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Short-term interleukin-37 treatment improves vascular endothelial function, endurance exercise capacity, and whole-body glucose metabolism in old mice

Ballak, Dov B., Brunt, Vienna E., Sapinsley, Zachary J., Ziemba, Brian P., Richey, James J., Zigler, Melanie C., Johnson, Lawrence C., Gioscia‐Ryan, Rachel A., Culp‐Hill, Rachel, Eisenmesser, Elan Z., D'Alessandro, Angelo, Dinarello, Charles A., Seals, Douglas R.·2020·Aging Cell
DOI 10.1111/acel.13074PMC PMC6974720
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Abstract

Aging is associated with vascular endothelial dysfunction, reduced exercise tolerance, and impaired whole‐body glucose metabolism. Interleukin‐37 (IL‐37), an anti‐inflammatory cytokine of the interleukin‐1 family, exerts salutary physiological effects in young mice independent of its inflammation‐suppressing properties. Here, we assess the efficacy of IL‐37 treatment for improving physiological function in older age. Old mice (26–28 months) received daily intraperitoneal injections of recombinant human IL‐37 (recIL‐37; 1 µg/200 ml PBS) or vehicle (200 ml PBS) for 10–14 days. Vascular endothelial function (ex vivo carotid artery dilation to increasing doses of acetylcholine, ACh) was enhanced in recIL‐37 vs. vehicle‐treated mice via increased nitric oxide (NO) bioavailability (all p < .05); this effect was accompanied by enhanced ACh‐stimulated NO production and reduced levels of reactive oxygen species in endothelial cells cultured with plasma from IL‐37‐treated animals (p < .05 vs. vehicle plasma). RecIL‐37 treatment increased endurance exercise capacity by 2.4‐fold, which was accompanied by a 2.9‐fold increase in the phosphorylated AMP‐activated kinase (AMPK) to AMPK ratio (i.e., AMPK activation) in quadriceps muscle. RecIL‐37 treatment also improved whole‐body insulin sensitivity and glucose tolerance (p < .05 vs. vehicle). Improvements in physiological function occurred without significant changes in plasma, aortic, and skeletal muscle pro‐inflammatory proteins (under resting conditions), whereas pro‐/anti‐inflammatory IL‐6 was greater in recIL‐37‐treated animals. Plasma metabolomics analysis revealed that recIL‐37 treatment altered metabolites related to pathways involved in NO synthesis (e.g., increased L‐arginine and citrulline/arginine ratio) and fatty acid metabolism (e.g., increased pantothenol and free fatty acids). Our findings provide experimental support for IL‐37 therapy as a novel strategy to improve diverse physiological functions in old age.

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Figure 1 RecIL‐37 treatment improves endothelial function. (a) Endothelium‐dependent dilation (EDD) in isolated carotid arteries to increasing doses of acetylcholine (ACh) alone and in the presence of NG‐nitro‐L‐arginine methyl ester (L‐NAME). (b) Peak EDD to acetylcholine. (c) The component of EDD mediated by nitric oxide (NO‐mediated dilation), determined as the difference between peak EDD to ACh alone and EDD in the presence of L‐NAME. Data are mean ± SEM. N = 9–11 mice per group. *p < .05 vs. vehicle treatment (within time point)
Figure 1 RecIL‐37 treatment improves endothelial function. (a) Endothelium‐dependent dilation (EDD) in isolated carotid arteries to increasing doses of acetylcholine (ACh) alone and in the presence of NG‐nitro‐L‐arginine methyl ester (L‐NAME). (b) Peak EDD to acetylcholine. (c) The component of EDD mediated by nitric oxide (NO‐mediated dilation), determined as the difference between peak EDD to ACh alone and EDD in the presence of L‐NAME. Data are mean ± SEM. N = 9–11 mice per group. *p < .05 vs. vehicle treatment (within time point)CC BY · PubMed Central
Figure 2 Plasma from recIL‐37‐treated mice increases endothelial cell acetylcholine (ACh)‐stimulated nitric oxide (NO) production and reduces reactive oxygen species production. (a–c) NO production (DAF‐FM fluorescence) before and after incubation with ACh [presented at both time points (a) and as a fold change from basal (b), both normalized within plasma conditions] and reactive oxygen species production (c) (CellROX signal) in human umbilical vein endothelial cells cultured with plasma from vehicle‐ and recIL‐37‐treated old mice or with fetal cow serum (FCS; healthy control condition). Cells were also stained with Hoechst 33342 to indicate healthy nuclei. Data are mean ± SEM. N = 8–10/group. #p < .05 vs. basal conditions (pre‐ACh). *p < .05 vs. vehicle treatment. Each plasma sample was assayed twice over two days. For DAF‐FM experiments, 320 HUVECs were visualized after IL‐37‐treated plasma incubation and 256 cells were visualized after vehicle‐treated plasma incubation. For CellROX experiments, 240 cells were visualized after IL‐37‐treated plasma incubation and 192 cells were visualized after vehicle‐treated plasma incubation. (d) Representative fluorescent images taken at 20X optical zoom. Images are of the same wells incubated with plasma from vehicle‐ (top) or recIL‐37‐treated mice (bottom), before (left) and after (right) the addition of ACh. (e) Protein abundance of NADPH oxidase p67 in aortic lysates with representative Western blotting images generated from electropherograms from the automated capillary electrophoresis system shown below. Data are mean ± SEM. N = 8–10/group. †p < .10 vs. vehicle treatment
Figure 2 Plasma from recIL‐37‐treated mice increases endothelial cell acetylcholine (ACh)‐stimulated nitric oxide (NO) production and reduces reactive oxygen species production. (a–c) NO production (DAF‐FM fluorescence) before and after incubation with ACh [presented at both time points (a) and as a fold change from basal (b), both normalized within plasma conditions] and reactive oxygen species production (c) (CellROX signal) in human umbilical vein endothelial cells cultured with plasma from vehicle‐ and recIL‐37‐treated old mice or with fetal cow serum (FCS; healthy control condition). Cells were also stained with Hoechst 33342 to indicate healthy nuclei. Data are mean ± SEM. N = 8–10/group. #p < .05 vs. basal conditions (pre‐ACh). *p < .05 vs. vehicle treatment. Each plasma sample was assayed twice over two days. For DAF‐FM experiments, 320 HUVECs were visualized after IL‐37‐treated plasma incubation and 256 cells were visualized after vehicle‐treated plasma incubation. For CellROX experiments, 240 cells were visualized after IL‐37‐treated plasma incubation and 192 cells were visualized after vehicle‐treated plasma incubation. (d) Representative fluorescent images taken at 20X optical zoom. Images are of the same wells incubated with plasma from vehicle‐ (top) or recIL‐37‐treated mice (bottom), before (left) and after (right) the addition of ACh. (e) Protein abundance of NADPH oxidase p67 in aortic lysates with representative Western blotting images generated from electropherograms from the automated capillary electrophoresis system shown below. Data are mean ± SEM. N = 8–10/group. †p < .10 vs. vehicle treatmentCC BY · PubMed Central
Figure 3 RecIL‐37 treatment improves skeletal muscle function. (a) Time to fatigue on the endurance rota‐rod test and (b) normalized grip strength, measured pre‐ and post‐vehicle or recIL‐37 treatment. N = 10–13 mice per group. (c) Relative ratio of phosphorylated‐AMPK (p‐172) and total AMPK in quadriceps lysate from vehicle‐ and recIL‐37‐treated mice, with representative Western blot images shown to the right. Western blot analyses were performed in duplicate in tissue lysates from 10–12 mice per group. (d) ADP/ATP ratio in collagenase‐separated quadriceps muscle cells from vehicle‐ or recIL‐37‐treated mice. Enzymatic assays were performed in duplicated for each muscle tissue sample from 10 to 12 mice per group. All data are mean ± SEM. *p < .05 vs. vehicle. #p < .05 vs. pre‐intervention
Figure 3 RecIL‐37 treatment improves skeletal muscle function. (a) Time to fatigue on the endurance rota‐rod test and (b) normalized grip strength, measured pre‐ and post‐vehicle or recIL‐37 treatment. N = 10–13 mice per group. (c) Relative ratio of phosphorylated‐AMPK (p‐172) and total AMPK in quadriceps lysate from vehicle‐ and recIL‐37‐treated mice, with representative Western blot images shown to the right. Western blot analyses were performed in duplicate in tissue lysates from 10–12 mice per group. (d) ADP/ATP ratio in collagenase‐separated quadriceps muscle cells from vehicle‐ or recIL‐37‐treated mice. Enzymatic assays were performed in duplicated for each muscle tissue sample from 10 to 12 mice per group. All data are mean ± SEM. *p < .05 vs. vehicle. #p < .05 vs. pre‐interventionCC BY · PubMed Central
Figure 4 RecIL‐37 treatment improves insulin sensitivity and glucose tolerance. (a) Blood glucose concentrations over 90 min following intraperitoneal injection of insulin (0.75 U/kg body weight). N = 12–14 mice per group. (b) Blood glucose concentrations over 120 min following intraperitoneal injection of glucose (2 mg/g body weight). N = 8–10 mice per group. (c) Area under the curve blood glucose concentration over the 120‐min glucose tolerance test. Data are mean ± SEM. *p < .05 vs. vehicle treatment (within time point). †p < .10 vs. vehicle treatment (within time point)
Figure 4 RecIL‐37 treatment improves insulin sensitivity and glucose tolerance. (a) Blood glucose concentrations over 90 min following intraperitoneal injection of insulin (0.75 U/kg body weight). N = 12–14 mice per group. (b) Blood glucose concentrations over 120 min following intraperitoneal injection of glucose (2 mg/g body weight). N = 8–10 mice per group. (c) Area under the curve blood glucose concentration over the 120‐min glucose tolerance test. Data are mean ± SEM. *p < .05 vs. vehicle treatment (within time point). †p < .10 vs. vehicle treatment (within time point)CC BY · PubMed Central
Figure 5 Treatment with recIL‐37 affects levels of certain circulating metabolites. (a) Heat map of key metabolites in plasma. (b) Principal component analysis based on partial least squares‐discriminant analysis. Plasma levels of (c) L‐arginine and related metabolites, (d) pantothenol (bottom right of panel) and related metabolites, (e) key free fatty acids, including docosahexaenoic acid (DHA, first graph in panel), (f) select amino acids and their metabolites and (g) lysophosphatidic acid. Data are mean ± SEM of the integrated peak areas (i.e., area under the curve) from ion chromatograms for each metabolite. N = 6–8 mice per group. *p < .05 across groups (Student's unpaired t test)
Figure 5 Treatment with recIL‐37 affects levels of certain circulating metabolites. (a) Heat map of key metabolites in plasma. (b) Principal component analysis based on partial least squares‐discriminant analysis. Plasma levels of (c) L‐arginine and related metabolites, (d) pantothenol (bottom right of panel) and related metabolites, (e) key free fatty acids, including docosahexaenoic acid (DHA, first graph in panel), (f) select amino acids and their metabolites and (g) lysophosphatidic acid. Data are mean ± SEM of the integrated peak areas (i.e., area under the curve) from ion chromatograms for each metabolite. N = 6–8 mice per group. *p < .05 across groups (Student's unpaired t test)CC BY · PubMed Central

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Metadata

Authors
Ballak, D. B., Brunt, V. E., Sapinsley, Z. J., Ziemba, B. P., Richey, J. J., Zigler, M. C., Johnson, L. C., Gioscia‐Ryan, R. A., Culp‐Hill, R., Eisenmesser, E. Z., D'Alessandro, A., Dinarello, C. A., & Seals, D. R.
Affiliations
  • Department of Integrative Physiology University of Colorado Boulder Boulder CO USA
  • Department of Medicine University of Colorado Denver Aurora CO USA
ORCID
Brunt, Vienna E.
Venue
Aging Cell
Publisher
Wiley
Published
2019-11-21
Volume
19
Issue
1
ISSN
  • 1474-9718
  • 1474-9726
DOI
10.1111/acel.13074
PMC
PMC6974720
Cited by
44
References
6
Language
en
Licence
creativecommons.org/licenses/by/4.0/ (vor)
Funders
  • National Heart, Lung, and Blood Institute (HL007822, HL140875)
  • National Institute on Aging (AG053804)
  • National Institute of Allergy and Infectious Diseases (AI‐15614)

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