Tizhong Shan

Myostatin knockout drives browning of white adipose tissue through activating the AMPK-PGC1α-Fndc5 pathway in muscle

Myostatin (Mstn) is predominantly expressed in skeletal muscles and plays important roles in regulating muscle growth and development, as well as fat deposition. Mstn‐knockout (Mstn–/–) mice exhibit increased muscle mass due to both hypertrophy and hyperplasia, and leaner body composition due to reduced fat mass. Here, we show that white adipose tissue (WAT) of Mstn–/– develops characteristics of brown adipose tissue (BAT) with dramatically increased expression of BAT signature genes, including Ucp1 and Pgc1α, and beige adipocyte markers Tmem26 and CD137. Strikingly, the observed browning phenotype is non‐cell autonomous and is instead driven by the newly defined myokine irisin (Fndc5) secreted from Mstn–/– skeletal muscle. Within the muscle, Mstn–/– leads to increased expression of AMPK and its phosphorylation, which subsequently activates PGC1α and Fndc5. Together, our study defines a paradigm of muscle‐fat crosstalk mediated by Fndc5, which is up‐regulated and secreted from muscle to induce beige cell markers and the browning of WAT in Mstn–/– mice. These results suggest that targeting muscle Mstn and its downstream signaling represents a therapeutic approach to treat obesity and type 2 diabetes.—Shan, T., Liang, X., Bi, P., Kuang, S. Myostatin knockout drives browning of white adipose tissue through activating the AMPK‐PGC1α‐Fndc5 pathway in muscle. FASEB J. 27, 1981–1989 (2013). www.fasebj.org

Inhibition of Notch signaling promotes browning of white adipose tissue and ameliorates obesity

Pengpeng Bi1, Tizhong Shan1, Weiyi Liu1, Feng Yue1, Xin Yang1, Xin-Rong Liang1, Jinghua Wang1, Jie Li2, Nadia Carlesso3, Xiaoqi Liu2,4, and Shihuan Kuang1,4,* 1Department of Animal Sciences, Purdue University, West Lafayette, IN 47907, USA. 2Department of Biochemistry, Purdue University, West Lafayette, IN 47907, USA. 3Department of Pediatrics, Indiana University School of Medicine, Indianapolis, IN 46202, USA. 4Center for Cancer Research, Purdue University, West Lafayette, IN 47907, USA.Beige adipocytes in white adipose tissue (WAT) are similar to classical brown adipocytes in that they can burn lipids to produce heat. Thus, an increase in beige adipocyte content in WAT browning would raise energy expenditure and reduce adiposity. Here we report that adipose-specific inactivation of Notch1 or its signaling mediator Rbpj in mice results in browning of WAT and elevated expression of uncoupling protein 1 (Ucp1), a key regulator of thermogenesis. Consequently, as compared to wild-type mice, Notch mutants exhibit elevated energy expenditure, better glucose tolerance and improved insulin sensitivity and are more resistant to high fat diet–induced obesity. By contrast, adipose-specific activation of Notch1 leads to the opposite phenotypes. At the molecular level, constitutive activation of Notch signaling inhibits, whereas Notch inhibition induces, Ppargc1a and Prdm16 transcription in white adipocytes. Notably, pharmacological inhibition of Notch signaling in obese mice ameliorates obesity, reduces blood glucose and increases Ucp1 expression in white fat. Therefore, Notch signaling may be therapeutically targeted to treat obesity and type 2 diabetes.

Fatty acid binding protein 4 expression marks a population of adipocyte progenitors in white and brown adipose tissues

Adipose tissues regulate metabolism, reproduction, and life span. The development and growth of adipose tissue are due to increases of both adipocyte cell size and cell number; the latter is mediated by adipocyte progenitors. Various markers have been used to identify either adipocyte progenitors or mature adipocytes. The fatty acid binding protein 4 (FABP4), commonly known as adipocyte protein 2 (aP2), has been extensively used as a marker for differentiated adipocytes. However, whether aP2 is expressed in adipogenic progenitors is controversial. Using Cre/LoxP‐based cell lineage tracing in mice, we have identified a population of aP2‐expressing progenitors in the stromal vascular fraction (SVF) of both white and brown adipose tissues. The aP2‐lineage progenitors reside in the adipose stem cell niche and express adipocyte progenitor markers, including CD34, Sca1, Dlk1, and PDGFRα. When isolated and grown in culture, the aP2‐expressing SVF cells proliferate and differentiate into adipocytes upon induction. Conversely, ablation of the aP2 lineage greatly reduces the adipogenic potential of SVF cells. When grafted into wild‐type mice, the aP2‐lineage progenitors give rise to adipose depots in recipient mice. Therefore, the expression of aP2 is not limited to mature adipocytes, but also marks a pool of undifferentiated progenitors associated with the vasculature of adipose tissues. Our finding adds to the repertoire of adipose progenitor markers and points to a new regulator of adipose plasticity.—Shan, T., Liu, W., Kuang, S. Fatty acid‐binding protein 4 expression marks a population of adipocyte progenitors in white and brown adipose tissues. FASEB J. 27, 277–287 (2013). www.fasebj.org

miR-133a Regulates Adipocyte Browning In Vivo

Prdm16 determines the bidirectional fate switch of skeletal muscle/brown adipose tissue (BAT) and regulates the thermogenic gene program of subcutaneous white adipose tissue (SAT) in mice. Here we show that miR-133a, a microRNA that is expressed in both BAT and SATs, directly targets the 3′ UTR of Prdm16. The expression of miR-133a dramatically decreases along the commitment and differentiation of brown preadipocytes, accompanied by the upregulation of Prdm16. Overexpression of miR-133a in BAT and SAT cells significantly inhibits, and conversely inhibition of miR-133a upregulates, Prdm16 and brown adipogenesis. More importantly, double knockout of miR-133a1 and miR-133a2 in mice leads to elevations of the brown and thermogenic gene programs in SAT. Even 75% deletion of miR-133a (a1−/−a2+/−) genes results in browning of SAT, manifested by the appearance of numerous multilocular UCP1-expressing adipocytes within SAT. Additionally, compared to wildtype mice, miR-133a1−/−a2+/− mice exhibit increased insulin sensitivity and glucose tolerance, and activate the thermogenic gene program more robustly upon cold exposure. These results together elucidate a crucial role of miR-133a in the regulation of adipocyte browning in vivo.

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.

A heterogeneous lineage origin underlies the phenotypic and molecular differences of white and beige adipocytes

Summary A worldwide epidemic of obesity and its associated metabolic disorders raise the significance of adipocytes, their origins and characteristics. Our previous study has demonstrated that interscapular brown adipose tissue (BAT), but not intramuscular adipose, is derived from the Pax3-expressing cell lineage. Here, we show that various depots of subcutaneous (SAT) and visceral adipose tissue (VAT) are highly heterogeneous in the Pax3 lineage origin. Interestingly, the relative abundance of Pax3 lineage cells in SAT depots is inversely correlated to expression of BAT signature genes including Prdm16, Pgc1a (Ppargc1a) and Ucp1. FACS analysis further demonstrates that adipocytes differentiated from non-Pax3 lineage preadipocytes express higher levels of BAT and beige adipocyte signature genes compared with the Pax3 lineage adipocytes within the same depots. Although both Pax3 and non-Pax3 lineage preadipocytes can give rise to beige adipocytes, the latter contributes more significantly. Consistently, genetic ablation of Pax3 lineage cells in SAT leads to increased expression of beige cell markers. Finally, non-Pax3 lineage beige adipocytes are more responsive to cAMP-agonist-induced Ucp1 expression. Taken together, these results demonstrate widespread heterogeneity in Pax3 lineage origin, and its inverse association with BAT gene expression within and among subcutaneous adipose depots.

Lkb1 controls brown adipose tissue growth and thermogenesis by regulating the intracellular localization of CRTC3

Brown adipose tissue (BAT) dissipates energy through Ucp1-mediated uncoupled respiration and its activation may represent a therapeutic strategy to combat obesity. Here we show that Lkb1 controls BAT expansion and UCP1 expression in mice. We generate adipocyte-specific Lkb1 knockout mice and show that, compared with wild-type littermates, these mice exhibit elevated UCP1 expression in BAT and subcutaneous white adipose tissue, have increased BAT mass and higher energy expenditure. Consequently, KO mice have improved glucose tolerance and insulin sensitivity, and are more resistant to high-fat diet (HFD)-induced obesity. Deletion of Lkb1 results in a cytoplasm to nuclear translocation of CRTC3 in brown adipocytes, where it recruits C/EBPβ to enhance Ucp1 transcription. In parallel, the absence of Lkb1 also suppresses AMPK activity, leading to activation of the mTOR signalling pathway and subsequent BAT expansion. These data suggest that inhibition of Lkb1 or its downstream signalling in adipocytes could be a novel strategy to increase energy expenditure in the context of obesity, diabetes and other metabolic diseases.

mTOR is necessary for proper satellite cell activity and skeletal muscle regeneration

The serine/threonine kinase mammalian target of rapamycin (mTOR) is a key regulator of protein synthesis, cell proliferation and energy metabolism. As constitutive deletion of Mtor gene results in embryonic lethality, the function of mTOR in muscle stem cells (satellite cells) and skeletal muscle regeneration remains to be determined. In this study, we established a satellite cell specific Mtor conditional knockout (cKO) mouse model by crossing Pax7(CreER) and Mtor(flox/flox) mice. Skeletal muscle regeneration after injury was severely compromised in the absence of Mtor, indicated by increased number of necrotic myofibers infiltrated by Evans blue dye, and reduced number and size of regenerated myofibers in the Mtor cKO mice compared to wild type (WT) littermates. To dissect the cellular mechanism, we analyzed satellite cell-derived primary myoblasts grown on single myofibers or adhered to culture plates. The Mtor cKO myoblasts exhibited defective proliferation and differentiation kinetics when compared to myoblasts derived from WT littermates. At the mRNA and protein levels, the Mtor cKO myoblasts expressed lower levels of key myogenic determinant genes Pax7, Myf5, Myod, Myog than did the WT myoblasts. These results suggest that mTOR is essential for satellite cell function and skeletal muscle regeneration through controlling the expression of myogenic genes.

Lkb1 is indispensable for skeletal muscle development, regeneration, and satellite cell homeostasis.

Serine/threonine kinase 11, commonly known as liver kinase b1 (Lkb1), is a tumor suppressor that regulates cellular energy metabolism and stem cell function. Satellite cells are skeletal muscle resident stem cells that maintain postnatal muscle growth and repair. Here, we used MyoDCre/Lkb1flox/flox mice (called MyoD‐Lkb1) to delete Lkb1 in embryonic myogenic progenitors and their descendant satellite cells and myofibers. The MyoD‐Lkb1 mice exhibit a severe myopathy characterized by central nucleated myofibers, reduced mobility, growth retardation, and premature death. Although tamoxifen‐induced postnatal deletion of Lkb1 in satellite cells using Pax7CreER mice bypasses the developmental defects and early death, Lkb1 null satellite cells lose their regenerative capacity cell‐autonomously. Strikingly, Lkb1 null satellite cells fail to maintain quiescence in noninjured resting muscles and exhibit accelerated proliferation but reduced differentiation kinetics. At the molecular level, Lkb1 limits satellite cell proliferation through the canonical AMP‐activated protein kinase/mammalian target of rapamycin pathway, but facilitates differentiation through phosphorylation of GSK‐3β, a key component of the WNT signaling pathway. Together, these results establish a central role of Lkb1 in muscle stem cell homeostasis, muscle development, and regeneration. Stem Cells 2014;32:2893–2907

Adipocyte-specific deletion of mTOR inhibits adipose tissue development and causes insulin resistance in mice

Aims/hypothesisThe in vivo role of mechanistic target of rapamycin (mTOR) in the development and function of adipose tissue, especially brown adipose tissue (BAT), is not well understood. Here, we aimed to assess the effect of mTOR (also known as Mtor) knockout on adipose tissues and systemic energy metabolism.MethodsWe generated adipocyte-specific mTOR-knockout mice (Adipoq-mTOR) by crossing adiponectin-Cre (Adipoq-Cre) mice with mTORflox/flox mice. The mice were then subjected to morphological, physiological (indirect calorimetry, glucose and insulin tolerance tests) and gene expression analyses to determine the role of mTOR in adipose tissues.ResultsWe provide in vivo evidence that mTOR is essential for adipose tissue development and growth. Deletion of mTOR decreased the mass of both BAT and white adipose tissues (WAT) and induced browning of WAT. In addition, ablation of mTOR in adipose tissues caused insulin resistance and fatty liver in the Adipoq-mTOR mice. Furthermore, mTOR was required for adipocyte differentiation in vivo and activation of PPARγ ameliorated the differentiation deficiency of the mTOR-null adipocytes.Conclusions/interpretationOur findings demonstrate that mTOR is a critical regulator of adipogenesis and systemic energy metabolism. Our study provides key insights into the role of mTOR in adipose tissues; such knowledge may facilitate the development of novel strategies with which to treat obesity and related metabolic diseases.