Thermogenesis in brown adipose tissue

Watch the heat while your browning agent does the work! The calScreener ™ gives you the true measure of thermogenic capacity in heat (W, J/s)

Direct measurement of brown adipose tissue's thermogenic capacity

Searching for a true functional readout of total energy expenditure, label-free and in real-time in your quest for novel compounds with therapeutic potential in addressing the global epidemics of obesity and diabetes?

There is no better instrument to measure direct heat production and browning in adipose tissue than the calScreener™, the first cell biology adapted microcalorimeter.

Solid data from intact adipose tissue as well as from primary cultures of UCP1 (Uncoupling Protein 1) positive adipocytes clearly demonstrate the calScreener as the optimal and highly sensitive method for measuring stimulation of heat production and energy expenditure as a direct function of drug stimulation.

This opens up the field of developing drugs for obesity indications directly scored using energy expenditure as the readout and not proxy parameters or indirect measurements.

The epidemic of metabolic disorders

The epidemic of obesity and diabetes presents significant global health concerns. World-wide obesity has triplicated since 1975 with a total amount of 1.9 billion of adults being overweight. As such there is an urgent need for novel treatment strategies of metabolic disorders, but also for novel technologies to facilitate screening and drug discovery in the field.

The promise of Brown Adipocytes – heat expenditure

The need for novel and accurate screening assays has escalated since the emergence of Brown Adipose Tissue (BAT) as a potential therapeutic target in thanks to its fundamental role in maintaining body temperature and regulating energy homeostasis by producing heat. Brown adipose tissue provides a new therapeutic strategy, as its activity is negatively correlated with the human body mass index (BMI).

In addition to BAT, brown adipocytes residing within white adipose tissue, so called beige/brite adipocytes, have been demonstrated to take part in whole body metabolism. Importantly, multiple lines of evidence demonstrate that both brown adipose tissue and beige/brite adipocytes can reverse obesity as well as improve insulin sensitivity. Collectively, these findings have opened up for the new promising field of future clinical application in the recruitment and activation of beige/brite cells and brown adipose tissue to treat obesity and metabolic diseases.

Thermogenesis of brown adipose tissue and the discovery of ‘browning agents’

Brown adipose tissue import and combust triglycerides from the circulation delivering substrate for continued thermogenesis. In addition to brown adipose tissue being a lipid-combusting tissue, it is also major tissue for glucose disposal, where the glucose, just like lipids, will be combusted.

It is this thermogenic capacity of brown adipose tissue, and its avidity for glucose, that makes it a therapeutic target for inducing weight loss or improving blood sugar control. Advances in the area of brown adipose tissue have already led to the discovery of ‘browning’ agents, commonly defined as agents that increase the amount and activity of UCP1, and that stimulate brown adipogenesis or beige cell induction under certain conditions.

Isothermal microcalorimetry, the perfect measure of true thermogenesis

Until now the field has suffered from the lack of functional assays with a true energy expenditure readout. Conclusions are primarily reached by measuring basic molecular data, i.e. relative UCP1 mRNA and protein levels, expressed per unit of RNA or protein. By trusting only in molecular markers during standard browning studies, there is also a risk that that new browning agents may be overlooked.

The calScreener ™ is the only technology measuring heat flow as the direct readout in any biological system. Therefore, it’s the perfect match in the search for novel candidates selectively acting on adipocytes and providing the intended metabolic benefits, i.e enhanced fat oxidation, reduced body fat, and improved blood sugar control.

The calScreener ™ assay is based on isothermal microcalorimetry and opens up the field of developing drugs that activate brown adipose tissue or recruit beige cells, as it uses energy expenditure as the readout, instead of proxy parameters or indirect measurements.

Using the powerful calScreener ™ phenotypic screening assay you will increase the predictive value in drug development and significantly minimize the leap of faith when going from in-vitro to in-vivo.

Scientific References

The effects of the sequential addition of glucose, noradrenaline, propranolol and oleic acid on the rates of O2 consumption and heat production by isolated interscapular brown adipocytes from control and cafeteria-fed rats were compared. Although the chemical agents produced very similar changes in oxidative metabolism, the actual rates of O2 uptake and heat output in adipocytes from the cafeteria-fed rats, when expressed per g dry wt. of cells, were approx. 65% less than those obtained with cells from the control rats. However, when the same results were expressed per 10(8) multiloccular brown adipocytes, rather than gravimetrically, rates of O2 consumption and heat production were equivalent. Further interpretation of these data is complicated, because the average volume of multiloccular brown adipocytes from cafeteria-fed rats was 2.5 times that for multiloccular cells from control animals.

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The identification of brown adipose deposits in adults has led to significant interest in targeting this metabolically active tissue for treatment of obesity and diabetes. Improved methods for the direct measurement of heat production as the signature function of brown adipocytes (BAs), particularly at the single cell level, would be of substantial benefit to these ongoing efforts. Here, we report the first application of a small molecule-type thermosensitive fluorescent dye, ERthermAC, to monitor thermogenesis in BAs derived from murine brown fat precursors and in human brown fat cells differentiated from human neck brown preadipocytes. ERthermAC accumulated in the endoplasmic reticulum of BAs and displayed a marked change in fluorescence intensity in response to adrenergic stimulation of cells, which corresponded to temperature change. ERthermAC fluorescence intensity profiles were congruent with mitochondrial depolarisation events visualised by the JC-1 probe. Moreover, the averaged fluorescence intensity changes across a population of cells correlated well with dynamic changes such as thermal power, oxygen consumption, and extracellular acidification rates. These findings suggest ERthermAC as a promising new tool for studying thermogenic function in brown adipocytes of both murine and human origins.
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The possibility that brown adipose tissue thermogenesis can be recruited in order to combat the development of obesity has led to a high interest in the identification of “browning agents”, i.e. agents that increase the amount and activity of UCP1 in brown and brite/beige adipose tissues. However, functional analysis of the browning process yields confusingly different results when the analysis is performed in one of two alternative steps. Thus, in one of the steps, using cold acclimation as a potent model browning agent, we find that if the browning process is followed in mice initially housed at 21 °C (the most common procedure), there is only weak molecular evidence for increases in UCP1 gene expression or UCP1 protein abundance in classical brown adipose tissue; however, in brite/beige adipose depots, there are large increases, apparently associating functional browning with events only in the brite/beige tissues. Contrastingly, in another step, if the process is followed starting with mice initially housed at 30 °C (thermoneutrality for mice, thus similar to normal human conditions), large increases in UCP1 gene expression and UCP1 protein abundance are observed in the classical brown adipose tissue depots; there is then practically no observable UCP1 gene expression in brite/beige tissues. This apparent conundrum can be resolved when it is realized that the classical brown adipose tissue at 21 °C is already essentially fully differentiated and thus expands extensively through proliferation upon further browning induction, rather than by further enhancing cellular differentiation. When the limiting factor for thermogenesis, i.e. the total amount of UCP1 protein per depot, is analyzed, classical brown adipose tissue is by far the predominant site for the browning process, irrespective of which of the two steps is analyzed. There are to date no published data demonstrating that alternative browning agents would selectively promote brite/beige tissues versus classical brown tissue to a higher degree than does cold acclimation. Thus, to restrict investigations to examine adipose tissue depots where only a limited part of the adaptation process occurs (i.e. the brite/beige tissues) and to use initial conditions different from the thermoneutrality normally experienced by adult humans may seriously hamper the identification of therapeutically valid browning agents. The data presented here have therefore important implications for the analysis of the potential of browning agents and the nature of human brown adipose tissue.

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Nonshivering thermogenesis is the process of biological heat production in mammals and is primarily mediated by brown adipose tissue (BAT). Through ubiquitous expression of uncoupling protein 1 (Ucp1) on the mitochondrial inner membrane, BAT displays uncoupling of fuel combustion and ATP production in order to dissipate energy as heat. Because of its crucial role in regulating energy homeostasis, ongoing exploration of BAT has emphasized its therapeutic potential in addressing the global epidemics of obesity and diabetes. The recent appreciation that adult humans possess functional BAT strengthens this prospect. Furthermore, it has been identified that there are both classical brown adipocytes residing in dedicated BAT depots and “beige” adipocytes residing in white adipose tissue depots that can acquire BAT-like characteristics in response to environmental cues. This review aims to provide a brief overview of BAT research and summarize recent findings concerning the physiological, cellular, and developmental characteristics of brown adipocytes. In addition, some key genetic, molecular, and pharmacologic targets of BAT/Beige cells that have been reported to have therapeutic potential to combat obesity will be discussed.

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