Next Article in Journal
Anaerobic Digestion of the Residue (Combination of Wastewater and Solid Waste) from a New Olive-Oil Manufacturing Process Based on an Olive Cold-Pressing System: Kinetic Approach and Process Performance
Previous Article in Journal
Study of the Possibilities of Improving Maintenance of Technological Equipment Subject to Wear
 
 
Search for Articles:
Title / Keyword
Author / Affiliation / Email
Journal
Article Type
 
 
Advanced Search
 
Section
Special Issue
Volume
Issue
Number
Page
 
Journals
Processes
Volume 10
Issue 12
10.3390/pr10122551
processes-logo
Submit to this Journal Review for this Journal Propose a Special Issue
► Article Menu

Article Menu

share Share announcement Help format_quote Cite question_answer Discuss in SciProfiles thumb_up
...
Endorse textsms
...
Comment
first_page
settings
Order Article Reprints
Font Type:
Arial Georgia Verdana
Font Size:
Aa Aa Aa
Line Spacing:
Column Width:
Background:
Article

Dysregulation of Mir-193B and Mir-376A as a Biomarker of Prediabetes in Offspring of Gestational Diabetic Mice

by
Khawlah W Al-Bakr
1,
Abdullah M Alzahrani
1 and
Emad A Ahmed
1,2,*
1
Biological Sciences Department, College of Science, King Faisal University, Hofouf 31982, Saudi Arabia
2
Lab of Molecular Physiology, Zoology Department, Faculty of Science, Assiut University, Asyut 71515, Egypt
*
Author to whom correspondence should be addressed.
Processes 2022, 10(12), 2551; https://doi.org/10.3390/pr10122551
Submission received: 14 October 2022 / Revised: 11 November 2022 / Accepted: 21 November 2022 / Published: 1 December 2022
Browse Figures
Versions Notes

Abstract

:
Gestational diabetes mellitus (GDM) is a type of diabetes initiated during pregnancy and is characterized by maternal hyperglycemia that induces complications in mothers and children. In the current study, we used a GDM mouse model (through i.p. injection of a single dose of streptozocin, STZ, 60 mg/kg/bw) to investigate the biochemical and immunological changes in the blood and brain of diabetic mothers and their offspring relative to their appropriate controls. In addition, we estimated the expression levels of a set of microRNAs (miRNAs) to link between the dysregulation in the levels of miRNAs and the exposure to oxidative stress during embryonic development, as well as metabolic changes that occur after birth and during puberty in offspring (5-weeks-old). At the biochemical level, newborn pups appeared mostly to suffer from the same oxidative stress conditions of their mothers as shown by the significant increase in nitric oxide (NO) and malondialdehyde (MDA) in blood and brain of diabetic mothers and their pups. However, the 5-week-old offspring showed a significant increase in proinflammatory cytokines, IL-1β, IL-6, and TNF-α, and based on their blood glucose levels, could be considered as prediabetic (with glucose mean value of 165 mg/dl). In the meantime, the tested miRNAs, especially miR-15b, miR-146a, and miR-138 showed mostly similar expression levels in diabetic mothers and newborn pups. In this regard, miR-15a and -15b, miR-146a, and miR-138 are downregulated in diabetic mothers and their newborn pups relative to their appropriate controls. However, in offspring of diabetic mothers at puberty age, these miRNAs displayed different expression levels relative to mothers and control offspring. Interestingly, miR-193 and miR-763 expression levels were significantly lower in diabetic mothers but upregulated in their 5-week-old offspring, suggesting that miR-193 and miR-763 could be used as biomarkers to differentiate between prediabetes and diabetes.

1. Introduction

Advances in identifying specific biomarkers may help in the early detection and classification of diabetic conditions, which could improve treatment protocols to reduce or prevent diabetes. Gestational diabetes mellitus (GDM) is a type of diabetes that is initiated during pregnancy (around the 24th week of pregnancy in humans) due to maternal hyperglycemia that affects both mothers and children. These complications include problems during delivery as a result of fetal overgrowth, and hence obesity seems to be another long-term complication in offspring born to GDM mothers [1]. In addition to the higher risk of developing obesity and overweight in offspring, GDM may lead to an impairment in glucose tolerance, type 2 diabetes (insulin resistance), and thus the development of other diseases [2,3,4,5,6,7,8,9]. Therefore, GDM is one of the most common metabolic risk factors that might cause brain development abnormalities in offspring.
Research demonstrated a significant correlation between diabetes during pregnancy and lower IQ in offspring [8]. For example, children younger than age 9 born to GDM mothers had an increased rate of attentions deficit, lower IQ, and lower gross and fine motors achievement compared with controls [10]. In addition, a close relationship between maternal diabetes and a higher incidence of psychological disorders such as schizophrenia in were reported in human offspring [11]. Moreover, offspring born to diabetic mothers had a 3-fold-increased risk of congenital anomalies when compared to the offspring of nondiabetic mothers [12]. In another study, it was shown that children of diabetic mothers were at high risk of developing diabetes in adulthood [13].
The evolutionarily conserved short non-coding RNAs (miRNAs) are pluripotent components involved in various physiological cellular processes, such as cell development and differentiation. Recently, miRNAs have been highlighted as master regulators of various pathological conditions during cancer and other chronic diseases [14,15]. These include prediabetes, diabetes, heart diseases, and other oxidative-stress-causing diseases [16,17,18]. In this regard, the deregulated plasma levels of miR-150, miR-30a-5p, miR-15a, and miR-375 were observed years before the onset of T2DM and have been suggested to reflect the risk of the disease development [19,20]. In addition, circulating miR-192 and miR-193b specifically identify prediabetes in humans, where their significant increase was seen in the prediabetic state but not in diabetic patients [21]. Consistent with that, miR-192 and miR-193b were shown to be potential predictive serum biomarkers of obesity and specifically recognize the prediabetic phenotype [22]. On the other hand, miR-146b and miR-15b were found to be correlated positively with fasting plasma glucose and thus could be used to discriminate T2D patients from healthy controls [23]. In addition, the altered expression levels of miR-376a-3p and other miRNAs was suggested to contribute to impaired fracture healing in patients with diabetes mellitus [24]. In the meantime, miR-376a was found be associated the risk of developing Alzheimer’s disease (AD) [25]. Another two miRNAs, miR-146a and miR-126, were significantly downregulated in people with prediabetes [26]. In addition, type 2 diabetic patients displayed a decreased serum level of miR-146a as a sign of chronic inflammation [27]. On the other hand, circulatory miR-29a, -192, and -126 are consistently dysregulated in prediabetic patients [28]. However, miR-138-5p was downregulated in the GDM placentas resulting in abnormal growth of placenta through promoting proliferation of trophoblasts [29]. In addition, recently, miR-182-5p was found to be associated with dysglycaemia and was suggested to potentially predict prediabetes [30]. The expression of miR-182-5p was found to decrease with the increased duration of T2DM in African setting [31]. Overexpression of miR-182-3p and miR-132-3p triggered impaired insulin secretion in pancreatic beta cells [32].
However, although the effect of gestational diabetes on offspring have been studied on human and animal models, the involvement of miRNAs in the risk of developing diabetes and associated diseases in offspring has not yet been completely clarified. To the best of our knowledge, only one study has tested miRNAs expression in adult human offspring of mothers who were diabetic during pregnancy, where the levels of miRNA-15a and miRNA-15b raised the skeletal muscle of these offspring [33]. The brain is one of the main organs that produces miRNAs and is affected by any changes in glucose homeostasis. To summarize, we have reviewed above some of the miRNAs that were reported to be dysregulated during diabetes and prediabetes, taking into consideration that differential expression is the key to being reliable biomarkers (i.e., upregulated in diabetes and down in prediabetes or vice versa).
In the present study, we hypothesized that exposure to maternal diabetes may dysregulate the expression levels of miRNAs in offspring blood and brain tissue. We have selected a set of miRNAs that are conserved in humans, including miR-15a, miR-15b, miR-138, miR-146a, miR-182, miR-193, miR-202, and miR-376a, to be estimated in brain tissues of mothers and offspring using a STZ-induced GDM mouse model. The biochemical and histological alterations were evaluated in diabetic mothers and their offspring (at two different stages of development: newborn pups and 5-week-old ones) relative to their appropriate controls. Our results highlighted, in mice, that two conserved miRNAs in humans, miR-193 and miR-763, could be used as biomarkers of prediabetes.

2. Materials and Methods

2.1. Materials and Reagents

The used kits are listed in Supplementary Table S1. Other materials were of the highest grade, including STZ from Biosynth (Beijing, China), Bouin’s Solution (HT10132), Formalin (HT501850), Glacial acetic acid (A6283), Hematoxylin (H3136), Eosin (HT110216), Thiobarbituric acid (T5500), 1% sulphanilamide (all from Sigma-Aldrich, Saint Louis, MO, USA), 0.1% N-[1-naphthyl]-ethylenediamine dihydrochloride in 5% phosphoric acid, EDTA, Alcohol, Paraffin, and Xylene.

2.2. Animals and Procedures

The animal model of Gestational diabetes (GDM) was induced in female mice using 12 females and 5 males (males were used for mating only), in addition to their offspring (newly born pups and 4–5 weeks old). All animal procedures were performed according to standard protocols and ethical rules (approved by King Faisal University, KFU, under number KFU2020-2-7). Animals were bred at the animal house of Biological Sciences Department, College of Science, King Faisal University. Mice were housed in polypropylene cages inside a well-ventilated room. Mice were allowed to acclimatize for 1 week before starting the experiment. Each cage included no more than three mice (2 females or 1 male and 2 females during mating). Mice were fed a standard commercial pellet diet (a protein content above 16% and fat content of 4–5%) and water and were maintained under standard laboratory conditions: temperature 25 °C, relative humidity 60–70% and a 12 h light/dark cycle. Mice were divided into two groups: the 1st group of 8 females and the 2nd group of 4 females. Five males (age 8–10 weeks) were used in mating and were not included in any further experimentation.

2.3. Induction of Gestational Diabetes in Mice Using Single Dose of Streptozocin

The mice of the 1st group (8 females) were fasted for 20 h before the induction of maternal gestational diabetes. These females were left with males (2 females and one male per cage) for 3 nights before the induction of diabetes. This occurred via intraperitoneal injection of a single dose of streptozocin (STZ from carbosynth; 60 mg/kg body weight. In 0.1 M citrate buffer, Ph 4.5). The mice of the 2nd group (4 females) were used as control and injected with 0.1 M citrate buffer, Ph 4.5. Therefore, we first bred males and females, and then STZ was injected. Herein, it is possible that there was a direct toxic effect of STZ on the developing embryos, indicating that offspring may suffer from oxidative stress related to STZ inducing toxic effect and diabetes. Pregnant female mice were housed individually in polypropylene cages under the conditions described above. Hyperglycemia was tracked during gestation by cutting off the tip of the tail and squeezing it gently to collect a blood sample. Blood glucose levels were measured every week from the day of STZ injection by testing a 5μL sample of tail-vein blood by glucose meter. Mice were found to be diabetic within 3–4 days of STZ induction.

2.4. Samples Collection and Preparation

At the 1st day of labor and 4–5 weeks later, 5 males’ offspring of both group 1 and 2 (STZ treated and control, respectively) were anaesthetized using diethyl ether before being sacrificed. In total, 20 mice offspring were used for subsequent experiments. The mice of age between 4 and 5 weeks old (named in later figures as 5 wk) were used as a prepuberty stage of development and all used offspring were older than 4 weeks and younger than 5 weeks.

2.5. Histological Alterations

The brain tissues of mothers and offspring of control and diabetic mice were isolated and samples were prepared for histological investigations according to the following: fixation on Bouin’s Solution for 24 h at 4 °C, dehydration by graded alcohol series from water through 70% -80%-90%-100% ethanol prior to embedding in paraffin. Then, samples were cut by microtome and sections of 5 µm were mounted on slides and dewaxed in xylene. Next, sections were hydrated in a graded series of alcohols 100%, 90%, 80%, 70%, and distilled water and stained for hematoxylin and eosin for histological evaluation. Remains of animal organs/tissues were packaged and kept frozen until disposed through the safety procedures and were delivered to the research station belonging to King Faisal University.

2.6. Biochemical Alterations

2.6.1. Determination of Plasma Cytokine Levels

The levels of plasma cytokine (IL-1β, IL-6, and TNF-α) were determined by ELISA using Invitrogen cytokines assay kits according to the manufacturer’s instructions. The immunoassay is formed of 1 × 96-well kit that includes coupled magnetic beads, detection antibodies, standards, assay buffer, wash buffer, sample diluent, standard diluent, detection antibody diluent, streptavidin-PE, a flat-bottom plate, and sealing tape. List of kits used are provided in Supplementary Table S1.

2.6.2. Estimation of Oxidative Stress Parameters in Plasma and Brain Tissues

To check if GDM could induce a stressful environment in mothers and offspring, the oxidative stress enzymes, nitric oxide (NO), and lipid peroxidation (LPO) levels were estimated in control and treated mothers and offspring using the manufacture’s protocols. Nitrate and nitrite concentration was measured with slight modification using the Griess reagent [34]. Diabetic tissue samples were examined for NO precursors (nitrate and nitrite) level), and this was determined by measuring nitrate and nitrite levels using the supernatant of brain tissue (100 μL) mixed with the same volume of Griess reagent (1% sulphanilamide and 0.1% N-[1-naphthyl]-ethylenediamine dihydrochloride in 5% phosphoric acid %) for 10 min, then adding 25 of TMB developer and incubating in the dark for 10 min. The reaction was stopped using stop solution and read absorbance at 540 nm using a Biotek microplate reader. Fresh culture media were used as blanks in all experiments.
For estimation of the end product of lipid peroxidation, malondialdehyde (MDA) is a useful measure of oxidative damage. MDA was examined using (BioVision Colorimetric/Fluorometric Assay Kit) where the MDA in the sample is reacted with Thiobarbituric Acid (TBA) to generate the MDA-TBA adduct which can be easily quantified calorimetrically (OD 532 nm). This assay detects MDA levels as low as 1 nmol/well calorimetrically.

2.6.3. RNA Isolation, cDNA Synthesis, and Real-Time qPCR

To determine the expression levels of the following miRNAs, Caspase and PPARg, we performed 3 main steps: RNA extraction, cDNA synthesis, and real-time qPCR. RNA was isolated from the brain tissue using TRIzol extraction and Thermo Fisher Scientific protocol (PureLink™ RNA Mini Kit, 12183018A). The RNA was precipitated from the aqueous phase by addition of an equal volume 2-propanol, then vortexed and incubated for 5 min at RT. RNA was pelleted by centrifugation at 12,000× g rpm for 15 min at 4 °C and the pellet was washed twice with 900 μL 75% ethanol. The pellets were suspended in nuclease-free water. Purity and integrity of RNA were evaluated using Nano Drop ND-2000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). Samples were stored at −80 °C until further use. Then, 10 ng of total RNA was reverse transcribed using the specific RT primers of miRNAs miR-15a, miR-15b, miR-138, miR-146a, miR-182, miR-193, miR-202, and miR-376a (Supplementary Table S2). This was carried out using high-capacity RT reagent Kit (Applied biosystems). The relative expression of mRNA of Caspase -3, PPARˠ and the miRNAs was compared to the internal control of β-actin and RU6, respectively. The sequences of real time PCR primers are shown in Table S2. The Quantitative real time qPCR was carried out on an Vii7a real time PCR system (Applied Biosystems, Waltham, MA, USA) using AB SYBR Master Mix instructions (Applied biosystems, Waltham, MA, USA). The PCR conditions were under the following thermal condition: preheating, 95 °C, 5 min, 40 cycles of denaturation: 95 °C, 15 s, annealing: 60 °C, 20 s, and extension: 72 °C, 20 s. Cycle threshold (Ct) values were used to calculate fold changes in gene expression using 2–ΔΔCt method.

2.6.4. Statistical Analysis

The significant difference between the control and treated groups was analyzed by the two ways analysis of variance. A value of p ≤ 0.05 was considered statistically significant. Results are presented as mean ± standard error of the mean (SEM) of data from at least three independent animals.

3. Results

3.1. Untreated Diabetes Induces Complications in Pregnant Females and Reduces the Offspring Typical Litter Size

We first checked the relation between maternal blood glucose level (in mg/dL) and the average number of given viable pups per mother (shown in Table 1). Around nine pups per mother were seen for the control mice (blood glucose level below 140 mg/dL). Almost half of this number was given by females with blood glucose level between 180 and 240 mg/dL. In addition, a lower yield of viable pups was given by mothers with blood glucose level between 240 and 340 (two pups/ mother). However, females with blood glucose level higher than 340 mg/dL failed to give viable pups. Here, we should mention that the mice selected to conduct the current model were of the same again and nearly the same weight, and then the difference in blood glucose level could be due to feeding or activity behaviours. For further biochemical and histopathological assays, we have considered offspring born to mothers with blood glucose level above 180 mg/dL as offspring of diabetic mothers without any splitting during the analysis. Together our data indicate that untreated diabetes in pregnant females induces complications that may cause embryonic lethality before birth, then reducing the typical litter size.

3.2. Offspring of Mothers with Untreated Diabetes Are at Higher Risk of Developing Future Diabetic

The data obtained after measuring the blood glucose in control and diabetic mothers and their offspring (5 weeks old) are shown in Table 2. As expected, diabetic mothers blood displayed higher glucose level relative to their offspring and control mice. In the meantime, the blood glucose of offspring born to diabetic mothers increased significantly relative to those born to control mothers. The mean value was around 165 mg/dL, indicating a higher level of blood sugar in the offspring classifying them as prediabetic or at higher risk of developing future diabetic offspring.

3.3. Untreated Diabetes Reduces the Weight of Diabetic Mothers but Do Not Change the Offspring Weight

The weights of offspring born to diabetic and control mothers are shown in Table 3, in gram (g). The mean value of the weight of newborn pups of diabetic mothers was around 1.33 g, while the weight of 5-week-old offspring of diabetic mothers was around 12.16. These results indicate a normal offspring (newborn and 5 weeks) body weight, which point out that untreated GDM in mothers does not affect the weight of the offspring.

3.4. Untreated Maternal Diabetes during Pregnancy Increases the Risk of Oxidative Damage Induction in Their Offspring

Gestational exposure to a higher glucose environment may induce oxidative damage in offspring that can be seen at adult age. Along this line, earlier studies showed that foetuses of mothers with GDM were at higher risk of developing oxidative stress associated with high level of glucose [35]. In the current model and as shown in Figure 1A, an elevation in LPO level was seen in the serum of both diabetic mothers and their 5-week-old offspring relative to control mothers and their offspring (p = 0.008 and p = 0.09, respectively), this elevation was significant between the diabetic and control mothers. However, NO is upregulated significantly in diabetic mothers relative to control mothers (p = 0.02). On the other hand, the upregulation of NO in the 5-week-old offspring relative to their control mice was insignificant. These results indicate that offspring of diabetic mothers are probably at a risk of developing oxidative damage.
We have also estimated the blood glucose level in the blood of the control and STZ-injected mice (60 mg/kg/bw) 28 h after injection of STZ (Figure 5). This is to check if STZ can induce oxidative damage in pregnant females after several hours of injection where the MDA in brain of the control and STZ-treated mice was estimated.

3.5. Untreated Maternal Diabetes Increases the Levels of Proinflammatory Cytokines in Mothers and Offspring

Maternal diabetes was reported to induce excessive inflammatory changes in the brain tissue of offspring [36]. In this regard, we evaluated the expression levels of proinflammatory cytokines (IL-1β, IL-6, TNF-α) in serum of mothers and their offspring (5 weeks old). Figure 1B shows that these cytokines are dysregulated in the serum of diabetic mothers and their 5-week-old offspring relative to their appropriate controls. In diabetic mothers, the increase in the proinflammatory cytokines level was highly significant relative to the control mothers. However, the upregulation in all the three proinflammatory cytokines, IL-6, IL-1β, and TNF-α was highly significant in diabetic mothers relative to their offspring (p = 0.02, 0.006 and 0.005, respectively). In the meantime, IL-1β and TNF-α are upregulated significantly in 5-week-old offspring relative to their control (p = 0.05 and 0.0001, respectively). These data indicate that GDM mothers may suffer from inflammation and oxidative damage resulting from the hyperglycemia, which appears to be moderate in their offspring.

3.6. Untreated Maternal Diabetes Increases the Risk of Oxidative Stress Induction in Offspring’ Brain

It is documented that maternal diabetes is associated with neuroanatomical deficits in developing fetuses of animal models and a higher incidence of psychological disorders in human offspring [11,37]. Since the brain is comparatively in demand of high oxygen utilization, it provides a rich environment for the free radical by-products; in addition, the antioxidants and the lipid-rich constitution are ready substrates for oxidation, which can reduce the potential of certain neurotransmitters and induce psychological disorder [11,37]. In this regard, we checked the lipid peroxidation products in brain tissues of diabetic mothers and their offspring relative to the control mice (Figure 2A). A significant increase in LPO was found in the brains of diabetic mothers and their newborn pups relative to control mothers and their offspring (p = 0.0002 and 0.0001, respectively). However, the 5-week-old offspring of diabetic mothers showed a significant increase of LPO products compared to the control young 5-week-old offspring, but displayed a significant decrease compared to diabetic mothers and their pups. This indicates that the brain of newborn pups is mostly affected by the stressed environment induced during pregnancy.

3.7. The Newborn Pups of Untreated Diabetic Mothers Display High Level of Caspase-3 Relative to Other Groups

In short, maternal hyperglycemia contributes to a proinflammatory state in mothers during pregnancy, which can influence the brain development and cognition function in offspring as a result of apoptosis induction. On the other hand, since caspase-3 is the major effector caspase involved in apoptotic pathways [38], we analyzed its level in brain tissue of diabetic mothers and their offspring at different age stages (newborn and 5 weeks old) relative to an appropriate control. According to our findings, caspase-3 is overexpressed noticeably in pups compared to the 5-week-old offspring (p = 0.003). In addition, caspase-3 level increased significantly in pups compared to their diabetic mothers (p = 0.01). In addition, there a significant variation between control and diabetic mothers was observed (p = 0.0005, Figure 2B). These data indicate that diabetic newborns exert a higher level of apoptosis than 5-week-old offspring born to untreated GDM.

3.8. The Newborn Pups Display Higher Level of PPAR Gamma Compared to Other Mice

The PPARγ has been reported to have a profound effect on metabolic regulation and brain development [39]. Then, we analyzed the expression levels of PPARγ in the brain tissue of untreated GDM mice and their offspring (newborn and 5 weeks old). As shown in Figure 2C, a significant increase is seen in GDM mothers (p = 0.0005) as well as in their newborn mice (p = 0.001) relative to the appropriate control mothers and pups. In addition, the difference between offspring at puberty age was not quite statistically significant between those of diabetic mothers and their control (p = 0.07). However, PPARγ expression levels between newborn and 5-week-old offspring born to diabetic mothers were highly significant (p = 0.003). These data suggest that PPARγ overexpression is associated with diabetes or oxidative stress in diabetic mothers and their newly born offspring.

3.9. Histological Alterations in Cerebral Hemisphere of Diabetic Mother and Their Offspring

To this end, oxidative stress was observed in the blood and brain of diabetic mothers and offspring, which was associated with a higher level of proinflammatory cytokines in the blood of these mice. Therefore, we next checked the histological alterations induced in diabetic mothers and their newborn and 5-week-old offspring relative to the appropriate control mice. As shown in Figure 3 and Figure 4, the cerebral hemisphere of control mothers showed a normal appearance of neuropil, high density of neurons with light normal nuclear stain, and myelinated neurons (Figure 3A,B).
In diabetic mothers scarified after giving birth, slight vacuolation in neuropil, shrunken neurons, and infiltrated cells were seen in cerebral hemispheres relative to those of control mothers (Figure 3C–E). In the meantime, diabetic mothers scarified 5 weeks after giving birth displayed a marked increase in vacuolation in neuropil and a significant decrease in cellularity associated hemorrhagic-like appearance and infiltrated inflammatory cells (Figure 3F,G). In newborn pups of diabetic mothers, more darkly stained nuclei were observed relative to the control, suggesting a higher number of apoptotic cells (Figure 4A,B). However, 5-week-old mice of diabetic mothers show some pathological changes characterized by slight vacuolization and infiltration of inflammatory cells (Figure 4E). On the other hand, pups and 5-week-old mice of control mothers showed normal histological appearance (Figure 4C,D).

3.10. Dysregulation of Micro-RNAs as A Biomarker of Prediabetes and Oxidative Stress in Brain Tissue of Offspring of Diabetic Mothers

The brain is one of the main organs that are affected by hyperglycemia, and it is of the major organs that produce miRNAs; therefore, we have tested the dysregulation of miRNAs in brain tissues of the diabetic model and the control. Initially, we checked the direct toxic effect of STZ on pregnant mice several hours after STZ injection. We tested the blood glucose level in the blood of these mice 28 h after STZ injection relative to the control (Figure 5). Data showed a non-significant increase in blood glucose of STZ-treated females. In the meantime, the difference in MDA level between the control and STZ-treated mice was not significant at this time point. Then, after 28 h of STZ injection, no direct oxidative stress from STZ was observed. On the other hand, miRNAs expression levels in brain tissue of the control and STZ-treated mice (28 h after i.p. injection) showed no statistical variation between groups (Figure 5).

3.10.1. miR-15a-3p and miR-15b-3p Are Dysregulated in Brain Tissue of Diabetic Mothers and their Newborn Offspring

According to a recent study, fetal exposure to maternal diabetes was found to be associated with an increase in the expression level of miR-15a and miR-15b in the skeletal muscle of offspring at 26–35 years old [33]. Then, we estimated the expression level of miR-15a and miR-15b in brain tissue of diabetic mothers and their offspring relative to their control. We found that miR-15a-3p expression decreased significantly in brain tissue of diabetic mothers (p = 0.0001). However, there were no significant differences between diabetic offspring (newborn and 5 weeks old) and their control (p = 0.24 and p = 0.79, respectively).
Furthermore, a significant increase in miR-15a-3p expression was seen in 5-week-old diabetic offspring relative to their diabetic mothers (p = 0.005, Figure 6A). Meanwhile, the 5-week-old diabetic offspring showed a significant increase in miR15a-3p expression in the brain compared to that of newly born pups of diabetic mothers (Figure 6A). These data indicate that offspring exposed to hyperglycemia in utero exhibit downregulation of miR-15a-3p expression in brain tissue. However, the miR-15a level of the 5-week-old offspring is higher in diabetic and control offspring, which is probably related to a developmental stage expression.
On the other hand, we found a significant downregulation miR-15b-3p levels in the brains of untreated diabetic mothers and their diabetic newborn offspring compared to their controls (p = 0.0001 and p = 0.01, respectively) (Figure 6B). In addition, a significant decrease was observed in diabetic mothers relative to their offspring (newborn and 5 weeks old, p-Value = 0.66 and 0.0001, respectively). These data point out that the miR-15b-3p expression level is downregulated significantly in brain tissue of untreated GDM mothers and their newborn offspring, and it is correlated with GDM.

3.10.2. miR-138-3p Is Downregulated in Brain Tissue of Untreated Diabetic Mothers and their Offspring

miR-138-5p plays a regulatory role in insulin resistance [40]. Therefore, we estimated the expression level of miR-138-5p in this diabetic model. As shown in Figure 6C, miR-138-5p decreased significantly in both diabetic mothers (p-value = 0.0001) and their offspring (newborn and 5 weeks, p-value ≤ 0.05) relative to their appropriate controls. These data indicate that miR-138-3p is downregulated significantly in untreated GDM mothers and their offspring (for both). In addition, miR-138-3p downregulation is strongly correlated to GDM and can be a potential biomarker for diabetes at adulthood.

3.10.3. The Expression Levels of miR-146a-3p Is Downregulated in Brain Tissue of Untreated Diabetic Mothers and Offspring

miR-146a-3p is recognized as an important transcriptional regulator of some inflammatory mediators in pregnancy [41], thus we examined its expression level in brain tissue of untreated GDM mothers and their offspring at different life stages. miR-146-5p expression levels in healthy conditions were higher in offspring than their mothers, especially in newborn offspring, whereas in hyperglycemia conditions, miR-146-5p expression levels were downregulated significantly in GDM mothers and their offspring (for both) (p = 0.0001, p = 0.03 and p = 0.03, respectively) (Figure 6D). These data revealed that the expression levels of miR-146-5p in the brain tissue of newborn and 5-week-old offspring whose mothers did not receive treatment for GDM are significantly reduced.

3.10.4. miR-182-5p Are Upregulated in Brain Tissue of Newborn Offspring of Untreated GDM Mothers

miR-182-5p plays a role in regulatory pathways in diabetes mellitus and potentially predicts diabetes [30]. In this regard, we estimate miR-182-5p expression in the brain tissue of untreated GDM mothers and their offspring. It was overexpressed noticeably in newborn offspring of untreated diabetic mothers (p = 0.002). In addition, there was significant variation of newborn and 5-week-old offspring relative to their mothers (p = 0.002 and p = 0.003, respectively) (Figure 6E). This points out that miR-182-5p is significantly upregulated in the brain tissue of newborns with untreated GDM mothers.

3.10.5. Differential Expression Levels of miR-193-3p in Untreated Diabetic Mothers and Offspring

Brain miR-193 acts as a tumor suppressor in different cancer types [42]. In addition, it is associated with neuroinflammation [43]. Thus, we examined and compared the expression level of miR-193-3p in GDM mice model (mothers and their offspring). Interestingly, we found that miR-193-3p is dramatically downregulated in diabetic mothers (p = 0.0001). However, the downregulated level in diabetic pups was insignificant compared to the control pups (p = 0.26, Figure 6F). On the other hand, the 5-week-old offspring born to diabetic mothers showed higher level of brain miR-193 relative to both diabetic mothers (p = 0.007) and the offspring of control mothers (p = 0.05). Interestingly, miR-193-3p, miR-146a-3p, and miR-15a-3p expression level in the brain showed insignificant variation in STZ-treated mice (28 h after STZ injection) relative to the control females (Figure 5C). These results indicate that miR-193-3p could also be used as a biomarker to predict prediabetes in offspring.

3.10.6. miR-202-5p Are Upregulated in Brain Tissue of Diabetic Mothers

miR-202-5p plays a pivotal function on cellular proliferation, migration, and invasion in multiple tumors [44,45]. Herein, we analyzed miR-202-5p level in the brain tissue of GDM mothers and their offspring. Interestingly, miR-202-5p was overexpressed significantly in diabetic mothers (p = 0.003). In the contrary, no significant variation was found in both the newborn and 5-week-old groups (p = 0.39 and p = 0.15, respectively) compared to controls (Figure 6G). These data show that miR-202-5p was overexpressed in brain tissue of untreated mothers and no significant variation in their offspring.

3.10.7. miR-376a-3p Is Dysregulated in Diabetic Mothers and Their 5 Weeks-Old Offspring

Dysregulation of miR-376a-3p was reported to be associated with chondrogenesis in fracture healing with diabetes [24] as well as synaptoneurosomes during prion disease [46]. As indicated in Figure 6H, we analyzed the expression of miR-376a-3p in the brain tissue of control and diabetic mothers and offspring. We found that miR-376a-3p decreased significantly in diabetic mothers relative to control mothers (p = 0.002) but showed noticeable increase in their 5-week-old mice relative to those of control mothers (p = 0.70 and p = 0.16, respectively). However, there was no significant variation between diabetic newborns and 5-week-old offspring (p = 0.11). These data indicate that miR-376a-3p expression is downregulated in diabetic mice but overexpressed in prediabetic conditions.

4. Discussion

In the present study, we found that a set of miRNAs (miR-15a, miR-15b, miR-182, miR-193, miR-138, miR-202, miR-146a and miR-376a) are altered in brain tissues of untreated diabetic mothers and their offspring (newborn and 5 weeks old). At the biochemical level, newborn pups appeared mostly to suffer from the similar oxidative stress conditions of diabetic mothers. However, the 5-week-old offspring seemed to be under a relatively moderate oxidative stress and could be considered prediabetic. In synchronization with this, the tested miRNAs in new pups showed mostly similar expression levels to that of their diabetic mothers. On the other hand, some of the tested miRNAs showed different expression levels in 5-week-old offspring relative to the diabetic mothers.
Based on the data presented in Table 2, we suggest that offspring born to diabetic mothers are at higher risk of developing diabetes. Our results agree with a long term follow up study that included a total of 150 offspring of diabetic mothers, born and followed up until the age of 20 years, where diabetes mellitus was diagnosed in 7.3% of born and followed-up children of GDM mothers (seven mothers). The study results showed that children of diabetic mothers were at high risk of developing diabetes in adulthood [13]. However, our data pointed out that untreated GDM does not affect the weight of offspring relative to the control offspring (Table 3), which is not the case in humans, where diabetes during pregnancy increases the body weight in childhood, a point of research which still remains to be deeply investigated [5]. Our data clarified that miR-15a and -15b, miR-146a and miR-138 are downregulated in diabetic mothers and their newborn pups relative to their appropriate controls. However, in offspring of diabetic mothers, at puberty age, miR-15a miRNAs increased significantly relative to different expression levels of their mothers and control offspring. For miR-15a and miR-15b, fetal exposure to maternal diabetes was found to be associated with their increased expression in the skeletal muscle in offspring of 26–35-year-olds [33], which is inconsistent with our results, probably due to estimating it in different organs. In the meantime, miR-193 expression was significantly lower in diabetic mothers but significantly higher in the 5-week-old offspring suggesting that it could be used as a biomarker to differentiate between prediabetes and diabetes. In addition, miR-376a is downregulated in brain tissue of diabetic mice but upregulated in the offspring at the prepuberty stage, indicating that miR-376a could be used as reliable biomarkers of prediabetes.
Herein, we found that caspase-3 is overexpressed in diabetic mothers but highly expressed in the newly born pup offspring. The higher apoptotic-related caspase-3 expression in newborn offspring could be related to the effect of oxidative stress on dividing brain cells at a stage of development sensitive to DNA damage, which is not the case at terminally differentiated cells in mature mothers. This also has been seen in histological section at brain tissue of pups of diabetic mothers. In agreement with our results, caspase-3 overexpression was reported in cerebral white matter of newborn rats born to GDM mothers, which induced white matter damage and demyelination [47]. However, other studies reported that caspase-3 downregulation in different experimental mice models was associated with an increase in the average weight of babies, which is not the case in the current model. For example, placental-Caspase-3 downregulation in GDM mothers resulted in elevated placentas and newborn weights in the GDM [48]. In addition, Piazza et al. (2019) reported that low level of caspase-3 is correlated with microsomia and neurodevelopmental delay in pups [49]. We also examined PPARγ overexpression, where PPARγ is overexpressed significantly in diabetic mothers and their offspring especially newborns. PPARγ is an important molecular factor participating in lipid and carbohydrate metabolism as well as inflammation [50]. In addition, it plays a significant role in the regulation of growth and glucose homeostasis in the nervous system [39]. Several studies have elucidated the PPARγ dysregulation with GDM. For example, leukocyte PPARγ overexpression was observed in hyperglycemia conditions in GDM patients at 24–33 weeks of gestation [50]. However, PPARγ downregulation in the placenta of GDM mothers was highlighted as a biomarker of hyperglycemia in the young adulthood offspring mice model [51]. In addition, PPARγ deregulation was observed in both adipose tissues and placenta tissues from patients with GDM, while PPARγ upregulation in mice was correlated with severe hyperglycemia [52].
Our data showed that diabetes increases the levels of proinflammatory cytokines including IL-1β, IL-6, TNF-α in the plasma of diabetic mothers. Meanwhile, and at the pathological level, diabetic mothers’ brains displayed a vacuolation in neuropil, shrunken neurons, and infiltrated inflammatory cells relative to the brain of control mothers, as a result of the induced inflammation. These results are in line with previous findings which reported that pro-inflammatory marker levels have increased in both placenta and brains in animal model of GDM fetuses [53]. In fact, elevated pro-inflammatory levels were found to not be limited to diabetic mothers, but also induced negative effect on the brain of their fetus [53]. These findings indicated that GDM can induce chronic inflammation in the brains of rat offspring [54]. IL-6 is a pleiotropic cytokine that is involved in the inflammatory response and is associated with insulin-resistant status. For example, IL-6 overexpression was reported, in the hippocampi of rats’ brains, to be related to diabetes [55]. Similarly, we found that IL-6 levels were overexpressed in diabetic offspring. IL-1β is the main pro-inflammatory cytokine and chemokine, which promotes induction of other pro-inflammatory cytokines. It is also known that IL-1β is important in the progression of insulin resistance and diabetes [55]. Herein, we observed that TNF-α was overexpressed, which is in agreement with previous findings [54,56]. Higher levels of pro-inflammatory cytokines such as interleukin IL-1β and TNF-α were found to be associated with impaired recognition memory in offspring in the object recognition test [56]. Overall, our findings demonstrate that GDM induce inflammation related to oxidative damage in mothers and cause a risk of inflammation and oxidative stress at the level of pro-inflammatory cytokines in the 5-week-old offspring.
Both miR-15a and miR-15b were shown to play a critical role in insulin signaling and synthesis, and numerous studies have reported that miR-15a is dysregulated in diabetes and related disease [33,57]. Fetal exposure to maternal diabetes was found to increase the expression level of miR-15a and miR-15b in biopsies of skeletal muscle from offspring at age 26–35, indicating that maternal diabetes may increase the risk of cardiometabolic disease in offspring [33]. Inconsistent with that, the expression levels miR-15a were significantly lower in prediabetic patients and in patients with T2D relative to healthy individuals [19,58], which may agree with our finding, but we could not show that miR-15a and 15b are useful markers for prediabetes in 5-week-old offspring. The expression levels of miR-182-5p are upregulated significantly in the brain tissue of untreated diabetic mothers and their offspring. Recently, miR-182-5p was found to be associated with dysglycaemia and was suggested to potentially predict prediabetes where it was overexpressed significantly in individuals with prediabetes or T2DM [30]. The expression of miR-182-5p was also found to decrease with the increased duration of T2DM [31] in one African setting. In addition, in vivo inhibition of miR-182-5p partially reverses the beneficial effects of colesevelam on glucose tolerance [59]. Neurologically, a high level of miR-182 was strongly associated with intrauterine infection and brain injury in premature infants [60]. Our data shows that the expression level of miR-202-5p is overexpressed in the brain tissue of diabetic mothers and their 5-week-old offspring, whereas the pups’ offspring showed lower expression level of miR-202-5p. miR-202-5p plays a pivotal function on cellular proliferation, migration, and invasion in multiple tumors [44,45]. In the diabetic model, miR-202-5p overexpression exerts a protective role to protect against proliferative diabetic retinopathy [61].
In the current study, miR-193b and miR-376a-3p expression levels were significantly lower in diabetic mothers but upregulated in the offspring at prepuberty stage, which indicates that these miRNAs could be used as a biomarker of prediabetes in offspring. The expression level of miR-376a-3p was found to contribute to impaired fracture healing in patients with diabetes mellitus [24]. However, miR-376a was not shown before to mark the prediabetic conditions. In agreement with our data, an increased expression level of miR-192 and miR-193b was found in the plasma of the prediabetic glucose-intolerant mice, but not in diabetic patients [22]. Interestingly, circulating levels of miR-192 and miR-193b returned to baseline in both prediabetic humans and glucose-intolerant mice undergoing chronic exercise that succeeded to normalize metabolic parameters [22]. However, a circulating level of miR-192 and -194 in humans was found to be associated with the induction of diabetes mellitus [62]. Recently, miR-193b-3p has been suggested to affect glucose metabolism via upregulating the transcription factor FOXO1 downstream of the PI3K-AKT pathway [63]. In addition, one study determined the multiple plasma metal levels associated with the plasma miRNAs and diabetes risk and found it to be positively correlated with miR-193b-3p (p = 0.002), and both were found to be associated with the risk of diabetes [64]. Together, these data suggest that miR-193b and miR-376a-3p could be used as biomarkers of prediabetes in offspring of mouse model of gestational diabetes.
However, although several recent non-invasive insulin delivery methods and applications have been developed to improve the quality of life among diabetic patients (reviewed in [65]) and to make it easier for pregnant mothers to accept temporal medication during pregnancy, more investigations about diagnostic diabetic biomarkers are needed. In addition, mechanistic studies include the mimics, and the inhibitors of the highlighted miRNAs is required in which targeted signaling molecules can be studied. Silencing or upregulating miRNAs levels may regulate glucose homeostasis and insulin delivery methods, the latter has also attracted significant research interest in designing and developing new insulin delivery technologies [65].

Supplementary Materials

The following are available online at https://www.mdpi.com/article/10.3390/pr10122551/s1, Table S1：List of Kits used in the current study; Table S2: The sequences of real time PCR primers.

Author Contributions

Conceptualization E.A.A. and A.M.A.; methodology K.W.A.-B. and E.A.A.; software K.W.A.-B. and E.A.A.; validation K.W.A.-B., A.M.A. and E.A.A.; formal analysis E.A.A.; investigation K.W.A.-B. and E.A.A.; resources, K.W.A.-B. and E.A.A. data curation E.A.A.; writing—original draft preparation K.W.A.-B. and E.A.A.; writing—review and editing, K.W.A.-B., A.M.A. and E.A.A.; visualization A.M.A.; supervision, A.M.A. and E.A.A.; project administration, A.M.A. and E.A.A. funding acquisition K.W.A.-B., A.M.A. and E.A.A. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by Deanship of Scientific Research of King Faisal University, Annual GRANT927.

Institutional Review Board Statement

All animal procedures were performed according to standard protocols and ethical rules (approved by King Faisal University, KFU, under number KFU2020-2-7).

Acknowledgments

Authors acknowledge Deanship of Scientific Research of King Faisal University, Saudi Arabia for providing the annual grant number GRANT927.

Conflicts of Interest

The authors declare no conflict of interest.

References

  1. American Diabetes Association. 2. Classification and Diagnosis of Diabetes: Standards of Medical Care in Diabetes-2018. Diabetes Care 2018, 41 (Suppl. S1), S13–S27. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  2. Metzger, B.E. Long-term Outcomes in Mothers Diagnosed with Gestational Diabetes Mellitus and Their Offspring. Clin. Obstet. Gynecol. 2007, 50, 972–979. [Google Scholar] [CrossRef]
  3. Bellamy, L.; Casas, J.-P.; Hingorani, A.D.; Williams, D. Type 2 diabetes mellitus after gestational diabetes: A systematic review and meta-analysis. Lancet 2009, 373, 1773–1779. [Google Scholar] [CrossRef] [PubMed]
  4. Kim, S.Y.; England, J.L.; Sharma, J.A.; Njoroge, T. Gestational Diabetes Mellitus and Risk of Childhood Overweight and Obesity in Offspring: A Systematic Review. Exp. Diabetes Res. 2011, 2011, 541308. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  5. Philipps, L.H.; Santhakumaran, S.; Gale, C.; Prior, E.; Logan, K.M.; Hyde, M.J.; Modi, N. The diabetic pregnancy and offspring BMI in childhood: A systematic review and meta-analysis. Diabetologia 2011, 54, 1957–1966. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  6. Buchanan, T.A.; Xiang, A.H.; Page, K.A. Gestational diabetes mellitus: Risks and management during and after pregnancy. Nat. Rev. Endocrinol. 2012, 8, 639–649. [Google Scholar] [CrossRef] [Green Version]
  7. Kawasaki, M.; Arata, N.; Miyazaki, C.; Mori, R.; Kikuchi, T.; Ogawa, Y.; Ota, E. Obesity and abnormal glucose tolerance in offspring of diabetic mothers: A systematic review and meta-analysis. PLoS ONE 2018, 13, e0190676. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  8. Shou, C.; Wei, Y.-M.; Wang, C.; Yang, H.-X. Updates in Long-term Maternal and Fetal Adverse Effects of Gestational Diabetes Mellitus. Matern.-Fetal Med. 2019, 1, 91–94. [Google Scholar] [CrossRef]
  9. Lowe, W.L., Jr.; Lowe, L.P.; Kuang, A.; Catalano, P.M.; Nodzenski, M.; Talbot, O.; Tam, W.H.; Sacks, D.A.; McCance, D.; Linder, B.; et al. Maternal glucose levels during pregnancy and childhood adiposity in the hyperglycemia and adverse pregnancy outcome follow-up study. Diabetologia 2019, 62, 598–610. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  10. Perna, R.; Loughan, A.R.; Le, J.; Tyson, K. Gestational Diabetes: Long-Term Central Nervous System Developmental and Cognitive Sequelae. Appl. Neuropsychol. Child 2014, 4, 217–220. [Google Scholar] [CrossRef] [PubMed]
  11. Van Lieshout, R.J.; Voruganti, L.P. Diabetes mellitus during pregnancy and increased risk of schizophrenia in offspring: A review of the evidence and putative mechanisms. J. Psychiatry Neurosci. 2008, 33, 395–404. [Google Scholar] [PubMed]
  12. Hami, J.; Shojae, F.; Vafaee-Nezhad, S.; Lotfi, N.; Kheradmand, H.; Haghir, H. Some of the experimental and clinical aspects of the effects of the maternal diabetes on developing hippocampus. World J. Diabetes 2015, 6, 412–422. [Google Scholar] [CrossRef]
  13. Pribylová, H.; Dvoráková, L.; Stroufová, A. Offspring of diabetic mothers in adulthood and their glucose tolerance. Czechoslov. Med. 1989, 12, 87–95. [Google Scholar]
  14. Condrat, C.E.; Thompson, D.C.; Barbu, M.G.; Bugnar, O.L.; Boboc, A.; Cretoiu, D.; Suciu, N.; Cretoiu, S.M.; Voinea, S.C. miRNAs as Biomarkers in Disease: Latest Findings Regarding Their Role in Diagnosis and Prognosis. Cells 2020, 9, 276. [Google Scholar] [CrossRef] [Green Version]
  15. Hanieh, H.; Ahmed, E.A.; Vishnubalaji, R.; Alajez, N.M. SOX4: Epigenetic regulation and role in tumorigenesis. Semin. Cancer Biol. 2020, 67 Pt 1, 91–104. [Google Scholar] [CrossRef]
  16. Durga, K.D.; Adhisivam, B.; Vidya, G.; Bhat, B.V.; Bobby, Z.; Chand, P. Oxidative stress and DNA damage in newborns born to mothers with hyperglycemia—A prospective cohort study. J. Matern. Fetal Neonatal Med. 2018, 31, 2396–2401. [Google Scholar] [CrossRef] [PubMed]
  17. Castilla-Peon, M.F.; Bravo, P.G.M.; Sánchez-Urbina, R.; Gallardo-Montoya, J.M.; Soriano-López, L.C.; Cruz, F.M.C. Diabetes and obesity during pregnancy are associated with oxidative stress genotoxicity in newborns. J. Périnat. Med. 2019, 47, 347–353. [Google Scholar] [CrossRef] [PubMed]
  18. Zamanian Azodi, M.; Rezaei-Tavirani, M.; Rezaei-Tavirani, M.; Robati, R.M. Gestational Diabetes Mellitus Regulatory Network Identifies hsa-miR-145-5p and hsa-miR-875-5p as Potential Biomarkers. Int. J. Endocrinol. Metab. 2019, 17, e86640. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  19. Al-Kafaji, G.; Al-Mahroos, G.; Alsayed, N.A.; Hasan, Z.A.; Nawaz, S.; Bakhiet, M. Peripheral blood microRNA-15a is a potential biomarker for type 2 diabetes mellitus and pre-diabetes. Mol. Med. Rep. 2015, 12, 7485–7490. [Google Scholar] [CrossRef] [Green Version]
  20. Jiménez-Lucena, R.; Rangel-Zúñiga, O.A.; Alcalá-Díaz, J.F.; López-Moreno, J.; Roncero-Ramos, I.; Molina-Abril, H.; Yubero-Serrano, E.M.; Caballero-Villarraso, J.; Delgado-Lista, J.; Castaño, J.P.; et al. Circulating miRNAs as Predictive Biomarkers of Type 2 Diabetes Mellitus Development in Coronary Heart Disease Patients from the CORDIOPREV Study. Mol. Ther. Nucleic Acids 2018, 12, 146–157. [Google Scholar] [CrossRef]
  21. Pescador, N.; Pérez-Barba, M.; Ibarra, J.M.; Corbatón, A.; Martínez-Larrad, M.T.; Serrano-Ríos, M. Serum Circulating microRNA Profiling for Identification of Potential Type 2 Diabetes and Obesity Biomarkers. PLoS ONE 2013, 8, e77251. [Google Scholar] [CrossRef]
  22. Parrizas, M.; Brugnara, L.; Esteban, Y.; Gonzalez-Franquesa, A.; Canivell, S.; Murillo, S.; Gordillo-Bastidas, E.; Cussó, R.; Cadefau, J.A.; Garcia-Roves, P.M.; et al. Circulating miR-192 and miR-193b Are Markers of Prediabetes and Are Modulated by an Exercise Intervention. J. Clin. Endocrinol. Metab. 2015, 100, E407–E415. [Google Scholar] [CrossRef] [Green Version]
  23. Cui, X.; You, L.; Zhu, L.; Wang, X.; Zhou, Y.; Li, Y.; Wen, J.; Xia, Y.; Wang, X.; Ji, C.; et al. Change in circulating microRNA profile of obese children indicates future risk of adult diabetes. Metabolism 2018, 78, 95–105. [Google Scholar] [CrossRef] [Green Version]
  24. Takahara, S.; Lee, S.Y.; Iwakura, T.; Oe, K.; Fukui, T.; Okumachi, E.; Arakura, M.; Sakai, Y.; Matsumoto, T.; Matsushita, T.; et al. Altered microRNA profile during fracture healing in rats with diabetes. J. Orthop. Surg. Res. 2020, 15, 135. [Google Scholar] [CrossRef]
  25. Shen, Y.; Chen, L.; Zhang, S.; Xie, L. Correlation Between SIRT2 3’UTR Gene Polymorphism and the Susceptibility to Alzheimer’s Disease. J. Mol. Neurosci. 2020, 70, 878–886. [Google Scholar] [CrossRef] [PubMed]
  26. Seyhan, A.A.; Lopez, Y.O.N.; Xie, H.; Yi, F.; Mathews, C.; Pasarica, M.; Pratley, R.E. Pancreas-enriched miRNAs are altered in the circulation of subjects with diabetes: A pilot cross-sectional study. Sci. Rep. 2016, 6, 31479. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  27. Baldeón, R.L.; Weigelt, K.; de Wit, H.; Özcan, B.; van Oudenaren, A.; Sempértegui, F.; Sijbrands, E.; Grosse, L.; Freire, W.; Drexhage, H.A.; et al. Decreased Serum Level of miR-146a as Sign of Chronic Inflammation in Type 2 Diabetic Patients. PLoS ONE 2014, 9, e115209. [Google Scholar] [CrossRef] [Green Version]
  28. Villard, A.; Marchand, L.; Thivolet, C.; Rome, S. Diagnostic Value of Cell-free Circulating MicroRNAs for Obesity and Type 2 Diabetes: A Meta-analysis. J. Mol. Biomark. Diagn. 2015, 6, 251. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  29. Ding, R.; Guo, F.; Zhang, Y.; Liu, X.-M.; Xiang, Y.-Q.; Zhang, C.; Liu, Z.-W.; Sheng, J.-Z.; Huang, H.-F.; Zhang, J.-Y.; et al. Integrated Transcriptome Sequencing Analysis Reveals Role of miR-138-5p/ TBL1X in Placenta from Gestational Diabetes Mellitus. Cell. Physiol. Biochem. 2018, 51, 630–646. [Google Scholar] [CrossRef]
  30. Weale, C.J.; Matshazi, D.M.; Davids, S.F.G.; Raghubeer, S.; Erasmus, R.T.; Kengne, A.P.; Davison, G.M.; Matsha, T.E. Circulating miR-30a-5p and miR-182-5p in Prediabetes and Screen-Detected Diabetes Mellitus. Diabetes Metab. Syndr. Obes. Targets Ther. 2020, 13, 5037–5047. [Google Scholar] [CrossRef]
  31. Weale, C.J.; Matshazi, D.M.; Davids, S.F.G.; Raghubeer, S.; Erasmus, R.T.; Kengne, A.P.; Davison, G.M.; Matsha, T.E. Expression Profiles of Circulating microRNAs in South African Type 2 Diabetic Individuals on Treatment. Front. Genet. 2021, 12, 702410. [Google Scholar] [CrossRef] [PubMed]
  32. Gillet, V.; Ouellet, A.; Stepanov, Y.; Rodosthenous, R.S.; Croft, E.K.; Brennan, K.; Abdelouahab, N.; Baccarelli, A.; Takser, L. miRNA Profiles in Extracellular Vesicles From Serum Early in Pregnancies Complicated by Gestational Diabetes Mellitus. J. Clin. Endocrinol. Metab. 2019, 104, 5157–5169. [Google Scholar] [CrossRef]
  33. Houshmand-Oeregaard, A.; Schrölkamp, M.; Kelstrup, L.; Hansen, N.S.; Hjort, L.; Thuesen, A.C.B.; Broholm, C.; Mathiesen, E.R.; Clausen, T.D.; Vaag, A.A.; et al. Increased expression of microRNA-15a and microRNA-15b in skeletal muscle from adult offspring of women with diabetes in pregnancy. Hum. Mol. Genet. 2018, 27, 1763–1771. [Google Scholar] [CrossRef] [PubMed]
  34. Csonka, C.; Szilvassy, Z.; Pali, T.; Blasig, I.E.; Tosaki, A.; Schulz, R.; Ferdinandy, P. Classic preconditioning decreases the harmful accumulation of nitric oxide during ischaemia and reperfusion in rat hearts. Circulation 1999, 100, 2260–2266. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  35. Kamath, U.; Rao, G.; Raghothama, C.; Rai, L.; Rao, P. Erythrocyte indicators of oxidative stress in gestational diabetes. Acta Paediatr. 1998, 87, 676–679. [Google Scholar] [CrossRef] [PubMed]
  36. Filardi, T.; Catanzaro, G.; Mardente, S.; Zicari, A.; Santangelo, C.; Lenzi, A.; Morano, S.; Ferretti, E. Non-Coding RNA: Role in Gestational Diabetes Pathophysiology and Complications. Int. J. Mol. Sci. 2020, 21, 4020. [Google Scholar] [CrossRef]
  37. Jiang, B.; Kumar, S.D.; Loh, W.T.; Manikandan, J.; Ling, E.-A.; Tay, S.S.W.; Dheen, S.T. Global gene expression analysis of cranial neural tubes in embryos of diabetic mice. J. Neurosci. Res. 2008, 86, 3481–3493. [Google Scholar] [CrossRef]
  38. Liadis, N.; Murakami, K.; Eweida, M.; Elford, A.R.; Sheu, L.; Gaisano, H.Y.; Hakem, R.; Ohashi, P.S.; Woo, M. Caspase-3-Dependent β-Cell Apoptosis in the Initiation of Autoimmune Diabetes Mellitus. Mol. Cell. Biol. 2005, 25, 3620–3629. [Google Scholar] [CrossRef] [Green Version]
  39. Stump, M.; Guo, D.-F.; Lu, K.-T.; Mukohda, M.; Cassell, M.D.; Norris, A.W.; Rahmouni, K.; Sigmund, C.D. Nervous System Expression of PPARγ and Mutant PPARγ Has Profound Effects on Metabolic Regulation and Brain Development. Endocrinology 2016, 157, 4266–4275. [Google Scholar] [CrossRef] [PubMed]
  40. Luan, B.; Sun, C. MiR-138-5p affects insulin resistance to regulate type 2 diabetes progression through inducing autophagy in HepG2 cells by regulating SIRT1. Nutr. Res. 2018, 59, 90–98. [Google Scholar] [CrossRef] [PubMed]
  41. Yang, C.; Song, G.; Lim, W. Effects of extracellular vesicles on placentation and pregnancy disorders. Reproduction 2019, 158, R189–R196. [Google Scholar] [CrossRef] [PubMed]
  42. Jian, B.; Li, Z.; Xiao, D.; He, G.; Bai, L.; Yang, Q. Downregulation of microRNA-193-3p inhibits tumor proliferation migration and chemoresistance in human gastric cancer by regulating PTEN gene. Tumor Biol. 2016, 37, 8941–8949. [Google Scholar] [CrossRef] [PubMed]
  43. Groot, M.; Lee, H. Sorting Mechanisms for MicroRNAs into Extracellular Vesicles and Their Associated Diseases. Cells 2020, 9, 1044. [Google Scholar] [CrossRef] [PubMed]
  44. Zhang, J.; Li, X.-Y.; Hu, P.; Ding, Y.-S. lncRNA NORAD Contributes to Colorectal Cancer Progression by Inhibition of miR-202-5p. Oncol. Res. Featur. Preclin. Clin. Cancer Ther. 2018, 26, 1411–1418. [Google Scholar] [CrossRef] [PubMed]
  45. Yu, H.-Y.; Pan, S.-S. MiR-202-5p suppressed cell proliferation, migration and invasion in ovarian cancer via regulating HOXB2. Eur. Rev. Med. Pharmacol. Sci. 2020, 24, 2256–2263. [Google Scholar] [PubMed]
  46. Boese, A.S.; Saba, R.; Campbell, K.; Majer, A.; Medina, S.; Burton, L.; Booth, T.F.; Chong, P.; Westmacott, G.; Dutta, S.M.; et al. MicroRNA abundance is altered in synaptoneurosomes during prion disease. Mol. Cell. Neurosci. 2016, 71, 13–24. [Google Scholar] [CrossRef] [PubMed]
  47. He, F.; Peng, Y.; Yang, Z.; Ge, Z.; Tian, Y.; Ma, T.; Li, H. Activated ClC-2 Inhibits p-Akt to Repress Myelination in GDM Newborn Rats. Int. J. Biol. Sci. 2017, 13, 179–188. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  48. Magee, T.R.; Ross, M.G.; Wedekind, L.; Desai, M.; Kjos, S.; Belkacemi, L. Gestational diabetes mellitus alters apoptotic and inflammatory gene expression of trophobasts from human term placenta. J. Diabetes its Complicat. 2014, 28, 448–459. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  49. Piazza, F.V.; Segabinazi, E.; de Meireles, A.L.F.; Mega, F.; Spindler, C.D.F.; Augustin, O.A.; Salvalaggio, G.D.S.; Achaval, M.; Kruse, M.S.; Coirini, H.; et al. Severe Uncontrolled Maternal Hyperglycemia Induces Microsomia and Neurodevelopment Delay Accompanied by Apoptosis, Cellular Survival, and Neuroinflammatory Deregulation in Rat Offspring Hippocampus. Cell. Mol. Neurobiol. 2019, 39, 401–414. [Google Scholar] [CrossRef] [PubMed]
  50. Wójcik, M.; Mac-Marcjanek, K.; Nadel, I.; Woźniak, L.; Cypryk, K. Clinical research Gestational diabetes mellitus is associated with increased leukocyte peroxisome proliferator-activated receptor γ expression. Arch. Med. Sci. 2015, 4, 779–787. [Google Scholar] [CrossRef] [Green Version]
  51. Zhao, Q.; Yang, D.; Gao, L.; Zhao, M.; He, X.; Zhu, M.; Tian, C.; Liu, G.; Li, L.; Hu, C. Downregulation of peroxisome proliferator-activated receptor gamma in the placenta correlates to hyperglycemia in offspring at young adulthood after exposure to gestational diabetes mellitus. J. Diabetes Investig. 2019, 10, 499–512. [Google Scholar] [CrossRef] [PubMed]
  52. Gao, Y.; She, R.; Sha, W. Gestational diabetes mellitus is associated with decreased adipose and placenta peroxisome proliferator-activator receptor γ expression in a Chinese population. Oncotarget 2017, 8, 113928–113937. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  53. Sousa, R.A.D.; Torres, Y.S.; Figueiredo, C.P.; Passos, G.F.; Clarke, J.R. Consequences of gestational diabetes to the brain and behavior of the offspring. An. Acad. Bras. Ciênc. 2018, 90 (Suppl. S1), 2279–2291. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  54. Vuong, B.; Odero, G.; Rozbacher, S.; Stevenson, M.; Kereliuk, S.M.; Pereira, T.J.; Dolinsky, V.W.; Kauppinen, T.M. Exposure to gestational diabetes mellitus induces neuroinflammation, derangement of hippocampal neurons, and cognitive changes in rat offspring. J. Neuroinflamm. 2017, 14, 80. [Google Scholar] [CrossRef] [Green Version]
  55. Głombik, K.; Trojan, E.; Kurek, A.; Budziszewska, B.; Basta-Kaim, A. Inflammatory Consequences of Maternal Diabetes on the Offspring Brain: A Hippocampal Organotypic Culture Study. Neurotox. Res. 2019, 36, 357–375. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  56. De Sousa, R.A.L. Animal models of gestational diabetes: Characteristics and consequences to the brain and behavior of the offspring. Metab. Brain Dis. 2021, 36, 199–204. [Google Scholar] [CrossRef] [PubMed]
  57. Sun, L.-L.; Jiang, B.-G.; Li, W.-T.; Zou, J.-J.; Shi, Y.-Q.; Liu, Z.-M. MicroRNA-15a positively regulates insulin synthesis by inhibiting uncoupling protein-2 expression. Diabetes Res. Clin. Pract. 2011, 91, 94–100. [Google Scholar] [CrossRef]
  58. Garcia-Diaz, D.F.; Camacho-Guillén, P.; Codner, E.; Pérez-Bravo, F. miR15a and miR16 in Chilean type 1 diabetes patients: Possible association with apoptosis, inflammatory, or autoimmunity markers. J. Endocrinol. Investig. 2018, 41, 1083–1088. [Google Scholar] [CrossRef]
  59. Sedgeman, L.R.; Beysen, C.; Allen, R.M.; Solano, M.A.R.; Turner, S.M.; Vickers, K.C. Intestinal bile acid sequestration improves glucose control by stimulating hepatic miR-182-5p in type 2 diabetes. Am. J. Physiol. Gastrointest. Liver Physiol. 2018, 315, G810–G823. [Google Scholar] [CrossRef] [PubMed]
  60. Gao, F.; Jiang, X.; Li, Q.; Fan, K. Association of miR-182 expression in amniotic fluid with intrauterine infection and brain injury in premature infants. Exp. Ther. Med. 2018, 16, 1905–1909. [Google Scholar] [CrossRef] [PubMed]
  61. Gu, S.; Liu, Y.; Zou, J.; Wang, W.; Wei, T.; Wang, X.; Zhu, L.; Zhang, M.; Zhu, J.; Xie, T.; et al. Retinal pigment epithelial cells secrete miR-202-5p-containing exosomes to protect against proliferative diabetic retinopathy. Exp. Eye Res. 2020, 201, 108271. [Google Scholar] [CrossRef] [PubMed]
  62. Jaeger, A.; Zollinger, L.; Saely, C.H.; Muendlein, A.; Evangelakos, I.; Nasias, D.; Charizopoulou, N.; Schofield, J.D.; Othman, A.; Soran, H.; et al. Circulating microRNAs-192 and -194 are associated with the presence and incidence of diabetes mellitus. Sci. Rep. 2018, 8, 14274. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  63. Hu, H.; Zhao, M.; Li, Z.; Nie, H.; He, J.; Chen, Z.; Yuan, J.; Guo, H.; Zhang, X.; Yang, H.; et al. Plasma miR-193b-3p Is Elevated in Type 2 Diabetes and Could Impair Glucose Metabolism. Front. Endocrinol. 2022, 13, 814347. [Google Scholar] [CrossRef] [PubMed]
  64. Nie, H.; Hu, H.; Li, Z.; Wang, R.; He, J.; Li, P.; Li, W.; Cheng, X.; An, J.; Zhang, Z.; et al. Associations of plasma metal levels with type 2 diabetes and the mediating effects of microRNAs. Environ. Pollut. 2022, 292, 118452. [Google Scholar] [CrossRef] [PubMed]
  65. Sabbagh, F.; Muhamad, I.I.; Niazmand, R.; Dikshit, P.K.; Kim, B.S. Recent progress in polymeric non-invasive insulin delivery. Int. J. Biol. Macromol. 2022, 203, 222–243. [Google Scholar] [CrossRef] [PubMed]
Processes 10 02551 g001 550
Figure 1. Untreated diabetes induces oxidative stress and inflammation in mothers and inflammatory response in 5-week-old offspring. (A). Lipid peroxidation products, MDA and NO induction in control and diabetic mothers and their offspring. (B). Untreated diabetes during pregnancy affects the proinflammatory cytokines level in serum of mothers and offspring. p ≤ 0.05 * significant variation, p ≤ 0.01 ** high significant variation. a versus appropriate control; (control mothers, Con M and 5-week-old offspring, con 5 wk), b relative to diabetic mothers (Diab M, n = 4–5.)
Figure 1. Untreated diabetes induces oxidative stress and inflammation in mothers and inflammatory response in 5-week-old offspring. (A). Lipid peroxidation products, MDA and NO induction in control and diabetic mothers and their offspring. (B). Untreated diabetes during pregnancy affects the proinflammatory cytokines level in serum of mothers and offspring. p ≤ 0.05 * significant variation, p ≤ 0.01 ** high significant variation. a versus appropriate control; (control mothers, Con M and 5-week-old offspring, con 5 wk), b relative to diabetic mothers (Diab M, n = 4–5.)
Processes 10 02551 g001
Processes 10 02551 g002a 550Processes 10 02551 g002b 550
Figure 2. Untreated diabetes during pregnancy induces oxidative stress and increases the rate of apoptosis and the expression levels of PPARˠ in brain tissue. p ≤ 0.05 * significant variation, p ≤ 0.01 ** high significant variation. (A) versus appropriate control (mothers, newborn pups and 5-week-old offspring), (B) relative to diabetic mothers, (C) relative to diabetic pups. n = 4.
Figure 2. Untreated diabetes during pregnancy induces oxidative stress and increases the rate of apoptosis and the expression levels of PPARˠ in brain tissue. p ≤ 0.05 * significant variation, p ≤ 0.01 ** high significant variation. (A) versus appropriate control (mothers, newborn pups and 5-week-old offspring), (B) relative to diabetic mothers, (C) relative to diabetic pups. n = 4.
Processes 10 02551 g002aProcesses 10 02551 g002b
Processes 10 02551 g003 550
Figure 3. Histopathological alterations in cerebral hemispheres of brain of control (A,B) and diabetic mothers (C–G). Vacuolated neuropil (VN), abnormal appearance or hemorrhage (HE), infiltration of inflammatory cells (arrowheads), blood capillary (B cap), myelinated nerve cells (Myl). n = 4–5, magnification bar equals 10 um.
Figure 3. Histopathological alterations in cerebral hemispheres of brain of control (A,B) and diabetic mothers (C–G). Vacuolated neuropil (VN), abnormal appearance or hemorrhage (HE), infiltration of inflammatory cells (arrowheads), blood capillary (B cap), myelinated nerve cells (Myl). n = 4–5, magnification bar equals 10 um.
Processes 10 02551 g003
Processes 10 02551 g004 550
Figure 4. Histopathological alterations in cerebral hemispheres of brain of newborn pups (normal, (A) and Diabetic (B)) and 5-week-old offspring (normal, (C,D) and Diabetic (E)). Normal histological appearance is shown with normal neuropil and lightly stained nuclei. (C,D). Section at the cerebral hemisphere of the brain of 5-week-old mice born for normal mothers. Darkly stained nuclei (apoptotic, arrows) and slightly vacuolated neuropil are shown. Slightly vacuolated Neuropil (VN), myelinated nerve cells (Myl). n = 4–5, magnification bar equals 10 um.
Figure 4. Histopathological alterations in cerebral hemispheres of brain of newborn pups (normal, (A) and Diabetic (B)) and 5-week-old offspring (normal, (C,D) and Diabetic (E)). Normal histological appearance is shown with normal neuropil and lightly stained nuclei. (C,D). Section at the cerebral hemisphere of the brain of 5-week-old mice born for normal mothers. Darkly stained nuclei (apoptotic, arrows) and slightly vacuolated neuropil are shown. Slightly vacuolated Neuropil (VN), myelinated nerve cells (Myl). n = 4–5, magnification bar equals 10 um.
Processes 10 02551 g004
Processes 10 02551 g005 550
Figure 5. (A). Glucose level in blood of control and STZ-injected mice (60 mg/kg/bw), 28 h after injection of STZ. (B). LPO products (MDA) in brain of control and STZ-treated mice. (C). miRNAs expression levels in brain tissue of control and STZ-treated mice. No statistical variation between group were found. Data are shown as mean ± SEM, n = 3 mice.
Figure 5. (A). Glucose level in blood of control and STZ-injected mice (60 mg/kg/bw), 28 h after injection of STZ. (B). LPO products (MDA) in brain of control and STZ-treated mice. (C). miRNAs expression levels in brain tissue of control and STZ-treated mice. No statistical variation between group were found. Data are shown as mean ± SEM, n = 3 mice.
Processes 10 02551 g005
Processes 10 02551 g006 550
Figure 6. miRNAs expression in brain tissues of control and diabetic mothers and their offspring. (A–H) The expression levels of the miRNAs are shown, (A) miR-15a-3p, (B) miR-15b-3p, (C) 138-3p, (D) miR-146a-3p, (E) miR-182-5p, (F) miR-193-3p, (G) miR-202-5p and (H) miR-376a-3p. p ≤ 0.05 * significant variation, p ≤ 0.01 ** high significant variation. a, versus appropriate control (mothers, newborn pups, and 5-week-old offspring), b, relative to diabetic mothers, c, relative to diabetic pups. n = 4.
Figure 6. miRNAs expression in brain tissues of control and diabetic mothers and their offspring. (A–H) The expression levels of the miRNAs are shown, (A) miR-15a-3p, (B) miR-15b-3p, (C) 138-3p, (D) miR-146a-3p, (E) miR-182-5p, (F) miR-193-3p, (G) miR-202-5p and (H) miR-376a-3p. p ≤ 0.05 * significant variation, p ≤ 0.01 ** high significant variation. a, versus appropriate control (mothers, newborn pups, and 5-week-old offspring), b, relative to diabetic mothers, c, relative to diabetic pups. n = 4.
Processes 10 02551 g006
Table
Table 1. Relationship Between the Mothers’ Blood Glucose Level and Average Number of Viable Pups.
Table 1. Relationship Between the Mothers’ Blood Glucose Level and Average Number of Viable Pups.
Blood Glucose Level (mg/dL)Number of MothersAverage Number of Viable Pups/Mother
Below 14059 ± 0.7
Between 180 & 24044 ± 0.9 **
Between 240 & 34042 ± 0.7 **
Above 34040
p ≤ 0.01 ** high significant variation.
Table
Table 2. Offspring of Mothers with Untreated Diabetes.
Table 2. Offspring of Mothers with Untreated Diabetes.
Blood Glucose Level (mg/dL)Average Level of Blood Glucose (mg/dL)Number of Mice
Control mother110 ± 24
Diabetic mothers 260 ± 20 ** a4
Offspring of Control mother98 ± 46
Offspring of diabetic mother165 ± 4 ** a6
p ≤ 0.01 ** high significant variation. a versus appropriate control (mothers, newborn pups and 5-week-old offspring).
Table
Table 3. Body Weight of Offspring of Diabetic Mother Versus Control.
Table 3. Body Weight of Offspring of Diabetic Mother Versus Control.
Mouse No.Newborn Pups of Control MothersNewborn Pups of Diabetic Mothers5-Week-Old Offspring of Control Mothers5-Week-Old Offspring of Diabetic Mothers
I1.41.3511.511
II1.271.41311.5
III1.31.2413.412.5
IV1.61.31413.2
V1.551.3613.512.6
Mean body weight (g) ± SD1.42 ± 0.151.33 ± 0.0613.08 ± 0.9512.16 ± 0.89
No significant difference in body weight was seen between diabetic mice and their appropriate control.
Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Share and Cite

MDPI and ACS Style

Al-Bakr, K.W.; Alzahrani, A.M.; Ahmed, E.A. Dysregulation of Mir-193B and Mir-376A as a Biomarker of Prediabetes in Offspring of Gestational Diabetic Mice. Processes 2022, 10, 2551. https://doi.org/10.3390/pr10122551

AMA Style

Al-Bakr KW, Alzahrani AM, Ahmed EA. Dysregulation of Mir-193B and Mir-376A as a Biomarker of Prediabetes in Offspring of Gestational Diabetic Mice. Processes. 2022; 10(12):2551. https://doi.org/10.3390/pr10122551

Chicago/Turabian Style

Al-Bakr, Khawlah W, Abdullah M Alzahrani, and Emad A Ahmed. 2022. "Dysregulation of Mir-193B and Mir-376A as a Biomarker of Prediabetes in Offspring of Gestational Diabetic Mice" Processes 10, no. 12: 2551. https://doi.org/10.3390/pr10122551

Note that from the first issue of 2016, this journal uses article numbers instead of page numbers. See further details here.

Article Metrics

Back to TopTop