Nine Amino Acids are Associated with Decreased Insulin Secretion and Elevated Glucose Levels in a 4.6-Year Follow-Up Study of 5,181 Finnish Men

2019

Nine Amino Acids are Associated with Decreased Insulin Secretion and

Elevated Glucose Levels in a 4.6-Year Follow-Up Study of 5,181 Finnish Men

Vangipurapu, J

American Diabetes Association

Tieteelliset aikakauslehtiartikkelit

© The American Diabetes Association All rights reserved

http://dx.doi.org/10.2337/db18-1076

https://erepo.uef.fi/handle/123456789/7641

Downloaded from University of Eastern Finland's eRepository

1 Manuscript DB18-1076 (2^nd revision)

Nine amino acids are associated with decreased insulin secretion and elevated glucose levels in a 4.6-year follow-up study of 5,181 Finnish Men

Running title: Amino acids and insulin secretion

Jagadish Vangipurapu¹, Alena Stancáková¹, Ulf Smith², Johanna Kuusisto^1,3, Markku Laakso^1,3

1Institute of Clinical Medicine, Internal Medicine, University of Eastern Finland, Kuopio, Finland

2The Lundberg Laboratory for Diabetes Research, Department of Molecular and Clinical Medicine, the Sahlgrenska Academy, University of Gothenburg, 405 30, Gothenburg, Sweden

3Department of Medicine, Kuopio University Hospital, Kuopio, Finland

Word count Abstract: 161 Main text: 1995

Correspondence

Markku Laakso, MD, PhD, Professor

Institute of Clinical Medicine, Internal Medicine,

University of Eastern Finland, and Kuopio University Hospital, 70210 Kuopio, Finland

email: markku.laakso@uef.fi phone: +358 40 672 3338

ABSTRACT

Several amino acids have been shown to be associated with insulin resistance and increased risk of type 2 diabetes, but none of previous studies has investigated the association of amino acids with insulin secretion in a longitudinal setting. Our study included 5181 participants of the cross-sectional METabolic Syndrome In Men study having metabolomics data on twenty amino acids. A total of 4851 of them had a 4.6-year follow-up visit. Nine amino acids (phenylalanine, tryptophan, tyrosine, alanine, isoleucine, leucine, valine, aspartate and glutamate) were significantly (P<5.8x10^-5) associated with decreases in insulin secretion (Disposition index) and the elevation of fasting or 2 hour glucose levels. Five of these amino acids (tyrosine, alanine, isoleucine, aspartate and glutamate) were also found to be significantly associated with an increased risk of incident type 2 diabetes after adjustment for confounding factors. Our study is the first population-based large cohort to report that amino acids are not only associated with insulin resistance but also with decreased insulin secretion.

Type 2 diabetes is often preceded by a long period of prediabetes, characterized by insulin resistance and impaired insulin secretion. Conversion to diabetes happens when insulin secretion from the pancreas is no longer able to compensate for insulin resistance in peripheral insulin-sensitive tissues (1).

Several metabolites, including especially branched chain and aromatic amino acids (AAs), have been reported to be associated with the risk of type 2 diabetes in previous studies (2-5).

However, most of these studies have been cross-sectional, and none of these studies has investigated the association of AAs with changes in insulin secretion in a longitudinal setting.

We investigated the associations of twenty AAs with insulin secretion, insulin resistance and glycemia in a large Finnish prospective population-based Metabolic Syndrome In Men (METSIM) cohort.

RESEARCH DESIGN AND METHODS Study Population, Methods and Calculations

The METSIM study comprises 10,197 Finnish men, aged from 45 to 73 years, randomly selected from the population register of Kuopio town, Eastern Finland. The study design and laboratory methods have been described previously (6,7). Glucose tolerance was evaluated with a 2-hour glucose tolerance test (OGTT, 75 g of glucose) including three time-points (glucose and insulin levels measured at 0, 30 and 120 min) according to the ADA criteria (8).

Out of 5,181 men without diabetes at entry included in the present analysis, 4,851 participated in the follow-up study (mean follow-up time 4.6 ± 1.2 years). This subset of 5,181 men had similar clinical and laboratory characteristics (S1 Table) as the entire METSIM population without diabetes, and therefore this subsample can be considered to present the entire METSIM cohort (7). A total of 522 participants developed incident type 2 diabetes. The study was

approved by the Ethics Committee of the University of Kuopio and Kuopio University Hospital. All study participants gave written informed consent.

Glucose and insulin areas under the curve (AUC) in an OGTT were calculated by the trapezoidal method. The Matsuda insulin sensitivity index (Matsuda ISI) was calculated as previously published (9). Insulin secretion index (InsAUC0-30/GluAUC0-30) was calculated based on an OGTT as follows: (insulin at 0 min + insulin at 30 min ) / (glucose at 0 min + glucose at 30 min). The selection of Matsuda ISI (among 6 insulin sensitivity indices compared to the M value from euglycemic clamp) and InsAUC0-30/GluAUC0-30 (among 11 insulin secretion indices compared to insulin secretion during a frequently sampled intravenous glucose tolerance test) were based on our previous validation study (7). Disposition Index (DI), a measure of insulin secretion adjusted for prevailing insulin sensitivity, was calculated as Matsuda ISI x (InsAUC0-30/GluAUC0-30) (7).

Metabolomics Analysis

Metabolites were measured as part of Metabolon Inc.'s untargeted Discovery HD4 platform using Ultrahigh Performance Liquid Chromatography-Tandem Mass Spectroscopy (UPLC- MS/MS). All methods utilized a Waters ACQUITY ultra-performance liquid chromatography (UPLC) and a Thermo Scientific Q-Exactive high resolution/accurate mass spectrometer interfaced with a heated electrospray ionization (HESI-II) source and Orbitrap mass analyzer operated at 35,000 mass resolution. Raw data was extracted, peak-identified and QC processed using Metabolon’s hardware and software. Peaks were quantified using area-under-the-curve.

For studies spanning multiple days, a data normalization step was performed to correct variation resulting from instrument inter-day tuning differences. Compounds were identified by comparison to library entries of purified standards or recurrent unknown entities. The metabolite levels were re-scaled to have median equal to 1.

The metabolomics analyses were performed in three batches. Batch 1 included 999 samples with 717 metabolites identified, batch 2 included 1,231 samples with 778 metabolites identified, and batch 3 included 3,000 samples with 843 metabolites identified. Twenty AAs from a total of 857 metabolites were included in current statistical analysis. The percentage of missing values for AAs was from 0 to 0.2 %.

Statistical Analysis

We conducted statistical analyses using IBM SPSS Statistics version 25. We log-transformed all continuous traits with the exception of age and follow-up time to correct for their skewed distribution. Inter-correlations between the AAs were calculated by Pearson correlations (Fig S1). We examined the association of AAs with Matsuda ISI, DI and glucose levels with linear regression, and presented the results as standardized regression coefficients (β, SE). We applied Cox regression to associate the levels of AAs with incident type 2 diabetes. P<5.8x10^-5 (corrected for 857 metabolites) was considered as statistically significant and P<0.05 as nominally significant.

RESULTS

Association of AAs with Insulin Sensitivity and Insulin Secretion

In our cross-sectional study all AAs, except for arginine, histidine and threonine, were significantly (P<5.8x10^-5) associated with insulin sensitivity (Matsuda ISI) (Table 1). Glycine, serine, glutamine and asparagine were associated with improved insulin sensitivity, whereas all other AAs were associated with decreased insulin sensitivity. Glutamate, tyrosine, isoleucine, and alanine had the largest effect on the reduction of insulin sensitivity. In a prospective study the effect sizes of AAs on Matsuda ISI were substantially smaller than those in the baseline study. Only six of 20 AAs showed significant changes in Matsuda ISI during

the follow-up. Glutamate, tyrosine, leucine, aspartate, and phenylalanine were associated with a decrease in Matsuda ISI, and glycine with an increase in Matsuda ISI.

In a cross-sectional study, fourteen AAs were significantly associated with a decrease in insulin secretion (DI), the most significant reductions in the DI were observed for glutamate, tyrosine, alanine, and isoleucine (Table 2). Glycine, glutamine, serine and asparagine were associated with a significant increase in the DI. Nine of the fourteen AAs that were significantly associated with the DI in the cross-sectional study, remained significant also in the prospective study. The largest effects on the DI were observed for leucine, isoleucine, tyrosine and glutamate.

In the cross-sectional study the effect sizes (beta) of the AAs were significantly larger for insulin resistance (except for glycine and serine which increased insulin sensitivity) than for reduction in insulin secretion (S2 Table, Fig S2). By contrast, in the prospective study effect sizes for the reduction of insulin secretion were numerically larger compared to effect sizes for insulin resistance, and showed a statistically significant difference for tryptophan, alanine, isoleucine, leucine and valine among the nine AAs which were associated with reduced insulin secretion during the follow-up.

Association of AAs with Fasting and 2-hour Glucose Levels and Incident Type 2 Diabetes in a Prospective Study

All AAs which were significantly associated with reduced insulin secretion (phenylalanine, tryptophan, tyrosine, alanine, leucine, isoleucine, valine, aspartate and glutamate) significantly increased fasting or 2-hour glucose levels. All these AAs were also associated with higher risk of incident type 2 diabetes (N=522), whereas glycine and glutamine were associated with a lower risk of type 2 diabetes (Table 3). In the follow-up study tryptophan, alanine, isoleucine and valine were associated with reduced insulin secretion and increased fasting and 2-hour glucose levels but not with impairment in insulin sensitivity.

DISCUSSION

We investigated the association of all twenty AAs with insulin secretion, insulin sensitivity, and fasting and 2-hour glucose levels in a large randomly selected population-based METSIM cohort. Our 4.6-year follow-up study reports novel findings: 1) several AAs were associated with both impaired insulin secretion and impaired insulin sensitivity, 2) only the AAs that were associated with impaired insulin secretion prospectively were associated with an increase in glucose levels.

The novel finding in our METSIM 4.6-year follow-up study was that nine AAs (phenylalanine, tryptophan, tyrosine, alanine, isoleucine, leucine, valine, aspartate and glutamate) were significantly associated with reduced insulin secretion, an important contributor in the conversion to diabetes. Among these nine AAs, five (phenylalanine, tyrosine, alanine, aspartate and glutamate) were significantly associated with decreases in both insulin secretion and insulin sensitivity and five with incident type 2 diabetes (tyrosine, alanine, isoleucine, aspartate and glutamate).

In our study 17 of 20 AAs were associated with insulin resistance in cross-sectional analyses in agreement with the results of earlier studies, but only six of them were associated with insulin resistance in our follow-up study. Our results agree with previous findings showing significant associations of branched-chain AAs (BCAAs) isoleucine, valine and leucine with insulin resistance (10-13). However, in our study BCAAs were also associated with reduced insulin secretion suggesting that elevated levels of BCAAs may over time result in a decrease in insulin secretion. Impairment in BCAA catabolism has been suggested to result in the accumulation of potentially toxic intermediates that contribute to β-cell mitochondrial dysfunction, and eventually to the apoptosis of β-cells (14).

We showed that phenylalanine and tyrosine were significantly associated with decreased insulin secretion and elevated levels of fasting and 2-hour glucose in our follow-up study. We

also confirmed the results of previous studies which have shown that phenylalanine and tyrosine are significantly associated with insulin resistance and diabetes (15,16).

Tryptophan was associated with impaired insulin secretion in our prospective study.

Tryptophan is an essential AA metabolized predominantly (about 95%) by the kynurenine pathway. Kynurenines are involved in inflammation, immune response, and excitatory neurotransmission (17). Previous studies have suggested that several metabolites of the kynurenine pathway are diabetogenic in humans (18). Tryptophan metabolites inhibit both proinsulin synthesis and glucose- and leucine-induced insulin release from rat pancreatic islets (19).

Glutamate and aspartate were significantly associated with decreased insulin secretion and increased insulin resistance in our follow-up study. A previous study has reported increased levels of glutamate in insulin resistance (20), but previous studies have not investigated the association of glutamate with insulin secretion in a prospective setting. In transgenic mice with β-cell specific glutamate dehydrogenase deletion glucose-stimulated insulin secretion was reduced by 37% demonstrating the essential role of glutamate in the regulation of insulin secretion (21). The association of aspartate with increased risk of type 2 diabetes is a novel finding.

Alanine, one of the most abundant AAs in the circulation (22), was associated in our follow-up study with impaired insulin secretion and type 2 diabetes, but not with insulin resistance. Glucagon is an important regulator of amino acid metabolism, and hyperglucagonemia is associated with increased levels of amino acids, including alanine, aspartate and glutamate (23). Unfortunately, we did not measure glucagon levels in our study.

Glycine was significantly associated with increases in insulin sensitivity and insulin secretion as previously published (24). Glycine stimulates insulin secretion by acting on glycine receptors, and N-methyl-D-aspartate receptors on β-cells (24). Glycine also decreased

fasting glucose nominally and 2-hour glucose significantly, as previously reported (25), in agreement with its beneficial effects on insulin secretion and insulin sensitivity.

We summarize our results nine AAs (isoleucine, leucine, valine, phenylalanine, tyrosine, tryptophan, alanine, aspartate and glutamate) on insulin sensitivity and insulin secretion in Fig 1. The early effect of AAs on glucose metabolism seems to be an increase in insulin resistance given the fact that the effect sizes of AAs (negative beta) on insulin resistance were considerably larger than those on insulin secretion in a cross-sectional analysis. However, in the follow-up study the effects sizes of AAs (negative beta) were consistently larger on reduction of insulin secretion than on insulin sensitivity. This suggest that AAs may have adverse effects on insulin secretion, and consecutively on the risk of hyperglycemia and type 2 diabetes. Our study is, however, a prospective population-based study, and cannot prove causality.

The strengths of this study are a large and homogeneous study population, and the validation of the methods used as surrogate markers for insulin sensitivity and secretion. We used a very conservative threshold for statistical significance to increase credibility of our conclusions. The limitation of the study is that only middle-aged and elderly Finnish men were included in the study, and therefore we do not know if the results are valid for women, all age groups and other ethnic and racial groups. We are not aware of any unselected population sample where insulin and glucose measurements at 0, 30 and 120 min in an OGTT and metabolomics including all 20 AAs had been performed. Therefore, we could not replicate our findings in other populations.

In conclusion, we demonstrate that nine AAs were significantly associated with reduced insulin secretion and elevated glucose levels in a prospective population-based study of 5,181 Finnish men. Further studies are, however, needed to investigate the role of insulin secretion in diabetogenic effects of AAs.

ACKNOWLEDGEMENTS

The research leading to these results has received support from the Innovative Medicines Initiative Joint Undertaking under EMIF grant agreement no. 115372 (M.L. and U.S.). The METSIM study was supported by grants from Academy of Finland (321428), Sigrid Juselius Foundation, Finnish Foundation for Cardiovascular Research, Kuopio University Hospital, and Centre of Excellence of Cardiovascular and Metabolic Diseases supported by the Academy of Finland (M.L.).

No potential conflicts of interest relevant to this article were reported.

J.V. conceived the study, performed metabolomics and genetic data analyses, wrote and revised the manuscript. A.S. performed metabolomics data analyses and revised the manuscript. U.S., and J.K. contributed to the discussion and revised the manuscript. M.L. conceived the study, wrote, reviewed the manuscript, supervised the entire study, and is a guarantor of this work.

REFERENCES

1. Laakso M, Kuusisto J. Insulin resistance and hyperglycaemia in cardiovascular disease development. Nat. Rev. Endocrinol. 2014;10:293-302

2. Newgard CB. Metabolomics and metabolic diseases: where do we stand? Cell Metab.

2017;25:43-56

3. Stancáková A, Civelek M, Saleem NK, et al. Hyperglycemia and a common variant of GCKR are associated with the levels of eight amino acids in 9,369 Finnish men.

Diabetes 2012;61:1895-1902

4. Lotta LA, Scott RA, Sharp SJ, et al. Genetic predisposition to an impaired metabolism of the branched-chain amino acids and risk of type 2 diabetes: a Mendelian randomisation analysis. PLoS Med. 2016;29:e1002179

5. Guasch-Ferré M, Hruby A, Toledo E, et al. Metabolomics in prediabetes and diabetes:

a systematic review and meta-analysis. Diabetes Care 2016;39:833-846

6. Laakso M, Kuusisto J, Stančáková A, et al. The Metabolic Syndrome in Men study: a resource for studies of metabolic and cardiovascular diseases. J. Lipid Res.

2017;58:481-493

7. Stancáková A, Javorský M, Kuulasmaa T, Haffner SM, Kuusisto J, Laakso M. Changes in insulin sensitivity and insulin release in relation to glycemia and glucose tolerance in 6,414 Finnish men. Diabetes. 2009;58:1212-1221

8. Genuth S, Alberti KG, Bennett P, et al. Follow-up report on the diagnosis of diabetes mellitus. Diabetes Care. 2003;26:3160-3167

9. Matsuda M, DeFronzo RA. Insulin sensitivity indices obtained from oral glucose tolerance testing:Comparison with the euglycemic insulin clamp. Diabetes Care.

1999;22:1462-1470

10. Gannon MC, Nuttall FQ. Amino acid ingestion and glucose metabolism – a review.

IUBMB Life. 2010;62:660-668

11. Newgard CB, An J, Bain JR, et al. A branched-chain amino acid-related metabolic signature that differentiates obese and lean humans and contributes to insulin resistance.

Cell Metab. 2009;9:311-326

12. Würtz P, Tiainen M, Mäkinen VP, et al. Circulating metabolite predictors of glycemia in middle-aged men and women. Diabetes Care. 2012;35:1749-1756

13. Lee CC, Watkins SM, Lorenzo C, et al. Branched-Chain Amino Acids and Insulin Metabolism: The Insulin Resistance Atherosclerosis Study (IRAS). Diabetes Care.

2016;39:582-588

14. Lynch CJ, Adams SH. Branched-chain amino acids in metabolic signalling and insulin resistance. Nat. Rev. Endocrinol. 2014;10:723-736

15. Würtz P, Soininen P, Kangas AJ, et al. Branched-chain and aromatic amino acids are predictors of insulin resistance in young adults. Diabetes Care. 2013;36:648-655 16. Palmer ND, Stevens RD, Antinozzi PA, et al. Metabolomic profile associated with

insulin resistance and conversion to diabetes in the Insulin Resistance Atherosclerosis Study. J. Clin. Endocrinol. Metab. 2015;100:E463-E468

17. Cervenka I, Agudelo LZ, Ruas JL. Kynurenines: Tryptophan's metabolites in exercise, inflammation, and mental health. Science 2017;357:6349

18. Oxenkrug G. Insulin resistance and dysregulation of tryptophan-kynurenine and kynurenine-nicotinamide adenine dinucleotide metabolic pathways. Mol. Neurobiol.

19. Rogers KS, Evangelista SJ. 3-Hydroxykynurenine, 3-hydroxyanthranilic acid, and o- aminophenol inhibit leucine-stimulated insulin release from rat pancreatic islets. Proc.

Soc. Exp. Biol. Med. 1985;178:275-278

20. Davalli AM, Perego C, Folli FB. The potential role of glutamate in the current diabetes epidemic. Acta Diabetol. 2012;49:167-183

21. Carobbio S, Frigerio F, Rubi B, et al. Deletion of glutamate dehydrogenase in beta-cells abolishes part of the insulin secretory response not required for glucose homeostasis. J.

Biol. Chem. 2009;284:921-929

22. Salvucci M, Neufeld Z, Newsholme P. Mathematical model of metabolism and electrophysiology of amino acid and glucose stimulated insulin secretion: in vitro validation using a β-cell line. PLoS ONE. 2013;8:e52611

23. Wewer Albrechtsen NJ, Junker AE, Christensen M, et al. Hyperglucagonemia correlates with plasma levels of non-branched-chain amino acids in patients with liver disease independent of type 2 diabetes. Am J Physiol Gastrointest Liver Physiol.

2018;314:G91-G96

24. Floegel A, Stefan N, Yu Z, et al. Identification of serum metabolites associated with risk of type 2 diabetes using a targeted metabolomic approach. Diabetes. 2013;62:639- 648

25. Wang-Sattler R, Yu Z, Herder C, et al. Novel biomarkers for pre-diabetes identified by metabolomics. Mol. Syst. Biol. 2012;8:615

TABLE 1. Associations of amino-acids with Matsuda ISI in the cross-sectional and 4.6-year prospective studies of the METSIM cohort

Cross-sectional Prospective

Amino-acid

n Beta SE P n Beta SE P

Aromatic

phenylalanine 5169 -0.282 0.013 5.0E-95 4781 -0.049 0.010 9.4E-07 tryptophan 5169 -0.208 0.014 1.4E-51 4781 -0.024 0.010 0.013 tyrosine 5169 -0.374 0.013 6.2E-171 4781 -0.055 0.010 8.8E-08 Non-polar, aliphatic

alanine 5169 -0.341 0.013 1.7E-140 4781 0.020 0.010 0.046 glycine 5169 0.255 0.013 1.3E-77 4781 0.043 0.010 1.1E-05 isoleucine 5169 -0.365 0.013 2.5E-162 4781 -0.039 0.010 1.5E-04 leucine 5169 -0.289 0.013 5.5E-100 4781 -0.052 0.010 2.1E-07 proline 5169 -0.255 0.013 3.3E-77 4781 0.013 0.010 0.193 valine 5169 -0.313 0.013 1.8E-117 4781 -0.034 0.010 7.3E-04 Negatively charged

aspartate 5169 -0.307 0.013 2.5E-113 4781 -0.050 0.010 8.3E-07 glutamate 5169 -0.384 0.013 3.4E-181 4781 -0.060 0.010 5.9E-09 Positively charged

arginine 5169 -0.056 0.014 6.2E-05 4781 -0.003 0.010 0.765 histidine 5169 0.031 0.014 0.025 4781 0.009 0.010 0.330 lysine 5169 -0.119 0.014 8.1E-18 4781 -0.032 0.010 8.5E-04 Polar, non-charged

asparagine 5169 0.099 0.014 1.0E-12 4781 0.013 0.010 0.179 cysteine 5169 -0.143 0.014 4.6E-25 4781 0.005 0.010 0.598 glutamine 5169 0.132 0.015 1.6E-21 4781 0.004 0.010 0.643 methionine 5169 -0.193 0.014 1.8E-44 4781 -0.016 0.010 0.110 serine 5169 0.148 0.014 1.4E-26 4781 -0.003 0.010 0.744 threonine 5169 -0.026 0.014 0.057 4781 0.004 0.010 0.642

Linear regression analyses was applied to obtain standardized beta, SE and p values. In cross- sectional statistical analysis, adjustment was done for the batch effect, and in the prospective analysis, for the batch effect, follow-up time and the baseline level of Matsuda ISI. P<5.8x10^-

5 was considered as statistically significant (bold and underlining), and P<0.05 nominally significant (bold) given the 857 metabolites included in analyses. Participants with diabetes at baseline were excluded from cross-sectional analysis, additionally participants diagnosed with diabetes during the follow-up were excluded from the prospective analysis.

TABLE 2. Associations of amino-acids with the Disposition index in the cross-sectional and 4.6-year prospective studies of the METSIM cohort

Cross-sectional Prospective

Amino-acid

n Beta SE P n Beta SE P

Aromatic

phenylalanine 5169 -0.104 0.014 6.9E-14 4781 -0.064 0.011 1.9E-08 tryptophan 5169 -0.052 0.014 1.7E-04 4781 -0.053 0.011 3.3E-06 tyrosine 5169 -0.157 0.014 5.9E-30 4781 -0.072 0.011 2.7E-10 Non-polar, aliphatic

alanine 5169 -0.132 0.014 1.4E-21 4781 -0.055 0.011 1.5E-06

glycine 5169 0.163 0.014 2.6E-32 4781 0.038 0.011 0.001

isoleucine 5169 -0.123 0.014 5.6E-19 4781 -0.077 0.011 1.1E-11

leucine 5169 -0.107 0.014 8.9E-15 4781 -0.081 0.011 9.6E-13

proline 5169 -0.057 0.014 3.8E-05 4781 -0.038 0.011 7.3E-04

valine 5169 -0.124 0.014 4.0E-19 4781 -0.066 0.011 6.2E-09

Negatively charged

aspartate 5169 -0.117 0.014 3.0E-17 4781 -0.054 0.011 1.9E-06 glutamate 5169 -0.182 0.014 7.7E-40 4781 -0.072 0.011 3.2E-10 Positively charged

arginine 5169 0.022 0.014 0.121 4781 -0.026 0.011 0.023

histidine 5169 0.026 0.014 0.057 4781 0.003 0.011 0.824

lysine 5169 -0.044 0.014 0.001 4781 -0.045 0.011 8.0E-05

Polar, non-charged

asparagine 5169 0.065 0.014 2.4E-06 4781 0.005 0.011 0.676

cysteine 5169 -0.066 0.014 1.9E-06 4781 -0.024 0.011 0.036

glutamine 5169 0.077 0.014 2.8E-08 4781 0.007 0.012 0.542

methionine 5169 -0.042 0.014 0.003 4781 -0.041 0.011 2.7E-04

serine 5169 0.072 0.014 2.0E-07 4781 0.025 0.011 0.028

threonine 5169 -0.007 0.014 0.622 4781 -0.011 0.011 0.345

Linear regression analyses was applied to obtain standardized beta, SE and p values. In cross- sectional statistical analysis, adjustment was done for the batch effect, and in the prospective analysis, for the batch effect, follow-up time and the baseline level of the Disposition index.

P<5.8x10^-5 was considered as statistically significant (bold and underlining), and P<0.05 nominally significant (bold) given the 857 metabolites included in analyses. Participants with diabetes at baseline were excluded from cross-sectional analysis, additionally participants diagnosed with diabetes during the follow-up were excluded from the prospective analysis.

TABLE 3. Associations of amino acids with changes in glycemia and incident type 2 diabetes during the 4.6-year follow-up of the METSIM cohort

Glycemia (N=4801)

Fasting glucose 2 hr glucose Incident type 2 diabetes (N=522) Amino acid

Beta SE P Beta SE P HR 95% CI P P*

Aromatic

phenylalanine 0.065 0.012 2.6E-07 0.062 0.012 3.5E-07 1.27 1.17 - 1.37 3.9E-09 6.0E-04 tryptophan 0.052 0.012 4.6E-05 0.051 0.012 2.7E-05 1.14 1.04 - 1.24 0.003 0.076 tyrosine 0.074 0.012 5.5E-09 0.085 0.012 4.1E-12 1.35 1.24 - 1.46 2.1E-12 2.6E-05 Non-polar, aliphatic

alanine 0.064 0.012 6.2E-07 0.039 0.012 0.001 1.27 1.17 - 1.39 2.0E-08 3.0E-05 glycine -0.038 0.012 0.003 -0.069 0.012 2.4E-08 0.83 0.76 - 0.90 6.0E-06 0.019 isoleucine 0.09 0.012 1.3E-12 0.068 0.012 3.2E-08 1.32 1.21 - 1.44 2.6E-10 1.5E-05 leucine 0.11 0.012 2.0E-18 0.061 0.012 5.3E-07 1.26 1.15 - 1.37 1.9E-07 7.4E-04 proline 0.03 0.012 0.018 0.037 0.012 0.002 1.15 1.06 - 1.25 0.001 0.012 valine 0.09 0.012 9.8E-13 0.057 0.012 3.6E-06 1.27 1.17 - 1.39 2.1E-08 6.4E-04 Negatively charged

aspartate 0.051 0.012 6.7E-05 0.053 0.012 1.2E-05 1.36 1.25 - 1.48 2.5E-12 5.0E-06 glutamate 0.082 0.012 1.3E-10 0.076 0.012 5.4E-10 1.54 1.41 - 1.68 4.1E-22 4.6E-11 Positively charged

arginine 0.041 0.012 0.001 0.013 0.012 0.275 1.15 1.05 - 1.25 0.002 4.7E-04 histidine 0.011 0.012 0.367 -0.022 0.012 0.072 0.85 0.78 - 0.92 0.0001 0.017 lysine 0.079 0.012 3.8E-10 0.035 0.012 0.004 1.11 1.02 - 1.21 0.020 0.090 Polar, non-charged

asparagine -0.007 0.012 0.582 -0.029 0.012 0.017 0.91 0.84 - 0.99 0.033 0.726 cysteine 0.045 0.012 4.3E-04 0.022 0.012 0.07 1.12 1.03 - 1.22 0.006 0.028 glutamine -0.008 0.013 0.553 -0.006 0.013 0.614 0.78 0.72 - 0.85 8.3E-09 6.4E-05 methionine 0.038 0.012 0.002 0.035 0.012 0.004 1.11 1.02 - 1.22 0.017 0.055 serine -0.015 0.012 0.242 -0.038 0.012 0.002 0.94 0.87 - 1.03 0.175 0.575 threonine 0.021 0.012 0.100 0.016 0.012 0.183 1.03 0.95 - 1.12 0.493 0.636

Linear regression analysis was applied to obtain standardized beta, SE and P values.

Adjustment was done for the batch effect, follow-up time and the baseline levels of fasting or 2-hour glucose. P<5.8x10^-5 was considered as statistically significant (bold and underlining), and P<0.05 nominally significant (bold) given the 857 metabolites included in analyses.

Participants with diabetes at baseline were excluded from cross-sectional statistical analyses, and participants diagnosed with incident diabetes during the follow-up from prospective statistical analyses of fasting and 2 hr glucose. Cox regression was used to analyze incident diabetes. P was adjusted for batch only and P* was additionally adjusted for age, BMI, smoking and physical activity at baseline.

FIGURE LEGENDS

Figure 1:

Amino acids significantly associated with increased insulin resistance and reduced insulin secretion during a 4.6-year follow-up of the METSIM cohort. Amino acids significantly associated with type 2 diabetes after the adjustment for confounding factors are shown by red font.

Amino acids significantly associated with increased insulin resistance and reduced insulin secretion during a 4.6-year follow-up of the METSIM cohort. Amino acids significantly associated with type 2 diabetes after the

adjustment for confounding factors are shown by red font.

Ala, alanine; Asp, aspartate; Glu, glutamate; Ile, isoleucine; Leu, leucine; Phe, phenylalanine; Trp, tryptophan; Tyr, tyrosine; Val, valine

338x190mm (96 x 96 DPI)

Supplemental Figures and Tables

Nine amino acids are associated with decreased insulin secretion and elevated glucose levels in a 4.6-year follow-up study of 5,181 Finnish Men

Jagadish Vangipurapu, Alena Stancáková, Ulf Smith, Johanna Kuusisto, Markku Laakso

Supplementary Figure 1. Inter-correlation plot of twenty amino acids in the METSIM study (N=5181)

Supplementary Figure 2. Scatter plots showing associations of twenty amino acids with the Disposition index and Matsuda ISI in the cross-sectional and follow-up studies of the METSIM cohort

Beta-values based on Tables 1 and 2 were plotted for baseline and follow-up studies. Amino-acids marked in bold were significantly associated with Matsuda ISI or DI or both.

Supplementary Table 1. Clinical and laboratory characteristics of 5,181 METSIM participants without diabetes at the baseline and follow-up visits

Baseline Follow-up

Variable

N Mean ± SD N Mean ± SD

Age (years) 5181 57.3 ± 6.9 4851 62.2 ± 6.9

Body mass index (kg/m²) 5180 26.5 ± 3.5 4851 26.7 ± 3.6

Waist (cm) 5180 96.4 ± 9.8 4851 97.5 ± 10.2

Fasting plasma insulin (mU/l) 5181 7.5 ± 5.1 4850 9.1 ± 6.6

2-hour insulin (mU/l) 5179 47.3 ± 45.9 4808 58.6 ± 60.4

Fasting plasma glucose (mmol/l) 5181 5.6 ± 0.4 4851 5.7 ± 0.5

2-hour plasma glucose (mmol/l) 5181 5.9 ± 1.6 4808 6.0 ± 1.9

Glucose area under the curve (OGTT) (mmol/l *

min) 5178 866.1 ± 127.7 4798 882.2 ± 147.6

InsAUC0-30/GluAUC0-30 (pmol/mmol) 5171 30.4 ± 20.5 4798 34.5 ± 23.7

Matsuda ISI 5169 7.4 ± 4.2 4798 6.6 ± 4.4

Disposition index 5169 173.1 ± 72.9 4798 167.5 ± 75.4

Current smokers (%) 5181 15.1 4851 14.1

Low physical activity (%) 5181 32.2 4851 32.0

Supplementary Table 2. Testing of the differences of beta-coefficients for Matsuda ISI and Disposition index by Z-test in the cross-sectional and prospective studies

Cross-sectional study Prospective study

Amino-acid Matsuda ISI

Disposition

index Z-test Matsuda ISI

Disposition

index Z-test

Beta SE Beta SE P Beta SE Beta SE P

Aromatic

Phenylalanine -0.282 0.013 -0.104 0.014 6.0E-21 -0.049 0.010 -0.064 0.011 0.156 Tryptophan -0.208 0.014 -0.052 0.014 1.6E-15 -0.024 0.010 -0.053 0.011 0.026 Tyrosine -0.374 0.013 -0.157 0.014 3.4E-30 -0.055 0.010 -0.072 0.011 0.126 Non-polar, aliphatic

Alanine -0.341 0.013 -0.132 0.014 3.7E-28 0.020 0.010 -0.055 0.011 2.3E-07

Glycine 0.255 0.013 0.163 0.014 7.3E-07 0.043 0.010 0.038 0.011 0.368

Isoleucine -0.365 0.013 -0.123 0.014 4.5E-37 -0.039 0.010 -0.077 0.011 0.005 Leucine -0.289 0.013 -0.107 0.014 8.1E-22 -0.052 0.010 -0.081 0.011 0.026 Proline -0.255 0.013 -0.057 0.014 1.8E-25 0.013 0.010 -0.038 0.011 3.0E-04 Valine -0.313 0.013 -0.124 0.014 2.2E-23 -0.034 0.010 -0.066 0.011 0.016 Negatively charged

Aspartate -0.307 0.013 -0.117 0.014 1.3E-23 -0.050 0.010 -0.054 0.011 0.394 Glutamate -0.384 0.013 -0.182 0.014 2.0E-26 -0.060 0.010 -0.072 0.011 0.210 Positively charged

Arginine -0.056 0.014 0.022 0.014 4.1E-05 -0.003 0.010 -0.026 0.011 0.061

Histidine 0.031 0.014 0.026 0.014 0.400 0.009 0.010 0.003 0.011 0.343

Lysine -0.119 0.014 -0.044 0.014 7.6E-05 -0.032 0.010 -0.045 0.011 0.191 Polar, non-charged

Asparagine 0.099 0.014 0.065 0.014 0.043 0.013 0.010 0.005 0.011 0.295 Cysteine -0.143 0.014 -0.066 0.014 5.0E-05 0.005 0.010 -0.024 0.011 0.026

Glutamine 0.132 0.015 0.077 0.014 0.004 0.004 0.010 0.007 0.012 0.424

Methionine -0.193 0.014 -0.042 0.014 1.2E-14 -0.016 0.010 -0.041 0.011 0.046

Serine 0.148 0.014 0.072 0.014 6.2E-05 -0.003 0.010 0.025 0.011 0.030

Threonine -0.026 0.014 -0.007 0.014 0.169 0.004 0.010 -0.011 0.011 0.156