Abstract
Background
The roles of systemic and airway-specific epithelial energy metabolism in altering the developmental programming of airway epithelial cells (AECs) in early life are poorly understood.
Objective
To assess carbohydrate metabolism in developing AECs among children with and without wheeze and test the association of infant plasma energy biomarkers with subsequent recurrent wheeze and asthma outcomes.
Methods
We measured cellular carbohydrate metabolism in live nasal AECs collected at age 2-years from 15 male subjects with and without wheeze history and performed a principal component analysis to visually assess clustering of data on AEC metabolism of glycolitic metabolites and simple sugars. Among 237 children with available 1-year plasma samples, we tested the associations of 1-year plasma energy biomarkers and recurrent wheeze and asthma using generalized estimating equations and logistic regression.
Results
Children with a history of wheeze had lower utilization of glucose in nasal AECs than children with no wheeze. Systemically, higher plasma glucose concentration at year 1 (within the normal range) was associated with decreased odds of asthma at age 5-years (adjusted odds ratio [aOR] 0.56, 95% confidence interval [CI] 0.35–0.90). Insulin, glucose/insulin ratio, c-peptide, and leptin at year 1 were associated with recurrent wheeze from age 2–5 years.
Conclusion
These studies suggest that there is significant energy metabolism dysregulation in early life which likely impacts AEC development. These pertubations of epithelial cell metabolism in infancy may have lasting effects on lung development, which could render the airway more susceptible to allergic sensitization.
Keywords: glucose, insulin, energy metabolism, asthma, wheezing, childhood, development, airway epithelial cells
Capsule Summary:
These studies suggest that there is significant energy metabolism dysregulation in early life which likely impacts airway epithelial cell development.
Introduction
Asthma is in part an airway epithelial disorder, and its origin and clinical manifestations are tightly linked with altered airway epithelial cell (AEC) physical, metabolic, and functional barrier properties of the lung.1 We have shown that downregulation of the insulin receptor (INSR) signaling pathway and loss of differentiation in epithelial cells were conserved features of asthma.2 This was overrepresented by the downregulation of the insulin target genes INSR and IRS2, decrease in expression of pyruvate metabolism markers, as well as changes in mitochondrial respiratory chain genes.2, 3 Consequently, perturbation of epithelial cell metabolism in infancy may have a lasting effect on the differentiation of AEC via alteration of fundamental developmental AEC differentiation programs and/or epigenetic reprogramming.4 Such perturbation of lung development has the potential to result in lasting epithelial barrier dysfunction that may render the airway more susceptible to allergic sensitization. Systemically there is growing evidence of an important role of energy metabolism in inflammatory conditions, including asthma. High-fructose containing foods are associated with asthma, possibly because of the high fructose:glucose ratios which may relate to glucose utilization by the early life developing AEC.5, 6 Similarly, high-sucrose diets have been associated with increased eosinophil cytokine content and airway resistance in allergen-challenged mice.7 However, the roles of systemic and airway-specific epithelial energy metabolism in altering the developmental programming of AECs in early life are poorly understood. To investigate the possible role of intracellular glucose in AEC development and systemic energy metabolism on asthma risk, we 1) assessed carbohydrate metabolism in developing AECs among children with and without wheeze and 2) tested the association of infant plasma energy biomarkers with subsequent recurrent wheeze and asthma outcomes.
Results and Discussion
Our study population was drawn from a birth cohort (INSPIRE) of 1946 children,8 and included a nested cohort of children in whom nasal AECs were collected and 237 children with excess available 1-year plasma samples. Compared to the larger population-based birth cohort from which our study population was drawn (INSPIRE), our study population included a larger proportion of males and privately insured children (Table 1).
Table 1.
Clinical and demographic characteristics of nested study and INSPIRE cohort.
| With 1-year plasma samplea N (%) |
Without 1-year plasma sample N (%) |
Total INSPIRE cohort N (%) |
|
|---|---|---|---|
| Sample size | 237 | 1709 | 1946 |
| Infant sex | |||
| Male | 135 (57) | 884 (52) | 1019 (52) |
| Female | 102 (43) | 825 (48) | 927 (48) |
| Infant race/ethnicity | |||
| Non-Hispanic White | 163 (69) | 1104 (65) | 1267 (65) |
| Non-Hispanic Black | 40 (17) | 303 (18) | 343 (18) |
| Hispanic | 19 (8) | 151 (9) | 170 (9) |
| Other | 15 (6) | 151 (9) | 166 (9) |
| Gestational age (weeks)b | 39 (39–40) | 39 (39–40) | 39 (39–40) |
| Birthweight (grams)b | 3405 (3178–3774) | 3405 (3093–3717) | 3405 (3120–3740) |
| Mode of delivery | |||
| Vaginal | 172 (73) | 1163 (68) | 1335 (69) |
| Cesarean | 65 (27) | 546 (32) | 611 (31) |
| Insurance | |||
| Medicaid | 111 (47) | 944 (55) | 1055 (54) |
| Private | 124 (52) | 743 (43) | 867 (45) |
| Self pay | 0 (0) | 12 (1) | 12 (1) |
| Unknown | 0 (0) | 2 (0) | 2 (0) |
| Other | 2 (1) | 8 (0) | 10 (1) |
| Maternal smoking during pregnancy | 36 (15) | 314 (18) | 350 (18) |
| Secondhand smoke exposure at enrollment | 97 (41) | 814 (48) | 911 (47) |
| Maternal asthma | 48 (20) | 331 (19) | 379 (19) |
| Body mass index at one year | 17 (16–19) | 17 (16–18) | 17 (16–18) |
Clinical and demographic characteristics did not differ between children with and without 1-year plasma sample.
Data expressed as median (interquartile range).
Variables may contain missing data.
Measuring cellular carbohydrate metabolism of live nasal AECs collected at age 2 years from male subjects from a nested cohort of those with (7 independent donors) and without (8 independent donors) wheeze history, we found lower utilization of glucose in nasal AECs from children with history of wheeze compared to children with no wheeze (Figure 1). This indicates lower utilization of glucose in cellular bioenergetic reactions, which was measured as production of NADH in a colorimetric dye reduction kinetic assay (Biolog Inc., Hayward, CA). Overall, nasal AECs cultured from children with wheeze had lower utilization of various carbohydrates, as indicated by exploratory principal component analysis (PCA) of glycolytic metabolites and simple sugars (Figure 1A). However, specifically α-D-glucose was significantly different in measured uptake between the wheeze and no wheeze groups (Figure 1B).
Figure 1. Children with wheeze have lower airway epithelial cell (AEC) carbohydrate energy substrate utilizationat 2 years than children without wheeze. A) Nasal AECs cultured from children with wheeze had lower utilization of various carbohydrates, B) α-D-glucose showed statistically significant difference in measured uptake between wheeze and no wheeze groups.
Panel A: Principal component analysis was used to visually assess clustering of data on AEC metabolism of glycolytic metabolites and simple sugars, measured as NADH production in colorimetric reactions. The wheeze group (7 independent donors) is shown in magenta, and the no wheeze group (8 independent donors) is shown in blue. Panel B: Difference in α-D-glucose utilization between wheeze and no wheeze groups was assessed using a t-test. *p<0.05
To assess the role of systemic energy metabolism on subsequent childhood asthma development, we tested the association of one-year random plasma glucose, insulin, glucose/insulin ratio, c-peptide, and leptin with annual recurrent wheeze from age 2 to 5 years and asthma at age 5 years. Energy biomarker distributions are presented in Table 2. All were within the normal ranges for this age.9, 10 One year plasma glucose concentration was not associated with recurrent wheeze from age 2–5 years (Figure 2). A 23 mg/dL increase (interquartile range [IQR]) of plasma glucose concentration at year 1 was associated with decreased odds of asthma at age 5-years (adjusted odds ratio [aOR] 0.56, 95% confidence interval [CI] 0.35–0.90). Conversely, one year plasma insulin, glucose/insulin ratio, c-peptide, and leptin concentrations were associated with recurrent wheeze from age 2–5 years, but not asthma at age 5 years. A 283 μU/mL increase (IQR) in insulin, 954 pg/mL increase (IQR) in c-peptide, and 755 pg/mL increase (IQR) in leptin at 1-year were associated with decreased odds of recurrent wheeze from age 2–5 years (insulin: aOR 0.58, 95% CI 0.38–0.89; c-peptide: aOR 0.53, 95% CI 0.30–0.93; leptin: aOR 0.53, 95% CI 0.34–0.82), while a 5.9 unit increase (IQR) in glucose/insulin ratio was associated with increased odds of recurrent wheeze from age 2–5 years (aOR 1.39, 95% CI 1.17–1.64).
Table 2.
Energy biomarker distributions among children with and without recurrent wheeze from age 2–5 years and asthma at age 5 years.
| No recurrent wheeze at 2 years/Recurrent wheeze at 2 years | No recurrent wheeze at 3 years/Recurrent wheeze at 3 years | No recurrent wheeze at 4 years/Recurrent wheeze at 4 years | No recurrent wheeze at 5 years/Recurrent wheeze at 5 years | No asthma at 5 years/ Asthma at 5 years | |
|---|---|---|---|---|---|
| Energy biomarker | Mean (SD) | Mean (SD) | Mean (SD) | Mean (SD) | Mean (SD) |
| Glucose (mg/dL) | 61 (19) 57 (17) |
61 (19) 64 (23) |
61 (20) 54 (11) |
62 (20) 53 (12) |
63 (20) 54 (13) |
|
| |||||
| Insulin (μU/ml) | 11 (8) 11 (7) |
11 (7) 9 (5) |
11 (8) 7 (4) |
11 (8) 7 (4) |
11 (9) 9 (6) |
|
| |||||
| Glucose/insulin ratio | 9 (7) 12 (17) |
9 (7) 13 (14) |
8 (6) 14 (15) |
9 (7) 14 (12) |
9 (8) 9 (9) |
|
| |||||
| C-peptide (pg/mL) | 1212 (772) 1189 (520) |
1198 (704) 1025 (515) |
1231 (764) 862 (477) |
1261 (777) 779 (492) |
1264 (801) 1024 (599) |
|
| |||||
| Leptin (pg/mL) | 784 (737) 817 (585) |
769 (614) 835 (824) |
815 (744) 582 (460) |
804 (726) 556 (480) |
757 (568) 836 (1115) |
SD, standard deviation.
Figure 2. Energy biomarkers are associated with subsequent recurrent wheeze and asthma outcomes in childhood.
The association between each energy biomarker and recurrent wheeze was assessed longitudinally using generalized estimating equations fit with robust sandwich covariance matrices, adjusting for infant sex, maternal asthma, infant race/ethnicity, and smoking exposure in the home at enrollment. The association between each energy biomarker and asthma at age 5 years using logistic regression, adjusting for the same covariates.
Our understanding of the central mediators for asthma and the allergic airway disease phenotype has been limited by focus on downstream processes of the immune system, insufficient understanding of the complexity required to maintain AEC homeostasis, the dynamic nature of biological systems, and the extreme clinical heterogeneity of asthma and other allergic diseases. In a healthy birth cohort, we demonstrated differences in carbohydrate metabolism and glucose uptake between nasal AECs from 2 year old children who were and were not wheezers in the first year of life. This suggests that there is significant epithelial metabolic change detectable in early life associated with the phenotype of wheeze, a pattern which has potential to impact development of airway epithelial barrier. Moreover, energy biomarkers at age 1-year were associated with recurrent wheeze from age 2–5 years and asthma at age 5 years. These are intriguing observations that raise several important questions about early life origins of disease.
On the systemic level, changes in diet and mechanisms regulating central energy metabolism (e.g., endocrine regulation and energy storage versus expenditure) may dictate in vivo availability of glucose and other energy substrates for differentiating epithelial cells. Given the known association of fructose with asthma,5 we speculate that reduced glucose metabolism may reflect the possibility of increased fructose versus glucose dietary consumption among those at risk for wheeze and asthma, a hypothesis which requires further testing. Epidemiologic and experimental studies have demonstrated that nutrition is especially important in prenatal and the first few years of life. In particular, constituents of both maternal milk and milk formulas, which includes sugars, have been implicated in development of allergy and asthma via their effect on epigenetic reprogramming in early life.11 At the cellular level, it remains to be tested in newborns developing wheeze whether: 1) dysregulated glucose metabolism is accompanied by changes in insulin signaling pathway; 2) there are defects in insulin-dependent glucose transport mechanisms into epithelial cells; 3) glucose uptakes are sufficient but utilized in anabolic rather than catabolic reactions, which is a feature of adult asthmatic epithelium.3 Moreover, it remains to be determined whether such metabolic defects persist later in life or are sensitive to hormonal changes around puberty.
It is becoming apparent that epithelial barrier defects at birth are linked to development of atopic disease later in life. For example, skin barrier dysfunction measured by transepidermal water loss (TEWL) and dry skin after birth has been shown to pre-date and predict atopic dermatitis development later in life.12, 13 We hypothesize that dysregulated metabolism at birth may be an important upstream contributor to dysregulated airway epithelial barrier early in life. This is supported by the fact that energy metabolism, differentiation, and epigenetic changes are closely intertwined upstream processes in establishment and maintenance of epithelial homeostasis. AEC development is epigenetically orchestrated and is sensitive to metabolic change, especially early in life. Therefore, changes in metabolism have consequences for both establishment of competent epithelial barrier and immune response.14–17 Despite the growing recognition of immunometabolism in homeostasis and disease, there are very few studies examining the contribution of AEC metabolism to asthma disease pathogenesis.18–20 Among them, several important studies demonstrated significant changes in the mitochondria of asthmatic AECs, specifically pointing to arginine metabolism as fueling nitric oxide production and mitochondrial bioenergetics.21, 22 We have previously shown that dysregulation of insulin signaling is specific to type 2 high inflammation.3 Intriguingly, it is well documented that type 2 cytokines, IL-4 and IL-13, signal through insulin receptor substrates, IRS1 and IRS2, for activation of PI3K and other downstream pathways, which warrants investigation of the crosstalk between insulin and allergic inflammation.3, 23
Our study has many strengths, including assessment of both intracellular and systemic energy metabolism and the effect of early-life metabolism on longitudinal respiratory outcomes. However, there are some limitations. Carbohydrate metabolism in nasal AECs was only profiled in a small subset of male subjects. Although plasma glucose concentrations at 1-year did not differ by infant sex in our study population, we acknowledge that sex differences in metabolic readouts are likely and future studies will need to be performed in a larger sample including both males and females. Additionally, as the sample size was small and our outcomes were correlated, we did not perform multiplicity correction. However, we do present all statistically significant and insignificant results.
These new findings provide evidence that differences in glucose metabolism are a very early feature differentiating children who are and are not wheezers in the first year of life. Further, glucose concentration at age 1-year is associated with subsequent asthma development, suggesting a link between systemic glucose availability and differential glucose utilization by AECs. Overall, these hypothesis-generating studies suggest that there is significant energy metabolism dysregulation in early life which likely impacts AEC development. Whether this detectable metabolic dysregulation is genetically or environmentally determined will be important in further understanding and preventing asthma.
Supplementary Material
Key Messages.
The roles of systemic and airway-specific epithelial energy metabolism in altering the developmental programming of airway epithelial cells (AECs) in early life are poorly understood.
These new findings provide evidence that changes in airway epithelial and systemic energy metabolism are very early features of wheeze in the first year of life.
These studies suggest that there is significant energy metabolism dysregulation in early life which likely impacts AEC development and asthma risk.
Acknowledgements
We would like to thank the research nurses and coordinators, data managers, laboratory personnel, and other research staff at the Center for Asthma Research at Vanderbilt University Medical Center, and all the INSPIRE participants and their families for their involvement in and dedication to this study.
Funding Statement:
This work was supported by the National Institutes of Health (grant number U19 AI095227 to TVH). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
Abbreviations:
- AEC
airway epithelial cell
- INSR
insulin receptor
- PCA
principal component analysis
- aOR
adjusted odds ratio
- CI
confidence interval
- TEWL
transepidermal water loss
Footnotes
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Disclosure Statement: The authors have no reported conflicts of interest.
References
- 1.Shi W, Xu J, Warburton D. Development, repair and fibrosis: what is common and why it matters. Respirology 2009; 14:656–65. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 2.Loffredo LF, Abdala-Valencia H, Anekalla KR, Cuervo-Pardo L, Gottardi CJ, Berdnikovs S. Beyond epithelial-to-mesenchymal transition: Common suppression of differentiation programs underlies epithelial barrier dysfunction in mild, moderate, and severe asthma. Allergy 2017; 72:1988–2004. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 3.Queener AM, Chiarella SE, Cuervo-Pardo L, Coden ME, Abdala-Valencia H, Berdnikovs S. Metabolism of Epithelial Cells in Health and Allergic Disease: Collegium Internationale Allergologicum Update 2021. Int Arch Allergy Immunol 2021:1–16. [DOI] [PubMed] [Google Scholar]
- 4.Schleimer RP, Berdnikovs S. Etiology of epithelial barrier dysfunction in patients with type 2 inflammatory diseases. J Allergy Clin Immunol 2017; 139:1752–61. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 5.DeChristopher LR, Tucker KL. Excess free fructose, high-fructose corn syrup and adult asthma: the Framingham Offspring Cohort. Br J Nutr 2018; 119:1157–67. [DOI] [PubMed] [Google Scholar]
- 6.Gugliucci A Formation of Fructose-Mediated Advanced Glycation End Products and Their Roles in Metabolic and Inflammatory Diseases. Adv Nutr 2017; 8:54–62. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 7.Percopo CM, McCullough M, Limkar AR, Druey KM, Rosenberg HF. Impact of controlled high-sucrose and high-fat diets on eosinophil recruitment and cytokine content in allergen-challenged mice. PLoS One 2021; 16:e0255997. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 8.Larkin EK, Gebretsadik T, Moore ML, Anderson LJ, Dupont WD, Chappell JD, et al. Objectives, design and enrollment results from the Infant Susceptibility to Pulmonary Infections and Asthma Following RSV Exposure Study (INSPIRE). BMC Pulm Med 2015; 15:45. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 9.Children’s Hospital of Philadelphia Pathology & Laboratory Medicine. Reference ranges document Available at: https://www.chop.edu/centers-programs/pathology-and-laboratory-medicine. Accessed 20 September 2021.
- 10.Savino F, Rossi L, Benetti S, Petrucci E, Sorrenti M, Silvestro L. Serum reference values for leptin in healthy infants. PloS one 2014; 9:e113024-e. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 11.van Esch B, Porbahaie M, Abbring S, Garssen J, Potaczek DP, Savelkoul HFJ, et al. The Impact of Milk and Its Components on Epigenetic Programming of Immune Function in Early Life and Beyond: Implications for Allergy and Asthma. Front Immunol 2020; 11:2141. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 12.Horimukai K, Morita K, Narita M, Kondo M, Kabashima S, Inoue E, et al. Transepidermal water loss measurement during infancy can predict the subsequent development of atopic dermatitis regardless of filaggrin mutations. Allergol Int 2016; 65:103–8. [DOI] [PubMed] [Google Scholar]
- 13.Rehbinder EM, Advocaat Endre KM, Lødrup Carlsen KC, Asarnoj A, Stensby Bains KE, Berents TL, et al. Predicting Skin Barrier Dysfunction and Atopic Dermatitis in Early Infancy. J Allergy Clin Immunol Pract 2020; 8:664–73.e5. [DOI] [PubMed] [Google Scholar]
- 14.Kayama H, Takeda K. Manipulation of epithelial integrity and mucosal immunity by host and microbiota-derived metabolites. Eur J Immunol 2020; 50:921–31. [DOI] [PubMed] [Google Scholar]
- 15.Mavin E, Verdon B, Carrie S, Saint-Criq V, Powell J, Kuttruff CA, et al. Real-time measurement of cellular bioenergetics in fully differentiated human nasal epithelial cells grown at air-liquid-interface. Am J Physiol Lung Cell Mol Physiol 2020; 318:L1158–l64. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 16.Stuard WL, Titone R, Robertson DM. The IGF/Insulin-IGFBP Axis in Corneal Development, Wound Healing, and Disease. Front Endocrinol (Lausanne) 2020; 11:24. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 17.Aghapour M, Remels AHV, Pouwels SD, Bruder D, Hiemstra PS, Cloonan SM, et al. Mitochondria: at the crossroads of regulating lung epithelial cell function in chronic obstructive pulmonary disease. Am J Physiol Lung Cell Mol Physiol 2020; 318:L149–l64. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 18.Wang A, Luan HH, Medzhitov R. An evolutionary perspective on immunometabolism. Science 2019; 363. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 19.Pité H, Morais-Almeida M, Rocha SM. Metabolomics in asthma: where do we stand? Curr Opin Pulm Med 2018; 24:94–103. [DOI] [PubMed] [Google Scholar]
- 20.Holguin F, Grasemann H, Sharma S, Winnica D, Wasil K, Smith V, et al. L-Citrulline increases nitric oxide and improves control in obese asthmatics. JCI Insight 2019; 4. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 21.Xu W, Ghosh S, Comhair SA, Asosingh K, Janocha AJ, Mavrakis DA, et al. Increased mitochondrial arginine metabolism supports bioenergetics in asthma. J Clin Invest 2016; 126:2465–81. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 22.Winnica D, Corey C, Mullett S, Reynolds M, Hill G, Wendell S, et al. Bioenergetic Differences in the Airway Epithelium of Lean Versus Obese Asthmatics Are Driven by Nitric Oxide and Reflected in Circulating Platelets. Antioxid Redox Signal 2019; 31:673–86. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 23.Wills-Karp M, Finkelman FD. Untangling the complex web of IL-4- and IL-13-mediated signaling pathways. Sci Signal 2008; 1:pe55. [DOI] [PMC free article] [PubMed] [Google Scholar]
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