Isolation of Progenitors that Exhibit Myogenic/Osteogenic Bipotency In Vitro by Fluorescence-Activated Cell Sorting from Human Fetal Muscle

Summary Fluorescence-activated cell sorting (FACS) strategies to purify distinct cell types from the pool of fetal human myofiber-associated (hMFA) cells were developed. We demonstrate that cells expressing the satellite cell marker PAX7 are highly enriched within the subset of CD45−CD11b−GlyA−CD31−CD34−CD56intITGA7hi hMFA cells. These CD45−CD11b−GlyA−CD31−CD34−CD56intITGA7hi cells lack adipogenic capacity but exhibit robust, bipotent myogenic and osteogenic activity in vitro and engraft myofibers when transplanted into mouse muscle. In contrast, CD45−CD11b−GlyA−CD31−CD34+ fetal hMFA cells represent stromal constituents of muscle that do not express PAX7, lack myogenic function, and exhibit adipogenic and osteogenic capacity in vitro. Adult muscle likewise contains PAX7+ CD45−CD11b−GlyA−CD31−CD34−CD56intITGA7hi hMFA cells with in vitro myogenic and osteogenic activity, although these cells are present at lower frequency in comparison to their fetal counterparts. The ability to directly isolate functionally distinct progenitor cells from human muscle will enable novel insights into muscle lineage specification and homeostasis

Cardiolipin synthesis in brown and beige fat mitochondria is essential for systemic energy homeostasis

Activation of energy expenditure in thermogenic fat is a promising strategy to improve metabolic health, yet the dynamic processes that evoke this response are poorly understood. Here we show that synthesis of the mitochondrial phospholipid cardiolipin is indispensable for stimulating and sustaining thermogenic fat function. Cardiolipin biosynthesis is robustly induced in brown and beige adipose upon cold exposure. Mimicking this response through overexpression of cardiolipin synthase (Crls1) enhances energy consumption in mouse and human adipocytes. Crls1 deficiency in thermogenic adipocytes diminishes inducible mitochondrial uncoupling and elicits a nuclear transcriptional response through endoplasmic reticulum stress-mediated retrograde communication. Cardiolipin depletion in brown and beige fat abolishes adipose thermogenesis and glucose uptake, which renders animals insulin resistant. We further identify a rare human CRLS1 variant associated with insulin resistance and show that adipose CRLS1 levels positively correlate with insulin sensitivity. Thus, adipose cardiolipin has a powerful impact on organismal energy homeostasis through thermogenic fat bioenergetics

Clonal analyses and gene profiling identify genetic biomarkers of human brown and white preadipocyte thermogenic potential

Targeting brown adipose tissue (BAT) content or activity has therapeutic potential for treating obesity and the metabolic syndrome by increasing energy expenditure. Both inter- and intra-individual differences contribute to heterogeneity in human BAT and potentially to differential thermogenic capacity in human populations. Here, we demonstrated the generated clones of brown and white preadipocytes from human neck fat of four individuals and characterized their adipogenic differentiation and thermogenic function. Combining an uncoupling protein 1(UCP1) reporter system and expression profiling, we defined novel sets of gene signatures in human preadipocytes that could predict the thermogenic potential of the cells once they were maturated in culture. Knocking out the positive UCP1 regulators identified by this approach, PREX1 and EDNRB in brown preadipocytes using CRISPR/Cas9 markedly abolished the high level of UCP1 in brown adipocytes differentiated from the preadipocytes. Finally, we were able to prospectively isolate adipose progenitors with great thermogenic potential using cell surface marker CD29. These data provide new insights into the cellular heterogeneity in human fat and offer the identification of possible biomarkers of thermogenically competent preadipocytes

ADRA1A-Gα_q signalling potentiates adipocyte thermogenesis through CKB and TNAP

Noradrenaline (NA) regulates cold-stimulated adipocyte thermogenesis(1). Aside from cAMP signalling downstream of β-adrenergic receptor activation, how NA promotes thermogenic output is still not fully understood. Here, we show that coordinated α(1)-adrenergic receptor (AR) and β(3)-AR signalling induces the expression of thermogenic genes of the futile creatine cycle(2,3), and that early B cell factors, oestrogen-related receptors and PGC1α are required for this response in vivo. NA triggers physical and functional coupling between the α(1)-AR subtype (ADRA1A) and Gα(q) to promote adipocyte thermogenesis in a manner that is dependent on the effector proteins of the futile creatine cycle, creatine kinase B and tissue-non-specific alkaline phosphatase. Combined Gα(q) and Gα(s) signalling selectively in adipocytes promotes a continual rise in whole-body energy expenditure, and creatine kinase B is required for this effect. Thus, the ADRA1A–Gα(q)–futile creatine cycle axis is a key regulator of facultative and adaptive thermogenesis

Control of CD36 phosphorylation by global intestinal alkaline phosphatase mediates intestinal adaptation to high-fat diet

Thesis (Ph.D.)--Boston UniversityPLEASE NOTE: Boston University Libraries did not receive an Authorization To Manage form for this thesis or dissertation. It is therefore not openly accessible, though it may be available by request. If you are the author or principal advisor of this work and would like to request open access for it, please contact us at [email protected]. Thank you.The mechanisms by which diets high in saturated fat (HFD) contribute to intestinal adaptation and obesity are unknown. The hypothesis that functional changes in distal portions of small intestine are induced by HFD was tested in C57B1/6 mice. Specifically, it was examined whether the putative fatty acid translocase CD36 was phosphorylated in mouse intestinal epithelial cells and whether dephosphorylation of CD36 increased long chain fatty acid (LCFA) absorption. Co-immunoprecipitation was used to investigate specific intestinal alkaline phosphatases that might interact with CD36. It was also examined whether chronic ingestion of an HFD would lead to upregulation of the CD36 and/or one or more intestinal alkaline phosphatases that may activate CD36. CD36 was found to be phosphorylated on the surface of mouse enterocytes, indicating that there may be a phosphatase-sensitive pool of phospho-CD36 (pCD36) in mouse small intestinal tissue. CD36 was dephosphorylated by alkaline phosphatase and this treatment increased long chain but not short chain fatty acid uptake. Long chain fatty acid uptake was blocked with a specific CD36 inhibitor. CD36 from mouse small intestines physically interacted specifically with global intestinal alkaline phosphatase (gIAP) but not duodenal alkaline phosphatase (dIAP). As expected, HFD increased body weight, adiposity, and plasma triglycerides compared to control mice. CD36 and gIAP but not dIAP protein levels were significantly increased in distal but not proximal regions of intestines of HFD mice. Finally, HFD increased the absorptive capacity of the distal small intestine for LCFA in a CD36-dependent manner. It is concluded that HFD specifically upregulates gIAP protein in epithelial cells of the distal regions of the small intestine of mice, and that one of its substrates is pCD36, which has been implicated in transcellular fat transport. This diet also increases the absorptive capacity of the distal small intestine for LCFAs. Taken together, these results suggest that HFD causes intestinal adaptation that results in an increased capacity to absorb dietary fat. This effect is mediated in part by increasing the expression and activity of the fatty acid transporter CD36 and its regulatory enzyme gIAP.2031-01-0

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

A CRISPR Screen Identifies the E3 Ubiquitin Ligase Rfwd2 as a Negative Regulator of Glucose Uptake in Brown Adipocytes

Brown adipose tissue activation increases energy expenditure and has been shown to improve glucose tolerance, making it a promising target for the treatment of obesity and type 2 diabetes. Brown adipocytes differentiate into cells with multilocular lipid droplets, which can efficiently absorb and oxidize glucose; however, the mechanisms regulating these processes are not completely understood. We conducted a genome-wide loss-of-function screen using a CRISPR-based approach to identify genes that promote or inhibit adipogenesis and glucose uptake in brown adipocytes. We validated genes that negatively or positively regulated these pathways and verified that the E3-ubiquitin ligase Rfwd2 suppressed brown adipocyte glucose uptake. Brown adipocytes with CRISPR-targeted Rfwd2 deletion showed an altered proteomic landscape and increased basal, as well as insulin-stimulated, glucose uptake. These data reveal the complexity of genetic regulation of brown adipogenesis and glucose metabolism