Of 1,515 European and 1,419 South Asian male study participants from the Southall study centres, 1,423 (94%) and 1,111 (78%), respectively, did not have diabetes at baseline and were included in this study. Serum samples were available for 1,279 European men and 1,007 South Asian men, of whom 801 (63%) Europeans and 643 (64%) South Asians had known follow-up information for diabetes status (electronic supplementary material [ESM] Fig. 1).
Baseline cross-sectional analyses (Southall centre: 1988–1990)
South Asian men were more insulin resistant and more had diagnosed hypertension compared with European men. They were more centrally obese but had lower a BMI. They were less physically active, consumed less alcohol and smoked less. They also reported a lower consumption of meat, fish and dairy products (Table 1). The serum concentrations of isoleucine, phenylalanine, tyrosine and alanine were significantly higher in South Asian men (Table 2). The findings were almost identical in those with and without information on diabetes status at follow-up. No participants were receiving lipid-lowering medications at baseline. Among the South Asian men, the concentrations of branched chain AAs and histidine were higher in Muslims than in Sikhs or Hindus, while the glutamine and glycine concentrations were higher in the Hindu than the Sikh or Muslim participants; however, the ethnic differences in AA levels compared with Europeans were maintained regardless of the South Asian subgroup.
Positive and significant correlations with markers of glycaemia and insulin resistance were observed for isoleucine, leucine, valine, phenylalanine, tyrosine and alanine in both ethnic groups. The correlations were non-significantly weaker between AAs and glycaemia and insulin resistance in South Asian men. Histidine was weakly positively correlated with measures of glycaemia and insulin resistance, and glycine and glutamine were negatively correlated in both ethnic groups. Most AAs were less strongly correlated with obesity measures in South Asian men (significantly so for branched chain AAs and alanine) (Table 3).
Prospective analyses: AAs and incident diabetes (1988–2011)
The median duration of follow-up was 19 years. Diabetes developed in 227 South Asian men (35%) and 113 European men (14%). The median (interquartile range) number of years from baseline to the development of diabetes was 15 (11, 18) in Europeans and 14 (9, 18) in South Asians (p = 0.075), and the median (interquartile range) age at diagnosis of diabetes was 67 (60, 71) years in the European participants and 62 (57, 68) years in the South Asian participants (p < 0.0002).
In logistic regression analyses adjusted only for age, all AAs and AA combinations were associated with incident diabetes in both ethnic groups (Table 4, model 1), with the exception of phenylalanine, glutamine and histidine in European men and glycine in South Asian men. Similar results were obtained when the AAs were analysed in quartiles (see ESM Table 1). Multivariable analyses for the European men showed that, with the exception of glycine and isoleucine, all linear associations (per SD log-transformed AA level) were markedly attenuated following adjustments for obesity and further attenuated on adjustment for insulin resistance and other conventional risk factors (Table 4, models 2–5). Positive associations between incident diabetes and individual AAs and the three- and five-AA combinations were generally more marked in South Asian individuals and only partially attenuated on adjustment for obesity measures and fasting glucose levels (Table 4, models 2 and 5).
Additional adjustment for Matsuda-IR, smoking and HDL-cholesterol level and then alcohol consumption resulted in further attenuation for branched chain AAs, phenylalanine, alanine, glutamine and histidine (models 3 and 4). However, the association for tyrosine was particularly marked among the South Asian participants even after full adjustment (fully adjusted OR for a 1 SD increment in the log-transformed level: 1.47 [1.17, 1.85] vs Europeans: 1.10 [0.87, 1.39]; ethnicity × tyrosine interaction p = 0.045 (fully adjusted)). Similarly, the three- and five-AA combinations of isoleucine, leucine, valine, phenylalanine and tyrosine were also more strongly associated with incident diabetes in South Asian participants (fully adjusted ORs [model 4]: South Asians: 1.32 [1.04, 1.68] and 1.31[1.04, 1.66] vs European participants: 1.03 [0.82, 1.29] and 1.05 [0.84, 1.30]), with weak evidence of ethnicity interactions (p = 0.10 and 0.13, respectively) (Table 4, model 4). Glycine was negatively associated with incident diabetes in European but not in South Asian men (interaction p = 0.06).
Comparisons with the Framingham Offspring Study are shown following adjustment for age, fasting glucose level and BMI (Table 4, model 5). In European men, the model 5 adjusted ORs per 1 SD increment in the log-transformed three-AA (isoleucine, phenylalanine and tyrosine) and five-AA (isoleucine, leucine, valine, phenylalanine and tyrosine) combinations were virtually identical and weak: 1.10 (0.89, 1.36) and 1.10 (0.89, 1.35). In contrast, the ORs were greater in South Asian men (1.57 [1.26, 1.96] and 1.52 [1.22, 1.89], respectively; Table 4, model 5). In South Asian men, the ORs for the upper vs lowest quartile were 3.11 (1.71, 5.67) and 2.55 (1.46, 4.48) for the three- and five-AA combinations, respectively, with evidence of linear associations. The corresponding categorical associations for Europeans were weaker (ORs for the upper vs lower quartile 1.99 [1.07, 3.67] and 1.98 [1.05, 3.76], respectively) (ESM Table 1, model 5).
In aiming to predict incident diabetes, no improvements in the C statistic, NRI or IDI were observed in European men on the addition of tyrosine or either the three- or five-AA combination to models containing prespecified conventional risk factors. However, in South Asian men, the IDI and NRI significantly improved with the addition of tyrosine, although only discrimination (IDI) significantly improved with the addition of the three- and five-AA combinations (ESM Table 2).
Prospective analyses: AAs and ethnic differences in incidence of diabetes
In age-adjusted models, South Asian men had a 3.18-fold (95% CI 2.46, 4.12, p = 1.5 × 10−18) greater risk of developing diabetes than European men (Fig. 1). Additional adjustment for baseline tyrosine level reduced the ethnic difference in risk of diabetes to the greatest extent (OR 2.64 [2.02, 3.44]; 17% reduction). Adjustment for the prespecified combination of conventional risk factors reduced the ethnic OR to 2.10 (1.56, 2.82). Further adjustment for AAs modestly altered the ethnic difference in risk of diabetes; the addition of tyrosine to the full model led to the largest reduction in OR, to 1.99 (1.48, 2.69; 5% reduction) (Fig. 1).
Sensitivity analyses that included adjustment for diet, physical activity or serum creatinine, or excluded participants with impaired fasting glucose or impaired glucose tolerance, produced similar results to those reported above. There were no significant age-adjusted interactions between the main South Asian subgroups (Punjabi Sikh, Hindu and Muslim) and AAs in association with incident diabetes (all interaction p values > 0.10).
Prospective analyses using competing risks regression resulted in the loss of 23 cases of incident diabetes without dates of diagnosis, but demonstrated similar ethnic differences in the incidence of diabetes (age-adjusted subhazard ratio [SHR] for South Asian vs European participants 2.90 [2.33, 3.61]), which were again most markedly attenuated on the addition of baseline tyrosine (SHR 2.52 [2.00, 3.20]). Competing risks regression also demonstrated similar associations between AAs and incident diabetes, for example the age-adjusted SHR per SD log-transformed tyrosine in Europeans of 1.31 (1.05, 1.64) and in South Asians of 1.65 (1.44, 1.89).