Abstract
Factors that underlie the clustering of metabolic syndrome traits are not fully known. We performed whole-exome sequence analysis in kindreds with extreme phenotypes of early-onset atherosclerosis and metabolic syndrome, and identified novel loss-of-function mutations in the gene encoding the pancreatic elastase chymotrypsin-like elastase family member 2A (CELA2A). We further show that CELA2A is a circulating enzyme that reduces platelet hyperactivation, triggers both insulin secretion and degradation, and increases insulin sensitivity. CELA2A plasma levels rise postprandially and parallel insulin levels in humans. Loss of these functions by the mutant proteins provides insight into disease mechanisms and suggests that CELA2A could be an attractive therapeutic target.
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Data availability
Human variants and phenotypes have been reported to ClinVar under accession numbers SCV000916382, SCV000916383, SCV000916384 and SCV000916385. The data have also been reported to NIH with other identified variants in the Yale Center for Mendelian Genomics. Proteomics data are available on request.
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Acknowledgements
We thank T. Lam, J. Kanyo, W. Wang and N. Rauniar from the Yale Keck Mass Spectrometry and Proteomics Services for help with the proteomics analysis, and J. Murphy from the Department of Pharmacology at Yale University School of Medicine for preparing the ribbon diagrams. This work was supported by grants from the National Institutes of Health (NIH) (RHL135767A and P30 DK34989 to A.M., NIH R01DK095753 to M.S.-T. and NIH T32DK to F.E. (DK007356)), a grant from the NIH Centers for Mendelian Genomics (5U54HG006504) and a VA Merit Award to F.S.G. (NIH S10 (SIG) OD018034 awarded to the Mass Spectrometry and Proteomics Resource of the W.M. Keck Foundation Biotechnology Resource Laboratory at Yale University). The authors would like to thank the NHLBI GO Exome Sequencing Project and its ongoing studies, which produced and provided exome variant calls for comparison: the Lung GO Sequencing Project (HL-102923), the WHI Sequencing Project (HL-102924), the Broad GO Sequencing Project (HL-102925), the Seattle GO Sequencing Project (HL-102926) and the Heart GO Sequencing Project (HL-103010).
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F.E. contributed primarily to designing and performing the experiments, as well as to preparing the figures and manuscript. J.S.B., R.C., T.T., A.S., N.U., M.V.M., M.G., B.A., S.C., M.F. and A.A. were involved with performing the experiments. S.M., R.P.L., M.H.N., J.H., M.S.-T. and R.G.K. were involved in the design and supervision of certain aspects of the project. G.K., E.W., J.B. and E.S. were involved in patient recruitment and clinical characterizations. R.B.-D. carried out all of the OGTT and hyperglycemic clamp studies. N.U. was involved in the analysis of the genetic data. F.S.G. was involved in the design and supervision of aspects of the project, and participated in manuscript writing. A.M. designed the study and oversaw its implementation, supervised all aspects of the project from performing the experiments to the analysis of all data, and wrote the manuscript.
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Supplementary Figure 1 Selected laboratory and clinical data in p.D121N carriers vs. non-carriers of the kindred CAD-2001.
a, Plasma TG levels (mg/dl; mean ± s.e.m.; n = 14 non-carriers vs. n = 10 carriers; Student’s t-test, two-sided at **P < 0.0001). b, BMI (kg/m2) (mean ± s.e.m. ; n = 14 non-carriers vs. n = 13 carriers; Student’s t-test, two-sided at **P = 0.002). c, Plasma cortisol levels (μg/ml; mean ± s.e.m.; n = 8 non-carriers vs. n = 8 carriers; Student’s t-test, two-sided at *P = 0.011). d, Plasma GLP1 levels (pm/L; mean ± s.e.m; n = 8 non-carriers vs. n = 8 carriers; Student’s t-test, two-sided, P = n.s). e, Plasma globular adiponectin levels (ng/ml; mean ± s.e.m.; n = 8 non-carriers vs. n = 7 carriers, Student’s t-test, two-sided; P = n.s). TG, triglycerides; BMI, body mass index; GLP1, Glucagon-like peptide 1.
Supplementary Figure 2 Validation of CELA2A antibody used in western blot analysis and immunohistochemistry.
a, Western blot image of different quantities of rCela2a using CELA2A-specific antibody and CELA2A antibody (Sigma-Aldrich, SAB1104798) pre-blocked with rCela2a (MyBiosource, MBS1246487). The 25-kDa rCela2a bands are almost invisible in the western blot carried out using pre-blocked antibody (n = 2 independent experiments). b, Western blot analysis using the supernatants of the 293T cells overexpressing either empty vector, WT-CELA2A or p.D121N His-CELA2A. The 25-kDa and 75-kDa His-CELA2A bands are no longer visible in the western blot carried out using pre-blocked antibody; the MW marker (middle) has been greatly overexposed to allow visualization of the CELA2A bands (n = 2 independent experiments). c, Immunohistochemistry staining of mouse skeletal muscle for CELA2A in WT mice injected with rCela2a vs. saline. IgG staining was used as negative control (n = 5 independent experiments). d, Validation of the ELISA kit assay for human CELA2A using known concentrations of purified WT-CELA2A and p.D121N-CELA2A represented as bar charts with mean ± s.e.m. The vehicle served as a negative control (n = 3 independent experiments; triplicates of one experiments have been represented). e, Whole membrane of the western blot for Cela2a, shown in Fig. 3b.
Supplementary Figure 3 Tissue distribution of CELA2A.
a-c, Immunohistochemical staining of mouse adrenal gland cortex and medulla (a) (n = 3 independent experiments), small intestine (b) (n = 3 independent experiments), and exocrine pancreas (c) (n = 5 independent experiments). Scale bar, 100 μm. Arrows indicate lymphoid follicles in the small intestine. Different zones in the adrenal gland are shown. d, Western blot analysis of CELA2A in human cadaveric liver, white adipose tissue (WAT) and pancreas.
Supplementary Figure 4 Expression of CELA2A in disease states from Gene Expression Omnibus (GEO) datasets and insulin, glucose and glucagon levels before and after the meal in human subjects.
a, Relative expression of CELA2A mRNA in vastus lateralis muscle samples from insulin-resistant obese (n = 5) and insulin sensitive (n = 5) Pima Indians. b, Beta-cell enriched pancreatic tissues obtained from subjects with T2D (n = 10) and without T2D (n = 10), shown as dot plots (mean ± s.e.m). Data were derived from GEO database (see the references in the text). Statistical analyses were carried out using two-sided Student’s t-test; P = 0.0115 and P = 0.0150, respectively. c, Glucagon level (pg/dl; mean ± s.e.m) in p.D121N-CELA2A carriers compared to controls (n = 7 control samples vs. 8 p.D121N-carriers). Statistical analyses were carried out using two-sided Student’s t-test, P = 0.033. d,e, Plasma glucagon levels (pg/dl; mean ± s.e.m) in fast/fed healthy individuals and correlation with plasma CELA2A in healthy subjects before and after meal (n = 8 samples). Statistical analyses were carried out using two-sided Student’s t-test; P = 0.049. Correlation coefficient (r2 = -0.72) for e was performed using GraphPad. f, CELA2A to glucose ratios (mean ± s.e.m) at 60 min hyperglycemic clamp and OGTT represented as bar chart (n = 5 samples, Student’s t-test, two-sided, P = n.s.). g,h, Average values of CELA2A and glucagon (pg/dl; mean ± s.e.m) during hyperglycemic clamp and OGTT studies (n = 5 samples). OGTT, oral glucose tolerance test. AU, arbitrary units. All human studies were performed only once.
Supplementary Figure 5 rCela2a administration in vivo and in human islets.
a,b, Plasma insulin and glucose levels (mean ± s.e.m.) after intravenous administration of rCela2a to normoglycemic wild-type C57BL/6 mice (n = 5 in each group). Statistical analyses were carried out using two-sided Student’s t-test. *P < 0.05. c,d, Insulin and C-peptide secretion of human islets in response to WT-CELA2A. Response to KCl is used as a positive control and test of viability. Violin plots represent median, minimum and maximum. Statistical analyses were carried out using two-sided Student’s t-test per condition (n = 4). **P <0.01. e, Insulin secretion of human islets in response to WT-CELA2A (human) compared to rCela2a (mouse) represented in bar charts (mean ± s.e.m; n = 4 samples). Statistical analyses were carried out using two-sided Student’s t-test per conditions (***P <0.001). Response to KCl is used as a positive control and test of viability.
Supplementary Figure 6 Quantification of insulin signaling activation by WT-CELA2A and p.D121N-CELA2A protein.
a-e, Plots show relative intensities compared to controls for the phosphoproteins in the western blots shown in Fig. 5g displayed as bar charts (mean ± s.e.m.; n = 2 independent experiments). Statistical analyses were performed using one-way ANOVA. *P < 0.05, **P < 0.001.
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Esteghamat, F., Broughton, J.S., Smith, E. et al. CELA2A mutations predispose to early-onset atherosclerosis and metabolic syndrome and affect plasma insulin and platelet activation. Nat Genet 51, 1233–1243 (2019). https://doi.org/10.1038/s41588-019-0470-3
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DOI: https://doi.org/10.1038/s41588-019-0470-3
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