A Duplication CNV That Conveys Traits Reciprocal to Metabolic Syndrome and Protects against Diet-Induced Obesity in Mice and Men

The functional contribution of CNV to human biology and disease pathophysiology has undergone limited exploration. Recent observations in humans indicate a tentative link between CNV and weight regulation. Smith-Magenis syndrome (SMS), manifesting obesity and hypercholesterolemia, results from a deletion CNV at 17p11.2, but is sometimes due to haploinsufficiency of a single gene, RAI1. The reciprocal duplication in 17p11.2 causes Potocki-Lupski syndrome (PTLS). We previously constructed mouse strains with a deletion, Df(11)17, or duplication, Dp(11)17, of the mouse genomic interval syntenic to the SMS/PTLS region. We demonstrate that Dp(11)17 is obesity-opposing; it conveys a highly penetrant, strain-independent phenotype of reduced weight, leaner body composition, lower TC/LDL, and increased insulin sensitivity that is not due to alteration in food intake or activity level. When fed with a high-fat diet, Dp(11)17/+ mice display much less weight gain and metabolic change than WT mice, demonstrating that the Dp(11)17 CNV protects against metabolic syndrome. Reciprocally, Df(11)17/+ mice with the deletion CNV have increased weight, higher fat content, decreased HDL, and reduced insulin sensitivity, manifesting a bona fide metabolic syndrome. These observations in the deficiency animal model are supported by human data from 76 SMS subjects. Further, studies on knockout/transgenic mice showed that the metabolic consequences of Dp(11)17 and Df(11)17 CNVs are not only due to dosage alterations of Rai1, the predominant dosage-sensitive gene for SMS and likely also PTLS. Our experiments in chromosome-engineered mouse CNV models for human genomic disorders demonstrate that a CNV can be causative for weight/metabolic phenotypes. Furthermore, we explored the biology underlying the contribution of CNV to the physiology of weight control and energy metabolism. The high penetrance, strain independence, and resistance to dietary influences associated with the CNVs in this study are features distinct from most SNP–associated metabolic traits and further highlight the potential importance of CNV in the etiology of both obesity and MetS as well as in the protection from these traits

Notch Signaling Rescues Loss of Satellite Cells Lacking Pax7 and Promotes Brown Adipogenic Differentiation

SummaryPax7 is a nodal transcription factor that is essential for regulating the maintenance, expansion, and myogenic identity of satellite cells during both neonatal and adult myogenesis. Deletion of Pax7 results in loss of satellite cells and impaired muscle regeneration. Here, we show that ectopic expression of the constitutively active intracellular domain of Notch1 (NICD1) rescues the loss of Pax7-deficient satellite cells and restores their proliferative potential. Strikingly NICD1-expressing satellite cells do not undergo myogenic differentiation and instead acquire a brown adipogenic fate both in vivo and in vitro. NICD-expressing Pax7−/− satellite cells fail to upregulate MyoD and instead express the brown adipogenic marker PRDM16. Overall, these results show that Notch1 activation compensates for the loss of Pax7 in the quiescent state and acts as a molecular switch to promote brown adipogenesis in adult skeletal muscle

The dystrophin glycoprotein complex regulates the epigenetic activation of muscle stem cell commitment

Asymmetrically dividing muscle stem cells in skeletal muscle give rise to committed cells, where the myogenic determination factor Myf5 is transcriptionally activated by Pax7. This activation is dependent on Carm1, which methylates Pax7 on multiple arginine residues, to recruit the ASH2L:MLL1/2:WDR5:RBBP5 histone methyltransferase complex to the proximal promoter of Myf5. Here, we found that Carm1 is a specific substrate of p38γ/MAPK12 and that phosphorylation of Carm1 prevents its nuclear translocation. Basal localization of the p38γ/p-Carm1 complex in muscle stem cells occurs via binding to the dystrophin-glycoprotein complex (DGC) through β1-syntrophin. In dystrophin-deficient muscle stem cells undergoing asymmetric division, p38γ/β1-syntrophin interactions are abrogated, resulting in enhanced Carm1 phosphorylation. The resulting progenitors exhibit reduced Carm1 binding to Pax7, reduced H3K4-methylation of chromatin, and reduced transcription of Myf5 and other Pax7 target genes. Therefore, our experiments suggest that dysregulation of p38γ/Carm1 results in altered epigenetic gene regulation in Duchenne muscular dystrophy.We thank Drs. Jeffrey Dilworth and Lynn Megeney for careful reading of the manuscript. We also thank Jennifer Ritchie for animal husbandry, Dr. Lawrence Puente for mass spectrometry analysis, Dr. Chloë van Oostende for microscopy and imaging analysis, Paul Oleynik for FACS, and Fan Xiao, Natasha Mercier, and David Wilson for technical assistance. N.C.C. is a recipient of the Centre for Neuromuscular Disease Scholarship in Translational Research Award from the University of Ottawa Brain and Mind Research Institute and was supported by Postdoctoral fellowships from the Canadian Institutes of Health Research (CIHR) and the Ontario Institute for Regenerative Medicine (OIRM). F.P.C. was supported by a Postdoctoral fellowship from the French Muscular Dystrophy Association (AFM)-Téléthon (380782). C.E.B. is supported by a Postdoctoral fellowship from OIRM. M.L. was supported by a Postdoctoral fellowship from CIHR. P.M.-C. acknowledges support from ERC-2016-AdG-741966 (STEM-AGING) and SAF2015-67369-R. M.A.R. holds the Canada Research Chair in Molecular Genetics. These studies were carried out with support of grants to M.A.R. from the US NIH (R01AR044031), the Canadian Institutes of Health Research (FDN-148387), the Muscular Dystrophy Association (USA), E-Rare-2: Canadian Institutes of Health Research/Muscular Dystrophy Canada (ERA-132935), and the Stem Cell Network

<i>Df(11)17/+</i> mice (green) are obese, have reduced TC, HDL, and display reduced insulin sensitivity in comparison to WT mice (gray).

<p>(A) ECHO-MRI identified elevated fat mass (*p = 0.0041) and reduced lean mass (*p = 0.0034) in <i>Df(11)17/+</i> animals. (B) <i>Df(11)17/+</i> animals have lower serum TC (*p = 0.033) and lower HDL (*p = 0.039), but no significantly change in TC/HDL ratio. IP-GTT documented (C) similar blood glucose levels but (D) significantly higher insulin levels (*p = 0.015, 0.028, 0.012 and 0.013 at 0, 30, 60, 120 mins post injection and *p = 0.011 for AUC) in <i>Df(11)17/+</i> animals. During IP-ITT, <i>Df(11)17/+</i> mice retain higher blood glucose concentration, shown as both (E) actual concentration (*p = 0.026 and 0.008 for 60 and 120 mins post insulin injection and *p = 0.018 for AUC) and (F) percentage of the initial glucose concentration (*p = 0.01 for both 60 and 120 min after injection and *p = 0.0086 for AUC). All comparisons were made with two-tailed t-test; results are expressed as mean ± s.e.m. from measurements of (A) 5 <i>Df(11)17/+</i> and 7 WT mice at 32–36 wks (B) 6 <i>Df(11)17/+</i> and 6 WT mice at 34–37 wks (C, D) 5 <i>Df(11)17/+</i> and 5 WT mice at 37–41 wks (E, F) 5 <i>Df(11)17/+</i> and 6 WT mice at 33–37 wks. All AUCs are computed until 120 minutes, for the entire length of the time curves.</p

<i>Dp(11)17/+</i> mice (red) are also leaner and have reduced serum TC, LDL, TC/HDL ratio, and leptin.

<p>(A) Less relative total fat mass (*p = 0.000088) and more relative lean mass (*p = 0.00012) was identified in <i>Dp(11)17/+</i> mice with ECHO-MRI system. (B) <i>Dp(11)17/+</i> animals also possess smaller epididymal white adipose tissue pad (EWAT) (*p = 0.0020). Fasting serum profile revealed (C) reduced TC (*p = 0.021), LDL (*p = 0.01), TC/HDL ratio (*p = 0.0007) and (D) reduced leptin (*p = 0.021) in <i>Dp(11)17/+</i> mice. All comparisons were made with two-tailed t-test; results are expressed as mean ± s.e.m. from measurements of (A) 6 <i>Dp(11)17/+</i> and 10 WT at 21–22 wks (B) 6 <i>Dp(11)17/+</i>, 7 WT at 41 wks (C) 5 <i>Dp(11)17/+</i> and 6 WT at 20–22 wks (D) 6 <i>Dp(11)17/+</i> and 4 WT of 20–21 wks.</p

<i>Dp(11)17/+</i> mice (red) display resistance to diet-induced obesity compared to WT littermates (gray) after a high-fat diet (HF) feeding from 19 to 22 weeks.

<p>(A) Only WT, but not <i>Dp(11)17/+</i> mice have significant (*p = 0.00028) weight gain after three weeks of HF (19–22 wks) that is mainly due to fat mass increase (*p = 0.00030). (B) Body weight percentage of epididymal (EWAT), mesenteric (MWAT), retroperitoneal (RWAT) and inguinal (IWAT) white adipose tissues are all higher in WT mice post HF than <i>Dp(11)17/+</i> mice (*p = 0.000033 for EWAT, p = 0.00021 for MWAT, p = 0.000033 for RWAT, p = 0.000048 for IWAT). Liver and brown adipose tissues (BAT) remain similar. (C) Dissected EWAT, MWAT and liver are compared between WT and <i>Dp(11)17/+</i> mice post HF diet. EWAT and MWAT, but not the liver, are much smaller in <i>Dp(11)17/+</i> mice. (D) Histology of the EWAT adipocytes from <i>Dp(11)17/+</i> and WT mice, demonstrating smaller adipocytes in <i>Dp(11)17/+</i> mice after HF feeding. The measurements are from (A): 11 <i>Dp(11)17</i>/+ and 12 WT at 22 wks post HF feeding compared to 6 <i>Dp(11)17</i>/+ and 10 WT on RC at 21–22 wks (B) 8 <i>Dp(11)17</i>/+ and 9 WT post HF feeding. <i>Dp</i>/WT mice after HF diet: red/gray bars with dotted pattern; <i>Dp</i>/WT mice with RC: red/gray bars without pattern.</p

<i>Dp(11)17/+</i> mice (red) display improved insulin sensitivity compared to WT mice (gray).

<p>During IP-GTT (6 hr fasting, 1.5 mg glucose/g body weight), <i>Dp(11)17/+</i> mice demonstrate (A) lower blood glucose (*p = 0.006 for 120 minutes post injection; # p = 0.052 for the area under curve (AUC)) and (B) lower blood insulin level (*p = 0.0037, 0.0026, 0.0051, 0.0031 and 0.0015 for the time points 0, 15, 30, 60 and 120 minutes; *p = 0.002 for AUC). During IP-ITT (4–6 hrs fasting, 1 mU insulin/g body weight), <i>Dp(11)17/+</i> mice also demonstrate lower blood glucose concentration, shown as both actual concentration (C) (*p = 0.011, 0.004 and 0.037 for 0, 15 and 30 mins post insulin injection) and percentage of the initial glucose concentration (D) (*p = 0.038 for 15 mins post insulin injection). All comparisons were made with two-tailed t-test; results are expressed as mean ± s.e.m. from measurements of (A, B) n = 5 <i>Dp(11)17/+</i> and 6 WT at 30 wks (C, D) 4 <i>Dp(11)17/+</i> and 4 WT mice at 20–22 wks. All AUCs are computed until 120 minutes, for the entire length of the time curves.</p

<i>Dp(11)17/+</i> mice (red) have similar food intake and activity levels, but higher energy expenditure than WT mice (gray), which may be partially accounted for by the difference in expression levels of UCP1 in the BAT tissue.

<p>(A) <i>Dp(11)17/+</i> mice have similar amount daily food intake to WT mice after 4 wks of age, although they consume less food at 3 wks (*p = 0.001) and 4 wks (*p = 0.048). (B) VersaMax system (Accuscan Inc., Ohio) using the beam block technique implemented in home cages revealed no difference in horizontal activity level between <i>Dp(11)17/+</i> and WT animals. (C, D) Oxygen consumption measured using the CLAMS system (Columbus Ins., Ohio) for over three days documented higher energy expenditure of <i>Dp(11)17/+</i> mice in the light phases alone (*p = 0.0095) and during the entire day (*p = 0.044). (E, F) Respiratory exchange ratio (RER) measured using the CLAMS system for over three days again confirmed higher metabolic activity of <i>Dp(11)17/+</i> mice (*p = 0.00151). (G) Western blot for UCP1 expression in BAT tissue of three <i>Dp(11)17/+</i> and three WT mice with antibody AB3036 (Millipore). The same blot was normalized to actin blotting using MAB1501 (Millipore). (H) Normalized intensity of UCP1 signals in <i>Dp(11)17/+</i> vs. WT mice (17.13±10.21 vs. 4.74±2.05, p = 0.35). The measurements are from (A) 5–13 <i>Dp(11)17</i>/+ and 5–11 WT at different time points (B) to (F) 12 <i>Dp(11)17</i>/+ and 7–10 WT at 25–32 wks (G) 3 <i>Dp(11)17</i>/+ and 3 WT at 30 wks.</p