Mismatch between obesogenic intrauterine environment and low-fat postnatal diet may confer offspring metabolic advantage.

OBJECTIVE: Mismatch between a depleted intrauterine environment and a substrate-rich postnatal environment confers an increased risk of offspring obesity and metabolic syndrome. Maternal diet-induced obesity (MATOB) is associated with the same outcomes. These experiments tested the hypothesis that a mismatch between a nutrient-rich intrauterine environment and a low-fat postnatal environment would ameliorate offspring metabolic morbidity. METHODS: C57BL6/J female mice were fed either a 60% high-fat diet (HFD) or a 10% fat control diet (CD) for 14-week pre-breeding and during pregnancy/lactation. Offspring were weaned to CD. Weight was evaluated weekly; body composition was determined using EchoMRI. Serum fasting lipids and glucose and insulin tolerance tests were performed. Metabolic rate, locomotor, and sleep behavior were evaluated with indirect calorimetry. RESULTS: MATOB-exposed/CD-weaned offspring of both sexes had improved glucose tolerance and insulin sensitivity compared to controls. Males had improved fasting lipids. Females had significantly increased weight and body fat percentage in adulthood compared to sex-matched controls. Females also had significantly increased sleep duration and reduced locomotor activity compared to males. CONCLUSIONS: Reduced-fat dietary switch following intrauterine and lactational exposure to MATOB was associated with improved glucose handling and lipid profiles in adult offspring, more pronounced in males. A mismatch between a high-fat prenatal and low-fat postnatal environment may confer a metabolic advantage. The amelioration of deleterious metabolic programming by strict offspring adherence to a low-fat diet may have translational potential.

Established cell surface markers efficiently isolate highly overlapping populations of skeletal muscle satellite cells by fluorescence-activated cell sorting.

BACKGROUND: Fluorescent-activated cell sorting (FACS) has enabled the direct isolation of highly enriched skeletal muscle stem cell, or satellite cell, populations from postnatal tissue. Several distinct surface marker panels containing different positively selecting surface antigens have been used to distinguish muscle satellite cells from other non-myogenic cell types. Because functional and transcriptional heterogeneity is known to exist within the satellite cell population, a direct comparison of results obtained in different laboratories has been complicated by a lack of clarity as to whether commonly utilized surface marker combinations select for distinct or overlapping subsets of the satellite cell pool. This study therefore sought to evaluate phenotypic and functional overlap among popular satellite cell sorting paradigms. METHODS: Utilizing a transgenic Pax7-zsGreen reporter mouse, we compared the overlap between the fluorescent signal of canonical paired homeobox protein 7 (Pax7) expressing satellite cells to cells identified by combinations of surface markers previously published for satellite cells isolation. We designed two panels for mouse skeletal muscle analysis, each composed of markers that exclude hematopoietic and stromal cells (CD45, CD11b, Ter119, CD31, and Sca1), combined with previously published antibody clones recognizing surface markers present on satellite cells (ß1-integrin/CXCR4, α7-integrin/CD34, and Vcam1). Cell populations were comparatively analyzed by flow cytometry and FACS sorted for functional assessment of myogenic activity. RESULTS: Consistent with prior reports, each of the commonly used surface marker schemes evaluated here identified a highly enriched satellite cell population, with 89-90 % positivity for Pax7 expression based on zsGreen fluorescence. Distinct surface marker panels were also equivalent in their ability to identify the majority of the satellite cell pool, with 90-93 % of all Pax7-zsGreen positive cells marked by each of the surface marker schemes. The direct comparison among surface marker schemes validated their selection for highly overlapping subsets of cells. Functional analysis in vitro showed no differences in the abilities of cells sorted by these different methods to grow in culture and differentiate. CONCLUSIONS: This study demonstrates the equivalency of several previously published and widely utilized surface marker schemes for isolating a highly purified and myogenically active population of satellite cells from the mouse skeletal muscle, which should facilitate cross-comparison of data across laboratories.

In vivo gene editing in dystrophic mouse muscle and muscle stem cells.

Frame-disrupting mutations in the DMD gene, encoding dystrophin, compromise myofiber integrity and drive muscle deterioration in Duchenne muscular dystrophy (DMD). Removing one or more exons from the mutated transcript can produce an in-frame mRNA and a truncated, but still functional, protein. In this study, we developed and tested a direct gene-editing approach to induce exon deletion and recover dystrophin expression in the mdx mouse model of DMD. Delivery by adeno-associated virus (AAV) of clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 endonucleases coupled with paired guide RNAs flanking the mutated Dmd exon23 resulted in excision of intervening DNA and restored the Dmd reading frame in myofibers, cardiomyocytes, and muscle stem cells after local or systemic delivery. AAV-Dmd CRISPR treatment partially recovered muscle functional deficiencies and generated a pool of endogenously corrected myogenic precursors in mdx mouse muscle.