To our knowledge, this is the first study that assesses the effects of breastfeeding and formula feeding on the expression level of BDNF, LXR-α, PPAR-γ, ACACB, and PTEN genes in infants. Based on the results of this study, anthropometric indices and biochemical variables including 24th -month weight, 24th -month height, birth head circumference, 24th -month head circumference, insulin, TC, LDL-C, and HDL-C in infants that fed exclusively with breast-milk were lower than others (Tables 3 and 5). Moreover, the expression levels of BDNF, LXR-α, and PPAR-γ genes were higher in breastfeeding infants in comparison to others, while ACACB gene expression was lower (Table 7).
Breastfeeding has reciprocal health beneficial effects on mothers and their children. Different studies revealed that the risk of type 2 diabetes, metabolic and cardiovascular disease, ovarian, and breast cancer may reduce in mothers with regular breastfeeding. On the other hand, the risk of childhood and adolescent overweight in infants who exclusively breastfeed is 22–24% lower than formula-fed infants (39).
Numerous investigations have demonstrated that breastfeeding can prevent obesity and adiposity in adolescence and adulthood via desirable effects on anthropometric indices and biochemical parameters (40–42). In this way, Gopinath et al. showed that the duration of breastfeeding is significantly associated with lower BMI levels and each month the increase in breastfeeding is associated with an average reduction in BMI about 0.04 kg/m2 and 0.03 kg/m2 among children aged 1–2 years and 3–4 years, respectively. As well, each month increase in breastfeeding duration is associated with a 0.06 cm decrease in waist circumference in 12–24 month-year-old children (43). Moreover, different meta-analysis and high-quality studies showed that breastfeeding can reduce the risk of diabetes and significantly decrease the incidence of overweight/obesity by 13% in both high-income and low- or middle-income countries in childhood (40, 44, 45). Similarly, our investigation showed that exclusive breastfeeding can prevent obesity in early life via modification of anthropometric indices.
Besides, Kimpimäki et al. revealed that short-term exclusive breastfeeding and early initiation of formula feeding that is based on cow’s milk can trigger progressive signs of beta-cell autoimmunity in children who are genetically susceptible to type Ι diabetes (13). There are different proteins and constituents in the cow’s milk like; α- and β-lactoglobulin, Ig E, casein, and bovine serum albumin antibodies that stimulate the immune system in a damaging way on β-cells and trigger type Ι diabetes in the first year of life (46–49).
In addition, the results from a cohort study demonstrated that the risk of elevated weight gain at the age of 2 years between infants who breastfed for less than 6 months is more than infants who fed for 6 months or more. These findings showed that the duration of breastfeeding is highly related to the risk of overweight, so that breastfeeding for at least 6 months decreases the risk of weight gain in comparison to infants who were exclusively breastfed 1 month at most (50).
The first few months of life is an important period of life that growth is very fast and the body weight doubles during 4–6 months (51, 52). Therefore, in this period of life, the type of feeding has a key role in the healthy growth of infants and prevention of overweight and obesity in the future. According to different studies, fast weight gain during the first 4 months of life was associated with a higher risk of overweight at the age of 7 years, independent of birth weight and weight at the age of 1 year (53, 54).
The particular role of breastfeeding on future overweight and obesity is related to human milk’s exceptional biochemical constituents. The nutritive and non-nutritive components of breast milk provide all the requirements of infants and moderate their energy balance. The unique bioactive substances and lower protein, fat, and insulin content of breast milk can regulate appetite, fat deposition, and metabolic responses (50, 55). Moreover, some components of breast milk such as hormones, growth factors, neuropeptides, and anti-inflammatory and immune-modulating agents affect the growth, development, and function of the gastrointestinal (GI) tract during early infancy (55). One of the exclusive components of breast milk is the human milk oligosaccharides (HMOs) that do not exist in infant formula. These complex sugars improve the gut microbiome and immune system of infants and recently have been suggested as a possible link between breastfeeding and lower obesity risk (56).
Besides, the energy density of human milk is lower than formula milk, and children who breastfed have better self-control on energy intake compared to formula feeding infants (57–59). Some investigations suggested that the intake of high protein and nutrients reduces the basal metabolic rate and rose adiposity in rats and humans more than 10 percent (60, 61). Generally, formula-fed infants intake more protein than breastfed infants, which is related to an increased risk of obesity via higher adipogenic activity and adipocyte differentiation (62). High protein intake may decrease the secretion of growth hormone (GH) that leads to lower lipolysis of fat mass and affects the plasma free fatty acids (62, 63). Moreover, consuming formula has some growth accelerating factors that accelerate the growth rate of infants in early life and make them overweight in their later life (54, 64).
It seems that the most important factor that makes the difference between the two types of feeding is feeding behavior and mother-child contact, so that formula-fed infants have a lower frequency of meals, longer time between meals, and different suckling patterns in comparison to breastfed infants (60, 65, 66). On the other hand, breastfed infants can control the quantity, duration, and frequency of their meals based on their feeling of hunger and satiation (60, 67).
Besides, the type of feeding can influence the insulin level and lipid profile of infants. The results of our study showed a significant difference in the insulin and lipid profiles of infants based on their type of feeding. The exclusively breastfed infants have significantly lower insulin, TC, LDL-C, and HDL-C levels compared to formula-fed and mixed-fed groups.
The high protein content of the formula can stimulate the insulin secretion of formula-fed infants and is positively related to the risk of overweight. Different investigations showed that the infants who fed with the formula possess higher postprandial plasma insulin level and have a prolonged insulin response on day 6 of their life compared to breastfed infants (53, 68). Intake of higher protein levels lead to the production of higher insulin and insulin-like growth factor (IGF-I) concentrations, which raises adipose tissue deposition and the risk of overweight, obesity, and type 2 diabetes in the coming years (69).
In line with our results, Hui et al. revealed that breastfeeding for the first 3 months is able to decrease TC, LDL-C, and TG at adolescence. Furthermore, the difference between the lipid profile of mixed feeding and formula feeding groups wasn’t significant, except for HDL-C that was lower in the mix-fed group (70). On the contrary, Thorsdottir et al. showed that the LDL-C level in boy infants who breastfed for 48 months was significantly higher and the TC level was not significantly more in 12 months’ infants who breastfed at the ages of 2, 4, 6, 9, and 12 months. The HDL-C level was significantly lower in infants who had been breastfed at the ages of 4 and 9 months. As well, in this study, the TG level was not significantly associated with breastfeeding (71). Similarly, Harit et al. demonstrated that exclusively breastfed infants had significantly higher TC and LDL-C compared to mixed-fed infants at both 14 weeks and 6 months. Besides, at 14 weeks, TG was significantly higher in breastfed infants compared to mixed fed. Moreover, Teller et al. revealed that cholesterol and lipoprotein concentrations in breastfed infants were higher than formula-fed infants (72). It seems that the high lipid profile in exclusively breastfed infants is beneficial for cognitive growth and lipid metabolism (73). The results of our study showed that breastfeeding can significantly reduce TC and LDL-C in comparison to formula feeding and mix-feeding. The controversy between our findings and other studies may be due to different methodologies, type of blood sample (venous/capillary), the time interval between last feeding and sampling, type of formula, preparation of formula (dilution), and genetic variations. Furthermore, our findings showed that the expression levels of PPAR-γ and ACACB genes were significantly different between groups. In contrast, the statistical analysis between the three groups for BDNF, LXR-α, and PTEN genes was not significant (Table 7). The comparison of the breastfeeding group with formula feeding and mix-feeding groups indicated significant differences for ACACB gene expression (P < 0.0001). This comparison for PPAR-γ gene expression was significant just for breastfeeding and formula feeding groups (P = 0.008). The family of peroxisome proliferation-activated receptors consists of three isoforms: 1) PPAR-α, PPAR-β/δ, and PPAR-γ, which differ from each other in terms of their physiological roles, ligand specificity, and tissue distribution. PPAR-γ is abundantly expressed in white and brown adipose tissue, the large intestine and spleen (74). These isoforms play an important roles in controlling the expression of genes involved in glucose and lipid homeostasis, cell differentiation, morphogenesis, and inflammatory response (75). Due to the significant role of PPAR-γ in macronutrient metabolism, it is a good target for synthetic insulin sensitizers like; thiazolidinediones in the treatment of type 2 diabetes mellitus (76). Based on different studies, the secretion of some effective adipocytokines on insulin sensitivity such as leptin and adiponectin, was maintained via the activation of PPAR-γ in adipocytes (77).
Acetyl-CoA carboxylase (ACAC) is an important enzyme in the synthesis of malonyl-CoA that involves in fatty acid metabolism. Two isoforms of ACAC are ACACA and ACACB, which encode by genes located in chromosomes 17 and 12, respectively. ACACB is predominantly expressed in the heart and skeletal muscle and catalyzes the carboxylation of acetyl-CoA to malonyl-CoA, which regulates the amount of fatty acid entrance into the mitochondria and fatty acid oxidation via modulation of carnitine palmitoyltransferase-1. Therefore, ACACB plays an important role in fatty acid synthesis and oxidation pathways, and any disruption in these pathways is related to impaired insulin sensitivity and metabolic syndrome (MetS) (18, 78, 79). Our investigation showed that breastfeeding in comparison to formula feeding and mix-feeding can decrease the expression level of the ACACB gene. In this way, Ma et al. revealed that ACACB single-nucleotide polymorphism (SNP rs2268388) is related to BMI in general population and to obesity in subjects with type 2 diabetes and affects gene expression in adipose and hepatic tissue. They suggested that ACACB has a significant role in obesity and a potential role in lipid metabolism changes in type 2 diabetes (T2DM)-associated nephropathy (80). Moreover, Abu-Elheiga et al. demonstrated that ACACB knock-out mice are morphologically normal, grow at the expected rate, and breed normally, whereas they have higher fatty acid oxidation rate and lower fat and glycogen storage in their adipose tissue and liver respectively (24). Tang et al. showed that a SNP in the ACACB gene is associated with susceptibility to type 2 diabetic nephropathy (T2DN). They suggested that targeting this pathway can be a new treatment option for the prevention of diabetic nephropathy (81). In addition, Riancho et al. revealed that different polymorphisms of the ACACB gene play an important role in energy metabolism in postmenopausal women and predispose them to obesity and type 2 diabetes (18). It seems that the ACACB gene plays an important role in the development of obesity and diabetes by reducing fat oxidation, and breastfeeding in childhood can be a good way to prevent its adverse effects in adulthood. There are some effective biological compounds in humane milk that have epigenetic effects via triggering some processes such as; histone modification, DNA methylation, and chromatin remodeling. In addition, these active compounds are able to decrease or increase the expression level of different key genes in the metabolic pathways and may prevent weight gain or subsequent non-communicable diseases in later life. As well, our previous investigation revealed that breastfeeding can influence the key genes in the obesity pathway and decrease the expression level of fat mass and obesity-associated (FTO) and carnitine palmitoyltransferase IA (CPT1A) genes and increase the expression level of the PPAR-α gene in 5–6 month’s infants (82). Likewise, in the present study, we investigated the effects of breastfeeding on other important genes in obesity and diabetes pathways like; BDNF, LXR-α, PPAR-γ, ACACB, and PTEN and found that breastfeeding significantly affected PPAR-γ and ACACB genes that confirmed the epigenetic effects of humane milk which is deficient or absent in formula feeding. These findings elucidated some unclear mechanisms of humane milk against obesity and diabetes. There are some limitations with this study, including low sample size and recall bias. By assessment of more infants, we could acquire more reliable and accurate outcomes. Since the duration and type of feeding were obtained via recall, the possibility of recall bias may exist. Also, due to the nature of cross-sectional studies, we couldn’t found cause-effect relationships. In this study, the data about the volume, calories, and components of the consumed formula feeding was not available, and so we couldn’t achieve the association between calorie intake, formula components, and the expression level of investigated genes. In addition, due to financial constraints, we were unable to examine all obesity-related genes in infants. The strength of this study was the infants which consisted of both sexes, acceptable response and participation rate, and standardized anthropometric measurement protocol.