Background

Gestational diabetes mellitus (GDM), commonly defined as impaired glucose tolerance with onset or first recognition during pregnancy [1]. The prevalence of GDM was increasing globally and approximately 14.0% of live births worldwide were affected by hyperglycemia during pregnancy in 2021 according to an estimate of International Diabetes Federation [2, 3]. Several serious adverse pregnancy outcomes correlated highly with GDM, including macrosomia, hypoglycemia, fetal hypoxia, and fetal malformation for the infants [4,5,6]. Moreover, GDM might also be associated with developing type 2 diabetes, metabolic syndrome, and cardiovascular disease later in life for the pregnancy women [7]. As the age and pre-pregnant body mass index (BMI) increased gradually, the prevalence of GDM aroused great attention around the world, and the adverse effect of GDM became one of the important public health problems. Therefore, earlier identification of GDM for pregnant women became particularly crucial.

Branched chain amino acids (BCAAs), including leucine (Leu), isoleucine (Ile), and valine (Val), played critical roles in the regulation of energy homeostasis, nutrition metabolism, gut health, immunity and disease in humans and animals [8]. Recently, many animal experimentations and population-based studies reported that increased circulating BCAAs level owned the predictive values for type 2 diabetes [9,10,11,12]. There were also a few studies conducted to address the correction between elevated circulating BCAAs levels and increased risk of GDM, but the relevant conclusions were contradictory. For example, a case–control study found that circulating concentrations of BCAAs were independently and positively associated with GDM through modifying insulin resistance and secretion among the Korean population [13]. However, a nested case–control study reported that the levels of the BCAAs did not differ significantly between GDM and normal glucose tolerance [14]. And another study also showed that the increase in BCAAs occurred postpartum since the BCAAs did not differ during pregnancy, as compared to normoglycemic women [12].

Mammalian target of rapamycin (mTOR), an atypical serine/threonine kinase, was regulated by cellular nutrient and metabolic status [15]. The mTOR signaling pathway was a key component that regulated many major cellular processes and was implicated in various pathological conditions, including cancer, neurodegeneration, and metabolic diseases [16]. Previous studies suggested that placental protein kinase B (Akt)/mTOR signaling was substantially upregulated in GDM patients [17, 18]. And other studies indicated that paeoniflorin [19], olive oil [20] and 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) [21] could serve as a potential therapeutical strategy for patients with GDM through suppressing mTOR signaling. However, there were few studies to explore the association between circulating mTOR levels and risk of gestational diabetes mellitus (GDM). And our study might be useful for identifying biomarkers and understanding the mechanistic underpinnings of GDM.

Therefore, to investigate the associations between the serum levels of BCAAs and mTOR and risk of GDM, we conducted the 1:1 matched case–control study among 132 pregnant women (including 66 cases and 66 controls).

Methods

Study design and population

A 1:1 age- (± 3 years)-matched case–control study was conducted at the Third Affiliated Hospital of Zhengzhou University. Pregnant women who aged from 18 to 45 years, carried a singleton fetus, and decided to deliver in the study hospital were recruited at their first prenatal visit. The study protocol had been approved by the Ethics Committee of the Third Affiliated Hospital of Zhengzhou University. All the participants have signed the informed consent forms before enrollment. The study had been registered with on the Chinese Clinical Trial Registry (ChiCTR2000028811), and the Registration Date was January 4, 2020.

All participants had been visited from January 2019 to October 2019. Individuals were excluded if they met the following criteria: (a) had the history of smoking and drinking, (b) had pregnant complication or miscarried, (c) had the family history of thyroid disorders, (d) took the medicine influencing hormone secretion and glycol-metabolism, (e) with diabetes mellitus, hypertension, disease of heart, or renal disease in pre-pregnancy. Finally, 66 GDM cases were identified according to the criteria of the International Association of Diabetes and Pregnancy Study Groups (IADPSG) in 2011. For each case, one control was selected randomly from the remaining participants who were free of GDM, and matched by pregnant women’s age (± 3 years). Thus, a total of 66 cases and 66 controls were included in the study.

Measurements

Anthropometric measurement

Information of sociodemographic characteristics (e.g.age, residential area, and education) and other detailed information including parity, history of previous miscarriages, miscarriage times was obtained by a standard questionnaire. Professional trained researchers instructed pregnant women face to face to fill in the questionnaires, and then timely checked and corrected completed questionnaires. The weight and height of participants were measured by the InBody J30 (Biospace, Seoul, South Korea) and a stadiometer in the hospital without shoes and wearing light clothing, respectively. Pre-pregnancy BMI was calculated by weight (kg)/height2 (m2). Blood pressure was measured twice after 5 min of rest using an appropriate cuff according to arm size. The average of two blood pressure measurements was recorded as the final value. And waist-hip ratio was calculated as the waist (m) divided by the hip (m).

Biochemical measurement

Fasting bloods of pregnant women were collected in pregnancy at 24 ~ 28 weeks gestation. Blood samples were collected and serum was obtained by centrifugation at 3000 × rpm for 10 min at 4 °C. Separated serum samples were put into the liquid nitrogen container, and then stored in − 80 °C refrigerator until analysis.

Concentrations of BCAAs in serum were measured using high-performance liquid chromatography (HPLC) by L-8900 High speed amino acid analyzer (Hitachi High-Tech Science Corporation, Japan). Serum fasting insulin (Wuhan Elabcience Company) and mTOR (Wuhan CUSABIO Company) levels were both determined by enzyme-linked immunosorbent assay (ELISA) following the manufacture’s protocol. The levels of triglyceride (TG), total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C) and low-density lipoprotein cholesterol (LDL-C) in serum were determined using the GPO-PAP method by biochemical analyzer (Shanghai Kehua Bioengineering Co, LTD).

Definitions

Plasma glucose of pregnant women was measured during a 75-g oral glucose tolerance test (OGTT) between 24 and 28 weeks gestation as a routine prenatal examination. The diagnosis of GDM was based on the recommended criterion by IADPSG of exceeded glucose values of 5.1, 10.0, and 8.5 mmol/L at fasting, 1 h, and 2 h.

Education level was divided into three categories: low (junior school or below), middle (high school, technical school, or technical secondary school), and high (junior college, college or above). The homeostasis model assessment of insulin resistance (HOMA-IR) was calculated using the following formula: HOMA-IR = fasting plasma insulin (μIU/L) × fasting plasma glucose (FPG) (mmol/L)/22.5.

Statistical analysis

Continuous variables and categorical variables were presented as the mean ± SD and absolute value with percentage, respectively. The comparisons of continuous or categorical characteristics were examined using the t-tests or Chi-square tests.

Conditional logistic regression models were used to calculate odds ratios (ORs) and 95% confidence intervals (95% CIs) for the relationships of BCAAs and mTOR concentrations with the risk of GDM. The levels of BCAAs and mTOR were analyzed as categorical variables based on the tertile distributions among GDM and controls (the lowest tertile was defined as the referent group). Additionally, we conducted trend tests by using the median value of each tertile of BCAAs and mTOR. Covariates were retained in the models if they altered a > 10% change of regression parameter in the unadjusted model or had been associated with BCAAs or mTOR and GDM in previous studies. Finally, three models were fitted as follows: model 1 was an unadjusted analysis; model 2 was adjusted for age and pre-pregnancy BMI; model 3 was further adjusted for levels of HDL, LDL and TG.

Statistical analyses were performed using SPSS software (IBM SPSS 25.0, SPSS Inc), and two-sided P-values < 0.05 were considered statistically significant in all of the tests.

Results

Table 1 presented the baseline characteristics of case and control group. The average age and pre-pregnancy BMI of the 132 participants were 30.7 ± 5.9 years and 22.2 ± 3.2 kg/m2, respectively. Compared with the controls, GDM cases had higher waist-hip ratio (0.88 ± 0.05 vs. 0.85 ± 0.06, P = 0.002). The proportion of pregnant women from urban in GDM group were higher than that in control group (68.2% vs. 40.9%, P = 0.002). No significant differences were observed between the cases and controls in age, pre-pregnancy BMI, parity, educational level, pregnancy times, miscarriage times, history of previous miscarriages, systolic blood pressure (SBP) and diastolic blood pressure (DBP) (all P > 0.05).

Table 1 Baseline characteristics of case and control group
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Table 2 showed the biochemical index of case and control group in the second trimester of pregnancy. In the 75 g oral glucose tolerance test, the mean glucose levels of fasting (4.94 ± 0.55 mmol/L vs. 4.60 ± 0.35 mmol/L), 1 h (9.82 ± 1.95 mmol/L vs. 7.25 ± 1.46 mmol/L), and 2 h (8.56 ± 2.12 mmol/L vs. 6.57 ± 1.39 mmol/L) among cases were significantly higher than that among controls, respectively (all P < 0.001). The average of AUCGlucose (16.20 ± 3.82 mmol/ (L • h) vs. 12.84 ± 1.98 mmol/ (L • h)), TC (5.70 ± 1.04 mmol/L vs. 6.19 ± 1.35 mmol/L), Val (171.18 ± 50.42 μmol/L vs. 154.20 ± 39.40 μmol/L) and mTOR (20.13 ± 6.97 ng/mL vs. 17.84 ± 4.95 ng/mL) in serum concentrations among cases were significantly higher than that among controls, respectively (all P < 0.05). There were no statistically significant differences between two groups on Fasting insulin, HOMA-IR, HDL-C, LDL-C, TG, Ile and Leu (all P > 0.05).

Table 2 The serum index of case and control group in the second trimester of pregnancy
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Figure 1 presented the comparison of BCAAs and mTOR in GDM and healthy pregnant women among different tertiles of oral glucose tolerance. In Fig. 1a, the levels of Val among cases were significantly higher than that among controls in the second tertile of OGTT 1 h PG and third tertile of FPG, OGTT 1hPG and OGTT 2hPG (all P < 0.05). In Fig. 1b, the levels of Ile among cases were significantly higher than that among controls in the second tertile of OGTT 1hPG and the first and the third tertile of OGTT 2hPG (all P < 0.05). In Fig. 1c, the levels of Leu among cases were significantly higher than that among controls in the first tertile of FPG and the third tertile of OGTT 1hPG and OGTT 2hPG (all P < 0.05). In Fig. 1d, the levels of mTOR among cases were significantly higher than that among controls in the first tertile of FPG and OGTT 1hPG, and the second and the third tertile of OGTT 2hPG (all P < 0.05).

Fig. 1
figure 1

Comparison of glucose tolerance in GDM and control among different tertiles of BCAAs and mTOR. Abbreviation: GDM, gestational diabetes mellitus; BCAAs, Branched chain amino acids; mTOR, mammalian target of rapamycin; FPG, fasting plasma glucose; OGTT, oral glucose tolerance test, 1hPG, 1 h plasma glucose; 2hPG, 2 h plasma glucose; Val, valine; Ile, isoleuline; Leu, leucine.*: P < 0.05; **: P < 0.01; ***: P < 0.001

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Multivariate conditional logistic regression analysis ORs for GDM across tertiles of BCAAs and mTOR were reported in Table 3. In crude models, compared to the first tertile of mTOR, the crude ORs (95% CIs) of GDM risk were 11.358 (4.084–31.593) for the second tertile and 3.073 (1.260–7.497) for the third tertile. Compared to the first tertile of Leu, the crude OR (95% CI) of GDM risk were 0.393 (0.166–0.932) for the second tertile. In model 3 adjusted for confounding factors, serum mTOR level was still associated with a higher risk of GDM, and the adjusted ORs and 95% CIs of GDM for the subjects in T2 and T3 compared to the subjects in T1 of serum mTOR level were 11.771 (3.949–35.083) and 4.869 (1.742–13.611), respectively (both P < 0.05). Serum Val and Leu levels were associated with a lower risk of GDM, and the ORs and 95% CIs of GDM for the subjects in T2 compared to the subjects in T1 of serum Val and Leu level were 0.377 (0.149–0.954) and 0.322 (0.129–0.811), respectively (both P < 0.05). However, there was no significant association between the risk of GDM and serum levels of Ile (P = 0.989).

Table 3 Odd ratio for gestational diabetes mellitus in BCAAs and mTOR
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The restricted cubic spline indicated that after adjusted age, pre-pregnancy BMI, LDL-C, HDL-C, TC and TG, there was a significant nonlinear association between the serum levels of mTOR and the risk of GDM (Poverall = 0.001, Pnon-linearity = 0.005). However, no statistically significant non-linear association was found between the serum levels of Val, Ile, Leu and the risk of GDM (all P > 0.05. Figure 2).

Fig. 2
figure 2

The restricted cubic spline for the association between Val, Ile, Leu, mTOR concentration and risk of GDM. Abbreviation: GDM, gestational diabetes mellitus; Val, valine; Ile, isoleucine; Leu, leucine; mTOR, mammalian target of rapamycin; OR were displayed as a blue solid line, and the upper and lower limit of 95% confidence intervals were displayed as a dotted line. Adjustment factors were age, pre-pregnancy BMI, HDL-C, LDL-C, TC, TG

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The correlation heat map showed that there was significantly positive correlation between the serum levels of Val and FPG (r = 0.293, P = 0.001), OGTT 1hPG (r = 0.218, P = 0.212). However, no statistically significant association was found between the serum levels of Ile, Leu, mTOR and the oral glucose tolerance (all P > 0.05. Figure 3 and Supplementary Table 1).

Fig. 3
figure 3

The correlation heat map between BCAAs, mTOR and oral glucose tolerance. Abbreviation: BCAAs, Branched chain amino acids; mTOR, mammalian target of rapamycin; Val, valine; Ile, isoleucine; Leu, leucine; FPG, fasting plasma glucose; OGTT, oral glucose tolerance test, 1hPG, 1 h plasma glucose; 2hPG, 2 h plasma glucose.*: P < 0.05

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Discussion

In this study, we conducted an exploratory analysis on 132 pregnant women including 66 GDM cases and 66 normal controls. The present study found that the serum levels of val and mTOR were higher in GDM women than that in the control group. And the higher concentrations of serum BCAAs and mTOR levels were association with the higher glucose according to the OGTT results. The conditional logistic regression models showed that the higher serum level of mTOR in pregnant women was significantly correlated with increased risk of GDM. However, serum levels of Val and Leu were negatively associated with risk of GDM within a certain range.

The association between circulating BCAAs and the risk of type 2 diabetes (T2DM) was described, previously. A nested case–control study in the Framingham Offspring Study including 189 cases and 189 controls reported that for the branched-chain and aromatic amino acids, each s.d. increment in log marker was associated with a 57–102% increased odds of future diabetes [11]. Another two studies including a nested case–control study and a cohort study found that BCAAs were potential markers to be evaluated as predictors of T2DM after pregnancy complicated by GDM [22, 23]. A cohort study conducted in African-American women showed that amino acids and their derivatives were increased significantly in T2DM subjects [10]. What’s more, an animal experimentation showed that adding BCAAs to a high-fat diet contributed to development of obesity-associated insulin resistance in the context of a dietary pattern that includes high fat consumption in rodents [9].

Several previous studies investigated the association between levels of BCAAs and GDM in pregnant women. A case–control study found the association between plasma amino acids levels and higher risk of GDM in Iran [24]. A nest case–control study (n = 486) found that serum Val, Leu, Ile and total BCAAs in early pregnancy were positively associated with the risk of GDM in Chinese pregnant women [25]. A meta-analysis including eight studies with 432 subjects showed that the increase of individual plasma BCAAs concentration, as potential biomarkers, might be related to the increased risk of GDM [26]. In our present study, univariate analysis showed that BCAAs were the risk factors of GDM. However, the result of conditional logistic regression analysis showed that levels of Val and Leu were negatively associated with risk of GDM in the range of 149.94–176.37 μmol/L and 116.69–144.43 μmol/L, respectively. But the association of Val and Leu with GDM would disappear if the value of Val and Leu was out of the range, which might indicate that the range of 149.94–176.37 μmol/L for Val and 116.69–144.43 μmol/L for Leu was suitable for the pregnant women. And other studies also reported the normal range of Val and Leu, which supported our findings. For example, Li et al. [25] reported that the normal range of Val and Leu is 155.3–200.8 μmol/L and 122.0–155.3 μmol/L in pregnant women, respectively. And Kalliopi et al. [27] reported that the normal range of Val and Leu is 146.65–244.43 μmol/L and 81.44–142.08 μmol/L, respectively. Moreover, null results were also observed in two case–control studies involving the European population and American white person [12, 14]. The null results of the study in European may be due to small sample size (n = 56), especially the size of the GDM group (n = 11), and the sample source from two different countries in European [12]. Furthermore, the study results conducted in American white person was not suitable for Chinese region because of the variation of genetics and ethnic [14].

In the current study, we provided evidence that the level of serum mTOR is closely associated with the risk of GDM. And several published studies might corroborate our findings. Min Shang et al. found macrosomia born from women with GDM were associated with increased placental mTOR activity in Chinese pregnant women [28]. Kary Tsai et al. concluded that regulation of the mTOR pathway was uniquely involved in the development of some obstetric complications including GDM [29]. What’s more, Ke Xu et al. found that MiR-503 regulated functions of pancreatic β-cells by targeting the mTOR pathway, suggesting that targeting miR-503/mTOR axis could serve as a novel therapeutic target for GDM [30]. At the same time, Jie Wen and Xiaoxia Bai confirmed that the expression of mTOR in the GDM cell model was correlated with cell viability and cell apoptosis, including pancreatic islet β cells [31].

Pregnancy entails an increased demand for energy, including amino acids, to enable the fetus and placenta to grow. Thus, normal pregnancy might reduce the BCAAs levels in the circulation, potentially increase protein synthesis aimed at conservation and accretion of nitrogen by the woman and the fetus [32]. Increased levels of BCAAs, or other mitotoxic/lipotoxic metabolites from these amino acids, might increase risk for GDM. Two possible mechanisms might explain the relationship of BCAAs and GDM. Firstly, the higher levels of BCAAs might activate mammalian target of rapamycin complex 1 (mTORC1) through the phosphorylation of insulin receptor substrate 1 (IRS-1), leading to insulin resistance, and then increasing risk for GDM [33]. An experimental study found that BCAAs deprivation for 7 days could improve insulin sensitivity in wild type and insulin-resistant mice models, which is possibly mediated by decreasing mTOR pathway and increasing AMP-activated protein kinase (AMPK) signaling pathways [34]. Another possible mechanism is that the accumulation of precursor amino acids leucine, isoleucine and valine and their metabolites might result in induction of oxidative stress, mitochondrial dysfunction, impaired insulin action, and ultimately to perturbation of glucose homeostasis [35, 36].

The present study had several strengths. Firstly, it was the first study to investigate the association between serum mTOR level and glucose metabolism. What’s more, the cases and controls were matched by pregnant women’s age in order to reduce the potential effects of individual’s susceptibility and confounder. In addition, we not only collected detailed information of all participants on demographic characteristics, lifestyle factors and medical records, but also used the conditional logistic regression models to adjust for potential confounders and verify the stability of results. Several limitations should also be noted. Firstly, the possible causal relationship could not be established owing to the observational design. Moreover, the BCAA levels were influenced by dietary intake, but the information on dietary habits were not collected. Additionally, the small size of the study was another limitation. Lastly, since the participants in our study were enrolled from in Henan province of China, extrapolated results might not be an accurate description of the wider Chinese population.

Conclusions

In summary, our investigation confirmed the positive association between the serum level of mTOR and risk of GDM. And a certain range serum Val and Leu in second trimester of pregnancy were protective for the risk of GDM. These findings provided epidemiological evidence for prevention and control of GDM. However, future prospective researches on the correlation of circulation BCAAs and mTOR in GDM population with larger sample size and longer follow-up are still expected.