Substantial Metabolic Activity Of Human Brown Adipose Tissue During Warm Conditions And Cold-Induced Lipolysis Of Local Triglycerides
Background and Goals
Due to the exponential growth in cardiovascular medical cases such as obesity, analyzing the way brown adipose tissue (BAT) works has become crucial. The reason behind its relevance is the ability this tissue has to convert excess food energy into thermal energy. This unique process could be extremely helpful when investigating new ways or techniques to confront these medical conditions. In adults, throughout our thoracic cavities, but especially in the supraclavicular area, we have certain depots where BAT is stored. This tissue is mainly focused on regulating thermogenesis by the glucose and triglycerides uptake which causes energy dissipation in the form of heat. The adipocytes that conform BAT contain multiple lipid droplets and an abundant number of mitochondrias, that are responsible to generate heat instead of ATP. It is a very irrigated tissue and, unlike white adipose tissue (WAT), it is innervated by the sympathetic nervous system (SNS).
What scientists are looking for with this article is to take a deep look at BAT’s metabolism during certain conditions, such as high and cold temperatures, that is their main goal. They are trying to understand, with the use of a new technique which we will later talk about, the substrate utilization in metabolic activity of this tissue by comparing it to WAT. The exposure to warm and cold conditions could trigger the activation of BAT and consequently, the release and uptake of not only exogenous but also endogenous substrates.
Methodology
To carry this study, they divided the conducted analysis in two: an in vitro study and in vivo study.
In vitro study and cell culture: They obtained a small quantity of BAT from the central compartment of the neck to the clavicle and in WAT’s case, the sample was obtained from the superficial subcutaneous neck. After that, the tissue was frozen for a posterior qPCR and a vascular fraction isolated for cell cultures. Afterwards, the previously mentioned isolated adipose tissue was digested using collagenase type 1 for 45 min at 37ºC. The product was centrifugated, at 600g for 10 min at 20ºC, and the pellet was passed through a 100µm filter. Once again, the resulting pellet was centrifugated at 200g for 5 min at 20ºC. Finally, the pellet was cultured in 6 petri plates at 37ºC. Every 2-3 days, the mediums were changed due to cell confluence. At the end, confluent cells were differentiated.
In vivo study: Researchers recruited six healthy men by using a specific criteria, such as ages between 18-35, a normal BMI, no medical conditions and no use of medication. The mentioned subjects had to follow a specific protocol including an overnight fast, an alcohol and exercise avoidance (48hr prior to the visit). This study was divided in 2 visits with two different goals. During the first visit, named the BAT finding visit, a cannula was introduced in the antecubital vein and a CT needle guide was inserted at the scapular area to help in the CT guided insertions. Next, the patients were placed in a cooled room (~17ºC) for 2h and right after an hour of exposure, they received an intravenous injection with glucose (FDG). Finally, a PET/CT scanning of the upper body was executed to make sure that the introduced levels of glucose (FDG) were enough to proceed with the use of microdialysis. Researchers measured the FDG uptake by BAT as the mean uptake from all the pixels by the volume of active BAT.
In the second visit, the named microdialysis visit, a cannula was placed in the antecubital vein and they received an intravenous infusion of glucose and glycerol. After the injection, a CT scan was made and following this, a microdialysis catheter was inserted into the supraclavicular BAT. Thanks to the utilization of a CT-guidance, they made sure that the catheter was correctly positioned. At this time, xenon (133) was injected in the supraclavicular BAT depot by CT-guidance to measure adipose tissue blood flow with a gamma counter.
Then, a second microdialysis catheter was placed in the subcutaneous abdominal WAT and just like before xenon (133) was injected at the same depth as the catheter. After this, an arterial catheter was inserted so that they could collect blood sampling and pressure measurements. At t=0 min, microdialysis was started in BAT and WAT depots and energy expenditure was measured each hour by indirect calorimetry, using a ventilated hood indirect calorimeter, as well as the skin temperature. At t=180 min, subjects were moved into a cold room and the previous measurements were performed.
Achieved Results
After all six healthy men went through a FDG-PET/CT scanning, scientists analyzed the results obtained and classified them depending on temperature conditions. All results are based on the comparison between white and brown adipose tissue’s endogenous substrate utilization. Previously, they had identified and quantified FDG uptake by BAT to ensure they could proceed with the study. BAT’s activity was measured by using microdialysis. In warm conditions (~25ºC), researchers found that glucose and glycerol concentrations in BAT were lower than in WAT. Regarding the glucose levels, this could be because of a glucose uptake by BAT even in warm conditions not associated with thermogenesis. On the other hand, concerning about the glycerol levels, this might be explained because of a reduced lipolysis or glycerol’s recycling, which will later be explained. However, glutamate concentrations were higher in BAT due to a reduced consumption. In cold conditions (~17ºC), it was discovered that glucose concentration, only in BAT, decreased even more during this temperature condition.
Meanwhile, glycerol was not affected by this temperature change since its concentration remained almost the same. Similarly, glutamate concentration was reduced exclusively in BAT. The reduction in BAT glucose concentrations was associated with cold-induced thermogenesis. Results showed that the whole body energy expenditure was very increased under cold temperatures.
Innovations
One of the researchers greatest accomplishments in this study was introducing an adapted microdialysis technique to examine human BAT and WAT’s in vivo physiology in real time measurements. This interesting procedure consists in inserting a catheter with a semi-permeable membrane into the tissue we want to study. Then, an isotonic perfusate, that in this case corresponds to a mix of: glucose, pyruvate, lactate and glycerol, is slowly infused through the cannula to allow these to equilibrate with the interstitial concentrations.
Finally, the dialysate is collected to measure the previous compounds. In this case, two microdialysis catheters were placed into supraclavicular BAT and abdominal subcutaneous WAT. The combination of the arterio-interstitial measure, the real time measurement of adipose tissue blood flow and the glucose and glycerol tracer infusion (inserted into a radial artery with another catheter) was the key to quantify the substrate utilization and the release of metabolites in both tissues during temperature changes.
Conclusions
It is extremely important and necessary to understand how brown adipose tissue works, so it can offer the chance to treat metabolic diseases, such as obesity. The more we investigate and research about this extraordinary tissue, the more successful results we will achieve. Once researches collected all results from their studies, they ended up with very interesting conclusions about BAT’s metabolism depending on temperature conditions. The main discovery was that human BAT could be activated even in warm circumstances and how this process is not necessarily associated with thermogenesis. In glucose’s case, during 25ºC, there was a significant uptake in BAT but not WAT. Similarly, in cold conditions, out of all the substrates studied glucose was the primary one taken up by BAT. To finish up with glucose, scientists suggest that the majority of glucose uptake is not fully oxidized during BAT thermogenesis. Focusing on pyruvate now, they observed that, as expected, the release of this substrate was considerable due to the high glycolysis rates in BAT during warm conditions.
After analyzing these results, they proposed that, in brown adipose tissue, glucose may be used for glyceroneogenesis or fatty acids synthesis so that way when activated by cold, there are proper triglyceride stores. Another key point authors really investigate on is glycerol’s possible recycling. In WAT, this said recycling process does not occur to the fact that there is no glycerol kinase. However, after some tests, they found a significant higher expression of this enzyme in BAT.
Finally, the use of microdialysis allowed researchers to measure the activity of multiple endogenous analytes. A great advantage of this technique is the possible real time interventions to locally deliver drugs and determine the effect on BAT’s function.
