J Biol Chem. 2015 Dec 18;290(51):30514-29.
Lecithin:Cholesterol Acyltransferase (LCAT) Deficiency Promotes Differentiation of Satellite Cells to Brown Adipocytes in a Cholesterol-dependent Manner.
Nesan D, Tavallaee G, Koh D, Bashiri A, Abdin R, Ng DS.
- 1From the Keenan Research Centre, Li Ka Shing Knowledge Institute, Department of Medicine, St. Michael’s Hospital, Toronto, Ontario M5B 1W8, Canada and the Department of Physiology, Faculty of Medicine, and.
- 2From the Keenan Research Centre, Li Ka Shing Knowledge Institute, Department of Medicine, St. Michael’s Hospital, Toronto, Ontario M5B 1W8, Canada and.
- 3From the Keenan Research Centre, Li Ka Shing Knowledge Institute, Department of Medicine, St. Michael’s Hospital, Toronto, Ontario M5B 1W8, Canada and the Department of Physiology, Faculty of Medicine, and Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ontario M5S 1A8, Canada firstname.lastname@example.org.
Our laboratory previously reported that lecithin:cholesterol acyltransferase (LCAT) and LDL receptor double knock-out mice (Ldlr(-/-)xLcat(-/-) or DKO) spontaneously develop functioning ectopic brown adipose tissue (BAT) in skeletal muscle, putatively contributing to protection from the diet-induced obesity phenotype. Here we further investigated their developmental origin and the mechanistic role of LCAT deficiency. Gene profiling of skeletal muscle in DKO newborns and adults revealed a classical lineage. Primary quiescent satellite cells (SC) from chow-fed DKO mice, not in Ldlr(-/-)xLcat(+/+) single-knock-out (SKO) or C57BL/6 wild type, were found to (i) express exclusively classical BAT-selective genes, (ii) be primed to express key functional BAT genes, and (iii) exhibit markedly increased ex vivo adipogenic differentiation into brown adipocytes. This gene priming effect was abrogated upon feeding the mice a 2% high cholesterol diet in association with accumulation of excess intracellular cholesterol. Ex vivo cholesterol loading of chow-fed DKO SC recapitulated the effect, indicating that cellular cholesterol is a key regulator of SC-to-BAT differentiation. Comparing adipogenicity of Ldlr(+/+)xLcat(-/-) (LCAT-KO) SC with DKO SC identified a role for LCAT deficiency in priming SC to express BAT genes. Additionally, we found that reduced cellular cholesterol is important for adipogenic differentiation, evidenced by increased induction of adipogenesis in cholesterol-depleted SC from both LCAT-KO and SKO mice. Taken together, we conclude that ectopic BAT in DKO mice is classical in origin, and its development begins in utero. We further showed complementary roles of LCAT deficiency and cellular cholesterol reduction in the SC-to-BAT adipogenesis.
The prevalence of obesity continues to escalate worldwide, imposing increasing health burden to the society via multiple complications, including the metabolic syndrome, type 2 diabetes (T2D) and atherosclerotic cardiovascular diseases. Ever since the exciting discovery of abundant metabolically active BAT in adult humans in 2009, the notion of harvesting brown adipose tissue (BAT) to combat obesity and T2D through regulating thermogenesis has attracted intense interest (1).
The current paper was built on a serendipity observation in my lab, reporting the detection of islands of brown adipose tissues dispersed in inter-fiber areas in skeletal muscle of LCAT and LDL receptor double knockout mice, a phenotype attributable to the deletion of the LCAT gene and augmented by concurrent absence of the LDL receptors (2). These findings led to the following questions: (1) What is the developmental origin of the ectopic BAT? (2) To what extent are they dependent on either the absence of LCAT or the LDL receptors? (3) What could be the underlying mechanisms?
Studies in the biogenesis of BAT, based on rodent models, have identified two major classes of BAT, namely (i) the classical lineage which is derived from myf5+ myoblasts beginning in utero and (ii) the beige fat, which is present in adult white adipose depots and is primed for induction by a broad array of environmental cues, the most established of which include cold exposure and β-adrenergic agonism (3). In humans, the classical BAT is most often detected in infants but undergo involution shortly after birth. As a result, targeting of this class of BAT as therapeutics proves challenging. On the other hand, most of the thermogenically active BAT in adults share the same gene signature as beige fat (4), a finding that resulted in the intense interest to developing strategies to “brown” the white fat. In spite of the exciting prospect, the identification of safe and effective experimental strategies remains elusive (5). Very recently, a paper by Yin et al reported that, in mice, adult satellite cells (SC) can be induced to differentiate into active BAT upon injury-like activation followed by inhibition of microRNA133 (6), which regulates the expression of PRDM16, the master switch of the BAT gene program. This finding suggests another source of progenitors inducible to form BAT in adults and that the cells share the classical BAT gene signature.
We first determined the developmental origin of the ectopic BAT by two different sets of experiments. We first demonstrated abundant UCP1 gene expression signal in the hindlimb muscle of newborn LCAT/LDLR double knockout mice, in support of a classical BAT lineage as myf5+ myoblast-derived BAT begins in utero. We then showed that the UCP1+ muscle strongly expressed a panel of signature genes for classical BAT but not those for the beige fat. We concluded from these data that the ectopic BAT are of classical lineage.
We then investigated whether the ectopic BAT seen in adult LCAT/LDLR double knockout mice were preformed in utero or they could develop from progenitor cells in adulthood. The first experimental cue came from the observation that when the LCAT/LDLR double knockout mice were fed a high cholesterol diet, the high expression levels of both PRDM16 and UCP1 in whole skeletal muscle lysates in the mice were dramatically suppressed, raising the possibility of the BAT gene program being modulated by tissue loading of cholesterol. A second cue came from our observation that ectopic UCP1 signal by immunofluorescence co-localized with Pax7, a marker for both quiescent and activated SC and with laminin a marker for localizing where quiescent SC typically reside. We have thus provided anatomical evidence to link adult quiescent SC with strong expression of BAT genes.
We therefore hypothesized that adult SC can serve as progenitors to differentiate into functioning brown adipocytes and that cellular cholesterol plays a modulatory role of this adipogenesis. To test this hypothesis, we proceeded to first isolate quiescent SC from LCAT/LDLR double knockout mice vs their LDLR knockout control, each fed either a chow or a 2% high cholesterol diet. We examined the cells both in their quiescent states and also after a 10-day post-adipogenic stimulation ex vivo. We first noted that the expression levels of PRDM16 and UCP1 in quiescent SC of the LCAT/LDLR double knockout mice are already elevated and when compared to the undetectable levels seen in its LDLR single knockout control, suggestive of priming of the BAT gene program in the double knockout strain. This altered gene expression was found associated with a reduced cellular cholesterol level and an increased brown adipogenicity, namely formation of brown adipocytes upon pro-adipogenic differentiation on the basis of number of Oil red O positive cells or UCP1 positive cells per 100 nuclei. The role of cellular cholesterol in modulating the SC-to-brown adipocyte differentiation was further evidenced by SC isolated from high cholesterol fed double knockout mice have higher cellular cholesterol content whereas the expressions of UCP1 and PRDM16 being dramatically suppressed, in association with a reduced SC-to-brown adipocyte adipogenicity.
Having identified an important role of cellular cholesterol in modulating the SC-to-brown adipocyte differentiation in this unique LCAT/LDLR double knockout model, we asked the question whether this adipogenic process is dependent on the unique phenotype of LCAT deficiency. We tested the adipogenicity of SC from LDLR single knockout mice with and without depletion of cellular cholesterol. Satellite cells from LDLR knockout mice contains higher cellular cholesterol when compared to wild type control and showed near undetectable adipogenicity. However, lowering the cellular cholesterol prior to adipogenic stimulation resulted in a marked induction of brown adipogenesis, providing evidence that the ectopic SC-to-brown adipogenesis can be activated independent of LCAT deficiency.
The importance of this study. This study represents the first report of spontaneous development of functional BAT ectopically in skeletal muscle from SC secondary to a well-defined genetic disorder of lipid metabolism. Complete LCAT deficiency represents the first of such metabolic perturbations. In fact, the quiescent SC in LCAT null mice were found to be primed to express the BAT gene program and are more inducible to undergo brown adipogenesis ex vivo. Importantly, we further observed that the brown adipogenicity can be modulated by modifying the cellular cholesterol content, independent of other phenotypes of LCAT deficiency. These findings have provided a framework for recruiting BAT from SC through modulation of their lipid milieu.
- Saito M. Human brown adipose tissue: regulation and anti-obesity potential. Endocr J. 2014;61(5):409-16.
- Li L, Hossain MA, Sadat S, Hager L, Liu L, Tam L, Schroer S, Huogen L, Fantus IG, Connelly PW, Woo M, Ng DS. Lecithin cholesterol acyltransferase null mice are protected from diet-induced obesity and insulin resistance in a gender-specific manner through multiple pathways. J Biol Chem. 2011 May 20;286(20):17809-20.
- Kajimura S, Saito M. A new era in brown adipose tissue biology: molecular control of brown fat development and energy homeostasis. Annu Rev Physiol. 2014;76:225-49.
- Sharp LZ, Shinoda K, Ohno H, Scheel DW, Tomoda E, Ruiz L, Hu H, Wang L, Pavlova Z, Gilsanz V, Kajimura S. Human BAT possesses molecular signatures that resemble beige/brite cells. PLoS One. 2012;7(11):e49452.
- Sidossis L, Kajimura S. Brown and beige fat in humans: thermogenic adipocytes that control energy and glucose homeostasis. J Clin Invest. 2015 Feb;125(2):478-86.
- Yin H, Pasut A, Soleimani VD, Bentzinger CF, Antoun G, Thorn S, Seale P, Fernando P, van Ijcken W, Grosveld F, Dekemp RA, Boushel R, Harper ME, Rudnicki MA. MicroRNA-133 controls brown adipose determination in skeletal muscle satellite cells by targeting Prdm16. Cell Metab. 2013 Feb 5;17(2):210-24.
Acknowledgements: This work was supported by a Banting & Best Diabetes Centre Fellowship in Diabetes Care (funded by Eli Lilly and Boehringer Ingelheim) (to D. N.) and Canadian Institutes of Health Research Operating Grant MOP 275369 (to D. S. N.).