Neuroendocrine Control of Brown Adipocytes: Functions, Beiging, Whitening and Autophagy

Adipose tissue has emerged as a fundamental player in metabolic and energy processes. Beyond its fat storage capacity, it displays adaptive mechanisms to react to metabolic challenges throughout life. Three types of adipocytes, white, brown and beige, share common and yet fundamentally different roles and molecular regulation. This review focuses on brain and endocrine control of the function and plasticity of brown and beige adipocytes. The involvement of brain circuits controlling brown adipocytes and thermogenesis via the sympathetic nervous system is described. This autonomic control is mainly exerted by the hypothalamus and brainstem, and noradrenaline released from sympathetic efferent postganglionic bers interacts with brown or beige b3 adrenoreceptors (b3Adr) to trigger a cascade of events favorable to thermogenesis. Metabolic plasticity in response to environmental or hormonal cues, such as high fat diets, temperature, or hormones and aging, has been identied as a hallmark feature of brown/beige adipocytes. Besides the plastic response of WAT beiging, which is the generation of brown-like adipocytes within white adipose depots, diet-induced obesity and other cues evoke vascular remodeling and functional hypoxia in BAT leading to a whitening phenotype, characterized by mitochondrial dysfunction and loss, lipid droplet accumulation, and decreased thermogenesis. A detailed analysis of the participation of autophagy, and the endocrine signals relied by prolactin and growth hormone uncovers potential avenues of intervention to improve the metabolic function of BAT. Because BAT dysfunction in rodents is linked to fat gain, and glucose and lipid disorders, targeting BAT function and WAT beiging holds promise for obesity prevention and metabolic improvement.


Introduction
Adipose tissue has emerged as a fundamental player in the regulation of metabolic and energetic processes. Beyond its fat storage capacity, it displays adaptive mechanisms or plasticity to react to metabolic challenges throughout life. Three types of adipocytes, white, brown and beige, share common and yet fundamentally different roles and molecular regulation. White adipocytes have a large unilocular lipid compartment, store excess energy as triglycerides, and in times of caloric scarcity release free fatty acids (FFA) to support energy metabolism. In addition, white adipose tissue (WAT) is an endocrine organ which produces and secretes adipokines such as leptin, adiponectin and resistin, among other endocrine signals which participate in the regulation of food intake and energetic metabolism.
Brown adipose tissue (BAT), on the other hand, is composed of adipocytes with numerous small lipid droplets and large number of mitochondria. It provides protection against hypothermia and obesity, due to its capacity to oxidize FFAs and glucose to produce heat, actively participating in thermogenesis. This mechanism relies on the presence of the proton transporter uncoupling protein 1 (UCP1) found in the inner membrane of the adipocyte mitochondria, which uncouples electron transport in the respiratory chain, from ATP synthesis. Several batokines, such as protein peptides, metabolites, or miRNAs have been identi ed in BAT, though their action as secreted factors has not been completely settled (Nishio and Saeki, 2020).
Thermogenic brown-like adipocytes named brite/beige adipocytes in rodents and humans are interspersed within WAT, in rodents mainly in the inguinal, but also in other fat depots (Cannon and Nedergaard, 2004;Giordano et al., 2004;Gouon-Evans and Pollard, 2002;Lizcano, 2019). Both brown and beige adipocytes participate in thermoregulatory processes and have been put forward as potential targets in obesity control.
This review focuses on brain and endocrine control of the function and plasticity of brown and beige adipocytes. It has become clear that BAT dysfunction in rodents is linked to obesity, and glucose and lipid disorders. Therefore, understanding BAT physiology and WAT beiging is paramount in obesity prevention and metabolic improvement. On the other hand, it has to be kept in mind that hyperactivation of BAT may In rodents, BAT is found mainly in the interscapular and subscapular regions, but also in cervical, axillary, perirenal, and pericardic regions and mammary gland (Cannon and Nedergaard, 2004;Giordano et al., 2004;Gouon-Evans and Pollard, 2002); while in humans, it is mainly located in supraclavicular and cervical regions, but also in paravertebral, mediastinal, pericardial and periadrenal regions (Cannon and Nedergaard, 2004;Lidell et al., 2014;Nishio and Saeki, 2020;Virtanen et al., 2009). BAT is more abundantly found in new born babies, and markedly decreases with age (Ponrartana et al., 2013); nevertheless, some adults have traceable BAT, and in some individuals with very low BAT, cold acclimation induces its mass increase and the related non shivering thermogenesis (Marken Lichtenbelt et al., 2009), suggesting that BAT is recruitable. Adult human BAT contains both brown and beige cells (Wu et al., 2012).

Function
BAT is involved in thermoregulatory processes, including non-shivering cold-induced thermogenesis, dietinduced thermogenesis, and as part of the febrile response (Cannon et al., 1998;Morrison et al., 2012;Nakamura and Morrison, 2011). Thermogenesis is activated in cold environments allowing protection against hypothermia, and depends mainly on the presence of UCP1 (Carre and Binart, 2014).
For this purpose, brown adipocytes utilize fatty acids liberated by intracellular lipolysis which activate UCP1, and consequently lead to heat production (Townsend and Tseng, 2014). Studies in rodents further reveal that cell death-inducing DFFA-like effector A (Cidea), peroxisome proliferator activated receptor γ coactivator 1α (Pgc1a), CCAAT/enhancer-binding protein alpha (C/Ebpa), peroxisome proliferatoractivated receptor (Ppar), PR/SET Domain 16 (Prdm16) and other genes expressed in brown and beige adipocytes, are also critical regulators of BAT activity in response to environmental stimuli (Sharma et al., 2014;Townsend and Tseng, 2014).
The thermogenic capacity of BAT is engaged not only during cold exposure, but also in response to stress or energetically rich diets. Forced immobilization stress (Shibata and Nagasaka, 1982), or psychosocial stress (Kataoka et al., 2014) increase heat production by BAT, effect which relies on the sympathetic innervation of BAT. On the other hand, BAT has also an important role in lipid and glucose regulation (Maliszewska and Kretowski, 2021), and therefore its function is triggered by energetically rich diets (Bachman et al., 2002). Importantly, clearance of 50% of ingested triglycerides and 75% of ingested glucose can be attributed to BAT (Bartelt et al., 2011). In this context, subcutaneous transplant of embryonic BAT reversed type I diabetes in mice (Gunawardana and Piston, 2012). Therefore, considering that transplant of BAT or human pluripotent stem cell derived brown adipocytes (Nishio et al., 2012;Stanford et al., 2013) improve lipid and glucose metabolism in experimental settings, and that energy expenditure is associated to the thermogenic process, targeting BAT function can be envisaged as an anti-obesity strategy.

Brain Control Of Bat Function
The involvement of brain neuronal circuits controlling brown adipocytes and thermogenesis via the sympathetic nervous system (SNS) has been conclusively demonstrated (Bartness et al., 2010; Bartness and Ryu, 2015;Chechi et al., 2013;Labbe et al., 2015). Brain centers, mainly the hypothalamus and brainstem, participate in this autonomic control of BAT (Cannon and Nedergaard, 2004;Labbe et al., 2015) and denervation causes atrophy of BAT and decreased thermogenic capacity.
Brown adipocytes are richly innervated with strong immunopositivity for tyrosine hydroxylase (Murano et al., 2009) and beta adrenoceptors (bAdr), the main receptors involved in BAT thermogenesis, in particular b3Adr (Bartness et al., 2010;Cannon and Nedergaard, 2004). The release of noradrenaline from SNS efferent postganglionic bers, activates the b3Adr in brown adipocytes and leads to substrate oxidation and heat production (Bachman et al., 2002;Jimenez et al., 2002). The cascade of events is initiated by increased cyclic adenosine monophosphate levels, activation of protein kinase A (PKA), phosphorylation of hormone sensitive lipase, and nally lipolysis of triglycerides into FFA which are energy substrates for beta oxidation in mitochondria, and UCP1 substrates for H + transport across the membrane with the generation of heat in the process (Cannon and Nedergaard, 2004). Simultaneously the number of brown adipocytes increase, there is mitochondrial biogenesis, and thermogenic proteins are upregulated.
Hypothalamic centers participating in BAT thermogenic activity Using pseudorabies virus as polysynaptic retrograde tracers, brain areas which modulate the sympathetic innervation of BAT and WAT have been delineated. Injection of pseudo rabies virus in BAT mapped the distribution and neurochemical characteristics of viral infected neurons in the hypothalamus, mainly in the para ventricular hypothalamus (PVH), arcuate nucleus (ARC), dorsomedial hypothalamus (DMH), lateral hypothalamus (LH) and preoptic area (POA) (Bamshad et al., 1999;Old eld et al., 2002;Ryu et al., 2015).
Hypothalamic components of cold activated pathways, which shape the thermoregulatory pathway, mainly include the POA, DMH, the rostral raphe pallidus (rPA) and also VMH and PVH. The POA is considered a major coordinator, receiving inputs from the brain and skin thermoreceptors ( (Morrison et al., 2014). Concordantly, desinhibition of DMH neurons using a GABA antagonist increases BAT and body temperature (Zaretskaia et al., 2002). DMH neurons do not directly stimulate BAT, but project to the rPA, which is a main site of BAT SNS premotor neurons, (Nakamura et al., 2005). Desinhibition of DMH neurons, activates glutamatergic descending signaling to the rPA and subsequently sympathetic activity in BAT is increased (Zhang and Bi, 2015). POA also projects to the VMH and PVH, which may participate in the thermogenic response to cold. The DMH is a key nucleus in thermogenesis. Besides DMH-GABAergic involvement in this process, there is an inverse relationship of NPY expression in the DMH, and BAT thermogenesis. Overexpression of NPY in the DMH leads not only to hyperphagia and obesity, but also to decreased BAT activity and BAT-Ucp1  (Chao et al., 2011). Furthermore, during cold exposure there is a decrease in DMH-Npy expression (Park et al., 2007).
The VMH also participates in the SNS control of BAT activity increasing thermogenesis, as indicated by electrical stimulation or lesion of this nucleus (Holt et al., 1987;Monda et al., 1997;Perkins et al., 1981;Yoshida and Bray, 1984). Importantly, deletion of SF1 which is speci cally expressed in the VMH evoked a decrease in BAT UCP1 (Kim et al., 2011a). Even though retrograde viral studies did not point to the VMH as a main hypothalamic center in the connection of BAT to the brain (Bamshad et al., 1999), a participation of rPA and the inferior olive has been suggested in VMH modulation of SNS out ow to BAT (Morrison 1999). GLP1 and BMP8R activation of VMH may also mediate the action of VMH on BAT function (

Bat Plasticity
Metabolic plasticity in response to environmental or hormonal cues, such as high fat diets or changes in temperature, or hormones, has been identi ed as a hallmark feature of brown adipose tissue (Galic et al., 2010). Besides the plastic response of WAT beiging, which is the generation of brown-like adipocytes in white adipose depots, diet-induced obesity in mice evokes vascular remodeling and functional hypoxia in BAT leading to a whitening phenotype, characterized by mitochondrial dysfunction and loss, lipid droplet accumulation, and decreased expression of thermogenic markers (Shimizu et al., 2014b). BAT whitening is associated to decreased thermogenic capacity and impaired energy balance of this tissue, while WAT beiging increases thermogenesis in this depot.

Beiging
Though beiging in rodents is classically studied in subcutaneous depots, it may also occur, depending on mouse strain, in visceral depots such as mesenteric, periaortic, mediastinal or perirrenal depots (Vitali et al., 2012). Brown but not white or beige adipocytes derive from myogenic factor 5 (Myf5)-expressing precursors and therefore share a common lineage with skeletal myocytes (

Whitening
In contrast to beiging which occurs in WAT, whitening is a phenomenon evidenced mainly in BAT, though it also may occur in beige adipocytes. Physiological inactivation of brown/beige adipose tissues by the whitening process is observed during obesity, aging, lactation, or increased environmental temperature, . Beige adipocytes within WAT "disappear" when returning to thermoneutrality: they lose their multilocular phenotype and UCP1 expression.
Conversely, BAT inactivation includes protein degradation, protein synthesis inhibition, and lipid accumulation, even though brown adipocytes do not disappear. In obese subjects, or HFD-fed rodents, an increase in BAT weight may be found, explained by higher lipid content, and a reduction in the thermogenic function of the tissue. In this sense, heavier BAT depots do not necessarily indicate a greater thermogenic capacity but are associated with a defective tissue, as triglycerides are stored instead of being used for heat production (Shimizu et al., 2014b).
During BAT whitening, cells with multilocular lipid droplets evolve to unilocular cells, resembling white adipocytes. There is a proneness to tissue in ammation and adipocyte death (Kotzbeck et al., 2018). Tissue in ammation leads to macrophage in ltration, and the appearance of crown like structure formations, enlarged endoplasmic reticulum, cholesterol crystals, degenerating or dysfunctional mitochondria, decreased lipid oxidation and increased collagen brils (Kotzbeck et al., 2018). Importantly, reduced insulin stimulated glucose uptake by BAT can be found, suggesting insulin resistance of the tissue (Kuipers et al., 2019;Roberts-Toler et al., 2015). A reduced uptake of triglycerides derived from FFA, and marked reduction of thermogenic biomarkers are also key features of whitening. As a result, thermogenesis is compromised and the ability to maintain body temperature when exposed to cold is impaired.
The whitened brown adipocytes still retain some brown like typical mitochondria, and weak UCP1 expression, suggesting a conversion of brown to white in stressful conditions such as lipid overload, or inhibition of oxidation ( The occurrence of whitening in response to exogenous compounds may be involved in their capacity to induce obesity. And therefore the inhibition of this process is a promising target in the treatment or prevention of obesity. For example, glucocorticoids which induce obesity, decrease BAT UCP1 expression and hinder thermogenesis in rodents (Deng et al., 2020;Mousovich-Neto et al., 2019). In humans they acutely increase or chronically suppress BAT activity (Ryu et al., 2015). In this respect, it has been shown that dexamethasone induces BAT whitening in vivo and in vitro (Deng et al., 2020) decreasing thermogenic markers, and increasing the expression of WAT markers within brown adipocytes. This effect has been related to whitening due to enhanced autophagy, mediated by the antiproliferative gene B cell translocation gene 1 (Btg1). Therefore, knocking down either Btg1 or the autophagy related gene Atg7 In macroautophagy, hereafter referred to as "autophagy", the cytoplasmic cargo is engulfed by a double-membrane vesicle (autophagosome), which fuses to a lysosome to form an autolysosome; this process has been extensively reviewed (Bento et al., 2016).
Autophagy may participate in BAT plasticity dynamically turning on or off, depending on the thermogenic status of adipocytes, in order to respond to different cues, adapting not only intracellular processes (i.e. mitochondrial biogenesis/degradation) but also tissue remodeling (i.e. hypertrophy/hyperplasia) In this sense, acute cold-induced thermogenesis stabilizes PINK1 at the surface of depolarized (but healthy) mitochondria, but strongly represses Parkin at transcriptional level through a PPARa-dependent mechanisms and inhibits its translocation by PKA (Cairo et al., 2019). This mechanism allows active mitochondria to avoid degradation even when physiological depolarization stabilizes PINK1. Nevertheless, and in contrast with the previous a rmations, cultured brown adipocytes with acute thermogenic activation and mice chronically exposed to cold showed increased mitophagy (Lu et al., 2018b). These discrepant results could imply that despite thermogenic stimulation some mitochondria escape the "protective" role of Parkin downregulation, and have unfailingly the destiny to undergo mitophagy.
In general, lowering the expression of adipocyte Parkin or other autophagy mediators, can be envisaged as probable targets in obesity treatment, by preventing mitochondrial degradation and enhancing thermogenesis.

Autophagy and the adaptive inactivation of BAT by whitening
During the plastic process of whitening, autophagy is required for the mitochondrial protein degradation promoting the transition from beige and brown to white adipose tissue. This requirement was evidenced using mice with speci c deletion of the autophagic genes Atg5 and Atg12 in brown and beige adipocytes, as well as Parkin KO  However, a recent study in mice with adipose tissue-speci c ablation of Parkin did not reproduce this observation (Corsa et al., 2019), further pointing to the complexity of the process.
Interestingly, in short-term adaptive inhibition of BAT activity during re-acclimation to a thermoneutral temperature, accumulation of defective mitochondria in BAT was found, while long-term inactivation of BAT elicited normalization in mitochondria degradation probably through Parkin-independent mitophagic mechanisms (Cairo et al., 2019).
In summary, autophagy has a key role in regulating body lipid accumulation by controlling the thermogenic capacity of brown adipocytes, which is enhanced when autophagy or mitophagy is inhibited, allowing the net increase in mitochondrial mass and the consequent enhancement in beta oxidation. Nevertheless, the relation of autophagy in brown and beige human adipocytes is far from being resolved.

Endocrine Regulation Of Bat Function
Several homeostatic hormones affect BAT thermogenesis, by acting directly at the tissue or modulating regulatory systems and pathways which intervene in the process.

a) Prolactin
Prolactin is a pituitary-derived hormone whose main function is to facilitate lactation, but that has multiple metabolic roles, associated to the widespread location of its receptors. Prlr mRNA has been documented in both white and brown adipocytes (Ling et al., 2003;Royster et al., 1995;Wittmann et al., 2002). ; and in hamsters BAT weight and activity decreases throughout pregnancy (Wade et al., 1986). These data suggest a negative association between high prolactin and the thermogenic capacity of BAT during pregnancy and lactation, when glucose and triglycerides should not be burned to produce heat because they are needed for the pregnant and lactating mother, and glucose for the developing fetus.
Furthermore, lactation performance is in uenced by the capacity to dissipate body heat, and prolactin may participate in this process limiting thermogenesis by the downregulation of BAT UCP1 (Krol et al., 2011).
Of note, larger depots of intrascapular BAT related to BW with higher UCP1 content are found in female compared to male rats as adults (Quevedo et al., 1998;Rodriguez et al., 2001), and it has been postulated that this sex difference might arise from sex differences in serum prolactin levels during development The GH receptor (GHR), a class 1 cytokine receptor, is expressed in many tissues including brown and white adipocytes, and pre-adipocytes (Vikman et al., 1991;Zou et al., 1997). But even though the effects of GH on WAT have been well studied, its impact on BAT function or in the process of WAT beiging is not yet a settled matter. In particular, there are only few clinical data on BAT activity in patients with acromegaly or GH de ciency. Nevertheless, increasing data using mouse models unravel the intricate and sometimes contradictory role of GH on BAT function and WAT beiging.

GH impact on brown and beige adipocytes
The role of GH on BAT function has been studied using several mouse models. Namely the Ames and Snell dwarfs, the GHRKO, the GHA, the FaGHRKO, and the AdGHRKO mice. The Ames dwarfs harbor a recessive Prophet of Pituitary Factor 1 (Prop1) loss-of-function mutation, and the Snell dwarfs have a point mutation within the Pit1 gene, therefore both mice have de ciencies of GH, thyroid stimulating hormone and prolactin levels. The GHRHKO mouse harbors a disruption in the GH receptor/GH binding protein gene, and is resistant to the action of GH (Liang et al., 2003). GH receptor antagonist (GHA) transgenic mice express a GH analog which competes with GH for binding to the GHR, thus decreasing, but not entirely eliminating, GH signaling (Coschigano et al., 2003). In addition to studies using these global knockouts, mouse models with tissue speci c deletion of GHR have also provided insightful data on the role of GH in BAT function (Li et  GH and beiging within WAT Studies using dwarf mouse models, global GHRKO, and GH overexpressing, as well as adipose tissue speci c GHR knockout mice yield con icting results on GH action on WAT beiging. Some data favor a positive action of GH on WAT beiging. For example, in transgenic mice in which GH is unable to activate STAT5 (the GHR-391 mouse) a marked de ciency of beta oxidation was described in inguinal WAT, associated to a decrease in beiging transcripts including Ucp1, Cidea, Pgca1, Ppara, and Prdm16 (Nelson et al., 2018). Besides, in the GHR-391 mouse as well as in GHRKO mice there was a marked decrease in the transcript and protein levels of b3Adr, a receptor required for beiging of inguinal WAT, preventing beiging induced by a beta adrenergic speci c agonist in WAT from both mouse models, unlike the robust response found in wildtype mice (Nelson et al., 2018). Furthermore, a transcriptome study comparing WAT of wildtype and GHRKO mice featured a decreased expression of mitochondrial genes in response to the loss of GH signaling, in a divergent manner to the signature found in BAT (Stout et al., 2015). In addition, increased GH action in the bovine GH transgenic mice showed the expected opposite pattern in WAT beiging, there was an increase in transcripts involved in fatty acid oxidation, as well as increments in protein expression of UCP1 and PRDM16 (Nelson et al., 2018). These studies coalesce to demonstrate that intact GH signaling may be supportive to the beiging process in WAT. Nevertheless, opposite data have also been published. In a recent report Ucp1 mRNA and protein expression was increased in WAT of GHRKO compared to wildtype mice (Li et al., 2020), and this nding was replicated in Snell mice (Li et al., 2020). Results showcase the complexity of analyzing different mouse models and assays (transcriptome, protein, mRNA, heat production, etc).
Studies using adipocyte speci c deletion of GHR yield concordant results in some though not all aspects of the WAT beiging response to the loss of adipocyte GHR. The FaGHRKO mouse showed decreased expression of genes associated with beige adipocyte differentiation, such as Ucp1, Prdm16, CYp1b, and b3Adr, in inguinal WAT (Nelson et al., 2018), and similarly in the AdGHRKO mouse there was decreased beiging, adaptive thermogenesis response to cold exposure, and lack of Ucp1 or Pgc1a upregulation by cold (Ran et al., 2019). These data suggest a positive action of adipocyte GHR in WAT beiging, as also described above using the global knockouts. But on the other hand, in FaGHRKO mice an increase in UCP1 in inguinal WAT has been described in males, and not in females (Li et al., 2020), further adding to the controversy of the exact role played by GH on WAT beiging.
Even so, a positive action of GH on WAT beiging, and a negative or restraining effect on BAT thermogenic function is in accordance with whole-genome microarrays which demonstrate divergent response patterns of BAT and WAT to the loss of GH signaling (Stout et al., 2015).

Conclusions
Brain control is fundamental in the function of brown adipose tissue, and speci c brain centers within the hypothalamus and brainstem participate in thermogenic and metabolic pathways which activate BAT function and WAT beiging. Furthermore, adequate autophagy promotes a healthy tissue; a decrease in autophagy is generally needed for BAT activation, and impaired authophagy is associated to BAT whitening and dysfunction. Whitening of BAT is a plastic response to stressful conditions such as chronic over nutrition, or increase in environmental temperature, among other stimuli. During this process BAT weight may be increased, but its thermogenic function decreased, implying that organ weight may not a good readout of thermogenic capacity, and the need of assessing thermogenic markers.
Beiging or browning of white adipocytes within WAT is also a plastic response which opposes adipose accretion, by enhancing thermogenesis. This reveals an outstanding reaction of the organism to combat obesity by enhancing energy dissipation in an energy storing depot. Therefore, efforts have been channeled into the development of genetic or pharmacological tools to improve the recruitment of beiging in WAT, in an attempt to ameliorate overweight.
Finally, several endocrine mediators are involved in BAT control, such as leptin, insulin, prolactin and GH.
In the present review, we focused on GH and prolactin actions, and, overall, chronic high prolactin levels are associated with BAT whitening, and loss of function, while GH has a negative action on BAT thermogenesis. Nevertheless, several contradictory data have been published which hints the use of caution when forwarding a hypothesis, as endocrine control of BAT may differ among species, transgenic mouse models, and metabolic status.
Since the description of an active and recruitable brown adipose tissue in humans, increasing interest in its physiology and pathology has positioned this tissue in a new hierarchy, as a possible target to tackle metabolic dysfunction. Within the genetic causes to obesity predisposition, UCP1 polymorphisms have been pointed (Murray et al., 2004), as UCP1 has a secondary fat and calorie burning capacity which provides a useful advantage in a high calorie nutritional environment. BAT has turned out to be more dynamic and complex than once thought. Therefore unraveling the complexities of brown adipocytes may yield valuable tools in the effort to curtail obesity or accelerate weight loss.

Declarations
Funding: Partial nancial support was received from: This work was supported by Argentinean Agency