Response to Comment on Foussard et al. Skin Autofluorescence of Pregnant Women With Diabetes Predicts the Macrosomia of Their Children. Diabetes 2019;68:1663–1669

Longitudinal Metabolome-Wide Signals Prior to the Appearance of a First Islet Autoantibody in Children Participating in the TEDDY Study


Discussion

The TEDDY study offers a robust analysis of the metabolome in 417 infants who developed IA, with primarily either IAA only (49%) or GADA only (33%) as the first appearing autoantibodies. The subjects who experienced both IAA and GADA (14%) as the first appearing autoantibodies were not considered in the current analysis. Our aim in the current study was to discover longitudinal metabolic patterns preceding different first appearing IA in the presence of the well-known age effect on metabolic profiles (3,5).

Our data suggest that IAA-first and GADA-first differ in the way that metabolites and lipids precede seroconversion. The significantly lower abundance of isoleucine and valine prior to seroconversion in IAA-first subjects (Fig. 2) is of interest, as isoleucine and valine are BCAAs widely known to potentiate glucose-stimulated insulin secretion (38,39). On the other hand, proline as a nonessential amino acid produced from glutamate cyclization (40) remained at a reduced level across multiple visits before IA (Fig. 2). In addition to the differentiated trajectory, proline was also found to be negatively associated with the risk of GADA-first in independent time point analysis. Proline biosynthesis involves l-glutamate, which interacts with the metabolic pathway of glutamic acid (41), GAD, and GABA (42), as illustrated in Fig. 3. Another critical metabolite derived from glutamic acid, i.e., α-ketoglutarate (42), also displayed lower concentration in GADA-first case compared with control subjects over time before seroconversion (Fig. 4). These results suggest that an enduring decrease in proline and lower α-ketoglutarate level may imply an abnormal glutamic acid metabolism causally related to the underlying change of GAD enzymatic activity in subjects who later developed GADA-first, with relatively higher glutamic acid levels prior to seroconversion (Fig. 4). The elevated level of glutamic acid may also be an indicator for the emergence of GADA-first.

Figure 3

Metabolic pathways involving trajectory signals and independent time point biomarkers for IA in TEDDY. TCA, tricarboxylic acid.

Figure 4
Figure 4

Mean abundance of preseroconversion GABA, glutamic acid, glutamine, α-ketoglutarate, leucine, and plasmenyl-PC (ether PC) per age point for case and control subjects in GADA-first and IAA-first groups.

Another major finding in the current study was the association between plasma GABA after birth and the future appearance of IAA-first. This finding was based on both independent time point analysis (Table 2) and enrichment analysis results (Table 4). Why GABA levels would be associated with IAA-first and not GADA-first may be explained by the fact that β-cell GAD65, which produces GABA, may not be affected before GADA onset. The impact of GABA on the pancreatic β-cell function has been thoroughly investigated (43,44). The odds ratio for plasma GABA (Table 2) for IAA-first indicates that higher levels of GABA immediately after birth may be related to β-cell dysfunction, with the appearance of IAA-first possibly related to abnormal insulin synthesis or secretion at early ages. In contrast, we did not find the contribution of GABA to future risk of seroconversion in GADA-first children, providing evidence that GABA has no causal influence on the appearance of GADA-first.

A third major finding of biomarkers for the risk of IA in TEDDY was DHAA after birth. DHAA identified at 3 months of age did not discriminate between GADA-first and IAA-first but showed statistical significance for both autoantibodies. Elevated DHAA or oxidized vitamin C was found to inhibit insulin secretion in mice (45–47), and exposure of isolated mouse pancreatic islets to DHAA or vitamin C reduced the responsiveness of the islets (48) or led to inhibition of insulin secretion from the pancreatic β-cells (47). The results for DHAA in TEDDY samples would seem to be consistent with the existing findings, showing a possible suppressive effect on human pancreatic islet β-cells. Another recent study in TEDDY found immunoassay measurements of plasma vitamin C levels to be associated with lower risk of IAA but not GADA. Further analyses would therefore be required to include immunoassay measurement of DHAA to detail the possible importance of the vitamin C/DHAA ratio and its regulation. Other compounds identified as contributing to IA development in TEDDY were also found to be important metabolic features in existing T1D-related studies, such as amino acids alanine (4) and methionine (5), fatty acids (49), vitamin E (45), sugar alcohols, and unsaturated TGs (5,50). Furthermore, we observed (Table 2) 5-methoxytryptamine at ages 6 months and 9 months contributing to the risk of IAA-first with association altered from positive to negative between 6 and 9 months, prior to and near the age of population-wide IAA-first incidence peak (10). 5-methoxytryptamine is a metabolite of melatonin and serotonin, which have been linked to diabetes and autoimmune disorders in previous studies (51,52).

The current study in TEDDY represents the largest prospective cohort analysis of metabolomes in children at increased genetic risk for T1D and identifies biomarkers for islet autoimmunity (stage I and II) that precedes the clinical onset of diabetes (stage III) (53). Similar observations were found in the Type 1 Diabetes Prediction and Prevention (DIPP) study (4) reporting that changes in GABA, glutamic acid, glutamine, α-ketoglutarate, leucine, and plasmenyl-PCs (ether PCs) were age dependent and could be associated with the onset of GADA and IAA. Based on the TEDDY longitudinal metabolome profiles, we not only confirmed the DIPP findings using average abundance of these compounds across time points but also separated the age and time-to-seroconversion effects (Fig. 4 and Supplementary Fig. 3). The trend of ether PC between 1 and 2 years of age in TEDDY subjects was similar to the change over time before GADA-first onset observed in DIPP, which was the result of overlapping effects of age and time to seroconversion. On the other hand, the decrease in GABA levels within 1 year before seroconversion observed in DIPP was a pattern determined by time to seroconversion instead of age. Furthermore, our results in metabolite enrichment analysis (Table 4) not only agreed with the reduced level of PC, TGs and plasmenyl- (or ether) phospholipids found in individuals who developed T1D (4) but also revealed lower PE and sugar alcohols during infancy associated with future onset of IA.

A limitation to the current study is the quarterly time sampling from 3 months of age and onward. The effect of age on metabolites and complex lipids (4,5) would have been better understood with a more frequent blood sampling, especially in relation to IAA as the first appearing autoantibody. IAA-first has been related to prior infectious episodes both in DIPP (54) and TEDDY (55), and at this early age, statistical analyses will have to take both age-related effects and environmental exposures into account to further delineate the mechanisms that trigger an autoimmune response against insulin.

Current analyses focused on metabolic markers for IA prior to seroconversion and included TEDDY participants who only developed IAA or GADA as the first-appearing autoantibody. It is worthwhile to extend future analyses to participants who experienced multiple autoantibodies either at seroconversion or throughout the follow-up, since the age at development of multiple autoantibodies has been found associated with the risk of progression to T1D (16). Genetics or environmental causes leading to metabolic signals (such as DHAA, GABA, and proline) identified in present analyses were still unknown and should be investigated further jointly with genome-wide SNP data, gut microbiome, and dietary patterns.

Conclusion

These results from metabolome-wide trajectory, independent time point, and enrichment analyses support the notion that the onset of IA as GADA-first or IAA-first in TEDDY children is heralded by distinct metabolic precursors in plasma after birth. The precursory signals for each autoantibody include DHAA; GABA; amino acids proline, alanine, and methionine; and compounds in BCAA metabolism as well as fatty acids. Unsaturated TGs and PEs at infant age were found to be decreased before appearance of either autoantibody. The distinct metabolic patterns for these autoantibodies support the idea that the causes of each type of initial autoimmunity may be different, and may account for the earlier incidence peak of IAA-first compared with that of GADA-first in TEDDY.

Appendix

The members of the TEDDY Study Group are listed below. The numbers listed correspond with the committees as follows: 1Ancillary Studies, 2Diet, 3Genetics, 4Human Subjects/Publicity/Publications, 5Immune Markers, 6Infectious Agents, 7Laboratory Implementation, 8Psychosocial, 9Quality Assurance, 10Steering, 11Study Coordinators, 12Celiac Disease, and 13Clinical Implementation.

Colorado Clinical Center. Marian Rewers, Principal Investigator (PI),1,4,5,6,9,10 Aaron Barbour, Kimberly Bautista,11 Judith Baxter,8,9,11 Daniel Felipe-Morales, Kimberly Driscoll,8 Brigitte I. Frohnert,2,13 Marisa Stahl,12 Patricia Gesualdo,2,6,11,13 Michelle Hoffman,11,12,13 Rachel Karban,11 Edwin Liu,12 Jill Norris,2,3,11 Stesha Peacock, Hanan Shorrosh, Andrea Steck,3,13 Megan Stern, Erica Villegas,2 and Kathleen Waugh6,7,11: Barbara Davis Center for Childhood Diabetes.

Finland Clinical Center. Jorma Toppari, PI,¥^1,4,10,13, Olli G. Simell, Annika Adamsson,^11 Suvi Ahonen,*±§ Mari Åkerlund,*±§ Leena Hakola,* Anne Hekkala,µ† Henna Holappa,µ† Heikki Hyöty,*±6 Anni Ikonen,µ† Jorma Ilonen,¥¶3 Sinikka Jäminki,*± Sanna Jokipuu,^ Leena Karlsson,^ Jukka Kero,¥^ Miia Kähönen,µ†11,13 Mikael Knip,*±5 Minna-Liisa Koivikko,µ† Merja Koskinen,*± Mirva Koreasalo,*±§2 Kalle Kurppa,*±12 Jarita Kytölä,*± Tiina Latva-aho,µ† Katri Lindfors,*12 Maria Lönnrot,*±6 Elina Mäntymäki,^ Markus Mattila,* Maija Miettinen,§2 Katja Multasuo,µ† Teija Mykkänen,µ† Tiina Niininen,±*11 Sari Niinistö,±§2 Mia Nyblom,*± Sami Oikarinen,*± Paula Ollikainen,µ† Zhian Othmani,^ Sirpa Pohjola,µ† Petra Rajala,^ Jenna Rautanen,±§ Anne Riikonen,*±§2 Eija Riski,^ Miia Pekkola,*± Minna Romo,^ Satu Ruohonen,^ Satu Simell,¥12 Maija Sjöberg,^ Aino Stenius,µ†11 Päivi Tossavainen,µ† Mari Vähä-Mäkilä,¥ Sini Vainionpää,^ Eeva Varjonen,^11 Riitta Veijola,µ†13 Irene Viinikangas,µ† and Suvi M. Virtanen*±§2: ¥University of Turku; *Tampere University; µUniversity of Oulu; ^Turku University Hospital; Hospital District of Southwest Finland; ±Tampere University Hospital; Oulu University Hospital; §National Institute for Health and Welfare, Finland; and University of Kuopio.

Georgia/Florida Clinical Center. Jin-Xiong She, PI,,1,3,4,10 Desmond Schatz,*4,5,7,8 Diane Hopkins,11 Leigh Steed,11,12,13 Jennifer Bryant,11 Katherine Silvis,2 Michael Haller,*13 Melissa Gardiner,11 Richard McIndoe, Ashok Sharma, Stephen W. Anderson,^ Laura Jacobsen,*13 John Marks,*11,13 and P.D. Towe*: Center for Biotechnology and Genomic Medicine, Augusta University; *University of Florida; and ^Pediatric Endocrine Associates, Atlanta.

Germany Clinical Center. Anette G. Ziegler, PI,1,3,4,10 Ezio Bonifacio,*5 Anita Gavrisan, Cigdem Gezginci, Anja Heublein, Verena Hoffmann,2 Sandra Hummel,2 Andrea Keimer,¥2 Annette Knopff,7 Charlotte Koch, Sibylle Koletzko,¶12 Claudia Ramminger,11 Roswith Roth,8 Marlon Scholz, Joanna Stock,8,11,13 Katharina Warncke,13 Lorena Wendel, and Christiane Winkler2,11 from: Forschergruppe Diabetes e.V. and Institute of Diabetes Research, Helmholtz Zentrum München, Forschergruppe Diabetes, and Klinikum rechts der Isar, Technische Universität München; *Center for Regenerative Therapies, TU Dresden; Department of Gastroenterology, Dr. von Hauner Children’s Hospital, Ludwig Maximillians University Munich; and ¥Department of Nutritional Epidemiology, University of Bonn.

Sweden Clinical Center. Åke Lernmark, PI,1,3,4,5,6,8,9,10 Daniel Agardh,6,12 Carin Andrén Aronsson,2,11,12 Maria Ask, Rasmus Bennet, Corrado Cilio,5,6 Helene Engqvist, Emelie Ericson-Hallström, Annika Fors, Lina Fransson, Thomas Gard, Monika Hansen, Hanna Jisser, Fredrik Johansen, Berglind Jonsdottir,11 Silvija Jovic, Helena Elding Larsson,6,13 Marielle Lindström, Markus Lundgren,13 Marlena Maziarz, Maria Månsson-Martinez, Maria Markan, Jessica Melin,11 Zeliha Mestan, Caroline Nilsson, Karin Ottosson, Kobra Rahmati, Anita Ramelius, Falastin Salami, Anette Sjöberg, Birgitta Sjöberg, Malin Svensson, Carina Törn,3 Anne Wallin, Åsa Wimar13, and Sofie Åberg: Lund University.

Washington Clinical Center. William A. Hagopian, PI,1,3,4,5,6,7,10,12,13 Michael Killian,6,7,11,12 Claire Cowen Crouch,11,13 Jennifer Skidmore,2 Masumeh Chavoshi, Rachel Hervey, Rachel Lyons, Arlene Meyer, Denise Mulenga,11 Jared Radtke, Matei Romancik, Davey Schmitt, and Sarah Zink: Pacific Northwest Research Institute.

Pennsylvania Satellite Center. Dorothy Becker, Margaret Franciscus, MaryEllen Dalmagro-Elias Smith,2 Ashi Daftary, Mary Beth Klein, and Chrystal Yates: UPMC Children’s Hospital of Pittsburgh.

Data Coordinating Center. Jeffrey P. Krischer, PI, 1,4,5,9,10 Sarah Austin-Gonzalez, Maryouri Avendano, Sandra Baethke, Rasheedah Brown,11 Brant Burkhardt,5,6 Martha Butterworth,2 Joanna Clasen, David Cuthbertson, Stephen Dankyi, Christopher Eberhard, Steven Fiske,8 Jennifer Garmeson, Veena Gowda, Kathleen Heyman, Belinda Hsiao, Christina Karges, Francisco Perez Laras, Hye-Seung Lee,1,2,3,12 Qian Li,5,12 Shu Liu, Xiang Liu,2,3,8,13 Kristian Lynch,5,6,8 Colleen Maguire, Jamie Malloy, Cristina McCarthy,11 Aubrie Merrell, Hemang Parikh,3 Ryan Quigley, Cassandra Remedios, Chris Shaffer, Laura Smith,8,11 Susan Smith,11 Noah Sulman, Roy Tamura,1,2,11,12,13 Dena Tewey, Michael Toth, Ulla Uusitalo,2 Kendra Vehik,4,5,6,8,13 Ponni Vijayakandipan, Keith Wood, and Jimin Yang2; past staff, Michael Abbondondolo, Lori Ballard, David Hadley, Wendy McLeod, and Steven Meulemans: University of South Florida.

Project Scientist. Beena Akolkar1,3,4,5,6,7,9,10: National Institutes of Diabetes and Digestive and Kidney Diseases.

Other Contributors. Kasia Bourcier5: National Institutes of Allergy and Infectious Diseases. Thomas Briese6: Columbia University. Suzanne Bennett Johnson8,11: Florida State University. Eric Triplett6: University of Florida.

Autoantibody Reference Laboratories. Liping Yu,^5 Dongmei Miao,^ Polly Bingley,*5 Alistair Williams,* Kyla Chandler,* Olivia Ball,* Ilana Kelland,* and Sian Grace*: ^Barbara Davis Center for Childhood Diabetes and *Bristol Medical School, University of Bristol, U.K.

HLA Reference Laboratory. William Hagopian,3 Masumeh Chavoshi, Jared Radtke, and Sarah Zink: Pacific Northwest Research Institute, Seattle, WA (previously Henry Erlich,3 Steven J. Mack, and Anna Lisa Fear: Center for Genetics, Children’s Hospital Oakland Research Institute).

Metabolomics Laboratory. Oliver Fiehn, Bill Wikoff, Brian Defelice, Dmitry Grapov, Tobias Kind, Mine Palazoglu, Luis Valdiviez, Benjamin Wancewicz, Gert Wohlgemuth, and Joyce Wong: West Coast Metabolomics Center.

SNP Laboratory. Stephen S. Rich,3 Wei-Min Chen,3 Suna Onengut-Gumuscu,3 Emily Farber, Rebecca Roche Pickin, Jonathan Davis, Jordan Davis, Dan Gallo, Jessica Bonnie, and Paul Campolieto: Center for Public Health Genomics, University of Virginia.



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