Comparative proteomics reveals that lipid droplet-anchored mitochondria are more sensitive to cold in brown adipocytes.

Brown adipose tissue (BAT) is specialized for uncoupled heat production through mitochondrion fueled majorly from fatty acids (FAs) of lipid droplets (LDs). How the interaction between the two organelles contributes the generation of heat remains elusive. Here, we report that LD-anchored mitochondria (LDAM) were observed in the BAT of mice raised at three different temperatures, 30 °C, 23 °C, and 6 °C. The biochemical analyses including Western blotting of electron transport chain subunits showed that LDAM were functional. Comparative proteomics analysis was conducted, which revealed differential expressions of proteins between LDAM and cytoplasmic mitochondria (CM) at different temperatures. Higher expressions of proteins at low temperature were observed for i) FA ß-oxidation in LDAM including FA synthesis and uncoupling, ii) pseudo-futile cycle in CM, and iii) two shuttle systems: glycerol 3-phosphate in both CM and LDAM and citrate malate in CM. Together, these results suggest that LDs and LDAM form a preorganized and functional organelle complex that permits the rapid response to cold.

Structural models of mitochondrial uncoupling proteins obtained in DPC micelles are not functionally relevant.

Uncoupling protein 1 (UCP1) is found in the inner mitochondrial membrane of brown adipocytes. In the presence of long-chain fatty acids (LCFAs), UCP1 increases the proton conductance, which, in turn, increases fatty acid oxidation and energy release as heat. Atomic models of UCP1 and UCP2 have been generated based on the NMR backbone structure of UCP2 in dodecylphosphocholine (DPC), a detergent known to inactivate UCP1. Based on NMR titration experiments on UCP1 with LCFA, it has been proposed that K56 and K269 are crucial for LCFA binding and UCP1 activation. Given the numerous controversies on the use of DPC for structure-function analyses of membrane proteins, we revisited those UCP1 mutants in a more physiological context by expressing them in the mitochondria of Saccharomyces cerevisiae. Mitochondrial respiration, assayed on permeabilized spheroplasts, enables the determination of UCP1 activation and inhibition. The K56S, K269S, and K56S/K269S mutants did not display any default in activation, which shows that the NMR titration experiments in DPC detergent are not relevant to UCP1 function.

Ad36 promotes differentiation of hADSCs into brown adipocytes by up-regulating LncRNA ROR.

AIMS: This study is to investigate the role of adenovirus type 36 (Ad36) in inducing differentiation of human adipose-derived stem cells (hADSCs) into brown adipocytes. MAIN METHODS: The hADSCs were induced to differentiate into adipocytes by a cocktail method and Ad36, respectively. They were collected on the 2nd, 4th, 6th, and 8th day, respectively. LncRNA ROR was silenced by siRNA. RT-qPCR and Western-blot were used to detect the mRNA and protein levels. Transmission electron microscopy was used to observe the mitochondria. KEY FINDINGS: The mRNA and protein expression levels of LncRNA ROR, Cidea, Dio2, Fgf21, Ucp1, Prdm16, Cox5b, Atp5o, Atp6, and Nd2 in the Ad36 induction group were significantly higher than those in the cocktail induction group. The expression levels of Leptin mRNA and protein in the Ad36 induction group were significantly lower than those in the cocktail induction group. After siRNA knockdown of LncRNA ROR, mRNA and protein expression levels of Cidea, Dio2, Fgf21, Ucp1, Prdm16, Cox5b, Atp5o, Atp6 and Nd2 were significantly lower than the control group during the induction of hADSC differentiation into adipocytes by Ad36. Additionally, mitochondria in the Ad36 induction group was increased compared to that in the cocktail induction group. SIGNIFICANCE: Ad36 may promote the differentiation of hADSCs into brown adipocytes by up-regulating LncRNA ROR.

GADD45α drives brown adipose tissue formation through upregulating PPARγ in mice.

Stress can lead to obesity and metabolic dysfunction, but the underlying mechanisms are unclear. Here we identify GADD45α, a stress-inducible histone folding protein, as a potential regulator for brown adipose tissue biogenesis. Unbiased transcriptomics data indicate a positive correlation between adipose Gadd45a mRNA level and obesity. At the cellular level, Gadd45a knockdown promoted proliferation and lipolysis of brown adipocytes, while Gadd45a overexpression had the opposite effects. Consistently, using a knockout (Gadd45a-/-) mouse line, we found that GADD45α deficiency inhibited lipid accumulation and promoted expression of thermogenic genes in brown adipocytes, leading to improvements in insulin sensitivity, glucose uptake, energy expenditure. At the molecular level, GADD45α deficiency increased proliferation through upregulating expression of cell cycle related genes. GADD45α promoted brown adipogenesis via interacting with PPARγ and upregulating its transcriptional activity. Our new data suggest that GADD45α may be targeted to promote non-shivering thermogenesis and metabolism while counteracting obesity.

BAF60a deficiency uncouples chromatin accessibility and cold sensitivity from white fat browning.

Brown and beige fat share a remarkably similar transcriptional program that supports fuel oxidation and thermogenesis. The chromatin-remodeling machinery that governs genome accessibility and renders adipocytes poised for thermogenic activation remains elusive. Here we show that BAF60a, a subunit of the SWI/SNF chromatin-remodeling complexes, serves an indispensable role in cold-induced thermogenesis in brown fat. BAF60a maintains chromatin accessibility at PPARγ and EBF2 binding sites for key thermogenic genes. Surprisingly, fat-specific BAF60a inactivation triggers more pronounced cold-induced browning of inguinal white adipose tissue that is linked to induction of MC2R, a receptor for the pituitary hormone ACTH. Elevated MC2R expression sensitizes adipocytes and BAF60a-deficient adipose tissue to thermogenic activation in response to ACTH stimulation. These observations reveal an unexpected dichotomous role of BAF60a-mediated chromatin remodeling in transcriptional control of brown and beige gene programs and illustrate a pituitary-adipose signaling axis in the control of thermogenesis.

Agonist Antibody Converts Stem Cells into Migrating Brown Adipocyte-Like Cells in Heart.

We present data showing that Iodotyrosine Deiodinase (IYD) is a dual-function enzyme acting as a catalyst in metabolism and a receptor for cooperative stem cell differentiation. IYD is present both in thyroid cells where it is critical for scavenging iodine from halogenated by-products of thyroid hormone production and on hematopoietic stem cells. To close the cooperative loop, the mono- and di-Iodotyrosine (MIT and DIT) substrates of IYD in the thyroid are also agonists for IYD now acting as a receptor on bone marrow stem cells. While studying intracellular combinatorial antibody libraries, we discovered an agonist antibody, H3 Ab, of which the target is the enzyme IYD. When agonized by H3 Ab, IYD expressed on stem cells induces differentiation of the cells into brown adipocyte-like cells, which selectively migrate to mouse heart tissue. H3 Ab also binds to IYD expressed on human myocardium. Thus, one has a single enzyme acting in different ways on different cells for the cooperative purpose of enhancing thermogenesis or of regenerating damaged heart tissue.

Elucidating the Regulatory Role of Melatonin in Brown, White, and Beige Adipocytes.

The high prevalence of obesity and its associated metabolic diseases has heightened the importance of understanding control of adipose tissue development and energy metabolism. In mammals, 3 types of adipocytes with different characteristics and origins have been identified: white, brown, and beige. Beige and brown adipocytes contain numerous mitochondria and have the capability to burn energy and counteract obesity, while white adipocytes store energy and are closely associated with metabolic disorders and obesity. Thus, regulation of the development and function of different adipocytes is important for controlling energy balance and combating obesity and related metabolic disorders. Melatonin is a neurohormone, which plays multiple roles in regulating inflammation, blood pressure, insulin actions, and energy metabolism. This article summarizes and discusses the role of melatonin in white, beige, and brown adipocytes, especially in affecting adipogenesis, inducing beige formation or white adipose tissue browning, enhancing brown adipose tissue mass and activities, improving anti-inflammatory and antioxidative effects, regulating adipokine secretion, and preventing body weight gain. Based on the current findings, melatonin is a potential therapeutic agent to control energy metabolism, adipogenesis, fat deposition, adiposity, and related metabolic diseases.

Glycogen Dynamics Drives Lipid Droplet Biogenesis during Brown Adipocyte Differentiation.

Browning induction or transplantation of brown adipose tissue (BAT) or brown/beige adipocytes derived from progenitor or induced pluripotent stem cells (iPSCs) can represent a powerful strategy to treat metabolic diseases. However, our poor understanding of the mechanisms that govern the differentiation and activation of brown adipocytes limits the development of such therapy. Various genetic factors controlling the differentiation of brown adipocytes have been identified, although most studies have been performed using in vitro cultured pre-adipocytes. We investigate here the differentiation of brown adipocytes from adipose progenitors in the mouse embryo. We demonstrate that the formation of multiple lipid droplets (LDs) is initiated within clusters of glycogen, which is degraded through glycophagy to provide the metabolic substrates essential for de novo lipogenesis and LD formation. Therefore, this study uncovers the role of glycogen in the generation of LDs.

Brown adipocytes and ß3-stimulant-induced brown-like adipocytes contribute to the prevention of renal crystal formation.

According to recent studies, kidney stones are associated with metabolic syndrome. We focused on brown adipocytes and ß3-stimulant-induced brown-like adipocytes to investigate how these adipocytes influence kidney stone disease. For the interscapular brown adipose tissue (iBAT) removal experiment, mice were subjected to either iBAT removal or sham operation (X-BAT group or sham group), and, after 3 wk, renal crystal deposition was induced by intra-abdominal injection of glyoxylate (GOX) for 6 days. For the ß3-stimulant experiment, mice were administered intra-abdominal injections of the ß3-stimulant (ß3-group) or saline (control group) for 6 days. Thereafter, renal crystal deposition was induced by intra-abdominal injection of GOX for 6 days. iBAT removal decreased the expression of Sod1 and increased that of chemokine (C-C motif) ligand 2 (Ccl2), EGF module-containing mucin-like receptor 1 (Emr1), and tumor necrosis factor (Tnf) in the kidneys. Renal crystal deposition was 2.06-fold higher in the X-BAT group than in the sham group. The ß3-stimulant caused differentiation of white adipocytes into brown-like adipocytes. In the kidneys of the ß3-group, the expression of Ccl2 and Emr1 decreased and that of Sod1 increased. Renal crystal deposition was 0.17-fold lower in the ß3-group than in the control group. In summary, iBAT removal promoted kidney inflammation and renal crystal formation. ß3-Stimulant-induced brown-like adipocytes reduced inflammation and improved antioxidant action in the kidneys, which suppressed renal crystal formation. This is the first report on the therapeutic role of brown and brown-like adipocytes for kidney stone formation.

Sucrose Nonfermenting-Related Kinase Regulates Both Adipose Inflammation and Energy Homeostasis in Mice and Humans.

Sucrose nonfermenting-related kinase (SNRK) is a member of the AMPK-related kinase family, and its physiological role in adipose energy homeostasis and inflammation remains unknown. We previously reported that SNRK is ubiquitously and abundantly expressed in both white adipose tissue (WAT) and brown adipose tissue (BAT), but SNRK expression diminishes in adipose tissue in obesity. In this study we report novel experimental findings from both animal models and human genetics. SNRK is essential for survival; SNRK globally deficient pups die within 24 h after birth. Heterozygous mice are characterized by inflamed WAT and less BAT. Adipocyte-specific ablation of SNRK causes inflammation in WAT, ectopic lipid deposition in liver and muscle, and impaired adaptive thermogenesis in BAT. These metabolic disorders subsequently lead to decreased energy expenditure, higher body weight, and insulin resistance. We further confirm the significant association of common variants of the SNRK gene with obesity risk in humans. Through applying a phosphoproteomic approach, we identified eukaryotic elongation factor 1δ and histone deacetylase 1/2 as potential SNRK substrates. Taking these data together, we conclude that SNRK represses WAT inflammation and is essential to maintain BAT thermogenesis, making it a novel therapeutic target for treating obesity and associated metabolic disorders.

Medicarpin induces lipolysis via activation of Protein Kinase A in brown adipocytes.

Natural pterocarpan Medicarpin (Med) has been shown to have various beneficial biological roles, including inhibition of osteoclastogenesis, stimulation of bone regeneration and induction of apoptosis. However, the effect of the Med on lipolysis in adipocytes has not been reported. Here, we show the effect of Med on lipolysis in different mouse adipocytes and elucidate the underlying mechanism. We observed that Med treatment promoted release of glycerol in the media. Differentiated mouse brown adipose tissue cells were treated with Med. RNA-Seq analysis was performed to elucidate the effect of med and subsequently was confirmed by qRT-PCR and western blotting analyses. Med treatment increased both protein and gene expression levels of hormone-sensitive lipase (Hsl) and adipose triglyceride lipase (Atgl), which are two critical enzymes necessary for lipolysis. Mechanistic study showed that Med activates Protein Kinase A (PKA) and phosphorylates Hsl at PKA target position at Serine660. Silencing of PKA gene by short interfering RNA attenuated the Med-induced increase in glycerol release and Hsl phosphorylation. The results unveil that Med boosts lipolysis via a PKA-dependent pathway in adipocytes and may provide a possible avenue of further research of Med mediated reduction of body fat. [BMB Reports 2018; 51(5): 249-254].

An unexpected role for the transcriptional coactivator isoform NT-PGC-1α in the regulation of mitochondrial respiration in brown adipocytes.

Brown adipose tissue dissipates energy as heat, a process that relies on a high abundance of mitochondria and high levels of electron transport chain (ETC) complexes within these mitochondria. Two regulators of mitochondrial respiration and heat production in brown adipocytes are the transcriptional coactivator PGC-1α and its splicing isoform NT-PGC-1α, which control mitochondrial gene expression in the nucleus. Surprisingly, we found that, in brown adipocytes, some NT-PGC-1α localizes to mitochondria, whereas PGC-1α resides in the nucleus. Here we sought to investigate the role of NT-PGC-1α in brown adipocyte mitochondria. Immunocytochemistry, immunotransmission electron microscopy, and biochemical analyses indicated that NT-PGC-1α was located in the mitochondrial matrix in brown adipocytes. NT-PGC-1α was specifically enriched at the D-loop region of the mtDNA, which contains the promoters for several essential ETC complex genes, and was associated with LRP130, an activator of mitochondrial transcription. Selective expression of NT-PGC-1α and PGC-1α in PGC-1α-/- brown adipocytes similarly induced expression of nuclear DNA-encoded mitochondrial ETC genes, including the key mitochondrial transcription factor A (TFAM). Despite having comparable levels of TFAM expression, PGC-1α-/- brown adipocytes expressing NT-PGC-1α had higher expression of mtDNA-encoded ETC genes than PGC-1α-/- brown adipocytes expressing PGC-1α, suggesting a direct effect of NT-PGC-1α on mtDNA transcription. Moreover, this increase in mtDNA-encoded ETC gene expression was associated with enhanced respiration in NT-PGC-1α-expressing PGC-1α-/- brown adipocytes. Our findings reveal a previously unappreciated and isoform-specific role for NT-PGC-1α in the regulation of mitochondrial transcription in brown adipocytes and provide new insight into the transcriptional control of mitochondrial respiration.

Mammary alveolar epithelial cells convert to brown adipocytes in post-lactating mice.

During pregnancy and lactation, subcutaneous white adipocytes in the mouse mammary gland transdifferentiate reversibly to milk-secreting epithelial cells. In this study, we demonstrate by transmission electron microscopy that in the post-lactating mammary gland interscapular multilocular adipocytes found close to the mammary alveoli contain milk protein granules. Use of the Cre-loxP recombination system allowed showing that the involuting mammary gland of whey acidic protein-Cre/R26R mice, whose secretory alveolar cells express the lacZ gene during pregnancy, contains some X-Gal-stained and uncoupling protein 1-positive interscapular multilocular adipocytes. These data suggest that during mammary gland involution some milk-secreting epithelial cells in the anterior subcutaneous depot may transdifferentiate to brown adipocytes, highlighting a hitherto unappreciated feature of mouse adipose organ plasticity.

Human White Adipocytes Convert Into "Rainbow" Adipocytes In Vitro.

White adipocytes are plastic cells able to reversibly transdifferentiate into brown adipocytes and into epithelial glandular cells under physiologic stimuli in vivo. These plastic properties could be used in future for regenerative medicine, but are incompletely explored in their details. Here, we focused on plastic properties of human mature adipocytes (MA) combining gene expression profile through microarray analysis with morphologic data obtained by electron and time lapse microscopy. Primary MA showed the classic morphology and gene expression profile of functional mature adipocytes. Notably, despite their committed status, MA expressed high levels of reprogramming genes. MA from ceiling cultures underwent transdifferentiation toward fibroblast-like cells with a well-differentiated morphology and maintaining stem cell gene signatures. The main morphologic aspect of the transdifferentiation process was the secretion of large lipid droplets and the development of organelles necessary for exocrine secretion further supported the liposecretion process. Of note, electron microscope findings suggesting liposecretion phenomena were found also in explants of human fat and rarely in vivo in fat biopsies from obese patients. In conclusion, both MA and post-liposecretion adipocytes show a well-differentiated phenotype with stem cell properties in line with the extraordinary plasticity of adipocytes in vivo. J. Cell. Physiol. 232: 2887-2899, 2017. © 2016 Wiley Periodicals, Inc.

Constitutive adipocyte mTORC1 activation enhances mitochondrial activity and reduces visceral adiposity in mice.

Mechanistic target of rapamycin complex 1 (mTORC1) loss of function reduces adiposity whereas partial mTORC1 inhibition enhances fat deposition. Herein we evaluated how constitutive mTORC1 activation in adipocytes modulates adiposity in vivo. Mice with constitutive mTORC1 activation in adipocytes induced by tuberous sclerosis complex (Tsc)1 deletion and littermate controls were evaluated for body mass, energy expenditure, glucose and fatty acid metabolism, mitochondrial function, mRNA and protein contents. Adipocyte-specific Tsc1 deletion reduced visceral, but not subcutaneous, fat mass, as well as adipocyte number and diameter, phenotypes that were associated with increased lipolysis, UCP-1 content (browning) and mRNA levels of pro-browning transcriptional factors C/EBPß and ERRα. Adipocyte Tsc1 deletion enhanced mitochondrial oxidative activity, fatty acid oxidation and the expression of PGC-1α and PPARα in both visceral and subcutaneous fat. In brown adipocytes, however, Tsc1 deletion did not affect UCP-1 content and basal respiration. Adipocyte Tsc1 deletion also reduced visceral adiposity and enhanced glucose tolerance, liver and muscle insulin signaling and adiponectin secretion in mice fed with purified low- or high-fat diet. In conclusion, adipocyte-specific Tsc1 deletion enhances mitochondrial activity, induces browning and reduces visceral adiposity in mice.

1,25-Dihydroxyvitamin D3/vitamin D receptor suppresses brown adipocyte differentiation and mitochondrial respiration.

PURPOSE: The vitamin D system plays a role in metabolism regulation. 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) suppressed 3T3-L1 white adipocyte differentiation. Vitamin D receptor (VDR) knockout mice showed increased energy expenditure, whereas mice with adipose-specific VDR over-expression showed decreased energy expenditure. Brown adipose tissue (BAT), now known to be present in adult humans, functions in non-shivering thermogenesis by uncoupling ATP synthesis from respiration and plays an important role in energy expenditure. However, the effects of 1,25(OH)2D3/VDR on brown adipocyte differentiation and mitochondrial respiration have not been reported. METHODS: mRNA expression of VDR and the metabolizing enzymes 1α-hydroxylase (CYP27B1) and 24-hydroxylase (CYP24A1) were examined in BAT of mice models of obesity and during brown adipocyte differentiation. The effects of 1,25(OH)2D3 and VDR over-expression on brown adipocyte differentiation and functional outcomes were evaluated. RESULTS: No significant changes in mRNA of VDR and CYP27B1 were noted in both diet-induced obese (DIO) and ob/ob mice, whereas uncoupling protein 1 mRNA was downregulated in BAT of ob/ob, but not DIO mice when compared to the controls. In contrast, mRNA of VDR, CYP24A1, and CYP27B1 were downregulated during brown adipocyte differentiation in vitro. 1,25(OH)2D3 dose-dependently suppressed brown adipocyte differentiation, accompanied by suppressed isoproterenol-stimulated oxygen consumption rates (OCR), maximal OCR and OCR from proton leak. Consistently, over-expression of VDR also suppressed brown adipocyte differentiation. Further, both 1,25(OH)2D3 and VDR over-expression suppressed PPARγ transactivation in brown preadipocytes. CONCLUSION: Our results demonstrate the suppressive effects of 1,25(OH)2D3/VDR signaling on brown adipocyte differentiation and mitochondrial respiration. The role of 1,25(OH)2D3/VDR system in regulating BAT development and function in obesity warrant further investigation.

MicroRNA-26 family is required for human adipogenesis and drives characteristics of brown adipocytes.

Adipose tissue contains thermogenic adipocytes (i.e., brown and brite/beige) that oxidize nutrients at exceptionally high rates via nonshivering thermogenesis. Its recent discovery in adult humans has opened up new avenues to fight obesity and related disorders such as diabetes. Here, we identified miR-26a and -26b as key regulators of human white and brite adipocyte differentiation. Both microRNAs are upregulated in early adipogenesis, and their inhibition prevented lipid accumulation while their overexpression accelerated it. Intriguingly, miR-26a significantly induced pathways related to energy dissipation, shifted mitochondrial morphology toward that seen in brown adipocytes, and promoted uncoupled respiration by markedly increasing the hallmark protein of brown fat, uncoupling protein 1. By combining in silico target prediction, transcriptomics, and an RNA interference screen, we identified the sheddase ADAM metallopeptidase domain 17 (ADAM17) as a direct target of miR-26 that mediated the observed effects on white and brite adipogenesis. These results point to a novel, critical role for the miR-26 family and its downstream effector ADAM17 in human adipocyte differentiation by promoting characteristics of energy-dissipating thermogenic adipocytes.

Clinical, morphologic, and immunohistochemical features of canine orbital hibernomas.

Hibernomas are uncommon benign tumors of brown fat that occur in humans and various animal species. They have not been observed in the orbit of dogs, humans, or other animals. Here we report clinical, light and electron microscopic, and immunohistochemical features of a series of 7 hibernomas arising in the orbital region of dogs. These neoplasms occurred in adult dogs with no breed predilection. The mean age of the affected dogs was 10.4 years (range, 8-13 years). All neoplasms presented as soft lobular masses composed of predominantly round or polygonal neoplastic cells with granular eosinophilic and vacuolated cytoplasm resembling adipocytes. The cytoplasm contained large numbers of pleomorphic mitochondria with dense matrices and indistinct cristae. Immunohistochemical evaluation confirmed positive labeling of neoplastic cells from all cases with uncoupling protein 1 (UCP-1) consistent with brown fat differentiation. Interestingly, rare neoplastic cells also expressed myogenin and myoD, possibly suggesting a common progenitor cell for neoplastic brown adipose and skeletal muscle cells.

The origin of lipofuscin in brown adipocytes of hyperinsulinaemic rats: the role of lipid peroxidation and iron.

The aim of this study was to investigate lipofuscin origin in brown adipocytes of hyperinsulinaemic rats and the possible role of lipid peroxidation and iron in this process. Ultrastructural examination revealed hyperinsulinaemia-induced enhancement in the lipofuscin production, accompanied by an increase of mitochondrial damage in brown adipocytes. Extensive fusions of lipid droplets and mitochondria with lysosomes were also observed. Confocal microscopy showed lipofuscin autofluorescence emission in brown adipose tissue (BAT) after excitation at 488 nm and 633 nm, particularly in the insulin-treated groups. The presence and distribution of lipid peroxidation product, 4-hydroxy-2-nonenal (4-HNE), in brown adipocytes was assessed by immunohistochemical examination revealing its higher content after treatment with insulin. The iron content was quantified by electron dispersive X-ray analysis (EDX) showing its higher content in the hyperinsulinaemic groups. The ultrastucture of the majority of lipofuscin granules suggests their mitochondrial origin, which was additionally confirmed by their co-localization with ATP synthase. In conclusion, our results suggest that increased lipofuscinogenesis in the brown adipocytes of hyperinsulinaemic rats is a consequence of lipid peroxidation, mitochondrial damage and iron accumulation.