Background and Aim: Altered gluconeogenesis from alanine (i.e. glucose-alanine cycle) that increases the levels of serum alanine aminotransferase (ALT) are linked to the development of type II diabetes (T2DM) [1,2]. A recent study also revealed that elevated serum ALT levels in early pregnancy are associated with the risk of subsequent development of gestational diabetes mellitus (GDM) and preeclampsia in late pregnancy [3]. So based on this, we hypothesized that the circulatory alanine and alanine to glucose ratio (AGR) would be altered in GDM and may serve as an indicative biomarker to improve the clinical diagnosis of GDM. Methods: The circulatory levels of alanine and glucose were estimated for 50 GDM patients and 49 age matched healthy female subjects using 800 MHz NMR spectroscopy. The NMR spectra were analyzed using NMR suite of commercial software (CHENOMX). The estimated circulatory levels of alanine and glucose were used to determine the circulatory AGR levels as [Alanine in µM]/[Glucose in mM] and the values are reported as Mean ± SEM (SEM is standard error in mean). Results: The present study aims to compare the serum metabolic profiles of alanine (Ala) and alanine to glucose ratio (AGR) between age and sex matched GDM patients (N=50; pre term =32, post term=18) and healthy normal control (NC) female subjects (N=49). The comparison revealed that the circulatory levels of alanine and AGR are significantly decreased in GDM patients (Ala=397.5 ± 28.12| AGR=94.65 ± 9.06) compared to NC subjects (Ala =515.1 ± 24.03 | AGR=166.3 ± 7.577) (Figure 1A and 1B). The respective p-values for comparison of alanine and AGR found to be 0.002 (**) and <0.0001, ****). The disturbed glucose-alanine metabolic cycle in GDM patients was further corroborated through performing metabolic Pearson r correlation analysis between alanine and glucose (Figure 1C and 1D). For NC subjects, the circulatory metabolites alanine and glucose found to be strongly correlated (r=0.61, 95% CI=0.40 to 0.76; R square =0.37) with two-tailed p-value <0.0001 (see Table depicted in Figure 1E). Whereas for GDM subjects, the circulatory metabolites alanine and glucose found to be moderately correlated (r=0.45, 95% CI=0.19 to 0.64; R square =0.20, Fig. 1E) with two tailed p-value =0.002 suggesting that the glucose-alanine metabolic cycle is altered in GDM. Overall, the observed changes in circulatory metabolites and metabolic ratios clearly suggested that the gluconeogenesis from alanine is disturbed in GDM and this abnormal glucose-alanine metabolism is possibly associated with the development of disease and its progression. References: [1] N. Sattar, O. Scherbakova, I. Ford, D.S. O'Reilly, A. Stanley, E. Forrest, P.W. MacFarlane, C.J. Packard, S.M. Cobbe, J. Shepherd, Elevated alanine aminotransferase predicts new-onset type 2 diabetes independently of classical risk factors, metabolic syndrome, and C-reactive protein in the west of Scotland coronary prevention study, Diabetes 53 (2004) 2855-2860. [2] N.H. Cho, H.C. Jang, S.H. Choi, H.R. Kim, H.K. Lee, J.C. Chan, S. Lim, Abnormal liver function test predicts type 2 diabetes: a community-based prospective study, Diabetes care 30 (2007) 2566-2568. [3] S.M. Lee, J.S. Park, Y.J. Han, W. Kim, S.H. Bang, B.J. Kim, C.W. Park, M.Y. Kim, Elevated alanine aminotransferase in early pregnancy and subsequent development of gestational diabetes and preeclampsia, Journal of Korean Medical Science 35 (2020)

{"references": ["N. Sattar, O. Scherbakova, I. Ford, D.S. O'Reilly, A. Stanley, E. Forrest, P.W. MacFarlane, C.J. Packard, S.M. Cobbe, J. Shepherd, Elevated alanine aminotransferase predicts new-onset type 2 diabetes independently of classical risk factors, metabolic syndrome, and C-reactive protein in the west of Scotland coronary prevention study, Diabetes 53 (2004) 2855-2860.", "N.H. Cho, H.C. Jang, S.H. Choi, H.R. Kim, H.K. Lee, J.C. Chan, S. Lim, Abnormal liver function test predicts type 2 diabetes: a community-based prospective study, Diabetes care 30 (2007) 2566-2568.", "S.M. Lee, J.S. Park, Y.J. Han, W. Kim, S.H. Bang, B.J. Kim, C.W. Park, M.Y. Kim, Elevated alanine aminotransferase in early pregnancy and subsequent development of gestational diabetes and preeclampsia, Journal of Korean Medical Science 35 (2020)"]}

Compared to normal healthy female subjects, the decreased circulatory levels of alanine and alanine-to-glucose ratio (AGR) in gestational diabetes mellitus (GDM) patients suggested that the disturbed glucose-alanine cycle (or gluconeogenesis from alanine) in GDM possibly underlies the development of gestational diabetes and its progression during pregnancy.