Mitochondrial lipidomes are tissue specific - low cholesterol contents relate to UCP1 activity.

Lipid composition is conserved within sub-cellular compartments to maintain cell function. Lipidomic analyses of liver, muscle, white and brown adipose tissue (BAT) mitochondria revealed substantial differences in their glycerophospholipid (GPL) and free cholesterol (FC) contents. The GPL to FC ratio was 50-fold higher in brown than white adipose tissue mitochondria. Their purity was verified by comparison of proteomes with ER and mitochondria-associated membranes. A lipid signature containing PC and FC, calculated from the lipidomic profiles, allowed differentiation of mitochondria from BAT of mice housed at different temperatures. Elevating FC in BAT mitochondria prevented uncoupling protein (UCP) 1 function, whereas increasing GPL boosted it. Similarly, STARD3 overexpression facilitating mitochondrial FC import inhibited UCP1 function in primary brown adipocytes, whereas a knockdown promoted it. We conclude that the mitochondrial GPL/FC ratio is key for BAT function and propose that targeting it might be a promising strategy to promote UCP1 activity.

PAT2 regulates vATPase assembly and lysosomal acidification in brown adipocytes.

OBJECTIVE: Brown adipocytes play a key role in maintaining body temperature as well as glucose and lipid homeostasis. However, brown adipocytes need to adapt their thermogenic activity and substrate utilization to changes in nutrient availability. Amongst the multiple factors influencing brown adipocyte activity, autophagy is an important regulatory element of thermogenic capacity and activity. Nevertheless, a specific sensing mechanism of extracellular amino acid availability linking autophagy to nutrient availability in brown adipocytes is unknown. METHODS: To characterize the role of the amino acid transporter PAT2/SLC36A2 in brown adipocytes, loss or gain of function of PAT2 were studied with respect to differentiation, subcellular localization, lysosomal activity and autophagy. Activity of vATPase was evaluated by quenching of EGFP fused to LC3 or FITC-dextran loaded lysosomes in brown adipocytes upon amino acid starvation, whereas the effect of PAT2 on assembly of the vATPase was investigated by Native-PAGE. RESULTS: We show that PAT2 translocates from the plasma membrane to the lysosome in response to amino acid withdrawal. Loss or overexpression of PAT2 impair lysosomal acidification and starvation-induced S6K re-phosphorylation, as PAT2 facilitates the assembly of the lysosomal vATPase, by recruitment of the cytoplasmic V1 subunit to the lysosome. CONCLUSIONS: PAT2 is an important sensor of extracellular amino acids and regulator of lysosomal acidification in brown adipocytes.

Loss of UCP1 function augments recruitment of futile lipid cycling for thermogenesis in murine brown fat.

OBJECTIVE: Classical ATP-independent non-shivering thermogenesis enabled by uncoupling protein 1 (UCP1) in brown adipose tissue (BAT) is activated, but not essential for survival, in the cold. It has long been suspected that futile ATP-consuming substrate cycles also contribute to thermogenesis and can partially compensate for the genetic ablation of UCP1 in mouse models. Futile ATP-dependent thermogenesis could thereby enable survival in the cold even when brown fat is less abundant or missing. METHODS: In this study, we explore different potential sources of UCP1-independent thermogenesis and identify a futile ATP-consuming triglyceride/fatty acid cycle as the main contributor to cellular heat production in brown adipocytes lacking UCP1. We uncover the mechanism on a molecular level and pinpoint the key enzymes involved using pharmacological and genetic interference. RESULTS: ATGL is the most important lipase in terms of releasing fatty acids from lipid droplets, while DGAT1 accounts for the majority of fatty acid re-esterification in UCP1-ablated brown adipocytes. Furthermore, we demonstrate that chronic cold exposure causes a pronounced remodeling of adipose tissues and leads to the recruitment of lipid cycling capacity specifically in BAT of UCP1-knockout mice, possibly fueled by fatty acids from white fat. Quantification of triglyceride/fatty acid cycling clearly shows that UCP1-ablated animals significantly increase turnover rates at room temperature and below. CONCLUSION: Our results suggest an important role for futile lipid cycling in adaptive thermogenesis and total energy expenditure.

Fibroblast growth factor induced Ucp1 expression in preadipocytes requires PGE2 biosynthesis and glycolytic flux.

High uncoupling protein 1 (Ucp1) expression is a characteristic of differentiated brown adipocytes and is linked to adipogenic differentiation. Paracrine fibroblast growth factor 8b (FGF8b) strongly induces Ucp1 transcription in white adipocytes independent of adipogenesis. Here, we report that FGF8b and other paracrine FGFs act on brown and white preadipocytes to upregulate Ucp1 expression via a FGFR1-MEK1/2-ERK1/2 axis, independent of adipogenesis. Transcriptomic analysis revealed an upregulation of prostaglandin biosynthesis and glycolysis upon Fgf8b treatment of preadipocytes. Oxylipin measurement by LC-MS/MS in FGF8b conditioned media identified prostaglandin E2 as a putative mediator of FGF8b induced Ucp1 transcription. RNA interference and pharmacological inhibition of the prostaglandin E2 biosynthetic pathway confirmed that PGE2 is causally involved in the control over Ucp1 transcription. Importantly, impairment of or failure to induce glycolytic flux blunted the induction of Ucp1, even in the presence of PGE2 . Lastly, a screening of transcription factors identified Nrf1 and Hes1 as required regulators of FGF8b induced Ucp1 expression. Thus, we conclude that paracrine FGFs co-regulate prostaglandin and glucose metabolism to induce Ucp1 expression in a Nrf1/Hes1-dependent manner in preadipocytes, revealing a novel regulatory network in control of Ucp1 expression in a formerly unrecognized cell type.

Isolation, Culture, and Functional Analysis of Murine Thermogenic Adipocytes.

Studying brown and brite adipose tissue requires precise and reliable quantification of cellular thermogenesis. This protocol describes the isolation of primary murine pre-adipocytes, differentiation into thermogenic brown and brite adipocytes, and subsequent oxygen consumption analysis. Commonly applied procedures only measure basal and maximal proton leak-linked oxygen consumption but not explicitly uncoupling protein 1 (UCP1)-dependent respiration. Meaningful oxygen consumption analyses require (1) the activation of UCP1, (2) control over intracellular free-fatty-acid levels, and (3) inhibition of ATP-consuming futile cycles. For complete details on the use and execution of this protocol, please refer to Li et al. (2014, 2017, 2018) and Schweizer et al. (2018).

The lipidome of primary murine white, brite, and brown adipocytes-Impact of beta-adrenergic stimulation.

Lipid species patterns are conserved within cells to maintain physicochemical properties of membranes and cellular functions. We present the lipidome, including sterols, glycerolipids (GLs), glycerophospholipids (GPLs), and sphingolipids (SLs), of primary ex vivo differentiated (I) white, (II) brite, and (III) brown adipocytes derived from primary preadipocytes isolated from (I) epididymal white, (II) inguinal white, and (III) intrascapular brown adipose tissue. Quantitative lipidomics revealed significantly decreased fractions of phosphatidylcholine (PC) and phosphatidylethanolamine (PE), with longer (C > 36) and more polyunsaturated species, as well as lower levels of cardiolipin (CL) in white than in brite and brown adipocytes. Together, the brite and brown lipidome was comparable and indicates differences in membrane lipid packing density compared with white adipocytes. Changes in ceramide species profile could be related to the degree of browning. Beta-adrenergic stimulation of brown adipocytes led to generation of saturated lyso-PC (LPC) increasing uncoupling protein (UCP) 1-mediated leak respiration. Application of stable isotope labeling showed that LPC formation was balanced by an increased de novo synthesis of PC.

Substrate fluxes in brown adipocytes upon adrenergic stimulation and uncoupling protein 1 ablation.

Brown adipocytes are highly specialized cells with the unique metabolic ability to dissipate chemical energy in the form of heat. We determined and inferred the flux of a number of key catabolic metabolites, their changes in response to adrenergic stimulation, and the dependency on the presence of the thermogenic uncoupling protein 1 and/or oxidative phosphorylation. This study provides reference values to approximate flux rates from a limited set of measured parameters in the future and thereby allows to evaluate the plausibility of claims about the capacity of metabolic adaptations or manipulations. From the resulting model, we delineate that in brown adipocytes (1) free fatty acids are a significant contributor to extracellular acidification, (2) glycogen is the dominant glycolytic substrate source in the acute response to an adrenergic stimulus, and (3) the futile cycling of free fatty acids between lipolysis and re-esterification into triglyceride provides a mechanism for uncoupling protein 1-independent, non-shivering thermogenesis in brown adipocytes.

Non-adrenergic control of lipolysis and thermogenesis in adipose tissues.

The enormous plasticity of adipose tissues, to rapidly adapt to altered physiological states of energy demand, is under neuronal and endocrine control. In energy balance, lipolysis of triacylglycerols and re-esterification of free fatty acids are opposing processes operating in parallel at identical rates, thus allowing a more dynamic transition from anabolism to catabolism, and vice versa. In response to alterations in the state of energy balance, one of the two processes predominates, enabling the efficient mobilization or storage of energy in a negative or positive energy balance, respectively. The release of noradrenaline from the sympathetic nervous system activates lipolysis in a depot-specific manner by initiating the canonical adrenergic receptor-Gs-protein-adenylyl cyclase-cyclic adenosine monophosphate-protein kinase A pathway, targeting proteins of the lipolytic machinery associated with the interface of the lipid droplets. In brown and brite adipocytes, lipolysis stimulated by this signaling pathway is a prerequisite for the activation of non-shivering thermogenesis. Free fatty acids released by lipolysis are direct activators of uncoupling protein 1-mediated leak respiration. Thus, pro- and anti-lipolytic mediators are bona fide modulators of thermogenesis in brown and brite adipocytes. In this Review, we discuss adrenergic and non-adrenergic mechanisms controlling lipolysis and thermogenesis and provide a comprehensive overview of pro- and anti-lipolytic mediators.