Association between thermogenic brown fat and genes under positive natural selection in circumpolar populations.

BACKGROUND: Adaptation to cold was essential for human migration across Eurasia. Non-shivering thermogenesis through brown adipose tissue (BAT) participates in cold adaptation because some genes involved in the differentiation and function of BAT exhibit signatures of positive natural selection in populations at high latitudes. Whether these genes are associated with the inter-individual variability in BAT thermogenesis remains unclear. In this study, we evaluated the potential associations between BAT activity and single nucleotide polymorphisms (SNPs) in candidate gene regions in East Asian populations. METHODS: BAT activity induced by mild cold exposure was measured in 399 healthy Japanese men and women using fluorodeoxyglucose-positron emission tomography and computed tomography (FDG-PET/CT). The capacity for cold-induced thermogenesis and fat oxidation was measured in 56 men. Association analyses with physiological traits were performed for 11 SNPs at six loci (LEPR, ANGPTL8, PLA2G2A, PLIN1, TBX15-WARS2, and FADS1) reported to be under positive natural selection. Associations found in the FDG-PET/CT population were further validated in 84 healthy East Asian men and women, in whom BAT activity was measured using infrared thermography. Associations between the SNP genotypes and BAT activity or other related traits were tested using multiple logistic and linear regression models. RESULTS: Of the 11 putative adaptive alleles of the six genes, two intronic SNPs in LEPR (rs1022981 and rs12405556) tended to be associated with higher BAT activity. However, these did not survive multiple test comparisons. Associations with lower body fat percentage, plasma triglyceride, insulin, and HOMA-IR levels were observed in the FDG-PET/CT population (P < 0.05). Other loci, including TBX15-WARS2, which is speculated to mediate cold adaptation in Greenland Inuits, did not show significant differences in BAT thermogenesis. CONCLUSIONS: Our results suggest a marginal but significant association between LEPR SNPs. However, robust supporting evidence was not established for the involvement of other loci under positive natural selection in cold adaptation through BAT thermogenesis in East Asian adults. Given the pleiotropic function of these genes, factors other than cold adaptation through BAT thermogenesis, such as diet adaptation, may contribute to positive natural selection at these loci.

Genetic evidence for involvement of ß2-adrenergic receptor in brown adipose tissue thermogenesis in humans.

BACKGROUND: Sympathetic activation of brown adipose tissue (BAT) thermogenesis can ameliorate obesity and related metabolic abnormalities. However, crucial subtypes of the ß-adrenergic receptor (AR), as well as effects of its genetic variants on functions of BAT, remains unclear in humans. We conducted association analyses of genes encoding ß-ARs and BAT activity in human adults. METHODS: Single nucleotide polymorphisms (SNPs) in ß1-, ß2-, and ß3-AR genes (ADRB1, ADRB2, and ADRB3) were tested for the association with BAT activity under mild cold exposure (19 °C, 2 h) in 399 healthy Japanese adults. BAT activity was measured using fluorodeoxyglucose-positron emission tomography and computed tomography (FDG-PET/CT). To validate the results, we assessed the effects of SNPs in the two independent populations comprising 277 healthy East Asian adults using near-infrared time-resolved spectroscopy (NIRTRS) or infrared thermography (IRT). Effects of SNPs on physiological responses to intensive cold exposure were tested in 42 healthy Japanese adult males using an artificial climate chamber. RESULTS: We found a significant association between a functional SNP (rs1042718) in ADRB2 and BAT activity assessed with FDG-PET/CT (p < 0.001). This SNP also showed an association with cold-induced thermogenesis in the population subset. Furthermore, the association was replicated in the two other independent populations; BAT activity was evaluated by NIRTRS or IRT (p < 0.05). This SNP did not show associations with oxygen consumption and cold-induced thermogenesis under intensive cold exposure, suggesting the irrelevance of shivering thermogenesis. The SNPs of ADRB1 and ADRB3 were not associated with these BAT-related traits. CONCLUSIONS: The present study supports the importance of ß2-AR in the sympathetic regulation of BAT thermogenesis in humans. The present collection of DNA samples is the largest to which information on the donor's BAT activity has been assigned and can serve as a reference for further in-depth understanding of human BAT function.

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.

Mitochondrial biogenesis in white adipose tissue mediated by JMJD1A-PGC-1 axis limits age-related metabolic disease.

Mitochondria play a vital role in non-shivering thermogenesis in both brown and subcutaneous white adipose tissues (BAT and scWAT, respectively). However, specific regulatory mechanisms driving mitochondrial function in these tissues have been unclear. Here we demonstrate that prolonged activation of ß-adrenergic signaling induces epigenetic modifications in scWAT, specifically targeting the enhancers for the mitochondria master regulator genes Pgc1a/b. This is mediated at least partially through JMJD1A, a histone demethylase that in response to ß-adrenergic signals, facilitates H3K9 demethylation of the Pgc1a/b enhancers, promoting mitochondrial biogenesis and the formation of beige adipocytes. Disruption of demethylation activity of JMJD1A in mice impairs activation of Pgc1a/b driven mitochondrial biogenesis and limits scWAT beiging, contributing to reduced energy expenditure, obesity, insulin resistance, and metabolic disorders. Notably, JMJD1A demethylase activity is not required for Pgc1a/b dependent thermogenic capacity of BAT especially during acute cold stress, emphasizing the importance of scWAT thermogenesis in overall energy metabolism.

Brown fat thermogenesis and branched-chain amino acids in metabolic disease.

Since the 1960s, researchers have recognized an association between elevated plasma branched chain amino acids (BCAA) and metabolic disease, including type 2 diabetes mellitus and obesity, but the cause for it remained poorly understood. Recent advances in metabolomics, advanced imaging techniques, and genetic analyses over the past decade have enabled newfound insights into the mechanism of BCAA metabolic dysregulation across a variety of peripheral tissues and its impact on metabolic disease, suggesting a key role for brown adipose tissue (BAT) in determining BCAA metabolic homeostasis. Previous investigations into BAT have emphasized fatty acids and glucose as substrates for BAT thermogenesis. Here, we address the importance of BAT in systemic BCAA metabolism, driven via the newly identified mitochondrial BCAA carrier (MBC), as well as the impact of BAT-driven BCAA clearance on glucose homeostasis and metabolic disease. The newly identified MBC offers new therapeutic avenues by which BAT activity may be enhanced to improve metabolic and cardiovascular health, as well as other diseases in which increases of circulating BCAA may play a role in pathogenicity.

Brown fat-associated postprandial thermogenesis in humans: Different effects of isocaloric meals rich in carbohydrate, fat, and protein.

The increase of whole-body energy expenditure seen after a single meal ingestion, referred to as diet-induced thermogenesis (DIT), substantially varies depending on the meal's macronutrient composition. Brown adipose tissue (BAT), a site of non-shivering thermogenesis, was reported to be involved in DIT. To examine the effects of meal composition on BAT-associated DIT in humans, healthy male participants underwent fluorodeoxyglucose-positron emission tomography to assess BAT activity, and respiratory gas analysis for 2 h after ingestion of a carbohydrate-, protein-, or fat-rich meal (C-meal, P-meal, and F-meal, respectively). The calculated DIT at 2 h was 6.44 ± 2.01%, 3.49 ± 2.00%, and 2.32 ± 0.90% of the ingested energy after the P-meal, C-meal, and F-meal, respectively. The DIT after C-meal ingestion correlated positively with BAT activity (P = 0.011), and was approximately twice greater in the group with high-BAT activity than in the group with low-BAT activity (4.35 ± 1.74% vs. 2.12 ± 1.76%, P < 0.035). Conversely, the DIT after F-meal or P-meal ingestion did not correlate with BAT activity, with no difference between the two groups. Thus, BAT has a significant role in DIT after ingestion of a carbohydrate-rich meal, but hardly after ingestion either protein- or fat-rich meal.

MYPT1-PP1ß phosphatase negatively regulates both chromatin landscape and co-activator recruitment for beige adipogenesis.

Protein kinase A promotes beige adipogenesis downstream from ß-adrenergic receptor signaling by phosphorylating proteins, including histone H3 lysine 9 (H3K9) demethylase JMJD1A. To ensure homeostasis, this process needs to be reversible however, this step is not well understood. We show that myosin phosphatase target subunit 1- protein phosphatase 1ß (MYPT1-PP1ß) phosphatase activity is inhibited via PKA-dependent phosphorylation, which increases phosphorylated JMJD1A and beige adipogenesis. Mechanistically, MYPT1-PP1ß depletion results in JMJD1A-mediated H3K9 demethylation and activation of the Ucp1 enhancer/promoter regions. Interestingly, MYPT1-PP1ß also dephosphorylates myosin light chain which regulates actomyosin tension-mediated activation of YAP/TAZ which directly stimulates Ucp1 gene expression. Pre-adipocyte specific Mypt1 deficiency increases cold tolerance with higher Ucp1 levels in subcutaneous white adipose tissues compared to control mice, confirming this regulatory mechanism in vivo. Thus, we have uncovered regulatory cross-talk involved in beige adipogenesis that coordinates epigenetic regulation with direct activation of the mechano-sensitive YAP/TAZ transcriptional co-activators.

The Effects of 10-Week Strength Training in the Winter on Brown-like Adipose Tissue Vascular Density.

There is no evidence of the effect of exercise training on human brown-like adipose tissue vascular density (BAT-d). Here, we report whether whole-body strength training (ST) in a cold environment increased BAT-d. The participants were 18 men aged 20-31 years. They were randomly assigned to two groups: one that performed ST twice a week at 75% intensity of one-repetition maximum for 10 weeks during winter (EX; n = 9) and a control group that did not perform ST (CT; n = 9). The total hemoglobin concentration in the supraclavicular region determined by time-resolved near-infrared spectroscopy was used as a parameter of BAT-d. ST volume (Tvol) was defined as the mean of the weight × repetition × sets of seven training movements. The number of occasions where the room temperature was lower than the median (NRcold) was counted as an index of potential cold exposure during ST. There was no significant between-group difference in BAT-d. Multiple regression analysis using body mass index, body fat percentage, NRcold, and Tvol as independent variables revealed that NRcold and Tvol were determined as predictive of changes in BAT-d. An appropriate combination of ST with cold environments could be an effective strategy for modulating BAT.

Post-translational control of beige fat biogenesis by PRDM16 stabilization.

Compelling evidence shows that brown and beige adipose tissue are protective against metabolic diseases1,2. PR domain-containing 16 (PRDM16) is a dominant activator of the biogenesis of beige adipocytes by forming a complex with transcriptional and epigenetic factors and is therefore an attractive target for improving metabolic health3-8. However, a lack of knowledge surrounding the regulation of PRDM16 protein expression hampered us from selectively targeting this transcriptional pathway. Here we identify CUL2-APPBP2 as the ubiquitin E3 ligase that determines PRDM16 protein stability by catalysing its polyubiquitination. Inhibition of CUL2-APPBP2 sufficiently extended the half-life of PRDM16 protein and promoted beige adipocyte biogenesis. By contrast, elevated CUL2-APPBP2 expression was found in aged adipose tissues and repressed adipocyte thermogenesis by degrading PRDM16 protein. Importantly, extended PRDM16 protein stability by adipocyte-specific deletion of CUL2-APPBP2 counteracted diet-induced obesity, glucose intolerance, insulin resistance and dyslipidaemia in mice. These results offer a cell-autonomous route to selectively activate the PRDM16 pathway in adipose tissues.

Melanin-concentrating hormone-producing neurons in the hypothalamus regulate brown adipose tissue and thus contribute to energy expenditure.

KEY POINTS: Melanin-concentrating hormone (MCH) neuron-ablated mice exhibit increased energy expenditure and reduced fat weight. Increased brown adipose tissue (BAT) activity and locomotor activity-independent energy expenditure contributed to body weight reduction in MCH neuron-ablated mice. MCH neurons send inhibitory input to the medullary raphe nucleus to modulate BAT activity. ABSTRACT: Hypothalamic melanin-concentrating hormone (MCH) peptide robustly affects energy homeostasis. However, it is unclear whether and how MCH-producing neurons, which contain and release a variety of neuropeptides/transmitters, regulate energy expenditure in the central nervous system and peripheral tissues. We thus examined the regulation of energy expenditure by MCH neurons, focusing on interscapular brown adipose tissue (BAT) activity. MCH neuron-ablated mice exhibited reduced body weight, increased oxygen consumption, and increased BAT activity, which improved locomotor activity-independent energy expenditure. Trans-neuronal retrograde tracing with the recombinant pseudorabies virus revealed that MCH neurons innervate BAT via the sympathetic premotor region in the medullary raphe nucleus (MRN). MRN neurons were activated by MCH neuron ablation. Therefore, endogenous MCH neuron activity negatively modulates energy expenditure via BAT inhibition. MRN neurons might receive inhibitory input from MCH neurons to suppress BAT activity.

Spatiotemporal dynamics of SETD5-containing NCoR-HDAC3 complex determines enhancer activation for adipogenesis.

Enhancer activation is essential for cell-type specific gene expression during cellular differentiation, however, how enhancers transition from a hypoacetylated "primed" state to a hyperacetylated-active state is incompletely understood. Here, we show SET domain-containing 5 (SETD5) forms a complex with NCoR-HDAC3 co-repressor that prevents histone acetylation of enhancers for two master adipogenic regulatory genes Cebpa and Pparg early during adipogenesis. The loss of SETD5 from the complex is followed by enhancer hyperacetylation. SETD5 protein levels were transiently increased and rapidly degraded prior to enhancer activation providing a mechanism for the loss of SETD5 during the transition. We show that induction of the CDC20 co-activator of the ubiquitin ligase leads to APC/C mediated degradation of SETD5 during the transition and this operates as a molecular switch that facilitates adipogenesis.

Bacteroides spp. promotes branched-chain amino acid catabolism in brown fat and inhibits obesity.

The gut microbiome has emerged as a key regulator of obesity; however, its role in brown adipose tissue (BAT) metabolism and association with obesity remain to be elucidated. We found that the levels of circulating branched-chain amino acids (BCAA) and their cognate α-ketoacids (BCKA) were significantly correlated with the body weight in humans and mice and that BCAA catabolic defects in BAT were associated with obesity in diet-induced obesity (DIO) mice. Pharmacological systemic enhancement of BCAA catabolic activity reduced plasma BCAA and BCKA levels and protected against obesity; these effects were reduced in BATectomized mice. DIO mice gavaged with Bacteroides dorei and Bacteroides vulgatus exhibited improved BAT BCAA catabolism and attenuated body weight gain, which were not observed in BATectomized DIO mice. Our data have highlighted a possible link between the gut microbiota and BAT BCAA catabolism and suggest that Bacteroides probiotics could be used for treating obesity.

Prolonged Treatment with Grains of Paradise (Aframomum melegueta) Extract Recruits Adaptive Thermogenesis and Reduces Body Fat in Humans with Low Brown Fat Activity.

Increasing adaptive thermogenesis through the activation of brown adipose tissue (BAT) is a promising practical strategy for preventing obesity and related disorders. Ingestion of a single dose of 40 mg of an extract of Grains of Paradise (GP), a ginger family species, reportedly triggers BAT thermogenesis in individuals with high but not in those with low BAT activity. We hypothesized that prolonged treatment with GP might revive BAT in individuals who have lost active BAT. In the present study, we recruited 9 healthy young male volunteers with reduced BAT that was assessed by fluorodeoxyglucose positron emission tomography and computed tomography (FDG-PET/CT) following 2-h cold exposure at 19ºC. The subjects ingested GP extract (40 mg/d) or placebo every day for 5 wk. Before and after the treatment with either GP or placebo, their body composition and BAT-dependent cold-induced thermogenesis (CIT)-a non-invasive index of BAT-were measured in a single-blinded, randomized, placebo-controlled cross-over design. Their whole-body resting energy expenditure at a thermoneutral condition remained unchanged following GP treatment. However, CIT after treatment was significantly higher in GP-treated individuals than in placebo-treated individuals. Body weight and fat-free mass did not change significantly following GP or placebo treatment. Notably, body fat percentage slightly but significantly decreased after GP treatment but not after placebo treatment. These results suggest that repeated ingestion of GP elevates adaptive thermogenesis through the re-activation of BAT, thereby reducing body fat in individuals with low BAT activity.

Metabolic flexibility via mitochondrial BCAA carrier SLC25A44 is required for optimal fever.

Importing necessary metabolites into the mitochondrial matrix is a crucial step of fuel choice during stress adaptation. Branched chain-amino acids (BCAAs) are essential amino acids needed for anabolic processes, but they are also imported into the mitochondria for catabolic reactions. What controls the distinct subcellular BCAA utilization during stress adaptation is insufficiently understood. The present study reports the role of SLC25A44, a recently identified mitochondrial BCAA carrier (MBC), in the regulation of mitochondrial BCAA catabolism and adaptive response to fever in rodents. We found that mitochondrial BCAA oxidation in brown adipose tissue (BAT) is significantly enhanced during fever in response to the pyrogenic mediator prostaglandin E2 (PGE2) and psychological stress in mice and rats. Genetic deletion of MBC in a BAT-specific manner blunts mitochondrial BCAA oxidation and non-shivering thermogenesis following intracerebroventricular PGE2 administration. At a cellular level, MBC is required for mitochondrial BCAA deamination as well as the synthesis of mitochondrial amino acids and TCA intermediates. Together, these results illuminate the role of MBC as a determinant of metabolic flexibility to mitochondrial BCAA catabolism and optimal febrile responses. This study also offers an opportunity to control fever by rewiring the subcellular BCAA fate.

Branched-chain α-ketoacids are preferentially reaminated and activate protein synthesis in the heart.

Branched-chain amino acids (BCAA) and their cognate α-ketoacids (BCKA) are elevated in an array of cardiometabolic diseases. Here we demonstrate that the major metabolic fate of uniformly-13C-labeled α-ketoisovalerate ([U-13C]KIV) in the heart is reamination to valine. Activation of cardiac branched-chain α-ketoacid dehydrogenase (BCKDH) by treatment with the BCKDH kinase inhibitor, BT2, does not impede the strong flux of [U-13C]KIV to valine. Sequestration of BCAA and BCKA away from mitochondrial oxidation is likely due to low levels of expression of the mitochondrial BCAA transporter SLC25A44 in the heart, as its overexpression significantly lowers accumulation of [13C]-labeled valine from [U-13C]KIV. Finally, exposure of perfused hearts to levels of BCKA found in obese rats increases phosphorylation of the translational repressor 4E-BP1 as well as multiple proteins in the MEK-ERK pathway, leading to a doubling of total protein synthesis. These data suggest that elevated BCKA levels found in obesity may contribute to pathologic cardiac hypertrophy via chronic activation of protein synthesis.

Kruppel-like factor 15 regulates fuel switching between glucose and fatty acids in brown adipocytes.

AIMS/INTRODUCTION: Brown adipose tissue (BAT) utilizes large amounts of fuel for thermogenesis, but the mechanism by which fuel substrates are switched in response to changes in energy status is poorly understood. We have now investigated the role of Kruppel-like factor 15 (KLF15), a transcription factor expressed at a high level in adipose tissue, in the regulation of fuel utilization in BAT. MATERIALS AND METHODS: Depletion or overexpression of KLF15 in HB2 differentiated brown adipocytes was achieved by adenoviral infection. Glucose and fatty acid oxidation were measured with radioactive substrates, pyruvate dehydrogenase complex activity was determined with a colorimetric assay, and gene expression was examined by reverse transcription and real-time polymerase chain reaction analysis. RESULTS: Knockdown of KLF15 in HB2 cells attenuated fatty acid oxidation in association with downregulation of the expression of genes related to this process including Acox1 and Fatp1, whereas it increased glucose oxidation. Expression of the gene for pyruvate dehydrogenase kinase 4 (PDK4), a negative regulator of pyruvate dehydrogenase complex, was increased or decreased by KLF15 overexpression or knockdown, respectively, in HB2 cells, with these changes being accompanied by a respective decrease or increase in pyruvate dehydrogenase complex activity. Chromatin immunoprecipitation showed that Pdk4 is a direct target of KLF15 in HB2 cells. Finally, fasting increased expression of KLf15, Pdk4 and genes involved in fatty acid utilization in BAT of mice, whereas refeeding suppressed Klf15 and Pdk4 expression. CONCLUSIONS: Our results implicate KLF15 in the regulation of fuel switching between glucose and fatty acids in response to changes in energy status in BAT.

The regulation of glucose and lipid homeostasis via PLTP as a mediator of BAT-liver communication.

While brown adipose tissue (BAT) is well-recognized for its ability to dissipate energy in the form of heat, recent studies suggest multifaced roles of BAT in the regulation of glucose and lipid homeostasis beyond stimulating thermogenesis. One of the functions involves interorgan communication with metabolic organs, such as the liver, through BAT-derived secretory factors, a.k.a., batokine. However, the identity and the roles of such mediators remain insufficiently understood. Here, we employed proteomics and transcriptomics in human thermogenic adipocytes and identified previously unappreciated batokines, including phospholipid transfer protein (PLTP). We found that increased circulating levels of PLTP, via systemic or BAT-specific overexpression, significantly improve glucose tolerance and insulin sensitivity, increased energy expenditure, and decrease the circulating levels of cholesterol, phospholipids, and sphingolipids. Such changes were accompanied by increased bile acids in the circulation, which in turn enhances glucose uptake and thermogenesis in BAT. Our data suggest that PLTP is a batokine that contributes to the regulation of systemic glucose and lipid homeostasis as a mediator of BAT-liver interorgan communication.

CD81 Controls Beige Fat Progenitor Cell Growth and Energy Balance via FAK Signaling.

Adipose tissues dynamically remodel their cellular composition in response to external cues by stimulating beige adipocyte biogenesis; however, the developmental origin and pathways regulating this process remain insufficiently understood owing to adipose tissue heterogeneity. Here, we employed single-cell RNA-seq and identified a unique subset of adipocyte progenitor cells (APCs) that possessed the cell-intrinsic plasticity to give rise to beige fat. This beige APC population is proliferative and marked by cell-surface proteins, including PDGFRα, Sca1, and CD81. Notably, CD81 is not only a beige APC marker but also required for de novo beige fat biogenesis following cold exposure. CD81 forms a complex with αV/ß1 and αV/ß5 integrins and mediates the activation of integrin-FAK signaling in response to irisin. Importantly, CD81 loss causes diet-induced obesity, insulin resistance, and adipose tissue inflammation. These results suggest that CD81 functions as a key sensor of external inputs and controls beige APC proliferation and whole-body energy homeostasis.