Diabetes mellitus (DM) is a syndrome that is a growing health problem, accounting for 10.4% of global mortality. In 2015, hyperglycemia during pregnancy was observed in 16.2% of women (Cho et al., 2018). In the first few weeks of pregnancy, maternal diabetes is intensely linked to higher number of spontaneous abortions and major congenital malformations (Kitzmiller et al., 1996; Ray et al., 2001).

Fetal programming is a theory that suggests that the environment around the developing fetus plays an important role in determining the risk of disease in childhood and adulthood (Entringer et al., 2012). In this sense, factors such as overweight, obesity, and maternal diabetes during pregnancy are known to be effective agents leading to chronic-disease development in offspring (Yessoufou and Moutairou, 2011), showing the relevance of intrauterine environment for health of descendants in the future.

To reproduce maternal hyperglycemia found in Type 2 DM in animal models, streptozotocin (STZ) induction can be performed in the neonatal period of rats (Tsuji et al., 1988; Jawerbaum and White, 2010; Santos et al., 2015; Bequer et al., 2018; Bueno et al., 2020). This type of experimental diabetes is termed as “mild diabetes” (Hauschildt et al., 2018; Machado et al., 2020). Besides diabetic status, a change in lifestyle, especially in dietary patterns related with growing consumption of industrialized foods (high in calories and fat), affects diabetic progress at long term (Hu, 2011; Ley et al., 2014; Popkin, 2015; Krishan et al., 2018). Animals that are fed high-fat (HF) and/or high-sugar (HS) diets showed metabolic changes, such as increased concentrations of glucose, triglycerides (TG), total cholesterol (TC), and obesity (Matias et al., 2018; Zhao et al., 2019). The offspring of mothers who consumed the HF/HS diet had greater fat tissue, glycemia, TG, and TC levels (Martins Terra et al., 2020). Despite the knowledge about the single effect of HF/HS diet and diabetes on the metabolic response in animals, there are still few studies exploring the association of these two variables during pregnancy. Considering that pregnancy is a critical period, where maternal conditions and habits can lead to persistent changes in offspring (Fleming et al., 2015), it is important that the studies are conducted in a manner where new care strategies can be taken.

Thus, the hypothesis of this study is that diabetic rats submitted to the HF/HS diet before and during pregnancy will present exacerbated damage on a biochemical profile, leading to impaired maternal-fetal relationship. Therefore, the aim of this study was to evaluate maternal and fetal repercussions of the diabetes associated with an HF/HS diet offered before and during pregnancy of rats.

The streptozotocin-induced mild diabetes in rat offspring after birth caused a diabetic status. This was confirmed by oral glucose tolerance test (OGTT) and higher area under curve (AUC) data. Before pregnancy, there was no significant difference in fertility rates among the groups. In the experimental model of mild diabetes induction, used in the present study, the fertility rate was around 90%, corroborating Sinzato et al. (2021). In the groups that consumed HS/HF diet, the treatment time was not enough to change the fertility rate. During pregnancy of these rats, the hyperglycemia led to impairment on embryonic development, contributing to embryo losses as verified at term pregnancy. The rats that received high-fat diet and sugar in drinking water at adulthood presented with greater Lee index values, confirming obesity. In addition, these female rats showed higher periovarian and visceral fat weight and hypertriglyceridemia. In this maternal condition, there was higher incidence of small fetuses for gestational age, indicating intrauterine growth restriction (IUGR) associated with obesity. The use of animal models to study maternal association between diabetes and obesity helps understand the functional, biochemical, and morphological changes caused by these connected diseases. Our findings showed that diabetes and obesity status caused maternal hyperglycemia and an abnormal leukocyte profile, contributing to IUGR. In addition, this association led to embryo-fetal losses and the onset of anomalies in the fetuses at the end of pregnancy, confirming the maternal, fetal, and perinatal complications induced by diabetes and obesity during pregnancy.

Metabolic disorders, such as diabetes, may cause hyperphagia condition, but the mechanisms involved are not fully understood (Li et al., 2019). The regulator of food intake is influenced by the balance among appetite, satiety, and energy expenditure, and this biological process is called “energy homeostasis” (Morton et al., 2014; Deemer et al., 2019). This balance is regulated by the central nervous system (Deemer et al., 2019) and multiple metabolic signals, such as leptin (Zhang and Chua, 2017), insulin (Brüning et al., 2000), glucagon-like peptide 1 (GLP1) (Ong et al., 2017), and cholecystokinin (CCK) (Woods et al., 2018). In this study, diabetes and high-fat/high-sugar diet alone did not interfere with daily caloric intake. However, the association between diabetes and abnormal diet might activate neurocircuits, which impaired the controller system of energy homeostasis, influencing in body weight (Morton et al., 2014) and the regulation of caloric intake. Then, even the rats eating less high-fat diet but drinking more sugar in the water presented obesity status. The deregulation of energy consumption is one of the major causes of obesity (Erlanson-Albertsson, 2005), confirmed by body weight (de Almeida et al., 2016), body composition and fat deposits, especially visceral fat (Poirier et al., 2006). Our findings showed that diabetic rats submitted to a high-fat/high-sugar diet showed a reduction in body weight gain during pregnancy, but, even so, they developed obesity, as verified by Lee index, which is a murinometric parameter for obesity classification used in experimental studies (Bernardis and Patterson, 1968; Fernandes et al., 2012). Concomitantly, there was an increased weight of periovarian and visceral fat. In experimental models, the carcass relative fat is one of the variables to indicate obesity (Nascimento et al., 2008; Kim et al., 2017).

The oral glucose tolerance test (OGTT) determines degree of glucose tolerance, expressing the ability of β-pancreatic cells to secrete insulin and tissue sensitivity to this hormone (American Diabetes Association, 2020). There were higher blood glucose values in the OGTT in diabetic groups and consequent increase in the area under the curve (AUC), leading to glucose intolerance and, later, diabetes. Then, once diabetes and obesity have been confirmed, it was demonstrated that diabetic and obese rats presented higher levels of blood total protein and albumin. These biochemical parameters are used in animal nutrition research to evaluate its health (Luca and Reis, 2004). Several processes regulate plasma albumin concentration, including synthesis, distribution, and exogenous albumin loss (Dom and Kaysen, 2003). Roche et al. (2008) and Guerin-Dubourg et al. (2012) described albumin as an antioxidant, and it might be elevated in our animals to compensate the higher levels of reactive oxygen species (ROS) induced by diabetes (Raza et al., 2011; Patche et al., 2017; Sinzato et al., 2019) and obesity (Diniz et al., 2004; Burneiko et al., 2006; De Sibio et al., 2013). Our results showed that obesity, alone or associated with diabetes, causes dyslipidemia. Other authors also verified dyslipidemia in experimental animals (Panchal et al., 2011; Zhou et al., 2014; Hao et al., 2015; Senaphan et al., 2015), and different types of diets influence the lipid profile (Desroches et al., 2006). Lipid metabolism, including lipid absorption, transport, synthesis, and degradation, is a complex process, which can lead to other diseases (Huang and Freter, 2015). Among these, diabetes (Dong et al., 2017), inflammation, atherosclerosis (Joseph et al., 2003), obesity (Kaess et al., 2014), and hypertension (Siri-Tarino and Krauss, 2016) are related. The male rats feeding HFD showed increased blood pressure (Sá et al., 2019), and Hsu et al. (2019) showed that the consumption of HFD during pregnancy of rats was responsible for inducing hypertension in adult offspring.

Considering the hematological profile, the association between diabetes and obesity increased the number of total leukocytes, segmented (mature neutrophils), monocytes, and eosinophil. The total and differential leukocyte count is an important parameter to evaluate conditions related with inflammatory processes (George-Gay and Parker, 2003). Increased leukocyte amount due to deregulation of immune activity caused by adipose tissue expansion contributes to obesity-induced inflammation (Trellakis et al., 2012; Poret et al., 2018). Obesity may cause immunomodulation, inducing a higher ratio from neutrophils to lymphocytes due to increased recruitment and activation of peripheral blood neutrophils to adipose tissue (Elgazar-Carmon et al., 2008; Trellakis et al., 2012). In addition, it can stimulate mobilization of bone marrow monocytes so that they fall into the bloodstream and reach adipose tissue as macrophages (Ghigliotti et al., 2014). In this study, the diabetic rats presented lower levels of monocytes. Monocyte is one of the main leukocyte subtypes and is considered an inflammatory biomarker (Badr et al., 2019), and its influx in perivascular regions and retinal pigment epithelium has been verified (Benhar et al., 2016). Decreased peripheral blood monocyte levels were related to diabetic retinopathy in diabetic adults without potential confounders (Wan et al., 2020), suggesting the onset of the diabetes-induced retinal complication in these dams. Eosinophils are the main regulators of the physiological processes and immune function of perivascular adipose tissue (Withers et al., 2017). According to Maizels and Allen (2011), eosinophils prevent inflammation caused by obesity because it possibly increases the numbers of eosinophils or Th2 cells. This might be explained because the IL-4 and IL-13 secretion signal gamma peroxisome proliferator activated receptor (PPARγ), which, if activated by appropriate lipids, inhibits the expression of genes that promote inflammation (Szanto et al., 2010). Therefore, it is supposed that eosinophilia present in the diabetic and obese group was due to the homeostatic mechanism, tending to minimize the possible inflammation caused by obesity.

The reproductive analysis of the animals in this study showed embryonic losses before and after implantation, which were higher in both diabetic groups, demonstrating the influence of hyperglycemia on the implantation process. Regardless of the degree of severity, hyperglycemia is related to pre- and postimplantation losses in the intrauterine environment (Sinzato et al., 2011; Bequer et al., 2018; Gallego et al., 2018). Moreover, problems with cytokine regulation, which occurs in diabetic pregnancy, can lead to damage during early embryonic development, such as pre-implant failure, leading to a reduced number of implants and postimplantation losses indicated by an increased rate of resorption and a reduced number of live fetuses (Sinzato et al., 2011; Dela Justina et al., 2017). These findings contributed to a lesser weight gain during pregnancy and maternal final weight at term pregnancy. However, obesity did not increase embryo loss rates in the animals.

For the success of pregnancy, it is essential that, during the implantation period, the physiological and molecular processes are coordinated, involving close interactions between the uterus and the blastocyst (Cha et al., 2012). Then, the dams presenting biochemical alterations induced by diabetes, obesity, and both contributed to impaired reproductive outcomes. In relation to fetal development and growth from diabetic and/or obese dams, our study demonstrated that only altered diet caused intrauterine growth restriction, which was confirmed by reduction of the fetal weight, higher percentage of small fetuses for the gestational age, and decline of ossification sites. These findings corroborate other authors since diabetes (Damasceno et al., 2014) and maternal obesity and high-calorie intake (Zou et al., 2017) may impair fetal development. Growth restriction may be related to different maternal adaptations to diet and diet components, with maternal nutrition being a possible factor in intrauterine growth restriction (Howie et al., 2009; Setia and Sridhar, 2009; Mark et al., 2011; Tellechea et al., 2017). The animals that received a high fat/sugar diet had altered placental weights, which may be related to functional or morphological placental alterations. These placental changes may have contributed to the decrease in fetal weight. Intrauterine growth restriction leads to a variety of phenotypes related to the metabolic syndrome in adult children, including hypertension (Tain et al., 2017; Bendix et al., 2020). In addition, the obesity induced by high-fat/high-sugar diet, whether associated or not associated with diabetes, decreased the frequency of fetuses without anomalies. The diabetes status increased skeletal anomalies. The rats presenting diabetes and obesity showed an exacerbated percentage of fetal abnormalities, with an increased frequency of visceral anomalies. Bueno et al. (2020) already demonstrated that maternal hyperglycemia causes an abnormal fetal metabolism, contributing to an increase of visceral anomalies in the offspring of diabetic rats. Besides, fetal metabolic dysregulation may also occur due to the maternal consumption of a carbohydrate and lipid-rich diet (Musial et al., 2017), which interferes with various pathways of the developing organs, such as the liver, skeletal muscle, adipose tissue, brain, and pancreas (Heerwagen et al., 2010).

This study points out strengths as several variables and biomarkers evaluated, using a solid and structured experimental model; however, it is a study performed in laboratory animals and needs more attention for human application. For limitation, we have the lack of measurement of leptin, since it could be used during the discussion of appetite and feed intake. Another limiting factor is the determination of the free fat acid levels to relate to other lipid parameters.

In conclusion, the association between maternal diabetes and obesity induces metabolic, leukocyte, and biochemical alterations that contribute to affect fetal development and growth. Further studies are needed to clear more the mechanisms involved during diabesity in pregnancy to prevent the fetal/neonatal outcomes in humans. Then, the experimental model employed in our study helps understand some pathophysiological mechanisms linked to this association, allowing interventionist methods to avoid maternal changes and, consequently, fetal repercussions as found in this study.

The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author/s.

The animal study was reviewed and approved by The Ethical Committee for Animal Research of Araguaia—UFMT, Brazil (Protocol number 23108.022251/2019-61).

VA-S and GV: conception and design, data acquisition, data analysis and interpretation, and manuscript writing. AS-S, AL, and CB-B: data acquisition, data analysis and interpretation, and manuscript writing. RM-S, TS, VP, and YS: data interpretation and manuscript writing. BK: manuscript writing. DD: data analysis and interpretation and manuscript writing. All authors have reviewed the manuscript, agreed with its contents, consented to its publication, and that there are no other persons who satisfied the criteria for authorship. We further confirm that all of us have approved the order of the authors listed in the manuscript.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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