Isolation of Nuclei from Human Intermuscular Adipose Tissue and Downstream Single-Nuclei RNA Sequencing.

Intermuscular adipose tissue (IMAT) is a relatively understudied adipose depot located between muscle fibers. IMAT content increases with age and BMI and is associated with metabolic and muscle degenerative diseases; however, an understanding of the biological properties of IMAT and its interplay with the surrounding muscle fibers is severely lacking. In recent years, single-cell and nuclei RNA sequencing have provided us with cell type-specific atlases of several human tissues. However, the cellular composition of human IMAT remains largely unexplored due to the inherent challenges of its accessibility from biopsy collection in humans. In addition to the limited amount of tissue collected, the processing of human IMAT is complicated due to its proximity to skeletal muscle tissue and fascia. The lipid-laden nature of the adipocytes makes it incompatible with single-cell isolation. Hence, single nuclei RNA sequencing is optimal for obtaining high-dimensional transcriptomics at single-cell resolution and provides the potential to uncover the biology of this depot, including the exact cellular composition of IMAT. Here, we present a detailed protocol for nuclei isolation and library preparation of frozen human IMAT for single nuclei RNA sequencing. This protocol allows for the profiling of thousands of nuclei using a droplet-based approach, thus providing the capacity to detect rare and low-abundant cell types.

Distinct subpopulations of human subcutaneous adipose tissue precursor cells revealed by single-cell RNA sequencing.

Adipose-derived stem cells (ADSCs) play an important role in the differential capacity for excess energy storage between upper body abdominal (ABD) adipose tissue (AT) and lower body gluteofemoral (GF) AT. We cultured ADSCs from subcutaneous ABD AT and GF AT isolated from eight women with differential body fat distribution and performed single-cell RNA sequencing. Six populations of ADSCs were identified and segregated according to their anatomical origin. The three ADSC subpopulations in GF AT were characterized by strong cholesterol/fatty acid (FA) storage and proliferation signatures. The two ABD subpopulations, differentiated by higher expression of committed preadipocyte marker genes, were set apart by differential expression of extracellular matrix and ribosomal genes. The last population, identified in both depots, was similar to smooth muscle cells and when individually isolated and cultured in vitro they differentiated less than the other subpopulations. This work provides important insight into the use of ADSC as an in vitro model of adipogenesis and suggests that specific subpopulations of GF-ADSCs contribute to the more robust capacity for GF-AT to expand and grow compared with ABD-AT in women.NEW & NOTEWORTHY Identification of distinct subpopulations of adipose-derived stem cells (ADSCs) in upper body abdominal subcutaneous (ABD) and lower body gluteofemoral subcutaneous (GF) adipose tissue depots. In ABD-ADSCs, subpopulations are more committed to adipocyte lineage. GF-ADSC subpopulations are enriched for genes involved in lipids and cholesterol metabolism. Similar depot differences were found in stem cell population identified in freshly isolated stoma vascular fraction. The repertoire of ADSCs subpopulations was different in apple-shaped versus pear-shaped women.

Human skeletal muscle tissue chip autonomous payload reveals changes in fiber type and metabolic gene expression due to spaceflight.

Microphysiological systems provide the opportunity to model accelerated changes at the human tissue level in the extreme space environment. Spaceflight-induced muscle atrophy experienced by astronauts shares similar physiological changes to muscle wasting in older adults, known as sarcopenia. These shared attributes provide a rationale for investigating molecular changes in muscle cells exposed to spaceflight that may mimic the underlying pathophysiology of sarcopenia. We report the results from three-dimensional myobundles derived from muscle biopsies from young and older adults, integrated into an autonomous CubeLab™, and flown to the International Space Station (ISS) aboard SpaceX CRS-21 as part of the NIH/NASA funded Tissue Chips in Space program. Global transcriptomic RNA-Seq analyses comparing the myobundles in space and on the ground revealed downregulation of shared transcripts related to myoblast proliferation and muscle differentiation. The analyses also revealed downregulated differentially expressed gene pathways related to muscle metabolism unique to myobundles derived from the older cohort exposed to the space environment compared to ground controls. Gene classes related to inflammatory pathways were downregulated in flight samples cultured from the younger cohort compared to ground controls. Our muscle tissue chip platform provides an approach to studying the cell autonomous effects of spaceflight on muscle cell biology that may not be appreciated on the whole organ or organism level and sets the stage for continued data collection from muscle tissue chip experimentation in microgravity. We also report on the challenges and opportunities for conducting autonomous tissue-on-chip CubeLabTM payloads on the ISS.

Validation of Human Skeletal Muscle Tissue Chip Autonomous Platform to Model Age-Related Muscle Wasting in Microgravity.

Microgravity-induced muscle atrophy experienced by astronauts shares similar physiological changes to muscle wasting experienced by older adults, known as sarcopenia. These shared attributes provide a rationale for investigating microgravity-induced molecular changes in human bioengineered muscle cells that may also mimic the progressive underlying pathophysiology of sarcopenia. Here, we report the results of an experiment that incorporated three-dimensional myobundles derived from muscle biopsies from young and older adults, that were integrated into an autonomous CubeLabâ"¢, and flown to the International Space Station (ISS) aboard SpaceX CRS-21 in December 2020 as part of the NIH/NASA funded Tissue Chips in Space program. Global transcriptomic RNA-Seq analysis comparing the myobundles in space and on the ground revealed downregulation of shared transcripts related to myoblast proliferation and muscle differentiation for those in space. The analysis also revealed differentially expressed gene pathways related to muscle metabolism unique to myobundles derived from the older cohort exposed to the space environment compared to ground controls. Gene classes related to inflammatory pathways were uniquely modulated in flight samples cultured from the younger cohort compared to ground controls. Our muscle tissue chip platform provides a novel approach to studying the cell autonomous effects of microgravity on muscle cell biology that may not be appreciated on the whole organ or organism level and sets the stage for continued data collection from muscle tissue chip experimentation in microgravity. Thus, we also report on the challenges and opportunities for conducting autonomous tissue-on-chip CubeLab TM payloads on the ISS.

Isolation of nuclei from frozen human subcutaneous adipose tissue for full-length single-nuclei transcriptional profiling.

Automated single-cell dispensing is incompatible with white adipose tissue (WAT) due to lipid-laden adipocytes. Single-nuclei RNA-Seq permits transcriptional profiling of all cells from WAT. Human WAT faces unique technical challenges in isolating nuclei compared to rodent tissue due to greater extra-cellular matrix content and larger lipid droplets. In this protocol, we detail how to isolate nuclei from frozen subcutaneous human WAT for single-nuclei RNA-Seq. For complete information on the generation and use of this protocol, please refer to Whytock et al. (2022).1.

Optimization of a urea-containing series of nicotinamide phosphoribosyltransferase (NAMPT) activators.

NAD+ is a crucial cellular factor that plays multifaceted roles in wide ranging biological processes. Low levels of NAD+ have been linked to numerous diseases including metabolic disorders, cardiovascular disease, neurodegeneration, and muscle wasting disorders. A novel strategy to boost NAD+ is to activate nicotinamide phosphoribosyltransferase (NAMPT), the putative rate-limiting step in the NAD+ salvage pathway. We previously showed that NAMPT activators increase NAD+ levels in vitro and in vivo. Herein we describe the optimization of our NAMPT activator prototype (SBI-0797812) leading to the identification of 1-(4-((4-chlorophenyl)sulfonyl)phenyl)-3-(oxazol-5-ylmethyl)urea (34) that showed far more potent NAMPT activation and improved oral bioavailability.

Fat Distribution in Women Is Associated With Depot-Specific Transcriptomic Signatures and Chromatin Structure.

BACKGROUND: Preferential accumulation of fat in the upper body (apple shape) is associated with higher risk of developing metabolic syndrome relative to lower body fat (pear shape). We previously discovered that chromatin openness partially defined the transcriptome of preadipocytes isolated from abdominal and gluteofemoral fat. However, the molecular mechanisms underlying interindividual variation in body shape are unknown. METHODS: Adipocyte fraction was isolated from abdominal and gluteofemoral fat biopsies of premenopausal women (age and body mass index matched) segregated initially only by their waist-to-hip ratio. We evaluated transcriptomic and chromatin accessibility using RNA sequencing and assay for transposase-accessible chromatin using sequencing (ATAC-seq) along with key clinical parameters. RESULTS: Our data showed that higher lower body fat mass was associated with better lipid profile and free fatty acid decrease after glucose administration. Lipid and glucose metabolic pathways genes were expressed at higher levels in gluteofemoral adipocyte fraction in pears, whereas genes associated with inflammation were higher both in abdominal and gluteofemoral apple adipocyte fraction. Gluteofemoral adipocyte chromatin from pear-shaped women contained a significantly higher number of differentially open ATAC-seq peaks relative to chromatin from the apple-shaped gluteofemoral adipocytes. In contrast, abdominal adipocyte chromatin openness showed few differences between apple- and pear-shaped women. We revealed a correlation between gene transcription and open chromatin at the proximity of the transcriptional start site of some of the differentially expressed genes. CONCLUSIONS: Integration of data from all 3 approaches suggests that chromatin openness partially governs the transcriptome of gluteofemoral adipocytes and may be involved in the early metabolic syndrome predisposition associated with body shape.

Boosting NAD+ with a small molecule that activates NAMPT.

Pharmacological strategies that boost intracellular NAD+ are highly coveted for their therapeutic potential. One approach is activation of nicotinamide phosphoribosyltransferase (NAMPT) to increase production of nicotinamide mononucleotide (NMN), the predominant NAD+ precursor in mammalian cells. A high-throughput screen for NAMPT activators and hit-to-lead campaign yielded SBI-797812, a compound that is structurally similar to active-site directed NAMPT inhibitors and blocks binding of these inhibitors to NAMPT. SBI-797812 shifts the NAMPT reaction equilibrium towards NMN formation, increases NAMPT affinity for ATP, stabilizes phosphorylated NAMPT at His247, promotes consumption of the pyrophosphate by-product, and blunts feedback inhibition by NAD+. These effects of SBI-797812 turn NAMPT into a "super catalyst" that more efficiently generates NMN. Treatment of cultured cells with SBI-797812 increases intracellular NMN and NAD+. Dosing of mice with SBI-797812 elevates liver NAD+. Small molecule NAMPT activators such as SBI-797812 are a pioneering approach to raise intracellular NAD+ and realize its associated salutary effects.

Elevated Nicotinamide Phosphoribosyl Transferase in Skeletal Muscle Augments Exercise Performance and Mitochondrial Respiratory Capacity Following Exercise Training.

Mice overexpressing NAMPT in skeletal muscle (NamptTg mice) develop higher exercise endurance and maximal aerobic capacity (VO2max) following voluntary exercise training compared to wild-type (WT) mice. Here, we aimed to investigate the mechanisms underlying by determining skeletal muscle mitochondrial respiratory capacity in NamptTg and WT mice. Body weight and body composition, tissue weight (gastrocnemius, quadriceps, soleus, heart, liver, and epididymal white adipose tissue), skeletal muscle and liver glycogen content, VO2max, skeletal muscle mitochondrial respiratory capacity (measured by high-resolution respirometry), skeletal muscle gene expression (measured by microarray and qPCR), and skeletal muscle protein content (measured by Western blot) were determined following 6 weeks of voluntary exercise training (access to running wheel) in 13-week-old male NamptTg (exercised NamptTg) mice and WT (exercised WT) mice. Daily running distance and running time during the voluntary exercise training protocol were recorded. Daily running distance (p = 0.51) and running time (p = 0.85) were not significantly different between exercised NamptTg mice and exercised WT mice. VO2max was higher in exercised NamptTg mice compared to exercised WT mice (p = 0.02). Body weight (p = 0.92), fat mass (p = 0.49), lean mass (p = 0.91), tissue weight (all p > 0.05), and skeletal muscle (p = 0.72) and liver (p = 0.94) glycogen content were not significantly different between exercised NamptTg mice and exercised WT mice. Complex I oxidative phosphorylation (OXPHOS) respiratory capacity supported by fatty acid substrates (p < 0.01), maximal (complex I+II) OXPHOS respiratory capacity supported by glycolytic (p = 0.02) and fatty acid (p < 0.01) substrates, and maximal uncoupled respiratory capacity supported by fatty acid substrates (p < 0.01) was higher in exercised NamptTg mice compared to exercised WT mice. Transcriptomic analyses revealed differential expression for genes involved in oxidative metabolism in exercised NamptTg mice compared to exercised WT mice, specifically, enrichment for the gene set related to the SIRT3-mediated signaling pathway. SIRT3 protein content correlated with NAMPT protein content (r = 0.61, p = 0.04). In conclusion, NamptTg mice develop higher exercise capacity following voluntary exercise training compared to WT mice, which is paralleled by higher mitochondrial respiratory capacity in skeletal muscle. The changes in SIRT3 targets suggest that these effects are due to remodeling of mitochondrial function.

Skeletal muscle overexpression of nicotinamide phosphoribosyl transferase in mice coupled with voluntary exercise augments exercise endurance.

OBJECTIVE: Nicotinamide phosphoribosyl transferase (NAMPT) is the rate-limiting enzyme in the salvage pathway that produces nicotinamide adenine dinucleotide (NAD+), an essential co-substrate regulating a myriad of signaling pathways. We produced a mouse that overexpressed NAMPT in skeletal muscle (NamptTg) and hypothesized that NamptTg mice would have increased oxidative capacity, endurance performance, and mitochondrial gene expression, and would be rescued from metabolic abnormalities that developed with high fat diet (HFD) feeding. METHODS: Insulin sensitivity (hyperinsulinemic-euglycemic clamp) was assessed in NamptTg and WT mice fed very high fat diet (VHFD, 60% by kcal) or chow diet (CD). The aerobic capacity (VO2max) and endurance performance of NamptTg and WT mice before and after 7 weeks of voluntary exercise training (running wheel in home cage) or sedentary conditions (no running wheel) were measured. Skeletal muscle mitochondrial gene expression was also measured in exercised and sedentary mice and in mice fed HFD (45% by kcal) or low fat diet (LFD, 10% by kcal). RESULTS: NAMPT enzyme activity in skeletal muscle was 7-fold higher in NamptTg mice versus WT mice. There was a concomitant 1.6-fold elevation of skeletal muscle NAD+. NamptTg mice fed VHFD were partially protected against body weight gain, but not against insulin resistance. Notably, voluntary exercise training elicited a 3-fold higher exercise endurance in NamptTg versus WT mice. Mitochondrial gene expression was higher in NamptTg mice compared to WT mice, especially when fed HFD. Mitochondrial gene expression was higher in exercised NamptTg mice than in sedentary WT mice. CONCLUSIONS: Our studies have unveiled a fascinating interaction between elevated NAMPT activity in skeletal muscle and voluntary exercise that was manifest as a striking improvement in exercise endurance.