Myf5 Lineage Research Articles

Novel discovery of Averrhoa bilimbi ethanolic leaf extract in the stimulation of brown fat differentiation program in combating diet-induced obesity

BackgroundBrown adipocytes are known to promote energy expenditure and limit weight gain to combat obesity. Averrhoa bilimbi, locally called belimbing buluh (DBB), is mainly used as an ethnomedicine in the treatment of metabolic disorders including diabetes mellitus, hypertension and obesity. The present study aims to investigate the browning activity on white adipocytes by A. bilimbi leaf extract and to evaluate the potential mechanisms.MethodsEthanolic leaf extract of A. bilimbi was exposed to Myf5 lineage precursor cells to stimulate adipocyte differentiation. Protein expressions of brown adipocyte markers were determined through high content screening analysis and validated through western blotting. Mito Stress Test assay was conducted to evaluate the cellular oxygen consumption rate upon A. bilimbi treatment.ResultsA. bilimbi ethanolic leaf extract exhibited an adipogenesis effect similar to a PPARgamma agonist. It also demonstrated brown adipocyte differentiation in myoblastic Myf5-positive precursor cells. Expression of UCP1 and PRDM16 were induced. The basal metabolic rate and respiratory capacity of mitochondria were increased upon A. bilimbi treatment.ConclusionsThe findings suggest that Averrhoa bilimbi ethanolic leaf extract induces adipocyte browning through PRDM16 activation and enhances mitochondria activity due to UCP1 up-regulation.

312-OR: Functional Decline in Brown Adipose Progenitors during BAT Remodeling

In humans, there is a striking decrease in the prevalence and activity of brown adipose tissue (BAT) with age and obesity; however, the cellular and molecular mechanisms of BAT remodeling are almost completely unknown. Rodent models have been widely used to study BAT but they possess constitutively active BAT throughout life even at thermoneutrality; therefore, complementary animal models that possess characteristics of human BAT remodeling are needed. Here, we found that BAT progressively whitened in rabbits in a similar manner as in humans. As rabbits mature, preadipocytes in BAT displayed reduced capacity to be differentiated into brown adipocytes, when transplanted into immune-deficient mice. RNA-sequencing of brown preadipocytes identified that, during BAT remodeling, genes enriched for muscle contraction and mitochondrial function were down-regulated, while genes involved in lipid and amino acid metabolism were up-regulated. Single-cell RNA sequencing discovered a unique population of stromal vascular fraction cells specifically expressing genes regulating muscle and mitochondrial function. Knocking down these genes in rabbit brown preadipocytes blunted their ability to become mature brown adipocytes. We also identified follistatin like 1 (FSTL1) as a novel “batokine” that is specifically expressed by brown preadipocytes but not mature brown adipocytes. FSTL1 gradually reduced its expression when BAT whitens in rabbits, but was stably expressed in mouse BAT during life. Deleting FSTL1 specifically in the Myf5 lineage in mice reduced BAT size, UCP1 expression, and body temperature in newborns, suggesting the constitutive expression of FSTL1 prevents BAT whitening in mice. Taken together, we identify potential molecular mechanisms underlying functional decline in the browning ability of preadipocytes that contribute to BAT remodeling in rabbits and possibly humans. Disclosure Z. Huang: None. Z. Zhang: None. H. Ruan: None. Funding American Diabetes Association (1-18-IBS-167 to H-B.R.)

Functional decline in brown adipose progenitors during BAT remodeling

In humans, there is a striking decrease in the prevalence and activity of brown adipose tissue (BAT) with age and obesity; however, the cellular and molecular mechanisms of BAT remodeling are almost completely unknown. Rodent models have been widely used to study BAT but they possess constitutively active BAT throughout life even at thermoneutrality; therefore, complementary animal models that possess characteristics of human BAT remodeling are needed. Here, we found that BAT progressively whitened in rabbits in a similar manner as in humans. As rabbits mature, preadipocytes in BAT displayed reduced capacity to be differentiated into brown adipocytes, when transplanted into immune‐deficient mice. RNA‐sequencing of brown preadipocytes identified that, during BAT remodeling, genes enriched for muscle contraction and mitochondrial function were down‐regulated, while genes involved in lipid and amino acid metabolism were up‐regulated. Knocking down genes regulating muscle and mitochondrial function in rabbit brown preadipocytes blunted their ability to become mature brown adipocytes in vitro and in vivo. We also identified follistatin like 1 (FSTL1) as a novel “batokine” that is specifically expressed by brown preadipocytes but not mature brown adipocytes. FSTL1 gradually reduced its expression when BAT whitens in rabbits, but was stably expressed in mouse BAT during life. Deleting FSTL1 specifically in the Myf5 lineage in mice reduced BAT size, UCP1 expression, and body temperature in newborns, suggesting the constitutive expression of FSTL1 prevents BAT whitening in mice. Taken together, we identify potential molecular mechanisms underlying functional decline in the browning ability of preadipocytes that contribute to BAT remodeling in rabbits and possibly humans.Support or Funding InformationAmerican Diabetes Association (18‐IBS‐167)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Paracrine osteoprotegerin and β-catenin stabilization support synovial sarcomagenesis in periosteal cells.

Synovial sarcoma (SS) is an aggressive soft-tissue sarcoma that is often discovered during adolescence and young adulthood. Despite the name, synovial sarcoma does not typically arise from a synoviocyte but instead arises in close proximity to bones. Previous work demonstrated that mice expressing the characteristic SS18-SSX fusion oncogene in myogenic factor 5-expressing (Myf5-expressing) cells develop fully penetrant sarcomagenesis, suggesting skeletal muscle progenitor cell origin. However, Myf5 is not restricted to committed myoblasts in embryos but is also expressed in multipotent mesenchymal progenitors. Here, we demonstrated that human SS and mouse tumors arising from SS18-SSX expression in the embryonic, but not postnatal, Myf5 lineage share an anatomic location that is frequently adjacent to bone. Additionally, we showed that SS can originate from periosteal cells expressing SS18-SSX alone and from preosteoblasts expressing the fusion oncogene accompanied by the added stabilization of β-catenin, which is a common secondary change in SS. Expression and secretion of the osteoclastogenesis inhibitory factor osteoprotegerin enabled early growth of SS18-SSX2-transformed cells, indicating a paracrine link between the bone and synovial sarcomagenesis. These findings explain the skeletal contact frequently observed in human SS and may provide alternate means of enabling SS18-SSX-driven oncogenesis in cells as differentiated as preosteoblasts.

Schwann Cells in the Ventral Dermis Do Not Derive from Myf5-Expressing Precursors.

SummaryThe embryonic origin of lineage precursors of the trunk dermis is somewhat controversial. Precursor cells traced by Myf5 and Twist2 (Dermo1) promoter activation (i.e., cells of presumed dermomyotomal lineage) have been reported to generate Schwann cells. On the other hand, abundant data demonstrate that dermal Schwann cells derive from the neural crest. This is relevant because dermal precursors give rise to neural lineages, and multilineage differentiation potential qualifies them as adult stem cells. However, it is currently unclear whether neural lineages arise from dedifferentiated Schwann cells instead of mesodermally derived dermal precursor cells. To clarify these discrepancies, we traced SOX2+ adult dermal precursor cells by two independent Myf5 lineage tracing strains. We demonstrate that dermal Schwann cells do not belong to the Myf5+ cell lineage, indicating that previous tracing data reflected aberrant cre recombinase expression and that bona fide Myf5+ dermal precursors cannot transdifferentiate to neural lineages in physiological conditions.

Loss of MyoD Promotes Fate Transdifferentiation of Myoblasts Into Brown Adipocytes

Brown adipose tissue (BAT) represents a promising agent to ameliorate obesity and other metabolic disorders. However, the abundance of BAT decreases with age and BAT paucity is a common feature of obese subjects. As brown adipocytes and myoblasts share a common Myf5 lineage origin, elucidating the molecular mechanisms underlying the fate choices of brown adipocytes versus myoblasts may lead to novel approaches to expand BAT mass. Here we identify MyoD as a key negative regulator of brown adipocyte development. CRISPR/CAS9-mediated deletion of MyoD in C2C12 myoblasts facilitates their adipogenic transdifferentiation. MyoD knockout downregulates miR-133 and upregulates the miR-133 target Igf1r, leading to amplification of PI3K–Akt signaling. Accordingly, inhibition of PI3K or Akt abolishes the adipogenic gene expression of MyoD null myoblasts. Strikingly, loss of MyoD converts satellite cell-derived primary myoblasts to brown adipocytes through upregulation of Prdm16, a target of miR-133 and key determinant of brown adipocyte fate. Conversely, forced expression of MyoD in brown preadipocytes blocks brown adipogenesis and upregulates the expression of myogenic genes. Importantly, miR-133a knockout significantly blunts the inhibitory effect of MyoD on brown adipogenesis. Our results establish MyoD as a negative regulator of brown adipocyte development by upregulating miR-133 to suppress Akt signaling and Prdm16.

Disruption of insulin signaling in Myf5-expressing progenitors leads to marked paucity of brown fat but normal muscle development.

Insulin exerts pleiotropic effects on cell growth, survival, and metabolism, and its role in multiple tissues has been dissected using conditional knockout mice; however, its role in development has not been studied. Lineage tracing experiments have demonstrated that interscapular brown adipose tissue (BAT) arises from a Myf5-positive lineage shared with skeletal muscle and distinct from the majority of white adipose tissue (WAT) precursors. In this study, we sought to investigate the effects of impaired insulin signaling in the Myf5-expressing precursor cells by deleting the insulin receptor gene. Mice lacking insulin receptor in the Myf5 lineage (Myf5IRKO) have a decrease of interscapular BAT mass; however, muscle development appeared normal. Histologically, the residual BAT had decreased cell size but appeared mature and potentially functional. Expression of adipogenic inhibitors preadipocyte factor-1, Necdin, and wingless-type MMTV integration site member 10a in the residual BAT tissue was nonetheless increased compared with controls, and there was an enrichment of progenitor cells with impaired adipogenic differentiation capacity, suggesting a suppression of adipogenesis in BAT. Surprisingly, when cold challenged, Myf5IRKO mice did not show impaired thermogenesis. This resistance to cold could be attributed to an increased presence of uncoupling protein 1-positive brown adipocytes in sc WAT as well as increased expression of lipolytic activity in BAT. These data suggest a critical role of insulin signaling in the development of interscapular BAT from Myf5-positive progenitor cells, but it appears to be dispensable for muscle development. They also underscore the importance of compensatory browning of sc WAT in the absence of BAT for thermoregulation.

Rictor/mTORC2 Loss in the Myf5 Lineage Reprograms Brown Fat Metabolism and Protects Mice against Obesity and Metabolic Disease

The in vivo functions of mechanistic target of rapamycin complex 2 (mTORC2) and the signaling mechanisms that control brown adipose tissue (BAT) fuel utilization and activity are not well understood. Here, by conditionally deleting Rictor in the Myf5 lineage, we provide in vivo evidence that mTORC2 is dispensable for skeletal muscle development and regeneration but essential for BAT growth. Furthermore, deleting Rictor in Myf5 precursors shifts BAT metabolism to a more oxidative and less lipogenic state and protects mice from obesity and metabolic disease at thermoneutrality. We additionally find that Rictor is required for brown adipocyte differentiation in vitro and that the mechanism specifically requires AKT1 hydrophobic motif phosphorylation but is independent of pan-AKT signaling and is rescued with BMP7. Our findings provide insights into the signaling circuitry that regulates brown adipocytes and could have important implications for developing therapies aimed at increasing energy expenditure as a means to combat human obesity.

Adipocytes arise from multiple lineages that are heterogeneously and dynamically distributed.

Adipose tissue development is poorly understood. Here we use a lineage-tracing strategy optimal for adipocytes to provide evidence that Myf5 precursors are not the exclusive source of brown adipocytes and contribute more to the mature white and brite adipocyte populations than previously thought. Moreover, Myf5 lineage distribution in adipose tissue changes in response to modifiable and non-modifiable factors. We also find that the Pax3 lineage largely overlaps with the Myf5 lineage in brown fat and subcutaneous white fat, but exhibits gender-linked divergence in visceral white fat while the MyoD1 lineage does not give rise to any adipocytes. Finally, by deleting insulin receptor beta in the Myf5 lineage, we provide in vivo evidence that the insulin receptor is essential for adipogenesis and that adipocyte lineages have plasticity. These data establish a conceptual framework for adipose tissue development and could explain body fat patterning variations in healthy and lipodystrophic or obese humans.

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.

PTEN Loss in the Myf5 Lineage Redistributes Body Fat and Reveals Subsets of White Adipocytes that Arise from Myf5 Precursors

The developmental origin of adipose tissue and what controls its distribution is poorly understood. By lineage tracing and gene expression analysis in mice, we provide evidence that mesenchymal precursors expressing Myf5--which are thought to give rise only to brown adipocytes and skeletal muscle--also give rise to a subset of white adipocytes. Furthermore, individual brown and white fats contain a mixture of adipocyte progenitor cells derived from Myf5(+) and Myf5(neg) lineages, the number of which varies with depot location. Subsets of white adipocytes originating from both Myf5(+) and Myf5(neg) precursors respond to β(3)-adrenoreceptor stimulation, suggesting "brite" adipocytes may also have multiple origins. We additionally find that deleting PTEN with myf5-cre causes lipomatosis and partial lipodystrophy by selectively expanding the Myf5(+) adipocyte lineages. Thus, the spectrum of adipocytes arising from Myf5(+) precursors is broader than previously thought, and differences in PI3K activity between adipocyte lineages alter body fat distribution.

Two Cell Lineages, myf5 and myf5-Independent, Participate in Mouse Skeletal Myogenesis

In skeletal muscle development, the myogenic regulatory factors myf5 and myoD play redundant roles in the specification and maintenance of myoblasts, whereas myf6 has a downstream role in differentiating myocytes and myofibers. It is not clear whether the redundancy between myf5 and myoD is within the same cell lineage or between distinct lineages. Using lineage tracing and conditional cell ablation in mice, we demonstrate the existence of two distinct lineages in myogenesis: a myf5 lineage and a myf5-independent lineage. Ablating the myf5 lineage is compatible with myogenesis sustained by myf5-independent, myoD-expressing myoblasts, whereas ablation of the myf6 lineage leads to an absence of all differentiated myofibers, although early myogenesis appears to be unaffected. We also demonstrate here the existence of a significant myf5 lineage within ribs that has an important role in rib development, suggested by severe rib defects upon ablating the myf5 lineage.