Milk fat globule membrane and its component phosphatidylcholine induce adipose browning both in vivo and in vitro

The functional induction of brown-like adipocytes in white adipose tissue (WAT) provides a defense against obesity. The aim of this study was to analyze the effects of milk fat globule membrane (MFGM) and its component phosphatidylcholine (PC) on the brown remodeling of WAT. Male C57BL/6 J mice were fed a high-fat diet (HFD) for 8 weeks and then fed HFD for another 8 weeks with MFGM. In vitro studies were performed in C3H10T1/2 pluripotent stem cells, 3T3-L1 pre-adipocytes and differentiated inguinal WAT stromal vascular cells (SVCs) to determine the role of MFGM and PC on the formation of brown-like adipocytes. MFGM decreased fasting glucose and serum insulin levels in HFD-fed mice. MFGM improved glucose tolerance and insulin sensitivity, and induced browning of inguinal WAT. MFGM and its component PC stimulated transformation of brown-like adipocytes in C3H10T1/2 pluripotent stem cells, 3T3-L1 adipocytes and SVCs by increasing the protein expression of UCP1, PGC-1α, PRDM16 as well as the mRNA expression of other thermogenic genes and beige cell markers. MFGM and PC also increased mitochondrial DNA (mtDNA) copy number, mitochondrial density and oxygen consumption rate and up-regulated the mRNA expression of mitochondria-biogenesis-related genes in vitro. PPARα inhibitor GW6471 treatment or knockdown of PPARα using lentivirus-expressing shRNA inhibited the PC-induced increase in the protein expression of UCP1, PGC-1α and PRDM16 in C3H10T1/2 pluripotent stem cells and 3T3-L1 adipocytes, indicating the potential role of PPARα in PC-mediated brown-like adipocyte formation. In conclusion, MFGM and milk PC induced adipose browning, which has major protective effects against obesity and metabolic dysfunction.     

Thermogenic Ability of Uncoupling Protein 1 in Beige Adipocytes in Mice

Chronic adrenergic activation leads to the emergence of beige adipocytes in some depots of white adipose tissue in mice. Despite their morphological similarities to brown adipocytes and their expression of uncoupling protein 1 (UCP1), a thermogenic protein exclusively expressed in brown adipocytes, the beige adipocytes have a gene expression pattern distinct from that of brown adipocytes. However, it is unclear whether the thermogenic function of beige adipocytes is different from that of classical brown adipocytes existing in brown adipose tissue. To examine the thermogenic ability of UCP1 expressed in beige and brown adipocytes, the adipocytes were isolated from the fat depots of C57BL/6J mice housed at 24°C (control group) or 10°C (cold-acclimated group) for 3 weeks. Morphological and gene expression analyses revealed that the adipocytes isolated from brown adipose tissue of both the control and cold-acclimated groups consisted mainly of brown adipocytes. These brown adipocytes contained large amounts of UCP1 and increased their oxygen consumption when stimulated with norepinephirine. Adipocytes isolated from the perigonadal white adipose tissues of both groups and the inguinal white adipose tissue of the control group were white adipocytes that showed no increase in oxygen consumption after norepinephrine stimulation. Adipocytes isolated from the inguinal white adipose tissue of the cold-acclimated group were a mixture of white and beige adipocytes, which expressed UCP1 and increased their oxygen consumption in response to norepinephrine. The UCP1 content and thermogenic ability of beige adipocytes estimated on the basis of their abundance in the cell mixture were similar to those of brown adipocytes. These results revealed that the inducible beige adipocytes have potent thermogenic ability comparable to classical brown adipocytes.     

Eicosapentaenoic acid regulates brown adipose tissue metabolism in high-fat-fed mice and in clonal brown adipocytes

Brown adipose tissue (BAT) plays a key role in energy expenditure through its specialized thermogenic function. Therefore, BAT activation may help prevent and/or treat obesity. Interestingly, subcutaneous white adipose tissue (WAT) also has the ability to differentiate into brown-like adipocytes and may potentially contribute to increased thermogenesis. We have previously reported that eicosapentaenoic acid (EPA) reduces high-fat (HF)-diet-induced obesity and insulin resistance in mice. Whether BAT mediates some of these beneficial effects of EPA has not been determined. We hypothesized that EPA activates BAT thermogenic program, contributing to its antiobesity effects. BAT and WAT were harvested from B6 male mice fed HF diets supplemented with or without EPA. HIB 1B clonal brown adipocytes treated with or without EPA were also used. Gene and protein expressions were measured in adipose tissues and H1B 1B cells by quantitative polymerase chain reaction and immunoblotting, respectively. Our results show that BAT from EPA-supplemented mice expressed significantly higher levels of thermogenic genes such as PRDM16 and PGC1α and higher levels of uncoupling protein 1 compared to HF-fed mice. By contrast, both WATs (subcutaneous and visceral) had undetectable levels of these markers with no up regulation by EPA. HIB 1B cells treated with EPA showed significantly higher mRNA expression of PGC1α and SIRT2. EPA treatment significantly increased maximum oxidative and peak glycolytic metabolism in H1B 1B cells. Our results demonstrate a novel and promising role for EPA in preventing obesity via activation of BAT, adding to its known beneficial anti-inflammatory effects.     

Distinct populations of adipogenic and myogenic Myf5-lineage progenitors in white adipose tissues

Brown adipose tissues (BAT) are derived from a myogenic factor 5 (Myf5)-expressing cell lineage and white adipose tissues (WAT) predominantly arise from non-Myf5 lineages, although a subpopulation of adipocytes in some WAT depots can be derived from the Myf5 lineage. However, the functional implication of the Myf5- and non-Myf5-lineage cells in WAT is unclear. We found that the Myf5-lineage constitution in subcutaneous WAT depots is negatively correlated to the expression of classical BAT and newly defined beige/brite adipocyte-specific genes. Consistently, fluorescent-activated cell sorting (FACS)-purified Myf5-lineage adipo-progenitors give rise to adipocytes expressing lower levels of BAT-specific Ucp1, Prdm16, Cidea, and Ppargc1a genes and beige adipocyte-specific CD137, Tmem26, and Tbx1 genes compared with the non-Myf5-lineage adipocytes from the same depots. Ablation of the Myf5-lineage progenitors in WAT stromal vascular cell (SVC) cultures leads to increased expression of BAT and beige cell signature genes. Strikingly, the Myf5-lineage cells in WAT are heterogeneous and contain distinct adipogenic [stem cell antigen 1(Sca1)-positive] and myogenic (Sca1-negative) progenitors. The latter differentiate robustly into myofibers in vitro and in vivo, and they restore dystrophin expression after transplantation into mdx mouse, a model for Duchenne muscular dystrophy. These results demonstrate the heterogeneity and functional differences of the Myf5- and non-Myf5-lineage cells in the white adipose tissue.     

The PPARγ agonist rosiglitazone promotes the induction of brite adipocytes,increasing β-adrenoceptor-mediated mitochondrial function and glucose uptake

Recruitment and activation of brite (or beige) adipocytes has been advocated as a potential avenue for manipulating whole-body energy expenditure. Despite numerous studies illustrating the differences in gene and protein markers between brown, brite and white adipocytes, there is very little information on the adrenergic regulation and function of these brite adipocytes. We have compared the functional (cyclic AMP accumulation, oxygen consumption rates, mitochondrial function, glucose uptake, extracellular acidification rates, calcium influx) profiles of mouse adipocytes cultured from three contrasting depots, namely interscapular brown adipose tissue, and inguinal or epididymal white adipose tissues, following chronic treatment with the peroxisome proliferator-activated receptor γ (PPARγ) agonist rosiglitazone. Prototypical brown adipocytes readily express β₃-adrenoceptors, and β₃-adrenoceptor stimulation increases cyclic AMP accumulation, oxygen consumption rates, mitochondrial function, glucose uptake, and extracellular acidification rates. Treatment of brown adipocytes with rosiglitazone increases uncoupling protein 1 (UCP1) levels, and increases β₃-adrenoceptor mitochondrial function but does not affect glucose uptake responses. In contrast, inguinal white adipocytes only express UCP1 and β₃-adrenoceptors following rosiglitazone treatment, which results in an increase in all β₃-adrenoceptor-mediated functions. The effect of rosiglitazone in epididymal white adipocytes, was much lower compared to inguinal white adipocytes. Rosiglitazone also increased α₁-adrenoceptor mediated increases in calcium influx and glucose uptake (but not mitochondrial function) in inguinal and epididymal white adipocytes. In conclusion, the PPARγ agonist rosiglitazone promotes the induction and function of brite adipocytes cultured from inguinal and epididymal white adipose depots.     

Beige adipocytes are a distinct type of thermogenic fat cell in mouse and human

Brown fat generates heat via the mitochondrial uncoupling protein UCP1, defending against hypothermia and obesity. Recent data suggest that there are two distinct types of brown fat: classical brown fat derived from a myf-5 cellular lineage and UCP1-positive cells that emerge in white fat from a non-myf-5 lineage. Here, we report the isolation of "beige" cells from murine white fat depots. Beige cells resemble white fat cells in having extremely low basal expression of UCP1, but, like classical brown fat, they respond to cyclic AMP stimulation with high UCP1 expression and respiration rates. Beige cells have a gene expression pattern distinct from either white or brown fat and are preferentially sensitive to the polypeptide hormone irisin. Finally, we provide evidence that previously identified brown fat deposits in adult humans are composed of beige adipocytes. These data provide a foundation for studying this mammalian cell type with therapeutic potential. PAPERCLIP:     

Brite/beige fat and UCP1 — is it thermogenesis?

The presence of two distinct types of adipose tissue, which have opposing functions, has been known for decades. White adipose tissue (WAT) is the main tissue of energy storage, while brown adipose tissue (BAT) dissipates energy as heat and is required for non-shivering thermoregulation. In the last few years, a third type of adipocyte was identified, termed the brite (“brown and white”) or beige adipocyte. Their physiological control and role, however, are not fully clarified. Brite/beige adipocytes have a positive impact on systemic metabolism that is generally explained by the thermogenesis of brite/beige adipocytes; although thermogenesis has not been directly measured but is mostly inferred by gene expression data of typical thermogenic genes such as uncoupling protein 1 (UCP1). Here we critically review functional evidence for the thermogenic potential of brite/beige adipocytes, leading to the conclusion that direct measurements of brite/beige adipocyte bioenergetics, beyond gene regulation, are pivotal to quantify their thermogenic potential. In particular, we exemplified that the massive induction of UCP1 mRNA during the browning of isolated subcutaneous adipocytes in vitro is not reflected in significant alterations of cellular bioenergetics. Herein, we demonstrate that increases in mitochondrial respiration in response to beta-adrenergic stimulus can be independent of UCP1. Using HEK293 cells expressing UCP1, we show how to directly assess UCP1 function by adequate activation in intact cells. Finally, we provide a guide on the interpretation of UCP1 activity and the pitfalls by solely using respiration measurements. The functional analysis of beige adipocyte bioenergetics will assist to delineate the impact of browning on thermogenesis, possibly elucidating additional physiological roles and its contribution to systemic metabolism, highlighting possible avenues for future research. This article is part of a Special Issue entitled: 18th European Bioenergetic Conference.     

White-to-brown transdifferentiation of omental adipocytes in patients affected by pheochromocytoma

In all mammals, white adipose tissue (WAT) and brown adipose tissue (BAT) are found together in several fat depots, forming a multi-depot organ. Adrenergic stimulation induces an increase in BAT usually referred to as “browning”. This phenomenon is important because of its potential use in curbing obesity and related disorders; thus, understanding its cellular mechanisms in humans may be useful for the development of new therapeutic strategies. Data in rodents have supported the direct transformation of white into brown adipocytes. Biopsies of pure white omental fat were collected from 12 patients affected by the catecholamine-secreting tumor pheochromocytoma (pheo-patients) and compared with biopsies from controls. Half of the omental fat samples from pheo-patients contained uncoupling protein 1 (UCP1)-immunoreactive-(ir) multilocular cells that were often arranged in a BAT-like pattern endowed with noradrenergic fibers and dense capillary network. Many UCP1-ir adipocytes showed the characteristic morphology of paucilocular cells, which we have been described as cytological marker of transdifferentiation. Electron microscopy showed increased mitochondrial density in multi- and paucilocular cells and disclosed the presence of perivascular brown adipocyte precursors. Brown fat genes, such as UCP1, PR domain containing 16 (PRDM16) and β3-adrenoreceptor, were highly expressed in the omentum of pheo-patients and in those cases without visible morphologic re-arrangement. Of note, the brown determinant PRDM16 was detected by immunohistochemistry only in nuclei of multi- and paucilocular adipocytes. Quantitative electron microscopy and immunohistochemistry for Ki67 suggest an unlikely contribution of proliferative events to the phenomenon. The data support the idea that, in adult humans, white adipocytes of pure white fat that are subjected to adrenergic stimulation are able to undergo a process of direct transformation into brown adipocytes. This article is part of a Special Issue entitled Brown and White Fat: From Signaling to Disease.     

UCP1 mRNA does not produce heat

Because of the possible role of brown adipose tissue and UCP1 in metabolic regulation, even in adult humans, there is presently considerable interest in quantifying, from in-vitro data, the thermogenic capacities of brown and brite/beige adipose tissues. An important issue is therefore to establish which parameters are the most adequate for this. A particularly important issue is the relevance of UCP1 mRNA levels as estimates of the degree of recruitment and of the thermogenic capacity resulting from differences in physiological conditions and from experimental manipulations. By solely following UCP1 mRNA levels in brown adipose tissue, the conclusion would be made that the tissue's highest activation occurs after only 6 h in the cold and then successively decreases to being only some 50% elevated after 1 month in the cold. However, measurement of total UCP1 protein levels per depot ("mouse") reveals that the maximal thermogenic capacity estimated in this way is reached first after 1 month but represents an approx. 10-fold increase in thermogenic capacity. Since this in-vitro measure correlates quantitatively and temporally with the acquisition of nonshivering thermogenesis, this must be considered the most physiologically relevant parameter. Similarly, observations that cold acclimation barely increases UCP1 mRNA levels in classical brown adipose tissue but leads to a 200-fold increase in UCP1 mRNA levels in brite/beige adipose tissue depots may overemphasise the physiological significance of these depots, as the high fold-increases are due to very low initial levels, and the UCP1 mRNA levels reached are at least an order of magnitude lower than in brown adipose tissue; furthermore, based on total UCP1 protein amounts, the brite/beige depots attain only about 10% of the thermogenic capacity of the classical brown adipose tissue depots. Consequently, inadequate conclusions may be reached if UCP1 mRNA levels are used as a proxy for the metabolic significance of recruited versus non-recruited brown adipose tissue and for estimating the metabolic significance of brown versus brite/beige adipose tissues. This article is part of a Special Issue entitled Brown and White Fat: From Signaling to Disease.     

Pharmacological and nutritional agents promoting browning of white adipose tissue

The role of brown adipose tissue in the regulation of energy balance and maintenance of body weight is well known in rodents. Recently, interest in this tissue has re-emerged due to the realization of active brown-like adipose tissue in adult humans and inducible brown-like adipocytes in white adipose tissue depots in response to appropriate stimuli (“browning process”). Brown-like adipocytes that appear in white fat depots have been called “brite” (from brown-in-white) or “beige” adipocytes and have characteristics similar to brown adipocytes, in particular the capacity for uncoupled respiration. There is controversy as to the origin of these brite/beige adipocytes, but regardless of this, induction of the browning of white fat represents an attractive potential strategy for the management and treatment of obesity and related complications. Here, the different physiological, pharmacological and dietary determinants that have been linked to white-to-brown fat remodeling and the molecular mechanisms involved are reviewed in detail. In the light of available data, interesting therapeutic perspectives can be expected from the use of specific drugs or food compounds able to induce a program of brown fat differentiation including uncoupling protein 1 expression and enhancing oxidative metabolism in white adipose cells. However, additional research is needed, mainly focused on the physiological relevance of browning and its dietary control, where the use of ferrets and other non-rodent animal models with a more similar adipose tissue organization and metabolism to humans could be of much help. This article is part of a Special Issue entitled Brown and White Fat: From Signaling to Disease.     

ERRγ enhances UCP1 expression and fatty acid oxidation in brown adipocytes

Objective:

Estrogen‐related receptors (ERRs) are important regulators of energy metabolism. Here we investigated the hypothesis that ERRγ impacts on differentiation and function of brown adipocytes.

Design and Methods:

We characterize the expression of ERRγ in adipose tissues and cell models and investigate the effects of modulating ERR? activity on UCP1 gene expression and metabolic features of brown and white adipocytes.

Results:

ERRγ was preferentially expressed in brown compared to white fat depots, and ERRγ was induced during cold‐induced browning of subcutaneous white adipose tissue and brown adipogenesis. Overexpression of ERRγ positively regulated uncoupling protein 1 (UCP1) expression levels during brown adipogenesis. This ERRγ‐induced augmentation of UCP1 expression was independent of the presence of peroxisome proliferator‐activated receptor coactivator‐1 (PGC‐1α) but was associated with increased rates of fatty acid oxidation in adrenergically stimulated cells. ERR? did not influence mitochondrial biogenesis, and its reduced expression in white adipocytes could not explain their low expression level of UCP1.

Conclusions:

Through its augmenting effect on expression of UCP1, ERRγ may physiologically be involved in increasing the potential for energy expenditure in brown adipocytes, a function that is becoming of therapeutic interest.

     

PEX13 is required for thermogenesis of white adipose tissue in cold-exposed mice

Non-shivering thermogenesis (NST) is a heat generating process controlled by the mitochondria of brown adipose tissue (BAT). In the recent decade, ‘functionally’ acting brown adipocytes in white adipose tissue (WAT) has been identified as well: the so-called process of the ‘browning’ of WAT. While the importance of uncoupling protein 1 (UCP1)-oriented mitochondrial activation has been intensely studied, the role of peroxisomes during the browning of white adipocytes is poorly understood. Here, we assess the change in peroxisomal membrane proteins, or peroxins (PEXs), during cold stimulation and importantly, the role of PEX13 in the cold-induced remodeling of white adipocytes. PEX13, a protein that originally functions as a docking factor and is involved in protein import into peroxisome matrix, was highly increased during cold-induced recruitment of beige adipocytes within the inguinal WAT of C57BL/6 mice. Moreover, beige-induced 3 T3-L1 adipocytes and stromal vascular fraction (SVF) cells by exposure to the peroxisome proliferator-activated receptor gamma (PPARγ) agonist rosiglitazone showed a significant increase in mitochondrial thermogenic factors along with peroxisomal proteins including PEX13, and these were confirmed in SVF cells with the beta 3 adrenergic receptor (β3AR)-selective agonist CL316,243. To verify the relevance of PEX13, we used the RNA silencing method targeting the Pex13 gene and evaluated the subsequent beige development in SVF cells. Interestingly, siPex13 treatment suppressed expression of thermogenic proteins such as UCP1 and PPARγ coactivator 1 alpha (PGC1α). Overall, our data provide evidence supporting the role of peroxisomal proteins, in particular PEX13, during beige remodeling of white adipocytes.     

Leptin deficiency impairs adipogenesis and browning response in mouse mesenchymal progenitors

Although phenotypically different, brown adipose tissue (BAT) and inguinal white adipose tissue (iWAT) are able to produce heat through non-shivering thermogenesis due to the presence of mitochondrial uncoupling protein 1 (UCP1). The appearance of thermogenically active beige adipocytes in iWAT is known as browning. Both brown and beige cells originate from mesenchymal stem cells (MSCs), and in culture conditions a browning response can be induced with hypothermia (i.e. 32 °C) during which nuclear leptin immunodetection was observed. The central role of leptin in regulating food intake and energy consumption is well recognised, but its importance in the browning process at the cellular level is unclear. Here, immunocytochemical analysis of MSC-derived adipocytes established nuclear localization of both leptin and leptin receptor suggesting an involvement of the leptin pathway in the browning response. In order to elucidate whether leptin modulates the expression of brown and beige adipocyte markers, BAT and iWAT samples from leptin-deficient (ob/ob) mice were analysed and exhibited reduced brown/beige marker expression compared to wild-type controls. When MSCs were isolated and differentiated into adipocytes, leptin deficiency was observed to induce a white phenotype, especially when incubated at 32 °C. These adaptations were accompanied with morphological signs of impaired adipogenic differentiation. Overall, our results indicate that leptin supports adipocyte browning and suggest a potential role for leptin in adipogenesis and browning.     

FABP3 and brown adipocyte-characteristic mitochondrial fatty acid oxidation enzymes are induced in beige cells in a different pathway from UCP1

Cold exposure and β₃-adrenergic receptor agonist (CL316,243) treatment induce the production of beige cells, which express brown adipocytes(BA)-specific UCP1 protein, in white adipose tissue (WAT). It remains unclear whether the beige cells, which have different gene expression patterns from BA, express BA-characteristic fatty acid oxidation (FAO) proteins. Here we found that 5 day cold exposure and CL316,243 treatment of WAT, but not CL316,243 treatment of primary adipocytes of C57BL/6J mice, increased mRNA levels of BA-characteristic FAO proteins. These results suggest that BA-characteristic FAO proteins are induced in beige cells in a different pathway from UCP1.