Isolation, Culture, Characterization, and Differentiation of Human Muscle Progenitor Cells from the Skeletal Muscle Biopsy Procedure

The use of primary human tissue and cells is ideal for the investigation of biological and physiological processes such as the skeletal muscle ...

Cornell University

Previous studies have used indirect measures of insulin sensitivity to link circulating amino acids with insulin resistance and identify potential biomarkers of diabetes risk. Using direct measures (i.e., hyperinsulinemic-euglycemic clamps), we examined the relationships between the metabolomic amino acid profile and insulin action (i.e., glucose disposal rate [GDR]). Relationships between GDR and serum amino acids were determined among insulin-sensitive, insulin-resistant, and type 2 diabetic (T2DM) individuals. In all subjects, glycine (Gly) had the strongest correlation with GDR (positive association), followed by leucine/isoleucine (Leu/Ile) (negative association). These relationships were dramatically influenced by BMI, the resting respiratory quotient (RQ), T2DM, and sex. Gly had a strong positive correlation with GDR regardless of BMI, RQ, or sex but became nonsignificant in T2DM. In contrast, Leu/Ile was negatively associated with GDR in nonobese and T2DM subjects. Increased resting fat metabolism (i.e., low RQ) and obesity were observed to independently promote and negate the association between Leu/Ile and insulin resistance, respectively. Additionally, the relationship between Leu/Ile and GDR was magnified in T2DM males. Future studies are needed to determine whether Gly has a mechanistic role in glucose homeostasis and whether dietary Gly enrichment may be an effective intervention in diseases characterized by insulin resistance. 

BMI, RQ, diabetes, and sex affect the relationships between amino acids and clamp measures of insulin action in humans.

Dietary protein and amino acids are necessary for overall human health. Insufficient protein intake induces a negative protein balance with adverse outcomes such as muscle atrophy and functional decline--outcomes that are worsened in older adults. Furthermore, during inactivity, such as bed rest/hospitalization, skeletal muscle protein synthesis is reduced, protein balance is negative, and older adults lose significant amounts of muscle. Dietary protein and amino acid supplementation (∼ 30 g protein and ∼ 3 g leucine) stimulate skeletal muscle protein anabolism in healthy, community-dwelling older adults and may be considered as possible nutritional interventions to improve the muscle protein balance and potentially support skeletal muscle maintenance in hospitalized older adults. The following is a timely review of metabolic and dietary challenges faced by hospitalized older adults and potential dietary protein and amino acids solutions for maintaining skeletal muscle health during hospitalization-induced inactivity in this population.

The importance of dietary protein for muscle health in inactive, hospitalized older adults.

S-adenosylmethionine (SAM) and S-adenosylhomocysteine (SAH) link one-carbon metabolism to methylation status. However, it is unknown whether regulation of SAM and SAH by nutrient availability can be directly sensed to alter the kinetics of key histone methylation marks. We provide evidence that the status of methionine metabolism is sufficient to determine levels of histone methylation by modulating SAM and SAH. This dynamic interaction led to rapid changes in H3K4me3, altered gene transcription, provided feedback regulation to one-carbon metabolism, and could be fully recovered upon restoration of methionine. Modulation of methionine in diet led to changes in metabolism and histone methylation in the liver. In humans, methionine variability in fasting serum was commensurate with concentrations needed for these dynamics and could be partly explained by diet. Together these findings demonstrate that flux through methionine metabolism and the sensing of methionine availability may allow direct communication to the chromatin state in cells. 

Histone Methylation Dynamics and Gene Regulation Occur through the Sensing of One-Carbon Metabolism.

HOXB13, a member of the homeobox proteins family, is a key regulator of the epithelial differentiation in the prostate gland. HOXB13 is overexpressed during malignant progression of the prostatic tissue and suspected to contribute in the pathogenesis of the prostate gland. In androgen deprived conditions, HOXB13 is thought to act through inhibition of the tumour suppressor protein p21. Since HOXB13 has a multifaceted role in ventral prostate development, its critical partners in the cascade need to be elucidated for a further understanding of its role in prostate malignancy. In this report, we review the functions attributed to HOXB13, by highlighting the most recent findings supporting the hypothesis that HOXB13 might serve as a novel biomarker for the prognosis of prostate cancer. 

Hoxb13, a potential prognostic biomarker for prostate cancer.

BRIP1 encodes a protein belonging to the RecQ DEAH helicase family. It interacts with BRCA1, and is involved in the repair of DNA damage and tumor suppression. Aberrations in BRIP1 have been mainly associated with the development of breast cancer (BC), ovarian cancer, and type J Fanconi anemia. Based on recent work, we hypothesize that BRIP1 might be the gene involved in the onset of BC in families that do not show BRACA1/2 mutations. This review will focus on the findings supporting this hypothesis, the mechanisms linking BRIP1 to the onset of BC, and the potential clinical relevance of its various inhibitors. 

BRIP1, a potential candidate gene in development of non-BRCA1/2 breast cancer.

Life expectancy in the U.S. and globally continues to increase. Despite increased life expectancy quality of life is not enhanced, and older adults often experience chronic age-related disease and functional disability, including frailty. Additionally, changes in body composition such as the involuntary loss of skeletal muscle mass (i.e. sarcopenia) and subsequent increases in adipose tissue can augment disease and disability in this population. Furthermore, increased oxidative stress and decreased antioxidant concentrations may also lead to metabolic dysfunction in older adults. Specific amino acids, including leucine, cysteine and its derivative taurine, and arginine can play various roles in healthy aging, especially in regards to skeletal muscle health. Leucine and arginine play important roles in muscle protein synthesis and cell growth while cysteine and arginine play important roles in quenching oxidative stress. Evidence suggests that supplemental doses of each of these amino acids may improve the aging phenotype. However, additional research is required to establish the doses required to achieve positive outcomes in humans. 

Amino acids in healthy aging skeletal muscle.

Skeletal muscle is the largest metabolic organ system in the human body. As such, metabolic dysfunction occurring in skeletal muscle impacts whole-body nutrient homeostasis. Macronutrient metabolism changes within the skeletal muscle with aging, and these changes are associated in part with age-related skeletal muscle remodeling. Moreover, age-related changes in skeletal muscle metabolism are affected differentially between males and females and are likely driven by changes in sex hormones. Intrinsic and extrinsic factors impact observed age-related changes and sex-related differences in skeletal muscle metabolism. Despite some support for sex-specific differences in skeletal muscle metabolism with aging, more research is necessary to identify underlying differences in mechanisms. Understanding sex-specific aging skeletal muscle will assist with the development of therapies to attenuate adverse metabolic and functional outcomes.

Understanding Age-Related Changes in Skeletal Muscle Metabolism: Differences Between Females and Males.

Trimethylamine-N-oxide (TMAO) is associated with chronic disease risk. However, little is known about the metabolic fate of dietary TMAO. This study sought to quantitatively elucidate the metabolic fate of orally consumed TMAO in humans. As part of a crossover feeding study, healthy young men (n=40) consumed 50-mg deuterium-labeled methyl d9-TMAO (d9-TMAO), and enrichments of TMAO and its derivatives were measured in blood for 6 h, urine and stool, as well as skeletal muscle in a subset of men (n=6). Plasma d9-TMAO was detected as early as 15 min, increased until 1 h and remained elevated through the 6-h period. TMAO exhibited an estimated turnover time of 5.3 h, and ~96% of the dose was eliminated in urine by 24 h, mainly as d9-TMAO. No d9-TMAO was detected in feces. Notably, d9-TMAO and d9-trimethylamine were detected in skeletal muscle (n=6) at 6 h, and the enrichment ratio of d9-TMAO to d9-trimethylamine was influenced by a genetic variant in flavin-containing monooxygenase isoform 3 (FMO3 G472A). These results suggest that the absorption of orally consumed TMAO is near complete and does not require processing by gut microbes. TMAO exhibits fast turnover in the circulation with the majority being eliminated in urine within 24 h. A small portion of the dose, however, is taken up by extrahepatic tissue in a manner that appears to be under the influence of FMO3 G472A polymorphism. This trial was registered at clinicaltrials.gov as NCT02558673.

The metabolic fate of isotopically labeled trimethylamine-N-oxide (TMAO) in humans.

Primary human muscle progenitor cells (hMPCs) are commonly used to understand skeletal muscle biology, including the regenerative process. Variability from unknown origin in hMPC expansion capacity occurs independently of disease, age, or sex of the donor. We sought to determine the transcript profile that distinguishes hMPC cultures with greater expansion capacity and to identify biological underpinnings of these transcriptome profile differences. Sorted (CD56+/CD29+) hMPC cultures were clustered by unbiased, K-means cluster analysis into FAST and SLOW based on growth parameters (saturation density and population doubling time). FAST had greater expansion capacity indicated by significantly reduced population doubling time (-60%) and greater saturation density (+200%), nuclei area under the curve (AUC, +250%), and confluence AUC (+120%). Additionally, FAST had fewer % dead cells AUC (-44%, P < 0.05). RNA sequencing was conducted on RNA extracted during the expansion phase. Principal component analysis distinguished FAST and SLOW based on the transcript profiles. There were 2,205 differentially expressed genes (DEgenes) between FAST and SLOW (q value ≤ 0.05); 362 DEgenes met a more stringent cut-off (q value ≤ 0.001 and 2.0 fold-change). DEgene enrichment suggested FAST (vs. SLOW) had promotion of the cell cycle, reduced apoptosis and cellular senescence, and enhanced DNA replication. Novel (RABL6, IRGM1, and AREG) and known (FOXM1, CDKN1A, Rb) genes emerged as regulators of identified functional pathways. Collectively the data suggest that variation in hMPC expansion capacity occurs independently of age and sex and is driven, in part, by intrinsic mechanisms that support the cell cycle.

Transcript profile distinguishes variability in human myogenic progenitor cell expansion capacity.

Activation of satellite cells and expansion of the muscle progenitor cell (MPC) population are essential to generate a sufficient number of cells to repair damaged skeletal muscle. Proliferating MPCs have high energetic and biosynthetic material requirements, and the ability to utilize oxidative phosphorylation (OXPHOS) and/or glycolysis may affect expansion capacity of MPCs. In the present study, we investigated the effect of donor age and sex on human (h)MPC expansion capacity and metabolic fuel preference. hMPCs from young and old male and female donors were grown for 408 h (17 days). Percent confluence, live nuclei count, and dead cell count were measured every 24 h. Metabolic phenotype was assessed by glucose uptake, expression of genes related to glycolysis and OXPHOS, and the Seahorse XF24 Phenotype Test Kit during the exponential phase of growth. hMPCs from old male donors had impaired expansion capacity secondary to heightened cell death early in expansion compared with hMPCs from young male donors, an effect not observed in female hMPCs. Age-related differences in metabolism were also sex dependent; markers of OXPHOS were altered in old (vs. young) male hMPCs, whereas markers of metabolism were largely unaffected by age in female hMPCs. For the first time, we identify sex-specific differences in cell death and OXPHOS that contribute to impaired expansion capacity of hMPC cell populations with age.

Expansion capacity of human muscle progenitor cells differs by age, sex, and metabolic fuel preference.

Peptide YY (PYY) is considered a gut peptide with roles in post-prandial appetite and glucose regulation. Circulating PYY protein levels increase during aerobic exercise. Furthermore, people who have greater increases in muscle progenitor cells (MPCs), the adult stem cell population responsible for skeletal muscle (SkM) repair, after resistance training have higher PYY transcript levels in SkM prior to training. Currently, examination of PYY expression patterns in SkM and/or MPCs is lacking. Our objective was to identify the expression patterns of PYY in SkM and MPCs. PYY and the associated Y receptors were analyzed in SkM biopsy tissue and cultured MPCs from young and old human participants. Additional experiments to assess the role and regulation of PYY in MPCs were performed. In SkM, PYY and one of the three Y receptors (Y1r) were detectable, but expression patterns were not affected by age. In expanding MPCs, PYY and all three Y receptor (Y1r, Y2r, and Y5r) proteins were expressed in a temporal fashion with young MPCs having greater levels of Y receptors at various time points. Exogenous PYY did not affect MPC population expansion. MPC PYY levels increased following the metabolic stimulus, 5-Aminoimidazole-4-carboxamide ribonucleotide (AICAR), but were not affected by the inflammatory stimulus, tumor necrosis factor alpha (TNFα). In conclusion, PYY and Y receptor expression are not impacted by age in SkM tissue but are reduced in old  young expanding MPCs. Furthermore, endogenous PYY production is stimulated by low energy states and thus may be integral for skeletal muscle and MPC responses to metabolic stimuli.

Anna Thalacker-Mercer

Division of Nutritional Sciences

Cornell University

Isolation, Culture, Characterization, and Differentiation of Human Muscle Progenitor Cells from the Skeletal Muscle Biopsy Procedure

Anna Thalacker-Mercer

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Cornell University

Isolation, Culture, Characterization, and Differentiation of Human Muscle Progenitor Cells from the Skeletal Muscle Biopsy Procedure

Division of Nutritional Sciences