Human Milk Oligosaccharides in Maternal Serum Respond to Oral Glucose Load and Are Associated with Insulin Sensitivity

Abstract

(1) Background: Pregnancy presents a challenge to maternal glucose homeostasis; suboptimal adaptations can lead to gestational diabetes mellitus (GDM). Human milk oligosaccharides (HMOs) circulate in maternal blood in pregnancy and are altered with GDM, suggesting influence of glucose homeostasis on HMOs. We thus assessed the HMO response to glucose load during an oral glucose tolerance test (OGTT) and investigated HMO associations with glucose tolerance/insulin sensitivity in healthy pregnant women. (2) Methods: Serum of 99 women, collected at 0 h, 1 h and 2 h during a 75 g OGTT at 24-28 gestational weeks was analyzed for HMOs (2'FL, 3'SLN, LDFT, 3'SL) by HPLC; plasma glucose, insulin and C-peptide were analyzed by standard biochemistry methods. (3) Results: Serum 3'SL concentrations significantly increased from fasting to 1 h after glucose load, while concentrations of the other HMOs were unaltered. Higher 3'SL at all OGTT time points was associated with a generally more diabetogenic profile, with higher hepatic insulin resistance (HOMA-IR), lower insulin sensitivity (Matsuda index) and higher insulin secretion (C-peptide index 1). (4) Conclusions: Rapid increase in serum 3'SL post-oral glucose load (fasted-fed transition) indicates utilization of plasma glucose, potentially for sialylation of lactose. Associations of sialylated HMOs with a more diabetogenic profile suggest sustained adaptations to impaired glucose homeostasis in pregnancy. Underlying mechanisms or potential consequences of observed HMO changes remain to be elucidated.

Keywords: 3′-sialyllactose; gestational diabetes mellitus; hexosamine biosynthetic pathway; human milk oligosaccharides; insulin sensitivity; oral glucose tolerance test; pregnancy; sialic acid.

Figure 1

Human milk oligosaccharide concentrations in maternal serum change during a 75g-OGTT. 2′-Fucosyllactose (2′FL), lactodifucotetraose (LDFT), 3′-sialyllactosamine (3′SLN) and 3-sialyllactose (3′SL) concentrations are shown for each time point of the OGTT: 0 h, fasting state; 1 h, one-hour post-glucose load; 2 h, two hours post-glucose load. Significant differences between time points were determined using one-way ANOVA with Dunn‘s multiple comparison (**** p < 0.0001, *** p < 0.001, * p < 0.01). 3′SLconcentrations were significantly higher 1 h and 2 h post-load, 3′SLN concentrations was significantly lower 2 h post-load. The total study group was analyzed (n = 90).

Figure 2

3′SL and 3′SLN are correlated with glucometabolic parameters. Heat map shows Spearman correlations between the four HMOs 2′FL, LDFT, 3′SL and 3′SLN and glucose, insulin and C-peptide (at 0 h, 1 h, 2 h post-glucose challenge during OGTT) and glucometabolic indices. The glucometabolic indices were calculated as described in Supplementary Table S1. Significant correlations are highlighted with asterisks (* p < 0.05, ** p < 0.01). The sialylated HMOs 3′SL and 3′SLN revealed numerous significant positive associations with glucose and C-peptide at several time points. Strong negative associations were found between 3′SL and Matsuda C-peptide as well as the oral disposition index (ODI). 3′SLN was also negatively associated with Matsuda C-peptide and showed additional strong positive associations with the insulin clearance (IC).

Figure 3

HMO dynamics during the OGTT differed among metabolic clusters. (A–D): HMO trajectories during OGTT in Clusters 1 (diabetogenic) and 2 (low risk) (median HMO concentration in nmol/mL with IQR). (A) 2′FL and (B) LDFT increased after 1 h, 2 h and from 1–2 h in Cluster 1 while remaining relatively stable in Cluster 2. (C) 3′SL concentration was significantly higher in Cluster 1 than in Cluster 2 at all time points (indicated by lower case letters a, b) and significantly increased in both clusters (indicated by asterisks). (D) 3′SLN concentration decreased 2 h post-load in Cluster 2 while slightly increasing in Cluster 2. 3′SLN concentration was significantly higher at 1 h in Cluster 1 (indicated by a). Significant differences between time points within the same cluster were determined using two-way ANOVA with Fisher‘s LSD testing (* p < 0.05; ** p < 0.005; *** p < 0.0005). Significant differences between Clusters 1 and 2 at the respective time points were determined using two-way ANOVA with Sidak’s multiple comparison (a = p < 0.05; b = p < 0.005); secretor-positive subcohort (n = 61). (E–H): Area under the curve (AUC) of the four HMOs 2′FL (E), LDFT (F), 3′SL (G) and 3′SLN (H) in the two metabolic clusters. Cluster 1 showed significantly higher 3′SL and 3′SLN AUC values. Significant differences were determined using Mann–Whitney Test (* p = 0.0381; *** p = 0.0002); total study cohort (n = 89).

Figure 4

Proposed model of HMO regulation by glucose availability. According to our hypothesis, two scenarios contribute to the transient rise in 3′SL: Plasma glucose levels are elevated due to the 75 g glucose uptake in the OGTT (blue arrow), and individuals with generally decreased insulin sensitivity (diabetogenic profile) have subclinically increased basal glucose availability (red arrow). (1) Liu et al. showed that increased availability of glucose leads to an increased expression of β-1,4-galactosyltransferase (B1,4GalT), leading to the increased production of lactose [34]; however, as described by Neville et al., lactose production is independent of the short-term glucose availability in humans [26]. Considering these facts, we speculate that elevated lactose levels are predominantly observed in the case of decreased insulin sensitivity (red arrow), with elevated basal glucose levels. (2) Similarly, the hexosamine biosynthetic pathway (HBP) depends on glucose availability as described by Chari et al. [44]. Higher glucose availability leads to higher flux through the HBP, leading to higher CMP-NeuAc5 sialic acid donor substrate. (3) Ultimately, the sialyltransferase activity (ST3Gal1) was shown to be enhanced under high glucose concentrations [35] which could further contribute to the observed rise in 3′SL. The additive effect of altered insulin sensitivity through this hypothetic regulatory pathway would explain the observed differences in 3′SL between the clusters. The proposed pathways for HMO synthesis are located in the lactocytes of the mammary gland; however, HBP can also be activated in other peripheral tissues.