Early supplementation of folate and vitamin B12 improves insulin resistance in intrauterine growth retardation rats
Original Article

Early supplementation of folate and vitamin B12 improves insulin resistance in intrauterine growth retardation rats

Hui Zhang1#, Xinli Wang1#, Jin Zhang2, Yuhong Guan3, Yan Xing1

1Department of Pediatrics, Peking University Third Hospital, Beijing, China; 2Department of Pediatrics, Beijing Jishuitan Hospital, Beijing, China; 3Department of Pulmonary, Beijing Children’s Hospital, Capital Medical University, Beijing, China

Contributions: (I) Conception and design: Y Xing, H Zhang, X Wang; (II) Administrative support: Y Xing; (III) Provision of study materials or patients: Y Xing, H Zhang, X Wang; (IV) Collection and assembly of data: H Zhang, J Zhang, X Wang, Y Xing; (V) Data analysis and interpretation: Y Xing, H Zhang, Y Guan; (VI) Manuscript writing: All authors; (VII) Final approval of manuscript: All authors.

#These authors contributed equally to this work.

Correspondence to: Yan Xing. Department of Pediatrics, Peking University Third Hospital, Beijing 100191, China. Email: yxsxz@outlook.com.

Background: Insulin sensitivity is changed during the neonatal period in small for gestational age (SGA) infants. Yet, the interventional strategies are still limited. We aimed to investigate the effects of supplementation with high folate and vitamin B12 diets in the early postnatal period on the changes in insulin sensitivity in an intrauterine growth retardation (IUGR) rat model.

Methods: IUGR rat model was established by both low-protein diet feeding and daily diet restriction. High folate and vitamin B12 diet was supplied in IUGR as nutritional interventional group (IUGR-I), otherwise, the non-intervened IUGR group (IUGR-NI). In this study, male rats were studied in order to avoid hormonal and gender influence. At 21, 60 and 120 days, fasting plasma glucose, insulin, triglyceride, cholesterol, and homocysteine levels were measured among the control, IUGR-I, and IUGR-NI groups. Pearson analysis was used to evaluate the correlation between homocysteine and fasting blood glucose, insulin, HOMA-IR, triglyceride, and cholesterol levels.

Results: We established IUGR rat model by both low protein and restricted diet feeding during pregnancy and the incidence of IUGR pups was 93.33%. There was no difference in fasting glucose, insulin, HOMA-IR, triglyceride and cholesterol levels between the control, the IUGR-NI and the IUGR-I group at day 21. At day 60, insulin, HOMA-IR and triglyceride levels in the IUGR-I group were remarkably lower than those in the IUGR-NI group, but still higher than those in the control group (F=38.34, P=0.02; F=49.48, P=0.02; F=17.93, P<0.001, respectively). At day 120, glucose, insulin, HOMA-IR and Hcy levels in the IUGR-I group were obviously lower than those in the IUGR-NI group, although still higher than those in the control group (F=21.60, P<0.001; F=164.46, P<0.001; F=75.15, P<0.001; F=35.46, P<0.001, respectively). There were no significant differences in triglyceride and cholesterol levels between the IUGR-I group and the control group at day 120. At 120-day, homocysteine in IUGR-I group was highly positively correlated with fasting glucose and HOMA-IR (r=0.863, P=0.006; r=0.725, P=0.042, respectively); Only homocysteine was positively correlated with fasting glucose in IUGR-NI group (r=0.721, P=0.044).

Conclusions: Early supplementation of folate and vitamin B12 improved insulin resistance and lipid levels in IUGR rats to some extent, along with decreasing homocysteine levels, but not enough to completely repair glucose and lipid metabolism.

Keywords: Insulin resistance; folate; vitamin B12; intrauterine growth restriction


Submitted Oct 21, 2021. Accepted for publication Feb 09, 2022.

doi: 10.21037/tp-21-498


Introduction

Infants whose weight is < the 10th percentile for gestational age are classified as small for gestational age (SGA), often caused by intrauterine growth retardation (IUGR) (1). Low birth weight infants with IUGR are closely related to adulthood insulin resistance (2,3). Recent studies have shown that SGA infants already had changes in insulin sensitivity during the neonatal period (4,5).

At present, there are many studies on the harm of IUGR, yet the interventional strategies are still limited. Epigenetic modifications such as DNA methylation may be the key mechanism of insulin resistance in IUGR (6). In the same genetic background, food is closely related to epigenetic changes. Animals in the early postnatal period are in a period of rapid development, and epigenetic modifications can be affected by changing the external environment such as food (7,8). Epigenetic forms established during the fetal period can be altered by nutritional interventions early in the postnatal life to improve the metabolic system that is not yet fully mature (9). Folate, vitamin B12, and homocysteine (Hcy), the main members of the methionine cycle, participate in nucleic acid synthesis and protein metabolism in the body (10). It has been reported that raised maternal plasma Hcy concentrations can predict small size at birth, a risk factor for type 2 diabetes mellitus. An association between maternal vitamin B12, folate and Hcy status during pregnancy was studied, showing in utero vitamin B12 and folate deficiency could predict about propensity for greater adiposity and insulin resistance in the offspring (11). Therefore, early nutrition may have a certain effect on later childhood insulin sensitivity (12).

In this study, we established IUGR rat model by both low protein and restricted diet feeding during pregnancy, as well as investigated the effects of supplementation with high folate and vitamin B12 diets in the early postnatal period on the changes in blood glucose, insulin, Hcy, triglyceride and cholesterol levels in IUGR rats. To explore whether early supplementation of folic acid and vitamin B12 would improve insulin resistance in IUGR rats. We present the following article in accordance with the ARRIVE reporting checklist (available at https://tp.amegroups.com/article/view/10.21037/tp-21-498/rc).


Methods

Subjects and grouping

This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the Peking University. The protocol was approved by the Committee on the Ethics of Animal Experiments of the Peking University Third Hospital (protocol number: LA2015190). Healthy Sprague-Dawley rats were purchased from the Laboratory Animal Science Department of Peking University Health Science Center (Beijing, China). In the previous study, the IUGR rat model was established by low protein diet throughout pregnancy, but some fetal rats were completely restricted in development and could not survive, which reduced the success rate of the experiment. Therefore, this study was improved and we successfully established IUGR rat model by both low protein and restricted diet feeding during the second and third trimester of pregnancy. Eighteen females, 2.5 months old and weighing 200–250 g, were mated with males, and caged at a ratio of 1:2. The first day of conception was to see the vaginal plug. On the 12th day of conception, 18 pregnant rats were randomly divided into a low (8%) protein diet group (n=12) for IUGR and a standard (20%) diet group (n=6) in a 2:1 ratio using a computer based random order generator. All of rats were kept in individually ventilated cages at a temperature of 24 ℃, humidity of 50%, and a 12/12-hour light/dark cycle with the lights. The standard diet group had unlimited free diet, while the low-protein diet group was restricted to 40% of the diet amount required by the standard diet group.

After the pregnant rats gave birth naturally, the weight of newborn pups within 6 hours of birth was weighed using an electronic balance to the nearest 0.01 g. There were 49 pups with the mean weight of 7.01±0.35 g in the control group, and 90 pups with the mean weight of 4.44±0.58 g in the low-protein group. Pups with the body wight of 2 standard deviations (SD) below the mean of the control group were defined as the IUGR pups, resulting in a total of 84 IUGR pups, and the remaining 6 pubs were excluded because of their high birth weight. IUGR pups were randomly divided into two groups (42 rats/group) in a 1:1 ratio using a computer based random order generator: IUGR intervention group (IUGR-I) and IUGR non-intervention group (IUGR-NI). The lactating female rats in the IUGR-I group were fed with high folate (32 mg/kg) and vitamin B12 (0.16 mg/kg) diet, 4 times that of the standard diet (Table 1). The lactating female rats in the IUGR-NI and in the control were fed with standard diet (folate, 8 mg/kg; vitamin B12, 0.04 mg/kg). The offspring of the three groups were weaned 21 days after birth, and fed with the standard diet to 120 days. Dietary assignments were done by an animal technician who was not involved in the outcome evaluation.

Table 1

Nutrient composition of diet for control, IUGR and treated IUGR

Ingredient Standard diet Low-protein diet Standard diet with high folate and vitamin B12
Corn (%) 38.95 38.95 38.95
Corn starch (%) 31.11 43.49 31.11
Casein (%) 16.73 4.34 16.73
Flour (%) 5 5 5
Vegetable oil (%) 3.76 3.76 3.76
CaCO3 (%) 0.38 0.38 0.38
Ca(HCO3)2 (%) 3.23 3.23 3.23
Microelement (%) 0.5 0.5 0.5
Salt (%) 0.3 0.3 0.3
Fiber (%) 0.05 0.05 0.05
Protein (%) 20 8 20
Folic acid (mg/kg) 8 8 32
Vitamin B12 (mg/kg) 0.04 0.04 0.16
Calories (kcal/g) 30.50 30.50 30.50

IUGR, intrauterine growth retardation.

Date collection and measurement

In this study, male rats were studied in order to avoid hormonal and gender influence. At 21, 60 and 120 days, 8 male pups were randomly picked from each group. We have chosen a small sample size because this study studied the effects of folic acid and vitamin B12 on insulin resistance in IUGR rats for the first time, which was a preliminary exploration of the follow-up mechanism. Isoflurane (2% inhalation) was used for anesthesia, and all efforts were made to minimize pain. After fasting for 12h, blood was collected from the inner canthal vein for fasting plasma glucose (FBG), fasting insulin (FINS), triglyceride and cholesterol. Hcy was measured at 120 days. FBG and FINS levels were measured using a blood glucose meter (Roche, German) and an enzyme-linked immunosorbent insulin assay kit (Mercodia, Sweden), respectively. Hcy, triglyceride and cholesterol were measured using the HITACHI 7150 automatic biochemical analyzer (Hitachi, Japan). To calculate insulin resistance index, we used the HOMA-IR (Homeostasis Model of Assessment-Insulin Resistance) score [FPG (mmol/L) × FINS (mU/L)]/22.5.

Statistical analysis

Statistical analysis was performed using SPSS 20.0 software. Model assumptions were checked by using the Shapiro-Wilk normality test. Data are presented with mean ± SD. Continuous variables between two groups were compared by Student’s t-test. The comparison among three groups was performed using analysis of variance, and the further pairwise comparison was performed using the Student-Newman-Keuls method. Correlation between homocysteine and fasting blood glucose, insulin, HOMA-IR, triglyceride, and cholesterol levels was assessed using Pearson analysis. P<0.05 indicates that the difference is statistically significant.


Results

Birth weight of offspring rat

We established IUGR rat model by both low protein and restricted diet feeding during pregnancy. The average birth weight of offspring rat in the low protein and restricted diet group [(4.44±0.58) g] was lower than that in the control group [(7.03±0.56) g]. The difference was statistically significant (t=15.75, P<0.05). There were 84 IUGR pups, and the incidence of IUGR pups was 93.33% in this study.

Comparisons of serum parameters between the control and the IUGR offspring

IUGR pups who were supplemented with high folate and vitamin B12 during lactation were classified as the intervened IUGR group (IUGR-I); otherwise, the non-intervened IUGR group (IUGR-NI). Comparisons of fasting glucose, insulin, HOMA-IR, triglyceride, cholesterol and Hcy levels between the control, the IUGR-NI and the IUGR-I group are shown in Figure 1. At day 21, there was no difference in fasting glucose, insulin, HOMA-IR, triglyceride, and cholesterol levels across the three groups (P>0.05).

Figure 1 Comparisons of fasting glucose, insulin, HOMA-IR, triglyceride, cholesterol and Hcy levels between the control, the IUGR-NI and the IUGR-I group. *, P<0.05 vs. Control; #, P<0.05 vs. IUGR-I. HOMA-IR, Homeostasis Model of Assessment-Insulin Resistance; Hcy, homocysteine; IUGR, intrauterine growth retardation; IUGR-I, IUGR pups were intervened by supplementation with high folate and vitamin B12 during lactation; IUGR-NI, non-intervened IUGR pups.

At day 60, insulin, HOMA-IR and triglyceride levels in the IUGR-I group were remarkably lower than those in the IUGR-NI group, although they were still higher than those in the control group (F=38.34, P=0.02; F=49.48, P=0.02; F=17.93, P<0.001, respectively). Fasting glucose and cholesterol levels in the IUGR-I and the IUGR-NI group were higher than those in the control group, but there was no significant difference between the IUGR-I and IUGR-NI group (F=17.14, P=0.01; F=8.34, P<0.001, respectively).

At day 120, the levels of fasting glucose, insulin, HOMA-IR and Hcy in the IUGR-I group were obviously lower than those in the IUGR-NI group, but still higher than those in the control group (F=21.60, P<0.001; F=164.46, P<0.001; F=75.15, P<0.001; F=35.46, P<0.001, respectively). The levels of triglyceride and cholesterol in the IUGR-NI group were dramatically higher than those in the IUGR-I group and control group, but there was no significant difference between the IUGR-I and control group (F=9.80, P<0.001; F=6.12, P<0.001, respectively).

Correlation between homocysteine and fasting glucose, insulin, HOMA-IR, triglyceride, and cholesterol levels in IUGR-I group and IUGR-NI group

Homocysteine levels were positively correlated with fasting glucose, HOMA-IR levels (r=0.863, P=0.006; r=0.725, P=0.042, respectively) in IUGR-I group at day 120 (Table 2). Homocysteine levels were not significantly correlated with insulin, triglyceride and cholesterol levels in IUGR-I group at day 120 (Table 2).

Table 2

Correlations between homocysteine and metabolic parameters at day 120 in IUGR-I rats

Pearson correlation Homocysteine
r P value
Fasting plasma glucose 0.863* 0.006
Fasting insulin 0.165 0.697
Total cholesterol 0.646 0.083
Total triglyceride 0.604 0.113
HOMA-IR index 0.725* 0.042

*, correlation is significant at the 0.05 level. HOMA-IR, Homeostasis Model of Assessment-Insulin Resistance; IUGR-I, intrauterine growth retardation pups were intervened by supplementation with high folate and vitamin B12 during lactation.

There was a positive correlation between homocysteine and fasting glucose levels (r=0.721, P=0.044) in IUGR-NI group at day 120 (Table 3). Homocysteine levels were not significantly correlated with HOMA-IR, insulin, triglyceride, and cholesterol levels in IUGR-NI group at day 120 (Table 3).

Table 3

Correlations between homocysteine and metabolic parameters at day 120 in IUGR-NI rats

Pearson correlation Homocysteine
r P value
Fasting plasma glucose 0.721* 0.044
Fasting insulin 0.147 0.728
Total cholesterol 0.469 0.241
Total triglyceride 0.424 0.296
HOMA-IR index 0.585 0.127

*, correlation is significant at the 0.05 level. HOMA-IR, Homeostasis Model of Assessment-Insulin Resistance; IUGR-NI, non-intervened intrauterine growth retardation pups.


Discussion

Infants born SGA are at an increased risk of metabolic syndrome in later life (13). SGA infants may have metabolic abnormalities at birth, but not all will develop insulin resistance in the future (14). Therefore, the implementation of nutritional intervention at this stage may change the body’s immature metabolic system and improve insulin resistance. Yet, the potential mechanism and interventional strategies are still limited. In our study, high amounts of folate and vitamin B12 were supplemented during lactation for IUGR rats. At 21 days after birth, there was no difference in fasting glucose, insulin, HOMA-IR, triglyceride and cholesterol levels among the control, IUGR-I, and IUGR-NI groups. This suggests that IUGR offspring still maintained normal glucose and lipid metabolism in the early postnatal period. From 60 days after birth, insulin resistance and hyperlipidemia occurred in IUGR offspring. This manifests that the compensatory secretion of insulin increases with age, but not enough to maintain the normal balance of glucose and lipid metabolism, leading to insulin resistance. But insulin resistance and hyperlipidemia were improved significantly by folate and vitamin B12 supplementation, indicating that nutritional intervention in the early postnatal period can improve the immature metabolic system established during the fetal period, and the lactation period can be used as a window of early nutritional intervention.

Specifically, studies in both humans and rats have shown that maternal B12 insufficiency is associated with insulin resistance in offspring (15,16). Huang et al. have suggested that maternal supplementation with high folic acid (4 times the normal diet) during pregnancy and lactation improves glucose intolerance and insulin resistance in male mice offspring fed a high-fat diet (17). Girard et al. have indicated that feed-restricted dairy cows receiving a combined supplement of folic acid and vitamin B12 might have enhanced insulin sensitivity (18).

At present, the mechanism by which folate and vitamin B12 improve insulin resistance is still unclear. It has been shown that high Hcy level might be highly correlated with insulin resistance (19-21). Folate and vitamin B12 may alter insulin sensitivity by regulating Hcy level. Setola et al. have shown that supplementation of folate and vitamin B12 improved insulin resistance, along with decreasing homocysteine levels in patients with metabolic syndrome (22). The main metabolic pathway of Hcy in vivo is the synthesis of methionine dependent on vitamin B12 and folic acid (23). When folic acid and vitamin B12 are deficient, the methionine cycle is impaired, leading to hyperhomocysteine (24,25). In this study, the highest serum Hcy concentration was found in the IUGR group, which was reduced significantly by folate and vitamin B12 supplementation. Moreover, Hcy in IUGR-I group was highly positively correlated with fasting glucose and HOMA-IR. Some clinical studies also showed that supplementation of folic acid and vitamin B12, rather than folic acid alone, is likely to be much more effective at lowering of Hcy concentrations, with potential benefits for improved insulin resistance and reduction of risk of vascular disease (26,27). Liu et al. believed that increased homocysteine level enhanced the activity of protein tyrosine phosphatase and inhibited the phosphorylation of insulin β receptor. Protein tyrosine phosphatase played a negative regulatory role in insulin signaling pathway, resulting in reduced glucose uptake ability of liver cells and insulin resistance (28). These data suggest that a fortification policy based on folate and vitamin B12 during lactation or at early neonatal period may improve insulin resistance by reducing Hcy concentration in IUGR offspring.

However, it was reported that insulin resistance induced by hyperglycemia in patients with type 2 diabetes would not change plasma Hcy level, and there was no correlation between insulin resistance and Hcy level (29). Jimmy has suggested that offspring of mothers with low vitamin B12 and high folate were the most insulin resistant (30). Therefore, there is no consistent conclusion as to whether folic acid and vitamin B12 supplementation can improve insulin resistance. The reason may be different from the design of the experimental scheme, or the animal experiment is different from the clinical situation, or folic acid and vitamin B12 have synergistic or antagonistic effect. In addition, the mechanism of the effect of folic acid and vitamin B12 supplementation on insulin sensitivity needs to be confirmed by further studies.


Conclusions

Taken together, our data indicated that early supplementation of folate and vitamin B12 improved insulin resistance and lipid levels in IUGR rats to some extent, along with decreasing homocysteine levels, but not enough to completely repair glucose and lipid metabolism.


Acknowledgments

Funding: This study was financially supported by National Nature and Science Foundation of China (No. 30901614 to YX, No. 81241026 to XW), Beijing Natural Science Foundation (No. S170003 to YX), Peking University Medical cross research seed fund (Medical-X, No. BMU2020MX010), and Peking University Third Hospital Research Fund for outstanding overseas returnees (No. BYSYLXHG2019005 to YX). The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.


Footnote

Reporting Checklist: The authors have completed the ARRIVE reporting checklist. Available at https://tp.amegroups.com/article/view/10.21037/tp-21-498/rc

Data Sharing Statement: Available at https://tp.amegroups.com/article/view/10.21037/tp-21-498/dss

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://tp.amegroups.com/article/view/10.21037/tp-21-498/coif). The authors have no conflicts of interest to declare.

Ethical Statement:The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. The protocol was approved by the Committee on the Ethics of Animal Experiments of the Peking University Third Hospital (Protocol Number: LA2015190). This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the Peking University.

Open Access Statement: This is an Open Access article distributed in accordance with the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License (CC BY-NC-ND 4.0), which permits the non-commercial replication and distribution of the article with the strict proviso that no changes or edits are made and the original work is properly cited (including links to both the formal publication through the relevant DOI and the license). See: https://creativecommons.org/licenses/by-nc-nd/4.0/.


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Cite this article as: Zhang H, Wang X, Zhang J, Guan Y, Xing Y. Early supplementation of folate and vitamin B12 improves insulin resistance in intrauterine growth retardation rats. Transl Pediatr 2022;11(4):466-473. doi: 10.21037/tp-21-498