Whey protein preloading can alleviate stress adaptation disorder and improve hyperglycemia in women with gestational diabetes mellitus

Yan Feng Qindao University Medical College A liated Yantai Yuhuangding Hospital https://orcid.org/00000002-8884-8668 Qi Feng Chinese PLA General Hospital Xinna Song Qindao University Medical College A liated Yantai Yuhuangding Hospital Xiaoyan Li Qindao University Medical College A liated Yantai Yuhuangding Hospital Yinghong Zhang Qindao University Medical College A liated Yantai Yuhuangding Hospital Jianwei Hu (  yyfy190085@163.com )


Introduction
Gestational diabetes mellitus (GDM) is characterized by abnormal glucose tolerance during pregnancy, and it is a common complication of pregnancy, which canincreases the risk of occurrence of type 2 diabetes mellitus (DM2) in both mothers and their offspring, also cause adverse effects on the outcome of mothers and fetus [1,2]. In recent years, the incidence of global GDM is on the rise. The incidence of GDM in pregnant women in China is as high as 22.94%, while the pathogenesis of GDM is not clear [3].
How to prevent and treat GDM and reduce the risks it brings?
Several studies show that Whey protein preloading can increase insulin sensitivity and have a positive response to the control of hyperglycemia [4,5]. We previously reported that stress adaptation disorder exists in GDM women and may beassociated with the pathogenesis of GDM [6,7]. The purpose of our study is to explore the effect of whey protein supplementation on stress hormones in GDM women, and to observe the effect of whey protein preloading on postprandial blood glucose control in GDM patients, in an attempt to gain insights into the prevention and treatment of GDM.

Materials And Methods
Subjects 60 pregnant women patients with GDM at 24-28 weeks were included in this study.
According to the American Diabetes Association criteria, a 75-g oral glucose tolerance test (OGTT) at 24-28 weeks of pregnancy was used, GDM was diagnosed with the cutoff value being >5.1 mmol/L at fasting, >10.0 mmol/L at 1 h, and >8.5 mmol/L at 2 h [8]. They were diveded into 2 groups consisting of 30 pregnant women each: GDM-W group receive 250ml preload drink containing 20 g whey protein isolate (73Kcal; Agropur inc, Le Sueur MN, America) 30 min before lunch and GDM group receive 250ml preload drink containing 20 g control avoring (6.5 kcal; Cottee's, Southbank, Australia) 30 min before lunch.
Blood samples were collected on days 3, 5, 7 and 14. The experiment lasts for14 days.
Inclusion criteria were 1) single pregnancies; and 2) fasting plasma glucose (FPG) <6.1mmol/L before pregnancy. Exclusion criteria were 1) pregnant women with previously known medical complications during pregnancy such as DM 1 or 2, polycystic ovary syndrome, hyperthyreosis, hypothyroidism; and 2) women who were treated with hormones or drugs that may affect blood glucose.
The study protocol was approved by Yantai yuhuangding hospital committee, and informed consents of all participants were obtained.

Characteristics of GDM women
Clinical features including maternal age, pre-gestational body mass index (BMI) and gestational weeks.
Blood glucose, stress hormones, oxidative stress injury markers and HOMA-IR in GDM women Roche automatic biochemical analyzer (Roche Diagnostics, Mannheim, Germany) was used to detect blood glucose, and electrochemical luminescence immunoassay (Roche Diagnostics, Mannheim, Germany) was used to test fasting and plasma 2h insulin levels. Insulin resistance (IR) was assessed by homeostatic model assessment of insulin resistance (HOMA-IR) formula [9].
Plasma epinephrine (E), noradrenaline (NE) and cortisol were used as markers of stress hormones. E and NE levels were determined by radioimmunoassay (RIA) (Roche Diagnostics, Mannheim, Germany). Electrochemical luminescence immunoassay (Roche Diagnostics, Mannheim, Germany) was used to detecte cortisol levels.
Malondialdehyde (MDA), superoxide dismutase (SOD) and glutathione (GSH) were used as markers of oxidative stress. MDA, SOD and GSH were detected by their correspongding assay kits (Jiancheng Bioengineering Institute, Nanjing, China). Results are expressed as umol/L, U/ml and mg/L.

Statistical analysis
Continuous variables of normal distribution data are presented as mean ± standard error. Independent-Samples t test was used to identify between-group differences. Data not normally distributed were logtransformed before analysis. P < 0.05 was considered statistically signi cant. Statistical analyses were performed with SPSS 19.0 (SPSS, Inc., Chicago, IL, USA).

Maternal characteristics
The maternal characteristics including the maternal age, pre-gestational BMI and gestational weeks were similar in GDM and GDM-W groups at entry to the study, there was no signi cant difference between two groups (p > 0.05)( Table 1).

Effects of whey preload on blood glucose related indexes in GDM women
There was no signi cant difference in fasting blood glucose bewteen two groups during the entire experimental session (p > 0.05) ( Fig. 2A).

Discussion
Our study showed that whey protein perload can reduce hyperglycemia during 14 days exposure in GDM women. Compared to GDM group, there was no signi cant difference in fasting blood glucose bewteen two groups, but postprandial blood glucose was decreased signi cantly during the entire experimental session. The results are consistent with previous studies that used whey protein preload [5,[10][11][12], but the mechanism is different. Some studies suggest that the hyperglycemia-reducing effect of whey protein preload may relate to insulin stimulation and delaying of glucose absorption in intestinal [13][14][15], and we have got similar result, plasma 2 h insulin was increased on days 5, 7 and 14 in GDM-W group, HOMA-IR decreased signi cantly on days 14 in the experimental session. Some studies found that slower gastric emptying may be responsible for hyperglycemia-reducing [12,16,17].
E, NE and cortisol are markers of stress hormones, especially cortisol. Cortisol could increase hepatic glucose production, decrease insulin secretion and aggravate β cell function, all of which could lead to hyperglycemi [18].
Pregnancy is a process of slight and chronic stress. Our previous sutdy showed that GDM women exists stress adaptive disorder, which is related to hyperglycemia and IR [6]. This study found that stress hormones E and cortisol were decreased by 10.8% and 19.8% respectively on days 14 after whey protein perload. Several studies have also found that cortisol was reduced after whey protein supplementation, which is similar to our resul [19,20]. It indicates that whey protein supplementation can reduce the stress adaptation disorder of GDM patients.

Effects of whey preload on oxidative stress injury in GDM women
The concentration of MDA in GDM-W group was increased by 20.7% on days 14, and showing signi cant difference between two groups (P < 0.01) (Fig. 2E).
Anti-oxidative enzymes SOD in GDM-W group was increased by 13.4% on days 14 (P < 0.01) (Fig. 2F), and same trend was observed in GSH, compared with GDM group, the content of GSH in GDM-W group was increased by 16.7% and 29.1% on days 7 and 14 (both P < 0.05) (Fig. 2G).

Effects of whey preload on plasma stress hormones in GDM women
Compared with GDM group, stress hormones E and cortisol were decreased by 10.8% and 19.8% respectively on days 14(p < 0.05) (Fig. 2H, 2J). However no signi cant difference between the two groups in NE (p > 0.05) (Fig. 2I).
Previous studies on the relation of oxidative stress damage and whey protein supplementation have different conclusions. Some studies believe that supplement whey protein can increase the levels of oxidative stress products [21,22], while others showed whey protein can reduce oxidative damage [23][24][25]. During our experiment session, oxidative stress products MDA was increased by 20.7% on days 14, anti-oxidative enzymes SOD was increased by 13.4% on days 14, and GSH was increased by 16.7% and 29.1% on days 7 and 14 after whey preload. So our results suggest that oxidative stress injury was decreased after supplement of whey protein.
Our study indicates that, whey protein preloading improved hyperglycemia and blood glucose related indexes, which may be related to the increase of insulin secretion, the improvement of IR, the delay of stress adaptation disorder, and the reduce of oxidative stress injury in GDM women.

Conclusions
Studies assessing effects of whey protein supplementation on diabetes are relatively few in number, and none of these studies focus on GDM. Only 60 GDM patients were included in our study, and one GDM patients quit because of protein falvour during the experiment. Our observations support the conlusion of whey protein preloads could improve stress adaptation disorder, oxidative stress injury and HOMA-IR, which should be explored with larger clinical trials in patients with GDM.

Conflicts of interest
None of the authors has a con ict of interest to declare.

Figure 1
Flowchart of enrollment and allocation of Whey protein preloading on GDM women