Cold‐induced beigeing of stem cell‐derived adipocytes is not fully reversible after return to normothermia

Abstract Beige adipocytes possess the morphological and biochemical characteristics of brown adipocytes, including the mitochondrial uncoupling protein (UCP)1. Mesenchymal stem cells (MSCs) are somatic multipotent progenitors which differentiate into lipid‐laden adipocytes. Induction of MSC adipogenesis under hypothermic culture conditions (ie 32°C) promotes the appearance of a beige adipogenic phenotype, but the stability of this phenotypic switch after cells are returned to normothermic conditions of 37°C has not been fully examined. Here, cells transferred from 32°C to 37°C retained their multilocular beige‐like morphology and exhibited an intermediate gene expression profile, with both beige‐like and white adipocyte characteristics while maintaining UCP1 protein expression. Metabolic profile analysis indicated that the bioenergetic status of cells initially differentiated at 32°C adapted post‐transfer to 37°C, showing an increase in mitochondrial respiration and glycolysis. The ability of the transferred cells to respond under stress conditions (eg carbonyl cyanide‐4‐phenylhydrazone (FCCP) treatment) demonstrated higher functional capacity of enzymes involved in the electron transport chain and capability to supply substrate to the mitochondria. Overall, MSC‐derived adipocytes incubated at 32°C were able to remain metabolically active and retain brown‐like features after 3 weeks of acclimatization at 37°C, indicating these phenotypic characteristics acquired in response to environmental conditions are not fully reversible.

the free flow of protons across the inner mitochondrial membrane without phosphorylating ADP, 2,5 conferring a high potential thermogenic capacity to brown adipose tissue. 6 In addition to brown and white adipocytes, beige adipocytes have been reported to display a similar morphology to white cells in the basal state, but can be stimulated by cold exposure to acquire an activated brown-like morphology with significant UCP1 expression. [7][8][9][10] The extent to which temperature-induced stimulation of beige fat can be maintained upon return to a warm environment is unclear. In vivo, it appears that beige adipocytes from cold-stimulated mice revert to white fat within six weeks of warm acclimatization. 11 Similarly, in vitro differentiation of brown-like adipocytes mediated by culture at 27-33°C for 10 days was not maintained after the temperature was restored to 37°C. 7 Once established, however, classical brown adipocytes have been reported to maintain their multilocular morphology for one month in the same culture conditions. 12 This longer term adaptation appears to be mediated by a different mechanism from promoting the initial appearance of UCP1 and could include adaptations in the rate of biogenesis 12 and mitophagy. 13 Mesenchymal stem cells (MSCs), which are progenitors of adipocytes among other lineages, 9,[14][15][16][17][18] can respond to cold exposure during in vitro differentiation by forming UCP1-expressing adipocytes, 10

| MATERIAL S AND ME THODS
All reagents were purchased from Thermo Fisher Scientific (Loughborough, UK) unless otherwise stated.
The culture medium was changed every 2-3 days, and cells were sampled at 14, 21 or 28 days for analysis.
F I G U R E 1 Schematic diagram of MSCs adipogenesis treatments. MSCs were cultured in adipogenic medium at 37°C or 32°C for 7 days and then either maintained at this temperature or transferred to 37°C. Green arrows indicate samples' time-points at 14, 21 or 28 days

| Metabolic activity assay
Presto Blue Cell Viability Reagent was used as previously described 10 to measure metabolic activity at day 14, 21 and 28.
Fluorescence measurements were taken on 100 µL triplicate samples using a multimode microplate reader (Tecan Infinite 200 PRO, Tecan, Switzerland) using an excitation and emission wavelengths of 560 nm and 590 nm, respectively.

| Oil Red O (ORO) imaging and quantification
Fixed cells were stained with ORO as previously described. 10 The staining intensity was quantified by extracting ORO staining with 100% isopropanol, before measuring absorbance in triplicates at 510 nm with a multimode microplate reader (Tecan Infinite 200 PRO, Tecan, Switzerland).

| Mitochondrial staining
Live cells were stained for 45 min with MitoTracker Deep Red (100 nM) and Hoechst 33 258 (20 μg/mL), according to the manufacturer's protocol, before fixation. Samples were mounted with FluoroGel mounting medium (GeneTex, Irvine, CA, USA) and analysed with a Zeiss Elyra PS.1 microscope (Cambridge, UK). Mitochondria numbers were evaluated using a total of 15 randomly selected images per condition of three biological replicates. Relative fluorescence intensities were measured using the ImageJ software (https://imagej.nih.gov/ij/).

| Oxygen consumption analysis
Oxygen consumption was measured using a Seahorse XF96 Extracellular Flux Analyzer as previously outlined. 10 Basal respiration rate, ATP-linked respiration, proton leak respiration, maximal oxygen consumption rate (OCR), extracellular acidification rate (ECAR), mitochondrial reserve capacity and coupling efficiency were calculated as previously described. 10,20

| Cold-induced adipocytes retain beige-like cellular and morphological features when returned to a normothermic environment
To analyse the stability of the beigeing traits induced by hypothermic conditions, MSC were exposed to different culture environments ( Figure 1) and their phenotypic, metabolic and molecular response was assessed over 28 days. When compared to normothermic control cultures, cells transferred from 32°C to 37°C showed similar expression of UCP1 protein and morphological features associated with hypothermic conditions, such as multilocular lipid droplets (LDs) and increased mitochondrial abundance which were maintained until day 21 ( Figure 2).
Compared with normothermic controls, metabolic activity was increased in cells maintained at 32°C, but not in cells subsequently transferred back to 37°C ( Figure 3A). By contrast, lipid content increased over time in cells maintained at 32°C and in those subsequently transferred to 37°C ( Figure 3B). The same response was observed when percentage of differentiated cells in each treatment ( Figure 3C) and lipid droplet size ( Figure 3D) were analysed. These changes in lipid content were reflected by differences in cell morphology, as MSCs differentiated at 37°C contained large perinuclear LDs consistent with a white phenotype, while those differentiated at 32°C were mostly multilocular with smaller LDs on days 14 and 21 and then exhibited larger lipid vacuoles by day 28. Cells transferred from 32°C to 37°C displayed mixed uni-and multilocular cells, which became more unilocular by 28 days ( Figure S1). and Figure S4). An overview of changes in TRPV expression pattern under the different treatments can be observed in Figure 5D.

| Metabolic function of cold-induced adipocytes is modified when returned to a thermoneutral environment
When metabolic function was analysed, the higher rate of oxygen consumption in cells maintained at 32°C was not sustained when transferred to 37°C ( Figure 6A). Transfer cells were, however, able to up-regulate glycolysis to meet the requirement for ATP upon FCCP stimulation ( Figure 6B). No differences were observed in basal respiration, ATP production, proton leak and non-mitochondrial oxygen consumption when Transfer cells were compared with normothermic conditions.
The bioenergetic status of Transfer cells displayed a maximal respiration and spare capacity higher than cells differentiated at 37°C, but significantly lower than cells differentiated at 32°C.
Additionally, coupling efficiency of Transfer cells showed no difference between hypothermic 32°C cultures when compared to 37°C ( Figure 6C). When plotting basal ECAR against OCR, Transfer adipocytes displayed increased glycolysis and mitochondrial respiration ( Figure 6D). The bioenergetic analysis further showed that Transfer adipocytes could be activated when the mitochondrial uncoupler FCCP 23 was added. Higher OCR, ECAR, maximal respiration, spare capacity and metabolic profile than adipocytes differentiated at 37°C reflect a transitional bioenergetic status in which glycolysis rather than mitochondrial respiration was enhanced. Glycolysis has been confirmed to support full adipocyte thermogenesis in BAT by providing pyruvate formation and other intermediates for metabolic pathways as well as maintaining OCR. 24 In addition, in vivo evidence shows that intracellular glycolysis acts by controlling short-term non-shivering thermogenesis. 25 These results suggest that Transfer cells maintain a high glycolytic rate in order to replenish ATP when uncoupled oxidative phosphorylation and thermogenic activation are occurring. 24 Together with the mitochondrial measurements and increased UCP1 protein expression 26 show that Transfer cells are metabolically activated by enhancing lipolysis adipocytes treated with FCCP. 23 These observations also suggest that MSC-derived beige adipocytes demonstrate a distinctive bioenergetic response, as they can both store lipids and produce heat when stimulated, 27 adding to previous in vitro 7,10 and in vivo 28,29 reports to highlight MSCs as a valuable resource to study brown/ beige adipogenesis.

| The induced beige-like phenotype is not fully reversible
When evaluating the phenotype of Transfer cells, they exhibited an intermediary amount of lipid content, with a morphology and percentage of differentiated cells more similarly to cells kept at 32°C than cells differentiated under normothermic culture conditions. This is in accord with the two main processes of brown adipocyte generation, that is de novo and trans-differentiation. 30 Additionally, lipogenesis and lipolysis, two important mechanisms in brown adipocytes, are required for UCP1 proton transport 31,32 and could explain why adipocytes differentiated in hypothermic and in Transfer conditions contained more lipid and differentiated cells. Even though Transfer cells possessed less mitochondria than hypothermic differentiated adipocytes, their ability to retain UCP1 protein could be due to the capacity to maintain a brown/beige morphology with multilocular LDs, which provided more substrate availability than possessing larger or unilocular LDs.

| Beige-brown marker expression
Following reports that human BAT may be essentially comprised by beige adipocytes, 33  shared mechanism of calcium-mediated UCP1-independent thermogenesis. 43 Furthermore, high expression of the classical beige and brown adipogenic markers CIDEA 42

| Temperature sensing channels
This study revealed a complex interaction between TRPV1, 2 and 4 over the course of the differentiation treatments. TRPV1 increases when cold stimulation is applied to adipocytes, 7,10 and its activation F I G U R E 6 Effect of incubation temperature on the bioenergetics status of cultures after 28 days. (A) Oxygen consumption rate (OCR), (B) Extracellular acidification rate (ECAR), (C) mitochondrial function calculated from the bioenergetics profile (basal respiration, ATP-linked respiration, proton leak, non-mitochondrial respiration, maximal respiration, spare respiratory capacity and coupling efficiency) and (D) metabolic profile. Data shown as mean ± SEM, n = 6 individual experiments. *P < .05, **P < .01, ***P < .001 at a pre-adipocyte stage could be clinically beneficial by increasing intracellular calcium levels and preventing adipogenesis. 50 Furthermore, TRPV2 mRNA expression is known to increase in differentiated mouse brown adipocytes. 51,52 Here, it could be that a cascade of TRPV channels is necessary to retain the bioenergetics response, starting with the activation of TRPV1, maintenance of TRPV2 and finally the activation of TRPV4. Sun and colleagues showed that TRPV2 is not only the highest expressed thermoreceptor channel member, but also essential in brown adipocytes thermogenic function. [51][52][53] Taking into account the importance of calcium in UCP1-dependent 54 and UCP1-independent adipocyte thermogenesis, 43,54,55 it is possible that TRPV2 is one of the main regulators for beigeing maintenance and/or temperature sensing mechanism of cells. 52 Although the role of TRPV4 is debated, 56  cultures. The persistence of these cellular features suggests cell responses to environmental changes are influenced by a mechanism of differentiation memory, which requires further investigation.

| CON CLUS I ON & PER S PEC TIVE S
The in vivo behaviour of such conditioned adipocytes remains to be evaluated in order to determine whether their browning properties may be retained upon transplantation into recipient tissue.
If confirmed in vivo, this prolonged maintenance of brown/beige adipocytes might provide an experimental approach to promote energy expenditure, possibly combined with additional browning stimuli, which may be relevant for obesity research.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.