BCAA-nitrogen flux in brown fat controls metabolic health independent of thermogenesis.

Brown adipose tissue (BAT) is best known for thermogenesis. Rodent studies demonstrated that enhanced BAT thermogenesis is tightly associated with increased energy expenditure, reduced body weight, and improved glucose homeostasis. However, human BAT is protective against type 2 diabetes, independent of body weight. The mechanism underlying this dissociation remains unclear. Here, we report that impaired mitochondrial catabolism of branched-chain amino acids (BCAAs) in BAT, by deleting mitochondrial BCAA carriers (MBCs), caused systemic insulin resistance without affecting energy expenditure and body weight. Brown adipocytes catabolized BCAA in the mitochondria as nitrogen donors for the biosynthesis of non-essential amino acids and glutathione. Impaired mitochondrial BCAA-nitrogen flux in BAT resulted in increased oxidative stress, decreased hepatic insulin signaling, and decreased circulating BCAA-derived metabolites. A high-fat diet attenuated BCAA-nitrogen flux and metabolite synthesis in BAT, whereas cold-activated BAT enhanced the synthesis. This work uncovers a metabolite-mediated pathway through which BAT controls metabolic health beyond thermogenesis.

Structural basis of respiratory complex adaptation to cold temperatures.

In response to cold, mammals activate brown fat for respiratory-dependent thermogenesis reliant on the electron transport chain. Yet, the structural basis of respiratory complex adaptation upon cold exposure remains elusive. Herein, we combined thermoregulatory physiology and cryoelectron microscopy (cryo-EM) to study endogenous respiratory supercomplexes from mice exposed to different temperatures. A cold-induced conformation of CI:III2 (termed type 2) supercomplex was identified with a ∼25° rotation of CIII2 around its inter-dimer axis, shortening inter-complex Q exchange space, and exhibiting catalytic states that favor electron transfer. Large-scale supercomplex simulations in mitochondrial membranes reveal how lipid-protein arrangements stabilize type 2 complexes to enhance catalytic activity. Together, our cryo-EM studies, multiscale simulations, and biochemical analyses unveil the thermoregulatory mechanisms and dynamics of increased respiratory capacity in brown fat at the structural and energetic level.

Light modulates glucose metabolism by a retina-hypothalamus-brown adipose tissue axis.

Public health studies indicate that artificial light is a high-risk factor for metabolic disorders. However, the neural mechanism underlying metabolic modulation by light remains elusive. Here, we found that light can acutely decrease glucose tolerance (GT) in mice by activation of intrinsically photosensitive retinal ganglion cells (ipRGCs) innervating the hypothalamic supraoptic nucleus (SON). Vasopressin neurons in the SON project to the paraventricular nucleus, then to the GABAergic neurons in the solitary tract nucleus, and eventually to brown adipose tissue (BAT). Light activation of this neural circuit directly blocks adaptive thermogenesis in BAT, thereby decreasing GT. In humans, light also modulates GT at the temperature where BAT is active. Thus, our work unveils a retina-SON-BAT axis that mediates the effect of light on glucose metabolism, which may explain the connection between artificial light and metabolic dysregulation, suggesting a potential prevention and treatment strategy for managing glucose metabolic disorders.

Lipolysis drives expression of the constitutively active receptor GPR3 to induce adipose thermogenesis.

Thermogenic adipocytes possess a therapeutically appealing, energy-expending capacity, which is canonically cold-induced by ligand-dependent activation of ß-adrenergic G protein-coupled receptors (GPCRs). Here, we uncover an alternate paradigm of GPCR-mediated adipose thermogenesis through the constitutively active receptor, GPR3. We show that the N terminus of GPR3 confers intrinsic signaling activity, resulting in continuous Gs-coupling and cAMP production without an exogenous ligand. Thus, transcriptional induction of Gpr3 represents the regulatory parallel to ligand-binding of conventional GPCRs. Consequently, increasing Gpr3 expression in thermogenic adipocytes is alone sufficient to drive energy expenditure and counteract metabolic disease in mice. Gpr3 transcription is cold-stimulated by a lipolytic signal, and dietary fat potentiates GPR3-dependent thermogenesis to amplify the response to caloric excess. Moreover, we find GPR3 to be an essential, adrenergic-independent regulator of human brown adipocytes. Taken together, our findings reveal a noncanonical mechanism of GPCR control and thermogenic activation through the lipolysis-induced expression of constitutively active GPR3.

Origin and Function of Stress-Induced IL-6 in Murine Models.

Acute psychological stress has long been known to decrease host fitness to inflammation in a wide variety of diseases, but how this occurs is incompletely understood. Using mouse models, we show that interleukin-6 (IL-6) is the dominant cytokine inducible upon acute stress alone. Stress-inducible IL-6 is produced from brown adipocytes in a beta-3-adrenergic-receptor-dependent fashion. During stress, endocrine IL-6 is the required instructive signal for mediating hyperglycemia through hepatic gluconeogenesis, which is necessary for anticipating and fueling "fight or flight" responses. This adaptation comes at the cost of enhancing mortality to a subsequent inflammatory challenge. These findings provide a mechanistic understanding of the ontogeny and adaptive purpose of IL-6 as a bona fide stress hormone coordinating systemic immunometabolic reprogramming. This brain-brown fat-liver axis might provide new insights into brown adipose tissue as a stress-responsive endocrine organ and mechanistic insight into targeting this axis in the treatment of inflammatory and neuropsychiatric diseases.

Immunomodulatory leptin receptor+ sympathetic perineurial barrier cells protect against obesity by facilitating brown adipose tissue thermogenesis.

Adipose tissues (ATs) are innervated by sympathetic nerves, which drive reduction of fat mass via lipolysis and thermogenesis. Here, we report a population of immunomodulatory leptin receptor-positive (LepR+) sympathetic perineurial barrier cells (SPCs) present in mice and humans, which uniquely co-express Lepr and interleukin-33 (Il33) and ensheath AT sympathetic axon bundles. Brown ATs (BATs) of mice lacking IL-33 in SPCs (SPCΔIl33) had fewer regulatory T (Treg) cells and eosinophils, resulting in increased BAT inflammation. SPCΔIl33 mice were more susceptible to diet-induced obesity, independently of food intake. Furthermore, SPCΔIl33 mice had impaired adaptive thermogenesis and were unresponsive to leptin-induced rescue of metabolic adaptation. We therefore identify LepR+ SPCs as a source of IL-33, which orchestrate an anti-inflammatory BAT environment, preserving sympathetic-mediated thermogenesis and body weight homeostasis. LepR+IL-33+ SPCs provide a cellular link between leptin and immune regulation of body weight, unifying neuroendocrinology and immunometabolism as previously disconnected fields of obesity research.

Structural mechanisms of mitochondrial uncoupling protein 1 regulation in thermogenesis.

In mitochondria, the oxidation of nutrients is coupled to ATP synthesis by the generation of a protonmotive force across the mitochondrial inner membrane. In mammalian brown adipose tissue (BAT), uncoupling protein 1 (UCP1, SLC25A7), a member of the SLC25 mitochondrial carrier family, dissipates the protonmotive force by facilitating the return of protons to the mitochondrial matrix. This process short-circuits the mitochondrion, generating heat for non-shivering thermogenesis. Recent cryo-electron microscopy (cryo-EM) structures of human UCP1 have provided new molecular insights into the inhibition and activation of thermogenesis. Here, we discuss these structures, describing how purine nucleotides lock UCP1 in a proton-impermeable conformation and rationalizing potential conformational changes of this carrier in response to fatty acid activators that enable proton leak for thermogenesis.

Brown adipose tissue CoQ deficiency activates the integrated stress response and FGF21-dependent mitohormesis.

Coenzyme Q (CoQ) is essential for mitochondrial respiration and required for thermogenic activity in brown adipose tissues (BAT). CoQ deficiency leads to a wide range of pathological manifestations, but mechanistic consequences of CoQ deficiency in specific tissues, such as BAT, remain poorly understood. Here, we show that pharmacological or genetic CoQ deficiency in BAT leads to stress signals causing accumulation of cytosolic mitochondrial RNAs and activation of the eIF2α kinase PKR, resulting in activation of the integrated stress response (ISR) with suppression of UCP1 but induction of FGF21 expression. Strikingly, despite diminished UCP1 levels, BAT CoQ deficiency displays increased whole-body metabolic rates at room temperature and thermoneutrality resulting in decreased weight gain on high-fat diets (HFD). In line with enhanced metabolic rates, BAT and inguinal white adipose tissue (iWAT) interorgan crosstalk caused increased browning of iWAT in BAT-specific CoQ deficient animals. This mitohormesis-like effect depends on the ATF4-FGF21 axis and BAT-secreted FGF21, revealing an unexpected role for CoQ in the modulation of whole-body energy expenditure with wide-ranging implications for primary and secondary CoQ deficiencies.

Non-canonical mTORC2 Signaling Regulates Brown Adipocyte Lipid Catabolism through SIRT6-FoxO1.

mTORC2 controls glucose and lipid metabolism, but the mechanisms are unclear. Here, we show that conditionally deleting the essential mTORC2 subunit Rictor in murine brown adipocytes inhibits de novo lipid synthesis, promotes lipid catabolism and thermogenesis, and protects against diet-induced obesity and hepatic steatosis. AKT kinases are the canonical mTORC2 substrates; however, deleting Rictor in brown adipocytes appears to drive lipid catabolism by promoting FoxO1 deacetylation independently of AKT, and in a pathway distinct from its positive role in anabolic lipid synthesis. This facilitates FoxO1 nuclear retention, enhances lipid uptake and lipolysis, and potentiates UCP1 expression. We provide evidence that SIRT6 is the FoxO1 deacetylase suppressed by mTORC2 and show an endogenous interaction between SIRT6 and mTORC2 in both mouse and human cells. Our findings suggest a new paradigm of mTORC2 function filling an important gap in our understanding of this more mysterious mTOR complex.

CLCF1 inhibits energy expenditure via suppressing brown fat thermogenesis.

Brown adipose tissue (BAT) is the main site of nonshivering thermogenesis which plays an important role in thermogenesis and energy metabolism. However, the regulatory factors that inhibit BAT activity remain largely unknown. Here, cardiotrophin-like cytokine factor 1 (CLCF1) is identified as a negative regulator of thermogenesis in BAT. Adenovirus-mediated overexpression of CLCF1 in BAT greatly impairs the thermogenic capacity of BAT and reduces the metabolic rate. Consistently, BAT-specific ablation of CLCF1 enhances the BAT function and energy expenditure under both thermoneutral and cold conditions. Mechanistically, adenylate cyclase 3 (ADCY3) is identified as a downstream target of CLCF1 to mediate its role in regulating thermogenesis. Furthermore, CLCF1 is identified to negatively regulate the PERK-ATF4 signaling axis to modulate the transcriptional activity of ADCY3, which activates the PKA substrate phosphorylation. Moreover, CLCF1 deletion in BAT protects the mice against diet-induced obesity by promoting BAT activation and further attenuating impaired glucose and lipid metabolism. Therefore, our results reveal the essential role of CLCF1 in regulating BAT thermogenesis and suggest that inhibiting CLCF1 signaling might be a potential therapeutic strategy for improving obesity-related metabolic disorders.

The thermoneutral zone in women takes an "arctic" shift compared to men.

Conventionally, women are perceived to feel colder than men, but controlled comparisons are sparse. We measured the response of healthy, lean, young women and men to a range of ambient temperatures typical of the daily environment (17 to 31 °C). The Scholander model of thermoregulation defines the lower critical temperature as threshold of the thermoneutral zone, below which additional heat production is required to defend core body temperature. This parameter can be used to characterize the thermoregulatory phenotypes of endotherms on a spectrum from "arctic" to "tropical." We found that women had a cooler lower critical temperature (mean ± SD: 21.9 ± 1.3 °C vs. 22.9 ± 1.2 °C, P = 0.047), resembling an "arctic" shift compared to men. The more arctic profile of women was predominantly driven by higher insulation associated with more body fat compared to men, countering the lower basal metabolic rate associated with their smaller body size, which typically favors a "tropical" shift. We did not detect sex-based differences in secondary measures of thermoregulation including brown adipose tissue glucose uptake, muscle electrical activity, skin temperatures, cold-induced thermogenesis, or self-reported thermal comfort. In conclusion, the principal contributors to individual differences in human thermoregulation are physical attributes, including body size and composition, which may be partly mediated by sex.

Integrating adipocyte insulin signaling and metabolism in the multi-omics era.

Insulin stimulates glucose uptake into adipocytes via mTORC2/AKT signaling and GLUT4 translocation and directs glucose carbons into glycolysis, glycerol for TAG synthesis, and de novo lipogenesis. Adipocyte insulin resistance is an early indicator of type 2 diabetes in obesity, a worldwide health crisis. Thus, understanding the interplay between insulin signaling and central carbon metabolism pathways that maintains adipocyte function, blood glucose levels, and metabolic homeostasis is critical. While classically viewed through the lens of individual enzyme-substrate interactions, advances in mass spectrometry are beginning to illuminate adipocyte signaling and metabolic networks on an unprecedented scale, yet this is just the tip of the iceberg. Here, we review how 'omics approaches help to elucidate adipocyte insulin action in cellular time and space.

NAMPT-dependent NAD+ biosynthesis controls circadian metabolism in a tissue-specific manner.

Molecular clocks in the periphery coordinate tissue-specific daily biorhythms by integrating input from the hypothalamic master clock and intracellular metabolic signals. One such key metabolic signal is the cellular concentration of NAD+, which oscillates along with its biosynthetic enzyme, nicotinamide phosphoribosyltransferase (NAMPT). NAD+ levels feed back into the clock to influence rhythmicity of biological functions, yet whether this metabolic fine-tuning occurs ubiquitously across cell types and is a core clock feature is unknown. Here, we show that NAMPT-dependent control over the molecular clock varies substantially between tissues. Brown adipose tissue (BAT) requires NAMPT to sustain the amplitude of the core clock, whereas rhythmicity in white adipose tissue (WAT) is only moderately dependent on NAD+ biosynthesis, and the skeletal muscle clock is completely refractory to loss of NAMPT. In BAT and WAT, NAMPT differentially orchestrates oscillation of clock-controlled gene networks and the diurnality of metabolite levels. NAMPT coordinates the rhythmicity of TCA cycle intermediates in BAT, but not in WAT, and loss of NAD+ abolishes these oscillations similarly to high-fat diet-induced circadian disruption. Moreover, adipose NAMPT depletion improved the ability of animals to defend body temperature during cold stress but in a time-of-day-independent manner. Thus, our findings reveal that peripheral molecular clocks and metabolic biorhythms are shaped in a highly tissue-specific manner by NAMPT-dependent NAD+ synthesis.

Intracapillary LPL levels in brown adipose tissue, visualized with an antibody-based approach, are regulated by ANGPTL4 at thermoneutral temperatures.

Lipoprotein lipase (LPL) is secreted into the interstitial spaces by parenchymal cells and then transported into capillaries by GPIHBP1. LPL carries out the lipolytic processing of triglyceride (TG)-rich lipoproteins (TRLs), but the tissue-specific regulation of LPL is incompletely understood. Plasma levels of TG hydrolase activity after heparin injection are often used to draw inferences about intravascular LPL levels, but the validity of these inferences is unclear. Moreover, plasma TG hydrolase activity levels are not helpful for understanding LPL regulation in specific tissues. Here, we sought to elucidate LPL regulation under thermoneutral conditions (30 °C). To pursue this objective, we developed an antibody-based method to quantify (in a direct fashion) LPL levels inside capillaries. At 30 °C, intracapillary LPL levels fell sharply in brown adipose tissue (BAT) but not heart. The reduced intracapillary LPL levels were accompanied by reduced margination of TRLs along capillaries. ANGPTL4 expression in BAT increased fourfold at 30 °C, suggesting a potential explanation for the lower intracapillary LPL levels. Consistent with that idea, Angptl4 deficiency normalized both LPL levels and TRL margination in BAT at 30 °C. In Gpihbp1-/- mice housed at 30 °C, we observed an ANGPTL4-dependent decrease in LPL levels within the interstitial spaces of BAT, providing in vivo proof that ANGPTL4 regulates LPL levels before LPL transport into capillaries. In conclusion, our studies have illuminated intracapillary LPL regulation under thermoneutral conditions. Our approaches will be useful for defining the impact of genetic variation and metabolic disease on intracapillary LPL levels and TRL processing.

CLCF1 signaling restrains thermogenesis and disrupts metabolic homeostasis by inhibiting mitochondrial biogenesis in brown adipocytes.

Great progress has been made in identifying positive regulators that activate adipocyte thermogenesis, but negative regulatory signaling of thermogenesis remains poorly understood. Here, we found that cardiotrophin-like cytokine factor 1 (CLCF1) signaling led to loss of brown fat identity, which impaired thermogenic capacity. CLCF1 levels decreased during thermogenic stimulation but were considerably increased in obesity. Adipocyte-specific CLCF1 transgenic (CLCF1-ATG) mice showed impaired energy expenditure and severe cold intolerance. Elevated CLCF1 triggered whitening of brown adipose tissue by suppressing mitochondrial biogenesis. Mechanistically, CLCF1 bound and activated ciliary neurotrophic factor receptor (CNTFR) and augmented signal transducer and activator of transcription 3 (STAT3) signaling. STAT3 transcriptionally inhibited both peroxisome proliferator-activated receptor-γ coactivator (PGC) 1α and 1ß, which thereafter restrained mitochondrial biogenesis in adipocytes. Inhibition of CNTFR or STAT3 could diminish the inhibitory effects of CLCF1 on mitochondrial biogenesis and thermogenesis. As a result, CLCF1-TG mice were predisposed to develop metabolic dysfunction even without external metabolic stress. Our findings revealed a brake signal on nonshivering thermogenesis and suggested that targeting this pathway could be used to restore brown fat activity and systemic metabolic homeostasis in obesity.

Differential responses to UCP1 ablation in classical brown versus beige fat, despite a parallel increase in sympathetic innervation.

In the cold, the absence of the mitochondrial uncoupling protein 1 (UCP1) results in hyper-recruitment of beige fat, but classical brown fat becomes atrophied. Here we examine possible mechanisms underlying this phenomenon. We confirm that in brown fat from UCP1-knockout (UCP1-KO) mice acclimated to the cold, the levels of mitochondrial respiratory chain proteins were diminished; however, in beige fat, the mitochondria seemed to be unaffected. The macrophages that accumulated massively not only in brown fat but also in beige fat of the UCP1-KO mice acclimated to cold did not express tyrosine hydroxylase, the norepinephrine transporter (NET) and monoamine oxidase-A (MAO-A). Consequently, they could not influence the tissues through the synthesis or degradation of norepinephrine. Unexpectedly, in the cold, both brown and beige adipocytes from UCP1-KO mice acquired an ability to express MAO-A. Adipose tissue norepinephrine was exclusively of sympathetic origin, and sympathetic innervation significantly increased in both tissues of UCP1-KO mice. Importantly, the magnitude of sympathetic innervation and the expression levels of genes induced by adrenergic stimulation were much higher in brown fat. Therefore, we conclude that no qualitative differences in innervation or macrophage character could explain the contrasting reactions of brown versus beige adipose tissues to UCP1-ablation. Instead, these contrasting responses may be explained by quantitative differences in sympathetic innervation: the beige adipose depot from the UCP1-KO mice responded to cold acclimation in a canonical manner and displayed enhanced recruitment, while the atrophy of brown fat lacking UCP1 may be seen as a consequence of supraphysiological adrenergic stimulation in this tissue.

mTORC1 and 2 adrenergic regulation and function in brown adipose tissue.

Brown adipose tissue (BAT) thermogenesis results from the uncoupling of mitochondrial inner membrane proton gradient mediated by the uncoupling protein 1 (UCP-1), which is activated by lipolysis-derived fatty acids. Norepinephrine (NE) secreted by sympathetic innervation not only activates BAT lipolysis and UCP-1, but uniquely in brown adipocytes, promotes "futile" metabolic cycles and enhances BAT thermogenic capacity by increasing UCP-1 content, mitochondrial biogenesis and brown adipocyte hyperplasia. NE exerts these actions by triggering signaling in the canonical G protein coupled b adrenergic receptors, cAMP and protein kinase A (PKA) pathway which, in brown adipocyte, is under a complex and intricated crosstalk with important growth-promoting signaling pathways such as those of mechanistic target of rapamycin (mTOR) complexes 1 (mTORC1) and 2 (mTORC2). This article reviews evidence suggesting that mTOR complexes are modulated by and participate in the thermogenic, metabolic, and growth-promoting effects elicited by NE in BAT and discusses current gaps and future directions in this field of research.

Tracers and Imaging of Fatty Acid and Energy Metabolism of Human Adipose Tissues.

White adipose tissue and brown adipose tissue (WAT and BAT) regulate fatty acid metabolism and control lipid fluxes to other organs. Dysfunction of these key metabolic processes contributes to organ insulin resistance and inflammation leading to chronic diseases such as type 2 diabetes, metabolic dysfunction-associated steatohepatitis, and cardiovascular diseases. Metabolic tracers combined with molecular imaging methods are powerful tools for the investigation of these pathogenic mechanisms. Herein, I review some of the positron emission tomography and magnetic resonance imaging methods combined with stable isotopic metabolic tracers to investigate fatty acid and energy metabolism, focusing on human WAT and BAT metabolism. I will discuss the complementary strengths offered by these methods for human investigations and current gaps in the field.

Terrestrial Birth and Body Size Tune UCP1 Functionality in Seals.

The molecular evolution of the mammalian heater protein UCP1 is a powerful biomarker to understand thermoregulatory strategies during species radiation into extreme climates, such as aquatic life with high thermal conductivity. While fully aquatic mammals lost UCP1, most semiaquatic seals display intact UCP1 genes, apart from large elephant seals. Here, we show that UCP1 thermogenic activity of the small-bodied harbor seal is equally potent compared to terrestrial orthologs, emphasizing its importance for neonatal survival on land. In contrast, elephant seal UCP1 does not display thermogenic activity, not even when translating a repaired or a recently highlighted truncated version. Thus, the thermogenic benefits for neonatal survival during terrestrial birth in semiaquatic pinnipeds maintained evolutionary selection pressure on UCP1 function and were only outweighed by extreme body sizes among elephant seals, fully eliminating UCP1-dependent thermogenesis.

Brown adipose tissue-derived exosomes improve polycystic ovary syndrome in mice via STAT3/GPX4 signaling pathway.

Polycystic ovary syndrome (PCOS) is associated with impaired adipose tissue physiology. Elevated brown adipose tissue (BAT) mass or activity has shown potential in the treatment of PCOS. In this study, we aimed to investigate whether BAT-derived exosomes (BAT-Exos), as potential biomarkers of BAT activity, exert similar benefits as BAT in the treatment of PCOS. PCOS was induced in female C57BL/6J mice orally administered 1 mg/kg of letrozole for 21 days. Subsequently, the animals underwent transplantation with BAT or administered BAT-Exos (200 µg) isolated from young healthy mice via the tail vein; healthy female mice were used as controls. The results indicate that BAT-Exos treatment significantly reduced body weight and improved insulin resistance in PCOS mice. In addition, BAT-Exos improved ovulation function by reversing the acyclicity of the estrous cycle, decreasing circulating luteinizing hormone and testosterone, recovering ovarian performance, and improving oocyte quality, leading to a higher pregnancy rate and litter size. Furthermore, western blotting revealed reduced expression of signal transducer and activator of transcription 3 (STAT3) and increased expression of glutathione peroxidase 4 (GPX4) in the ovaries of mice in the BAT-Exos group. To further explore the role of the STAT3/GPX4 signaling pathway in PCOS mice, we treated the mice with an intraperitoneal injection of 5 mg/kg stattic, a STAT3 inhibitor. Consistent with BAT-Exos treatment, the administration of stattic rescued letrozole-induced PCOS phenotypes. These findings suggest that BAT-Exos treatment might be a potential therapeutic strategy for PCOS and that the STAT3/GPX4 signaling pathway is a critical therapeutic target for PCOS.