Adipocyte lipolysis: from molecular mechanisms of regulation to disease and therapeutics.

Fatty acids (FAs) are stored safely in the form of triacylglycerol (TAG) in lipid droplet (LD) organelles by professional storage cells called adipocytes. These lipids are mobilized during adipocyte lipolysis, the fundamental process of hydrolyzing TAG to FAs for internal or systemic energy use. Our understanding of adipocyte lipolysis has greatly increased over the past 50 years from a basic enzymatic process to a dynamic regulatory one, involving the assembly and disassembly of protein complexes on the surface of LDs. These dynamic interactions are regulated by hormonal signals such as catecholamines and insulin which have opposing effects on lipolysis. Upon stimulation, patatin-like phospholipase domain containing 2 (PNPLA2)/adipocyte triglyceride lipase (ATGL), the rate limiting enzyme for TAG hydrolysis, is activated by the interaction with its co-activator, alpha/beta hydrolase domain-containing protein 5 (ABHD5), which is normally bound to perilipin 1 (PLIN1). Recently identified negative regulators of lipolysis include G0/G1 switch gene 2 (G0S2) and PNPLA3 which interact with PNPLA2 and ABHD5, respectively. This review focuses on the dynamic protein-protein interactions involved in lipolysis and discusses some of the emerging concepts in the control of lipolysis that include allosteric regulation and protein turnover. Furthermore, recent research demonstrates that many of the proteins involved in adipocyte lipolysis are multifunctional enzymes and that lipolysis can mediate homeostatic metabolic signals at both the cellular and whole-body level to promote inter-organ communication. Finally, adipocyte lipolysis is involved in various diseases such as cancer, type 2 diabetes and fatty liver disease, and targeting adipocyte lipolysis is of therapeutic interest.

Adipocyte lipolysis-stimulated interleukin-6 production requires sphingosine kinase 1 activity.

Adipocyte lipolysis can increase the production of inflammatory cytokines such as interleukin-6 (IL-6) that promote insulin resistance. However, the mechanisms that link lipolysis with inflammation remain elusive. Acute activation of ß3-adrenergic receptors (ADRB3) triggers lipolysis and up-regulates production of IL-6 in adipocytes, and both of these effects are blocked by pharmacological inhibition of hormone-sensitive lipase. We report that stimulation of ADRB3 induces expression of sphingosine kinase 1 (SphK1) and increases sphingosine 1-phosphate production in adipocytes in a manner that also depends on hormone-sensitive lipase activity. Mechanistically, we found that adipose lipolysis-induced SphK1 up-regulation is mediated by the c-Jun N-terminal kinase (JNK)/activating protein-1 signaling pathway. Inhibition of SphK1 by sphingosine kinase inhibitor 2 diminished the ADRB3-induced IL-6 production both in vitro and in vivo. Induction of IL-6 by ADRB3 activation was suppressed by siRNA knockdown of Sphk1 in cultured adipocytes and was severely attenuated in Sphk1 null mice. Conversely, ectopic expression of SphK1 increased IL-6 expression in adipocytes. Collectively, these data demonstrate that SphK1 is a critical mediator in lipolysis-triggered inflammation in adipocytes.

Adipose tissue plasticity from WAT to BAT and in between.

Adipose tissue plays an essential role in regulating energy balance through its metabolic, cellular and endocrine functions. Adipose tissue has been historically classified into anabolic white adipose tissue and catabolic brown adipose tissue. An explosion of new data, however, points to the remarkable heterogeneity among the cells types that can become adipocytes, as well as the inherent metabolic plasticity of mature cells. These data indicate that targeting cellular and metabolic plasticity of adipose tissue might provide new avenues for treatment of obesity-related diseases. This review will discuss the developmental origins of adipose tissue, the cellular complexity of adipose tissues, and the identification of progenitors that contribute to adipogenesis throughout development. We will touch upon the pathological remodeling of adipose tissue and discuss how our understanding of adipose tissue remodeling can uncover new therapeutic targets. This article is part of a Special Issue entitled: Modulation of Adipose Tissue in Health and Disease.

Coupling of lipolysis and de novo lipogenesis in brown, beige, and white adipose tissues during chronic ß3-adrenergic receptor activation.

Chronic activation of ß3-adrenergic receptors (ß3-ARs) expands the catabolic activity of both brown and white adipose tissue by engaging uncoupling protein 1 (UCP1)-dependent and UCP1-independent processes. The present work examined de novo lipogenesis (DNL) and TG/glycerol dynamics in classic brown, subcutaneous "beige," and classic white adipose tissues during sustained ß3-AR activation by CL 316,243 (CL) and also addressed the contribution of TG hydrolysis to these dynamics. CL treatment for 7 days dramatically increased DNL and TG turnover similarly in all adipose depots, despite great differences in UCP1 abundance. Increased lipid turnover was accompanied by the simultaneous upregulation of genes involved in FAS, glycerol metabolism, and FA oxidation. Inducible, adipocyte-specific deletion of adipose TG lipase (ATGL), the rate-limiting enzyme for lipolysis, demonstrates that TG hydrolysis is required for CL-induced increases in DNL, TG turnover, and mitochondrial electron transport in all depots. Interestingly, the effect of ATGL deletion on induction of specific genes involved in FA oxidation and synthesis varied among fat depots. Overall, these studies indicate that FAS and FA oxidation are tightly coupled in adipose tissues during chronic adrenergic activation, and this effect critically depends on the activity of adipocyte ATGL.

Inducible brown adipocytes in subcutaneous inguinal white fat: the role of continuous sympathetic stimulation.

Brown adipocytes (BA) generate heat in response to sympathetic activation and are the main site of nonshivering thermogenesis in mammals. Although most BA are located in classic brown adipose tissue depots, BA are also abundant in the inguinal white adipose tissue (iWAT) before weaning. The number of BA is correlated with the density of sympathetic innervation in iWAT; however, the role of continuous sympathetic tone in the establishment and maintenance of BA in WAT has not been investigated. BA marker expression in iWAT was abundant in weaning mice but was greatly reduced by 8 wk of age. Nonetheless, BA phenotype could be rapidly reinstated by acute ß3-adrenergic stimulation with CL-316,243 (CL). Genetic tagging of adipocytes with adiponectin-CreER(T2) demonstrated that CL reinstates uncoupling protein 1 (UCP1) expression in adipocytes that were present before weaning. Chronic surgical denervation dramatically reduced the ability of CL to induce the expression of UCP1 and other BA markers in the tissue as a whole, and this loss of responsiveness was prevented by concurrent treatment with CL. These results indicate that ongoing sympathetic activity is critical to preserve the ability of iWAT fat cells to express a BA phenotype upon adrenergic stimulation.

The Intersection of Genetic Factors, Aberrant Nutrient Metabolism and Oxidative Stress in the Progression of Cardiometabolic Disease.

Cardiometabolic disease (CMD), which encompasses metabolic-associated fatty liver disease (MAFLD), chronic kidney disease (CKD) and cardiovascular disease (CVD), has been increasing considerably in the past 50 years. CMD is a complex disease that can be influenced by genetics and environmental factors such as diet. With the increased reliance on processed foods containing saturated fats, fructose and cholesterol, a mechanistic understanding of how these molecules cause metabolic disease is required. A major pathway by which excessive nutrients contribute to CMD is through oxidative stress. In this review, we discuss how oxidative stress can drive CMD and the role of aberrant nutrient metabolism and genetic risk factors and how they potentially interact to promote progression of MAFLD, CVD and CKD. This review will focus on genetic mutations that are known to alter nutrient metabolism. We discuss the major genetic risk factors for MAFLD, which include Patatin-like phospholipase domain-containing protein 3 (PNPLA3), Membrane Bound O-Acyltransferase Domain Containing 7 (MBOAT7) and Transmembrane 6 Superfamily Member 2 (TM6SF2). In addition, mutations that prevent nutrient uptake cause hypercholesterolemia that contributes to CVD. We also discuss the mechanisms by which MAFLD, CKD and CVD are mutually associated with one another. In addition, some of the genetic risk factors which are associated with MAFLD and CVD are also associated with CKD, while some genetic risk factors seem to dissociate one disease from the other. Through a better understanding of the causative effect of genetic mutations in CMD and how aberrant nutrient metabolism intersects with our genetics, novel therapies and precision approaches can be developed for treating CMD.

Direct effects of adipocyte lipolysis on AMPK through intracellular long-chain acyl-CoA signaling.

Long-chain acyl-CoAs (LC-acyl-CoAs) are important intermediary metabolites and are also thought to function as intracellular signaling molecules; however, the direct effects of LC-acyl-CoAs have been difficult to determine in real-time and dissociate from Protein Kinase A (PKA) signaling. Here, we examined the direct role of lipolysis in generating intracellular LC-acyl-CoAs and activating AMPK in white adipocytes by pharmacological activation of ABHD5 (also known as CGI-58), a lipase co-activator. Activation of lipolysis in 3T3-L1 adipocytes independent of PKA with synthetic ABHD5 ligands, resulted in greater activation of AMPK compared to receptor-mediated activation with isoproterenol, a ß-adrenergic receptor agonist. Importantly, the effect of pharmacological activation of ABHD5 on AMPK activation was blocked by inhibiting ATGL, the rate-limiting enzyme for triacylglycerol hydrolysis. Utilizing a novel FRET sensor to detect intracellular LC-acyl-CoAs, we demonstrate that stimulation of lipolysis in 3T3-L1 adipocytes increased the production of LC-acyl-CoAs, an effect which was blocked by inhibition of ATGL. Moreover, ATGL inhibition blocked AMPKß1 S108 phosphorylation, a site required for allosteric regulation. Increasing intracellular LC-acyl-CoAs by removal of BSA in the media and pharmacological inhibition of DGAT1 and 2 resulted in greater activation of AMPK. Finally, inhibiting LC-acyl-CoA generation reduced activation of AMPK; however, did not lower energy charge. Overall, results demonstrate that lipolysis in white adipocytes directly results in allosteric activation of AMPK through the generation of LC-acyl-CoAs.

Lipolytic products activate peroxisome proliferator-activated receptor (PPAR) α and δ in brown adipocytes to match fatty acid oxidation with supply.

ß-Adrenergic receptors (ß-ARs) promote brown adipose tissue (BAT) thermogenesis by mobilizing fatty acids and inducing the expression of oxidative genes. ß-AR activation increases the expression of oxidative genes by elevating cAMP, but whether lipolytic products can modulate gene expression is not known. This study examined the role that adipose triglyceride lipase (ATGL) and hormone-sensitive lipase (HSL) plays in the induction of gene expression. Activation of brown adipocytes by ß-AR agonism or 8-bromo-cyclic AMP increased the expression of PGC1α, PDK4, PPARα, uncoupling protein 1 (UCP1), and neuron-derived orphan receptor-1 (NOR-1), and concurrent inhibition of HSL reduced the induction of PGC1α, PDK4, PPARα, and UCP1 but not NOR-1. Similar results were observed in the BAT of mice following pharmacological or genetic inhibition of HSL and in brown adipocytes with stable knockdown of ATGL. Conversely, treatments that increase endogenous fatty acids elevated the expression of oxidative genes. Pharmacological antagonism and siRNA knockdown indicate that PPARα and PPARδ modulate the induction of oxidative genes by ß-AR agonism. Using a live cell fluorescent reporter assay of PPAR activation, we demonstrated that ligands for PPARα and -δ, but not PPARγ, were rapidly generated at the lipid droplet surface and could transcriptionally activate PPARα and -δ. Knockdown of ATGL reduced cAMP-mediated induction of genes involved in fatty acid oxidation and oxidative phosphorylation. Consequently, ATGL knockdown reduced maximal oxidation of fatty acids, but not pyruvate, in response to cAMP stimulation. Overall, the results indicate that lipolytic products can activate PPARα and PPARδ in brown adipocytes, thereby expanding the oxidative capacity to match enhanced fatty acid supply.

Thermogenic Adipose Redox Mechanisms: Potential Targets for Metabolic Disease Therapies.

Metabolic diseases, such as diabetes and non-alcoholic fatty liver disease (NAFLD), have several negative health outcomes on affected humans. Dysregulated energy metabolism is a key component underlying the pathophysiology of these conditions. Adipose tissue is a fundamental regulator of energy homeostasis that utilizes several redox reactions to carry out the metabolism. Brown and beige adipose tissues, in particular, perform highly oxidative reactions during non-shivering thermogenesis to dissipate energy as heat. The appropriate regulation of energy metabolism then requires coordinated antioxidant mechanisms to counterbalance the oxidation reactions. Indeed, non-shivering thermogenesis activation can cause striking changes in concentrations of both oxidants and antioxidants in order to adapt to various oxidative environments. Current therapeutic options for metabolic diseases either translate poorly from rodent models to humans (in part due to the challenges of creating a physiologically relevant rodent model) or tend to have numerous side effects, necessitating novel therapies. As increased brown adipose tissue activity results in enhanced energy expenditure and is associated with beneficial effects on metabolic health, such as decreased obesity, it has gathered great interest as a modulator of metabolic disease. One potential reason for the beneficial health effects may be that although non-shivering thermogenesis is enormously oxidative, it is also associated with decreased oxidant formation after its activation. However, targeting its redox mechanisms specifically to alter metabolic disease remains an underexplored area. Therefore, this review will discuss the role of adipose tissue in energy homeostasis, non-shivering thermogenesis in adults, and redox mechanisms that may serve as novel therapeutic targets of metabolic disease.

A FRET sensor for the real-time detection of long chain acyl-CoAs and synthetic ABHD5 ligands.

Intracellular long-chain acyl-coenzyme As (LC-acyl-CoAs) are thought to be under tight spatial and temporal controls, yet the ability to image LC-acyl-CoAs in live cells is lacking. Here, we developed a fluorescence resonance energy transfer (FRET) sensor for LC-acyl-CoAs based on the allosterically regulated interaction between α/ß hydrolase domain-containing 5 (ABHD5) and Perilipin 5. The genetically encoded sensor rapidly detects intracellular LC-acyl-CoAs generated from exogenous and endogenous fatty acids (FAs), as well as synthetic ABHD5 ligands. Stimulation of lipolysis in brown adipocytes elevated intracellular LC-acyl-CoAs in a cyclic fashion, which was eliminated by inhibiting PNPLA2 (ATGL), the major triglyceride lipase. Interestingly, inhibition of LC-acyl-CoA transport into mitochondria elevated intracellular LC-acyl-CoAs and dampened their cycling. Together, these observations reveal an intimate feedback control between LC-acyl-CoA generation from lipolysis and utilization in mitochondria. We anticipate that this sensor will be an important tool to dissect intracellular LC-acyl-CoA dynamics as well to discover novel synthetic ABHD5 ligands.

Interactions of perilipin-5 (Plin5) with adipose triglyceride lipase.

Members of the perilipin family of lipid droplet scaffold proteins are thought to play important roles in tissue-specific regulation of triglyceride metabolism, but the mechanisms involved are not fully understood. Present results indicate that adipose triglyceride lipase (Atgl) interacts with perilipin-5 (Plin5) but not perilipin-1 (Plin1). Protein interaction assays in live cells and in situ binding experiments showed that Atgl and its protein activator, α-ß-hydrolase domain-containing 5 (Abhd5), each bind Plin5. Surprisingly, competition experiments indicated that individual Plin5 molecules bind Atgl or Abhd5 but not both simultaneously. Thus, the ability of Plin5 to concentrate these proteins at droplet surfaces involves binding to different Plin5 molecules, possibly in an oligomeric complex. The association of Plin5-Abhd5 complexes on lipid droplet surfaces was more stable than Plin5-Atgl complexes, and oleic acid treatment selectively promoted the interaction of Plin5 and Abhd5. Analysis of chimeric and mutant perilipin proteins demonstrated that amino acids 200-463 are necessary and sufficient to bind both Atgl and Abhd5 and that the C-terminal 64 amino acids of Plin5 are critical for the differential binding of Atgl to Plin5 and Plin1. Mutant Plin5 that binds Abhd5 but not Atgl was defective in preventing neutral lipid accumulation compared with wild type Plin5, indicating that the ability of Plin5 to concentrate these proteins on lipid droplets is critical to functional Atgl activity in cells.

Fluorescent and Luminescent Methods to Detect Lipolysis.

Intracellular lipolysis, the hydrolysis of stored triacylglycerol to fatty acids and glycerol, is a core metabolic function of brown and white adipocytes. In brown adipocytes, mobilized fatty acids directly activate uncoupling protein 1, provide fuel for heat generation, and ligands of nuclear receptors that expand the thermogenic gene expression program. Lipolysis in white adipocytes mobilizes lipid energy for systemic use, including both shivering and non-shivering thermogenesis. In addition, most metabolic tissues, including muscle and liver, have the ability to store triacylglycerol and release fatty acids; thus, there is a general interest in measuring lipolysis in a wide array of cell types. Here we describe detailed protocols for the enzymatic detection of cellular fatty acid and glycerol efflux via fluorescent and colorimetric means, respectively. In addition, we also describe a genetically encoded luminescent detection system for intracellular fatty acids that is amenable to high-throughput analysis.

beta3-adrenergic receptor induction of adipocyte inflammation requires lipolytic activation of stress kinases p38 and JNK.

Activation of beta-adrenergic receptors (AR) in adipocytes triggers acute changes in metabolism that can alter patterns of gene expression. This work examined the mechanisms by which activation of hormone sensitive lipase (HSL) induces expression of inflammatory cytokines in adipocytes in vivo and model adipocytes in vitro. beta3-AR activation in mice triggered expression of inflammatory genes CCL2, IL-6, and PAI-1, as well as endoplasmic reticulum (ER) stress markers GRP78 and CHOP. Pharmacological inhibition of HSL blocked induction of inflammatory genes, but not ER stress markers. Promoting intracellular accumulation of free fatty acids (FFAs) in 3T3-L1 adipocytes increased expression of inflammatory cytokines, whereas inhibiting ceramide synthesis partly blocked PAI-1 expression, but not IL-6. Induction of inflammatory markers in vivo and in vitro was preceded by phosphorylation of p38 and JNK, and inhibition of HSL prevented activation of these kinases. Experiments with pharmacological inhibitors of specific MAP kinases demonstrated the importance of p38 MAPK as a mediator of lipolysis-induced inflammation in vivo and in vitro. Together, these results demonstrate that FFAs liberated by HSL activate p38 and JNK, and p38 mediates pro-inflammatory cytokine expression in adipose tissue.

Posttranslational regulation of membrane type 1-matrix metalloproteinase (MT1-MMP) in mouse PTEN null prostate cancer cells: Enhanced surface expression and differential O-glycosylation of MT1-MMP.

Membrane type 1 (MT1)-matrix metalloproteinase (MT1-MMP) is a membrane-tethered MMP that has been shown to play a key role in promoting cancer cell invasion. MT1-MMP is highly expressed in bone metastasis of prostate cancer (PC) patients and promotes intraosseous tumor growth of PC cells in mice. The majority of metastatic prostate cancers harbor loss-of-function mutations or deletions of the tumor suppressor PTEN (phosphatase and tensin homologue deleted on chromosome ten). However, the role of PTEN inactivation in MT1-MMP expression in PC cells has not been examined. In this study, prostate epithelial cell lines derived from mice that are either heterozygous (PTEN(+/-)) or homozygous (PTEN(-/-)) for PTEN deletion or harboring a wild-type PTEN (PTEN(+/+)) were used to investigate the expression of MT1-MMP. We found that biallelic loss of PTEN is associated with posttranslational regulation of MT1-MMP protein in mouse PC cells. PTEN(-/-) PC cells display higher levels of MT1-MMP at the cell surface when compared to PTEN(+/+) and PTEN(+/-) cells and consequently exhibited enhanced migratory and collagen-invasive activities. MT1-MMP displayed by PTEN(-/-) cells is differentially O-glycosylated and exhibits a slow rate of turnover. MT1-MMP expression in PTEN(-/-) cells is under control of the PI3K/AKT signaling pathway, as determined using pharmacological inhibitors. Interestingly, rapamycin, an mTOR inhibitor, upregulates MT1-MMP expression in PTEN(+/+) cells via PI3K activity. Collectively, these data in a mouse prostate cell system uncover for the first time a novel and complex relationship between PTEN loss-mediated PI3K/AKT activation and posttranslational regulation of MT1-MMP, which may play a role in PC progression.

Intracellular fatty acids suppress ß-adrenergic induction of PKA-targeted gene expression in white adipocytes.

ß-Adrenergic receptor (ß-AR) activation elevates cAMP levels in fat cells and triggers both metabolic and transcriptional responses; however, the potential interactions between these pathways are poorly understood. This study investigated whether lipolysis affects ß-AR-mediated gene expression in adipocytes. Acute ß(3)-adrenergic receptor (ß(3)-AR) stimulation with CL 316,243 (CL) increased expression of PKA-targeted genes PCG-1α, UCP1, and NOR-1 in mouse white fat. Limiting lipolysis via inhibition of hormone-sensitive lipase (HSL), a direct target of PKA, sharply potentiated CL induction of PCG-1α, UCP1, and NOR-1. CL also induced greater expression of PKA-targeted genes in white fat of HSL-null mice compared with wild-type littermates, further indicating that HSL activity limits PKA-mediated gene expression. Inhibiting HSL in 3T3-L1 adipocytes also potentiated the induction of PGC-1α, UCP1, and NOR-1 by ß-AR activation, as did siRNA knockdown of adipose triglyceride lipase, the rate-limiting enzyme for lipolysis. Conversely, treatments that promote intracellular fatty acid accumulation suppressed induction of PGC-1α and UCP1 through ß-AR stimulation. Analysis of ß-adrenergic signaling indicated that excessive intracellular fatty acid production inhibits adenylyl cyclase activity and thereby reduces PKA signaling to the nucleus. Lastly, partially limiting lipolysis by inhibition of HSL increased the induction of oxidative gene expression and mitochondrial electron transport chain activity in white adipose tissue and facilitated fat loss in mice treated for 5 days with CL. Overall, our results demonstrate that fatty acids limit the upregulation of ß-AR-responsive genes in white adipocytes and suggest that limiting lipolysis may be a novel means of enhancing ß-AR signaling.

Dynamic interactions of ABHD5 with PNPLA3 regulate triacylglycerol metabolism in brown adipocytes.

Patatin-Like Phospholipase Domain Containing 2 (PNPLA2)/Adipose Triglyceride Lipase (ATGL) and PNPLA3/Adiponutrin are close paralogs that appear to have opposite functions on triacylglycerol (TAG) mobilization and storage. PNPLA2/ATGL is a major triglyceride lipase in adipose tissue and liver, whereas a common human variant of PNPLA3, I148M, greatly increases risk of hepatosteatosis. Nonetheless, the function of PNPLA3 and the mechanism by which the I148M variant promotes TAG accumulation are poorly understood. Here we demonstrate that PNPLA3 strongly interacts with α/ß hydrolase domain-containing 5 (ABHD5/CGI-58), an essential co-activator of PNPLA2/ATGL. Molecular imaging experiments demonstrate that PNPLA3 effectively competes with PNPLA2/ATGL for ABHD5, and that PNPLA3 I148M is more effective in this regard. Inducible overexpression of PNPLA3 I148M greatly suppressed PNPLA2/ATGL-dependent lipolysis and triggered massive TAG accumulation in brown adipocytes, and these effects were dependent on ABHD5. The interaction of PNPLA3 and ABHD5 can be regulated by fatty acid supplementation and synthetic ABHD5 ligands, raising the possibility that this interaction might be targeted for treatment of fatty liver disease.

Genetically-encoded sensors to detect fatty acid production and trafficking.

OBJECTIVE: Fatty acids are important for biological function; however, in excess, they can cause metabolic dysregulation. Methods to image and detect fatty acids in real time are lacking. Therefore, the current study examined the dynamics of fatty acid trafficking and signaling utilizing novel fluorescent and luminescent approaches. METHODS: We generated fluorescent and luminescent-based genetically-encoded sensors based upon the ligand-dependent interaction between PPARα and SRC-1 to image and detect cellular dynamics of fatty acid trafficking. RESULTS: The use of a fluorescent sensor demonstrates that fatty acids traffic rapidly from lipid droplets to the nucleus. Both major lipases ATGL and HSL contribute to fatty acid signaling from lipid droplet to nucleus, however, their dynamics differ. Furthermore, direct activation of lipolysis, independent of receptor-mediated signaling is sufficient to promote lipid droplet to nuclear trafficking of fatty acids. A luminescent-based sensor that reports intracellular fatty acid levels is amenable to high-throughput analysis. CONCLUSIONS: Fatty acids traffic from lipid droplets to the nucleus within minutes of stimulated lipolysis. Genetically-encoded fluorescent and luminescent based sensors can be used to probe the dynamics of fatty acid trafficking and signaling.

SERCA2b Cycles Its Way to UCP1-Independent Thermogenesis in Beige Fat.

A new study in Nature Medicine, by Ikeda et al. (2017), reports that calcium cycling in beige adipocytes elevates energy expenditure and glucose oxidation in the absence of uncoupling protein 1. Thermogenic calcium cycling in beige fat is mediated by SERCA2b and improves cold tolerance and metabolic status.