothalamic functions is still unclear. Here we investigated the metabolic asymmetry between the left and right hypothalamic sides of ovariectomized female rats by measuring mitochondrial respiration rates, a parameter that reflects the intensity of cell and tissue metabolism. Ovariectomized (saline injected) and ovariectomized+estrogen injected animals were fed ad libitum or fasted to determine 1) the contribution of estrogen to metabolic asymmetry of hypothalamus; and 2) whether the hypothalamic asymmetry is modulated by the satiety state. Results show that estrogen-priming significantly increased both the proportion of animals with detected hypothalamic lateralization and the degree of metabolic difference between the hypothalamic sides causing a right-sided dominance during state 3 mitochondrial respiration (St3) in ad libitum fed animals. After 24 hours of fasting, lateralization in St3 values was clearly maintained; however, instead of the observed right-sided dominance that was detected in ad libitum fed animals here appeared in form of either right- or left-sidedness. In conclusion, our results revealed estrogen- and satiety state-dependent metabolic differences between the two hypothalamic hemispheres in female rats showing that the hypothalamic hemispheres drive the reproductive and satiety state related functions in an asymmetric manner.


II. Buffer solutions
To set pH 7.2 use the 5M KOH and the 37% HCl solutions. These buffers should be stored at 4°C for not more than a couple days. ADP, malate and pyruvate should be fresh made on the day of the experiment while FCCP and oligomycin can be made up two several months and stored at -20 °C before use.

Dissection and tissue homogenization
The experimental design is also summed up on Fig. 1.
After quick guillotine decapitation, the hypothalamus was extracted in ice-cold environment as follows. After removing the skin and muscles, the skull was opened as described earlier [1], and the cranial part of the brain was slightly lifted with a cold spatula in order to cut the optic nerve. After cutting, the brain was gently removed, and placed into an ice-cold brain matrix. The connecting tissue from the basal part of the hypothalamus was removed with a fine forceps. Then, vertical incisions were made using ice-cold blades, for a coronal section of the entire hypothalamus: an incision right behind the rostral part of the chiasma opticum (Bregma -0.25), and an other one through the corpus mamillare (Bregma -5.0). The coronal sections were placed on the rostral surface, and the piriform and entorhinal cortex, then the thalamic area dorsal of the fornices were cut off. Finally, the hypothalamus was cut into left and right sides along the 3 rd ventricle. The tissue blocks (30-35mg) were put into 750μl ice-cold isolation buffer, and stored until homogenization. Dissected brain samples were placed and further processed in icecold buffer starting from approximately 30 seconds after the decapitation.
The homogenization was performed in a motorized teflon-on-glass tissue homogenizer (Potter-Elvehjem, 600-800rpm) by moving the glass tube firmly up and down. After the homogenization, all buffer and foam was recollected and put into a 1.5ml Eppendorf tube. The homogenate was kept on ice until all other tissue samples were homogenized. Between to samples, homogenizer was cleaned with isolation buffer.

Fractionation procedure
All fractionation steps were carried out at 4ºC. A summary of the procedure is shown on Fig.  2 for better understanding.

Preparing crude mitochondrial fraction from brain tissue
Homogenized samples were spun at 1300rcf (3700rpm) for 4 minutes. The supernatant was collected in an empty Eppendorf tube, while the pellet was resuspended in 750µl isolation buffer (with EGTA), then it was spun again with the same settings in order to release mitochondria from large cell debris. After the second spin, the supernatant was put together with the former supernatant collected from the first centrifugation step, and the pellet was discarded. The next step, the two supernatants collected in one tube are spun together at 13000rcf (11800rpm) for 11 minutes. The mitochondria containing pellet was saved, and resuspended in 500µl isolation buffer. This stage is called "crude mitochondrial fraction" that still contains contaminating particles (cell organelles, myelin, cell debris, etc).

Percoll gradient fractionation procedure
For further purification, we used a simplified discontinuous Percoll gradient that merely consists of a 15% and a 0% Percoll layer (filtered Percoll stock solution is diluted to 15% with isolation buffer). Using a gradient centrifugation step, mitochondria and synaptosomes were separated from other, non-useful elements; on the other hand, easy enough to prepare even in a small-sized Eppendorf tube. The crude mitochondrial fraction was layered on 500µl of 15% Percoll solution in a special "Percoll tube" (2ml, conical shape). The Percoll gradient containing tubes were gently put into the centrifuge and spun at 22000rcf (15400rpm) for 7 minutes 40 seconds. In order to save the layers during the centrifugation, we used the lowest possible acceleration and the break was turned off. The two layers at the bottom (somal and synaptosomal mitochondria) were collected before the last steps by a fine pipette, while the top layer (cell membrane and myelin debris) was discarded.
Percoll, although considered as a harmless compound, has to be cleared off the sample. In order to obtain uninjured, coupled, viable mitochondria, the following cleaning steps were used before the final utilization: entirely filled tubes (of the resuspended sample) were spun at 22000rcf (15400rpm) for 11 minutes (full acceleration and break). After the centrifugation, the supernatant was carefully poured off. As the last step, the remaining, minimal amount of Percoll and the EGTA was removed by diluting it with 1ml of isolation buffer without EGTA. The tubes were centrifuged at 13000rcf (11800rpm) for 11 minutes, and the samples were stored as pellet in isolation buffer (without EGTA) on 4°C until the measurements.

Mitochondrial respiration rate measurements
Mitochondria containing fraction was transferred into respiration buffer and put into a Clark-type oxygen electrode chamber (Hansatech Instruments, Norfolk, UK) to measure their activity at 37°C. The electrode groove was filled with potassium chloride for establishing the electrode bridge between cathode and anode. Calibration was fulfilled by air saturated, deionized distilled water in order to establish the air line, while sodium dithionite for zero oxygen line. Using the protocol described by Toth et al [2], we measured the oxygen consumption by consecutively adding 5µl pyruvate together with 2.5µl malate, 2.5µl ADP, 1µl oligomycin and 2.5µl carbonylcyanide-4-(trifluoromethoxy)-phenylhydrazone to 50 µl of resuspended samples diluted with 450 µl respiration buffer in the electrode chamber.
The oxygen consumption was measured real time, and the results are expressed as consumed oxygen per minute (nmol O2/ml). Five stages (each measured for 60 seconds) were distinguished according to the subsequently added respiration modifiers [3]. therefore, by binding to and blocking cytochrome C oxidase, depletes all remaining oxygen from the sample (also acts as uncoupler, [6,7]). Decrease of oxygen level under such conditions depends on the initial (in vivo) metabolic state of the sampled tissue, and the amount of oxygen consumed during states 1-4 respiration. Therefore, this experimental setup is also known as total mitochondrial respiratory capacity.